20

Cytokines and cytokine receptors

20

Cytokines and cytokine receptors

20

Cytokines and cytokine receptors

20

Cytokines and cytokine receptors

20.1 Definition, classification, structure and function of cytokines

Lothar Thomas

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.

20.1.1 Cytokines

The term cytokine is derived from the Greek words cyto (cell) and kinesis (movement) and means “moving between cells” /1/. 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) /2/.

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) /2/. 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.

Cytokines are classified based on their first described biological function (Tab. 20.1-1 – Functional classification of cytokines) as well as based on structural features (Tab. 20.1-2 – Classification of cytokines based on structural features):

  • 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.

20.1.2 Cytokine receptors

A cytokine receptor consists of at least three parts /13/:

  • 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.

Refer to Fig. 15.10-6 – Homodimer erythropoietin receptor.

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.

20.1.2.1 Soluble cytokine receptors (sCR)

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 /2/. 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.

20.1.3 Signal transduction

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 (Fig. 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). Class I and class II receptors signal through STAT3.

20.1.4 Function of cytokines

Cytokines play an important role in the differentiation of cells and in regulating and coordinating the immune system /12/.

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 (Fig. 20.1-2 – Regulation of hematopoiesis by cytokines). 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 (Fig. 20.1-3 – Function of cytokines in innate and adaptive immunity). 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.

Pleiotropy

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.

Redundancy

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.

20.1.5 Synthesis and mechanism of action of cytokines

Cytokines are produced by certain cell types upon stimulation /12/. 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 (Fig. 20.1-3 – Function of cytokines in innate and adaptive immunity).

20.1.6 Cytokine network

Under pathologic conditions, multiple cytokines are produced at the same time, and their actions in total have a protagonistic or antagonistic effect /12/. 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.

20.1.6.1 Synergistic and antagonistic effects

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 (Fig. 20.1-4 – Development of the subpopulations of T-helper cells (Th1 and Th2) under the influence of IL-4 and IL-12).

20.1.6.2 Receptor effects

Receptor expression

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 /12/.

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

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.

20.1.7 Cytokine action

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 (Tab. 20.1-3 – Characteristics and function of 30 human cytokines). 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).

20.1.8 Diagnostic value of cytokines

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.

An overview of established diagnostic indications for assaying cytokines is provided in Tab. 20.1-4 – Indications for the determination of cytokines and cytokine receptors.

20.1.8.1 Indication

  • 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).

20.1.8.2 Specimen

Plasma, serum, cerebrospinal fluid and other extravascular fluids: 1 mL

20.1.8.3 Method of determination

Immunoassays such as enzyme-linked immunoassays (ELISAs).

20.1.8.4 Clinical significance

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) /4/. 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 /5/.

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% /6/.

20.1.8.5 Comments and problems

Blood sampling

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 /6/.

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.

Hemolysis

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.

Stability

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.

References

1. Schooltink H, Rose-John St. Das Zytokinnetzwerk. Bundesgesundheitsbl, Gesundheitsforsch- Gesundheitsschutz 2003; 46: 188–96.

2. Fernandez-Botran F. Soluble cytokine receptors: basic immunology and clinical applications. Crit Rev Clin Lab Sci 1999; 36: 165–224.

3. Theze J. Cytokine receptors: a combinative family of molecules. Eur Cytoline Netw 1994; 5: 353–68.

4. Lamont AG, Adorini L. IL-12: a key cytokine in immune regulation. Immunol Today 1996; 17: 214–7.

5. Hirano T. Interleukin 6 in autoimmune and inflammatory diseases: a personal memoir. Proc Jpn Acad Ser B Phys Biol Sci 2010; 86: 717–30.

6. Kreuzer KA, Rockstroh JK, Sauerbruch T, Spengler U. A comparative study of different enzyme immunosorbent assays for human tumor necrosis factor-alpha. J Immunol Methods 1996; 195: 49–54.

7. Setrerrahmane S, Xu H. Tumor-related interleukins: old validated targets for new anti-cancer drug development. Molecular Cancer 2017; 16: 153. https://doi.org/10.1186/s12943-017-0721-9.

8. Smith KA. Interleukin-2. Curr Opin Immunol 1992; 4: 271–6.

9. Koppelainen EI, Gamble JR, Vadas MA, Lopez AF. IL-3 receptor expression, regulation and function in cells of the vasculature. Immunol Cell Biol 1996; 74: 1–7.

10. Ryan JJ. Interleukin-4 and its receptor: essential mediators of the allergic response. J Allergy Clin Immunol 1997; 99: 1–5.

11. Egan RW, Umland SP, Cuss FM, Chapman RW. Biology of interleukin-5 and its relevance to allergic disease. Allergy 1996; 51: 71–81.

12. Fry TJ, Mackall CJ. Interleukin-7: from bench to clinic. Blood 2002; 99: 3892–4.

13. Renauld JC, Kermouni A, Vink A, Louahed J, van Snick J. Interleukin-9 and its receptor: involvement in mast cell differentiation and T cell oncogenesis. J Leukocyte Biology 1995; 57: 353–60.

14. Opal SM, Wherry JC, Grint P. Interleukin-10: potential benefits and possible risks in clinical infectious diseases. Clin Infect Dis 1998; 7: 1497–507.

15. Du X, Williams DA. Interleukin-11: review of molecular, cell biology, and clinical use. Blood 1997; 89: 3897–908.

16. Vignali DAA Kuchroo VK. IL-12 family cytokines: immunological playmakers. Nat Immunol 2014; 13: 722–8.

17. Fehniger TA, Caligiuri MA. Interleukin-15: biology and relevance to human disease. Blood 2001; 97: 14–32.

18. Center DM, Kornfeld H, Cruikshank WW. Interleukin-16. Int J Biochem Cell Biol 1997; 29: 1231.

19. Moseley TA, Haudenschild DR, Rose L, Reddi AH. Interleukin-17 family and IL-17 receptors. Cytokine Growth Factor Rev 2003; 14: 155–74.

20. Hsing CH, Li HH, Hsu YH, Ho CL, Chuang SS, Lan KM, et al. The distribution of interleukin-19 in healthy and neoplastic tissue. Cytokine 2008; 44: 221–8.

21. Blumberg H, Conklin D, Xu WF, Grossmann A, Brender T, Carollo S, et al. Interleukin-20: discovery, receptor identification, and role in epidermal function. Cell 2001; 104: 9–19.

22. Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL 23, with biologic activities similar as well as distinct from IL-12. Immunity 2001; 13: 715–25.

23. Wang M, Liang P. Interleukin-24 and its receptors. Immunology 2005; 114: 166–70.

24. Fort MM, Cheung J, Yen D, Li J, Zurawski SM, Lo S, et al. IL-25 induces IL-4, IL-5, and IL-13 and TH2-associated pathologies. Immunity 2001; 15: 985–95.

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.

26. Larousserie F, Bardel E, Pflanz S, Arnulf B, Lome-Moldonado C, Hermine O, et al. Analysis of interleukin-27 (EB13/p28) expression in Epstein-Barr virus- and human T-cell leukemia virus type-1 associated lymphomas: heterogenous expression of EB13 subunit by tumoral cells. J Pathol 2005; 166: 1217–28.

27. Kempuraj D, Donelan J, Frydas S, Iezzi T, Conti F, Boucher W, et al. Interleukin-28 and 29: new cytokines with antiviral activities. Int J Immunopathol Pharmacol 2004; 17: 103–6.

28. Volk HD, Keyßer G, Burmester GR. Zytokine und Zytokinrezeptoren. In: Thomas L, ed. Labor und Diagnose, 7. Aufl. Frankfurt, 2008, TH-Books.

20.2 Interleukin-6 (IL-6)

Lothar Thomas

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 /123 /. 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 homodimerization of the gp130 molecule, and a high functional receptor complex of IL-6, IL-6R, and gp 130 is formed.

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.

20.2.1 Indication

  • 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.

20.2.2 Method of determination

Immunoassay, predominantly ELISA.

20.2.3 Specimen

Plasma, serum, cerebrospinal fluid, bronchoalveolar lavage fluid: 0.5–1 mL

20.2.4 Reference interval

Plasma: less than 10 ng/L

20.2.5 Clinical significance

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 /12/:

  • 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.

Refer also to Tab. 20.2-1 – Activating functions of IL-6.

