19

Inflammation

19

Inflammation

519

Inflammation

19

Inflammation

19.1 Inflammation

Lothar Thomas

Inflammation is an important physiological response for the maintenance of tissue homeostasis, protecting the host against foreign substances, invading microorganisms and host self disturbances, such as the molecules derived from metabolically stressed or even dying cells. After the host has been incited, important microcirculatory events occur in response to local release of pro inflammatory mediators (prostaglandines, histamine, leukotrienes, chemokines, cytokines) leading to increased vascular permeability and leukocyte recruitment. Inflammatory cells, such as neutrophils and macrophages release further inflammatory mediators and acting as effector cells and phagocytes to remove the inflammatory agents and stimuli /1/.

The involvement of the whole body in the acute inflammatory response is known as an acute-phase reaction (APR). During an APR, mechanisms that operate remote from the site of local inflammation can overwhelm the normal homeostasis of the organism. Many organ systems become involved in the host-defense process, primarily through systemically active inflammatory cytokines /2/.

Continuous inflammatory stimuli (longer than three months), also known as chronic inflammation, can be detrimental to the organism and are predisposing factors for many chronic diseases. Chronic inflammation is a predictor of disease and mortality, even in the absence of clinical symptoms. This applies in particular to individuals with low-grade systemic inflammation, who have slightly elevated inflammatory markers such as C-reactive protein compared to the rest of the population. The secretion of mediators of inflammation by white adipose tissue in obesity, for example, results in low-grade inflammation and an increased long-term risk of cardiovascular disease, stroke and type 2 diabetes /34/.

In patients with severe trauma, survival rates depend on whether inflammatory states such as acute lung injury (ALI), adult respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome (MODS) and their associated complications are present.

19.1.1 Local inflammatory response

Complex organisms have three mechanisms to defend against foreign substances and invading pathogens:

  • The skin and mucous membranes act as the first barrier
  • The innate immune system provides the second line of defense
  • The last line of defense is the antigen-dependent acquired immune system.

Inflammatory response

Inflammation is the innate immune system’s response to tissue injury. Because the response to a particular foreign agent is always the same (qualitatively and quantitatively) in the same patient, the response to re-exposure is similar to the initial response.

Inflammatory stimuli

Examples of inflammatory stimuli include bacteria, viruses, parasites, allergens, immune complexes, and mechanical and surgical tissue damage (myocardial infarction, bone fracture, foreign agents, erythrocyte shear stress, hydrostatic pressure, urate crystals, and uremia).

Symptoms of local inflammation

Pain (dolor), heat (calor), redness (rubor), swelling (tumor), and loss of function (functio laesa).

19.1.2 Course of inflammatory response

At microscopic level, local clinical symptoms are the result of /5/:

  • Microcirculatory events; dilation of arterioles, capillaries, and venules, associated with increased blood flow lead to higher vascular permeability
  • Exudation of fluid and plasma proteins into the tissues
  • Leukocyte recruitment and migration to the inflammatory focus.

From a pathophysiological perspective, local inflammation comprises a broad range of cellular, intercellular, and intracellular processes that facilitate the phagocytosis of the foreign agent and recruit additional cells to the inflammatory process. The type of inflammation that is triggered by each different foreign agent represents a complex, concerted, finely-tuned process that is focused on the inflammatory stimulus, in which soluble inflammatory mediators, immune cells, and local tissue cells interact with one another. If local inflammation develops into an acute-phase response, the local process is then controlled by the systemic metabolic changes associated with the acute-phase reaction /6/. The inflammatory response is highly individualized. For example, one individual might experience a bacterial peritoneal infection as a well-encapsulated abscess, while another might develop diffuse peritonitis and possibly multi organ failure /5/.

Local inflammatory effects

Various stimuli trigger a cellular, inter cellular, and intracellular inflammatory response at the site of tissue damage via the following mechanisms:

  • Activation of the complement system, kinin system, and coagulation cascade. The products of these systems, such as C5a, bradykinin, and thrombin, increase vascular permeability, attract leukocytes, and alter vasomotor tone at the inflammatory site. These soluble mediators induce cellular reactions. Local macrophages are activated and produce pro inflammatory mediators such as TNF-α, IL-1β, and chemokines. Chemokines recruit leukocytes and determine which leukocytes will accumulate at a particular site at a particular time.
  • Stimulation of inflammatory cells such as polymorphonuclear neutrophils, eosinophils, monocytes/macrophages, dendritic cells, mast cells, fibroblasts, smooth muscle cells, thrombocytes, and lymphocytes. Intercellular actions occur: inflammatory cells interact with each other and with vascular endothelial cells. Lipid mediators (eicosanoids), reactive oxygen species, heat shock proteins, and selectins are produced.
  • Activation of intracellular communication in inflammatory cells. This is a prerequisite for synthesizing new mediators such as cytokines, chemokines, and adhesion molecules, which in turn involve polymorphonuclear neutrophils, monocytes, T-cells, and dendritic cells in the inflammatory process. The intracellular processes for producing inflammatory proteins depend on the transcription of particular DNA sequences under the control of transcription factors. Nuclear factor kappa B (NF-kB) is a pro inflammatory transcription factor that has an important role in regulating the synthesis of RNA and, therefore, proteins /7/. In non-activated cells, NF-kB is present in the cytoplasm bound to the inhibitor kappa B (IkB) and prevents NF-kB from permeating into the nucleus. When an inflammatory cell becomes activated (e.g., by TNF-α) IkB is phosphorylated and degraded. This enables NF-kB to enter the nucleus, where it binds to the promoter region of a target gene and initiates the synthesis of inflammatory mediators (Fig. 19.1-1 – The activation of NFκB takes place through phosphorylation and subsequent proteolytic degradation of the inhibitor protein IκB by IκB kinases). NF-kB is essential for normal immune function, however, over stimulation can lead to inflammation and tumorigenesis /7/.

Fig. 19.1-2 – Activation of the pro inflammatory cascade by lipopolysaccharide (LPS) shows the pro inflammatory cascade that is activated when a monocyte is stimulated by a lipopolysaccharide.

19.1.3 Recognition mechanisms in the inflammatory response

The innate immunity represents the first barrier in the host immune defense; it identifies pathogens or other harmful triggers inducing an inflammatory process with the aim of blocking their diffusion, and activates adaptive immunity. The effectors are:

  • The complement system
  • Inflammatory proteins e.g., C-reactive protein
  • Effector cells of innate immunity, e.g., macrophages, dendritic cells and other antigen presenting cells (APC).

The innate immune system acts through pattern recognition receptors which bind to highly conserved structures expressed by pathogens (pathogens associated molecular patterns, PAMPs) or by damaged cells (damage associated molecular patterns DAMPs) /8/. More specific recognition (immune recognition) takes place subsequently by cellular (T and B-lymphocytes) and humoral components (antibodies) of the adaptive immune system.

Three classes of pattern recognition receptors have been identified /9/:

  • Toll-like receptors (TLRs)
  • Retinoic acid-inducible gene-I (RIG-I-) like receptors (RLRs)
  • Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs).

19.1.3.1 Recognition by the complement system

The alternative complement pathway is activated by complex structured molecules and by denatured foreign agents. These include lipopolysaccharides in the cell walls of Gram-negative bacteria (endotoxins), viral envelopes, and denatured erythrocyte membranes. Inflammation is initiated by /10/:

  • The formation of membrane attack complex C5b-9, which punches holes in the bacterial and cellular lipid membranes. The membrane attack complex is particularly effective against Gram-negative bacteria. It is formed following activation of the complement cascade through the deposition of complement component C3b on the surface of the target cell. Target cells coated with C3b are recognized by macrophages, which engulf the target cell and digest it.
  • Generation of anaphylatoxins C3a and C5a. Both of these induce contraction of smooth muscles in vessel walls, increased vascular permeability, and mast cell degranulation. C5a is a chemoattractant and induces the accumulation of neutrophilic granulocytes and macrophages at the site of tissue damage.

Refer to Fig. 24-1 – Pathways activating the complement system

19.1.3.2 Recognition by the adaptive immune system

The adaptive immune system recognizes foreign agents using surface receptors on macrophages, T-cells, and B-cells as well as antibodies produced by B-cells. After binding of an antigen to the B-cell receptor the cells differentiate into plasma cells, which synthesize IgM and IgG antibodies against the corresponding antigen. Immune complexes are formed, which activate the classic pathway of the complement system and trigger an inflammatory response.

19.1.3.3 Production of heat shock proteins (HSP)

HSP are usually found in various intracellular compartments and some have chaperone and protease functions /11/. Chaperones are proteins that are involved in the assembly, folding, and stabilization of oligomeric proteins. HSPs are classified according to their molecular weight (e.g., 60 kDa HSP or 70 kDa HSP).

HSPs are released into the extracellular space by necrotic and stressed (but not apoptotic) cells, where they usually constitute 5–10% of the total protein. An increased presence always indicates cellular compromise or unnatural cell death. HSPs have a pro inflammatory effect in the extracellular compartment. They act as intercellular signaling molecules to induce the production of pro inflammatory mediators, and can trigger an inflammatory response. They cause vascular endothelial cells to express E-selectin, adhesion molecules such as ICAM-1 and VCAM-1, and interleukin 6 (IL-6). Bacterial HSPs also induce the production of pro inflammatory mediators.

The stimulation of inflammatory cells by HSP to produce pro inflammatory cytokines occur after their uptake by macrophages and dendritic cells. These cells present HSPs together with MHC class I proteins on their cell surfaces. There they are recognized by CD4+T-cell receptors, which activate the cells to produce inflammatory cytokins. Fig. 19.1-3 – Induction and regulation of heat shock protein (Hsp) expression depicts the induction and regulation of HSP expression.

19.1.4 Mediators of inflammation

The recognition of foreign molecules is followed by the activation of intracellular signal transduction pathways, which induces the expression of genes for the production of inflammatory mediators. These molecules enhance the inflammatory response. Their role is to recruit other effectors of the host-defense mechanisms into the inflammatory response. Mediators are humoral substances that are produced from endogenous lipids or peptides, are secreted by activated inflammatory cells, or can even be released by invading bacteria. Initially small stimuli can be strongly amplified by mediators.

The main task of inflammatory mediators is to recruit inflammatory cells to the site where the foreign agent is localized or where tissue damage is present. The main mediators of the inflammatory response are presented in Tab. 19.1-1 – Mediators of inflammation.

The eicosanoids are essential mediators of inflammation:

19.1.5 Inflammatory cells

The mediators of inflammation recruit inflammatory cells to accumulate at the site of tissue damage; this is an essential step in the inflammatory response /12/.

The recruitment sequence starts with the extravasation of polymorphonuclear neutrophils (PMNs), followed by monocytes (Fig. 19.1-7 – Sequential interaction between leukocytes and the vascular endothelium, regulated by adhesion molecules).

Molecules involved in the recruitment of inflammatory cells are shown in Tab. 19.1-3 – Chemokine groups and their effects on immune cells.

Monocytes are recruited as follows:

  • Proteins are released from the granules of activated PMNs. These induce the migration of activated monocytes to the vascular region of the inflammation site. The β2 integrins and formyl peptide receptors play an important role in this.
  • PMNs modify the chemokine network and their granular proteins create an environment that facilitates monocyte extravasation.
  • Accumulated PMNs at the site of inflammation are subject to rapid apoptosis, which leads to the release of lysophosphatidylcholine. As a result, monocytes are captured by G2A receptors.

Monocytes are extravasated as follows:

  • Selectins on the vascular endothelium (P-selectins) and on monocytes (L-selectins) allow the monocyte to attach to the vascular wall and roll along its surface
  • When it slows down, the monocyte can detect endothelium-bound chemokines and chemoattractants such as PAF and leukotriene B4, which activate the monocyte
  • Monocyte activation leads to the release of integrins, which interact with adhesion molecules of the vessel wall (ICAM-1, VCAM-1) to fix the monocyte to the endothelium
  • Chemokines than induce cytoskeletal changes in the monocyte, which lead to trans epithelial migration and extravasation.

Other important effector cells in inflammation (in addition to PMNs and monocytes) are:

  • Platelets, which are recruited to the damaged vessel wall by the interaction of platelet glycoprotein (GP) Ib with von Willebrand factor and the subendothelial matrix
  • Dendritic cells, which are important in chronic inflammation
  • Tissue-specific mast cells and basophils, which have a role in the immediate hypersensitivity response.

The adhesion molecules involved in leukocyte extravasation are shown in Tab. 19.1-4 – Molecules involved in leukocyte recruitment.

19.1.6 Acute phase reaction (APR)

The acute phase response is a highly orchestrated defense mechanism against infectious and inflammatory insults. The initiation of this response is predominantly driven by the endogenous cytokines interleukin-1β, interleukin-6 and tumor necrosis factor (TNF)-α, induced in macrophages and other leukocytes by exogenous pathogens binding to toll-like receptors. Besides systemic and metabolic changes, such as fever and anorexia, these cytokines also provide the induction of acute phase proteins in the liver /2/.

The systemic changes control the local inflammatory response through the following reactions (Fig. 19.1-8 – Systemic changes in an acute phase response/6/:

  • Fever: increased temperature increases enzymatic reactions in inflammatory cells that do not occur optimally at normal temperature due to their potentially damaging effects
  • Leukocytosis: a large number of phagocytic cells become available
  • Increased hormone secretion to mobilize energy from glucose, free fatty acids, and amino acids
  • Synthesis of acute phase proteins (APP) and restricted synthesis of albumin, transferrin, and lipoproteins (negative acute phase proteins) to supply tissues with inflammatory proteins
  • Production of hepcidin to reduce functional iron by increasing its storage in macrophages and inhibiting enteral iron absorption and hemoglobin synthesis; iron withdrawal restricts the growth of microorganisms
  • Initiation of counter-regulatory mechanisms. Thus, the potentially destructive effects of the inflammation are modulated and reduced through an ACTH induced increased release of cortisol.

The APR is an integrated reaction between pro inflammatory and anti inflammatory stimuli. The systemic changes signaled in the tissues in an APR are mediated by inflammatory cytokines that are produced in inflammatory cells. Distinctions are made between (i) pro inflammatory cytokines (TNF-α, IL-1β, IL-6, transforming growth factor β, interferon-γ) and (ii)anti-inflammatory cytokines (IL-4, IL-10, IL-13).

APRs can be different. For example, comparable degrees of tissue damage caused by different agents can trigger APRs with different degrees of severity. The same injury (e.g., infection) can lead to a normal inflammatory response in one patient and an inappropriate response in another. Genetic polymorphism of cytokine production is an important cause of this phenomenon.

19.1.7 Compensatory anti-inflammatory response syndrome (CARS)

The goal of the acute phase reaction (APR) is to achieve an appropriate balance between activating and down-regulating mechanisms that benefits the organism as a whole /13/. An inappropriate inflammatory response to harmless localized inflammation can prevent healing and has negative consequences for the organism that can culminate in SIRS, sepsis, or MODS, for example. The nature and dose of the antigen that is presented by the antigen-presenting cell and the genetic polymorphism of cytokine production determine the course of the APR. The extent to which the response of the T-helper cell system (also known as the T-helper cell (Th1/Th2) paradigm) is influenced is also important. The Th1 system has pro inflammatory effects while the Th2 system down-regulates inflammation (Fig. 19.1-9 – Th1/Th2 paradigm in immune-mediated inflammation). If these systems are not in balance, this can have lethal consequences for the patient.

An appropriate balance between APR and CARS is the goal of acute inflammation. Systemically mechanisms of CARS that counteract the APR are:

  • Dilution of pro inflammatory cytokines when they escape from the area of injury; they are diluted in the large volumes of the interstitial fluid and the circulation
  • Production of soluble receptors (e.g., for TNF-α and IL-1β). These circulating receptors bind their ligands and thereby inhibit the binding at the receptors on target cells.

19.1.8 Acute phase proteins (APPs)

APPs are proteins whose concentration rises (positive APPs) or falls (negative APPs) by more than 25% during the course of an inflammatory disease /14/. IL-6 is the main stimulator of APP synthesis and can induce exactly the same pattern of APP synthesis in cell cultures as in inflammatory reactions. APPs are synthesized in the hepatocytes. At the peak of the acute phase response (APR), around 20% of the protein synthesis capacity of the liver is used to produce APPs.

The positive APPs consist of a family of around 30 proteins. The peak increase fluctuates depending on the protein, from a 0.5-fold increase for ceruloplasmin to a 1,000 fold increase for C-reactive protein (CRP) /15/. Although the APPs usually rise together in concert during an APR, there are significant differences between the response time, maximum rise, and half-life of the decline since the individual proteins are regulated separately. Refer to:

Fast-reacting APPs such as CRP increase rapidly following an infection and also decrease again quickly following successful treatment (Fig. 19.1-11 – Time profile of CRP and body temperature).

In sterile inflammation, the increase in the APR is determined by the extent of tissue damage (Fig. 19.1-12 – CRP increase in relation to the extent of surgical intervention).

An imbalance between the acute phase proteins and the acute phase response (APR) occurs if the APR is associated with other pathologies. This is the case for /6/:

  • Fibrinogen: its concentration can be normal or reduced in an APR if disseminated intravascular coagulation is also present
  • Haptoglobin (Hp) with a combination of APR and intravascular hemolysis. Furthermore, 20% of African Americans have reduced Hp α-chain synthesis, which means that the normal increase in Hp during an APR is moderate.
  • α1-antitrypsin: if a polymorphism with reduced synthesis is present, the protein does not increase, or does not increase in proportion to the APR. Prevalences of the genetic polymorphism: Type ZZ, 1 : 2080; type SZ, 1 : 1160; both types combined, 1 : 750.
  • Complement protein C3: this does not increase appropriately with a combination of APR and immune complex disease.

The nature of the inflammatory stimuli is an important determinant of APP activity (Tab. 19.1-6 – Acute phase response induced by various inflammatory stimuli/6/.

19.1.8.1 Function of acute phase proteins

During an APR, the serum concentration of most positive APPs, with the exception of serum amyloid A, increases much less than that of CRP. The concentration of the negative APPs declines. Directly or indirectly, all positive and negative APPs have an inflammatory or anti-inflammatory effect.

The following pathophysiological effects can be assumed for the individual APPs /15/:

  • Binding of exogenous and endogenous ligands and promotion of their removal from the circulation by facilitation of opsonization
  • Because substances that contain serum amyloid A (SAA) increase the phagocytic activity of macrophages, SAA has a pro inflammatory effect
  • Haptoglobin and hemopexin are positive APPs and protect against reactive oxygen species; haptoglobin also promotes wound healing by stimulating angiogenesis
  • α1-antichymotrypsin and α1-proteinase inhibitors are positive APPs that inhibit the activity of proteolytic enzymes; α1-antichymotrypsin also inhibits the production of superoxide anions
  • Transthyretin, a positive APP, inhibits the production of IL-1 by monocytes and endothelial cells
  • Fibrinogen, a positive APP, mediates the binding of inflammatory cells, in particular platelets, to the vascular endothelium and plays an important role in wound healing
  • Transferrin, a negative APP, reduces the functional iron so that less iron is supplied to the tissues and invading microorganisms
  • Lipoproteins are negative APPs as well as the substrate for eicosanoid production via arachidonic acid metabolism and the cyclooxygenase and lipoxygenase pathways
  • Albumin is a negative APP whose synthesis is inhibited by pro inflammatory cytokines. The pathophysiological effect of reduced albumin during an APR is not known.

19.1.9 Non resolving inflammation

The usual result of inflammation is protection from the spread of infection, followed by resolution (the restoration of affected tissues to their normal structural and functional state). Non resolving inflammation is a major driver of disease. Perpetuation of inflammation is an inherent risk because inflammation can damage tissue and necrosis can provoke inflammation /3/.

Inflammation sometimes progresses from acute to chronic and then stalls for a prolonged period, although signs of acute inflammation, such as accumulation of neutrophils, may reappear later. Classic examples involve persistent infections. The most straightforward cause of non resolving inflammation is the persistence of inflammatory stimuli of exogeneous origin e.g., persistent infections by M. tuberculosis, H. pylori, schistosomes and hepatitis viruses /3/.

Often the host inflammatory response, not the pathogen, is chiefly responsible for the damage of the host. Some chronic inflammatory diseases appear to begin with repeated exposure to a pathogen, leading to tissue injury that prevokes an autoimmune reaction. The autoimmune reaction may then perpetuate the inflammation. This is the view of chronic obstructive pulmonary disease /3/.

Nondegradable particles of asbestos and silica that trigger inflammation promote chronic inflammation.

Acute and chronic inflammation can coexist over long periods, implying continual reinitiation. Examples are found in rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, multiple sclerosis, Crohn’s disease, ulcerative colitis and cancers whose stroma is infiltrated both by macrophages and immature myeloid cells /16/.

Non resolving inflammation is not a primary cause of atherosclerosis, obesity, cancer, chronic obstructive pulmonary disease, asthma, inflammatory bowel disease, neurodegenerative disease, multiple sclerosis, or rheumatoid arthritis, but it contributes significantly to their pathogenesis /3/.

Acute phase proteins behave differently in non resolving inflammation /6/:

  • In rheumatoid arthritis, psoriatic arthropathy, and Reiter’s syndrome, CRP elevation indicates a persistent inflammatory process and reflects the mass of inflamed tissue. In degenerative inflammatory processes such as osteoarthritis, CRP is not usually elevated.
  • Poly myalgia rheumatica is associated with a significant elevation in CRP at diagnosis, whereas fibromyalgia is not
  • While CRP can be increased in immune complex vasculitides such as Wegener’s granulomatosis and polyarteritis nodosa, it is not a direct biomarker of inflammatory activity. It is only useful for adjusting corticosteroid therapy to minimize side effects.
  • In inflammatory small bowel disease, CRP is significantly increased in active ulcerative colitis but only slightly increased in Crohn’s disease. Because of the significant overlap in values, CRP is not suitable for differentiating between these two diseases. However, it can be used to differentiate inflammatory bowel disease from irritable bowel syndrome, in which CRP is always normal.

Successful pro inflammatory tissue repair requires the coordinated restitution of different cell types and structures, including epithelial and mesenchymal cells but also extracellular matrix and vasculature. Without appropriate restitution of vasculature, reduced tissue oxygenation may preclude normal tissue repair resulting in atrophy or fibrosis. Atrophy is often accompanied by expansion of extracellular tissue elements, particularly collagen, resulting in fibrosis, the deposition of excess connective tissue. Fibrosis sufficient to interfere with organ function can arise without known preceding inflammation /3/.

19.1.10 Auto inflammation

Auto inflammatory diseases (AIDs) refer to a group of rare, hereditary recurrent unprovoked inflammatory disorders which occur in the absence of infection. What is auto inflammation from the pathogenetic point of view /8/?

  • The pathological process is directed against self but the patients do not have autoantibodies or auto reactive antigen-specific T cells and B cells driving the disease process
  • Monocytes and macrophages rather than T and B cells are drivers for inflammation and damage
  • The chronic activation of the immune system, which eventually leads to tissue inflammation occurs in genetically predisposed individuals
  • Auto inflammation includes monogenic and polygenic diseases
  • Frequently the skin and musculoskeletal systems are involved
  • The innate immune system directly causes tissue inflammation
  • Patients affected with AID do not have associations with the major histocompatibility (MHC) class II haplotypes.

Mutations in inflammasome-related proteins, particularly in NOD-like receptor (NLR) genes are associated with the occurrence of AIDs. Inflammasomes are large protein complexes serving as a molecular platform which mediates the activation of pro-caspase-1, which cleaves the pro form of IL-1β to the active form. Activation of NLR proteins (NLRPs) results in the formation of inflammasomes. Inflammasome activation is crucial for host defense of pathogens.

AIDs are strongly linked to mutations in inflammasome-forming NLRs. The majority of patients with AIDs have mutations in either pyrin, cryopyrin (NLRP3) or tumor necrosis factor receptor super family genes /8/.

IL-1β is not only triggered by infectious signals but also by signals released from metabolically stressed or even dying cells. NLRP3 containing inflammasomes are intracellular receptors that are triggered not only in response to exogenous pathogens but also to endogenous stress molecules and coordinate processing and IL-1 secretion through caspase-1 activation. Metabolic substrates that accumulate in target tissues can stimulate the NLRP3 inflammasome to release IL-1β. Metabolic substrates that accumulate in target tissues are urate in gout, islet amyloid polypeptide and oxidized low-density lipoprotein (oxLDL) in diabetes, ceramide and others in obesity, and cholesterol crystals in atherosclerosis /17/.

The majority of monogenic AIDs clinically occur in neonatal period or early infancy. Polygenic AIDs usually develop during adolescence. Rarely AIDs occur in the elderly.

AIDs are multi systemic diseases, in which skin, musculoskeletal or gastrointestinal involvement is commonly observed. Auto inflammatory diseases are shown in Tab. 19.1-7 – Auto inflammatory diseases.

Reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Antiinflammatory therapy targeting the IL-1β innate immunity pathway with canakinumab leads to a significantly lower rate of recurrent cardiovascular events than placebo /18/.

Best Practice Guidelines for the determination of 4 genes (ADA2, NOD2, PSTPIP1, and TNFAIP3) of monogenic autoinflammatory diseases are published in Ref. /40/.

19.1.11 Graft-versus-host disease

Acute graft-versus-host disease (GVHD) occurs when immunocompetent T cells in the donated tissue (the graft) recognize the recipient (the host) as foreign. The resulting immune response activates donor T cells to gain cytolytic capacity and to the attack the recipient to eliminate foreign antigen-bearing cells. Important target organs are the skin, the gastrointestinal tract, and the liver. In the early phase of acute GVHD inflammation triggers can drive both innate and adaptive immune responses /19/.

The triggers are divided into two categories /19/:

  • Sterile damage associated molecular pattern (DAMP) molecules. These molecules are released into the extracellular space only when there is tissue damage that causes immune activation.
  • Pathogen associated molecular pattern (PAMP) molecules. Bacterial components engage immune system pattern recognition receptors, such as toll-like receptors and NOD receptors to activate antigen presenting cells and promote acute GVHD.

Risk factors for acute GVHD include the degree of HLA mismatch, receipt of a transplant from an unrelated donor, a female donor for a male recipient, the use of peripheral blood stem-cell grafts, and the intensity of the conditioning regimen /19/.

Signs of typical acute GVHD include maculopapular rash, jaundice, nausea, vomiting, anorexia, watery or blood diarrhea and cramp abdominal pain /19/.

To predict the risk of GVHD in recipients of allogenic transplantation an early biomarker algorithm predicts lethal GVHD and survival /20/.

19.1.12 Anaphylactoid reaction

An anaphylactoid reaction is an acute allergic reaction with the symptoms of anaphylaxis. Three different pathogenetic mechanisms of antigen exposure that lead to anaphylaxis are distinguished /21/:

  • Immediate allergic reaction mediated by IgE (type 1 in Gell-Coombs classification). Re exposure to an antigen or hapten weeks after initial exposure results in the release of vasoactive substances from IgE-containing mast cells within minutes. The granules in these cells are filled with histamine and tryptase. Each cell has more than 100,000 IgE receptors. The binding of IgE to the receptors stimulates degranulation and, since mast cells are located perivascularly in the skin, lung, and intestine, the surrounding tissue is involved immediately in the reaction. In addition to the release of vasoactive substances such as histamine that cause increased capillary permeability, vasodilatation, and bronchoconstriction, lymphocytes, monocytes, and polymorphonuclear neutrophils are also attracted by chemoattractants to act as amplifiers of the inflammatory reaction. An acute phase reaction is also triggered.
  • Complement-mediated anaphylaxis (Gell-Coombs type III). The complement system is activated by immune complexes and activated factors C3a and C5a cause vasoactive mediators of inflammation to be released from mast cells.
  • Chemicals such as water-soluble X-ray contrast medium can provoke an anaphylactoid reaction mediated by IgE or the complement system.

19.1.13 Systemic inflammatory response syndrome (SIRS)

A 1991 consensus conference developed initial definitions that focused on the then-prevailing view that sepsis resulted from a host’s systemic inflammatory response syndrome to infection. Sepsis complicated by organ dysfunction was termed severe sepsis, which could progress to septic shock, defined as sepsis induced hypotension persisting despite adequate fluid resuscitation /22/. Components of SIRS include tachycardia, tachypnea, hyperthermia or hypothermia and abnormalities in peripheral blood cell count. However, the presence of SIRS is nearly ubiquitous in hospitalized patients and occurs in many benign conditions, both related to and not related to infection, and thus is not adequately specific for the diagnosis of sepsis. According to the new consensus definition of sepsis no longer includes SIRS /23/. SIRS is defined as two or more of the variables presented in Tab. 19.1-8 – Diagnostic variables of SIRS.

19.1.14 Sepsis and septic shock

A fundamental component of the new definitions for sepsis and septic shock remains the presence of infection. Yet negative microbiologic cultures from blood or relevant anatomic sites are frequent in patients clinically identified as being septic /23/. However, new techniques thus as those using matrix-associated laser de sorption ionization-time of flight (MALDI-TOF) /24/, metabolites like myristic acid in blood /25/ or circulating microRNAs /26/ are likely to enhance the current ability to diagnose infection.

Criteria for organ dysfunction in sepsis represented by the SOFA score are listed in Tab. 19.1-9 – SOFA score. The score grades abnormality of six organs in five degrees of severity (0–4) including laboratory tests, namely PaO2, platelet count, creatinine concentration and bilirubin level are needed for full computation. A severely limited function is rated with four points, normal function is rated with zero points. In total 0–24 points are possible. A score of 2 points or more is associated with an in-hospital mortality greater than 10% /27/.

Sepsis

According to the new definitions, sepsis is now defined as evidence of a life threatening organ dysfunction caused by deregulated host response to infection plus life-threatening organ dysfunction, clinically characterized by an acute change of 2 points or greater in the SOFA score. The consensus document introduced a new index, called qSOFA, which helps to identify patients with suspected infection without using laboratory tests. The quick SOFA (qSOFA) requires at least ≥ 2 of the following 3 risk variables /27/:

  • Respiratory rate of 22 or more breaths per minute
  • Systolic blood pressure of 100 mmHg or less
  • Altered mental status (Glasgow coma scale < 15).

