Cytokines and cytokine receptors
The cytokine network consists of the cytokines, the cytokine receptors of the cell membrane, and a signal transduction system which transmits the information of the cytokines into the center of the cell, predominantly the nucleus.
The term cytokine is derived from the Greek words cyto (cell) and kinesis (movement) and means “moving between cells” . Cytokines are regulatory proteins of 100–200 amino acids and are produced by many types of cells of both hematopoietic and non hematopoietic lineages. Cytokines encompass those regulatory proteins secreted by T and B lymphocytes (lymphokines) monocytic cells (monokines), interferons, and hemopoietic colony stimulating factors. They act as messengers, triggering specific signals either in the same cell (endocrine) or, after being carried through the circulation, on distant target cells (paracrine) .
Cytokines exert a hormone-like effect and are usually secreted only upon stimulation of the cells. They share a number of functional characteristics, including the ability to act on many different cells (pleiotropy) and to mimic other cytokines in their activity (redundancy) . Cytokines are regulatory proteins which are involved in the regulation of inflammation, immune defense, tissue repair, contractility of the heart and vessels, maintaining body functions, and activation of apoptosis and cell death.
- The first class of cytokines have the four-helix bundle structure made up of four α-helices, arranged in an up-up-down-down topology. Four-helical cytokines have a preferential role in hematopoiesis and in innate and adaptive immunity.
- The second class of cytokines have a pleated sheet structure. These cytokines preferentially regulate the growth and differentiation of cells, but are also responsible for immune regulation (IL-1 and TNF-α).
- The cytokines of the TNF-α family have a β-jelly-roll structure (cytokines with a ring or cylindrical structure) and are usually present as trimers.
- The proteins of the IL-1 family have a β-trefoil structure.
- Only few cytokines within a structural class have more than 20–30% homology.
- An extracellular domain provides the binding site for the cytokine and creates the specificity for a particular ligand
- A transmembrane region spans the phospholipid bilayer of the plasma membrane
- The intracellular domain responsible for signal transduction has either enzymatic activity or binds other molecules, so that the signal is delivered inside the cell in response to the cytokine ligand.
Cytokines act on their target cells through receptors. All cytokine receptors are type 1 membrane proteins. The ligands bind to the extracellular portion of receptor proteins and, via di- or multimerization of receptor proteins, induce a functionally active receptor. This receptor transmits the information into the cell (e.g., the nucleus) via a signal transduction pathway.
Some of the cytokine receptors have a death domain or are decoy receptors, meaning that they bind the cytokine but do not transmit signals. Cytokines are thus removed from the circulation and can no longer perform a function.
Analogously to the structural classification of the cytokines, their receptors can also be divided into different classes.
Class I cytokine receptors
These receptors bind the four-helical cytokines. The receptor complex is formed in one of two ways:
- The receptor consists of a single protein (e.g., the gp130 of the IL-6 family). Class I receptors often form heterodimers upon ligand binding. The intracellular domain of the receptor protein triggers the signal transduction cascade.
- The receptor complex consists of a single protein (α-subunit) which, upon cytokine binding, associates with another protein unit (β-subunit). The β-subunit carries out the signal transduction. Some cytokines (IL-2, IL-7, IL-4, IL-9, IL-15 and IL-21) share a common gamma chain (γc) rather than through αβ-subunit. The ligands of the class I receptors, the four-helical cytokines, constitute a large group. It is therefore not surprising that many of these cytokines have similar functional activities.
Class II cytokine receptors
The receptors are the interferon receptor family. In contrast to class I receptors, which have two extracellular domains of 100 amino acids each, class II receptors have only one domain with 210 amino acids.
Class III cytokine receptors
Class III receptors bind cytokines of the TNF-α family. They consist of two proteins (p55, p75), both of which independently bind the ligand as a trimer and trigger an intracellular signal. Class III receptors are found on many cells and their activities partly overlap. The p55 receptor predominantly transmits inflammatory signals, while the p75 receptor transmits signals that are important for T-cell proliferation.
Class IV cytokine receptors
These receptors bind cytokines of the IL-1 family. They have three immunoglobulin-like domains. There are two receptor types: IL-1-RI and IL-1-RII. IL-1-RI relays the IL-1 signal into the cell with the aid of a co receptor (IL-1 receptor accessory protein, IL-RacP). IL-1-RII is a decoy receptor. It recruits IL-RacP into a non-signaling complex. Thus, co receptors are sequestered from the signaling pathway and IL-1 action is limited.
IL-1 receptors are related to the toll-like receptors (TLR), which are expressed on monocytes, dendritic cells and endothelial cells. TLRs are activated by binding bacterial structures (lipopolysaccharides, bacterial DNA, flagellin) and form cytokines.
Expression of cytokine receptors
The distribution and density of the cytokine receptors in the tissues determines the specificity of cytokine action. The individual tissues have only a limited spectrum of receptors that can be targeted by the cytokines. Like the production of cytokines, the receptor density is also subject to regulatory mechanisms. Receptors are often not expressed constitutively, but appear on the cell membrane only after corresponding stimulation. Some cytokines, such as IL-2, can induce the expression of their own (autologous) receptor. However, receptors can also be expressed by heterologous cytokines. For example, IL-1 also induces the expression of IL2R.
In addition to membrane-bound receptors there are sCR (sIL-2R, sIL-4R, sIL-6R). They are generated:
- Through shedding (i.e., limited proteolysis of membrane-bound proteins in the extracellular space). The cell-bound receptors thus become soluble proteins and appear in the circulation.
- Through a separate process of synthesis (alternative mRNA splicing), which directly leads to the secretion of sCR into the circulation.
Basically, sCR function by competing with the cell bound surface receptors for binding of the free cytokine molecules . As a result, sCR prevent their ligands from reaching the specific membrane receptors generating a signal, leading to inhibition of cytokine activity. An exception of this rule, however, is that receptor-bound cytokine is carried to a distant site where it enhances rather than inhibits the activity of its ligands. The soluble IL-6 receptor (sIL-6R) can interact in the presence of its ligands with its signal transducing subunit, thus generating signal (i.e., the sIL6R and gp130). The soluble receptor can also compete for the cytokine with the membrane-bound receptors, thus acting as an antagonist, as in the case of sIL-4R. SCR can be induced as a result of cell activation and thus correlate with the activity of immune-mediated diseases.