IL-6 is also involved in:

  • Anemia of chronic disease. IL-6 stimulates hepcidin production and causes functional iron deficiency (Section. 7.6 – Hepcidin). 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 /4/.
  • 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 (Section. 6.1 – Bone metabolism).

20.2.5.1 Acute inflammation

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 (Fig. 20.2-1 – Increase and decline of IL-6 upon inflammatory stimulation by injection of lipopolysaccharide).

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 /5/.

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.

20.2.5.2 Neonatal sepsis

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.

20.2.5.3 Chronic 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.

20.2.5.4 Non-inflammatory IL-6 response

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 /6/.

During sterile surgical operations, an increase in serum IL-6 concentration precedes elevation of body temperature and serum acute phase protein concentration.

20.2.6 Comments and problems

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.

Influence factors

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.

Stability

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.

20.2.7 Pathophysiology

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 Fig. 20.2-2 – Cytokine activated transformation of naive T-helper cells). It also stimulates endothelial cells to produce chemokines and adhesion molecules, thus recruiting leukocytes to the site of inflammation /5/.

IL-6 is a key cytokine involved in the regulation of T- cells /7/:

  • 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.

References

1. Nishimoto N, Terao K, Mirna T, Nakahara H, Tagaki N, Kakehi T,et al. Mechanisms and pathologic significances in increase in serum IL-6 and soluble IL-6b receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease. Blood 2008; 112 (109: 3959–64.

2. Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol 2014; 6: a016295

3. Rose-John S. Interleukin-6 family cytokines. Clod Spring Harbor Perspektives in Biology 2018; 10: a028415

4. Ganz T. Anemia of inflammation. N Engl J Med 2019; 381: 1148–57.

5. Nishimito N, Kishimoto T. Interleukin 6: from bench to beside. Nat Clin Pract Rheumatol 2006; 2: 619–26.

6. Hirano T. Interleukin 6 in autoimmune and inflammatory diseases: a personal memoir. Proc Jpn Acad Ser B Phys Biol Sci 2010; 86: 717–30.

7. Miossec P, Korn T, Kuchroo VK. Interleukin-17 and type 17 helper T cells. N Engl J Med 2009; 361: 888–98.

8. Jawa RS, Anillo S, Huntoon K, Baumann H, Kulayat M. Interleukin-6 in surgery, trauma, and critical care: part I: basic science. J Intensive Care Med 2011; 26: 3–12.

9. Gebhard F, Pfetsch H, Steinbach G, Strecker W, Kinzl L, Bruckner UB. Is interleukin-6 an early marker of injury severity following major trauma in humans? Arch Surg 2000; 135: 291–5.

10. Maier B, Lefering R, Lehnert M, et al. Early versus late onset of multiple organ failure is associated with differing patterns of plasma cytokine biomarker expression and outcome after severe trauma. Shock 2007; 28: 668–74.

11. Spindler-Vesel A, Wraber B, Vovk I, Kompan L. Intestinal permeability and cytokine inflammatory response in multiply injured patients. J Interferon Cytokine Res 2006; 26: 771–6.

12 Selberg O, Hecker H, Martin M, Klos A, Bautsch W, Kohl J. Discrimination of sepsis and systemic inflammatory response syndrome by determination of circulating plasma concentrations of procalcitonin protein complement 3a, and interleukin-6. Crit Care Med 2000; 28: 2793–8.

13. Habarth S, Holeckova K, Froidevaux C, et al. Diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected sepsis. Am J Respir Crit Care Med 2001; 164: 396–402.

14. Taniguchi M, Nakada T, Shinozaki K, Mizushima Y, MatsuokaT. Association between increased blood interleukin-6 levels on emergency department arrival and prolonged length of intensive care unit stay for blunt trauma. World J of Emergency Surgery 2016; 11: 6. https://doi.org/10.1186/s13017-016-0063-8.

15. Stoll BJ, Hansen NI, Adams-Chapman I, et al. Neurodevelopmental and growth impairment among extremely low birth-weight infants with neonatal infection. JAMA 2004; 292: 2357–65.

16. Malik A, Hui CPS, Pennie RA, Kirpalani H. Beyond the complete blood count and C-reactive protein. A systematic review of modern diagnostic tests for neonatal sepsis. Arch Pediatr Adolesc Med 2003; 157: 511–6.

17. Panero A, Pacifico L, Rossi N, Mancuso G, Stegagno M, Chiesa C. Interleukin-6 in neonates with early and late onset infection. Pediatr Infect Dis J 1997; 16: 370–5.

18. Ng PC, Lam HS. Diagnostic markers for neonatal sepsis. Curr Opin Pediatr 2006; 18: 125–31.

19. Schütte H, Lohmeyer J, Rosseau S, Ziegler S, Siebert C, Kielisch H, et al. Bronchoalveolar and systemic cytokine profiles in patients with ARDS, severe pneumonia and cardiogenic pulmonary oedema. Eur Resp J 1996; 9: 1858–67.

20. Schoch B, Regel JP, Nierhaus A, Wichert M, Mueller OM, Sandalcioglu IE, et al. Predictive value of intrathecal interleukin-6 for ventriculostomy-related infection. Zentralbl Neurochir 2008; 69: 80–6.

21. White UA, Stephens JM. The gp130 receptor cytokine family: regulation of adipocyte development and function. Curr Pharm Des 2011; 17: 340–6.

22. Grivennikov SI, Karin M. Inflammatory cytokines in cancer: tumour necrosis factor and interleukin-6 take the stage. Ann Rheum Dis 2011; 70 (suppl 1): i104–i108.

23. Madhok R, Crilly A, Watson J, Capell HA. Serum IL-6 levels in rheumatoid arthritis: correlations with clinical and laboratoy indices of disease activity. Ann Rheumat Dis 1993; 52: 232–4.

24. Robak T, Gladalska A, Stepien H, Robak E. Serum levels of IL-6 type cytokines and soluble interleukin-6 receptor in patients with rheumatoid arthritis. Mediators of Inflammation 1998; 7: 347–53.

25. Sieber S, Tsoukas A, Robertson J, McInnes I. Cytokines as therapeutic targets in rheumatoid arthritis and other inflammatory diseases. Pharmacol Rev 2015; 67: 280–309.

20.3 Interleukin-8 (IL-8)

Lothar Thomas

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 /1/.

Hemolysis significantly influences the concentration in plasma. The total concentration of IL-8 in the blood is determined in hemolysate.

20.3.1 Indication

  • 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.

20.3.2 Method of determination

Immunoassay, predominantly ELISA.

20.3.3 Specimen

  • 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

20.3.4 Reference interval

Plasma/Serum

≤ 10 ng/L

EDTA blood (hemolysate)

≤ 200 ng/L

20.3.5 Clinical significance

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. Tab. 20.3-1 – Behavior of IL-8 in diseases and different conditions lists examples of diseases and conditions in which the measurement of IL-8 is or could become of diagnostic value.

20.3.6 Comments and problems

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 /2/.

Stability

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.

20.3.7 Pathophysiology

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) /3/. 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.

References

1. Remick DG. Interleukin-8. Crit Care Med 2005; 33: S466-S467.

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.

3. Darbonne WC, Rice GC, Mohler MA, Apple T, Hebert CA, Valente AJ, et al. Red blood cells are a sink for interleukin-8, a leukocyte chemotaxin. J Clin Invest 1991; 88: 1362–9.

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.

6. Baines KJ, Simpson JI, Gibson PG. Innate immune responses are increased in chronic obstructive pulmonary disease. Plos One 2011; 6 (3) e18426.

7. Kobawala TP, Patel GH, Gajjar DR, Patel KN, Thakor PB, Parekh UB, et al. Clinical utility of serum interleukin-8 and interferon-alpha in thyroid diseases. J Thyroid Res 2011 (März); https://doi.org/10.4061/2011/270149.

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).

20.4 Tumor necrosis factor-α (TNF-α)

Lothar Thomas

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 /1/. TNF-α has a significant role in cell apoptosis.

20.4.1 Indication

  • 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).

20.4.2 Method of determination

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).

20.4.3 Specimen

  • Heparinized or EDTA plasma, serum: 1 mL
  • CSF: 0.5 mL

20.4.4 Reference interval

Total TNF-α (plasma)

≤ 20 ng/L

Bioactive TNF-α (sTNF) (plasma)

≤ 5 ng/L

20.4.5 Clinical significance

Local TNF-α -mediated signals are required for the normal development and function of the immune system /2/.