Septic shock

Septic shock should be defined as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone /27/. Patients with septic shock can be clinically identified by a vasopressor requirement to maintain a mean arterial pressure of 65 mmHg or greater and serum lactate levels greater than 18 mg/dL (2 mmol/L) in the absence of hypovolemia. This combination is associated with hospital mortality rates greater than 40%.

19.1.14.1 Laboratory tests in SIRS and sepsis

Laboratory investigations to detect clinically suspected focal infection, SIRS and sepsis are listed in Tab. 19.1-10 – Laboratory tests in SIRS and sepsis and disease monitoring.

19.1.15 Effect of anti-inflammatory drugs

19.1.15.1 Nonsteroidal anti-inflammatory drugs

Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents in the treatment of arthritis and other musculoskeletal disorders, and as analgesics in a variety of clinical scenarios. It is generally accepted that NSAIDs exert their anti-inflammatory effects by inhibiting prostaglandin synthesis. Unfortunately the use of COX-2 inhibitors has been limited by their association with mucosal injury to the upper gastrointestinal tract, including the development of peptic ulcer disease and its complications, most notably upper gastrointestinal hemorrhage, and perforation /28/.

However, individual NSAIDs have different targets:

  • Indomethacin, fenoprofen, and ibuprofen inhibit the enzyme cyclooxygenase (COX) through competitive displacement of arachidonic acid from the substrate binding site
  • Imidazole and dazoxiben inhibit COX leading to a significant decrease in prostaglandin production. COX exist as two isoenzymes, COX-1 and COX-2. COX-1 is a constitutive enzyme and exists in many tissues. COX-2 is an inducible enzyme and is associated with inflammation in the joints. The selective inhibition of COX-2 leads to decreased inflammation in musculoskeletal tissues.
  • Acetylsalicylic acid (ASA) acts by irreversibly acetylating a serine residue within the active center of cyclooxygenase. This irreversible inhibition of cyclooxygenase is the basis for using low-dose ASA for atherosclerosis prophylaxis. Cyclooxygenase is the enzyme responsible for TXA2 synthesis in platelets and for PGI2 synthesis in the vascular endothelium. Studies have shown that the effect of ASA on vascular endothelial cells is only short-term and therefore incomplete while its effects on platelets last 8–10 days. This is because vascular endothelial cells have a nucleus and can quickly regenerate new cyclooxygenase whereas platelets do not have a nucleus and cannot generate the enzyme, which means that the effect of ASA lasts for the duration of the life of the platelet. TXA2 has a proaggregatory effect on thrombocytes and has a vasoconstrictor effect on the vascular system, while PGI2 has an effect in the opposite direction, so that ASA has an anti thrombotic effect.

19.1.15.2 Glucocorticoids

Glucocorticoids (GCs) are used in pharmacological concentrations (cortisol 0.5–1 mg/L) in the following situations /29/:

  • Treatment of inflammatory diseases
  • Suppression of inflammation and immune responses associated with organ transplantation
  • Any disease that is associated with increased expression of pro inflammatory cytokines, chemokines, and adhesion molecules.

At pharmacological concentrations GCs /30/:

  • Inhibit the release of pro inflammatory cytokines and stimulate the production of antiinflammatory cytokines such as IL-10. Overall, inflammation is down-regulated through inhibition of the Th1 immune response and stimulation of the Th2 immune response (Fig. 19.1-9 – Th1/Th2 paradigm in immune-mediated inflammation)
  • Inhibit inflammatory mediators. GCs decrease the production of eicosanoids by inhibiting COX-2; leukotriene catabolism is accelerated. Nitric oxide (NO) synthesis is reduced through the inhibition of inducible NO synthase (iNOS) in macrophages, polymorphonuclear neutrophils, and endothelial cells.
  • Inhibit cell surface markers. GCs down-regulate the expression of surface markers such as the CD14 endotoxin receptor and adhesion molecules ELAM-1 and ICAM-1 on endothelial cells. This prevents polymorphonuclear neutrophils from binding to the endothelium and migrating through the vascular wall to the inflammatory focus. The migration of lymphocytes is also slowed down due to the inhibition of the expression of LFA-1 and CD2.

GCs exert their effects on the cells via the glucocorticoid receptor, which forms a complex with a heat shock protein. The receptor becomes dimerized and permeates the nuclear membrane (Fig. 19.1-13 – Regulation of gene expression by glucocorticoids (GCs)). An overview of the effects of glucocorticoids is shown in Fig. 19.1-14 – Actions of glucocorticoids (GCs).

At physiological concentrations (cortisol 50–200 μg/L), glucocorticoids (GCs) support the inflammatory response as follows /30/:

  • Pro inflammatory cytokines (TNF-α, IL-1, IL-6) stimulate the production of GCs via the hypothalamic-pituitary-adrenal axis, which in turn promotes the synthesis of acute phase proteins in the liver
  • GCs increase the expression of cytokine receptors on their target cells (e.g., T-cells) which makes the target cells more sensitive to cytokines
  • GCs have a stimulatory effect on the release of macrophage migration inhibitory factor (MIF). It is released by lipopolysaccharide-stimulated macrophages and has an inflammatory effect.
  • GCs induce apoptosis in lymphocytes and eosinophils, to a lesser extent in monocytes, and do not induce apoptosis in polymorphonuclear neutrophils.

The effect of physiological concentrations of GCs with an initial increase in receptors for pro inflammatory cytokines followed by inhibition of their synthesis serves to trigger an inflammatory response quickly but also end it again quickly.

References

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3. Nathan C, Ding A. Nonresolving inflammation. Cell 2010; 140: 871–82.

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6. Whicher J. The acute phase response. In: Ritchie RF, ed. Serum proteins in clinical medicine, Vol II. Scarborough; Foundation for Blood Research 1999: 105.001–21.

7. Aggarwal BB. Nuclear factor kB: the enemy within. Cancer cell 2004; 6: 203–8.

8. Doria A, Zen S, Bettio M, Getto N, Bassi L, Nalotto A, et al. Autoinflammation and autoimmunity: bridging the divide. Autoimmunity Rev 2012; 12: 22–30.

9. Martinon F, Mayor A, Tschopp J. The inflammasomes; guardians of the body. Annu Rev Immunol 2009; 27: 229–65.

10. Ali H, Haribabu B, Richardson RM, Snyderman R. Mechanisms of inflammation and leukocyte activation. Med Clin North Am 1997; 81: 1–28.

11. Pockley G. Heat shock proteins as regulators of the immune response. The Lancet 2003; 362: 469–76.

12. Soehnlein O, Lindbom L, Weber C. Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood 2009; 114: 4613–23.

13. Bone RC. Sir Isaac Newton, sepsis, SIRS and CARS. Crit Care Med 1996; 24: 1125–9.

14. Morley JJ, Kushner I. Serum C-reactive protein levels in disease. Ann NY Acad Sci 1982; 389: 406–18.

15. Gabay C, Kushner I. Acute phase proteins and other systemic responses to inflammation. N Engl J Med 1999; 340: 448–55.

16. Mantovani A, Aliavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature 2008; 454: 436–44.

17. Goldbach-Mansky R. Immunology in clinic review series; focus on autoinflammatory diseases: update on monogenic autoinflammatory diseases: the role of interleukin (IL)-1 and an emerging role of cytokines beyond IL-1. Clinical and Experimental Immunology 2011; 167: 391–404.

18. Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, et al. Antiinflammatory therapy with canakinumab for atherosclerotic disease. N Engl J Med 2017; 377 1119–31.

19. Zeiser R, Blazar BR. Acute graft-versus-host disease. Biologic process, prevention, and therapy. N Engl J Med 2017; 377: 2167–79.

20. Hartwill MJ, Özbek U, Holler E, Renteria AS, Major-Monfried H, Reddy P, Aziz M, et al. An early-biomarker algorithm predicts lethal graft-versus-host disease and survival. JCI Insght 2017: 2(3): e89789. https://insight.jci.org/articles/view/89798.

21. Walther A, Böttiger BW. Anaphylaktoide Reaktionen in der Prähospitalphase. Internist 2004; 45: 296–304.

22. Bone RC, Balk RA, Cerra FB. Definitions for sepsis and organ failure and guidelines for the use innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. Chest 1992; 101: 1644–55.

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24. Buehler SS, Madison B, Snyder SR, Derzon JH, Comish NE, Saubolle MA, et al. Effectiveness of practices to increase timeliness of providing targeted therapy for inpatients with bloodstream infections: a laboratory medicine best practices systematic review and meta-analysis. Microbiol Rev 2016; 29: 59–103.

25. Kauppi AM, Edin A, Ziegler I, Möling P, Sjöstedt A, Gylfe A, et al. Metabolites in blood for prediction of bacteremic sepsis in the emergency room. PloS One 2016, 11(1) e0147670

26. Benz F, Roy S, Trautwein C, Roderburg C Luedde T. Circulating microRNAs as biomarkers of sepsis. Int J Mol Sci 2016; 17,78 https://doi.org/10.3390/ijms17010078.

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19.2 Oxidative stress

Lothar Thomas

In the body, the oxidation-reduction reactions which effect the transfer of one species with reducing character to another with oxidizing character are particularly important because they activate or inactivate some biologically active compounds. The entirety of redox reactions may create particularly reactive secondary products, oxygen or nitrogen species of radical or non radical type, commonly referred to as reactive oxygen species (ROS) and reactive nitrogen species (RNS) /12/.

The body can counteract the high reactivity of ROS and RNS by synthesizing antioxidants. Antioxidants are species that prevent ROS and RNS formation and their interaction with biostructures.

Excess ROS and RNS causes oxidative stress, a pathogen condition when the antioxidant defense can no longer compensate for the production of ROS and RNS. Oxidative stress can generate multiple diseases e.g., degradation of cell membranes, DNA, lipids, proteins, and the distortion of cell proliferation, mitochondrial structure and function.

19.2.1 Free radicals

Free radicals are compounds that have an unpaired (and therefore, highly reactive) electron on their outer shell /2/. This definition includes: the hydrogen atom, most metal ions (which can exist in various states), and the oxygen molecule, which is a biradical, since its two external electrons are on different shells.

Free radicals can be positively or negatively charged. A dot is placed after the chemical symbol to represent the free electron and to indicate that a compound is a radical. For example: A·, A, A–·.

Free radicals are found in lipids, amino acids, nucleotides, and especially in oxygen compounds.

Radicals may be formed by three different mechanisms /2/:

  • Homolysis of covalent bonds:
    A:B A· + B·; this is very unusual in biological systems
  • Addition of a single electron (e) to a neutral atom:
    A + e A–· a common biological event
  • Loss of a single electron from a neutral atom:
    A A + e; rare in biological systems.

Radicals, especially those of low molecular weight, are extremely reactive and are, therefore, short-lived; having an unpaired electron, they are usually highly reactive electrophilic species. As a result, they attack sites of increased electron density such as the nitrogen atom (nitrogen contains two unpaired 2s electrons) carbon-carbon double bonds present in polyunsaturated fatty acids and phospholipids to produce additional free radical intermediates /2/.

The main endogenous source of free radical generation is cellular metabolism, while exogenous sources are pollution, smoking, some exogenous drugs, irradiation, the presence of some transition metals such as iron and manganese in the body /12/.

19.2.2 Formation of free reactive oxygen radicals (ROS)

The main reactive oxygen species (ROS) include:

1. free radicals

  • Superoxide anion; O2-
  • Peroxide anion; O22-
  • Hydoxyl radical; OH.

2. Non radical molecules

  • Hydrogen peroxide; H2O2
  • Hydroxyl anion; OH-

ROS are produced as a result of intracellular catabolism that requires oxygen as terminal electron acceptor (oxidant). During this process, ROS such as super oxide anion radical (O2–·), hydrogen peroxide (H2O2) and hydroxyl radicals (HO·) are produced as intermediates, even in healthy individuals. The removal of an electron from O2 leads to the formation of the super oxide anion radical (O2–·), an unstable free radical that reacts with other super oxide ions or oxygen-containing compounds. Under normal conditions there is a balance between ROS and the defense system of antioxidants (super oxide dismutase, catalase, glutathione and glutathione peroxidase, ascorbate, pyruvate, flavonoids, and carotinoids), thereby preventing or limiting oxidative damage /3/.

In addition to ROS, radicals that contain a nitrogen atom exist, for example, nitrogen monoxide (NO·).

Oxygen radicals are particularly important because they lead to the production of all other radicals /3/. Important intracellular ROS are:

  • The super oxide anion radical (O2–·)
  • The hydoxyl radical (HO·).

19.2.2.1 The super oxide anion radical

In biological systems, oxygen is involved primarily in the intracellular production of free radicals by various reactions, but are most frequently initiated by the addition of an electron to molecular oxygen to produce the super oxide anion radical /2/.

O2 + e O2–·

This reaction in cells results from electron leakage from the electron transport chains in mitochondria and from endoplasmic reticulum.

The super oxide anion radical is formed from a variety of reactions (Tab. 19.2-1 – Sources of reactive O2 species (ROS)):

  • Non catalytic oxidation of oxyhemoglobin to methemoglobin

Hb-Fe2+ + O2 Hb-Fe3+ + O2–·

The methemoglobin produced is then reduced to oxyhemoglobin catalyzed by methemoglobin reductase.

  • The autoxidation of reduced transition metals also generates the super oxide anion radical

Fe2+ + O2 Fe3+ + O2–·

19.2.2.2 The hydroxyl radical

Free hydroxyl radicals are produced by the homolytic dissociation of water by ionizing radiation to form hydrogen atom and hydroxyl radical

H2O H· + HO·

In the Haber-Weiss reaction super oxide anion radical is converted to hydroxyl radical via the following reactions /2/

O 2 · + Fe 3+ → O 2 + Fe 2+ Fe 2+ + H 2 O 2 → HO · + OH + Fe 3+ Sum: O 2 · + H 2 O 2 Iron catalyst HO · + OH + O 2

The respiratory burst as an example of ROS formation

The respiratory burst is a reaction that takes place in phagocytic cells such as polymorphonuclear neutrophils and monocytes/macrophages. Phagocytes ingest bacteria and destroy them intracellularly by producing HO· and HOCl. The process starts with the uptake of O2 and activation of NADPH oxidase to produce O2–·.

2O 2 + NADPH Oxidase 2O 2 · + NADP + H +

Subsequent dis mutation, either spontaneous or catalyzed by super oxide dis mutase (SOD), leads to the formation of H2O2:

2O 2 · + 2H + SOD H 2 O 2 + O 2

Potent oxidizing and antimicrobial substances such as HOCl and HO· are produced from H2O2 or O2–· as follows:

  • In the presence of Cl, myeloperoxidase (MPO) from polymorphonuclear neutrophils generates HOCl
H 2 O 2 + Cl + H + MPO HOCl + H 2 O
  • HO· is formed independently of MPO in the Fenton or Haber-Weiss reaction:
Fenton: H 2 O 2 + Fe 2+ HO · + OH + Fe 3+ Haber-Weiss: O 2 · + H 2 O 2 Iron catalyst HO · + OH + O 2

19.2.2.3 Physiological formation of ROS

ROS are produced physiologically:

  • During mitochondrial respiration, for which a cell consumes 1012 molecules of O2 per day, of which around 2% (2 × 1010 ROS) reach the circulation as intermediate stages in the form of O2–·, HO·, and H2O2
  • During granulocyte and macrophage activation, resulting in the production of O2–· and HO·
  • By the catalytic activity of oxidases such as xanthine oxidase, monoamine oxidase, L-amino acid oxidase, tyrosine hydroxylase, and NO synthase resulting in the production of O2–· and H2O2
  • By the Fenton reaction (radical production catalyzed by iron).

19.2.2.4 Effect of ROS on the tissues

ROS are produced physiologically in the processes shown in Tab. 19.2-1 – Sources of ROS. Increased concentrations of ROS result in structural and functional changes in lipids, proteins, and nucleic acids (Tab. 19.2-2 – Pathogenic effects of ROS) /2/.

19.2.3 Reactive nitrogen species

The main reactive nitrogen species are:

1. Radical molecules

  • Nitric oxide; NO.

2. Non radical anions

  • Peroxinitrite anion; ONOO-

Nitrosative stress results from an increase in reactive nitrogen species (RNS). Though the RNS, the radical molecule nitric oxide (NO·) and the non radical anion peroxynitrite (ONOO-) play pivotal physiological roles. At elevated concentrations, both moieties can be poisonous to cells due to their capacity to disrupt a variety of essential biological processes.

Nitric oxide (NO)

Post translational modifications involving RNS share a common progenitor: nitric oxide (NO·). Nitric oxide is produced from L-arginine by three main isoforms of nitric oxide synthase (NOS) /5/:

  • Neuronal NOS (nNOS), which is linked to intracellular signaling
  • Inducible NOS (iNOS) which has a variety of situational functions. The enzyme is activated in response to various endotoxin or cytokine signals, which can lead to a rapid production of large fluxes of NO·. The expression of iNOS is regulated by well characterized signal pathways suggesting that the inducible production of NO· must be tightly controlled.
  • Endothelial NOS (eNOS). This isoenzyme is present in all tissues and produces low concentrations of NO· following activation. The NO· formed by eNOS has a vasoprotective effect.
L-arginine + O 2 + NADPH NOS NO · + L-citrulline + NADP

The activity of eNOS and iNOS is controlled by the concentration of Ca2+. This enables endothelial cells to respond immediately to an extracellular signal by increasing NO· production, for example. Under physiological conditions, small quantities of NO· are released by eNOS and nNOS only, to regulate the blood pressure, for neurological processes, and to counter regulate pathological processes /6/.

19.2.3.1 Physiological effects of nitric oxide

NO fulfills important signaling and protective functions /3/. NO produced by eNOS has vasoprotective effects. It inhibits platelet aggregation and the proliferation of smooth muscle cells and promotes the dilatation of blood vessels, which reduces the blood pressure. NO prevents endothelial dysfunction in general. This includes functional injury to the vascular endothelium caused by disruption of the protective signaling pathway. Functional disruption can be caused by ROS, for example, since they can oxidize cofactor BH4, thereby disrupting the function of eNOS.

NO· can permeate freely through the cell membrane and intervene directly in signaling processes. Small quantities are sufficient to activate guanylate cyclase intracellularly. Cyclic GMP produced by guanylate cyclase activates protein kinases, phosphodiesterases, and ion channels, which then mediate the physiological functions of NO· /6/

Refer to:

19.2.3.2 Oxidative modifications by peroxynitrite anion

Nitrite oxide mediated post-translational modification through the reaction with super oxide produces the peroxynitrite anion.

ONOO NO · + O 2 ·

Reactive products of peroxynitrite anions have the following effects /6/:

  • In aqueous environment, the protonated peroxynitrous acid forms that rapidly decompose into nitrogen dioxide (NO2) and an extremely reactive hydroxyl radical (OH.)
  • The hydroxyl radical is reactive to remove electrons from nearly any biological molecule e.g., the reaction with tyrosine creates a tyrosyl radical which can than react with NO2 to form nitro tyrosine
  • Peroxynitrite anions directly react with CO2 yielding nitrosoperoxycarbonate anion (ONOOCO2-) which decompensates into NO2 and the carbonate radical (CO3-) that has a similar reactivity to the hydroxyl radical.
  • Peroxynitrite anion modifies proteins and other macromolecules. The ability is used by macrophages and polymorphonuclear neutrophils to kill invading bacteria. It is important to recognize that peroxynitrite anion is used to protect the organism from bacteria by releasing oxidative species in the respiratory burst /7/. The example demonstrates that overproduction or deregulation of these pathways is not only damaging but also beneficial for the organism /67/.

Peroxynitrite anion has disadvantages for functions of the organism /8/:

  • The concentration of NO· declines and the blood vessels cannot dilate adequately
  • Peroxynitrit anions play a role in neurodegenerative diseases, rheumatoid arthritis, and diseases of the organs. Enzymes such as manganese super oxide dis mutase (MnSOD) are inactivated by the irreversible conversion of tyrosine into nitro tyrosine. MnSOD neutralizes the superoxide anion radical· through its binding to H2O to form H2O2.
  • Under conditions of oxidative stress, production of α-methylated L-arginine metabolite (asymmetrical dimethylarginine, ADMA) in the cells is increased. ADMA competitively inhibits eNOS by binding to it instead of L-arginine /9/.

19.2.4 Antioxidant defense

Antioxidants are species that prevent both ROS/RNS formation and their interaction with biostructures. Free radicals are produced in all cells of the body. It is very important for the body to preserve the balance between the oxidant and antioxidant systems at the cellular level. Living cells manage to maintain a balance between the production and elimination of ROS so that, the concentration of ROS/RNS species is neither too high to produce stress nor too low to deprive the body from these species /1/. However, if the cell function is altered by various factors higher amounts of ROS/RNS are produced in the presence of insufficient amounts of antioxidants and the affected cells will develop pathologies by the per oxidation of proteins, lipids and DNA, leading to a variety of diseases like atherosclerosis, heart disease, neurodegenerative disorders, diabetes, autoimmune diseases, and cancer /101112/.

The first line of defense against oxidative stress are endogenous antioxidants:

A mitochondrial manganese-dependent super oxide dis mutase (SOD) and a cytosolic copper and zinc-dependent SOD that convert super oxide anion· radical to H2O2 which is not toxic to cells. However, in the presence of Fe2+ and Cu+ H2O2 is converted to toxic hydroxyl radical·. SOD is associated with manganese and other transition metals (MnSOD, CuZnSOD) and localized in the mitochondria to control the production of ROS. Around 70% of SOD activity is localized to the heart, of which 90% is contained in myocytes.

O2 ·  + 2H + SOD H 2 O 2
  • Glutathione peroxidase (GP): before H2O2 can oxidize metal ions, it is converted by GP or catalase into H2O. The activity of GP is selenium-dependent and GP have a major function in the protection of cells against peroxides and free radicals, thus against oxidative stress. GP and catalase reduce hydrogen peroxide to water and alcohol.
H 2 O 2 + 2 GSH GP GSSG + 2H 2 O 2H 2 O 2 Catalase 2H 2 O + O 2

GP, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione

  • Metal-binding proteins: these proteins bind transition metals such as iron and copper and prevent them from reacting with H2O2 in the Fenton or Haber-Weiss reaction and generation of free radicals. Metal-binding proteins include transferrin, lactoferrin, ferritin, hemoglobin, myoglobin, metallothionein, and the cytochrome oxidases.
  • The usual antioxidant free radical scavengers include bilirubin, uric acid, carotinoids, vitamins A, C, and E, thioles (R-SH), copper, glutathione (GSH), manganese, selenium, and zinc.

Along with super oxide dismutase and catalase and together with vitamins E (α-tocopherol) and C (ascorbic acid), glutathione per oxidases protect cells against the effects of ROS, improving the resistance and recovery of tissues, slowing the aging process and participating in detoxification processes in the body. Vitamin E acts as a trap for free radicals and prevents the formation of lipid hydroperoxides in cell membranes, while gutathione peroxidase destroys lipid hydroxy peroxides formed in the per oxidation of polyunsaturated fatty acids /113/.

19.2.5 Determination of ROS and RNS

Because of the nature and rapid reactivity ROS/RNS are difficult to measure directly with high accuracy and precision. A review of current methods for directly measuring these species and the secondary products they generate is presented in Ref. /14/.

The following methods are commonly used:

  • To determine the ROS: measurement of peroxides, malondialdehyde, and isoprostanes
  • To evaluate the antioxidant defense: the total antioxidant capacity and enzymes such as super oxide dis mutase, glutathione peroxidase, and catalase or the concentration of antioxidant substances such as vitamin E, vitamin C, reduced glutathione (GSH), and uric acid
  • Nitric oxide· can not be determined directly due to its short half-life. Indirect methods used include the determination of nitrite in the form of nitrate or nitrosylated tyrosine residues (3-nitrotyrosine) and of asymmetrical dimethylarginine (ADMA).

Refer to:

References

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5. Adams L, Franco MC, Estevez AG. Reactive nitrogen species in cellular signaling. Experimental Biology and Medicine 2015; 240: 711–7.

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8. Wingler K, Schmidt HHHW. Guter Stress, schlechter Stress – die feine Balance in Blutgefäßen. Dtsch Ärztebl. 2009; 106: 677–84.

9. Böger GI, Rudolph TK, Maas R, Schwedhelm E, Dumbadze E, Bierend A, et al. Asymmetric dimethylarginine determines the improvement of endothelium-dependent vasodilation by simvastatin. J Am Coll Cardiol 2007; 49: 2274–82.

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13. Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr J 2016; 15: 71. https://doi.org/10.1186/s12937-016-0186-5.

14. Griendling KK, Touyz RM, Zweier JL, Dikalov S, Chilian W, Chen YR, et al. Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association. Circ Res 2016; 119: e39-e75.

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19.3 Tests for the diagnosis of inflammation

Lothar Thomas

Inflammation can be detected based on the following tests (Tab. 19.3-1 – Diagnostic value of various tests for the detection of inflammation):

  • Temperature measurement
  • White blood cell (WBC) count and differential
  • Erythrocyte sedimentation rate (ESR)
  • Serum protein electrophoresis and assessment of the α1-, α2- and γ-globulins
  • Acute phase proteins, in particular CRP and serum amyloid A (SAA)
  • Inflammatory cytokines (IL-1, IL-6, TNF-α); refer to Chapter 20 – Cytokines and cytokine receptors.

19.3.1 Temperature measurement

The core body temperature is regulated by the temperature center of the anterior pituitary. Normal body temperature of an individual is an expression of the thermoregulatory set point. Temperatures above this value are referred to as fever and lower values as hypothermia. In routine diagnostics, fever is generally indicated by a rectal temperature of ≥ 38.0 °C (100.4 °F) in children or adults.

Normal temperature

Normal body temperature is 37 °C (98.6 °F) and fluctuates by ± 0.6 °C (1.08 °F). The rectal temperature is around 0.6 °C (1.08 °F) higher than the oral temperature. Body temperature exhibits diurnal variation, being higher in the evening than in the early morning /12/.

19.3.1.1 Fever

Fever is characterized by an elevated core temperature due to an increase in the thermoregulatory set point. This set point does not normally exceed 41 °C (105.8 °F). Temperatures higher than this indicate extreme fever. In adults, such extreme elevation is typically caused by central nervous system disorders, drug-induced disturbances, or heatstroke. In children, temperatures of 40–41 °C (104.0–105.8 °F) are not unusual and can result from viral infections. Persistent temperature elevation above 41 °C (105.8 °F) can cause irreversible brain damage /23/.

Etiology: the temperature can provide clues to the origin of a fever /4/:

  • Temperatures ≤ 39 °C (102.2 °F) suggests non-bacterial disease such as acute myocardial infarction, acute pulmonary embolism, acute pancreatitis, gastrointestinal hemorrhage, hematoma, phlebitis, uncomplicated wound infection, cystitis, cholecystitis, viral hepatitis, tracheobronchitis, osteomyelitis, decubitus ulcers, deep vein thrombosis, neoplasia, antibiotic induced diarrhea, systemic lupus erythematosus, or acute respiratory distress syndrome (ARDS).
  • Temperatures > 39 °C (102.2 °F) indicates an infectious origin such as pneumonia, sepsis, or pyelonephritis.

Fever of unknown origin

Fever of unknown origin is characterized by a temperature > 38.3 °C (100.9 °F) on several occasions for at least three weeks with failure to reach a diagnosis. It often occurs in intensive care units in patients with traumatic brain injury or neurological disorders, some of whom are mechanically ventilated, and those with some combination of urethral, central, and peripheral catheters. Findings include intermittent Staphylococcus positive blood cultures, pyuria with 10–20 thousand CFU/mL, or chronic watery stools with occasional proof of Clostridium difficile. CRP values fluctuate almost daily, making therapeutic decisions difficult, and patients have been repeatedly treated with antibiotics without success /5/. A diagnostic algorithm for fever of unknown origin in children is shown in Ref. /6/.

Hereditary periodic fever /7/

Hereditary periodic fever includes familial Mediterranean fever (FMF), hyper immunoglobulinemia D and periodic fever syndrome (HIDS), and tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS). All three diseases are thought to be caused by disturbed cytokine balance. Common features are recurring fever with pleuritis, pericarditis and/or peritonitis, arthralgia, and exanthemas, with initial onset in childhood or early adolescence. Genetic investigations are used to verify the diagnosis: detection of mutations in the MEFV gene in FMF, in the MVK gene in HIDS, and in the TNFRSF1A gene in TRAPS.

Clinical features of fever

Elevation of the body temperature leads to chills, shivering, cold clammy skin, pallor, and generalized vasoconstriction.

Types of fever

The following types of fever are distinguished /89/:

  • Remittent fever: the body temperature rises or declines by 1–2 °C (1.8–3.6 °F) over the course of a day; occurrence in patients, for example, with purulent processes and tuberculosis
  • Intermittent or septic fever: the temperature rises from normal values or a subnormal temperature level by 2–3 °C (3.6–5.4 °F), often in combination with shaking chills and then falls again; occurrence in patients, for example, with pneumonia, cystitis, or malaria
  • Continuous fever: the body temperature is continuously high and its daily variation does not exceed ± 1 °C (1.8 °F). This fever type was previously known as febris continua and is the classical fever type associated with microbial infection
  • Biphasic fever pattern: febrile periods are interrupted by one or more days without fever; occurrence in patients, for example, with Hodgkin’s disease, borreliosis, or malaria.