About 30% of cytokines signal through four Janus kinases and/or seven signal transducers and activators of transcription (STAT) factors. Class I and class II receptors are intracellularly associated with Janus kinases (JAKs). JAKs are tyrosine kinases. Upon cytokine binding, the JAKs are activated by dimerization of the receptor proteins, and both the JAKs and the tyrosine residues of the intracellular domain of the receptor proteins become phosphorylated. The phosphorylated receptor then becomes a docking site for STAT factors which, after dimerization and translocation into the nucleus of the cell, induce the expression of cytokine-specific genes (). Class I and class II receptors signal through STAT3.
Differentiation of cells
Cytokines act as regulatory proteins in embryo development. They prevent premature differentiation and thus maintain the pluripotency of stem cells. For example, they regulate the proliferation and differentiation of hematopoietic stem cells into mature blood cells (). In the same way, the differentiation of neuronal stem cells and cells of the germ line is regulated during embryo development.
Coordination of the immune system
When the body’s integrity is compromised in the presence of inflammation, cytokines act as mediators between the cells of the immune system and coordinate the innate and adaptive immune responses (). For example, the inflammatory response must be regulated carefully in order to prevent unnecessary tissue injury or systemic manifestation (systemic inflammatory response syndrome, SIRS).
Cytokines are usually produced transiently upon stimulation and act locally. They can act in an autocrine manner on the producing cell or cells of the same type, but can also stimulate other cell types in a paracrine manner.
Cytokines can have different effects on different cells. This phenomenon is referred to as pleiotropy. For example, IL-6 stimulates the synthesis of CRP on hepatocytes and inhibits the synthesis of hepcidin; in hematopoiesis it acts as a differentiation factor and for neuronal cells it is a proliferation factor.
Multiple cytokines can trigger the same response on a target cell and thus replace each other. This phenomenon is referred to as redundancy. One reason for this is the fact that different cytokines use the same receptor. For example, the synthesis of CRP can be activated by IL-6 as well as by oncostatin and IL-11. IL-1 and TNF-α are also redundant cytokines. They share many functions, stimulate the production of further cytokines, cause fever, and induce the proliferation of fibroblasts.
Cytokines are produced by certain cell types upon stimulation /, /. Stimulation occurs through antigens; and antigen-stimulated immune cells generate activating cytokines. For example, T-helper cells need to be activated to secrete IL-2, which has a stimulating effect on cells of the immune system. Activation of the T-helper cells occurs through the antigen and through the pro inflammatory cytokines TNF-α, IL-1 and IL-6, which are produced by monocytes. The key pro inflammatory cytokine is TNF-α, because it not only stimulates the synthesis of IL-1 and IL-6, but also inhibits the IL-4 and IL-10 cytokines, thus amplifying the pro inflammatory signals which lead to the activation of T-cells, mediated by macrophages and granulocytes ().
Under pathologic conditions, multiple cytokines are produced at the same time, and their actions in total have a protagonistic or antagonistic effect /, /. These effects induce the production of new cytokines or the up-/down regulation of the production of existing cytokines. Often a cytokine cascade develops. For example, cytokines like TNF-α or IL-1 induce the production of many more cytokines during an inflammatory response. The following basic mechanisms occur in a cytokine network:
- Synergistic or antagonistic effects
- Receptor effects
- Production of soluble receptors.
The presence of multiple cytokines can cause synergistic effects which lead to amplification of a signal. For example:
- While IFN-γ and IL-2 separately stimulate the expression von TNF-α only marginally, together they cause significant expression
- While IL-4 and IL-5 can both trigger the IgE class switch in B cells, they are markedly more effective in combination.
Negative modulation of the cytokine production can inhibit the synthesis of certain cytokines. IL-10, for example, reduces the production of tissue factor and the synthesis of IL-1 in monocytes. IFN-γ can inhibit the IL-4-induced IgE class switch, but at the same time promotes the synthesis of immunoglobulins of the class IgG2.
Cytokine specific antagonists also occur physiologically. The IL-1 system consists of the two structurally different molecules IL-1α and IL-1β, which bind to the IL-R1 receptor. The IL-1 receptor antagonist (IL-1Ra) is another ligand. The binding of IL-1Ra to an IL-1R does not trigger a signal. The receptor is blocked by the binding of IL-1Ra and is temporarily unavailable for signaling. IL-1Ra plays a role in limiting inflammatory responses. Another member of the IL-1 family, IL-18, can be blocked by a binding protein (IL-18 bp).
It depends on the antigen presented to the quiescent T cell (Th0) by the antigen-presenting cell (APC) whether a Th1 or Th2 cell subset is generated and which cytokines are produced. If a Th1 response is generated, the IL-2, IFN-γ and TNF-α cytokines are produced upon stimulation by IL-12, and the cell-mediated immune response dominates. If a Th2 response is generated, the IL-5, IL-6, IL-10 and IL-13 cytokines are produced upon stimulation by IL-4, and a humoral immune response is favored ().
The effects of cytokines are limited to certain cell populations. This occurs through regulation of the expression of their respective receptors. Since certain cell types only have or can only express receptors for certain cytokines, they can also only respond to these cytokines. Because receptors are often not expressed constitutively but only upon stimulation, the spectrum of cytokine receptors on the cell surface is limited, depending on the situation. For example, IL-2R is only synthesized by activated T cells, and IL-2 receptors are only expressed upon stimulation by IL-1 /, /.
Use of the same receptor by different cytokines
Due to the shared use of receptor populations by several cytokines, a cytokine signaling network is formed on the cell surface at the receptor level. The receptors have different ligand-binding subunits. For example, the β-subunits of the receptors of IL-3 and IL-5 are identical, and therefore both cytokines can bind to either receptor.
Effects of signal transduction
Upon binding of the cytokine to its receptor, signaling is initiated. Depending on the situation, the signals:
- Of multiple cytokines can trigger the same response. During an inflammation, many cytokines are produced, and the signaling of many cytokines focuses on a small number of intracellular signaling molecules. The response is therefore relatively uniform, because the four-helical cytokines, the interferons and the cytokines of the IL-10 family all use JAK and STAT molecules for signaling.