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 /3/. 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:

Tab. 20.4-1 – Behavior of TNF-α in diseases and different conditions.

The behavior of TNF-α in relation to other inflammatory markers in the assessment of inflammatory activity during treatment with methotrexate is shown in Tab. 20.4-2 – Levels of TNF-α and other inflammatory markers before and during treatment of rheumatoid arthritis (RA) with methotrexate.

20.4.6 Comments and problems

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 /4/.

Stability

Stable in CSF for approximately 5 years if stored at –70 °C and for 190 and 90 days if stored at –20 °C and +4 °C, respectively /5/.

20.4.7 Pathophysiology

TNF-α is member of a super family of cytokines with well characterized roles in cell survival, differentiation, apoptosis, and inflammatory response /25/.

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 /5/. 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 /25/. 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 /5/. Refer to Fig. 20.4-1 – Synthesis of TNF-α and action with target cells).

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) /6/.

References

1. Hsu H, Xiong J, Goeddel D. The TNF receptor-1-associated protein TRADD signals death and NF-kappa B activation. Cell 1995; 81: 495–504.

2. Bazzoni F, Beutler B. The tumor necrosis factor ligand and receptor families. N Engl J Med 1996; 334: 1717–25.

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.

4. Moreau E, Philippe J, Couvent S, Leroux-Roels G. Interference of soluble TNF-α receptors in immunological detection of tumor necrosis factor-α. Clin Chem 1996; 42: 1450–3.

5. Caminero A, Comabella M, Montalbau X. Tumor necrosis factor alpha (TNF-α), anti TNF-α and demyelinisation revisited: an ongoing story. J Neuroimmunol 2011; 234: 1–6.

6. Ruiz MC, Quijano FC, Cortes LFL, Duvison MH, Rubio RV. Determination of shelf life and activation energy for tumor necrosis factor-α in cerebrospinal fluid. Clin Chem 1996; 42: 670–4.

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.

8. Takakuwa T, Endo S, Nakae H, Kikichi M, Suzuki T, Inada K, Yoshida M. Plasma levels of TNF-α, endothelin and thrombomodulin in patients with sepsis. Res Comm Chem Pathol Pharmacol 1994; 84: 261–9.

9. Hamilton G, Hofbauer S, Hamilton B. Endotoxin, TNF-α, interleukin-6 and parameters of the cellular immune system in patients with intraabdominal sepsis. Scand J infect Dis 1992; 24: 361–8.

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.

12. Grewal HP, Kolb M, El Din S, Novak K, Martin J, Gaber LW, Gaber O. Elevated tumor necrosis factor levels are predictive for pancreas allocraft transplant rejection. Transplantation Proceedings 1993; 25: 132–5.

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.

14. Tak PP, Kalden JR. Advances in rheumatology: new targeted therapeutics. Arthritis Res Ther 2011; 13 (suppl 1). https://doi.org/10.1186/1478-6354-13-S1-S5.

15. Kang SY Kim MH, Lee WI. Measurement of inflammatory cytokines in patients with rheumatoid arthritis. Korean J Lab Med 2010; 30: 301–6.

20.5 Soluble IL-2 receptor (sIL-2R)

Lothar Thomas

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 /123/.

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.

20.5.1 Indication

  • 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).

20.5.2 Method of determination

Enzyme-linked immunoassay. Sandwich enzyme immunoassays employ two monoclonal antibodies to IL-2Rα.

20.5.3 Specimen

  • Heparinized plasma, serum: 1 mL
  • Cerebrospinal fluid: 0.5 mL

20.5.4 Reference interval

  • Plasma/serum: 112–502 U/mL /4/
  • Cerebrospinal fluid (in cytologically normal samples): ≤ 10 U/mL /3/

1 U corresponds to 3.3 pg.

20.5.5 Clinical significance

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 (Tab. 20.5-1 – Causes of elevated sIL-2R levels/4/. 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 Section 1.5 – Angiotensin-converting enzyme (ACE))
  • 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.

Tab. 20.5-2 – Behavior of sIL-2R in diseases and different conditions shows the behavior of IL-2R in selected diseases and conditions.

20.5.6 Comments and problems

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.

Reference interval

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 /5/. Elderly individuals tend to have higher levels than young adults.

Stability

Stable for 1 day if stored at –20 °C (recommended temperature).

20.5.7 Pathophysiology

Interleukin-2 (IL-2)

IL-2 is a marker of T-cell activation. It is produced predominantly by activated CD4+T cells (Fig. 20.1-4 – Development of the sub populations of T-helper cells under the influence of IL-4 and IL-12), 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 /6/. Treg cells are activated by IL-2 via the IL-2R.

Membrane-bound interleukin-2 receptor (IL-2R)

The IL-2R is located in the cell membrane of lymphatic cells (activated T cells, NK cells) monocytes and eosinophils /7/.

  • 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.

The IL-2R is shown in Fig. 20.5-1 – A model signaling by IL-2 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 /4/.

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 /4/.

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 /4/.

The sIL-2Rα has its highest diagnostic value in diseases that are associated with T cell stimulation. One of its advantages over IL-2Rβ and IL-2Rγ is its specificity for the IL-2R /2/.

References

1. Nayki C, Gunay M, Kulhan M, Nayki U, Cankaya M, Kulhan NG. Serum levels of soluble interleukin-2 receptor in association with oxidative stress index in patients with different types of HPV. Ginekologia Polska 2017; 88: 355–9.

2. Witkowska AM. On the role of sIL2R measurements in rheumatoid arthritis and cancers. Mediators Inflamm 2005; 3: 121–30.

3. Lee W, Kim SJ, Lee S, Kim J, Kim L, Lim J, et al. Significance of cerebrospinal fluid sIL-2R level as a marker of CNS involvement in acute lymphoblastic leukemia. Ann Clin Lab Sci 2005; 35: 407–12.

4. Morris JC, Waldmann TA. Advances in interleukin 2 receptor targeted treatment. Ann Rheum Dis 2000; 59 (suppl 1) i109–i114.

5. Sack U, Burkhardt U, Borte M, Schädlich H, Berg K, Emmrich F. Age-dependent levels of select immunological mediators in sera of healthy children. Clin Diag Lab Immunol 1998; 5: 28–32.

6. Fehervari Z, Yamaguchi T, Sakaguchi S. The dichotomous role of IL-2: tolerance versus immunity. Trends in Immunology 2006; 27: 109–11.

7. Nelson BH, Willerford DM. Biology of the interleukin-2 receptor. Advances in Immunology 1998; 70: 1–81.

8. Müller-Quernheim J. Sarcoidosis: immunopathogenetic concepts and their clinical application. Eur Respir J 1998; 12: 716–38.

9. Prasse A, Katic C, Germann M, Buchwald A, Zissel G, Müller-Quernheim J. Phenotyping sarcoidosis from a pulmonal perspective. Am J Respir Crit Care Med 2008; 177: 330–6.

10. Gundlach E, Hoffmann MM, Prasse A, Heinzelmann S, Nees T. Interleukin-2 receptor and angiotensin-converting enzyme as markers for ocular sarcoidosis. Plos One 2016; https://doi.org/10.1371.

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.

12. Bien E, Balcerska A. Serum soluble interleukin 2 receptor α in human cancer of adults and children: a review. Biomarkers 2008; 13: 1–26.

13. Viviani S, Camerini E, Bonfante V, Santoro A, Balzarotti M, Fornier M et al. Soluble interleukin-2 receptor (sIL-2R) in Hodgkin’s disease: outcome and clinical implications. Br J Cancer 1998; 77: 992–7.

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.

15. Sawada S, Hashimoto H, Iijama S, Tokano Y, Matsukawa Y, Takei M, et al. Increased soluble IL-2 receptor in serum of patients with systemic lupus erythematosus. Clin Rheumatol 1993; 12: 204–9.

16. Schmitt WH, Heesen C, Csernok E, Rautmann A, Gross WL. Elevated serum levels of soluble interleukin-2 receptor in patients with Wegners granulomatosis. Arthritis and Rheumatism 1992; 35: 1088–96.