Biochemistry and physiology

The temperature center in the hypothalamus regulates body temperature by controlling peripheral vasoconstriction and muscle metabolism. Fever results from the direct stimulation of the temperature regulation center by pyrogens, which are biochemical substances with various structures. Pyrogens include substances produced by microbes or exogenous substances (e.g., drugs). Endogenous pyrogens are produced following the activation of monocytes/macrophages and other inflammatory cells. The pro inflammatory cytokines synthesized by these cells (e.g., TNF-α, IL-1, IL-6) and chemokines such as prostaglandin E1, induce fever in the context of an acute phase response. The rationale for setting the body temperature higher is /10/:

  • To activate enzymatic reactions that run at normal activity at normal body temperature
  • To promote phagocytosis, bacterial killing, and the immune response
  • To increase energy provision.

19.3.2 Erythrocyte sedimentation rate (ESR)

The ESR is the relative rate of the red cell column in relation to the length of the total blood column in a graduated tube.

19.3.2.1 Indication

Screening test in a suspected inflammatory response as well as a method for monitoring such a reaction.

19.3.2.2 Method of determination

Test protocol

Westergren method: a citrated blood sample is aspirated into a glass or plastic pipette with millimeter graduation up to the 200 mm mark. The pipette remains in an upright position and the sedimentation of erythrocytes is read off in mm after one hour; in some tests, a result is also read off after two hours. The performance of the method follows an approved guideline /11/.

19.3.2.3 Specimen

Citrated blood (1.6 mL of blood + 0.4 mL of 3.8% sodium citrate solution): 2 mL

19.3.2.4 Reference interval

 

< 50 years /12/

≥ 50 years /12/

≤ 20

≤ 30

≤ 15

≤ 20

Values are expressed in mm for the first hour

19.3.2.5 Clinical significance

During an inflammatory response, the plasma concentration of acute phase proteins, fibrinogen, and immunoglobulins rises. However, as an indicator of the acute phase response, the ESR responds slowly. Accordingly, a rise in the ESR occurs at the earliest 24 h after the initiation of an inflammatory response and, after completion of the acute phase response, the ESR declines with a half-life of 96–144 h.

The ESR is often considered to be a general indicator of disease. This is incorrect because:

  • A normal ESR does not rule out non-inflammatory organ diseases, organ dysfunctions, or neoplasias. For instance, in a study /13/ of 1,000 asymptomatic soldiers in whom the ESR was measured monthly over the course of 15 years, 10 individuals developed a malignant tumor despite a normal ESR.
  • An elevated ESR is only of supportive value in relation to data from case history as well as clinical and laboratory findings; in rare cases (less than 0.1%), it is the only clue to an important diagnosis /14/.

A moderately elevated ESR should always be investigated if there is any association with a disease (i.e., in the medical history and/or clinically or laboratory-diagnostically relevant data or findings). Approximately 5% of all ESR elevations are essentially unexplainable. In one study /15/ involving more than 9,000 outpatients, 8% had an ESR elevation, whose underlying cause could not be determined in 43 patients. Follow-up ESR monitoring over the course of 10 years showed spontaneous normalization in 74% of these patients. Another study /13/ demonstrated that asymptomatic young individuals (18–33 years) with persistent, slight ESR elevation had an approximately 5-fold higher risk of disease than those with a normal ESR. The risk of disease was highest for myocardial infarction.

In comparison to the quantitative determination of an acute phase protein (e.g., CRP) the ESR is also raised by an increase in the concentration of immunoglobulins, immune complexes, and other proteins. It therefore covers a broader spectrum of diseases than CRP.

In the case of chronic inflammatory diseases such as SLE, polymyalgia rheumatica, and temporal arteritis, in which CRP is often normal or only slightly elevated, and for monitoring in patients with these diseases, the ESR is a better indicator of the inflammatory process. This is also supported by the fact that the ESR, due to its sluggishness, is subject to little influence by transient inflammatory diseases such as viral infections. In one study /16/ of patients with chronic inflammation and malignant tumors, however, the determination of CRP was shown to be slightly more advantageous. For the diagnosis of an inflammatory reaction, an ESR of 31 mm/h corresponded to a CRP concentration of 15 mg/L.

It must be kept in mind that the usefulness of the ESR is limited because it is a reliable indicator of inflammation only in diseases that cause moderate to strong dysproteinemia and have little to no effect on the volume and number of erythrocytes (Tab. 19.3-2 – Factors influencing the ESR).

In the absence of inflammation or anemia, ESR elevation in individuals over the age of 50 years suggests a monoclonal gammopathy. At monoclonal immunoglobulin concentrations of greater than 20 g/L, the ESR is often extremely high (above 120 mm/h). A normal ESR does not rule out the presence of multiple myeloma. This is especially true if the monoclonal immunoglobulin fraction is below 10 g/L or in light chain myeloma where polyclonal immunoglobulin synthesis is only moderately reduced.

19.3.2.6 Comments and problems

Blood sampling

An increase in the proportion of citrate overestimates the ESR while a reduction is associated with a decreased ESR. Attention must be paid to thoroughly mixing the sample /11/.

Test protocol

The ESR testing should be started no later than 2 h after the blood collection. The 2-h value does not provide additional information. If the opportunity to read the 1-h value was missed, the blood cannot be remixed and used again for the ESR. The test should be performed at room temperature. At a temperature lower than 18 °C, the result is of no value because of alterations to the erythrocyte membrane. Temperatures higher than 20–24 °C lead to a rise in the ESR; at 27 °C, the ESR is approximately twice as high as at 20 °C /14/.

The ESR can be determined within 3 minutes using quantitative centrifugation methods and photometric recording. The results obtained using these methods differ from those obtained using the Westergren method /18/, particularly if monoclonal immunoglobulins are present in the blood /19/.

Drugs

Anti-inflammatory drugs such as acetylsalicylic acid, cortisone, indomethacin, and phenylbutazone, exert an inhibitory effect on the erythrocyte sedimentation.

19.3.2.7 Biochemistry and physiology

The ESR is based on the sedimentation and aggregation of erythrocytes.

The erythrocyte density is 6–7% higher than that of plasma; the erythrocytes therefore sink to the bottom as a result of gravity. Simultaneously, plasma rises to the top and slows down the erythrocyte sedimentation. Since the erythrocyte surface is negatively charged (zeta potential), adjacent cells repel each other once the intercellular distance falls below a certain minimum and thus the erythrocytes stay afloat.

Plasma proteins attach to the erythrocyte surface. Depending on the presence of dysproteinemia in certain diseases, plasma proteins may reduce the zeta potential and thus cause the erythrocytes to approach each other more closely.

Plasma proteins such as fibrillary fibrinogen or pentameric IgM can adhere to two erythrocytes.

Overall, both effects of plasma proteins promote the formation of erythrocyte aggregates which, as large particles, undergo faster sedimentation than individual red cells. The contribution of the individual plasma proteins to the ESR are fibrinogen (55%), α2-macroglobulin (27%), immunoglobulins (11%), and albumin (7%).

Diseases that cause an increase in acute phase proteins (such as acute inflammation) and those that are associated with a polyclonal increase in immunoglobulins (chronic inflammatory diseases), but also monoclonal gammopathies, lead to a rise in the ESR. The rise in the ESR does not correspond to the extent of the inflammatory event when the fibrinogen has been consumed (e.g., as in the case of sepsis with hyperfibrinolysis and diffuse intravascular coagulation).

19.3.3 WBC and differential

An increase or decrease in the number of polymorphonuclear granulocytes with or without an increased number of precursors such as bands, immature granulocytes, and myelocytes may be a sign of an inflammatory reaction (refer also to Section 15.12 – Leukocyte count and Section 15.13 – Blood smear examination).

19.3.3.1 Neutropenia

With the exception of acute leukemia and bone marrow toxicity, neutropenia is caused mainly by infection with Gram-negative bacteria, especially if sepsis or septic shock is present. The bone marrow reservoir can also be depleted transiently in the early stage of a severe infection. The neutropenia that results can be associated with the release of immature granulocyte precursors including promyelocytes (leukemoid reaction). This can also happen if patients suffering from infections have reduced bone marrow reserves (e.g., neonates, the elderly, alcoholics, and immunosuppressed individuals) /20/.

19.3.3.2 Neutrophilia

Neutrophilia occurs within minutes of exposure to an inflammatory stimulus. The half-life of the subsequent increase in neutrophils is approximately 10 h. Neutrophilia may indicate an infection, but is a non-specific finding. Neutrophilia is also caused by /20/:

  • Inflammatory stimuli such as collagen disease, hypersensitivity reaction, tissue injury, necrosis, and neoplasia
  • Acute hemorrhage, hemolysis, intoxication, and poisoning
  • Metabolic diseases such as gout, uremia, ketoacidosis, and eclampsia
  • Seizures
  • Myeloproliferative diseases.

Acute purulent bacterial infection

Usually gives rise to a leukocytosis of greater than 15 × 109/L; more than 80% of the cells are granulocytes. In addition, the presence of a left shift is characteristic and may sometimes be the only sign. Granulocytes with toxic granulation are present in approximately three quarters of patients with sepsis but this is not specific for sepsis /21/.

Tissue necrosis and sterile inflammation

Only slight to moderate elevation of the granulocyte count; a left shift is rare.

Chronic inflammation

Normal leukocyte count or a slight rise, often monocytosis.

Acute allergic reaction, parasitosis

Normal leukocyte count or a slight rise, often eosinophilia

Viral infection

Normal, slightly increased or reduced leukocyte count, in most cases lymphocytosis.

19.3.4 Serum protein electrophoresis

The earliest sign of the acute phase response on protein electrophoresis is the increase in the α1-globulin fraction due to an increase in the concentration of α1-antitrypsin and α1-glycoprotein. This is followed by an elevation in the α2-globulin fraction due to an increase in the synthesis of haptoglobin and ceruloplasmin. Fibrinogen and CRP are located in the β-globulin fraction but are not associated with a fractional rise since serum is analyzed (fibrinogen is consumed) and CRP is not detectable because the concentration is too low. Significant changes in the α-globulins are not to be expected within the first 48–72 hours /22/.

Chronic inflammation is associated with a rise in γ-globulins while chronic active inflammation is associated with a rise in α- and γ-globulins.

References

1. Bernheim HA, Block LN, Atkins E. Fever: Pathogenesis, pathophysiology, and purpose. Ann Int Med 1979; 91: 261–70.

2. Saper CB, Breder CD. The neurologic basis of fever. N Engl J Med 1994; 330: 1860–6.

3. Ritchie R. Fever (pyrexia). In: Ritchie R, Navolotskaja O, eds. Serum proteins in clinical medicine. Scarborough; Association of Blood Banks 1999; 105.01–8.

4. Cunha BA. Fever in the critical care unit. Critical Care Clinics 1999; 14: 1–14.

5. Horowitz HD. Fever of unknown origin or fever of too many origins? N Engl J Med 2013; 368: 197–9.

6. Ishimine P. Fever without source in children 0 to 36 months of age. Pediatr Clin N Am 2006; 53: 167–94.

7. Lamprecht P, Timmann C, Ahmadi-Simab K, Gross WL. Hereditäres periodisches Fieber. Internist 2004; 45: 904–11.

8. Bock H. Fieberzustände – diagnostische und therapeutische Problematik. Medwelt 1989; 40: 1109–11.

9. Kaufmann W. Differentialdiagnostik und Differentialtherapie unklarer Fieberzustände. Medwelt 1989; 40: 1120–5.

10. Whicher J. The acute phase response. In: Ritchie R, Navolotskaja O (eds). Serum proteins in clinical medicine. Scarborough; Association of Blood Banks 1999; 105,001–8.

11. NCCLS. Methods for the erythrocyte sedimentation rate (ESR) test – third edition; approved standard. NCCLS Document H2–A3 Vol 13 No 8. Villanova, 1993.

12. Hanger HC, Sainsbury R, Gilchrist NL, Beard MEJ. Erythrocyte sedimentation rates in the elderly: a community study. New Zealand Med J 1991; 104: 134–6.

13. Froom P, Margaliot S, Cane Y, Benbassat J. Significance of erythrocyte sedimentation rate in young adults. Am J Clin Pathol 1984; 82: 198–200.

14. Reinhart WH. Die Blutsenkung – ein einfacher und nützlicher Test? Schweiz Med Wschr 1988; 118: 839– 44.

15. Liljestrand A, Olhagen B. Persistently high erythrocyte sedimentation rate. Acta Med Scand 1955; 151: 425–39.

16. Dinant JC, de Kock CA, van Wersch JWJ. Diagnostic value of C-reactive protein measurement does not justify replacement of the erythrocyte sedimentation rate in daily general practice. Eur J Clin Invest 1995; 25: 353–9.

17. Weitbrecht WU, Gmeiner HJ, Böcker F, Schult H. Blutsenkungsreaktion bei wiederholten Dextraninfusionen in der Therapie zerebraler Infarkte. Dtsch Med Wschr 1985; 110: 977–8.

18. Shelat SG, Chakosky D, Shibutani S. Differences in erythrocyte sedimentation rates using the Westergren method and a centrifugation method. Am J Clin Pathol 2008; 130: 127–30.

19. Raijmakers MTM, Kuijper PHM, Bakkeren DL, Vader HL. The effect of paraproteins on the erythrocyte sedimentation rate: a comparison between the StarrSed and Test 1. Ann Clin Biochem 2008; 45: 593–7.

20. Cornbleet PJ. Clinical utility of the band count. Clin Lab Med 2002; 22: 101–36.

21. Kroft SH. Infectious diseases manifested in the peripheral blood. Clin Lab Med 2002; 22: 253–77.

22. Thomas L (ed). Serumeiweiss-Elektrophorese. München: Urban und Schwarzenberg, 1981.

19.4 C-reactive protein (CRP)

Lothar Thomas

CRP is the classic biomarker used to diagnose the acute phase response of an inflammatory process. An increase in plasma concentration of CRP results from the stimulation of pro inflammatory cytokines, such as interleukin-6. Pro inflammatory cytokines trigger acute systemic inflammation, also known as the acute phase response (refer also to Fig. 19.1-8 – Systemic changes in an acute phase response). The acute phase protein CRP is an integral component of the innate immune system. It is synthesized in the liver and reaches via the circulation to the site of tissue damage or is produced locally by activated monocytes/macrophages and fibroblasts.

The acute phase response causes an increase in the serum CRP level that corresponds to the extent of the inflammatory process. Inflammation is caused by infections, sterile tissue damage (surgical procedures), malignant tumors (especially metastatic), systemic malignant diseases (Hodgkin and non-Hodgkin lymphomas), and some autoimmune diseases.

A distinction is made between high-grade, moderate, mild, and low-grade inflammation. Low-grade inflammation is associated with obesity, diabetes mellitus, atherosclerosis, and cardiovascular disease /1/. The CRP concentration is low in low-grade inflammation, which can be present for decades without clinical symptoms. For this reason, assays that could measure CRP in low-grade inflammation were also known in the past as high-sensitivity CRP (hs-CRP) assays. This designation will no longer be used here since most quantitative CRP assays can now detect low-grade inflammation.

19.4.1 Indication

To screen for acute phase response and monitor the course:

  • In fever, leukocytosis, and suspicion of infection
  • In the critical care setting and neonatology
  • In the postoperative period (the first 6 days in particular)
  • In premature rupture of membranes
  • During cytotoxic therapy-related episodes of neutropenia and in bone marrow transplantation.

Infections:

  • Differentiation between viral and bacterial febrile illnesses (e.g., in meningitis or pneumonia)
  • Monitoring response to antibiotic therapy
  • Identification of intercurrent infections in patients with connective tissue disease.

Diagnosis of inflammatory processes:

  • Confirmation of suspected acute organ disease (e.g., cholangitis, adnexitis, synovitis, and deep vein thrombosis)
  • Confirmation of chronic inflammatory disease (rheumatic, gastrointestinal, respiratory)
  • Differential diagnosis of arthralgia, myalgia, and atypical back pain.

To aid in the differential diagnosis of gastrointestinal complaints:

  • To distinguish irritable bowel syndrome from organic disease (inflammatory bowel syndrome)
  • To aid the differentiation of ulcerative colitis from Crohn’s disease.

To aid in the management of rheumatic diseases: evaluation of an optimal anti-inflammatory therapy (steroidal or non-steroidal) and to establish the minimal effective dose.

Management of patients with atherosclerosis:

  • Risk stratification in patients with cardiovascular disease
  • Prediction of future cardiovascular events in patients with myocardial infarction
  • Prognostic indicator in unstable angina pectoris.

19.4.2 Method of determination

Principle: many different analytical procedures can be used to quantitatively determine CRP. In routine diagnostics, the most commonly used methods are particle-enhanced immunoassays, with nephelometric or turbidimetric measurement. These assays have a low detection limit and broad measuring range, to measure CRP concentrations in both low-grade and high-grade inflammation (refer also to Section 52.1.5.3).

19.4.3 Specimen

Serum, plasma: 1 mL

19.4.4 Reference interval

Refer to Ref. /134/ and Tab. 19.4-1 – Upper threshold values for CRP (mg/L).

19.4.5 Clinical significance

Elevated serum levels of CRP always signal the presence of a disease condition.

19.4.5.1 CRP and systemic inflammation

Elevated CRP concentrations are indicators for:

  • Acute and chronic inflammation caused by microbial infections (bacterial infections are the most potent stimulators of CRP synthesis)
  • Acute tissue damage due to major surgery, trauma, or malignant tumors. Even the tissue damage caused by heavy smoking and marathon running can cause a minor elevation of CRP and indicate pathology.
  • Inflammation that accompanies autoimmune diseases such as rheumatoid arthritis and for immune-complex mediated diseases such as immune vasculitis.

Normal CRP levels do not exclude the presence of minor degrees of localized inflammation in which the acute phase response is minimal. Examples include chronic autoimmune diseases (inactive systemic lupus erythematosus, progressive systemic sclerosis, dermatomyositis, and ulcerative colitis).

A single CRP measurement is only useful to indicate inflammation within 3 days of an acute event. In such an event, plasma CRP concentrations are elevated after 6 h, reach a peak at 48 h, and decline with a half-life of 48 hours /5/.

Daily determinations provide fare more information in monitoring the progress than a one-time measurement. This is the case because:

  • The intraindividual variation of CRP within the reference interval is high, with a coefficient of variation of 30–60% /6/
  • The reference interval is very wide. Approximately 25% of healthy individuals have a CRP value below 1 mg/L, which means that the CRP concentration for some people has to increase by at least a factor of 5 to exceed the upper reference interval value. Approximately 14% of healthy individuals have values ≥ 10 mg/L, which are suggestive of inflammation /7/.

In many cases, the increase in CRP precedes the clinical symptoms. Therefore, the CRP level must always be interpreted in the light of the clinical picture.

The degree of elevation of CRP over the upper reference interval value reflects the product of the inflammatory activity and mass of the inflamed tissue and depends on the liver’s capacity for synthesis. Only in the light of the clinical picture is it possible to determine from the rise in CRP whether an inflammatory process is the primary component of the underlying disease or secondary to this disease. Examples of diseases in which inflammation is the primary component include adnexitis and rheumatoid arthritis, whereas inflammation is a secondary component in the case of a postoperative complication.

19.4.5.2 CRP and disease activity

The degree of elevation of CRP reflects the mass of the inflamed tissue and, in acute inflammation and infection, the level of CRP correlates well with inflammatory activity. This is less often the case in chronic infections, although in a few important diseases, such as rheumatoid arthritis, Crohn’s disease, and poly myalgia rheumatica, the correlation is used for therapeutic monitoring.

Disease activity is classified as follows based on the CRP concentration:

  • Low-grade inflammation (> 3–10 mg/L): the reasons include obesity, type 2 diabetes mellitus, and atherosclerotic disease
  • Mild inflammation (> 10–40 mg/L): causes include local abscesses, minor operative and accidental trauma, myocardial infarction, deep vein thrombosis, quiescent rheumatic disease, metastatic malignant tumors, and isolated viral infections
  • Moderate inflammation (> 40–100 mg/L): diseases include severe inflammatory processes such as purulent cystitis, bronchitis, purulent dental infections, urinary tract infections, and genital infections. These infections require treatment (e.g., with antibiotics).
  • High-grade inflammation (> 100 mg/L): diseases include acute generalized bacterial and fungal infections (sepsis) as well as serious tissue damage following multiple trauma or major surgery (Fig. 19.4-1 – Time profile of CRP concentration in relation to the extent of tissue damage caused by different types of surgery).

19.4.5.3 CRP in infections

In cases of suspected infection, it is essential to evaluate the clinical picture of the patient in relation to the CRP level, since non-inflammatory events like deep vein thrombosis and pulmonary embolus may also induce an acute phase response.

Bacterial endotoxin is the most potent stimulus of the acute phase response. The highest levels of CRP are seen in sepsis caused by Gram-negative bacteria and S. aureus, sometimes reaching more than 500 mg/L. Sepsis caused by fungi is also associated with levels of 100–200 mg/L. Parasitic infections usually cause a more modest acute phase response, typically not exceeding 50 mg/L and rarely more than 100 mg/L. Sequential CRP determination can therefore be used to detect intercurrent infection in high-risk surgical patients and following cytostatic therapy in order to detect intercurrent infections.

Viral and bacterial infections can often be differentiated on the basis of the extent of CRP elevation. For example, in meningitis and respiratory tract infection, CRP concentrations > 100 mg/L indicate a bacterial origin. The median CRP concentration in viral infections is 15 mg/L; values > 40 mg/L are rare.

CRP measurement is valuable since it can indicate anatomically closed infections that are not accessible to conventional microbiological diagnosis. The rapid response time of the rise and fall in CRP allows diagnosis before bacteriological tests provide a result.

The behavior of CRP in conditions involving mild to high-grade inflammation is shown in Tab. 19.4-2 – CRP in diseases associated with mild to high-grade inflammation. In a study chronic low level CRP concentrations (> 3 to 10 mg/L) were associated with increased risk of bacterial infections, and in particular Gram-negative infections /8/.

19.4.5.4 Monitoring of therapy with CRP

When using CRP for therapeutic monitoring, it is important to note that changes in plasma level lag behind changes in inflammatory activity by some 12–24 h. This is relatively unimportant, though, since changes in the clinical symptoms are usually much slower. For example, following antibiotic therapy for pyelonephritis or bacterial cystitis, improvement in the clinical picture is not expected for at least 24–48 hours and following anti-inflammatory therapy for rheumatoid arthritis, improvement is not expected for at least 4–6 weeks. In most cases of inflammatory disease, therefore, CRP provides a much earlier assessment of the therapeutic response than the change be made from the clinical picture. A persistently raised CRP concentration generally indicates that therapy is ineffective.

Serial measurements of CRP can be used to monitor therapy in the following situations:

  • Optimization of antibiotic therapy in acute infections
  • Optimization of antibiotic therapy in high-risk patients in the absence of a microbiological diagnosis
  • Dis continuation of antibiotic therapy when CRP has returned to normal
  • Selection of appropriate anti-inflammatory therapy in various rheumatic diseases that are difficult to assess clinically
  • Titration of the dose of anti-inflammatory drugs
  • Prediction of the development of complications (e.g., the development of giant cell arteritis in a patient with poly myalgia rheumatica).

19.4.5.5 CRP pattern and therapeutic response

The following four patterns have been observed /8/:

  • Simple infection: CRP declines precipitously or exponentially following antibiotic administration. The intensity of the decline is determined by the half-life of CRP. This pattern is observed in focal infection or in bacteremia.
  • Suppurative infection: there is a time delay between the administration of antibiotics and the decline in CRP. This pattern is observed in purulent effusions, suppurative bronchitis, and empyema in the third space and when antibiotic doses are inadequate. The source of a persistent infection must always be sought.
  • Complicated infection: the CRP concentration fails to decrease or it continues to increase despite antibiotic therapy. This can be caused by the wrong choice of antibiotic, surgical complications, or serious non-infectious disease.
  • Recurrent infection: CRP shows a bimodal pattern with an initial decline followed by a second rise. This suggests either reinfection with the same causative microorganism at the same site or a new infection.

19.4.5.6 Low-grade inflammation

In Europe and North America, two-thirds of the population over 45 years of age have CRP values below 3 mg/L, less than 5% have values above 10 mg/L, and the rest have values between 3 and 10 mg/L /9/. According to a statement issued by the Centers for Disease Control and Prevention and the American Heart Association, CRP determination in addition to the classical risk factors is beneficial in the risk stratification of cardiovascular disease. The following CRP threshold values for cardiovascular risk were specified /10/:

  • Below 1.0 mg/L = low risk
  • 1.0–3.0 mg/L = moderate (normal) risk
  • Above 3.0 mg/L = high risk.

Refer to Fig. 19.4-2 – Cardiovascular risk of apparently healthy individuals as a function of CRP concentration.

This recommendation is based on the following:

  • Inflammation plays an important role in the development and course of atherosclerosis
  • Population studies have shown an independent relationship between the basal CRP value and future coronary events /2/
  • The serum CRP is useful for detecting patients with metabolic syndrome or type 2 diabetes
  • Low-grade inflammation is associated with cardiovascular disease, the metabolic syndrome, type 2 diabetes, and vascular endothelial dysfunction /11/.

The association between CRP in low-grade inflammation and the body mass index is shown in:

The following should be taken into account when estimating cardiovascular risk in the presence of low-grade inflammation /12/:

  • In apparently healthy individuals, the intraindividual day-to-day variation in CRP is 46%, compared to just 9% for cholesterol. This means CRP measurements of samples collected on different days could differ by up to 84% and the individual could be assigned to more than one risk category. This background variation is attenuated and balanced out in large studies but is important at individual level. For example, 27.5% of women over 20 years of age in the USA have a CRP value of 3–10 mg/L /13/.
  • Non-inflammatory stimuli influence lower CRP concentrations such as genetic factors /13/, physical activity, high protein intake, alcohol consumption, depressive syndromes, chronic fatigue syndrome, and obesity in particular (Fig. 19.4-3 – Relationship between CRP concentration and body mass index (BMI) in the United States population/7/.

19.4.6 Comments and problems

Specimen

Serum and capillary blood provide comparable CRP values in the range from 5–173 mg/L /75/.

Method of determination

High concentrations of rheumatoid factor can mimic high CRP concentration because the rheumatoid factor can bind to CRP to form a CRP/anti-CRP complex.

Reference interval

In a large study in the adult population, the following cutoff values were determined /76/:

  • The 5th percentile ranged from 0.12 mg/L in men aged 25–34 years to 0.43 mg/L in women aged 65–74 years
  • The 95th percentile ranged from 4.9 mg/L in men aged 25–34 years to 15.5 mg/L in women aged 55–74 years.

Reference interval for adult women in the USA: 24.5% have CRP concentrations below 1 mg/L, 29.7% have concentrations of 1–3 mg/L, 31.8% have concentrations of 3–10 mg/L, and 14% have levels above 10 mg/L /13/.

Reference interval for children and young adults /77/: mean value 1.6 mg/L; median 0.4 mg/L. The CRP concentration increases with age. Women aged 16–19 years have higher concentrations than men of the same age. Individuals of Mexican origin have higher concentrations than black or white Americans.

Prevalence of CRP values above 3 mg/L

In individuals whose lipid values are within the recommended ranges, the prevalence of increased CRP values fluctuates between 28.8 and 35.3%, depending on the lipid profile /14/.

Biological variation /78/

The CRP concentration is influenced by age. Values increase from an average of 0.21 mg/L for individuals aged 5–13 years to 0.86 mg/L for men and 0.75 mg/L for women in the age group 50–75 years.

Intraindividual variation

The CRP level can fluctuate between 0.1 and 10 mg/L within 6 months /6/.

Body mass index (BMI)

Individuals who are considerably overweight have higher CRP values than those with normal BMI.

Hormonal therapy

CRP concentrations are two times higher in women receiving treatment with estrogens and progestogens than among women not receiving treatment. The effect of oral contraceptives on CRP and serum lipids is shown in Tab. 19.4-5 – The effect of hormonal contraceptives on CRP and lipids.

Smoking

Increases the CRP concentration by 16–52% in men aged 29–75 years.

CRP as an indicator of cardiovascular risk

CRP should be measured in two blood samples, taken two weeks apart. If a value greater than 10 mg/L is identified, the test should be repeated and, if the value is confirmed, a search should be initiated for any source of mild to moderate inflammation /10/.

19.4.7 Biochemistry and physiology

CRP is a member of the pentraxin family. The molecule consists of five linked monomers, each composed of 206 amino acids, arranged symmetrically around a central pore /79/.

Refer to Fig. 19.4-4 – Ribbon drawing of CRP monomer with β strands shown as arrows, helices as ribbons, and loops as knots.

The molecular weight of CRP is 118 kDa. CRP is a non glycosylated protein and the gene that encodes it is located on chromosome 1q21-q23. Two Ca2+ ions are bound to the protein chains and are an integral component of the binding site for ligands. The pneumococcal c-polysaccharide is a typical ligand. Phospho choline residues of the pneumococcal c-polysaccharide are the main determinants of Ca2+ ion-mediated binding to CRP. In the presence of Ca2+ ions, CRP also binds histone, chromatin, fibronectin, laminin, oxidized LDL, and other phospho choline-containing polysaccharides /80/.

CRP is synthesized in the hepatocytes under the control of IL-6 but its synthesis and secretion are also influenced by IL-1 and TNF-α. CRP is synthesized in the liver following induction by IL-6, and at the maximum point of an acute phase response, as much as 20% of the liver protein synthetic capacity may be directed toward its synthesis. The normal synthetic rate is 1–10 mg/day, increasing to more than 1 g/day in acute inflammation. In patients with surgical trauma, the doubling time is 8–10 h in the plasma. In the absence of an IL-6 stimulus, the synthetic rate normalizes within 2–4 h but the half-life in the blood of patients following surgery is 24–48 h; this does not reflect the half-life of CRP in blood, but rather the disappearance of a non-infection dependent inflammation. The biological half-life of CRP is around 19 h but it may be cleared more rapidly once it is bound to ligands.

The functions of CRP include the detection, clearing and elimination of apoptotic tissue cells and their products, such as DNA, which may be toxic or allergenic. CRP also acts as a non-adaptive defense mechanism by opsonizing invading microorganisms for phagocytosis. CRP only binds to cells when the normal structure of the lipid bilayer has been disrupted, leading to the exposure of internal phospholipids.