- Of one cytokine can trigger different responses in different target cells. For example, the TNF-α signal can trigger an inflammatory response or apoptosis. IL-1 has numerous functions in the different cell types.
Shedding has the following effects in the cytokine network:
- The ability of the cells to trigger a cell-specific signal is reduced
- The receptor-bound cytokine is transported to another site in the body where it can exert its effects. The cytokine bound to the soluble receptor can also compete with the cell membrane-bound cytokine.
- The soluble receptor/cytokine complex can bind to a new target cell with an incomplete receptor and thereby complete the receptor. This form of signaling is called trans-signaling. For example, soluble α-subunits of IL-6R which were generated by shedding form a new receptor with cell-bound gp130, thus expanding the potential spectrum of IL-6 signaling.
The action of cytokines can be assessed based on their biological effects on tissues of the body which release indicator molecules into the plasma upon cytokine stimulation (). However, no exact correlation can be made between the induction of such a molecule and the production of a specific cytokine.
Cytokines induce the production of the following indicator molecules (biomarkers), which provide the following clinical information:
- C-reactive protein (CRP); an increase in CRP is indicative of an acute-phase response triggered by inflammatory cytokines
- Neopterin; an increased concentration is indicative of IFN-γ synthesis and thus a marker of cellular immune system activation. It is therefore a useful parameter for monitoring patients with viral infections, transplants and tumors and for evaluating the efficacy of IFN-γ therapy.
- Procalcitonin; this inflammatory marker is induced by systemic action of TNF-α. Since systemic TNF-α action is limited to situations such as endotoxin invasion and especially bacterial sepsis, PCT is a sepsis marker.
- E-selectin: the plasma concentration of E-selectin is a measure of cytokine induced endothelial activation. High levels are seen in SIRS, vasculitis and heart failure.
- Soluble IL-2 receptor (sIL-2R); the plasma level of sIL-2R is a marker of cytokine induced T-cell activation. Elevated levels are an index of disease activity in sarcoidosis and are suitable for monitoring the treatment and progression of T-cell malignancies (acute T-cell leukemia).
Assaying a broad spectrum of cytokines has not gained any significance in clinical diagnostics due to the pleiotropy and redundancy of the cytokines. The diagnostic value of individual cytokines is limited to specific problems, in particular in the diagnosis of inflammation.
- Assessment of systemic inflammatory activity, infection and trauma-associated pathogeneses, transplant rejection, immune processes, and autoimmune diseases
- Prognostic marker in intensive care medicine (IL-6, IL-8, IL-10).
Plasma, serum, cerebrospinal fluid and other extravascular fluids: 1 mL
Immunoassays such as enzyme-linked immunoassays (ELISAs).
Cytokines exert their biological effects at concentrations in the picomolar range. Their biological half-life in blood and other body fluids is short; most cytokines have a half-life of minutes (with some exceptions, e.g. IL-12) . The short half-life is due to the binding to cell membrane receptors, to plasma proteins and soluble receptors, and due to the proteolytic degradation and elimination via the kidneys. Moreover, cytokine production usually only occurs for a few hours upon stimulation and is generally local, not systemic. Cytokine levels in plasma/serum and cerebrospinal fluid can be analyzed without problems. To determine the cytokine concentration in broncho alveolar lavage fluid, constant lavage volumes are required.
In routine diagnostics, cytokine assays have become established only in intensive care and transplantation.
One major challenge in intensive care is the early detection of the progression of local inflammation (infection- or trauma-induced) to SIRS. This development is accompanied by an increase in plasma levels of the pro inflammatory cytokines TNF-α, IL-1, IL-6 and IL-8. High concentrations of IL-6 and IL-8 are associated with SIRS, persistently high concentrations with a poor prognosis .
To enable early diagnosis of immunological complications following organ transplantation, IL-6, IFN-γ and TNF-α are analyzed to detect rejection episodes. The diagnostic sensitivity is 90–95% at a specificity of 40–60% .
The cytokine concentration increases upon activation of immune cells following blood coagulation and contact with the syringe material. It is therefore advisable to determine cytokine levels in plasma (heparinized, EDTA). The plasma should be separated from the blood cells within 4 h. Until then, samples should be refrigerated. Some tests are influenced by anticoagulants (especially EDTA). In general, the test kit manufacturers’ instructions must be followed.
Method of determination
Tests from different manufacturers often give different results for the same cytokine, although they were calibrated to international cytokine standards (NIH/WHO standards). This variation is due to the different epitope specificities and affinities of the antibodies used in the test kits.
Many cytokines are biologically active only as dimers or trimers. However, some ELISA assays also detect the biologically inactive monomers or proteolytic cleavage products. This is not necessarily a problem for diagnosis, since the in vivo half-life of the biologically active cytokine is so short that the cytokine often escapes detection. Cleavage products, however, remain detectable for a much longer period of time after the temporary release of cytokines, and thus their presence provides information on the history of cytokine release within the past hours or even days. This has been investigated thoroughly especially for the detection of TNF-α .
Since the detection limit of the immunoassays is less than 10 pg/mL, or in some cases even less than 1 pg/mL, cytokine concentrations are detectable even in the plasma/serum of healthy individuals.
IL-8 binds to the Duffy receptor of erythrocytes. Hemolysis causes the release of large amounts of IL-8 and leads to falsely high plasma/serum IL-8 levels.
The measurement should be performed within 4 h after blood collection. Cytokines in serum/plasma are stable for several years when stored at –70 °C.
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25. Hör S, Pirzer H, Dumoutier L, Bauer F, Wittmann S, Sticht H, et al. The T-cell lymphokine interleukin-26 targets epithelial cells through the interleukin-20 receptor 1 and interleukin-10 receptor 2 chains. J Biol Chem 2004; 279: 33343–51.
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IL-6 is a multifunction cytokine that has a wide range of biological activities in various target cells and regulates immune response, acute phase reactions, hematopoiesis, and bone metabolism /, , /. IL-6 signaling is mediated by a IL-6 receptor. The IL-6 receptor consists of two functional membrane proteins:
- An 80 kDa ligand-binding chain, known as IL-6R alpha chain or CD126
- A 130 kDa non-ligand binding signal transducing chain, known as glycoprotein 130 (gp130), IL-6R beta chain or CD130.