17. Prummel MF, Wiesinga WM, van der Gaag R, Mourits MP, Koornneff L. Soluble IL-2 receptor levels in patients with Grave´s opthalmopathy. Clin Exp Immunol 1992; 88: 405–9.

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.

Table 20.1-1 Functional classification of cytokines /1/

Classification of cytokines

Interferon (IFN)

IFNs are produced by virus-infected cells and provide protection against homologous and heterologous viruses. However, they also play an important role in immune regulation. IFNs are classified into type I interferons (IFN-α, IFN-β, IFN-τ, IFN-ω) and type II interferons (IFN-γ). IFN-α has 20 variants. Type I interferons are antiviral cytokines, type II interferons additionally have immunomodulatory activity. Type II interferons used to be termed macrophage-activating factor, since they are also capable of activating macrophages. All IFNs are capable of activating MHC class I molecules.

Interleukin (IL)

ILs act as mediators between leukocytes; there are over 30 known ILs. Their functions range from the stimulation to synthesize further ILs and other mediators to the initiation of cell proliferation and expression of enzymes.

Tumor necrosis factor (TNF)

TNFs were originally named after their ability to cause tumor necrosis when released into the plasma following treatment with BCG and lipopolysaccharide. TNF-α is the most important factor of this family. Other members include CD30L, CD40L, CD95L, FS7-associated surface antigen ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL), nerve growth factor (NGF), and lymphotoxin (LT α12). Their functions are antitumor activity (TNF-α), induction of apoptosis, (FasL, TRAIL, TNF-α), immune regulation (CD40L, TNF-α), regulation of lymph node development (LT α12). TNFs have a pleated sheet structure.

Colony-stimulating factor (CSF)

CSFs regulate the proliferation and differentiation of hematopoietic precursor cells in the bone marrow. They are differentiated into granulocyte macrophage CSF (GM-CSF), granulocyte CSF (G-CSF) and macrophage CSF (M-CSF). GM-CSF, for example, is able to stimulate the IL production of monocytes.

Growth factor (GF)

GFs are cytokines that influence the growth of non-hematopoietic cells. Known GFs include epidermal GF (EGF), insulin GF (IGF), platelet-derived GF (PDGF), vascular endothelial GF (VEGF)

Chemokine

Chemokines have a chemotactic effect on granulocytes and monocytes. They comprise more than 50 known proteins and are divided into the families C, CC, CXC and CX3C, depending on the position of relevant cysteines and the presence and number of amino acids between the first two cysteine molecules (see also Tab. 19.1-1 – Mediators of inflammation and Tab. 19.1-3 – Chemokine groups and their effects on immune cells).

Table 20.1-2 Classification of cytokines based on structural features /1/

Cytokine family

Four-helix bundle structure cytokines

IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-15, GM-CSF, G-CSF, M-CSF, ciliary neurotropic factor (CNTF), leukemia inhibiting factor (LIF), oncostatin M, new neurotropin-1 (NNT-1), cardiotropin-1 (CT-1) erythropoietin, hGH, prolactin.

Heterodimeric cytokines: IL-12, IL-23, IL-27, cardiotrophin-like cytokine/cytokine-like factor-1 (CLC/CLF1). Some of these cytokines (IFN-γ, IL-5, M-CSF) form dimers.

IL-6 family

IL-6, IL-11, IL-27, LIF, oncostatin M, CT-1, CNTF, CLC. They share gp130 as a signal transducer.

IL-10 family

IL-10, IL-19, IL-20, IL-22, IL-24, IL-26

IL-12 family

Comprises the only heterodimeric four-helical cytokines, which includes IL-12, IL-23, IL-27 and IL-35

Interferons

Type I: IFN-α, IFN-β, IFN-τ, IFN-ω, limitin

Type II: IFN-γ

TNF-α family

TNF-α, lymphotoxin, nerve growth factor (NGF), vascular endothelial growth inhibitor (VEGI)

IL-1 family

IL-1α, IL-1β, IL-1-Ra, IL-18

Table 20.1-3 Characteristics and function of 30 human cytokines

Interleukin
Synonym­

Origin
(selection)

Function

IL-1

Endogenous
pyrogen

Lymphocyte
activation factor

M,G, E, F

Target cells:
all body
cells

IL-1 is the central proinflammatory cytokine and important mediator of innate and adaptive immunity. IL-1 receptors (IL-1Rs) are expressed on the majotity of cell types including T cells, myeloid cells, fibroblasts and cancer cells.Il-1 has affinity to two types of receptors: IL-1R1 and IL-1R2. IL-1R1 is a signaling capable receptor whereas IL-1R2 is required for binding the soluble IL-1 and making its complex with IL-1R1 /7/.

IL-2

T-cell growth
factor

T

Target cells:
T, NK, B, M

IL-2 is a member of the γ-chain cytokine family, and a key cytokine in regulating the survival, proliferation, and differentiation of activated T cells and NK cells. IL-2 is a pivotal immunoactivator; it promotes T cell clonal expansion and effector differentiation. In the early phase of immune response, IL-2 acts by promoting the differentiation of naive CD8+T cells into effector and memory cells.

The IL-2 receptor (IL-2R) is composed by three subunits, α, β, γ. IL-2R is mainly expressed by immune cells (Treg, CD4+ and CD8+T cells, B cells, CD56hi NK cells, mature dendritic cells and endothelial cells). The IL-2Rγ subunit is expressed mainly by hematopoietic cells /78/. Refer to Section 20.5 – Soluble IL-2 receptor.

IL-3

Mast cell
growth factor

Multicolony-
stimulating
factor

T, Mo, F

Target cells:
progenitor
cells in
the bone
marrow

IL-3 is a hematopoietic growth factor which activates the precursor cells in the bone marrow to proliferate and differentiate. The IL-3 receptor consists of a ligand-specific α-unit and an inactive β-unit which, when associated with the α-unit, provides the receptor with high affinity for IL-3 /9/.

IL-4

B-cell
stimulating
factor

T, Bas, Mo

Target cells:
B, T, F, Bas

IL-4, the most important TH2 cytokine, is mainly produced by activated T cells, mast cells, basophils and eosinophils to regulate lymphocytes proliferation and survival. IL-4 stimulates Th2 cells to produce IL-5, IL-6, IL-10 and IL-13 and regulates the immune response to the production of IgE antibodies. Besides IL-4, IL-12 is a further important cytokine of the Th1/Th2 paradigm (Fig. 20.1-4 – Development of the subpopulations of T-helper cells (Th1 and Th2) under the influence of IL-4 and IL-12) and plays a key role in the development of allergies /10/. IL-4 and IL-13 share a common receptor, IL4R, for signal transduction. This receptor is composed of several subunits. Depending on the cell type, diverse binding configurations generate different types of IL-4/IL-4R complexes.

IL-5

Differentiation
factor:

– B cells

Eosinophils

T, Bas, Mo

Target cells:
Eos, Bas, B

IL-5 is a hematologic growth and differentiation factor for eosinophils /11/. These are activated by chemoattractants such as f-Met-Leu-Phe and respond with migration and degranulation. Signal transduction occurs through the IL-5R, which consists of an α- and a β-subunit.

IL-6

B-cell- and
hepatocyte-
stimulating
factor

M, G, T, F,
Endo

Target cells:
B, T M,
hepatocytes

IL-6 is a common multipotent cytokine which is involved in the early activation of T cells, in the differentiation of B cells, the regulation of the acute-phase response, and in hematopoiesis. Further information can be found in Section 20.2 – Interleukin-6 (IL-6).

IL-7

Lympho-
poietin 1

T, F, K

Thymus
epithelium

Target cells:
T, B

IL-7 is produced by non-bone-marrow-associated stromal cells and epithelial cells. The central role of this pleiotropic cytokine is to modulate the development of T and B cells as well as T-cell homeostasis /12/. Signal transduction occurs through the IL-7 receptor α-chain upon its heterodimerization with the γc protein, which is present on all lymphocytes, to form a new receptor. Signaling occurs via the γc protein. The cytokines IL-2, IL-4, IL-9, IL-15 and IL-21 also signal through γc.

IL-8

Neutrophil-
activating
protein

Chemotactic
factor

M, G, Endo,
T, E, K

Target cells:
N, (T)

IL-8 is a potent chemotactic cytokine for neutrophils and lymphocytes. Many cell types are capable of producing IL-8, especially upon stimulation by IL-1 and TNF-α. Signal transduction occurs through two receptors, IL-8R-A and IL-8R-B. See also Section 20.3 – Interleukin-8 (IL-8).