Once bound to one of these endogenous or exogenous ligands, CRP is able to activate a number of biological systems that result in removal of the ligand by the following processes:

  • Complement is activated by the classical pathway with the consecutive clearance of CRP ligand complexes by macrophages in tissues, the blood, and the spleen. C3b deposited on the surface of these complexes is recognized by macrophages. In addition, anaphylatoxin and chemokine production results in inflammation and macrophage activation.
  • CRP binds to both high and low-affinity receptors on phagocytes such as CD32. CRP is thus exposed on the cell surface, allowing direct cellular contact with the ligands, the activation of complement and the initiation of phagocytosis.
  • Spleen macrophages clear CRP-coated ligands and material from the circulation
  • CRP binds to IgG FcR on T, B, and null cells and activates natural killer cells (NK cells)
  • CRP bound to the cell membrane of neutrophils at the inflammatory focus is cleaved by proteases to generate small tuftsin-like fragments, which activate macrophages and inhibit neutrophil function
  • CRP cross-links soluble and particulate ligands whereby these precipitate and become localized in the tissues
  • CRP in the vascular wall has a dominant role in the development of atherosclerosis. It up regulates the expression of adhesion molecules, activates complement, and induces the expression of tissue factor by macrophages (Fig. 19.4-5 – Interaction between CRP and LDL at the CD32 macrophage receptor/80/.

Basal plasma levels of CRP depend on the genetic polymorphism of the CRP gene. The allele frequency of haplotype-associated single nucleotide polymorphisms (SNPs) varies significantly in ethnic groups. In the white population, for example, rs3093068, rs1130846, and rs1417938 are associated with higher CRP concentrations while rs1205 and rs1800947 are associated with lower CRP levels /81/. However, such variants are not significantly associated with an increased risk of diabetes in postmenopausal women /82/.

Pentameric CRP is found in the plasma whereas monomeric CRP is expressed in tissues and vascular walls. It is assumed that both forms have different biological activity at endothelial cells. CRP exerts its effects primarily via the Fc receptor CD32 (FCRII) and up regulates the LOX-1 receptor, which facilitates the uptake of oxidized LDL (oxLDL) by endothelial cells. In contrast, monomeric CRP inhibits the uptake of oxLDL in endothelial cells independently of LOX-1, CD32, or CD16 /83/.

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19.5 Procalcitonin (PCT)

Lothar Thomas

The CALC-1 gene encodes procalcitonin and calcitonin gene-related peptide. In healthy individuals, PCT is produced by the C cells of the thyroid and by neuroendocrine cells in the lung and pancreas. PCT undergoes successive cleavage to form three molecules:

  • Calcitonin with 32 amino acids
  • Katacalcin with 21 amino acids
  • An amino terminal end with 57 amino acids.

All parenchymal organs and differentiated cells in the body produce PCT in response to stimulation by bacterial toxins and mediators of inflammation. The plasma PCT concentration rises within a matter of hours in systemic bacterial infection but local bacterial infection, viral infection and bacterial colonization do not induce a rise in PCT.

19.5.1 Indication

PCT mirrors the severity of infection and is the most studied biomarker for the risk stratification of patients /1/:

  • In suspicion of sepsis
  • To guide the use of antibiotics in systemic bacterial infection
  • To determine whether or not antibiotics are indicated in patients with upper respiratory tract infection
  • Prognostic marker in infection

19.5.2 Method of determination

Immunoluminometric assay (ILMA) /2/

Principle: the PCT containing specimen is incubated in a test tube coated with monoclonal antibodies directed against PCT. After removal of the unbound material the solid-phase bound PCT-antibody immuncomplexes are labeled using a second monoclonal antibody coupled to an acridinium ester. After this step the addition of NaOH and H2O2 produces a light signal with an intensity corresponding to the concentration of PCT in the sample.

Assays of different functional sensitivity (lowest concentration with CV% below 20) are available:

  • Assays with functional activity of 0.3–0.5 ug/L are used for diagnosis and monitoring of sepsis
  • Ultra sensitive assays with functional activity of 0.06 ug/L are used for the differentiation of upper respiratory tract infection and pneumonia.

Semi quantitative solid phase Immunoassay /3/

Principle: the test is a semi quantitative one-step solid phase immunoassay. A polyclonal sheep anti-calcitonin antibody is immobilized on a solid phase and a monoclonal gold-conjugated mouse-anti-catacalcin antibody is used as a tracer in the soluble phase. The serum of the sample solubilizes the tracer when applied to the provided test area and proceeds into the test area. The PCT-antibody immuncomplex becomes visible when it is immobilized and binds the tracer. A red colored line develops at concentrations of about 0.5 µg/L. The non-bound tracer permeates into the area of the control line, developing a dark red second line, indicating the positive function of the assay. The following PCT levels by comparison with the reference scale are differentiated < 0.5 μg/L, 0.5 to < 2 μg/L, > 2 to ≤ 10 μg/L, and > 10 μg/L.

19.5.3 Specimen

Plasma (EDTA, citrate, heparin) or serum: 1 mL

19.5.4 Reference interval

Adults and children: ≤ 0.02 µg/L /3/

19.5.5 Clinical significance

Any attack against the body, whether inflammatory, traumatic, or chemical, stimulates the production of pro inflammatory cytokines. The PCT level is below 0.5 μg/L.

In severe systemic inflammation, especially that caused by bacteria, the body launches a vigorous immune response that triggers many changes such as the synthesis of proteins that can be used as biomarkers of inflammation and infection. The PCT level may increase to ≥ 0.5 μg/L.

PCT levels show a large scatter between patients with a seemingly similar clinical condition, hence single absolute values are difficult to interpret. However, there is overwhelming evidence that in most cases high PCT concentrations indicate bacterial infection. The shortcomings of PCT absolute values might be compensated when the kinetics of PCT is taken into account to indicate infection /4/.

Unlike CRP and IL-6, PCT concentrations do not increase ≥ 0.5 μg/L in sterile inflammatory processes such as connective tissue disease and rheumatic disease, postoperative inflammation, and many febrile conditions unless bacterial superinfection occurs.

19.5.5.1 PCT in infections

In systemic bacterial infection, plasma PCT rises rapidly and continues to rise if inflammation is uncontrolled, antibiotic therapy is not used, or the correct antibiotics are not applied. The sensitivity and specificity of PCT to indicate bacterial infection is 75–85%.

PCT is not elevated in viral infections (e.g., viral meningitis) and is only moderately elevated in systemic fungal infections. PCT cannot be used to reliably distinguish sepsis within the systemic inflammatory response syndrome (Tab. 19.5-1 – PCT in the differential diagnosis of inflammatory diseases).

Transient elevations in PCT can occur due to weakness of the intestinal barrier caused by hypotension, surgical intervention, or intestinal paralysis. In all of these cases, PCT declines again within 24 h.

Following contact with bacterial endotoxin, PCT is detectable within a few 4–6 h, reaches its peak within 24 h and then starts its decline in the case of adequate treatment with levels reducing by approximately 50% daily. In contrast, serum CRP is elevated only after 12 h and reaches a peak at 24–48 h. While increased PCT concentrations return below the threshold value within 2–3 days, CRP remains elevated for 3–7 days. The temporal course of the rise in PCT compared to other inflammatory markers is shown in Fig. 19.5-1 – Time profile of the increase in tumor necrosis factor (TNF), interleukins, C-reactive protein (CRP), and procalcitonin following surgical trauma.

19.5.5.2 Differentiation of bacterial infections from other systemic inflammatory disorders

In inflammatory disorders of unknown etiology, PCT indicates a possible bacterial etiology. In non bacterial disease (postoperative inflammation, viral infection, automimmune disease, transplant rejection), the PCT concentration is low (< 0.5 μg/L or rarely 0.5–2 μg/L) in cases of comparable clinical severity.

During the course of non bacterial inflammatory diseases, PCT concentrations may rise due to secondary bacterial infection, sepsis, or organ dysfunction. It is also important to bear in mind that PCT elevation can occur without infection in multiple trauma, burns, extensive surgery, or prolonged circulatory shock.

PCT is a more useful biomarker than CRP for distinguishing between bacterial infection and other non infectious causes of inflammation /5/. Refer to Fig. 19.5-2 – Receiver operator characteristic (ROC) curves for PCT and CRP as tools for differentiating between infectious and non-infectious etiology of inflammation.

19.5.5.3 Monitoring patients at high risk for infections

PCT is used as a biomarker for monitoring the course and treatment of inflammations with high-grade CRP elevation. In sepsis the PCT level correlates with disease severity and mortality. If the initial concentration is elevated, PCT should be determined daily for monitoring purposes. Consistent PCT elevations indicate an unfavorable prognosis. The pattern of the PCT concentration during the course of the disease provides information about the severity of inflammation. PCT is used as a biomarker to guide therapy and as a prognostic indicator.

Because of its short half-life, PCT is a useful parameter for monitoring the course of sepsis. It is therefore used to monitor infection in critically ill patients.

PCT has a high negative predictive value. Low levels rule out systemic bacterial infection with a high degree of certainty.

Since PCT does not rise in response to local inflammation and trivial infections, it is a better diagnostic tool than CRP, temperature measurement, or the WBC count.

19.5.5.4 Infections in children

PCT levels in healthy children are < 0.5 μg/L, with a slight increase (0.5–2 μg/L) during viral infections, non-infectious inflammation, stress situations, and focal bacterial infections. In systemic bacterial infections, PCT increases > 2 μg/L and can reach concentrations as high as 50–100 μg/L /6/.

In approximately 20% of children who attend the emergency department with fever, clinical examination fails to reveal a source for the fever /7/. Although the vast majority of these children have viral infections, 10–20%, in particular those under the age of 3 years, may have unrecognized serious bacterial infections such as pyelonephritis, pneumonia, osteomyelitis, or bacterial meningitis. The WBC count and CRP measurement have comparable diagnostic specificity and a sensitivity of only 70–86% /8/. The additional determination of PCT increases the positive likelihood ratio. In severe bacterial infection, the positive likelihood ratio is 10.6 by a combination of WBC > 15 × 109/L, CRP > 50 mg/L, and PCT > 2 μg/L /9/.

19.5.5.5 Neonatal infections

Bacterial infections are the main cause of neonatal morbidity and mortality, especially in premature infants. Neonatal infections are difficult to diagnose through medical examination alone since the clinical symptoms (hypoglycemia, respiratory distress syndrome) are either non specific or are not present at all if infection occurred shortly before delivery or is still in the early phase. Blood and CSF cultures are often negative and need an assay time up to 24 h. In such cases, it is often difficult to decide whether or not to initiate antibiotic therapy and to decide when antibiotics should be discontinued. Although PCT is an indicator of sepsis in adults and children, its use in suspicion of neonatal sepsis is not recommended. The reasons are:

Because of the afore mentioned reasons the IL-6 concentration should be determined if neonatal sepsis is suspected.

19.5.5.6 Inappropriately high or low PCT levels

Diseases and conditions in which PCT is elevated despite the absence of bacterial infection and those in which PCT levels are low in spite of obvious bacterial infection are listed in Tab. 19.5-2 – PCT levels that are not consistent with the corresponding disease or condition.

19.5.6 Comments and problems

Method of determination

In a study /12 /the evaluation of Diazyme PCT on Roche Cobas c702 (PCT-D), and Brahms PCT on Roche Cobas e602 (PCT-BR) analyzers was compared with Brahms PCT-sensitive Kryptor (PCT-BK). Almost 40% of the samples at concentration < 0.5 μg/L seem to show sample-related interferences using PCT-D. PCT-BR and PCT-BK showed a good correlation.

Reference interval

In healthy individuals, plasma concentrations of PCT are in the range of 0.005–0.05 μg/L. However, clinical studies have indicated a threshold value of < 0.5 μg/L to rule out sepsis.

Stability

In whole blood at room temperature, PCT decreases by 9% after 6 h and 13% after 24 h. At 4 °C, concentrations do not decline significantly within the first 6 h and have declined by 7% after 24 h. Repeated freezing (–20 °C) and thawing of samples does not lead to a loss in PCT concentration /13/. In cerebrospinal fluid (CSF) stored at 20 °C and 4 °C a degradation over the first 72 h of 5% and 8%, respectively was measured /14/.

19.5.7 Biochemistry and physiology

PCT is a protein with a molecular weight of 13 kDa that contains the amino acid sequence of human calcitonin (hCT, 32 amino acids) at position 60–91 /1516/. Refer to Fig. 19.5-4 – Procalcitonin (amino acids 1–116).

PCT is not glycosylated and, in plasma, the N-terminals of two amino acids are cleaved off by the enzyme dipeptidylpeptidase 4 (DPP4, CD26), which is present in renal, epithelial, and endothelial cells. Other fragments of PCT can also be detected in the plasma. PCT exists in two forms (PCT-I and PCT-II), which can be distinguished based on their eight C-terminal amino acids. PCT-I is the predominant form found in the plasma in sepsis. Commercial assays can detect both forms.

While endotoxin is a powerful stimulus for PCT synthesis, pro-inflammatory cytokines such as TNF-α and IL-6 can also induce the synthesis of PCT, albeit to a lesser extent. PCT synthesis is induced in various body cells in response to infection and inflammation but the greatest proportion is synthesized by the liver /17/. Its half-life in the plasma is 25–35 h, which may be prolonged by up to 30–40% in severe renal insufficiency without resulting in an accumulation of PCT /18/.

PCT is cleared by hemofiltration using PMSF 1200 membranes with a sieving coefficient of around 0.2 /19/. At normal filtration solution flow rates (< 1–2 L per hour), effects on the plasma level are minor but when high-flux systems and other membranes are used, a reduction in the plasma concentration can be observed.

The biological functions of PCT are not fully known. The protein may not have any measurable effects in healthy individuals but is associated with increased mortality in sepsis. Microbial infection generally causes increased expression of the CALC-1 gene, with the subsequent release of calcitonin-1 precursors in all body tissues. In bacterial infections, the serum PCT concentration can rise from values below the measurement range (0.005 μg/L) to as much as 1,000 μg/L. Such a rise correlates with the disease severity and mortality.

References

1. Christ-Crain M, Schuetz P, Huber AR, Müller B. Procalcitonin: importance for the diagnosis of bacterial infections. J Lab Med 2008; 32: 425–33.

2. Meisner M, Brunkhorst FM, Reith HB, Schmidt J, Lestin HG, Reinhart K. Clinical experiences with a new semi-quantitative solid phase immunoassay for rapid measurement of procalcitonin. Clin Chem Lab Med 2000; 38: 989–95.

3. Snider RH, Jr, Nylen ES, Becker KL. Procalcitonin and its component peptides in systemic inflammation: immunochemical characterization. J Investig Med 1997; 45: 552–60.

4. Trasy D, Molnar Z. Procalcitonin – assisted antibiotic strategy. eJIFCC 2017; 28: 104–13.

5. Simon L, Gauvin F, Amre DK, Saint-Louis P, Lacroix J. Serum procalcitonin and C-reactive protein levels as markers of bacterial infection: a systematic review and meta-analysis. Clin Infect Dis 2004; 39: 206–17.

6. Turner D, Hammerman C, Rudensky B, Schlesinger Y, Goia C, Schimmel MS. Procalcitonin in preterm infants during the first few days of life: introducing an age related nomogram. Arch Dis Child Fetal Neonatal Ed 2006; 91: F283–F286.

7. Baraff LJ. Management of fever without source in infants and children. Ann Emerg Med 2000; 36: 602–14.

8. Pullium PN, Attia MW, Cronan KM. C-reactive protein in febrile children 1 to 36 months of age with clinically undetectable serious bacterial infection. Pediatrics 2001; 108: 1275–9.

9. Thayyil S, Shenoy M, Hamaluba M, Gupta A, Frater J, Verber IG. Is procalcitonin useful in early diagnosis of serious bacterial infections in children? Acta Paediatrica 2005; 94: 155–8.

10. Guibourdenche J, Bedu A, Petzold L, Marchand M, Mariani-Kurdjian P, Hurtaud-Roux MF, et al. Biochemical markers of neonatal sepsis: value of procalcitonin in the emergency setting. Ann Clin Biochem 2002; 39: 130–5.

11. Chiesa C, Pellegrini G, Pandero A, Osborn JF, Signore F, Assumma M, Pacifico L. C-reactive protein, interleukin-6, and procalcitonin in the immediate postnatal period: influence of illness severity, risk status, antenatal and perinatal complications, and infection. Clin Chem 2003; 49: 60–8.

12. Ceriotti F, Marino I, Motta A, Carobene A. Analytical evaluation of the performances of Diazyme and Brahms procalcitonin applied to Roche Cobas in comparison with Brahms PCT.sensitive kryptor. Clin Chem Lab Med 2018; 56: 162–9.

13. Meisner M, Tschaikowsky K, Schnabel S, Schmidt J, Katalinic A, Schüttler J. Procalcitonin – influence of temperature, storage, anticoagulation and arterial or venous asservation of blood samples on procalcitonin concentrations. Eur J Clin Chem Clin Biochem 1997; 35: 597–601.

14. Dorresteijn KRIS, Jellema K, Ponjee GAE, Verheul RJ. Stability of procalcitonin in cerebrospinal fluid. Clin Chem Lab Med 2017; 55: e230-e232.

15. Meisner M. Procalcitonin: Erfahrungen mit einer neuen Messgrösse für bakterielle Infektionen und systemische Inflammation. J Lab Med 1999; 23: 263–72.

16. Le Moullec JM, Jullienne A, Chenais J, Lasmoles F, Guliana JM, Milhaud G, Moukhtar MS. The complete sequence of human preprocalcitonin. FEBS 1984; 167: 93–7.

17. Meisner M, Müller V, Khakpour Z, Toegel E, Redl H. Induction of procalcitonin and proinflammatory cytokines in an anhepatic baboon endotoxin shock model. Shock 2003; 19: 187–190.

18. Meisner M, Lohs T, Hüttemann E, Schmidt J, Hüller M, Reinhart K. The plasma elimination rate and urinary secretion of procalcitonin in patients with normal and impaired renal function. Eur J Anaesthesiol 2001; 18: 79–87.

19. Meisner M, Hüttemann E, Lohs T, Kasakov L, Reinhart K. Plasma concentrations and clearance of procalcitonin during continuous veno-venous hemofiltration in septic patients. Shock 2001; 15: 171–5.

20. Brunkhorst FM. Sepsismarker – was ist sinnvoll? Dtsch Med Wochenschr 2008; 133: 2512–5.

21. Sudhir U, Venkatachalaiah RK, Kumar TA, Rao MY, Kempegowda P. Significance of serum procalcitonin in sepsis. Indian J Crit Care Med 2011; 15: 1–5.

22. Uzzan B, Cohen R, Nicolas P, Cucherat M, Perret GY. Procalcitonin as a diagnostic test for sepsis in critically ill adults and after surgery or trauma: a systematic review and meta-analysis. Crit Care Med 2006; 34: 1996–2003.

23. Tang BMP, Eslick GD, Craig JC, McLean A. Accuracy of procalcitonin for sepsis diagnosis in critically ill patients: systemic review and meta-analysis. Lancet Infect Dis 2007; 7: 210–7.

24. Haasper C, Kalmbach M, Dikos GD, Meller R, Müller C, Krettek C, et al. Prognostic value of procalcitonin (PCT) and/or interleukin-6 (IL-6) plasma levels after multiple trauma for the development of multi organ dysfunction syndrome (MODS) or sepsis. Technol Health Care 2010; 18: 89–100.

25. Jensen JU, Heslet L, Jensen TH, Espersen K, Steffensen P, Tvede M. Procalcitonin increase in early identification of critically ill patients at high risk of mortality. Crit Care Med 2006; 34: 2596– 2602.

26. Luyt CE, Guerin V, Combes A, Trouillet JL, Ayed SB, Bernard M, et al. Procalcitonin kinetics as a prognostic marker of ventilator-associated pneumonia. Am J Respir Crit Care Med 2005; 171: 48–53.

27. Bouadma L, Luyt CE, Tubach F, Cracco C, Alvarez A, Schwebel C, et al. Use of procalcitonin to reduce patients’ exposure to antibiotics in intensive care units (PRORATA trial): a multicentre randomised controlled trial. Lancet 2010; 375: 463–74.

28. Charles PE, Tinel C, Barbar S, Aho S, Prin S, Doise JM, et al. Procalcitonin kinetics within the first days of sepsis: relationship with appropriateness of antibiotic therapy and outcome. Crit Care 2009; 13: R 38.

29. Rau B, Steinbach G, Gansauge F, Mayer JM, Grünert A, Beger HG. The potential role of procalcitonin and interleukin 8 in the prediction of infected necrosis in acute pancreatitis. Gut 1997; 41: 832–40.

30. Benador N, Siegrist CA, Gendrel D, Greder C, Benador D, Assicot M, et al. Procalcitonin is a marker of severity of renal lesions in pyelonephritis. Pediatrics 1998; 102: 1422–5.

31. Mantadakis E, Plessa E, Vouloumanou EK, Karageogopoulos DE, Chatzmichael A, Falagas ME. Serum precalcitonin for prediction of renal parenchymal involvement in children with urinary tract infections: a meta-analysis of prospective clinical studies. J Pediatr 2009; 155: 875–81.

32. Kuse ER, Langefeld I, Jaeger K, Külpmann WR. Procalcitonin in fever of unknown origin after liver transplantation: a variable to differentiate acute rejection from infection. Crit Care Med 2000; 28: 555–9.

33. Schuetz P, Christ-Crain M, Thomann R, Falconnier C, Wolbers M, Widmer I, et al. Effect of procalcitonin based guidelines on antibiotic use in lower respiratory tract infections. The ProHosp randomized controlled trial. JAMA 2009; 302: 1059–66.

34. Odermatt J, Friedli N, Kutz A, Briel M, Bucher HC, Christ-Crain M, et al. Effects of procalcitonin testing on antibiotic use and clinical outcomes in patients with upper respiratory tract infections. An indidividual patient data meta-analysis. Clin Chem Lab Med 2018; 56: 170–7.

35. Liu D, Su LX, Guan W, Xiao K, Xie LX. Prognostic value of procalcitonin in pneumonia: a systematic review and meta-analysis. Respirology 2016; 21: 280–8.

19.6 Serum amyloid A (SAA) protein

Lothar Thomas

SAA is an acute phase protein that is bound to plasma high-density lipoprotein (HDL). Like CRP, SAA is synthesized by the liver in response to stimulation by IL-6 as part of an acute phase response. SAA is also the precursor protein of reactive amyloid deposits in the organs. SAA is used to diagnose inflammation due to viral or bacterial infection in particular but also low-grade inflammation that is not caused by microbial infection.

19.6.1 Indication

  • Inflammatory marker in viral infections
  • Biomarker of transplant rejection
  • Investigation of patients with amyloidosis
  • Prediction of cardiovascular disease.

19.6.2 Method of determination

Enzyme immunoassay /1/, latex-enhanced immunonephelometric or immunoturbidimetric assay /2/, or surface-enhanced laser de sorption/ionization (SELDI) protein chip technology /3/.

19.6.3 Specimen

Serum, plasma: 1 mL

19.6.4 Reference interval

Refer to Ref. /4/ and Tab. 19.6-1 – Threshold values for serum amyloid A protein.

19.6.5 Clinical significance

The serum SAA concentration is a very sensitive, but non-specific marker both in diagnosis, prognosis and monitoring of inflammatory and infectious diseases. Under pathological conditions i.e., inflammatory or infectious disease or neoplasia SAA concentrations of more than 10 mg/L and up to 1,000 mg/l are found in serum. SAA rises approximately 8 h after the start of the inflammatory response but exceeds the upper reference interval value earlier than CRP. Whereas in the case of CRP, the median value of healthy individuals differs from the upper reference interval value by approximately a factor of 10, this factor is only 5 in the case of SAA. In milder infections (e.g., in many viral infections) SAA is therefore more commonly elevated than CRP /5/. In infections, the rise in SAA is higher than that of CRP (Tab. 19.6-2 – Comparison of SAA and CRP in viral infection).

Chronically elevated SAA concentrations (e.g., in patients with rheumatoid arthritis, tuberculosis, or leprosy) lead to the synthesis of amyloid A (AA) fibrils and secondary amyloidosis /6/.

19.6.5.1 SAA in infectious diseases

Since SAA is a more sensitive indicator of low-grade inflammation than CRP, it has an advantage over CRP in the diagnosis and differential diagnosis of infections. A positive correlation has been observed between SAA and CRP in bacterial infections when the CRP exceeds 100 mg/L, with the concentration of SAA being approximately 10–15 times higher than that of CRP /7/. SAA is more effective at discriminating between normal and disease states than CRP, especially in mild APRs (Tab. 19.6-3 – SAA in inflammatory diseases)

19.6.6 Comments and problems

Method of determination

Calibration is carried out using an SAA reference preparation created in 1997.

19.6.7 Biochemistry and physiology

SAA is the generic name of a family of proteins with 103–104 amino acids that share high levels of sequence homology, but are encoded by different genes. In humans there are 4 SAA genes (SAA1, SAA2, SAA3 and SAA4). Of these genes SAA1 and SAA2 code for acute-phase SAA proteins. SAA4 encodes a constitutively expressed SAA protein, and SAA3 is a pseudo gene /8/.

The N-terminal region of SAA is predicted to harbor an HDL binding site, whereas the C-terminal segment constrains the overall SAA structure such that its removal facilitates aggregation of the cleavage product AA, forming highly ordered β-sheets as in amyloid fibrils /8/.

SAA exists in the serum as two isoforms, SAA1 and SAA2, whose primary structures are 93% identical. SAA1 is the predominant isoform in the acute phase response. The SAA1/SAA2 ratio is about 3 to 1 and remains stable during the acute phase response /9/. Whereas the promoter genes for both SAA and CRP have a high sensitivity for IL-6, the SAA promoter genes are more sensitive than those of CRP to stimulation by IL-1β.

Like CRP, SAA is synthesized mainly by hepatocytes. After SAA is secreted, it binds to HDL, LDL, and VLDL but especially to HDL3. During the acute phase response, the SAA plasma concentration increases 100–1,000-fold as a result of increased incorporation of SAA into the HDL particles (Fig. 19.6-1 – Relationship between SAA and apo A-I in the lipid bilayer of HDL particles in healthy individuals and in those during an acute phase response).

SAA is an acute-phase protein and possesses pro inflammatory cytokine-like activity and is chemotactic for phagocytes. The results of a study /10/ indicate that the N- and C-terminal sequences contain structural determinants for the pro inflammatory and chemotactic activities of SAA, and their removal switches SAA to an antiinflammatory role.

SAA is catabolized in hepatocytes after it is taken up into these cells along with the HDL particle. During an acute phase response, this catabolism is reduced. This indicates that the SAA increase during an acute phase response is due to increased synthesis and reduced degradation of SAA /5/.

SAA is the main constituent of the fibrillary component of amyloid A deposits in the tissues. The homozygosity at the SAA1 allele is a strong predictor for type AA amyloidosis.

SAA has various biological functions /11/. The minimal effective SAA concentrations required to exert biological activity are shown in Tab. 19.6-4 – Various biological functions of SAA in diseases

References

1. Wilkins J, Gallimore JR, Tennent GA, Hawkins PN, Limburg PC, van Rijswijk MH, et al. Rapid automated enzyme immunoassay of serum amyloid A. Clin Chem 1994; 40: 1284–90.

2. Yamada T, Nomata Y, Sugita O, Okada M. A rapid method for measuring serum amyloid A protein by latex agglutination nephelometric immunoassay. Ann Clin Biochem 1993; 30: 72–6.

3. Pang RTK, Poon TCW, Chan KCA, Lee NLS, Chiu RWK, Tong YK, et al. Serum amyloid A is not useful in the diagnosis of severe acute respiratory syndrome. Clin Chem 2006; 52: 1202–4.

4. Lannergard A, Friman G, Ewald U, Lind L, Larsson A. Serum amyloid A (SAA) protein and high-sensitivity C-reactive protein (hsCRP) in healthy newborn infants and healthy young through elderly adults. Acta Paediatr 2005; 94. 1198–1202.

5. Whicher J, Chir B. Serum amyloid A. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research 1996: 7.02-1–5.

6. Husebekk A, Skogen B, Husby G, Marhang G. Transformation of amyloid precursor SAA to protein AA and incorporation of amyloid fibrils in vivo. Scand J Immunol 1985; 21: 283–7.

7. Lannergard A, Larsson A, Kragsbjerg P, Friman G. Correlations between serum amyloid A protein and C-reactive protein in infectious diseases. Scand J Clin Lab Invest 2003; 63: 267–72.

8. Sun L, Ye RD. Serum amyloid A1: structure, function ang gene polymorphism. Gene 2016; 25: 48–57.

9. Xu Y, Yamada T, Datoh T, Okuda Y. Measurement of serum amyloid A1 (SAA1), a major isotype of acute phase SAA. Clin Chem Lab Med 2006; 44: 59–63.

10. Zhou H, Chen M, Zhang G, Ye RD. Suppression of lipopolysaccharide-induced inflammatory response by fragments from serum amyloid. J immunol 2017; 199: 1105–12.

11. De Buck M, Gouwy M, Wang JM, van Snick J, Openakker G, Struyf S, van Damme J. Structure and expression of different serum amyloid A (SAA) variants and their concentration-dependent functions during host results. Current Medical Chemistry 2016; 23: 1725–55.

12. Peltola HO. C-reactive protein for rapid monitoring of infections of the central nervous system. Lancet 1982; i: 980–2.

13. Nakayama T, Sonoda S, Urano T, Yamada T, Okada M. Monitoring both serum amyloid protein A and C-reactive protein as inflammatory markers in infectious diseases. Clin Chem 1993; 39: 293–7.

14. Mangiarotti P, Moulin F, Palmer P, Ravilly S, Raymond J, Gendrel D. Interferon-alpha in viral and bacterial gastroenteritis: a comparison with C-reactive protein and interleukin-6. Acta Paediatr 1999; 88: 592–4.