In cells with sufficient membrane-bound IL-6R, IL-6 binds to these receptors, the IL-6/IL-6-R complex induces homo
In cells that do not express sufficient cell-surface IL-6R, soluble form of IL-6R, Il-6 signal transduction starts with the binding of IL-6 to the free soluble form of IL-6R (sIL-6R), which lacks the membrane and intracytoplasmic portion of the 89 kDa membrane-bound IL-6R molecule. Thus, either membrane bound or soluble IL-6R can mediate IL-6 signal into cells, as long as they express gp130.
Because IL-6 plays important physiologic roles, deregulated overproduction of IL-6 causes various pathologic conditions, including autoimmune, inflammatory and lymphoproliferative disorders. Toclizimab binds to the IL-6 binding site of IL-6R and competitively inhibits IL-6 signalling. Toclizimab is therapeutically effective in rheumatoid arthritis, juvenile rheumatoid arthritis and Castleman disease.
- Diagnostic and prognostic parameter in trauma, SIRS, sepsis and critically ill patients
- Suspected early onset bacterial infection
- After isolated traumatic brain injury
- Monitoring of ARDS and mechanical ventilation.
Immunoassay, predominantly ELISA.
Plasma, serum, cerebrospinal fluid, bronchoalveolar lavage fluid: 0.5–1 mL
Plasma: less than 10 ng/L
IL-6 is a multifunction cytokine that has a wide range of biological activities in various target cells and regulates immune response, acute phase reactions, hematopoiesis, and bone metabolism. IL-6 signaling is mediated by a IL-6 receptor. system consisting of two functional membrane proteins /, /:
- An 80 kDa ligand-binding chain, known as IL-6R alpha chain or CD126
- A 130 kDa non-ligand binding signal transducing chain, known as glycoprotein 1130, IL-6R beta chain or CD130.
IL-6 is also involved in:
- Anemia of chronic disease. IL-6 stimulates hepcidin production and causes functional iron deficiency (). Functional iron deficiency is a state of iron-poor erythropoiesis, in which there is insufficient mobilization of iron from the stores in the presence of increased demands. Hepcidin inhibits the iron transporter ferroportin on gut cells and iron release from storage cells (hepatocytes and reticuloendothelial cells), thus reducing iron level in serum .
- IL-6 enhances zinc importer ZIP14 expression on hepatocytes and so induces hypozincemia seen in inflammation
- In the bone marrow IL-6 promotes megakaryocyte maturation, thus leading to release of thrombocytes
- IL-6 promotes differentiation of naive CD4+Tcells linking the innate to acquired immune response.
- When IL-6 is generated in the bone marrow stromal cells, it stimulates RANKL, which is indispensable for the differentiation and activation of osteoclasts ().
Elevated levels can be detected as early as 24–48 hours before clinical symptoms (e.g., fever) appear. In the early phase of inflammation, IL-6 is superior to other inflammation markers such as leukocyte count, CRP and procalcitonin. In systemic infections, immune cells such as monocytes/macrophages and dendritic cells, through their toll-like receptors, detect molecular structures of Gram-positive bacteria (pathogen-associated molecular patterns, PAMPS) and the lipopolysaccharide of gram-negative bacteria. Immune cells produce IL-6, mediated by TNF-α. IL-6 rises after 1 h and can be elevated for up to 48 h ().
Non-immune cells such as endothelial cells, keratinocytes and fibroblasts also are stimulated directly by non-infectious factors such as hypoxia and lead to long-term (24–72 h) production of IL-6. An elevated IL-6 level can therefore be of infectious or non-infectious origin. High IL-6 levels with persistently low TNF-α is indicative of hypoxia and non-infectious factors rather than an infection .
While in complication-free inflammation due to tissue injury (postoperative) IL-6 decreases rapidly, persistent high levels indicate an infectious complication. The concentrations and behavior of IL-6 depend on the severity and cause of the inflammation. The relative change in the IL-6 concentration during the inflammation is diagnostically significant.
There are no clear IL-6 thresholds for the cause and extent of an acute inflammation because, due to the phased release of anti-inflammatory cytokines, the release of IL-6 is subject to considerable inter individual variation. IL-6 is therefore of significance mainly in individual monitoring. However, the following approximate thresholds apply in general:
- Levels < 10 ng/L exclude acute inflammation
- Concentrations ≤ 150 ng/L are due to local infections such as pyelonephritis, pneumonia or abscess
- Levels > 150 ng/L are indicative of systemic inflammation (SIRS, sepsis)
- Concentrations ≥ 1,000 ng/L characterize high-risk patients with severe sepsis, especially if levels persist for more than 3 days.
Serial measurements are more informative than single measurements, except in the case of neonatal sepsis and extremely high IL-6 levels after trauma, in SIRS and sepsis. They are associated with an unfavorable prognosis.
During the so-called cytokine storm a potentially fatal immune reaction induced by hyper activation of T cells, a major boost in IL-6 production is observed without comparable production of other inflammatory cytokines.
A valuable indicator of neonatal sepsis is a markedly elevated concentration of IL-6 in cord blood. This also applies to high plasma concentrations of IL-6 in critically ill children with suspected SIRS and sepsis. Elevations in CRP will not become clearly detectable until 24–36 h later. The measurement of IL-6 generally allows earlier intervention than other markers of inflammation.
The significance of IL-6 for diagnosing the activity of chronic inflammation is less obvious, since it has few advantages over indirect inflammatory biomarkers that remain elevated for a prolonged period of time, such as CRP.
Isolated elevated levels of IL-6 are indicative of the activation of non-immunological cells, especially if the elevation persists for several days. The elevation can result from a direct interaction between bacteria and their products (LPS) and endothelial cells and keratinocytes.
Tissue hypoxia and trauma also cause the release of IL-6 from non-immunological cells. The IL-6 concentration is therefore a good marker for assessing the extent of organ damage in SIRS and peripheral hypoxia. This explains why measurement of IL-6 is indicated especially in critical care.