IL-9

Mast cell factor
T-cell growth
factor III

T

Target cells:
T

Progenitor
cells in
the bone
marrow

IL-9 is a cytokine of the Th2 cell immune response (see Chapter 21 – Immune system) and is produced by activated CD4+ T cells /13/. IL-9 promotes the proliferation and differentiation of mast cells and stimulates activated T cells to activate B cells in the humoral immune response. The IL-9 receptor complex consists of the proteins IL-9R and γc.

IL-10

Cytokine
synthesis
inhibitory
factor

M, T, B, E, Mo

Target cells:
T, B, M

IL-10 has antiinflammatory properties. It down regulates the expression of IL-1, TNF-α and the C-X-C and C-C chemokine families /14/. In conditions of systemic immune activation, such as sepsis, it down regulates over expression of proinflammatory cytokines. IL-10 binds to a singular high-affinity receptor protein.

IL-11

Adipogenesis
inhibitory
factor

F

Target cells:
B, Mega, M

IL-11 has similar functional characteristics to IL-6 and is therefore classified as an IL-6-type cytokine. It synergizes with IL-3, IL-4, IL-7, IL-12 and IL-13 in stimulating the proliferation and differentiation of hematopoietic precursor cells. IL-11 is produced by epithelial cells of the gastrointestinal tract and the lungs and regulates normal growth of these epithelia. IL-11, IL-6 and oncostatin share the gp130 protein as a common receptor subunit /15/.

IL-12

Nk-cell-
stimulating
factor

M, B,
dendritic
cells

Target cells:
T, NK

IL-12 is a pro-inflammatory cytokine and is activated by macrophages that have phagocytized pathogens. IL-12 induces the production of IFN-γ and other cytokines in T and NK cells. IL-12 promotes the proliferation of NK cells and lysis of cytotoxic T cells and NK/lymphokine activated killer cells. IL-12 is a key cytokine of the Th1/Th2 paradigm (Fig. 20.1-4 – Development of the subpopulations of T-helper cells (Th1 and Th2) under the influence of IL-4 and IL-12). IL-12 activates the differentiation of antigen-stimulated Th0 cells in Th1 cells. These produce IL-2, IFN-γ and TNF-α and activate cell-mediated immunity. IL-12 signals through a high-affinity receptor, IL-12R,a heterodimer composed of the subunits β1 and β2, whose coexpression is required for IL-12 binding /16/.

IL-13

P600

T

Target cells:
B, M

IL-13 has similar functions to IL-4, since both bind to the α-chain of the IL-4Rα.

IL-14

High-molecular-weight B-cell growth factor

T, B

Target cells:
B

IL-14 is produced by germinal T cells, dendritic cells, and malignant B cells. It induces B-cell proliferation, inhibits antibody secretion and promotes the production of memory B cells.

IL-15

None

E, K, M

Target cells:
T, NK

IL-15 is a pleiotropic cytokine which shares many functions with IL-2. IL-15 activates NK-cell proliferation, cytotoxicity and cytokine production and regulates the interaction between NK cells and macrophages. It plays a pivotal role in linking innate and adaptive immune responses during infections /17/. Deregulated IL-15 has been associated with the development of autoimmune diseases such as rheumatoid arthritis. For signal transduction, IL-15 binds to the receptor unit IL-R15α, which is similar to IL-2Rα, although the binding affinity of IL-15 to IL-R15α is 1,000 times higher than that of IL-2 to IL-R2α.

IL-16

Lymphocyte chemotactic factor

T, Mz, F, Eos

Target cells:
T

IL-16 is a proinflammatory cytokine which is produced by CD8+ T cells, CD4+ T cells, mast cells, eosinophils and bronchial epithelia /18/. IL-16 induces the migration of CD4+ T cells, increases the intracellular concentrations of Ca2+ and 1,4,5-triphosphate and induces the production of proinflammatory cytokines.

IL-17

None

T, F

Target cells:
F, E, Endo

IL-17 is a family of so far 5 cytokines produced by activated T cells /19/. The prototype is IL-17A. The IL-17 family operates in tissues such as bone, cartilage, lungs, brain, kidneys and small intestine and appears to play a role in the homeostasis of these tissues, regardless of the immune system. The receptors are IL-17R, IL-17RH1 and IL-17Rl.

IL-18

Interferon-gamma-
inducing
factor

M, B,
dendritic
cells

Target cells:
T, NK

IL-18 is produced constitutively in dendritic cells, enterocytes and keratinocytes. It induces the synthesis of IFN-γ, which is primarily produced by NK cells and T-helper cells. IL-18 is a weak stimulator of IFN-γ synthesis. In the presence of IL-12, however, IFN-γ is produced abundantly. The receptor is composed of two molecules of the IL-1R family.

IL-19

None

M

Target cells:
E, M, K

IL-19 is a cytokine of the IL-10 family, which also includes IL-20, IL-22, IL-24 and IL-26. IL-19 is produced by monocytes/macrophages, epithelial and endothelial cells upon their activation by lipopolysaccharide or the granulocyte-macrophage colony-stimulating factor (GM-CSF). Signal transduction occurs through the receptor IL-20R, which is shared with IL-20 and IL-24. IL-19 is mitogenic and chemotactic for endothelial cells and has an antiinflammatory effect on these cells /20/.

IL-20

None

M

Target cells:
E, M, K

IL-20 is a cytokine of the IL-10 family. It is produced by activated keratinocytes and monocytes during inflammation and signals through a heterodimeric receptor on keratinocytes and epithelial cells which is comparable to the IL-10R. The number of receptors is highly upregulated in psoriasis /21/.

IL-21

None

T

Target cells: B, NK

IL-21 is produced by activated CD4+ T cells and by NK cells. The latter self-regulate by producing IL-21. IL-21 is also produced by cancer cells in Hodgkin’s lymphoma. IL-21 suppresses the synthesis of IgE antibodies and plays a role in controlling allergic responses. It is also thought to be involved in the coordinated response to viral infections.

IL-22

IL-10-related
T-cell-derived
inducible
factor

T, Mast

Target cells:
E, M, K, T

IL-22 belongs to the IL-10 family, which also includes IL-19, IL-20, IL-24 and IL-26. These are potent mediators of the cellular inflammatory response. IL-22 signals through a receptor composed of IL-22R1 and IL-10R2.

IL-23

Complex of
p40 subunit
of IL-12 and
IL6 G-CSF-
related
factor

M, D

Target cells:
T

This heterodimeric cytokine is composed of the two subunits p40, which is shared with IL-12, and p19, which is identical with the IL-23 α-subunit. IL-23 is an important antiinflammatory cytokine in infections and is produced by activated dendritic cells. It upregulates metalloprotease (MMP9) expression, activates angiogenesis and reduces infiltration of the site of inflammation with CD8+T cells. Together with IL-6 and TGF-β1, IL-23 stimulates CD4+T cells to differentiate into a new subpopulation, namely Th17 cells /22/. IL-23 is a cytoline of IL-12 family, but it diifers from IL-12 in playing a key role in Th17 development by stabilizing IL-17 development /16/.

IL-24

MDA-7
(melanoma
differentiation-
associated
gene 7) ST16
(suppression
of morgenicity-
16)

Meg, B, T, M

Progenitor
cells in
the bone
marrow

Target cells:
E, M, K,
carcinoma

IL-24 is a cytokine of the IL-10 family. It is produced by activated monocytes, macrophages and TH2 cells. Signal transduction occurs through two heterodimeric receptors, IL-20R1/IL-20R2 and IL-22R1/IL20R2. IL-24 regulates the proliferation and survival of cells via rapid induction of the transcription factors STAT-1 and STAT-3. IL-24 plays a role in wound healing, in malignant diseases and in psoriasis /23/.

IL-25

Cytokine of the
IL-17 family

Th2

Mast cells

IL-25 is secreted by TH2 cells and mast cells and is also known as IL-17E. It can induce nuclear factor kappa B activation and stimulate the production of IL-8. Both IL-25 and IL-17B signal through the IL-17 RB receptor. IL-25 induces the synthesis of IL-4, IL-5 and IL-13, all of which stimulate the proliferation of eosinophils. IL-25 is involved in regulating intestinal immunity and may play a role in inflammatory bowel disease /24/.