15. Lindback S, Hellgren U, Julander I, Hansson LO. The value of C-reactive protein as a marker of bacterial infection in patients with septicemia/endocarditis and influenza. Scand J Infect Dis 1989; 21: 543–9.

16. Whicher JT, Chambers RE, Higginson J, Nashel L, Higgins PG. Acute phase response of serum amyloid A protein and C-reactive protein to the common cold and influenza. J Clin Pathol 1985; 38: 312–6.

17. de Beer FC, Fagan EA, Hughes GRV, Mallya RK, Lanham JG, Pepys MB. Serum amyloid A protein concentration in inflammatory diseases and its relationship to the incidence of reactive systemic amyloidosis. Lancet 1982; ii: 231–3.

18. Lojnaric I, Casl T, Simic D, Lukac J. Serum amyloid A protein in colorectal carcinoma. Clin Chem Lab Med 2001; 39: 129–33

19. Maury CPJ, Teppo AM, Eklund B, Ahonen J. Serum amyloid A protein: a sensitive indicator of renal allograft rejection in humans. Transplantation 1983; 36: 501–4.

20. Müller TF, Grebe SO, Neumann MC, Lange H, Reibnegger G. Die Zeit als Schlüsselvariable beim immunologischen Monitoring nach Organtransplantation. Berichte der ÖGKC 1998; 21: 24–7.

21. Johnson DB, Kip KE, Marroquin OC, Ridker PM, Kelsey SF, Shaw LJ, et al. Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women. Circulation 2004; 109: 726–32.

22. Kang X, Xu Y, Wu X, Liang Y, Wang C, Guo J, et al. Proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome. Clin Chem 2005; 51: 56–64.

23. Real de Asua D, Costa R, Galvan JM, Filigheddu MT, Trujillo D, Cadinanos J. Systemic AA amyloidosis: epidemiology, diagnosis, and management. Clinical Epemiology 2014; 6: 369–77.

19.7 Polymorphonuclear neutrophil function

Gertrud M. Hänsch

Polymorphonuclear neutrophils (PMNs) play a key role in the clearance of pathogens. They are capable of affecting many aspects of both innate non-specific, non-adaptive cellular immune response and adaptive immunity. They are one of the front-line defenders involved in bacterial ingestion and subsequent killing but are also involved in defending the body against parasites, viruses, and tumors /1/.

Abnormal granulocyte function generally manifests as frequent, recurrent, or treatment-resistant bacterial infection, in some cases with banal or generally nonpathogenic microorganisms (Tab. 19.7-1 – Microorganisms in polymorphonuclear neutrophil deficiency). It makes sense to investigate granulocyte function if other causes of immunodeficiency such as antibody deficiency or complement deficiencies have been ruled out.

Because PMN function is influenced by many antibiotics (Tab. 19.7-2 – Impact of antibiotics on granulocyte function), investigation should be performed in a therapy-free interval.

19.7.1 Indication

  • Increased susceptibility to infections
  • Treatment-resistant infections
  • Recurrent infections with banal or generally harmless microorganisms
  • Severe periodontitis
  • Impaired wound healing.

19.7.2 Method of determination

The following can be determined:

  • Granulocyte count, morphology, and receptor expression
  • Functions such as chemotaxis, phagocytosis, and oxygen radical production
  • Genetic defects and genetic polymorphisms.

These investigations can be used to confirm or rule out neutropenia, PMN deficiencies, and important functional deficiencies. If the results indicate pathology, the investigations should be repeated after a few weeks and family members should also be tested if possible. If a disorder of PMN function is confirmed, further investigations are performed to determine the type of defect involved. A diagnostic algorithm is shown in Fig. 19.7-1 – Stepwise approach to diagnose functional PMN deficiency.

19.7.2.1 Granulocyte count and morphology

If the neutrophil count in the blood falls below the lower reference interval value, neutropenia is present. This can be caused by a range of factors, such as infections (Parvovirus), drugs (chemotherapy, antibiotics), or abnormal neutrophil maturation (refer to Section 15.12 – Leukocyte count). Molecular defects have been described in the case of congenital neutropenias. Mutations in the ELA2 gene occur in cyclic neutropenia, mutations in the Hax1 gene occur in Kostmann’s disease, and mutations in the WAS gene occur in X-linked neutropenia. Although the relationships between the gene defects and neutropenia have not yet been established, demonstration of the respective mutation results in a reliable diagnosis /4/.

Altered granulocyte morphology can be demonstrated using stains in a conventional blood smear. A deficiency of secondary specific granules is associated with increased susceptibility to infections. It is caused by a lack of transcription factor C/EBPε and results in reduced chemotaxis and reduced production of oxygen radicals /4/.

19.7.2.2 Testing of polymorphonuclear neutrophil function

A variety of in vitro assays are available for assessing PMN function, some using whole blood and some using isolated cells. Various procedures are used to isolate PMNs, such as density gradient sedimentation or centrifugation or hypotonic lysis of erythrocytes. Although contamination with monocytes and lymphocytes can occur, this does not affect fast functional assays.

19.7.2.2.1 Testing of spontaneous motility and chemotaxis

Principle of Boyden chamber technique /56/: in this technique, isolated granulocytes are allowed to migrate through a filter with the appropriate pore size toward a specific chemoattractant. This takes place in a chamber (Boyden chamber), which is separated by a filter (pore size 3 μm) into two compartments. The chemoattractant is placed in the lower compartment and the granulocytes (1 × 109/L) are contained in the upper compartment. Yeast-activated plasma is used as a chemoattractant and the chemoattractant peptide f-Met-Leu-Phe (formyl-methionine-leucine-phenylalanine) or interleukin 8 is used as a source of complement 5a.

The chambers are incubated for 2–3 h at 7 °C to migrate through the filter. The filter is then removed, and the cells are fixed, stained, and examined under a microscope. When granulocyte chemotaxis is assessed, a comparison must always be made with granulocytes donated by healthy controls.

By microscopic analysis, the following criteria are used to assess chemotaxis:

  • Number of cells on the surface and at different levels within the filter are counted. The number of cell at each level is plotted against the distance from the top. The area under the resulting curve is calculated and used as a chemotactic index.
  • Greatest granulocyte migration distance. In this method, known as the leading-front method, the greatest distance at which 5 granulocytes are still visible in the view field is determined.
  • Number of granulocytes that have reached the bottom of the filter.

Analytical evaluation: the testing of patient samples allow the following functions to be assessed:

  • Spontaneous motility of the PMN
  • Chemotactic activity of the PMN
  • Ability to generate chemotactic activity in the serum.

Medical assessment: a low level of spontaneous migration and/or chemotaxis suggests an adhesion defect. Flow cytometry is used to detect CD11/CD18 adhesion proteins. If expression of these proteins is normal, a possible deficiency can be investigated further by determining CD15, actin polymerization, complement receptors, and membrane signaling. Refer to Tab. 19.7-3 – Medical assessment of spontaneous granulocyte motility and chemotaxis.

19.7.2.2.2 Testing of phagocytosis and intracellular killing

Many methods have been described for determining phagocytosis (the internalization of bacteria) and intracellular killing /78/.

Principle des phagocytosis and killing assays: radioactively labeled, serum-resistant bacteria, (e.g., E. coli) are opsonized by incubation with serum and presented to isolated PMN for phagocytosis. After 20 min., the granulocytes are exposed to ultrasound to stimulate the release of phagocytosed bacteria. The phagocytosis rate is determined from the number of bacteria presented for phagocytosis and the number of bacteria actually phagocytosed. Intracellular killing is determined by plating the released bacteria and counting the colonies 20 hours later. The advantage of this method is that phagocytosis and killing can be determined simultaneously in the same assay. The disadvantage is that radioactive labeling is expensive, even with low levels of radioactivity.

Phagocytosis can also be detected using fluorescein (FITC)-labeled bacteria, using microscopy or flow cytometry. Heparinized blood can be used for this method, keeping experimental costs low, and the isolation procedure eliminates artefacts. Fixed, FITC-labeled bacteria are available commercially, which improves the standardization of the method. A disadvantage is that bacterial killing cannot be measured at the same time.

As in all functional assays, patient PMNs and PMNs donated by a healthy control are tested in parallel. If bacteria from patient isolates are used in this assay, their resistance to the body’s defense mechanisms can also be tested. This is relevant because bacterial variants have been described that, although they can be phagocytosed, cannot be killed.

Analytical evaluation: the ratio of phagocytosed bacteria to bacteria that are phagocytosed but not killed is determined.

Reference interval: 80–90%.

19.7.2.2.3 Nitro blue tetrazolium (NBT) test

Principle: this method allows the phagocytic capability of PMN and their ability to produce oxygen radicals to be investigated in parallel /9/. Isolated PMN are incubated with a suspension of Candida albicans, serum, and the dye NBT. If oxygen radicals are produced, the dye is reduced and a blue precipitate forms in the PMN. Light microscopy is used to detect any intracellular Candida or blue coloration. A blank test is also run, using a buffer solution instead of patient serum.

Analytical evaluation

The number of PMN with intracellular Candida particles (phagocytosis) and the number of PMN with blue coloration (phagocytosis and oxygen radical formation) are counted.

Reference interval

Test

Phagocytic PMN (%)

O2 radical producing PMN (%)

Spontaneous phagocytosis

60–80

40–60

Stimulated phagocytosis

80–100

80–100

Medical assessment

Refer to Tab. 19.7.4 – Medical assessment of phagocytosis and intracellular bacterial killing.

19.7.2.2.4 Determination of oxygen radicals

A number of methods are available to quickly and easily test the ability of PMN to produce oxygen radicals in response to stimulation. Depending on the laboratory equipment available, fluorescence or luminescence assays can be used.

Cytochrome c assay

Principle: isolated granulocytes are suspended in a cytochrome c solution and stimulated by PMA (phorbol 12-myristate 13-acetate), independently of receptor activation, to release oxygen radicals. The radicals produced reduce the cytochrome c and the resulting change in absorption is measured spectrophotometrically. The quantity of oxygen radicals produced is calculated from the change in absorption and the absorption coefficient for cytochrome c /10/.

Analytical evaluation: the ability of the patient’s PMN to produce oxygen radicals is compared with those of a control individual tested in parallel. The amount (in nmol) of reduced cytochrome c produced per 1 million cells per unit of time is measured.

Reference interval: 5–15 nmol/106 PMN

Medical assessment: reduced or absent oxygen radical production is suggestive of defects in the enzyme cascade that leads to the reduction of molecular oxygen. Some of these enzymes or coenzymes can be determined and used to investigate granulocyte function.

19.7.3 Specimen

Fresh whole blood (heparin, 50 IU/mL) that has been collected simultaneously from the patient and a healthy control and stored for the shortest time possible.

19.7.4 Clinical significance

Disorders of PMN function increase susceptibility to infections. Tab. 19.7-1 – Microorganisms in polymorphonuclear neutrophil deficiency lists the microorganisms detected in patients with abnormal PMN function. PMN deficiencies are extremely rare (less than 1 : 200,000) and mainly affect infants and young children; a number of hereditary disorders exist. In these cases, family studies can help to support the diagnosis. PMN deficiencies can be primary or secondary to other diseases or associated with particular underlying diseases, even though the association is not always clear at molecular level /1112/. Refer to Tab. 19.7-5 – Disorders of PMN function.

Disorders of PMN function may not manifest until adulthood, especially in cases where residual functions are preserved.

When evaluating the results of investigations, it is especially important to take the influence of medication into account. Antibiotics in particular can affect chemotaxis, phagocytosis, adhesion, and oxygen radical production /1314/. Refer to Tab. 19.7-2 – Impact of antibiotics on granulocyte function.

19.7.4.1 Leukocyte adhesion deficiency (LAD) syndromes

Leukocyte adhesion deficiency is divided into two subtypes: LAD I and LAD II.

LAD I is results from an autosomal recessive inherited deficiency of the β2-chain (CD18), which also results in reduced CD11 expression. In clinically severe cases, no CD11/CD18 is detectable on the granulocyte receptor, whereas in less severe cases, 3–10% of the normal quantity of CD11/CD18 is expressed. The cell adhesion deficiency is a distinct clinical entity /15/.

LAD II is caused by abnormal fucosylation that affects various ligands, including those for E-selectin. As a result, the initial binding of granulocytes to the endothelium fails to take place. This in turn leads to a reduced integrin reaction, reduced granulocyte adhesion, and finally, to reduced granulocyte migration out of the blood vessel. Children with LAD II also experience other severe symptoms in addition to increased susceptibility to infections /21/. The fucosylation defect can be detected by determining CD15 (Sialyl Lewis x) using cytofluorometry.

19.7.4.2 Other disorders of chemotaxis

Other disorders of chemotaxis and migration have been described for which the molecular causes are not yet evaluated. A variant of LAD I has been identified (referred to by some authors as LAD III) that is due to a reduction in the binding capacity of β2integrins /20/. Lazy leukocyte syndrome is another isolated deficiency that has only been described in individual case reports and is characterized by impaired PMN migration /21/.

Deficiencies in PMN chemotaxis are frequently associated with other diseases (Tab. 19.7-5 – Disorders of PMN function):

  • Chemotactic activity is reduced in children with Down’s syndrome, juvenile periodontitis, and pyoderma gangrenosum. This is thought to be due to defects in signal transmission.
  • Neutrophil-specific granule deficiency (SGD) is characterized by reduced chemotaxis. SGD is caused by a functional deficiency of a transcription factor (CCAA/enhancer binding protein epsilon), which is thought to lead to defective differentiation associated with an absence of granule protein synthesis. As well as a chemotaxis deficiency, granulocytes also exhibit other functional deficiencies (e.g., reduced bacterial killing and receptor expression) /422/.

19.7.4.3 Phagocytosis defects

Microbial infections lead to the production of chemoattractants, which promote the infiltration of PMN into the tissues. In the tissues, the PMN fulfill their most important function: the phagocytosis and intracellular killing of foreign organisms. A prerequisite for phagocytosis is the ability to recognize and bind to microorganisms. Pathogen-associated molecular patterns (PAMPs) are among the most important recognition structures present on bacteria. They are associated with groups of bacteria and include lipopolysaccharides, lipoteichoic acid, flagellar proteins etc. These PAMPs are recognized by pattern recognition receptors such as those from the Toll-like receptor family and by LPS receptor CD14 /2023/.

Bacterial recognition is increased significantly by opsonization, which involves the binding of endogenous proteins to bacteria so that they can be identified by special receptors on granulocytes. Opsonization with fibronectin, for example, promotes the recognition of S. aureus or Streptococcus spp. /24/.

Opsonization is improved by coating bacteria with specific IgG antibodies or the complement activation products C3b and iC3b. C3b is recognized by complement receptor CR1 and iC3b is recognized by CR3 (CD11b/CD18). Particles to which both C3b/iC3b and IgG are bound are phagocytosed optimally.

Specific receptors for IgG are found on granulocytes. These are called Fc receptors (FcγR) because they bind the constant fragment (Fc region) of the antibody.

Different types of FcγR are expressed on granulocytes: FcγRII (CD32), FcγRIII (CD16), and (following activation) the high-affinity Fc receptor FcγRI (CD64) /25/.

Although there is some overlap between Fc receptor functions, the receptors differ in their binding capacity and avidity and may bind monomeric IgG, aggregates of IgG, or particular IgG subclasses preferentially. Allotypes of CD32 and CD16 have also been described. The CD32 allotypes, for example, differ by a point mutation at position 131, which may be occupied by either arginine or histidine (FcγRIIa-R131 and FcγRIIa-H131).

Variations in the binding capacity of IgG2 have also been described, with FcγRIIa-H131 having high binding capacity and FcγRIIa-R131 binding only weakly. Because encapsulated bacteria (Streptococcus spp.group B, H. influenzae type b, N. meningitides type B, and K1-positive E. coli) stimulate the production of IgG2 mainly, the FcγII allotype may play a role in the phagocytosis of these bacteria. In fact, studies have shown that patients with FcγIIa-R131 are at higher risk of developing meningitis /26/.

Two isoforms of CD16 are also known: the FcγRIIIa isoform exists as an integral transmembrane protein on natural killer (NK) cells and macrophages and the FcγRIIIb isoform is bound to granulocytes via glycosylphosphatidylinositol (GPI). FcγRIII binds IgG aggregates, in particular IgG1 and IgG3, with a high degree of avidity. A CD16 polymorphism based on an amino acid substitution (NA1/NA2 polymorphism) has also been associated with different degrees of phagocytic efficacy but its effect on the immune system is not yet clear, especially since CD16 deficiencies are relatively common but do not seem to lead to an increased risk of infection /2627, 28, 2930/.

Bacterial binding to PMN induces intracellular signal transduction cascades, the quality and quantity of which depend on the recognition of bacterial structures (PAMPs) and opsonization. PMN activation leads to the formation of pseudopodia, which surround the bacterium and enclose it in a vesicle formed by the cell membrane (phagosome) within the granulocyte. Phagocytosis increases the oxygen requirements of the cell as well as glucose metabolism, which is associated with the reduction of molecular oxygen (Fig. 19.7-3 – Oxygen radical generation in granulocytes). Phagocytosis can only take place if cells are sufficiently motile and the cytoskeleton is intact. This is why disorders of phagocytosis are often associated with disorders of chemotaxis.

19.7.4.4 Disorders of bactericidal activity

The ultimate purpose of chemotaxis and phagocytosis is intracellular bacterial killing. Oxygen-dependent and oxygen-independent mechanisms can be distinguished. Ingested bacteria are enclosed in a membrane vesicle (phagosome), which then fuses with azurophilic granules (lysosomes) to form a phagolysosome. This leads to the release of lysosomal enzymes. Meanwhile, oxygen radicals are produced by the stepwise reduction of molecular oxygen. This reaction is known as an oxidative burst or respiratory burst and is crucial to the intracellular killing of pathogens (Fig. 19.7-3 – Oxygen radical generation in granulocytes). The oxygen radicals can also reach the area around the cells, where they are deactivated by serum proteins such as ceruloplasmin. Excessive production of oxygen radicals can lead to tissue damage and contribute to local inflammation.

Defects in the enzyme cascade leading to the production of oxygen radicals are rare (incidence 1/200,000) and manifest as recurrent bacterial infections, usually associated with cellular infiltrates and granuloma formation. This clinical syndrome is known as chronic granulomatous disease (CGD). Defects can affect the cytochrome b subunits p22phox or gp91phox or factors p47phox or p67phox of NADPH oxidase, which are located in the cytosol. Cytochrome defects are more common /31/.

Neutrophil proteins such as myeloperidase (MPO) and proteinase-3 play a key role in the development of autoimmune disease. The proteins act as auto antigens against which a pathogenic immune response has been generated /3/. MPO is an auto antigen in immune vasculitis and associated with MPO-ANCA (see Section 25.9 – ANCA associated vasculitides and pulmonary renal syndromes).

Myeloperoxidase (MPO) deficiencies, although relatively common (with an incidence of approximately 1 : 2000), are rarely clinically evident, which suggests that they are compensated effectively by other mechanisms.

Knowledge about bactericidal substances secreted by PMNs has grown in recent years. This applies to:

  • The production of NO·, which has bactericidal as well as vasoactive effects
  • Antibacterial proteins and peptides such as bacterial/permeability-increasing protein (BPI), defensins, and cathelins. The potency and mode of action of these natural antibiotics are comparable to those of classic antibiotics. Their role in defending against bacteria is difficult to assess.
  • Deficiencies of defensin and cathelin-LL37 in patients with congenital neutropenia (Kostmann syndrome) are responsible for the increased risk of infection that persists even after the granulocyte count has been increased using granulocyte colony-stimulating factor (G-CSF).

19.7.5 Comments and problems

PMN are active for a limited time only in the blood or following isolation. Complex activities such as chemotactic migration decline particularly quickly as a result of in vitro aging (2–6 h). When reduced PMN function is identified, therefore, it is important to check whether the blood or isolated cells have been stored for too long time. Isolation conditions, temperature, media, and buffers also influence PMN function and must therefore be kept constant and monitored.

Contamination by lipopolysaccharides (LPS) in particular must be avoided, because even very low concentrations of these substances can inhibit PMN function and the expression of surface receptors.

19.7.6 Biochemistry and physiology

About 5% of PMN are found circulating in the peripheral blood with a half-life of 6–8 h or attached to the vascular endothelium as a marginal pool. The rest remain in the bone marrow, from where they can be rapidly recruited as required (e.g., to sites of infection) resulting in leukocytosis.

PMNs have chemotactic, phagocytic, and bactericidal capabilities to defend the body against microbial infections (Fig. 19.7-2 – Infiltration of polymorphonuclear neutrophils (PMNs) to the focus of infection).

Effective defense against infections is largely dependent on the ability of PMN to migrate from the peripheral blood to the focus of infection. This requires /323321/:

  • Alteration in the shape and function of PMN to enable them to migrate actively instead of being transported passively in the blood
  • The ability to recognize the focus of infection.

Local alterations in the vascular endothelium are necessary to allow PMN to migrate through blood vessel walls. Mediators that are released at the site of inflammation (e.g., histamine) stimulate the expression of adhesion molecules (selectins) by endothelial cells. PMN are able to recognize selectins and bind to them via a surface glycoprotein, whose carbohydrate component, mainly fucose, plays a decisive role in the binding. This results in the (unstable) adhesion of PMN to the endothelial cells and the activation of PMN adhesion proteins known as β2-integrins.

Integrins are heterodimeric plasma membrane receptors, each consisting of one α and one β chain bound non covalently. Both chains are required for normal receptor expression and binding to the corresponding ligands. The main integrins involved in leukocyte adhesion to the vascular endothelium are the β2-integrins, which have the α chain CD18 in common: CD11a/CD18 (LFA-1), CD11b/CD18 (MAC-1), and CD11c/CD18 (gp150, 95). CD11b/CD18 is a complement receptor (CR3) that binds activated complement C3 (iC3b).

The contact between the PMN and the endothelium causes the PMN to proliferate and then migrate actively between the endothelial cells and through the tissues to the focus of inflammation. This directed movement is referred to as chemotaxis, to distinguish it from spontaneous migration (random migration of non stimulated leukocytes) and chemokinesis (random movement of leukocytes in response to chemicals).

Chemotaxis is triggered by soluble mediators produced at the site of infection known as chemotactic stimuli. The main chemotactic stimuli are anaphylatoxin C5a, formylated peptide chemoattractants produced by bacteria [in experiments, the corresponding synthetic peptide N-Formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) is used], leukotriene B4 (LTB4), platelet-activating factor (PAF), interleukin 8, and transforming growth factor β. Others include neuropeptides and fragments of matrix proteins /323321/.

Granulocyte receptors transmit signals and trigger increased adhesion, deformation, and migration. The chemoattractants released at the site of infection diffuse locally and bind to granulocyte receptors. At the point of maximum chemoattractant concentration, the chemotactic receptors become polarized, which determines the direction of active migration. Because the highest chemoattractant concentration is usually found at the infection site, the cells migrate to this location (leukocyte infiltration) (Fig. 19.7-2 – Infiltration of polymorphonuclear neutrophils (PMNs) to the focus of infection).

All currently known chemokine receptors are associated with G proteins, which transmit chemotactic signals to the cell. Phosphatidylinositol-3 kinase (PI3K) and p38 MAP kinase are important signaling molecules that ultimately control cytoskeletal dynamics and, therefore, cell motility /2122/.

Die Myeloperoxidase (MPO) is a cationic, heme-containing, glycosylated enzyme which is found in the azurophilic granules of PMN. The PMN contain the MPO/HOCl system for intracellular killing of bacteria and fungi (Fig. 19.7-3 – Oxygen radical generation in granulocytes). MPO is involved in neutrophil functions in innate and adaptive immunity /34/.Refer to Tab. 19.7-6 – Involvement of MPO in PMN functions in innate and adaptive immunity).

Activated PMN release NETs, structures composed of de condensed chromatin, histones, and various antimicrobial substances (e.g., myeloperoxidase, elastase) in the circulation, thus aiding in the overall elimination and spread of pathogens /2/. NETs can trap extracellular microbes and can kill some, but not all species.

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8. Quie PG, White JG, Holmes B, Good RA. In vitro bactericidal activity in chronic granulomatous disease of childhood. J Clin Invest 1967; 46: 668–79.

9. Baehner RL, Nathan DG. Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med 1968; 278: 971–6.

10. Babior BM, Kippnes RS, Curnutte JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 1973; 52: 741–4.

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12. Lekstrom-Himes JA, Gallin JI. Immunodeficiency diseases caused by defects in phagocytes. N Engl J Med 2000; 343: 1703–13.

13. Van Vlem B, Vanholder R, De PaepeP, Vogelaers D, Ringoir S. Immunomodulating effects of antibiotics: literature review. Infection 1996; 24: 275–91.

14. Gemmell CG. Antibiotics and neutrophil function – potential immunomodulating activities. J Antimicrob Chemother 1993; 31, Suppl B: 8–33.

15. Fischer A, Lisowska-Grospierre B, Anderson DC, Springer T. Leukocyte adhesion deficiency: molecular basis and functional consequences. Immunodefic. Rev 1988; 1: 39–54.

16. Etzioni A, Frydman M, Pollack S, Avidor I, Phillips ML, Paulson JC, Gershoni-Baruch R. Recurrent severe infections caused by a novel leukocyte adhesion deficiency. N Engl J Med 1992; 327: 1789–90.

17. Kuijpers TC, van Bruggen R, Kamerbeck N, Tool ATJ, Hicsonmez G, Gurgey, et al. Natural history and early diagnosis of LAD-1/variant syndrome. Blood 2007; 109: 3529–37.

18. Miller ME, Oski FA, Harris MB. Lazy-leucocyte syndrome. A new disorder of neutrophil function. Lancet 1971: 3: 665–9.

19. Gombart AF, Koeffler HP. Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP(epsilon). Curr Opin Hematol 2002; 9: 36–42.

20. Stuart LM, Ezekowitz AB. Phagocytosis: elegant complexity. Immunity 2005; 22: 539–50.

21. Igelias PA. Spatial regulation of PI3K signaling during chemotaxis. Syst Biol Med 2009; 1: 247–51.

22. Friedl P, Weigelin B. Interstitial leukocyte migration and immune function. Nature Immunology 2008; 9: 960–9.

23. Takeda K, Akira S: Toll-like receptors in innate immunity. International Immunology 2005; 17: 1–14

24. Lekstrom-Himes, Gallin JI. Immundeficiency diseases caused by defects in phagocytes. N Engl J Med 2000; 343: 1703-14.

25. Van de Winkel JG, Capel PJ. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunology Today 1993; 14: 215–21.

26. Bredius RGM, Fijen CAP, De Haas M, Kuijper EJ, Weening RS, van de Winkel JG, Out TA. Role of neutrophil FcRγIIa (CD32) and FcγRIIIb (CD16) polymorphic forms in phagocytosis of human IgG1- and IgG3-opsonized bacteria and erythrocytes. Immunol 1994; 83: 624–30.

27. Sanders LAM, Feldman RG, Voorhorst-Ogink MM, de Haas M, Rijkers GT, Capel PJA, et al. Human immunoglobulin G (IgG) FcγReceptor IIa (CD32) polymorphism and IgG2-mediated bacterial phagocytosis by neutrophils. Infect Immun 1995; 63: 73–81.

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30. Wagner C, Hänsch GM. Genetic deficiency of CD16, the low-affinity receptor for immunoglobulin G, has no impact on the functional capacity of polymorphonuclear neutrophils. Eur J Clin Invest 2004; 34: 149–55.

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32. Wong CHY, Heit B, Kubes P. Molecular regulators of leucocyte chemotaxis during inflammation. Cardiovascular Res 2010; 86: 183–91.

33. Kobayashi SD, Voyich JM, Burlak C, DeLeo F. Neutrophils in the innate immune response. Arch Immunol Ther Exp 2005; 53: 505–17.

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Table 19.1-1 Mediators of inflammation /10/

Mediators of inflammation

Complement derived inflammatory mediators

The complement fragments C4b, C2a, and C3b bind to the foreign agent and facilitate its opsonization by macrophages.

The fragments C3a, C4a, and C5a increase vascular permeability and cause contraction of the smooth muscle of the bronchi and venules.

C5a is an important chemoattractant and induces the accumulation of phagocytes at the site of inflammation.

N-formyl peptides

Bacterial protein synthesis starts with N-formyl methionine and many N-formylated methionyl peptides are chemoattractants for leukocytes. These chemoattractants, especially the prototype N-formylated methionyl-leucyl-phenylalanine (fMLP), exert their effect via receptors on the leukocyte surface. N-formylated peptide chemoattractants can also be synthesized by mitochondria, thus enabling damaged host cells to attract leukocytes.

Eicosanoids /12/

The stimulation of inflammatory cells causes the activation of phospholipases. These enzymes are ubiquitously present in tissue cells but especially in inflammatory cells. Fig. 19.1-4 – Hydrolysis of phospholipids by phospholipases shows the target points of phospholipases. Phospholipase A2 produces arachidonic acid and 1-0-alkyl-2-lyso-sn-glycerophosphocholine (lyso-PAF) from phospholipids. Arachidonic acid is then metabolized into eicosanoids by the following enzymes (Fig. 19.1-5 – Eicosanoid production):

  • Cyclooxygenases (COX): arachidonic acid is metabolized into prostanoids (prostaglandins, thromboxane) via the COX pathway. Monocytes and macrophages produce a wide range of prostanoids while other cell types produce only one dominant prostanoid (e.g., thrombocytes produce thromboxane, mast cells produce PGD2, and endothelial cells produce PGI2).
  • Lipoxygenases (LOX): arachidonic acid is metabolized into leukotrienes and hydroxyeicosatetraenoic acids (HETEs) via the LOX pathway. LXA4 and LXB4 are the most important lipoxygenases. They are the first anti-inflammatory lipid mediators at the site of inflammation.
  • Cytochrome P450 epoxygenases (CYPs): arachidonic acid is metabolized into epoxyeicosatetraenoic acids (EETs) and HETEs via the CYP pathway.