Other conditions associated with elevated IL-6 levels include pregnancy and ovarial hyper stimulation syndrome (OHSS). In pregnancy, IL-6 rises continuously during the first and second trimester. Levels in plasma average 50 ng/L, in amniotic fluid they are 100 times higher. In OHSS, levels in plasma are above 100 ng/L, and in peritoneal fluid or ascites they are 20–50 times higher .
During sterile surgical operations, an increase in serum IL-6 concentration precedes elevation of body temperature and serum acute phase protein concentration.
Method of determination
One reason for the variation in the results obtained with assays from different manufacturers is that IL-6 can exist as monomer, dimer or multimer or bound to other proteins such as CRP or soluble IL-6 receptor. It is questionable whether an assay detects only the unbound IL-6 or an additional portion of the IL-6 spectrum, and if so, what portion.
Antibody therapies (OKT 3, ATG) lead to elevated levels and thus false-positive results. Elevated concentrations with no obvious clinical relevance are also seen in the first 2–3 days after surgery.
Plasma and cells should be separated within 4 h. Stable for 1 day if stored at –20 °C (recommended temperature) or for 1 week if stored at –70 °C. Similar recommendations apply to samples from other body fluids.
Biological effects of IL-6
The IL-6 cytokine family includes IL-11, IL-27, IL-31, oncostatin (OSM), leukemia inhibitory factor (LIF) cardiotrophin-1 (CT-1) cardiotrophin-like cytokine CLC) , neuropoietin/cardiotrophin2 (NP) and CTNF.
IL-6 is a glycosylated protein of 21–28 kDa and has the typical four-helix bundle structure of all IL-6 type cytokines. The gene IL-6 is located on chromosome 7p21 and has glucocorticoid responsive elements. Elevation of the glucocorticoid concentration due to disease-related etiology or treatment with glucocorticoids suppresses the production of IL-6 and thus of CRP.
IL-6 is produced by a wide variety of immune cells and non immune cell types, especially monocytes/macrophages, dendritic cells, lymphocytes, endothelial cells, fibroblasts, and muscle cells. IL-6 targets multiple cell types and induces a broad array of responses which are often classified as pro- or anti-inflammatory in nature. A key function is mediation of the acute-phase response. However, IL-6 has further activities. For example, IL-6 is a major player in the immune system, where it is involved in the maturation of immune cells, activation of T cells, and the production of immunoglobulins by B cells. IL-6 activates mitogen stimulated T cells by inducing IL-2 production and IL-2 receptor expression and acts synergistically with IL-2 in propelling T cell differentiation into cytotoxic lymphocytes (see ). It also stimulates endothelial cells to produce chemokines and adhesion molecules, thus recruiting leukocytes to the site of inflammation .
- Th1 cells produce large quantities of interferon gamma, induce delayed hypersensitivity reactions and are essential for the defense against intracellular pathogens.
- TH2 cells produce mainly IL-4 and are important in including IgE production, recruiting eosinophils to sites of inflammation and helping to clear parasitic infections.
- Th17 constitute a distinct lineage and produce IL-17 but not interferon gamma or IL-4. IL-17 induces the production of IL-1 and TNF alpha.
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IL-8 is a member of the CXC family of cytokines. While it is produced by many cell types, leukocytes are the major source of secretion. IL-8 is stimulated by many different signals, including bacterial lipopolysaccharide or other pro-inflammatory signals. IL-8 plays a critical role in the immune response of the host by mediating the recruitment of neutrophils and monocytes to the sites of inflammation. IL-8 is associated with several systemic inflammatory diseases .
Hemolysis significantly influences the concentration in plasma. The total concentration of IL-8 in the blood is determined in hemolysate.
- Suspected early onset bacterial infection
- Prognostic parameter in trauma, SIRS and sepsis
- Prognostic significance of BAL levels for the early diagnosis of acute respiratory distress syndrome (ARDS) following trauma or burns.
Immunoassay, predominantly ELISA.
- Plasma: 0.1–1 mL
- EDTA blood: 0.05–1 mL
The hemolysate is prepared in the laboratory by mixing 0.05 mL of whole blood with 0.05 mL of hemolysis solution.
- Bronchoalveolar lavage (BAL) fluid: 5 mL
IL-8 is a marker of immune cell activation and indicates acute-phase response of various etiology. Like elevations of IL-6, increased levels of IL-8 have little diagnostic value with regard to a differential diagnosis. Serial measurements are more informative than single measurements, except in the case of neonatal sepsis and high IL-8 levels in BAL fluid in the early stages of ARDS.
Isolated high IL-8 levels with non-elevated or only mildly elevated TNF-α but combined with high levels of IL-6, are indicative of the activation of non-immunological cells, especially if the elevation persists for several days. Such elevations can be due to tissue hypoxia or a malignant tumor.
The significance of IL-8 for diagnosing the activity of chronic inflammation or organic diseases such as thyroid disease and chronic liver disease is less obvious. lists examples of diseases and conditions in which the measurement of IL-8 is or could become of diagnostic value.
Method of determination
Mild hemolysis leads to elevated IL-8 if the concentration is determined in plasma or serum. Moreover, IL-8 level is positively correlated with the hematocrit when measured in plasma, but not when measured in hemolysate .
Plasma and cells should be separated within 4 h. Stable for 1 day if stored at –20 °C (recommended temperature) or for 1 week if stored at –70 °C. Similar recommendations apply to samples from other body fluids.
IL-8 is a small protein (8.4 kDa) and produced by macrophages, endothelial cells and neutrophils. It belongs to the family of CXC chemokines and signals through the two cell membrane receptors CXCR1 and CXCR2. IL-8 is a neutrophil-specific chemoattractant and is involved in angiogenesis, cell proliferation and apoptosis. Increased expression of IL-8 and its receptors is measured in leukocyte tissue infiltration, in tumor-associated macrophages, and in cancer cells.
IL-8 is regulated analogously to IL-6, is secreted within 1–3 h of endotoxin invasion, and is immediately bound to two distinct high-affinity IL-8 receptors that are present on neutrophils. Its half-life is less than 4 h.