IL-26

AK-155

T

Target cells:
T

IL-26 is a cytokine of the IL-10 family. It is expressed by Herpesvirus transformed T cells, but not by primarily stimulated T cells. IL-26 signals through two proteins, IL-20 receptor 1 and IL-10 receptor 2. IL-26 accelerates the secretion of IL-10 and IL-8 and stimulates CD54 expression on epithelial cells /25/.

IL-27

None

M, D

IL-27 is a member of the IL-12 family and a heterodimeric protein with proinflammatory and immunosuppressive properties. It is composed of the protein units IL-27B, which is encoded by the Epstein-Barr virus-induced gene 3, and IL-27-p28. IL-27 is produced by antigen-presenting cells and regulates B and T cell activity. Signal transduction occurs through a receptor composed of the two units IL-27R and gp130 /26/.

IL-27 has pro-stimulatory and inhibitory function. However, the main functions seem to be in inhibiting immune responses. It is often generated during the resolution phase of an autoimmune response by local antigen presenting cells. IL-27 can inhibit development of Th17 producing cells /16/.

IL-28A

Interferon
lambda-2

IL-28B

Interferon
lambda-3

M

Target cells:
multiple
cell types

IL-28 has two isoforms and plays a role in immune defense against viruses. It is a cytokine of the IL-10 family, since the IL-28R receptor is composed of an IL-28 receptor α-chain and an IL-10 receptor β-chain. Both belong to the interferon-III family and are very similar to IL-29 /27/.

IL-29

Interferon
lambda-1

M

Target cells:
multiple
cell types

IL-29 is a member of the IFN-γ family, has similarities with type-I interferons and is a potent antiviral cytokine. IL-29 is secreted by dendritic cells and monocytes in response to viral infections and by stimulation by the toll-like receptor. The IL-29R receptor is composed of the protein IL-28Rα and IL-10Rβ and is expressed by virtually all non-hematologic cells /27/.

IL-30

 

Heterodimeric cytokine consisting of the proteins EB13 and p28. The gene of this protein is called IL-27 under HGNC guidelines.

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

Table 20.1-4 Indications for the determination of cytokines and cytokine receptors /28/

Clinical and laboratory findings

IL-6 in plasma/serum

Neonatal sepsis­

Accurate parameter for early diagnosis and prognosis. Rises within an hour. Declines after 10 h in response to successful antibiosis.­

Adult critical care­

Diagnosis and progression of systemic inflammation. Together with procalcitonin, it is a marker for diagnosis of SIRS and sepsis. Falls rapidly in response to successful antibiosis.­ No differentiation of SIRS and sepsis.

Trauma­

Prognostic parameter of clinical progression (duration of ventilation, incidence of pneumonia, mortality) within the first 2–48 h of traumatic brain injury, soft tissue injury and trauma.­

Heart failure­

Measure of peripheral hypoxia and prognostic marker.

Decline correlates with the success of mechanical heart support systems.­

IL-8 in plasma/serum, urine and bronchoalveolar lavage

Kidney transplantation­

Indicator of organ damage caused by ischemia/reperfusion.­

Neonatal sepsis­­

Early diagnosis of neonatal sepsis, although the parameter has been mostly replaced by the IL-6 biomarker.

Sepsis, adult poly- trauma­

Measure of systemic inflammation, although the parameter has been mostly replaced by the IL-6 biomarker.

Sepsis, polytrauma­

Prognostic marker for the development of adult respiratory distress syndrome (ARDS), also elevated in pneumonia.­

IL-10 in plasma/serum

Bypass surgery­

Prognostic marker for clinical progression after surgery (incidence, infections, duration of ventilation) 4–24 h post-surgery. ­

Sepsis, polytrauma, postoperative complications­

Measure of immunodepression, risk marker for infection.

TNF-α in cerebrospinal fluid and bronchoalveolar lavage

Sepsis, polytrauma­

Prognostic marker for the development of ARDS.­

Multiple sclerosis (MS) Meningitis­

Marker of MS activity, elevated in bacterial meningitis.

sTNF-α receptor in plasma/serum

­Multiple sclerosis(MS)­

Elevated in active MS, decreases during treatment with steroids.­

Heart failure­

Sepsis­

Correlates with the degree of heart failure, decreases with successful treatment. Elevated sTNF-α receptor 2 correlates with mortality risk.­

Independent predictor of the development of acute kidney failure, even in patients with normal creatinine levels.­

sIl-2R in plasma/serum

Sarcoidosis­

Marker of activity, not disease-specific.­

Table 20.2-1 Activating functions of IL-6 /5/

Activation of cells and regulatory systems

Hypothalamic-pituitary-adrenal axis (fever, release of hormones)

Immune cells (proliferation and differentiation of T cells and B cells for the production of antibodies)

Production of thrombocytes by megakaryocytes

Hematopoiesis (see Fig. 20.1-2 – Regulation of hematopoiesis by cytokines)

Endothelial cells

Differentiation of neuronal cells

Proliferation of keratinocytes

Myocardial hypertrophy

Osteoclasts

Table 20.2-2 Behavior of IL-6 in diseases and further conditions

Clinical and laboratory findings

Surgery /8/

Surgical trauma and anesthesia cause transient immune suppression, which can lead to postoperative infection. IL-6 concentrations have been shown to increase proportional to surgical stress in postoperative patients. For example, postoperative IL-6 levels are more elevated after open cholecystectomy and open colorectal resection than after laparoscopic surgery. The survival rate depends on the level of IL-6 on day 1. Patients who survived had median plasma IL-6 levels of 122 ng/L after surgery for ruptured aortic aneurysm, while patients who died had median levels of 543 ng/L.

Trauma

The greater the tissue damage, measured by the injury severity score (ISS), the greater the increase in IL-6 levels. After trauma, IL-6 levels rise 12 h earlier than CRP levels. In one study, in which trauma patients were divided into different groups according to their ISS 4 hours after trauma and had their IL-6 levels tested 6 h after trauma, the 4 ISS groups had the following average IL-6 levels: ISS below 9, 150 ng/L; ISS 9–17, approximately 200 ng/L; ISS 18–30 about 650 ng/L; ISS above 32, approximately 1,000 ng/L /9/. In another study /10/, IL-6 peaked on the first day, reached a nadir on day 4 and then decreased to baseline values on day 10. In patients with late-onset multiple organ failure (MOF), IL-6 levels rose again. Patients with multiple traumas and MOF had significantly higher IL-6 levels (145 ng/L) on day 2 compared to those without MOF (61.9 ng/L) /11/.

Critically ill /8/

Intensive care unit (ICU) patients represent a heterogenous group with a variety of acute and chronic diseases. The latter can be associated with deregulation of the IL-6–IL6R axis. Critically ill patients generally have elevated IL-6 levels, with the extent of elevation depending on the patient’s inflammatory status. If SIRS, sepsis or MODS are present, IL-6 is elevated. The level of IL-6 is a prognostic marker. Patients with sepsis have higher IL-6 levels than SIRS patients, and patients with septic shock have very high levels. In one study /12/, average levels of IL-6 within the first 8 h were 98 ng/L for SIRS, 382 ng/L for sepsis, and 520 ng/L for severe sepsis. In non-surviving patients, levels were in the range of 110–1004 ng/L (median 283 ng/L). Levels above 1,000 ng/L were an indicator of high sepsis-related mortality /13/. IL-6 is generally not useful for discriminating between SIRS and sepsis. Patients with prolonged ICU stay had significantly higher blood IL-6 levels on ICU arrival than the patients without prolonged ICU stay /14/.

Suspected onset of bacterial infection in the neonate

Neonatal sepsis is one of the main causes of morbidity and mortality in newborns. Most infections occur in the first 48 hours of life (early onset) and are of pre- or perinatal origin. Infections that occur after 48 h usually result from postpartum acquisition of pathogens (late onset). About one in five extremely low-birth-weight infants suffer at least one episode of culture-positive late-onset sepsis /15/. Early-onset infection can be diagnosed by serial IL-6 determinations within the first 48 h. The cutoff levels depend on the brand of test kit used. One review /16/ found that, at cutoff levels of 133 and 135 ng/L, the diagnostic sensitivities for sepsis were 81% and 93%, respectively, at specificities of 96% and 86%, respectively. Other authors /17/ report a diagnostic sensitivity of 69% and specificity of 36% for postnatal day 1 at a cutoff of 70 ng/L and a diagnostic sensitivity of 92% and specificity of 96% for postnatal day 2 at a cutoff of 50 ng/L.