Although eicosanoids are produced by every cell in the body, the particular eicosanoid expressed by a cell and the effect of the eicosanoid depend on the cell type. For example, eicosanoids produced via the COX pathway are synthesized preferentially by epithelial cells, endothelial cells, fibroblasts, and smooth muscle cells. Eicosanoids produced via the LOX pathway are synthesized preferentially in cells derived from bone marrow. Eicosanoids have a short half-life, ranging from seconds to minutes. Signal transmission from the eicosanoids to the immune cells occurs primarily via G-protein coupled receptors.

Cytokines

Cytokines regulate the growth, differentiation, and functions of inflammatory cells in an autocrine, paracrine, and endocrine manner. Cytokines are classified into interleukins (IL), interferons (IFN), tumor necrosis factors (TNF), growth factors (GF), and colony-stimulating factors (CF) (refer to Section 20.1 – Definition, classification, structure and function of cytokines). T-helper cells (Th), macrophages, and dendritic cells are important sources for cytokines. They produce pro inflammatory cytokines such as TNF-α, IL-1β, IL-6, TGF-β, and IFN-γ as well as anti-inflammatory cytokines such as IL-4, IL-10, and IL-13. Th cells are responsible for immune activation. A distinction is made between:

Inflammatory cytokines are multifunctional and play activating and inhibitory roles in inflammation. For example:

  • TGF-β is a potent chemoattractant for monocytes and plays a role in wound healing
  • IFN-α has both pro inflammatory and anti-inflammatory effects
  • IFN-γ exerts a pro inflammatory effect in the early stage of collagen-induced arthritis and an anti-inflammatory effect later in the disease
  • TNF-α exerts a pro inflammatory effect while also inducing the synthesis of anti-inflammatory IL-1 type II decoy receptors
  • IL-6 is pro inflammatory in the early phase and antiinflammatory in the late inflammation stage.

Chemokines /33/

Chemokines are a large group of proteins that play an important role in maintaining the inflammatory response. Although they are pleiotropic, their main role is to act as a chemoattractant. They have a molecular weight of 8–10 kDa. The chemokine super family consists of four main groups (C, CC, CXC, and CXC3), based on the position of the first two cysteine residues in a conserved four-cysteine topology. The CXC group, for example, has two conserved cysteine residues (C ) separated by a non conserved amino acid (X). The CC chemokines have two adjacent conserved cysteine residues. Chemokines are produced by all nucleated cells. In chronic inflammation, epithelial cells and fibroblasts are the most important locations for chemokine production. The main chemokine families are shown in Tab. 19.1-3. Chemokine receptors are G-protein coupled transmembrane receptors that are named after the chemokine that they bind.

Table 19.1-2 Metabolic products of eicosanoids and their function /3435/

Name,
Abbreviation

Function and
produced

Cyclooxygenase products

Prostaglandin G2

(PGG2, PGH 2)

Contraction of the smooth muscles of blood vessels, the eyes, the gastrointestinal tract, and the bronchi

Produced by all tissues

Prostaglandin E2

(PGE 2)

Vasodilation, bronchorelaxation, PMN inactivation, down-regulation of activated monocytes/macrophages and lymphocytes

Produced by monocytes/macrophages, fibroblasts, endothelial cells, adrenomedullary cells

Prostaglandin D2

(PGD2)

Released in the presence of allergies and other stimuli; systemic vasodilatation and pulmonary artery constriction

Produced by mast cells

Prostacyclin

(PGI2)

Refer to PGE2.

Thromboxane A2

(TXA2)

Platelet aggregation, vasoconstriction

Produced by platelets, PMN, monocytes/macrophages, fibroblasts, endothelial cells

Lipoxygenase products

HETE, HPETE

PMN activation, chemotaxis

Produced by cells derived from the bone marrow (with the exception of red blood cells)

Leukotrienes B4, C4, D4, E4

(LTB4, LTC4, LTD4, LTE 4)

Ligand formation between receptors on endothelial cells and leukocytes, leading to accumulation of all leukocyte subgroups at the site of inflammation

Produced by PMN, monocytes/macrophages, mast cells, endothelial cells, platelets

Lipoxins A4, B4

(LXA4, LXB4)

Inhibition of chemotaxis, adhesion, degranulation, and H2O2 production by PMN

Platelet-activating factor (PAF)

Platelet and granulocyte aggregation, vasodilatation, increase in vascular permeability, release of prostaglandins and leukotrienes

Produced by platelets, monocytes/macrophages, PMN, endothelial cells, NK cells

PMN, polymorphic mononuclear granulocytes; HETE, hydoxyeicosatetraenoic acid; HPETE, hydoxyeicosatetraenoic acid

Table 19.1-3 Chemokine groups and their effects on immune cells /36/

 

CXC

CC

C

CXXXC

Chemo-
kines

Interleukin 8
Growth factor
α, β, γ

Interferon-
inducible
protein 10

Monocyte
Chemoattractant
protein-1 (MCP-1)

Eotaxin

RANTES

Lympho-
taxin

Fractalkine

Chemo-
attractant

PMN

T-cells

Monocytes

T-cells

Eosinophils,
Basophils

Dendritic cells

Dormant T-cells

NK cells

RANTES, regulated on activation, normal T-cell expressed and secreted

Table 19.1-4 Molecules involved in leukocyte recruitment /37/

Molecules

Selectins

Selectins are a family of three adhesion molecules that facilitate the initial contact between leukocytes and vascular endothelial cells. Leukocyte L-selectin and endothelial E and P-selectin recognize ligands that carry specific carbohydrate residues (e.g., sialyl Lewis X). L-selectin is expressed permanently on most leukocytes. The expression of E-selectin on the vascular endothelium is induced by cytokines. P-selectin is stored in granules in vascular endothelial cells and expressed on the cell surface in response to induction by mediators of inflammation.

GlyCAM 1 and CD34 are ligands of L-selectin, P-selectin glycoprotein ligand 1 (PSGL-1) is a ligand of P-selectin, and E-selectin ligand 1 (ESL-1) is a ligand of E-selectin. The most important ligand is mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which has two N-terminal domains and is homologous to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), both of which are members of the immunoglobulin super family. Leukocyte L-selectin binds to the ligand MAdCAM-1.

Integrins

Integrins are proteins found on the leukocyte surface that cause stable adhesion in the leukocytes that roll along the vascular endothelium. Their functions are triggered by chemoattractants. Integrins are heterodimers that consist of an α-peptide chain and a β-peptide chain that are bound non-covalently to the cell membrane as heterodimers. 15 α-chains and 8 β-chains are recognized. Important integrins include LFAs (lymphocyte function-associated antigens), VLA (very late antigens), Mac-1 (CD11b), and p150/95 (CD11a/CD18).

In addition to the adhesion molecules of the immunoglobulin super family (ICAM, VCAM) of the vascular endothelium, other ligands of integrins include collagen, laminin, and fibrinogen.

Annexins /32/

The annexin super family is composed of 13 members, grouped in view of their Ca2+- binding site architecture, which enables them to peripherally attach to negatively charged membrane surfaces. Annexin A1, also known as annexin I or lipocortin I is an endogenous modulator of the inflammatory response. Annexin A1 inhibits neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. Annexin A1 promotes apoptotic neutrophil clearance by modulating monocyte recruitment and contributes to tissue homeostasis by inducing macrophage reprogramming toward a resolving phenotype.

Table 19.1-5 Classification of the acute phase proteins from Ref. /2/

Protein

Reference
interval
(g/L)

Reaction
time (h)

Rise
(x normal)

CRP

< 0.005

6–10

10–1,000

SAA

< 0.010

6–10

10–1,000

α1-anti-
chymotrypsin

0.3–0.6

10

10

Acid α1-
glycoprotein

0.5–1.4

24–48

2–3

α1-antitrypsin

1.9–3.5

24–48

2–3

Haptoglobin

0.7–3.8

24–48

2–3

Fibrinogen

2.0–4.5

24–48

2–3

C3

0.5–1.2

48–72

< 2

C4

0.2–0.5

48–72

< 2

Ceruloplasmin

0.15–0.60

48–72

< 2

Table 19.1-6 Acute phase response induced by various inflammatory stimuli /6/

Clinical and laboratory findings

Gram-negative bacterial Infection

Acute infections caused by Gram-negative bacteria such as enterobacteriaceae cause a marked rise in acute phase proteins (APPs) as a result of the direct activation of macrophages by bacterial endotoxin (CRP greater than 100 mg/L). Effective antibiotic therapy causes a rapid decline in the concentration of APPs (Fig. 19.1-11 – Time profile of CRP and body temperature in an immunosuppressed patient with bacterial infection following a positive response to antibiotic therapy).

Gram-positive bacterial Infection, parasitic, and fungal InFection

Acute infections caused by Gram-positive bacteria, parasites, and fungi (with the exception of sepsis) cause a moderate rise in APPs (CRP less than 100 mg/L).

Infection through viruses

Viruses (e.g., Adenovirus) cause a slight rise in APPs (CRP less than 30 mg/L).

Surgical intervention

Sterile inflammation such as that following surgical intervention leads to a rise in APPs in proportion to the extent of tissue damage (Fig. 19.1-12 – CRP increase in relation to the extent of surgical intervention). This is also the case for blunt trauma, myocardial infarction, gunshot wounds, and stab wounds as well as surgical intervention involving a greater degree of tissue damage. APPs that have a short reaction time (e.g., CRP) rise to more than 10 mg/L in 6 h and reach a peak of around 150 mg/L after 48 h.

Malignant tumor

Unless bacterial infection is present, malignant disease leads to only moderate rises in APPs, which are due to necrosis of metastatic tumors in particular rather than to cytokine formation (CRP less than 50 mg/L).

Local inflammation

Local infections such as appendicitis or low-grade coronary inflammation lead only to minor rises in certain APPs such as CRP or serum amyloid A protein that do not exceed the reference interval (CRP less than 10 mg/L).

Chronic inflammation

An increase in APPs in chronic inflammatory processes such as connective tissue diseases, rheumatic diseases, and autoimmune diseases indicates organic disease. The rise is usually mild to moderate (CRP less than 30 mg/L). Persistently elevated APPs indicate continuing inflammation and a probable increase in disease activity. A decline in APPs in response to anti-inflammatory drugs reflects clinical improvement.

Hyperinflammation, sepsis

The pathophysiology of sepsis suggests that some patients present with hyperinflammation, some present with immunosupression, and the rest lie somewhere between these two extremes. Macrophage activation syndrome (MAS), a state of hyperactivation of the innate immune responses is also known as hemophagocytotic lymphohistiocytosis (HLH). The hallmark of pathogenesis relies on the hyperactivation of tissue macrophages, leading to a cytokine storm /41/. The cytokine release is a life-threatening systemic inflammatory syndrome involving elevated levels of circulating cytokines, proteins (IL-6, IL-18, ferritin), and immune cell activation syndrome /42/. The MAS and cytokine storm are also described in SARS-CoV-2 patients /43/.

Autoimmune disease

In certain autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis, and polymyositis, the APR is less pronounced than expected and often occurs in the active phase only (mean CRP level of up to 30 mg/L).

Hormonal influences on acute phase proteins

Hormones have the following effect on the synthesis and serum levels of APPs /6/:

  • Corticosteroids cause an increase in α1-acid glycoprotein and haptoglobin
  • Androgens lead to an increase in α1-acid glycoprotein, α1-antitrypsin, and haptoglobin
  • Estrogens stimulate an increase in α1-antitrypsin and ceruloplasmin.

Table 19.1-7 Autoinflammatory diseases /17/

Monogenic diseases

Familial Meditarranean fever (FMF)

TNF receptor-associated periodic syndrome (TRAPS)

Cryopyrin-associated periodic syndrome (CAPS)

Familial cold auto-inflammatory syndrome (FACS)

Muckle-Wells syndrome (MWS)

Neonatal-onset multisystem inflammatory disease (NOMID)

Hyper immunoglobulinemia D syndrome (HIDS)

Blau’s syndrome

Pyogenic arthritis pyoderma gangrenosum and acne (PAPA)

Chronic recurrent multi focal osteomyelitis syndrome (CRMO)

Deficiency of the interleukin-1 receptor antagonist syndrome (DIRA)

Majeed’s syndrome

IL-10 deficiency

Polygenic diseases

Still’s disease

Crohn’s disease

Behcet’s disease

Gout

Systemic juvenile arthritis (sJlA)

Table 19.1-8 Diagnostic variables of SIRS

Variables

Fever more than 38 °C (100.4 °F) or less than 36 °C (96.8 °F)

Heart rate more than 90 beats per minute

Respiratory rate more than 20 breaths per minute or arterial CO2 (PaCO2) of less than 32 mmHg

Leukocytes > 12 × 109/L or < 4 × 109/L or > 10% immature (band) forms

Table 19.1-9 Sequential sepsis related organ failure assessment (SOFA) score /28/

Score

0

1

2

3

4

Respiration

PaO2/FI02
mmHg (kPa)

≥ 400
(53.3)

> 400
(53.3)

< 300
(40)

< 200*

< 200*

Coagulation

Platelets
(109/L)

≥ 150

< 150

< 100

< 50

< 20

Liver

Bilirubin
mg/dL
(umol/L)

 
< 1.2
(20)

 
1.2–1.9
(20–32)

 
2.0–5.9
(33–101)

 
6.0–11.9
(102–204)

 
> 12
(204)

Cardiovascular

MAP ≥ 70 mmHg

MAP < 70 mmHg

Dopamine (Dopa) < 5 or
dobutamine (any dose)

Dopa
5.1–15

Dopa
> 15

Central nervous system

Glagow coma
scale score

15

13–14

10–12

6–9

< 6

Renal

Creatinine
mg/dL
(umol/L)

 
< 1.2
(110)

 
1.2–1.9
(110–170)

 
2.0–3.4
(171–299)

 
3.5–4.9 (300–440)

 
> 5.0
(440)

Urine output

mL/day

< 500

< 200

FIO2 ,fraction of inspired oxygen; PaO2 ,partial pressure of oxygen; MAP, mean arterial pressure; *with respiratory support. The SOFA score is not meant to indicate the success or failure of interventions or to influence medical management.

Table 19.1-10 Laboratory tests in suspected SIRS and sepsis and disease monitoring

Investigations

Complete blood count with differential

Coagulation studies (PT, APTT)

Blood chemisty (sodium, chloride, calcium, phosphate, glucose, lactate)

Renal function tests (creatinine, albumin excretion)

Hepatic function tests (ALT, ALP, GGT, bilirubin)

Inflammation markers (CRP, PCT, IL-6)

Urinalysis and urine cultures

Blood cultures

Blood gas analysis

Table 19.2-1 Sources of reactive oxygen species (ROS) /15/

  • Normal leakage from electron transport chains of mitochondria, endoplasmic reticulum
  • NAD(P)H oxidase reaction in neutrophilic granulocytes, monocytes/macrophages, and vascular endothelium
  • Reactions mediated by flavin oxidase, xanthine oxidase, and monoamine oxidase
  • Arachidonic acid metabolism
  • Autooxidation of thiols such as glutathione
  • Oxyhemoglobin and oxymyoglobin

Table 19.2-2 Pathogenic effects of reactive oxygen species (ROS)

Clinical and laboratory findings

Lipid peroxidation

ROS oxidize a variety of unsaturated fatty acids and phospholipids in an autocatalytic process whereby polyunsaturated fatty acids and phospholipids undergo degradation by a chain reaction to form lipid peroxides. If this occurs in the lipid bilayer of the cell membrane which is highly susceptible to lipid per oxidation a variety of by products are formed. This results in membrane damage with impaired permeability and receptor expression and loss of communication with other cells /1/.

Lipid per oxidation results in the generation of a variety of relatively stable breakdown products, mainly hydrocarbons (pentane, ethane, ethene) ketones and α,β-unsaturated reactive aldehydes, such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), and 2-propenal (acrolein) and isoprostanes, which can be measured in the plasma and urine as indirect indicators of oxidative stress /1/.

Protein oxidation

Oxidative damage to proteins is mainly due to changes in their amino acids and the protein structure. They can be converted into carbonyl derivatives by ROS. Nitro tyrosine is produced as a result of the reaction of peroxynitrites with proteins. Enzyme activity is lost as a result and proteins are more susceptible to lysosomal proteolytic degradation. The sugar residues of glycoproteins are also oxidized to keto aldehydes and converted to Schiff bases following reaction with lysine.

MDA is a physiologic keto aldehyde. The excess MDA produced as a result of lipid per oxidation is a result of tissue injury. MDA combines with free amino groups of proteins, producing MDA modified protein adducts and alter their biological properties. The clinical relevance of the reaction between MDA and proteins is highlighted in atherosclerosis /1/. MDA modified proteins are immunogenic and autoantibodies are produced. MDA-LDL, in addition to oxidized LDL can mediate pro inflammatory and pro atherogenic processes which may lead to foam cell generation and promote the formation of atherosclerotic plaques.

DNA damage

Oxidative damage to DNA leads to fragmentation and strand shortening. This results in increased gene expression, increased DNA repair, and, ultimately, to persistence of DNA defects during DNA replication. The number of persistent pathological mutations increases in tissues with a high DNA replication rate, such as the hematopoietic system, mucous membrane of the small intestine, and glandular epithelium. The number of mutations caused by ROS also increases with age, particularly in the mitochondria. An inverse relationship is thought to exist between the prevalence of mitochondrial and nuclear DNA damage and longevity. Factors that increase the prevalence of DNA damage, such as an increased ROS load due to excessive calorie intake, smoking, pollution, and inflammation, reduce the life expectancy. The formation of 8-hydroxy-2·-deoxyguanosine is an index of oxidative damage to DNA, and its excretion in the urine provides an estimate of the repair rate of DNA /2/.

Endothelial dysfunction /3/

ROS causes direct injury to endothelial cells via oxidative reactions with lipids, proteins and DNA of the cell membranes. This results in endothelial cell swelling and detachment from the underlying basement membrane. Subendothelial structures and proteins, including tissue factor are exposed and generate a hyper coagulable state. In addition, through activation of nuclear factor kappa B, ROS stimulate gene expression and production of pro inflammatory cytokines (IL-1, IL-6, TNF-α) and adhesion molecules (intercellular vascular adhesion molecule-1, intercellular adhesion molecule-1).

iNOS activation

Inducible NO· synthase is activated as part of the inflammatory response and leads to the synthesis of large quantities of NO·. However, superoxide anion radical production is also increased and NO· acts as a radical scavenger. superoxide anion radical·· and NO· react to produce N2O3 and peroxynitrite (ONOO). The kinetics of NO· and superoxide anion radical·· production in inflammation determine the effect of ROS on the expression of genes for signaling molecules and are therefore critical for the activation of signaling cascades. The following counter regulatory mechanisms mediated by NO· take place during inflammation with respect to signal transmission (Fig. 19.2-2 – Temporal profile of radical production during an inflammatory reaction/17/:

  • (i) in primary response to an inflammatory stimulus, superoxide anion radical· is first produced and neutralizes basally synthesized NO·. The superoxide anion radical· exerts its effects as follows:
  • (ii) stimulation of iNOS leads to increased NO· synthesis in the second phase. The protein-attacking molecules N2O3 and peroxynitrite are produced. Proteins can be nitrosylated, tyrosine residues nitrated, and S-nitrosothiols produced. This affects signaling proteins and alters physiological signal transduction.
  • (iii) in a third phase, the activity of ROS-producing enzymes in the cells decreases, less superoxide anion radical· is released, and the synthesis of NO· dominates. This shifts signal transduction toward the effects of NO·.

Overall, the temporal profile of ROS and NO· production determines whether a cell involved in the inflammatory reaction dies as a result of necrosis or apoptosis or is restored to is previous function as a result of changes in gene expression /617/.

Table 19.2-3 Markers of the effects of reactive oxygen species (ROS) /18/

Marker

Estimation of ROS – Generally /1819/

Oxidative stress is quantified by measuring different biomarkers. This can be done by direct measurement of free radicals, the oxidized products of free radical damage, or the levels of individual and total antioxidants.

The commonly used markers are products of /16/:

  • Lipid per oxidation (malondialdehyde, 4-hydroxy-2’-nonenal, and isoprostanes)
  • Oxidized amino acid residues (cystine, methionine sulfoxide, 3-nitro tyrosine, and 3-chlor tyrosine).

A different approach to evaluation of oxidative stress is the analysis of the anti oxidative defence. A ROS attack can lead to a depletion of antioxidants such as vitamin E, vitamin C, reduced glutathione (GSH) and urate. GSH can be oxidized to glutathione disulfide (GSSG), or can form glutathionylated proteins (PSSG).

– Hydroxy peroxides

Hydroxy peroxides (HPs) are oxidation products generated by ROS oxidative changes in a variety of biological molecules, including lipids, proteins, carbohydrates and nucleic acids. The addition of peroxidases to the reaction mixture triggers the release of reactive oxygen from the peroxide ad ducts, which causes added tetramethylbenzidine to change color. In another assay, oxygen radicals are released from peroxide ad ducts using transition metals and cause N, N-dimethyl-p-phenylenediamine to change color /20/.

– MDA, 4-HNE, acrolein /2/ (TBARS)

Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and acrolein are the end products of enzymatic and non-enzymatic lipid per oxidation. These toxic aldehydes result from a radical attack against ω-6 unsaturated fatty acids (linoleic acid, linolenic acid, and arachidonic acid) and are biomarkers of tissue damage. MDA is produced in excess quantities as a by-product of tissue damage and the associated inflammatory process and also reacts with proteins. Lipid per oxidation products react with thiobarbituric acid (TBA) and are called thiobarbituric acid-reactive substances (TBARS). TBARS form colored ad ducts that absorb light maximally at 530–540 nm. MDA and 4-HNE can be measured directly by HPLC or as TBA adduct spectrophotometrically.

– Isoprostanes /21/

Isoprostanes are stable end products of lipid per oxidation of arachidonic acid and isomers of enzymatically derived products such as prostaglandins and leukotrienes. The F2-isoprostanes, which are thought to consist of 64 isomers, are biomarkers of oxidative stress and are most commonly measured using ELISA or LC-MS/MS. These methods measure the concentration of 8-iso-PGF. ELISA and LC-MS/MS do not produce comparable urinary values, for reasons that are related mainly to sample preparation.

Antioxidant defense – Generally

Because the determination of individual parameters of antioxidant defense does not always reflect the overall antioxidant spectrum, multiple parameters must be determined.

– Total oxidative state (TAS) /22/

The total antioxidant state (TAS) is a global assay used to measure the overall antioxidant capacity of the plasma. Contributors to the antioxidant capacity include: albumin (43%), uric acid (33%), ascorbic acid (9%), α-tocopherol (3%), bilirubin (2%), and other substances (10%). The principle of TAS determination is based on the plasma’s capacity to compensate for free radicals produced by 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). This assay has rarely been performed in studies.

– Glutathione /23/

An attack by ROS leads to a decrease in antioxidants such as vitamin E, vitamin C, uric acid, and reduced glutathione (GSH). The measurement of oxidized glutathione is a useful assay. In an attack by ROS, GSH is oxidized to glutathione disulfide (GSSG) and can combine with proteins to form glutathione ad ducts (PSSG). The measurement of GSH, GSSG, and PSSG and their ratio provides information about an individual’s oxidation status. Because this parameter varies so widely in the healthy population, it can be difficult to interpret test findings.

– Antioxidant enzymes

The activity of the enzymes glutathione peroxidase (GP) and super oxide dis mutase (SOD) is an indicator of antioxidant capacity and is reduced in an ROS attack.

Estimation of NO· production – Generally

Because of its short half-life, NO· can not be determined directly. However, it is estimated by measuring nitrite, nitrosylated tyrosine residues, and asymmetric dimethyl-L-arginine (ADMA).

– Nitrite and nitrate in serum and urine

Determination of nitrite and nitrate in body fluids like plasma and urine is widely used as a marker of NO· formation. As soon as it is produced, NO· is oxidized rapidly to nitrate. For determination nitrate must first be reduced to nitrite /24/. The Griess-Ilosvay reagent can then be used to measure serum nitrite in the supernatant following protein precipitation. Nitrite reacts with sulfanilic acid to form diazobenzenesulfonic acid, which combines with α-naphthylamine to produce a red color.

– 3-nitro tyrosine /16/

3-nitro tyrosine (NO2-Tyr) is a stable biomarker derived from NO· that is used to measure the products of oxidation. Oxidation products are usually determined using immunoassays. They can also be determined effectively using HPLC and electrochemical detection or GC-MS/MS.

– Asymmetric dimethly-L-arginine (ADMA) /9/

Plasma ADMA is determined using HPLC following prior derivatization with o-phthaldialdehyde. Values in healthy individuals without hypercholesterolemia are 1.09 ± 0.09 μmol/L. The improved endothelium-dependent vasodilation caused by simvastatin leads to a reduction in ADMA.

Table 19.2-4 Diseases in which ROS have a significant pathogenetic role

Clinical and laboratory findings

Tissue hypoxia /3/

Restoration of oxygen-rich blood flow after an episode of ischemia adds specifically to tissue damage. Decreased partial pressure of oxygen caused by reduced blood flow induces the generation of hypoxanthine and xanthine oxidase from ATP and xanthine dehydrogenase, respectively. After restoration of oxygen-rich blood flow, xanthine oxidase generates O2–· while catalyzing the conversion of hypoxanthine or xanthine to uric acid. Catalyzed by iron, the O2–· is ultimately converted to the toxic HO· catalyzed by iron. Although xanthin oxidase accounts for the oxidative damage in the early phase of re perfusion, NADPH oxidase, abundantly present in activated polymorphonuclear neutrophils (PMNs) and monocytes, acts in the later phase of re perfusion injury.

Atherosclerosis

ROS play an important role in the etiopathogenesis of atherosclerosis. Increased concentrations of ROS are found in the coronary vessels of patients with risk factors for atherosclerosis (smoking, diabetes mellitus, hypertension, and hypercholesterolemia). ROS activate mechanisms in the development of atherosclerosis such as endothelial cell apoptosis, proliferation of smooth muscle cells, and activation of metalloproteinases. The production of peroxynitrites also leads to NO· depletion, which reduces the effects of NO· such as vasodilatation and inhibition of platelet aggregation. This leads to vasoconstriction and increased thrombotic tendency.

Diabetes mellitus /17/

A close relationship exists between the complications of diabetes (micro- and macro angiopathy) and oxidative stress. Diabetics are predisposed to oxidative stress for a number of reasons. Under the hyperglycemic conditions associated with diabetes, glucose (as a reducing agent) reacts with the lysine residues of proteins to produce Schiff bases. Aldimines are converted to ketoamines by the Amadori rearrangement (refer to Fig. 3.6-1 – Glycation of N-terminal valine of hemoglobin with glucose and subsequent Amadori rearrangement) and further reactions lead to the formation of advanced glycation end products (AGEs). AGEs bind to specific receptors on vascular endothelial cells and smooth muscle cells, where they exert their signaling effect and initiate microvascular and macro vascular complications.

Pulmonary disease /2/

ROS have a role in the pathogenesis of adult respiratory distress syndrome (ARDS), asthma, emphysema, and chronic obstructive pulmonary disease (COPD).

ARDS, which can develop after a quiescent period lasting 12–48 h following multiple trauma or sepsis, is characterized by marked infiltration of the lungs by activated PMNs. These PMNs exhibit a high rate of oxidative metabolism, which leads to the production of ROS and the destruction of pulmonary endothelium.

COPD in smokers is thought to be the result of an imbalance between ROS and antioxidants caused by the increased presence of PMNs and macrophages in the alveoli of smokers.

Liver disease /2/

Carbon tetrachloride (CCl4) poisoning: homolytic cleavage of CCl4 by the cytochrome P450 system leads to the formation of the trichloromethyl radical (CCl3·) in the following reaction:

CCl 4 P450 CCl 3 · + Cl ·

The CCl3· reacts with oxygen to form the trichloromethyl peroxyl radical (CCl3O2·):

CCl3· + O2 CCl3O2·.

Hepatocyte damage results from the binding of CCl3· to macromolecules and the per oxidation of lipids by CCl3O2·.

Ethanol: one likely reason for the harmful effect of alcohol is the reduction of NAD to NADH during the oxidation of ethanol to acetaldehyde (refer to Fig. 18.6-1 – Alcohol is metabolized in the hepatocyte in two steps), which results in a deficiency of NAD. NADH inhibits xanthine dehydrogenase, which shifts the balance toward xanthine oxidase (XO) and thus purine oxidation with the formation of O2–·. This happens according to the following reaction:

Xanthine + H 2 O + O 2 XO Uric acid + 2O 2 –· + 2H +

The O2–· produced can in turn cause the production of hepatotoxic HO·.

Hemochromatosis: under reducing conditions, iron ions deposited in subcellular structures catalyze the Haber-Weiss reaction, resulting in the generation of HO·, which damages the cell membrane and intracellular membranes of hepatocytes.

Neurodegenerative disease /118/

Oxidative stress is thought to be involved in the development of many neurodegenerative diseases. It is known that the central nervous system (CNS) consumes a disproportionately large proportion of the body’s oxygen and that energy is produced mainly by the mitochondrial respiratory chain. On the other hand, compared to other organs, the CNS contains a relatively small amount of antioxidant enzymes such as super oxide dis mutase, glutathione peroxidase, and catalase.

Amyotrophic lateral sclerosis (ALS): some, but not all, patients with ALS have mutations in the gene that encodes cytosolic super oxide dis mutase (Cu/Zn SOD). Heterozygotes exhibit less than 50% of normal SOD activity. Increased amounts of ROS are produced and the concentration of 3-nitro tyrosine and peroxynitrites is increased in the cerebral cortex.

Alzheimer’s disease: ROS are thought to play a role in the etiopathogenesis of this disease. They are generated by β-amyloid, for example, and their elimination is reduced by a lack of antioxidants. An increased concentration of thiobarbituric acid reactive substance (TBARS) may be determined in the urine.