Only a small portion of the IL-8 in plasma is free IL-8, since 97% are bound to cellular receptors. Cell association is enhanced by chemokine binding and non IL-8-specific receptors. These are on various cell types such as Duffy antigen related chemokine receptors (DARC) . DARC-bound IL-8 is biologically inactive towards neutrophils, but can be released again by pathogens or due to replacement by other cytokines. Approximately 85% of IL-8 in the blood is bound to erythrocytes.
2. Neunhoeffer F, Lipponer D, Eichner M, Poets CF, Wacker A, Orlikowsky TW. Influence of gestational age, Cesarean section and hematocrit on interleukin-8 concentrations in plasma and detergent-lysed whole blood of non-infected newborns. Transfus Med Hemother 2011; 38: 183–9.
4. Orlikowsky TW, Neunhoeffer F, Goelz R, Eichner M, Henkel C, Zwirner M, et al. Evaluation of IL-8-concentrations in plasma and lysed EDTA-blood in healthy neonates and those with suspected early onset bacterial infection. Pediatr Res 2004; 56: 804–9.
5. Krueger M, Nauck MS, Sang S, Hentschel R, Wieland H, Berner R. Cord blood levels of interleukin-6 and interleukin-8 for the immediate diagnosis of early-onset infection in premature infants. Biol Neonate 2001; 80: 118–23.
8. Zimmermann HW, Seidler S, Gassler N, Nattermann J, Luedde T, Trautwein C, Tacke F. Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis. Plos One 2011; 6 (6) e21381
9. Pine SR, Mechanic LE, Enewold L, Chaturyedi AK, Katki HA, Bowman ED, et al. Increased levels of circulating interleukin 6, interleukin 8, C-reactive protein, and risk of lung cancer. J Natl Cancer Inst 2011; 103: 1112–22.
10. Sun L,Mao D, Cai Y, Tan W, Hao Y, Li L, Liu W. Association between higher expression of interleukin-8 (IL-8) and haplotype 353A/-251A/+678T of IL-8 gene with preeclampsia- Medicine 2016; 95: 52 (e5537).
TNF-α is a cytotoxic cytokine that induces apoptotic cell death by interacting with its membrane bound receptor TNFR1. TNF-α binding to TNFR1 allows TNF receptor 1 associated death domain (TRADD) to interact specifically with the death domain of TNFR1 and serve as a common platform for activating various signal molecules . TNF-α has a significant role in cell apoptosis.
- Systemic inflammatory response symptoms in cases of sepsis and trauma
- Diagnosis of the activity of chronic inflammatory processes (e.g., in rheumatoid arthritis)
- Multiple sclerosis (measurement in cerebrospinal fluid)
- Differentiation of bacterial meningitis from other forms of meningitis (measurement in cerebrospinal fluid).
ELISA or luminescence immunoassay. These assays use two antibodies which are directed against different epitopes of TNF-α. There are commercial tests which measure only bioactive TNF-α (sTNF) and tests which quantify total TNF-α (sTNF and tmTNF).
- Heparinized or EDTA plasma, serum: 1 mL
- CSF: 0.5 mL
If released systemically in large amounts, TNF-α activates neutrophils, induces the release of other inflammatory cytokines, and modifies the anticoagulant properties of endothelial cells.
Excessive production of TNF-α leads to severe inflammatory reactions, tissue injury, and shock.
Chronic mildly increased production of TNF-α contributes to chronic inflammation, fever, anemia and neurological diseases. In patients with heart failure, elevated TNF-α concentrations characterize a subgroup with marked cachexia and a considerably worse prognosis . An elevated concentration of TNF-α does not allow any differential diagnostic conclusions to be drawn, but is merely a marker of an inflammatory response of various etiology.
Upon acute activation, TNF-α is secreted only for a short period of time (< 6 h). If secreted by activated mast cells, it is released all at once. The half-life in plasma is < 5 min. If bioactive TNF-α homo monomer (sTNF) is detected, it must have been produced within the last 4–6 hours. tmTNF is often still detectable after 24 h.
The two markers (sTNF, tmTNF) determined with different assays thus provide different information:
- The increase of sTNF indicates that a systemic inflammatory response has just occurred or is occurring
- The increase of tmTNF alone indicates that an inflammatory reaction has occurred in the last 1–2 days.
Diseases and conditions associated with elevated TNF-α concentrations are shown in:
Method of determination
The presence of soluble TNF-α receptors has a variable effect on the ability of the various assays to detect TNF-α. Antigenic TNF-α concentrations in the presence of soluble receptors will be influenced by antibody properties and epitope location (i.e., near or distant from the receptor binding site). If both detection and capture antibodies recognize epitopes outside the receptor binding site, total TNF-α, whether complexed with its soluble receptors or not, will be measured. If, on the other hand, one or both antibodies interact with the receptor binding site, the antibody and receptor will compete for TNF-α binding .
TNF-α is member of a super family of cytokines with well characterized roles in cell survival, differentiation, apoptosis, and inflammatory response /, /.
TNF-α is synthesized as a monomeric type 2 transmembrane pro-protein (tmTNF) of 233 amino acids and has a molecular weight of 26 kDa. The cytoplasmic tail of tmTNF is then cleaved by the TNF-α converting enzyme (TACE), a metalloprotease, releasing the molecule as a 17 kDa soluble or circulating cytokine (sTNF). For signaling, the sTNF and tmTNF monomers must aggregate into groups of three, forming homotrimers . The gene encoding TNF-α is located within the MHC on chromosome 6 between 6p21.1 and 6p21.3.
Immune cells, in particular macrophages, but also dendritic cells, NK cells, T and B cells, microglia, astrocytes and certain neuronal cells produce both molecular forms of TNF-α (sTNF and tmTNF) upon activation. Both forms are biologically active, and their ratio in blood depends on the type of cell, its activation status and the stimulus which triggers the production of TNF-α.
TNF-α exerts pleiotropic functions in immunity, inflammation, cell proliferation and apoptosis.
The receptors of the TNF family are transmembrane proteins that consist of two identical subunits /, /. TNF-α (sTNF and tmTNF) interact with two structurally related but functionally different receptors (TNFR), TNFR1 (p55; CD120a) and TNFR2 (p75; CD120b).The trimeric structure of these receptors is similar to that of active TNF-α. TNFR1 and TNFR2 are type 1 transmembrane glycoproteins characterized by cysteine-rich extracellular domains, which can be proteolytically cleaved to generate soluble receptor fragments that may function as natural TNF-α antagonists . Refer to ).