The following diagnostic sensitivities and specificities (mean values) are reported for other markers at the time of suspected diagnosis:

Ratio of immature/mature granulocytes ≥ 25%: 76% sensitivity, 69% specificity. CD64 antigen activated granulocytes: 95–97% sensitivity, 88–90% specificity /18/.

C-reactive protein ≥ 10 mg/L: 45% sensitivity, 76% specificity.

Acute respiratory distress syndrome (ARDS)

Patients with ARDS often have elevated levels of IL-6 in plasma and broncho alveolar lavage (BAL) fluid. In addition, the mechanical ventilation causes increased release of inflammatory cytokines and influx of bacteria, which induces systemic inflammation. In ARDS and under mechanical ventilation, IL-6 levels in plasma and BAL fluid are persistently elevated. IL-6 levels are higher in BAL fluid than in plasma and higher in ventilated ARDS patients than in patients with cardiopulmonary edema. While in cardiopulmonary edema plasma IL-6 levels were only mildly elevated, in ARDS the median level was 500 ng/L and in BAL it was 3–5-fold higher /19/.

Traumatic brain injury /8/

Severe traumatic brain injury and ischemic stroke are associated with elevated IL-6 concentrations in plasma. The level of IL-6 correlates with the severity of injury. Since concentrations are higher in cerebrospinal fluid (CSF) than in plasma, the inflammation is compartmentalized.

The determination of IL-6 levels in CSF is a good marker for the early diagnosis of ventriculostomy-associated infection (VAI) and early initiation of therapy. Patients who developed VAI had significantly higher levels on the day prior to diagnosis than patients who did not develop VAI. A level > 2700 ng/L predicted VAI with a diagnostic sensitivity of 73.7% and specificity of 91.4% and a positive predictive value of 91.4% /20/.

Low-grade inflammation

IL-6 is a mediator of low-grade chronic inflammation in obesity. Obese individuals with a large hip circumference have elevated IL-6 levels, because visceral fat produces more IL-6 than subcutaneous fat. Elevated IL-6 levels are also associated with insulin resistance, but not always with type 2 diabetes mellitus, since slim type 2 diabetics do not have elevated IL-6 levels /21/.

Malignant tumor /22/

About 20% of malignant tumors arise in association with an infection or inflammation. IL-6 is a pro tumorigenic zytogene, because it activates the oncogenic transcription factors nuclear factor kappa B and STAT3 of the epithelial cells. IL-6 stimulates colon carcinoma and hepatocellular carcinoma cells. The role of IL-6 as a regulator of inflammation and tumorigenesis in cancer patients makes it an attractive target for adjuvant treatment in cancer.

Rheumatoid arthritis (RA)

RA is a chronic inflammatory arthritis characterized by inflammation of the synovial membrane within joints. Clinical symptoms are pain, swelling, and destruction of synovial joints, resulting in functional disability. The disease prevalence ranges from approximately 1% in Caucasians and up to 5% in certain North American indegenous groups. Women are more than 2–3 times affected than men. According to studies circulating IL-6 levels were:

  • In rheumatic patient 11 pg/mL (median) and 6–28 pg/mL (interquartile range); in healthy controls the levels were 3–6 pg /mL (interquartile range) /23/
  • In rheumatic patients stage the levels were 84 pg/ml (median) and 6–234 pg/mL (range) ; in healthy controls the levels were 0.5–17 pg /mL (range) /24/.

Therapeutic targets in rheumatoid arthritis are presented in Ref. /25/.

Table 20.2-3 Anti- and proinflammatory activities of the IL-6 receptor (IL-6R) /3/

Antiinflammatory
(membrane-bound IL-6R)

Proinflammatory
(soluble IL-6R)

Activation of STAT 3 leads to:

  • Intestinal endothelial cell proliferation
  • Inhibition of epithelial cell
  • apoptosis

Activation of the immune system leads to:

  • Recruitment of mononuclear cells
  • Inhibition of T-cell apoptosis
  • Inhibition of Treg development

Table 20.3-1 Behavior of IL-8 in diseases and different conditions

Clinical and laboratory findings

Suspected onset of bacterial infection

The diagnosis of neonatal sepsis by determining IL-8 levels depends on whether the parameter is assayed in plasma or in detergent-lysed whole blood (DLWB). In healthy newborns, the DLWB levels are 280-fold higher than corresponding plasma levels. One study /4/ investigated the significance of IL-8 levels in plasma versus those in DLWB in newborns with early-onset bacterial infection (EOBI). The plasma and DLWB cutoff levels were ≥ 60 ng/L and ≥ 18,000 ng/L, respectively. Comparison:

  • Plasma; 71% diagnostic sensitivity and 90% specificity after 6 h and 32% sensitivity and 99% specificity after 24 h
  • DLWB; 97% diagnostic sensitivity and 95% specificity after 6 h and 70% sensitivity and 92% specificity after 24 h
  • The determination in hemolyzed whole blood is therefore more reliable, even 24 h after the onset of EOBI.

Another study /5/ evaluated the significance of plasma IL-8 versus plasma IL-6. The following results were obtained for cord blood:

  • IL-8 at a cutoff level ≥ 90 ng/L; diagnostic sensitivity 87% and specificity 94%.
  • IL-6 at a cutoff level ≥ 80 ng/L; diagnostic sensitivity 96% and specificity 95%.

Trauma, SIRS, sepsis in children and adults

In general, the evaluation corresponds to that described for IL-6, which is mostly assayed in this case, too.

ARDS

The same evaluations as those described for IL-6 apply, see Tab. 20.2-3 – Anti- and proinflammatory activities of the IL-6 receptor.

Chronic obstructive pulmonary disease (COPD)

COPD is characterized by chronic obstruction of the airways, infiltration of the mucous membranes with polymorphonuclear granulocytes, and bacterial colonization. These patients have elevated concentrations of markers of the innate immune system, such as IL-8. Patients with COPD had average levels of 17 ng/L compared to controls with a mean plasma IL-8 concentration of 5 ng/L /6/.

Thyroid disease

Patients with struma, autoimmune thyroid disease and thyroid cancer have elevated plasma IL-8 concentrations compared to healthy controls. 57.1% of the patients with struma, 56.3% of those with autoimmune thyroid disease and 62.5% of the patients with thyroid carcinoma had plasma IL-8 concentrations above 7 ng/L. 40% of the patients with thyroid carcinoma were in an early stage and 82% were in an advanced stage of the disease /7/.

Chronic liver disease

Plasma IL-8 levels are increased in chronic liver diseases, especially in end-stage cirrhosis. Patients with cholestatic diseases exhibit high IL-8 serum concentrations. The concentration of IL-8 is up regulated in the circulation of patients with chronic liver disease depending on disease severity and etiology. IL-8 is thought to play a role in the genesis of chronic liver diseases. IL-8 may be associated with polymorphonuclear granulocyte infiltration in patients with primary biliary cirrhosis. In non cholestatic cirrhosis, the IL-8 concentration is associated with hepatic macrophage accumulation. In liver cirrhosis, IL-8 levels were elevated compared to healthy controls, with the degree of elevation varying according to etiology: 2-fold in viral diseases, 5-fold in biliary/autoimmune diseases, and 7-fold in alcoholic diseases /8/.

Lung carcinoma

The prospective Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) /9/ investigated the 10-year risk of developing lung carcinoma as a function of the IL-8 concentration. Based on IL-8 levels, the individuals were divided into 4 groups. The carcinoma risk was highest (8.01%) among smokers with IL-8 levels in the group with the highest IL-8 levels (above 23.3 ng/L) compared to individuals in the group with the lowest levels (less than 13.1 ng/L). The odds ratio was 1.49.

Preeclampsia

IL-8 expression is positively associated with the severity of preeclampsia. The serum IL-8 in preeclampsia patients was 180.3 ± 5.8 ng/L and 41.6 ± 5.7 ng/L in healthy controls /10/

Table 20.4-1 Behavior of TNF-α in diseases and different conditions

Clinical and laboratory findings

Newborn with sepsis

In a study /7/ TNF-α was assayed in comparison with IL-6 in newborns with sepsis. A positive test result for plasma concentration of TNF-α (> 70 ng/L), had a diagnostic sensitivity of 73% at a specificity of 94%. A positive test result for IL-6 (> 500 ng/L), had a diagnostic sensitivity of 80% and a specificity of 78%.