Parkinson’s disease: lipid per oxidation in the substantia nigra in particular is thought to have an etiological role in the development of this disease. Patients have increased blood concentrations of malondialdehyde and reduced antioxidant enzyme activity in the brain tissue.

Acute inflammation /19/

In acute inflammation, PMNs and monocytes migrate to the site of tissue damage during the pro inflammatory phase. This results in the phagocytosis of pathogens and triggers a number of independent processes, including the respiratory burst. Free HO·, HOCl, and enzymes such as elastase are released. In the event of PMN over stimulation (e.g., in sepsis) neither the enzymes nor the HO· can be neutralized. The body’s own tissue becomes damaged as a result of its defense against infection.

Chronic inflammatory states lead to the expression of adhesion molecules on vessel walls and inflammatory cells and to the activation of pro inflammatory cytokines and production of biomarkers of oxidative stress such as F2-isoprostanes.

Sickle-cell anemia /3/

Sickle-cell anemia is a hemoglobinopathy characterized by hemolytic anemia, increased susceptibility to infections, and vaso occlusive crises. There is evidence to support the use of ROS and their end products as biomarkers of disease activity.

Abdominal surgery

There is no difference between laparoscopic and open surgery with respect to the production of ROS /20/.

Heart failure /21/

Chronic heart failure is a chronic inflammatory condition with increased release of inflammatory cytokines. These induce iNOS, which leads to increased synthesis of NO·. Increased amounts of toxic peroxynitrites are produced as a result, leading to myocyte damage.

Table 19.3-1 Diagnostic value of various tests for the detection of inflammation

Test

Diagnostic
sensitivity

Diagnostic
specificity

Usefulness for
inflammation

Acute

Chronic

CRP/SAA

Very high

High

Very high

Moderate

SPE

High

Low

Moderate

High

ESR

High

Low

Moderate

Moderate

WBC
count

High

Low

High

Moderate

TM

High

Low

High

Low

ESR, erythrocyte sedimntation rate; SPE, serum protein electrophoresis; TM, temperature measurement

Table 19.3-2 Factors influencing the ESR /14/

Influence factor

Clinical and laboratory findings

Menstrual cycle

The ESR rises during the menstrual cycle, reaching its maximum during the premenstrual phase, and declines during menstruation.

Hormonal
contraceptives

The ESR is higher in women on hormonal contraceptives than in those without because of the hormones cause a rise in the fibrinogen concentration.

Pregnancy

From the 4th gestational week, the ESR rises continuously and reaches a maximum of up to 45 mm/h during the first postpartum week.

Neonates

Because of the high hematocrit and the low fibrinogen, the ESR is very low.

Hyper-
lipoproteinemia

Chylomicrons in particular lead to a rise in the ESR.

Dextrans

Rise in the ESR due to adsorption of dextrans to the red cell membrane. In the case of a dextran infusion over the course of 1 week, a cumulative increase of the ESR may occur up to a mean of 75 mm in the first hour /17/. Normal values are reached again 11–15 days after the termination of therapy.

Erythrocytosis

Sedimentation is slowed down and the ESR is decreased.

Macrocytosis

The sedimentation of erythrocytes is accelerated and the ESR is elevated.

Anemia

In the presence of a decreased erythrocyte count, the ESR is elevated. However, in the case of iron deficiency anemia, the rise in ESR does not correspond to the decrease in erythrocytes because the concomitant microcytosis slows down the ESR.

Erythrocyte
anomalies

A deviation from the disk-like shape (e.g., as seen in sickle-cell anemia, echinocytosis, poikilocytosis, stomacytosis, and acanthocytosis) leads to a decrease in the surface that is required for the aggregation of erythrocytes. This results in a decline of the ESR.

Table 19.4-1 Upper threshold values for CRP (mg/L)

Adults and children

Recommended threshold value in central Europe /4/

≤ 5.0

Recommended threshold value in North America, Scandinavia /1/

≤ 10.0

Age-related values

Young adults (20–24 yrs) /3/

< 5.1 (median 0.63)

Middle-aged adults (45–63 yrs) /3/

< 3.3 (median 0.74)

Older adults (65–72 yrs) /3/

< 9.3 (median 1.58)

Umbilical cord blood /3/

≤ 0.5 (median 0.16)

Neonates (3–7 days) /3/

≤ 12 (median 1.2)

Table 19.4-2 CRP in diseases associated with mild to high-grade inflammation

Clinical and laboratory findings

Infectious disease – Generally

Bacterial, parasitic, viral, and fungal infections lead to varying degrees of CRP elevation. Elevation may be minor (≤ 40 mg/L), moderate (40–100 mg/L), or marked (> 100 mg/L). CRP measurement is useful in detecting infection in situations where clinical and microbiological diagnosis is difficult but infection is likely. The CRP concentration correlates with the extent and intensity of infection and successful treatment leads to a decline in levels within three days. CRP levels > 40 mg/L indicate infection rather than another non-infectious cause of inflammation, except traumatic tissue damage and on postoperative days 1–5 /15/.

Bacterial infection

Bacterial endotoxin is the most potent stimulator of the release of pro inflammatory cytokines from monocytes/macrophages, which in turn induce the synthesis of acute phase proteins. Bacterial infections are therefore associated with some of the highest CRP concentrations. In a study of critically ill patients with a temperature above 38.2 °C (100.8 °F), a CRP concentration of greater than 87 mg/L was associated with an infectious cause, with a diagnostic sensitivity of 93.4% and a specificity of 100% /16/. In the same study, the CRP concentrations in sepsis, severe sepsis, and septic shock were 152 ± 82 mg/L, 203 ± 109 mg/L, and 233 ± 87 mg/L, respectively. Infections that are frequently associated with CRP concentrations > 100 mg/L include pneumonia, pyelonephritis, meningitis, purulent skin infections, septic arthritis, puerperal infection, and sepsis. CRP concentrations of 40–100 mg/L are commonly seen in acute bronchitis, bronchiectasis, tuberculosis, adnexitis (pelvic inflammatory disease), and sexually transmitted diseases (with the exception of uncomplicated chlamydial and gonococcal infections).

– Viral infection

CRP elevation is slight. In viral respiratory infections, for example, CRP concentrations rarely exceed 40 mg/L /17/. Mean CRP levels are around 15 mg/L on the second day of rubella infection, 22 mg/L in enterovirus infection, and 35 mg/L in the acute phase of Cytomegalovirus and Herpes simplex infections. In the acute phase of infection with Influenza virus A or B and Mycoplasma pneumoniae, the CRP concentration is 50–60 mg/L. In meningoencephalitis, CRP rarely exceeds 15 mg/L.

– Parasitic infection

Depending on the parasite in question, parasitic infestation can be associated with mild to moderate CRP elevations (below 40 mg/L).

– Fungal infection

Localized fungal infections are not associated with CRP elevation. However, systemic fungal infections in severely neutropenic patients are associated with CRP elevations similar to those seen in bacterial sepsis (greater than 100 mg/L) /18/.

– Sepsis /8/

It is difficult to diagnose sepsis based on a CRP value. For example, studies involving daily CRP measurement show the following: for a level > 50 mg/L, a diagnostic sensitivity of 98.5% with a specificity of 75% and for a threshold value of 79 mg/L, a diagnostic sensitivity of 71.8% with a specificity of 66.6%. If CRP is measured only once, levels > 100 mg/L are highly suggestive of sepsis in the presence of additional clinical evidence. A threshold value of ≤ 154 mg/L has a negative predictive value for excluding severe sepsis of only 63.5%. The following classification relates the severity of inflammation to the CRP value and classifies it in accordance with the ACCP/SCCM Consensus Conference criteria: mean CRP values are /19/: 70 mg/L in systemic inflammatory response syndrome; 98 mg/L in sepsis; 145 mg/L in severe sepsis; and 173 mg/L in septic shock. Elevated CRP in intensive care patients with sepsis or pneumonia are not associated with increased mortality.

In surgical and trauma patients, CRP > 130 mg/L indicates sepsis, with a diagnostic sensitivity of 85% and a specificity of 83%. Following pneumonectomy, peak CRP levels occur on days 3–6 and are < 50 mg/L by day 12 in uncomplicated cases. A value of > 100 mg/L after day 12 indicates sepsis, with a diagnostic sensitivity of 100% and a specificity of 94.8% /20/.

The main drawback of using CRP to diagnose sepsis is its sluggishness, since treatment needs to be initiated within 6 h of the onset of sepsis.

– Neonatal sepsis

The diagnosis of neonatal sepsis is a routine challenge in the neonatal intensive care unit. Within the first 48 h, it is often difficult to distinguish between infectious and non-infectious causes of inflammation in the newborn. Normal CRP concentrations in cord blood are lower than in the serum of healthy adults. The acute phase response is effective in the neonate and cord blood and circulating blood CRP levels are raised in infection. However, CRP is a questionable biomarker for diagnosing neonatal sepsis because, although its diagnostic specificity is high (90–96%), its sensitivity is low (33–44%) /21/. Its usefulness as a marker is therefore restricted to ruling out sepsis. Pathologies such as fetal asphyxia, distress, shock, cerebral hemorrhage, or meconium aspiration may all cause an elevation of CRP levels to as high as 70 mg/L in the absence of sepsis.

– Amniotic infection syndrome (AIS)

Pregnant women with premature rupture of the amnion are at risk from bacterial infection of the amnion and placenta (chorioamnionitis) with the risk of extension into the uterine musculature and fetus. The reported incidence of infection in this situation is 0.5–25% but during the gestational weeks 28–30, this may be as high as 20%. The diagnostic criteria are premature rupture of the amnion, fever, fetal and maternal tachycardia, leukocytosis, and uterine tenderness. There is controversy over the value of CRP as a predictor of AIS owing to the significant overlap between the CRP values found in the acute phase response that normally occurs postpartum and those found in AIS /22/. In one study /23/, however, if a maternal CRP value of greater than 20 mg/L was used as a criterion for the presence of AIS, the diagnostic sensitivity was 25.8%, with a specificity of 75.4%, a negative predictive value of 99%, and a positive predictive value of 47%. Therefore, a “positive” test would identify half the mothers with AIS. On the first postpartum day, CRP rises 2–3 times higher in the presence of AIS than in the normal acute phase response associated with delivery.

– Fever in children

In children, although fever is most often due to viral infections, this is difficult to distinguish from bacterial infections such as otitis media, bronchitis, tonsillitis, and cystitis, and antibiotics are often prescribed unnecessarily. It has been shown that, in children who have been ill for more than 12 h, a CRP level of greater than 40 mg/L has a diagnostic sensitivity of 79% and a specificity of 90% for the diagnosis of bacterial infection /24/. An ESR of greater than 30 mm/h, on the other hand, shows a sensitivity of 91% and specificity of 89%. In another study /25/, a CRP level below 40 mg/L was used to correctly rule out severe bacterial infections.

– Meningitis

Children with meningitis usually undergo lumbar puncture, which in most cases of bacterial infection gives the typical picture. However, in patients with negative microscopic evidence of infection and low-grade pleocytosis, serum CRP is a useful diagnostic adjunct. A CRP value > 20 mg/L in adults and children is highly suggestive of bacterial etiology while levels > 100 mg/L are diagnostic. Viral meningitis is associated with values of up to 19 mg/L /26/. Tuberculous meningitis is typically associated with values in the range of 20–50 mg/L.

– Pneumonia /27/

The Genomics to combat Resistance against Antibiotics in Community-acquired LRTI in Europe (GRACE) consortium has investigated how to distinguish acute bronchitis from pneumonia. This distinction is important because pneumonia requires antibiotic treatment whereas bronchitis does not. A score was used to classify the pneumonia risk of patients as low, intermediate, or high (Tab 19.4-4 – Pneumonia-risk classification in patients with bronchitis). Pneumonia was diagnosed by chest radiography in 5% of the 2820 patients presenting with acute cough. Signs and symptoms were useful in correctly identifying patients with a low (< 2.5%) or high (> 20%) diagnostic risk in 26% of patients; 74% were identified as intermediate-risk patients. At a CRP value of > 30 mg/L, the proportion of patients with pneumonia in the low, intermediate, and high-risk groups was 0.7%, 4%, and 18% respectively. In the patients with pneumonia, CRP concentrations (mg/L) were > 20, > 30, > 50 and > 100 in 85%, 74%, 58%, and 34% of patients respectively. 38% of patients had CRP concentrations of ≤ 20 mg/L. Addition of CRP at a threshold value of > 30 mg/L to the score allowed 48% of patients in the intermediate risk group to be reclassified as low risk and 3% as high risk (Tab 19.4-4 – Pneumonia-risk classification in patients with bronchitis).

– Appendicitis

Using a cutoff value of ≥ 10 mg/L, CRP has a diagnostic sensitivity of 68.2% and a specificity of 75.1%. In contrast, the values for a leukocyte count ≥ 6,3 × 109/L are 87.2% and 63%, respectively /12/.

– Genital infections

Uncomplicated chlamydial and gonococcal infections do not result in elevated CRP levels. However, extension into pelvic organs with acute or chronic pelvic inflammatory disease results in an acute phase response. Patients with adnexitis show elevated CRP levels in 81% of cases and leukocytosis in 52% of cases. CRP measurement is therefore a useful indicator in the therapeutic monitoring of such patients /28/.

Pregnancy and childbirth

The pre-pregnancy upper reference interval value of 8–10 mg/L increases to 18 mg/L shortly before delivery /29/. The CRP concentration rises to as much as 60 mg/L 24 h after vaginal delivery and returns to a mean value of 25 mg/L after 48 h. Following a cesarean section, mean CRP concentrations at 24 h, 48 h, and 72 h are 64 mg/L, 149 mg/L, and 113 mg/L, respectively /30/.

Postoperative phase

All surgical interventions cause an acute phase response roughly in proportion to the extent of tissue damage. In uncomplicated cases, CRP rises above 10 mg/L by 6 h, reaches a peak rarely greater than 150 mg/L at about 48 h, and declines thereafter to baseline values by 7–10 days. Postoperative complications such as infections, tissue necrosis, hematomas, and thromboses, depending on when they occur, maintain a raised CRP level after 48 h or result in a secondary increase. A CRP level greater than 75 mg/L on or beyond the 6th postoperative day is always a sign of complication. In many cases, the raised CRP precedes the clinical diagnosis of the complicating pathology by up to 24 h. Patients at risk of infection (e.g., up to 10% of patients following resections of parts of the colon) should be monitored by daily CRP measurements. In this situation, single measurements are of little value /31/.

CRP can be a useful adjunct to the diagnosis of deep vein thrombosis. In one study, CRP measurement had a diagnostic sensitivity of 100% and a specificity of 52% /32/.

Acute pancreatitis

Acute pancreatitis leads to CRP elevation during the first 24 h but in the absence of complications, the levels are declining by the end of the first week. Peak values on days 3–5 indicate complications such as interstitial edematous pancreatitis, sterile necrosis, or infected necrosis. If, within 48 h of the onset of symptoms, the CRP concentration is above 150 mg/L, acute necrotizing pancreatitis is likely (diagnostic sensitivity and specificity > 80%, diagnostic accuracy 86%) (refer also to Tab. 14.2-1 – Laboratory tests for the diagnosis and monitoring of acute pancreatitis).

Acute myocardial infarction (AMI)

AMI is usually associated with elevated CRP, often occurring within a few hours of the onset of pain and typically reaching a peak on the third or fourth day and reaching normal values again by 7–10 days. Elevated CRP in the presence of indicative symptoms is a sensitive indicator of this condition, being present in 49 out of 50 patients with AMI and in 100% of patients with significant Q-wave ECG changes. CRP levels > 50 mg/L after 10 days suggest the presence of complications and indicate a poor prognosis /33/.

Stroke

Following a major stroke, CRP concentrations at 0–8 h, 8–16 h, and 16–24 h after the acute event are 6–12 mg/L, 10–22 mg/L, and 18–35 mg/L, respectively. CRP elevation in the first 24 h correlates positively with 1-year mortality and can potentially reflect the vascular risk profile /34/.

Malignant tumor

Fever and an acute phase response are common features of malignant tumors. This is due to release of cytokines from the tumor itself, from invading monocytes/macrophages, or to concomitant tissue necrosis. Raised and increasing levels of CRP predict a poor prognosis and frequently indicate metastatic spread. CRP concentrations ranging from 8–328 mg/L have been measured in a broad spectrum of neoplastic diseases /35/.

CRP has been shown to be a useful (though nonspecific) biomarker for benign and malignant colorectal tumors. In preoperative staging, Dukes D tumors have been correctly ruled out on the basis of normal carcinoembryonic antigen (CEA) and CRP values, with a diagnostic sensitivity of 53%, specificity of 93%, and negative predictive value of 93%. Conversely, Dukes C and D colorectal carcinomas have been identified on the basis of elevated CRP and CEA concentrations with a sensitivity of 40%, specificity of 92%, and positive predictive value of 92%. Individuals > 45 years. whose baseline CRP concentrations are greater than 3 mg/L have a higher 10-year risk of developing colorectal carcinoma than individuals with lower CRP concentrations (odds ratio 2.5) /36/.

In malignant lymphatic diseases in which IL-6 is released from the tumor, notably multiple myeloma and Hodgkin’s disease, CRP levels correlate with prognosis and tumor spread if infection is excluded. Thus in asymptomatic Hodgkin’s disease, values are below 20 mg/L whereas in the presence of symptoms they are in the region of 150 mg/L.

CRP measurement is worthwhile in malignant disease both for monitoring tumor progression and response to treatment and for diagnosing infectious complications /37/.

In one study, there was no significant difference in CRP levels in men with localized prostate cancer or benign prostatic hypertrophy (usually below 10 mg/L) but levels were significantly higher in men with bone metastases /38/.

Rheumatic disease – Generally

The rheumatic diseases present with joint or soft tissue symptoms such as arthralgia, back pain, and myalgia. However, such symptoms may also commonly be due to local or psychogenic factors. One of the most important decisions is the distinction between organic and non-organic disease. The finding of elevated acute phase proteins confirms the presence of organic disease but a value within the reference range does not exclude it since mild local disease and some forms of connective tissue disease do not elicit a significant acute phase response. In some cases, such as ankylosing spondylitis, serum CRP may be elevated before the disease is clinically obvious.

– Rheumatoid arthritis

Increased CRP concentrations are found in > 90% of adults with this condition and in established disease, levels relate to severity. Values ≤ 50 mg/L are associated with mild inflammation and values of > 100 mg/L indicate more severe disease. Unfortunately, measurements made at the onset of disease have little predictive value for either the functional outcome or mortality. It seems that in those patients in whom a consistent therapeutic suppression of CRP is achieved, radiological progression is sustainably reduced. Normalization of CRP levels rarely occurs. The CRP concentration correlates more closely with radiologically determined joint damage than clinical symptoms or other tests such as ESR, rheumatoid factor, or soluble immune complexes. Rest, analgesics, and non-steroidal anti-inflammatory drugs have little effect on CRP levels whereas disease-modifying drugs such as gold, sulfasalazine, and D-penicillamine cause CRP to fall if a clinical response subsequently occurs. If a patient responds to a drug, the decline in the CRP concentration typically precedes the improvement in clinical symptoms by about 6 weeks and the radiological improvement by about 6 months /39/.

– Juvenile chronic arthritis

Active widespread inflammation is associated with increased CRP concentrations whereas in mild or local disease, CRP is normal or only slightly elevated. Patients in whom amyloidosis develops tend to have consistently high CRP levels for several years before serious and fatal complications arise. Reduction of CRP is therefore an important therapeutic objective. CRP is more useful than ESR in this regard /40/.

– Systemic lupus erythematosus

CRP levels are generally less than 15 mg/L but can range from 1 to 70 mg/L. Values greater than 15 mg/L are particularly associated with concomitant fever. Concentrations greater than 60 mg/L in these patients generally indicate intercurrent infection /41/.

– Ankylosing spondylitis

Back pain is a very common clinical symptom and the presence of a raised CRP concentration is a strong indication of organic disease such as ankylosing spondylitis. In established disease, CRP is often elevated long before the onset of clear-cut clinical symptoms but there is no consensus about the relationship between CRP or the ESR and disease activity /42/.

– Psoriatic arthropathy, Reiter’s syndrome

In both of these conditions, there is inflammation of synovium or connective tissues. The CRP concentration is elevated in proportion to disease activity.

– Crystal arthropathy

n gout, moderate elevations of CRP are usual whereas they are more modest in pseudo gout /43/.

– Osteoarthritis

Because this condition is primarily degenerative rather than inflammatory, the CRP is therefore normal.

– Polymyalgia rheumatica

This is a disease of the elderly characterized by morning stiffness and pain of the shoulder girdle and hip girdle associated with diffuse systemic symptoms such as depression and malaise. CRP and ESR are both markedly elevated, though not always in parallel, and are very useful in diagnosing this disease. Untreated, about 30% of patients will develop cranial arteritis with a serious risk to eyesight. CRP falls rapidly to normal levels as the disease responds to corticosteroid therapy and its measurement is widely used to monitor and evaluate treatment. The ESR changes more slowly. In a prospective serial study of serum CRP in poly myalgia rheumatica, elevated CRP concentrations of 10–140 mg/L (median 40 mg/L) at presentation decreased in response to prednisolone therapy to a mean value of 20 mg/L by the third day and were significantly lower by day 7 /44/.

– Systemic vasculitis

In immune vasculitis, polyarteritis nodosa, and Wegener’s granulomatosis, clinical assessment may be difficult. The measurement of CRP has proved useful for minimizing the effective dose of corticosteroids.

– Connective tissue disease

Systemic lupus erythematosus, polymyositis, and systemic sclerosis have in common that the acute phase response is minimal even in active disease and CRP concentrations are usually below 15 mg/L /45/. This fact is quite useful in that the CRP level can be used to distinguish these conditions from other rheumatic diseases and, in the presence of fever, to discriminate between intercurrent infection and disease exacerbation /44/. Although there is a considerable overlap in CRP values between these two latter situations, CRP concentrations of greater than 100 mg/L are strongly indicative of bacterial infection /46/.

Inflammatory bowel disease

Active Crohn’s disease is accompanied by an elevated CRP concentration. The median CRP level is 4 mg/L in mild disease, 15 mg/L in moderate disease, and 85 mg/L in severe disease. In ulcerative colitis, the median CRP concentration is 3 mg/L in mild to moderate disease and 12 mg/L in severe disease (range 2–33 mg/L) /47/. Irritable bowel syndrome is a functional condition that is not associated with inflammation and thus does not cause an elevation of CRP. Consistently raised levels of CRP therefore cast doubt on this diagnosis and necessitate further investigation /48/. The measurement of disease activity in inflammatory bowel disease is clinically significant and is generally carried out using scores such as the Crohn’s Disease Activity Index (CDAI), Harvey-Bradshaw Index (HBI), or the van Hees Index (VHI), which uses a combination of clinical data and laboratory findings (ESR, serum albumin). VHI values < 100 are associated with inactive disease while mild inflammatory activity is indicated by values between 100 and 150, moderate inflammatory activity is indicated by values between 150 and 210, and severe inflammatory activity is indicated by values > 210. In one study /49/ in which 59% of patients had CRP concentrations in excess of 20 mg/L, CRP correlated significantly with the VHI. A CRP cutoff value of 21.6 mg/L was associated with a VHI value ≥ 150.

Type A amyloidosis

The fibrils of this type of amyloidosis are derived from the proteolytic degradation of the acute phase protein, serum amyloid A protein (SAA). They are deposited in the basement membranes of blood vessels and in the sinusoids of the liver and spleen. The glomeruli and renal tubules are also frequently affected. The condition results from a sustained and profound acute phase reaction causing raised levels of the precursor protein, SAA. Typically, an active infection will have already been present in these patients for over 10 years. The most common predisposing diseases are juvenile rheumatoid arthritis, chronic suppuration, osteomyelitis, tuberculosis, and leprosy. Once established, the disease is inevitably fatal but the rate of progression can be slowed by reducing the serum concentration of SAA. It is difficult to measure SAA, but CRP levels parallel its changes and can therefore be used to monitor treatment with antibiotics or anti-inflammatory drugs /50/.

Immunocompromised patients (e.g., acute leukemia)

Fever in patients with leukemia and neutropenia can be caused by infection, the underlying disease process, or the administration of blood products. If CRP concentrations are less than 40 mg/L for 48 h after the onset of fever, infection is unlikely, whereas levels above 100 mg/L should be treated with antibiotics even in the absence of bacteriological confirmation. If levels do not fall below 100 mg/L after treatment, it must be assumed that a response has not occurred and the treatment must be changed. This applies to any patients with neutropenia following cytostatic therapy, including those with other forms of malignancy /51/.

Allogeneic bone marrow transplantation

Patients undergoing bone marrow transplantation are at particular risk of infection that can progress very rapidly to undiagnosed sepsis. In this situation, the clinical features of graft-versus-host-disease are difficult to distinguish from infection. The maximum CRP values seen in this condition rarely exceed 40 mg/L and values above this strongly suggest infection; values above 100 mg/L are only seen in infection /52/.

Treatment with erythropoiesis-stimulating agents (ESAs)

ESAs are used for the treatment of anemia in chronic hemodialysis patients. The response to ESAs is impaired in the presence of inflammation, which means that higher doses must be used. The CRP concentration appears to be an independent predictor of greater ESA dose requirements to achieve comparable hemoglobin levels. In one study /53/, patients with a CRP concentration of ≥ 32 mg/L required significantly higher ESA doses than patients with lower CRP levels to achieve the same hemoglobin increase within 3 months.

Treatment with Glucocorticoids

Dexamethsone, a synthetic glucocorticoid, has anti-inflammatory and immunosuppressive properties. In SARS-CoV-2 patients CRP levels decreased significantly following the start of dexamethasone from mean initial levels of 129.5 mg/L to 40.7 mg/L at time of discharge /84/.

In patients with early rheumatoid arthritis the release of cytokines, especially TNF-α, IL-6 and IL-1, causes synovial inflammation. In patients treated with a combined therapy of 15 mg of methotrexate weekly with a step-down scheme of daily oral prednisone (30 – 20 – 12 – 5 mg) CRP decreased from 20.1 mg/L to below 2.5 mg/L /85/.

In giant cell arteritis (GCA) glucocorticoids are the gold standard for induction of remission and toclizimab is now considered to be part of the standard treatment for GCA, particularly with relapsing disease. Most flares occure while patients are still receiving prednisone. CRP is not a reliable indicator of flare in patients treated with prednisone or toclizimab plus prednisone /86/

Table 19.4-3 Behavior of CRP in diseases associated with low-grade inflammation

Clinical and laboratory findings

Atherosclerosis

Atherosclerosis is a disease of the blood vessel wall characterized by pathological lipid deposits that trigger an inflammatory response. Macrophages have ingested and accumulated low-density lipoproteins (LDL), which after enzymatic or oxidative modification (oxLDL) were taken up by LOX-1 (lectin-like oxidized LDL receptor 1) scavenger receptors on endothelial cells. OxLDL have pro inflammatory properties and stimulate the synthesis of IL-6, which in turn activates the synthesis of CRP: in hepatocytes, in smooth muscle cells, in endothelial cells, in macrophages of the vessel wall, and in atherosclerotic plaques. CRP intensifies the effect of oxLDL by up regulating the LOX-1 receptor.

Cardiovascular disease (CVD) – Generalized

Atherosclerosis of the coronary vessels is the underlying cause cardiovascular disease. This process starts early in life and progresses slowly and insidiously, remaining asymptomatic for decades. As it progresses, atherosclerosis is associated with minor elevations of CRP within the reference interval. Measurement of CRP is recommended for:

  • Cardiovascular risk stratification in apparently healthy individuals
  • Secondary prevention in patients with cardiovascular disease
  • Therapeutic intervention: the fact that treatment with pravastatin decreases the CRP level shows that it also has an anti-inflammatory effect in addition to its ability to reduce LDL cholesterol.

– Primary prevention

According to the US Preventive Services Task Force recommendation statement /54/, an elevated CRP level predicts a higher risk for CVD independent of Framingham risk factors. Individuals with a CRP level greater than 3 mg/L on more than one occasion have a relative risk of 1.58 (confidence interval 1.37–1.83) for coronary events.

– Secondary prevention

CRP is a predictor of short-term and long-term risk after a coronary event or re vascularization procedure and predicts new coronary events in patients with unstable angina and acute myocardial infarction /10/.

Short-term follow-up: in one study /55/ of patients with severe unstable angina but without a history of myocardial infarction, CRP levels greater than 3 mg/L on admission were associated with an increased incidence of recurrent angina pectoris, myocardial infarction, and cardiovascular mortality. Another study /56/ confirmed that patients with unstable angina and CRP levels greater than 3 mg/L on admission experienced an increased number of ischemic events during their hospital stay while patients with lower levels did not. Other studies have shown that CRP levels above 5 mg/L on admission in patients with cardiovascular disease are associated with a poor prognosis, regardless of whether troponin levels are normal or elevated. Data from the Thrombolysis in Myocardial Infarction IIa (TIMI IIa) study /58/, a study of patients with unstable angina and non-Q-wave myocardial infarction, has shown that significant CRP elevation (median 15.5 mg/L) on admission is a reliable predictor of 14-day mortality.

Long-term follow-up: in a three-month follow-up study /59/ of patients with unstable angina, a CRP level > 3 mg/L had a positive predictive value (PPV) of 24% for predicting further cardiac events; this increased to 44% for a CRP cutoff value of > 10 mg/L. The negative predictive values were 96% and 92%, respectively. The Fragmin During Instability in Coronary Artery Disease (FRISC) study /60/ monitored patients with unstable angina for 37 months and showed that the mortality risk in patients with the highest troponin T concentrations (> 0.6 µg/L) and highest CRP levels (> 10 mg/L) was 16% compared to 0% in those with the lowest values.

Residual inflammatory risk dominates the clinical picture. Among the secondary prevention patients in the PROVE-IT trial treated with aggressive statin therapy, those who achieved LDLC levels < 70 mg/dl and CRP levels < 2 mg/l had substantly lower rates of recurrent vascular events when compared with those achieved only one or neither of these independent treatment targets /57/.