TNFR1 is constitutively expressed by nearly all body cells except erythrocytes and contains a 60-amino acid cytoplasmic sequence known as the death domain that couples this receptor to either of two distinct signaling pathways. sTNFs preferentially bind to TNFR1.
TNFR2 is generally inducible and is produced by endothelial cells, immune cells and some neuron populations. Signaling through this receptor activates pro inflammatory signals and survival signals. TNFR2 does not have a death domain and therefore does not activate apoptosis-inducing caspases. TmTNFs preferentially bind to TNFR2.
The principal signaling pathway leads to the activation of nuclear factor kappa-B and subsequently transcription of pro inflammatory and antiapoptotic genes, whereas a distinct pathway will lead to apoptosis via activation of caspases 8 and 3 (programmed cell death) .
3. Rauchhaus M, Doehner W, Darrel PF, Davos C, Kemp M, Liebenthal C, Niebauer J, et al. Plasma cytokine parameters and mortality in patients with chronic heart failure. Circulation. 2000; 102: 3060–7.
7. De Bont ESJM, Martens A, van Raan J, Samson G, Fetter WPF, Okken A, et al. Diagnostic value of plasma levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in newborns with sepsis. Acta Paediatr 1994; 83: 696–9.
10. Aukrust P, Liabakk NB, Müller F, Lien E, Espevik T, Froland SS. Serum levels of tumor necrosis factor-α (TNFα) and soluble TNF receptors in human immunodeficiency virus type 1 infection – correlations to clinical, immunologic, and virologic parameters. J Infect Dis 1994; 169: 420–4.
11. Dörge SE, Roux-Lombard P, Dayer JM, Koch KM, Frei U, Lonnemann G. Plasma levels of tumor necrosis factor (TNF) and soluble TNF receptors in kidney transplant recipients. Transplantation 1994; 58: 1000–8.
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13. Glimaker M, Kragsbjerg P, Forsgren M, Olcen P. Tumor necrosis factor-α (TNFα) in cerebrospinal fluid from patients with meningitis of different etiologies: high levels of TNFα indicate bacterial meningitis. J Infect Dis 1993; 167: 882–9.
Following activation, T cells release a soluble form of IL-2R (sIL-2R) and a number of cytokines such as IL-2 and IL-4, which are involved in amplifying and modulating immune cell networks and inducing B cell proliferation.
Elevated serum levels of sIL-2R are present in various autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus or Kawasaki disease, among transplant recipients who subsequently reject their graft and in individuals with viral infections, including HIV and hepatitis, or hematological and lung tumors, as well as pre cancer and cancer of the uterine cervix /, , /.
In most cases IL-2R is determined using immunoassays. The immunoassay employs antibodies against the α-chain of the sIL-2R. These tests are named IL2Rα assays.
- Monitoring organ transplantation (early detection of complications such as rejection reaction or infection)
- Assessment of disease activity in sarcoidosis and autoimmune diseases
- Assessment of disease activity in lymphoproliferative diseases (T- and B cell neoplasms).
Enzyme-linked immunoassay. Sandwich enzyme immunoassays employ two monoclonal antibodies to IL-2Rα.
- Heparinized plasma, serum: 1 mL
- Cerebrospinal fluid: 0.5 mL
1 U corresponds to 3.3 pg.
Elevated concentrations of sIL-2R are an indicator of an active cell-mediated immune response. The measurement of sIL-2R levels in plasma/serum is a valuable tool for diagnosing disease activity in certain diseases. Significant elevations are found in transplant rejection, autoimmune diseases, neoplasias, infections, treatment with IL-2, and in end-stage renal disease () . An elevated sIL-2R concentration does not allow any differential diagnostic conclusions to be drawn. It is merely a marker of cellular immune activation of various etiology.
sIL-2R is a useful marker in clinical diagnostics:
- In the non-invasive diagnosis of disease activity in patients with sarcoidosis, where it is a more reliable marker than angiotensin-converting enzyme (see )
- As an early warning sign of complications after organ transplantation, although it does not provide any information on the type of complication (infection, rejection)
- A good parameter for monitoring (detection of relapse) IL-2R-positive T-cell neoplasias.
Single measurements are useful only to a limited extent, often only serial measurements are informative.
Method of determination
All assays measure the α-chain of the IL-2R, regardless of whether they are called sIL-2R or sIL-2Rα. The results obtained with different commercial assays show acceptable comparability.
In children up to age 6, sIL-2Rα concentrations are higher than those of adults before declining significantly up to age 17 to reach adult levels thereafter . Elderly individuals tend to have higher levels than young adults.
Stable for 1 day if stored at –20 °C (recommended temperature).
IL-2 is a marker of T-cell activation. It is produced predominantly by activated CD4+T cells (), but can also be produced by non-stimulated CD8+T cells, dendritic cells and thymus cells. IL-2 is a cytokine with immunomodulatory activities which are important for maintaining immune function. It stimulates the proliferation and expansion of antigen-specific T cells in an autocrine (CD4+T cells) and paracrine fashion (CD8+T cells, B cells, NK cells, lymphokine activated cells, neutrophils, monocytes, γ/δ T cells). This also leads to the production of further cytokines.
IL-2 also plays an important role in promoting the survival of T-regulatory cells (CD4+CD25+ Tregs), which are responsible for maintaining self-tolerance. A lack of IL-2 action reduces the supportive effect of IL-2 on Treg cells, resulting in an immune-enhancing effect with proliferation of T and B cells and the development of autoantibodies . Treg cells are activated by IL-2 via the IL-2R.
Membrane-bound interleukin-2 receptor (IL-2R)
- The receptor consists of the obligate signaling subunits:
- β-chain (IL2Rβ; CD122; MW 75 kDa)
- γ-chain (IL2Rγ; CD132; MW 64 kDa)
- The variable expressed α-chain (IL2Rα; CD25; MW 55 kDa) which regulates the affinity for IL-2.
The β- and γ-subunits of the IL-2R belong to the hematopoietin super family. The β-chain is also shared by the IL-15R, and the γ-chain by IL-4R, IL-7R, IL-9R and IL-15R.