– Tertiary prevention

Intervention in patients with elevated CRP (tertiary prevention). Tertiary prevention is concerned with the management of established disease with the aim of preventing exacerbations. Aspirin or HMG-CoA reductase inhibitors (statins), for example, that are used to treat CVD have an anti-inflammatory effect. In the Physicians’ Health Study, the treatment with aspirin of apparently healthy men with CRP values above 3 mg/L reduced the risk of myocardial infarction by 60% /61/.

Another study showed that the use of HMG-CoA reductase inhibitors in individuals with average cholesterol levels reduced morbidity and mortality /62/.

The Long-Term Intervention with pravastatin in Ischemic Disease (LIPID) study showed a 24% reduction in cardiovascular disease mortality as a result of treatment with pravastatin in men and women with normal to slightly elevated total cholesterol /63/.

– Stent implantation

In patients with drug-eluting stents, the risk of stent thrombosis, death, myocardial infarction, death from myocardial infarction, or repeated re vascularization in the following 3.9 years depends on the CRP level before stent insertion. Patients with CRP levels > 3 mg/L, compared to those with lower levels, had an increased risk: of stent thrombosis (hazard ratio 3.86), of death (hazard ratio 1.61), of myocardial infarction (hazard ratio 1.63), and death from myocardial infarction (hazard ratio 1.61).

Mortality /65/

In the MONICA/KORA Augsburg cohort study, men aged 25–74 years with CRP levels > 3 mg/L had a mortality risk within 7.1 years that was twice as high as those with levels < 1 mg/L.

Insulin resistance /66/

In a study investigating the association of 10 surrogate markers of insulin resistance in nondiabetic adults, individuals with insulin resistance and a body mass index (BMI) ≥ 30 kg/m2 had the highest prevalence of insulin resistance (76.2%). Individuals with a BMI < 25 kg/m2 and CRP < 1 mg/L had a prevalence of only 6.6%.

Type 2 diabetes

In the MONICA/KORA Augsburg cohort study, men aged 25–74 years with CRP levels above 2.91 mg/L had a 2.7 times higher risk of type 2 diabetes within 7.1 years than those with levels ≤ 0.67 mg/L /67/.

In the Women’s Health Initiative observational study, postmenopausal women aged 50–79 years without diabetes mellitus or cardiovascular disease were monitored for diabetes over a period of 5.9 years. Women with a CRP level of greater than 3 mg/L at the start of the study were 3.46 times more likely to develop diabetes than those with a CRP concentration of less than 1 mg/L /68/.

Type 2 diabetics with a CRP concentration of greater than 3 mg/L were 1.72 times more likely to die from cardiac causes than those with lower CRP concentrations /69/. However, diabetics with a CRP level greater than 3 mg/L who were physically active had a lower mortality at 4 years than those who were sedentary /70/.

Asthma /71/

A CRP level > 2.21 mg/L is associated with respiratory symptoms and non-allergic asthma but not with allergic asthma, with an odds ratio of 3.57.

Cystatin C elevation /72/

In the absence of renal disease stage 3 or 4, patients with elevated cystatin C had a higher prevalence of low hemoglobin, elevated uric acid, phosphate, fibrinogen, and CRP than patients with a normal serum cystatin C. Elevated CRP levels above 10 mg/L were found in 6.5% of the patients with normal cystatin C and 22.5% of those with elevated cystatin C.

Hormonal contraceptives /73/

Most hormonal contraceptives contain the combination of an estrogen and a progestogen. The most commonly used estrogen is ethinyl estradiol (EE); the most commonly used progestogen in second-generation pills is levonorgestrel while progestogens such as norgestimate, desogestrel, or gestodene are used in the newer third-generation pills. To reduce the risk of thromboembolic events, only low-dose preparations (“micropills”) containing 15–35 μg EE are prescribed.

Hormonal contraceptives can affect cardiac function, lipid and carbohydrate metabolism, hemostasis, and have a direct effect on blood vessel walls. Estrogen and progestogen containing contraceptives may increase the risk of vascular disease in the presence of other risk factors.

The newer third-generation pills were developed with the aim of reducing side-effects (cardiovascular and thromboembolic in particular). In one study /74/, the effect of second and third-generation pills on CRP and lipid profile was investigated. CRP concentrations in healthy women who used third-generation pills were three times higher and CRP above 3 mg/L were more frequent than in the control group (Tab. 19.4-5). These findings suggest that apparently healthy women using hormonal contraceptives might not be free of cardiovascular risk.

Table 19.4-4 Diagnostic risk classification for pneumonia in patients with acute cough /27/

Scoring
criteria

Points

Score points and
risk category

Absence of
runny nose

1

Score 0 point of 7,
no risk for pneumonia

Breathlessness

1

Diminished
vesicular breathing

1

Score 1–2 points of 7,
intermediate risk for
pneumonia

Crackles

1

Pulse > 100/ min.

1

Score 3 points of 7,
high risk for pneumonia

Temperature
> 37.8 °C

1

CRP > 30 mg/L

1

Table 19.4-5 The effect of hormonal contraceptives (hC) on CRP and lipids /74/

Parameter

Control
group

hC second
generation

hC third
generation

CRP
(mg/L)

0.5
(0.2–1.15)

1.04
(0.4–3.5)

2.7
(1.8–4.1)

Triglycerides
(mg/dL)

60
(48–79)

89
(67–145)

96
(71–114)

LDL cholesterol
(mg/dL)

86
(69–117)

96
(81–111)

72
(63–86)

Values are median, 2.5 and 97.5 percentiles

Table 19.5-1 PCT in the differential diagnosis of inflammatory diseases

Clinical and laboratory findings

Post surgery

Following uncomplicated surgery, the PCT remains or declines rapidly in the range below 0,5 μg/L on days 3–4. Persistently elevated or increasing PCT levels are suggestive of systemic infection /1/.

Localized infection

In highly localized infections (pulmonary emphysema, abscess), PCT is not elevated or may be only slightly elevated below 0.5 μg/L /1/.

Sepsis

PCT is a more reliable biomarker for diagnosing sepsis than other infection markers such as the WBC count or CRP. PCT concentrations of 0.5–1.0 μg/L are both sensitive and specific. Concentrations greater than 1–2 μg/L are typically found in high-risk patients and concentrations > 10 μg/L indicate organ failure remote from the site of infection /20/. Three systematic reviews and meta-analyses of PCT as a diagnostic test for sepsis summarized the following results /21/:

  • Compared to CRP, PCT has a higher diagnostic sensitivity and specificity for distinguishing between bacterial and non bacterial causes of systemic inflammation /5/: the diagnostic sensitivities for PCT and CRP were 88% and 75% respectively, while the corresponding diagnostic specificities were 81% and 67%.
  • Odds ratios for the diagnosis of sepsis in critically ill adults and postoperative patients were 15.7 in the studies using PCT and only 5.4 in the studies using CRP /22/. PCT concentrations were 16 times higher on average in patients with infection than in those without. The CRP cutoff values for sepsis in the studies ranged from 39 mg/L to 180 mg/L.
  • PCT cannot reliably differentiate sepsis from other non infectious causes of SIRS and is therefore not suitable for widespread use in critical care settings /23/.

The advantages of PCT compared to CRP are that PCT has higher specificity and more rapid kinetics and is less influenced by steroid therapy /1/. Severe infections cause a marked increase in PCT levels (> 10 μg/L, occasionally as high as > 1,000 μg/L) whereas uncomplicated sepsis results in only slightly elevated PCT levels. In sepsis, therefore, the development and progression of multiple organ dysfunction syndrome is reflected by rising PCT levels /24/. A cutoff value of 6 μg/L on the first day predicts mortality with a diagnostic sensitivity of 87.5% and specificity of 45% in patients with septic shock /5/.

Mortality risk in critically ill patients

Measurement of the day-to-day variation in the PCT level and the absolute peak level are indicators that can be used to identify patients with increased 90-day mortality. In one study, the mortality rate for critically ill patients was 24.9% for those with peak levels of ≥ 1.0 μg/L and 33.5% for those with peak levels of ≥ 5 μg/l. A rise in PCT of ≥ 1.0 μg/L within a day was an independent predictor of mortality /25/.

Ventilator-associated pneumonia

PCT can be used as a biomarker to identify ventilator-associated pneumonia. The strongest predictor of pneumonia and an unfavorable outcome is a PCT level > 0.5 μg/L after the commencement of ventilation /26/.

Antibiotic monitoring in the critical care setting

Restricting the duration of antibiotic therapy in the critical care setting can help reduce bacterial resistance to antibiotics. The PRORATA (Use of procalcitonin to reduce patients’ exposure to antibiotics in intensive care units) randomized, multicenter study showed that PCT could be used to guide the use of antibiotics in the critical care setting /27/. When the PCT concentration was measured daily before and during antibiotic treatment and treatment was discontinued as soon as the PCT concentration was ≤ 0.5 μg/L, patients in the PCT group had significantly more treatment-free days than those in the control group (14.3 ± 9.1 compared to 11.6 ± 8.2), with a comparable clinical outcome.

Following a clinical diagnosis of sepsis, antibiotics must be started immediately, even before the pathogen responsible and its sensitivity to antibiotics are known. To determine the appropriateness of empirical antibiotic therapy, one study /28/ monitored patients with sepsis by measuring PCT daily over four days. Appropriate antibiotic therapy was associated with a 36% decrease in PCT between day 2 and day 3 of treatment, which was found to be an independent predictor of survival. In patients receiving inappropriate antibiotics, PCT rose by 30% during the same period. The PCT concentration on day 1 did not predict survival but patients with higher PCT had a worse prognosis than those with low PCT.

Acute pancreatitis

The most common cause of death in acute pancreatitis is infected pancreatic necrosis. Although the infection rate is less than 10%, diagnosis of infection is extremely important since surgical intervention can be necessary in these cases. PCT and IL-8 are better criteria than CRP for diagnosing infected necrosis. A PCT concentration ≥ 1.8 μg/L can differentiate between infected necrosis and edematous pancreatitis with a diagnostic sensitivity of 94%, specificity of 91%, and accuracy of 92% /29/.

Pyelonephritis

Febrile urinary tract infection (UTI) is a common problem, especially in pediatrics. Pyelonephritis must be distinguished from lower urinary tract infection because it can lead to chronic renal insufficiency and hypertension. PCT is useful in differentiating UTI from pyelonephritis and estimating the severity of renal lesions in pyelonephritis. In one study /30/ of children with UTI or pyelonephritis:

  • In UTI, the WBC count was (10.939 ± 0.834) × 109/L, CRP was 30.3 ± 7.6 mg/L, and PCT was 0.38 ± 0.19 μg/L
  • In pyelonephritis, the WBC count was (17.429 ± 0.994) × 109/L, CRP was 120.8 ± 8.9 mg/L, and PCT was 5.16 ± 2.33 μg/L
  • CRP measured at admission had a diagnostic sensitivity of 100% and specificity of 26.1% for predicting renal damage whereas PCT had a sensitivity of only 70.3% but a specificity of 82.6%.

According to a meta-analysis /31/ a PCT threshold value of 0.5–0.6 μg/L can differentiate between pyelonephritis and UTI in children, with an odds ratio of 14.25 (4.7–43.2).

Fever of unknown origin following organ transplantation

PCT can be used to differentiate between transplant rejection and infection in patients with fever of unknown origin. Following liver transplantation, PCT increases to 5.2 ± 1.23 μg/L and returns to normal within the first week. Local wound infection is associated with elevated PCT even in the absence of graft-versus-host-disease. In one study, patients with systemic infection had PCT concentrations of ≥ 0.8 to 41 μg/L /32/.

Upper respiratory tract infection

Upper respiratory tract infections (URTIs) are the most common reason for prescribing antibiotics in the Northern Hemisphere. URTIs include acute pharyngitis, acute tonsillitis, acute otitis media, acute laryngitis/tracheitis, acute sinusitis, and the common cold. Although most of these infections are viral, antibiotics are prescribed to about 75% of patients. The ProHOSP study /33/ and a further study /34/ examined whether PCT measurement on admission could be used to filter out patients with pneumonia and restrict antibiotic therapy to these patients. The results of the studies showed that antibiotic treatment should be recommended for PCT values greater than 0.25 μg/L and should be urgently required for values > 0.50 μg/L. In 15% of patients, antibiotics were not required based on low PCT values of ≤ 0.25 μg/L. In patients with pneumonia, PCT monitoring on days 3, 5, and 7 allowed the duration of antibiotic therapy to be reduced from 8.7 to 5.7 days compared to a control group if antibiotics were discontinued as soon as PCT had fallen to ≤ 0.25 μg/L. The rate of antibiotic-related side effects decreased from 28.1% to 19.8%.

Pneumonia

In a meta-analysis the accuracy of PCT in predicting mortality in pneumonia patients with different pathogenic features was studied. An elevated PCT level was a risk factor for death from community acquired pneumonia (risk ratio 4.38, 95% confidence interval 2.98–6.43), particularly in patients with a low CURB-65 score. The commonly used cutoff, 0.5 μg/L, had a low diagnostic sensitivity and was not able to identify patients at high risk for dying. However, the PCT assay with functional sensitivity < 0.1 μg/L was necessary to predict mortality in CAP in the clinic /35/.

Table 19.5-2 PCT levels that are not consistent with the corresponding disease or condition /1/

PCT elevation in the absence of bacterial infection

  • Neonatal period
  • Acute respiratory distress syndrome
  • Systemic fungal infection (PCT levels variable)
  • Severe trauma
  • Following major surgery
  • Severe burns and heatstroke
  • Pneumonitis
  • Calcitonin-producing tumor (medullary thyroid carcinoma, carcinoid, small cell lung cancer)
  • Treatment with anti-thymocyte globulin

Inappropriately low PCT elevation in bacterial infection

  • Very early stage of infection
  • Highly localized infection (abscess)
  • Subacute endocarditis

Table 19.6-1 Threshold values for serum amyloid A protein /4/

Adults and children

< 10 (20)

Young adults (20–24 years)

< 14.8 (median 2.3)

Middle-aged adults (45–63 years)

< 5.7 (median 2.5)

Older adults (65–72 years)

< 19.3 (median 3.7)

Umbilical cord blood

≤ 3.0 (median 0.76)

Neonates (3–7 days)

≤ 10.6 (median 1.5)

Values expressed in mg/L. Threshold values were determined in small groups of 27–80 individuals.

Table 19.6-2 Comparison of SAA and CRP in viral infection /4/

Infection

SAA (mg/L)

CRP (mg/L)

Hepatitis A

95 (13–222)

< 10 (< 10–40)

Hepatitis B

73 (68–73)

Cytomegalovirus

147 (87–783)

28 (< 10–115)

Varicella zoster

236 (11–1,105)

< 10 (< 10–78)

Mononucleosis

265 (145–1,001)

32 (17–73)

Influenza A

980 (59–1,620)

85 (18–132)

Median values and ranges (in parentheses) for 52 patients.

Table 19.6-3 SAA in inflammatory diseases

Clinical and laboratory findings

Viral infection

In viral infections, such as meningitis caused by Coxsackie B virus, Echovirus 30, Mumps virus, Cytomegalovirus, and Herpes simplex virus type 2, the CRP concentration is usually < 10 mg/L /12/. In Parainfluenza virus and Respiratory syncytial virus infections, the concentration is < 7 mg/L /13/, in gastroenteritis caused by Rotaviruses, it is < 17 mg/L /14/, and in Influenza virus A and B infections, it can be as high as 41 mg/L /15/. Concentrations of SAA are generally higher and are a more sensitive indicator of inflammation and its progression.

Common cold

Approximately two-thirds of individuals suffering from a common cold have increased SAA but less than half have increased CRP /16/.

Autoimmune disease

In many autoimmune diseases such as inactive systemic lupus erythematosus and ulcerative colitis, neither CRP nor SAA are elevated /17/.

Malignant tumor

In malignant tumors, the SAA concentration is generally higher than that of CRP and is a useful parameter for monitoring inflammation during chemotherapy. This has been shown to be the case for colorectal carcinoma, for example /18/.

Transplant rejection

SAA is a sensitive measure for identifying transplant rejection. In a study of renal transplant recipients, 97% of rejection episodes were associated with SAA elevation. The mean SAA concentration was 690 ± 29 mg/L in irreversible rejection and 271 ± 31 mg/L in reversible rejection /19/. A diagram based on the combined measurement of SAA and urine neopterin has been shown to distinguish reliably between transplant rejection and infection /20/.

Cardiovascular disease

An SAA concentration of ≥ 10 mg/L, like a CRP concentration of ≥ 3 mg/L, is a predictor for the development cardiovascular disease when other causes of inflammation are excluded /21/.

SARS

Severe acute respiratory syndrome (SARS) is a respiratory illness in humans that is caused by the SARS Coronavirus. SAA measurement is used to distinguish between SARS and non-SARS disease. In patients with SARS, the SAA concentration is 40 times higher than the upper reference interval value, whereas in non-SARS patients, it is 85 times higher /3/. However, there is significant overlap between the concentrations in the two groups. Some investigators consider SELDI protein chip technology to be a potential tool for distinguishing between SARS and other respiratory diseases /20/, whereas others disagree /3/.

AA amyloidosis (systemic)

The main subtypes of systemic amyloidosis are primary acute light chain (AL) amyloidosis, secondary amyloid A (AA) amyloidosis, familial amyloidosis, and β2 microglobulin-related amyloidosis.

Type AA amyloidosis, also known as inflammatory amyloidosis, is a complication of chronic inflammatory conditions and is characterized by the deposit of insoluble amyloid fibrils in the affected organs and tissues. The protein AA is mainly a degradation product of the acute phase SAA and the consequence of overproduction and aberrant processing of SAA. The AA consists of the N-terminal 76 amino acids of SAA1 and SAA2. Fibrillar AA derives mostly from circulating SAA1, which dissociates from HDL before its conversion to amyloid fibrils. This process occurs through an interaction with heparan sulfate, a glycosaminglycan component of the extracellular matrix /8/. The SAA genotype is an important determinant of amyloidogenesis in patients with rheumatoid arthritis or with familial Mediterranean fever. The presence of the SAA1 allele induces higher risk of developing AA amyloidosis in Caucasians, whereas homozygosity for the SAA3 allel is related to an increase among Japanese patients /23/.

Sustained abnormally high levels of SAA in the tissues, which is usually present at low concentrations in serum, are essential for the development of AA amyloidosis.

Clinical suspicion /23/: Proteinuria in up to 95% of patients leading to nephrotic syndrome is the most frequent clinical manifestation of AA in patients with chronic inflammation. The clinical significance of hepatosplenomegaly is relatively minor in the early stages of disease. The gastrointestinal tract may be affected, causing malabsorption, intestinal pseudo-obstruction, diarrhea or bleeding. Peripheral neuropathy, restrictive myocardiopathy and skin soft tissue involvement are uncommon when compared with other types of systemic amyloidosis. Diagnosis of AA amyloidosis is confirmed based on clinical organ involvement and histological demonstration of amyloid deposits /23/.

Table 19.6-4 Various biological functions of SAA /11/

Function

SAA (μg/L)*

Chemotaxis

12.5

Induction of chemokines

10

Induction of cytokines

500

Induction of matrix degradating enzymes

100

Inhibition of the oxidative burst in neutrophils

100

Opsonin for gram-negative bacteria

1,000

Formation of ion-channels in membranes

1,000

Inhibition of hepatitis C virus entry into hepatocytes

2,000

Retinol binding protein

3,200

Induction of M2 macrophages

6,000

Role in cholesterol transport

10,000–20,000

Stimulation of angiogenesis

10,000

Suppression of antibody production

20,000

Inhibition of platelet activation and aggregation

50,000

* Minimal effective SAA concentration required to exert biological activity

Table 19.7-1 Microorganisms in polymorphonuclear neutrophil deficiency

Common

Common

Rare

Staphylococcus spp.

Klebsiella spp.

E. coli

S. marcescens

C. albicans

Pseudomonas spp.

Aspergillus spp.

Proteus spp.

Salmonella spp.

Streptococcus spp.

Table 19.7-2 Impact of antibiotics on granulocyte function

PMN
function

Increased

Decreased

Not affected

Chemo-
taxis

Trimethoprim/
Sulfa­methoxazole

Clindamycin?

Cefotaxime

Imipenem

Erythromycin

Chlortetracycline

Gentamicin

Nitrofurantoin

Doxycycline

Minocycline

Erythromycin?

Rifampicin

Fusidic acid

Quinolone

Clindamycin

Ad-
hesion

Imipenem

Oxytetracycline?

Rifampicin

Colistin

Polymyxin B

Quinine

Chloroquine

Penicillin G

Nafcillin

Cefalotin

Bacitracin

Tetracycline?

Minocycline

Doxycycline

Chloramphenicol

Erythromycin

Lincomycin

Neomycin

Streptomycin

Acetylspiramycin

Flucytosine

Primaquine

Tinidazol

Sulphioxazole

Phago-
cytosis

Perfloxacin

Azlocillin

Aztreonam

Cefsulodin

Ceftriaxone

Clindamycin

Imipenem?

Spiramycin

Ticarcillin

Nitrofurantoin

Rifampicin

Chloramphenicol

Amikacin

Gentamicin

Tetracycline

Chloroquine

Penicillin

Ampicillin

Dicloxacillin

Neomycin

Streptomycin

Lincomycin

Erythromycin

Trimethoprim/Sulfamethoxazole

Imipenem?

Quinolone

Oxygen
radical
pro-
duction

Trimethoprim/
Sulfa­methoxazole?

Clindamycin

Cefotaxime

Ceftriaxone

Enoxazin

Norfloxacin

Isoniazid

Clofazimine

Chlortetracycline

Amphotericin B

Amoxicillin

Tetracycline

Doxycycline

Trimethoprim/
Sulfa­methoxazole

Clindamycin

Fusidic acid

Rifampicin

Isoniazid

Erythromycin

Tobramycin

Imipenem

Quinolone

Cefalexin

Penicillin

Chloramphenicol

Streptomycin

Cefamandole

Metronidazole

? = questionable; contradictory data

Table 19.7-3 Medical assessment of spontaneous granulocyte motility and chemotaxis

Findings

Disorder

Inhibition of spontaneous cell motility

Cytoskeletal defect, actin dysfunction, adhesion protein deficiency

Inhibition of chemotaxis

Cytoskeletal defect, actin dysfunction, adhesion protein deficiency

Inhibition of C5a-mediated chemotaxis with normal spontaneous motility

Chemotaxis receptor defect

Inhibition of chemotaxis (of control cells also) by own serum, with normal chemotaxis for C5a and control serum

Serum inhibitor/drugs

Absent chemotactic activity in patient plasma following treatment with zymosan

Complement deficiency

Table 19.7-4 Medical assessment of phagocytosis and intracellular bacterial killing

PMN
from

Bacteria
with serum
from

Phago-
cytosis

Killing

Interpretation

Patient

Patient

+

+

Normal findings

+

Killing defect, resistant bacteria

Defective phagocytosis, if opsonization is normal

Patient

Control

+

+

Normal findings

+

Killing defect, resistant bacteria

Defective phagocytosis, if opsonization is normal

Control

Patient

+

+

Normal findings

+

Defective control cells, error in test, or resistant bacteria

Defective control cells, error in test, or defective opsonization in patient serum

Control

Control

+

+

Normal findings

+

Defective control cells, error in test, or resistant bacteria

Defective control cells, error in test, or defective opsonization in control serum

Table 19.7-5 Disorders of polymorphonuclear neutrophil (PMN) function

Primary granulocyte disorders

Chemotaxis defects*

Leukocyte adhesion deficiency syndrome (LAD)

  • LAD I (CD18/CD11 deficiency)
  • LAD II (CD15 deficiency)

Lazy leukocyte syndrome (unknown cause)

Actin dysfunction?

  • Defective actin polymerization?

Defective triggering?

  • Receptor defects? Defective signal transduction?

Bactericidal dysfunction

  • Chronic granulomatous disease (CGD)
  • Defective production of oxygen radicals: glucose-6-phosphate dehydrogenase deficiency, Cytochrome b deficiency, NADPH oxidase deficiency
  • Myeloperoxidase deficiency (defense against infection rarely affected)
  • Cyclic neutropenia (elastase deficiency)
  • Specific granule deficiency
  • CD16 deficiency (risk of infection may be increased)
  • Gray platelet syndrome

Disorders of granulocyte function in other diseases

  • Chediak-Higashi syndrome
  • Glycogen storage disease type Ib
  • Down’s syndrome
  • Diabetes mellitus
  • Mannosidosis
  • Hypergammaglobulinemia E (Job’s syndrome)
  • Schwachman-Diamond syndrome
  • Gorlin-Goltz syndrome

Transient secondary granulocyte disorders

  • Infections (e.g., Influenza A, HIV)
  • Immunosuppressive or antibiotic therapy
  • Trauma, burns
  • Malnutrition, vitamin C deficiency
  • Alcohol intoxication
  • Tumors
  • Autoantibodies

Reduced granulocyte function

  • Neonates (up to approximately 6 months)
  • Elderly (> 80 years)

* Sometimes combined with disorders of phagocytosis

Table 19.7-6 Involvement of myeloperoxidase (MPO) in PMN function /34/

Clearance of microbes by intracellular function (via production of HOCl) and extracellularly (via release of NETs).

MPO released by PMN in lymph nodes may inhibit dendritic cell activation and thus generation of adaptive T cell responses, thus attenuating organ injury.

HOCl produced outside of activated PMN following MPO release can cause significant tissue damage.

The release of MPO-containing NETs can result in the generation of autoimmunity against MPO and subsequent development of ANCA.

NETs; structures composed of de condensed chromatin, histones, and various antimicrobial substances

Figure 19.1-1 The activation of NFκB takes place through phosphorylation and subsequent proteolytic degradation of the inhibitor protein IκB by IκB kinases. Free NFκB, a heterodimer composed of a 50 kDa and a 65 kDa unit, migrates to the nucleus, where it binds to specific sites on the promoter regions of genes that are responsible for the synthesis of inflammatory proteins such as cytokines, enzymes, and adhesion molecules. p, protein; mRNA, messenger RNA. Modified from Ref. /7/.

Inflammatory gene Cellmembrane mRNA Inflammatoryproteins Cytoplasm Nucleus IkB kinases Degradation IkBa IkBa NF-kB B p50 p65 p50 p65 p50 p65 p50 p65 IkBa Signalactivation

Figure 19.1-2 Activation of the pro inflammatory cascade by lipopolysaccharide (LPS). This interacts with the LPS-binding protein (LBP) and binds to the CD14 receptor on macrophages. Via the signal pathway of NF-κB, transcription of cytokine genes takes place, followed by translation and secretion of pro inflammatory cytokines and chemokines within 15 min. In addition, anti-inflammatory cytokines that down-regulate inflammatory activity such as IL-10 and TGF-β are activated. The inflammatory reaction is modulated by positive and negative feedback (+ve; -ve). Abnormal regulation of cytokine production leads to a disproportionate inflammatory response. Modified from Ref. /36/.

LPS CD14 LPB Monocyte TNFα and IL-1 IL-6 IL-10 TGFb Chemokines +ve –ve NF-κB

Figure 19.1-3 Induction and regulation of heat shock protein (Hsp) expression. Modified from Ref. /11/. Physical or chemical stress induces the synthesis of proteins that are not folded or that are folded incorrectly. Monomeric heat shock factor in the cytoplasm forms trimers, which are phosphorylated and trans located to the nucleus. In the nucleus, they bind to Hsp promoter regions and induce the synthesis of Hsp. Hsp is typically released upon cell destruction but can also be released by certain cells such as smooth muscle cells and islet cells in response to oxidative stress alone, without cell destruction.

Physical/chemical stress Non pleated ormisfolded protein Trimerization and phosphor-ylation (↑) of the heat shockprotein (Hsp) DNA bindingof HSF trimers Heat shock protein Cytoplasm Nucleus HSBP1Hsp70

Figure 19.1-4 Hydrolysis of phospholipids by phospholipases. As a result of the action by phospholipase A2, arachidonic acid and a corresponding lysophospholipid are produced. The residue X may represent: inositol (PI, phosphatidylinositol), choline (PC, phosphatidylcholine), ethanolamine (PE, phosphoethanolamine), or serine (PS, phosphatidylserine).

Phospholipase A 1 Phospholipase A 2 Phospholipase C Phospholipase D Phospholipid Lysophospholipid Arachidonic acid Arachidonicacid residue O O O O O O O O O O O O O O O O X X C C C C P P H CH OH H 31 C 19 H 2 O CH 2 CH 2 R 1 1 CH 2 2 CH R 1 H 31 C 19 3 CH 2

Figure 19.1-5 Eicosanoid production /37/. The cyclooxygenase and lipoxygenase pathways are depicted. For abbreviations, refer to Table 19.1-2.

Membranephospholipids Phospholipases A 2 Arachidonic acid Cyclooxygenase PGG 2 Peroxidase PGH 2 PGI 2 TXA 2 PGI synthetase TX synthetase Isomerases PGs of ”2“ series Prostaglandin synthetase P LTC 4 synthetase LTD 4 LTE 4 Gamma glutamyl-transpeptidase Aminopeptidase LTB 4 LTA 4 HPETE Lipoxygenase LTA-Hydrolase LTC 4

Figure 19.1-6 Cytoplasmic membrane with receptors, G-proteins (α, β, γ) and bound phospholipase A2 (PLA2). The figure depicts the metabolic pathways into HPETE/HETE, leukotrienes (LT), and lipoxins (LX) via lipoxygenases, into prostaglandins (PG), thromboxane A2 (TXA2), and prostacyclin (PGI2) via cyclooxygenase (CO), and into PAF via lyso-PAF and acetyltransferase. 20:4-AA, arachidonic acid; O-CO-R, fatty acid in an ester bond; alkyl, fatty acid in an ether bond (with kind permission from U. Tibes).