The complete receptor is a trimer consisting of the α-, β- and γ-chains. The β- and γ-chains are constitutively expressed by the lymphocytes. They have long intracytoplasmic domains. The α-chain, which is identical with the IL-2Rα, is inducible upon activation by IL-2. Since the IL-2Rα has only a short intracytoplasmic domain, it does not participate in signaling. Upon binding of IL-2 to IL2-Rα, the latter associates with the β-chain and γ-chain to form a signaling receptor.
Signaling begins upon oligomerization of the receptor subunits. The intracellular portions of the receptor chains associate with a variety of cytoplasmic proteins, including tyrosine kinases of the Jak family. Oligomerization of the receptor subunits brings these regulatory enzymes into close proximity, activating the signaling complex by phosphorylation of regulatory tyrosines on the kinases themselves, as well as on the cytoplasmic domains of IL2Rβ and IL2Rα .
Only 5% of the non-activated T cells in the blood express IL-2Rα. However, antigenic or mitogenic stimulation leads to expression of the receptor after 4–8 h, and after 48–96 h the T cell has 30–60 thousand IL-2Rα. The number then progressively decreases by 80–90% within 10–21 days of activation .
Soluble interleukin-2 receptor (sIL-2R)
The sIL-2R (CD25) is enzymatically cleaved from the receptor of expressing cells. It is a monomer with a MW of 45 kDa. sIL-2Rα shedding is proportional to the level of expression at the cell surface. It is excreted and catabolized by the kidneys and has a half-life of 0.62 hours. In renal insufficiency, the plasma concentration of sIL2Rα is increased .
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11. Weimann A, Eisele RE, Pawellek S, Hippler-Benscheid M, Rüggenberg A, Cammann H, et al. Diagnostic value of peripheral blood markers for acute rejection in liver-transplanted patients receiving anti IL-2R antibodies. J Lab Med 2008; 32: 148–57.
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14. Ambrosetti A, Nadali G, Vinante F, Ricetti MM, Todeschini G, Morosato L, de Sabata D, et al. Soluble interleukin-2 receptor in hairy-cell leukemia: a reliable marker of disease Int J Clin Lab Res 1993; 23: 34–7.
18. Tebib JG, Letroublon MC, Noel E, Bienvenu J, Bouvier M. sIL-2R levels in rheumatoid arthritis: poor correlation with clinical activity is due in part to disease duration. Br J Rheumatol 1995; 34: 1037–40.
19. Sivieri S, Ferrarini AM, Gallo P. Multiple sclerosis: IL-2 and sIL-2R levels in cerebrospinal fluid and serum. Review of literature and critical analysis of ELISA pitfalls. Multiple Sclerosis 1998; 4: 7–11.
T, T lymphocytes; B, B lymphocytes: G, granulocytes; N, neutrophils, Baso, basophils; Eos, eosinophils; M, monocytes; Mega, megakaryocytes; Mast, Mast cells; NK, natural killer cells; D, dendritic cells; O, osteoblasts; K, Keratinocytes; E, endothelial cells
Clinical and laboratory findings
Clinical and laboratory findings
Clinical and laboratory findings
Clinical and laboratory findings
Values expressed as x ± s; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate
Figure 20.1-1 The receptor complex for the transduction of the IL-6 signal consists of the receptor protein IL-6Rα and the signal transducer protein gp130. On target cells, IL-6 first binds to the α-chain of IL-6R. The IL-6-IL-6Rα complex then associates with two gp130 molecules and induces signaling, which basically occurs through activation of the two signaling pathways JAK/STAT and SHP2/Gab/MAPK. In each case gp130 is activated via its activation regions YYxxQ and Y759. The resulting signal complexes are activated by phosphorylation of both the JAK tyrosine kinases themselves and the tyrosines (Y) of the cytoplasmic domains of gp130. Modified according to Ref. .
Figure 20.1-2 Regulation of hematopoiesis by cytokines. Modified according to Ref. . The proliferation of the hematopoietic cell lines starts from the pluripotent stem cell. The differentiation is regulated by the cytokines. Flt-3L, Flt-3 ligand; SCF, stem-cell factor; gp130, glycoprotein 130; GM-CSF, granulocyte-macrophage-colony-stimulating factor; G-CSF, granulocyte-colony-stimulating factor; M-CSF, macrophage-colony-stimulating factor; EPO, erythropoietin, TPO, thrombopoietin
Figure 20.1-3 Function of cytokines in innate and adaptive immunity, modified according to Ref. . After antigen presentation to the quiescent T-helper (Th) cell by the macrophage MHC complex, the antigen-specific T-cell receptor of the Th cell is activated. The Th cell produces a multitude of cytokines, thereby activating the mechanisms of adaptive immunity.
Figure 20.2-2 Cytokine activated transformation of naive T-helper cells (Th0) into different CD4+T-cell subtypes . Treg, regulatory T cell; Foxp3 is a master regulator in the regulation of Treg cells; GATA-3, transcription factor which promotes the development of Th2 cells and the production of IL-4, IL-5 and IL-13; RORγt controls the genes for expression of IL-17; T-bet is a transcription factor which controls the genes of Th1 cells for the production of IFN-γ.
Figure 20.4-1 Synthesis of TNF-α and action with target cells. Modified with kind permission according to Ref. . TNF-α is synthesized as a monomeric transmembrane precursor protein (tmTNF). The cytoplasmic tail is cleaved by the TNF-α converting enzyme (TACE) releasing the molecule as a soluble cytokine (sTNF). To exhibit their biological functions, sTNF and tmTNF monomers must first form heterotrimers. sTNF and tmTNF interact with different types of heterotrimer transmembrane receptors (TNFR) to induce apoptosis or cell activation and survival.
Figure 20.5-1 A model signaling by IL-2 receptor. Of the 3 peptide chains α, β and γ, the latter two have domains extending into the cytoplasm and relay the signal after IL-2 binding to the γ-chain. The tyrosine residues, which are phosphorylated by the JAK kinases, are labeled Y (see figure on left). The tyrosine kinases and signal transducers associated with the receptor are shown on the right. Syk Lck, FYn and Lyn are kinases. Modified from Ref. .