Trauma, SIRS, sepsis in children and adults

TNF-α is the first cytokine to appear in the circulation after experimental sepsis or endotoxemia. Markedly elevated concentrations are measured after 45 min., and peak levels after 90 min., when clinical symptoms appear. Other cytokines such as IL-6 or IL-8 appear in the circulation shortly after. In a test with a TNF-α threshold of 4.5 ng/L, the TNF-α concentration was 98.5 ± 92.1 ng/L in sepsis /8/. In another study patients with intra-abdominal sepsis and endotoxin peak levels of 2.7 μg/L showed moderate TNF-α concentrations of 147 ± 41 ng/L at the onset of sepsis. In the patients with lethal outcome within the following 12 days, TNF-α levels fell below 75 ng/L while in those who survived the concentration was above 160 ng/L. The authors attributed the decrease to the development of an anergic immune status /9/.

HIV infection

Compared to healthy controls, seropositive HIV patients have elevated TNF-α concentrations. Asymptomatic patients had levels of 10–21 ng/L, and in AIDS patients levels were 14–50 ng/L (values are the 25th and 75th percentiles) /10/.

Transplant rejection

In kidney transplant recipients, it may be necessary to distinguish acute graft rejection from episodes of cisplatin A (CsA) toxicity. Using the 95th percentile of uncomplicated courses as a threshold, TNF-α detected graft rejection with a sensitivity of 40–60% and a specificity of 89% versus CsA toxicity /11/.

Patients with acute graft rejection within the first 10 days after pancreas transplantation had plasma TNF-α levels of 323–997 ng/L compared to concentrations of up to 41 ng/L in cases without rejection. The increase occurred two days earlier than the decrease in α-amylase /11/.

Multiple sclerosis

Patients with chronic progressive multiple sclerosis have elevated concentrations of TNF-α in cerebrospinal fluid compared to healthy controls. The plasma level correlates positively with disease activity and the neurological disorders in the following 24 months /5/.

Meningitis

Based on the concentration of TNF-α in cerebrospinal fluid, purulent bacterial meningitis can be differentiated from non-bacterial meningitis as well as meningitis due to Mycobacterium tuberculosis or Borrelia burgdorferi, and the viral types of meningitis. A threshold of 67 ng/L (median + 2 SD of controls) differentiated purulent from a purulent meningitis /13/.

Inflammatory arthritis therapy /14/

Inflammatory arthritides include rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA). The pro inflammatory mechanisms of these diseases are associated with progressive joint destruction early in the disease course. TNF-α plays a dominant role in the inflammatory cascade. Three TNF-targeting agents dominate the biologic management of RA, AS and PsA:

  • Etanercept, a dimeric fusion protein, consists of the extracellular portion of the human p75 TNF receptor linked to the Fc region of human IgG1
  • Infliximab, a chimeric human-murine monoclonal antibody, binds to TNF and consists of human constant and murine variable regions
  • Adalimumab is a recombinant human monoclonal antibody specific to TNF.

Inflammatory activity in rheumatoid arthritis (RA) during treatment with methotrexate can be better assessed with IL-6 than with TNF-α, if the efficacy of treatment cannot be consistently demonstrated based on the erythrocyte sedimentation rate and CRP (Tab. 20.4-2 – Levels of TNF-α and other inflammatory markers before and during treatment of rheumatoid arthritis (RA) with methotrexate/15/.

Table 20.4-2 Levels of TNF-α and other inflammatory markers before and during treatment of rheumatoid arthritis (RA) with methotrexate /15/

Marker

Controls

Inactive
RA

Active
RA

MTX therapy
(inactive RA)

MTX therapy
(active RA)

TNF-α (ng/L)

7.5 ± 2.8

16.0 ± 37.2

24.0 ± 67.1

14.7 ± 39.5

19.2 ± 51.1

IL-6 (ng/L)

8.0 ± 3.8

9.7 ± 11.3

35.4 ± 64.1

11.0 ± 12.4

30.7 ±41.6

RF (IU/mL)

7.8 ± 2.3

107 ± 169

128 ± 166

61 ± 78

120 ± 155

ESR

10 ± 8

23 ± 21

35 ± 24

25 ± 25

34 ± 24

CRP (mg/L)

1.3 ± 0.3

4 ± 8

19 ± 27

3 ± 4

16 ± 21

Values expressed as x ± s; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate

Table 20.5-1 Causes of elevated sIL-2Rα levels /5/

Rejection reactions

Infections

  • Bone marrow
  • Heart, liver, kidney
  • HIV/AIDS, rubella, infectious mononucleosis, pulmonary tuberculosis, sepsis

Neoplasias

Autoimmune diseases

  • Acute myeloic leukemia
  • Anaplastic large cell lymphoma
  • Adult T-cell leukemia
  • Chronic lymphatic leukemia
  • Chronic myeloic leukemia
  • Cutaneous T-cell lymphoma/
  • Mycosis fungoides
  • Hairy cell leukemia
  • Hodgkin lymphoma
  • Non-Hodgkin lymphoma
  • Peripheral T-cell lymphomas
  • Sarcoidosis
  • Aplastic anemia
  • Behcet’s syndrome
  • Crohn’s disease
  • Giant cell arteritis
  • Juvenile rheumatoid arthritis
  • Kawasaki syndrome
  • Multiple sclerosis
  • Polymyalgia rheumatica
  • Rheumatoid arthritis
  • Scleroderma
  • Sjögren’s syndrome
  • Systemic lupus erythematosus
  • Vasculitis
  • Wegener’s granulomatosis

In-vivo treatment with IL-2

End-stage renal disease

Table 20.5-2 Behavior of sIL-2R in diseases and different conditions

Clinical and laboratory findings

Sarcoidosis

Sarcoidosis is a chronic granulomatous disease characterized by an accumulation of lymphocytes and macrophages in the alveoli and with non-caseating epithelioid cellular granulomas which, in over 90% of cases, manifests in the lungs. Diagnosis is made by trans bronchial biopsy and bronchoalveolar lavage (BAL). BAL findings of sarcoidosis patients show a lymphocyte count above 20% and a CD4+/CD8+T-cell ratio above 3 (5). Sarcoidosis cannot be diagnosed by BAL alone /8/.

Clinically, sarcoidosis is classified into acute-onset and subacute-onset course. If left untreated, both courses will, over the years, lead to a distortion of the micro architecture of the lower respiratory tract.

Acute sarcoidosis is characterized by sudden onset, sub febrile temperature, night sweats, fatigue and exhaustion. There is systemic inflammation with T-cell response and an accumulation of lymphocytes and monocytes/macrophages in the alveoli. These produce sIL-2Rα, whose plasma concentration is an indicator of disease activity. In acute sarcoidosis, the median concentrations are 1,000–2,000 U/mL and indicate the need for treatment. Levels above 1,400 U/mL are indicative of extra pulmonary manifestations /9/.

Patients with non-acute sarcoidosis who require treatment also have elevated sIL-2Rα concentrations, but not to the extent as those with acute-onset sarcoidosis. In a study the median concentration was about 1,000 U/mL /9/.

The eye is a potential primary and/or presenting site for the manifestation of sarcoidosis and its clinical manifestation is easily overlooked. In patients with uveitis tested for sarcoidosis sIL-2R was a better marker of sarcoidosis than angiotensin converting enzyme (ACE). The sIL-2R (threshold > 639 U/L) diagnostic specificity was 94% with 98% sensitivity. The corresponding values for ACE (> 82 U/L; using the synthetic tripeptide substrate FAPGG) were 99.5% and only 22% /10/. For the determination of ACE refer to Section 1.5.2 – Method of determination.

Acute transplant rejection

Acute transplant rejection (e.g., after kidney and liver transplantation) are associated with an increase in sIL-2R. In a study of liver-transplant recipients, all patients had elevated sIL-2R levels in the 20 days following the transplantation /11/. However, patients with an acute rejection episode had higher concentrations than those with bacterial and viral infections but no rejection. The change in the sIL-2R concentration from baseline (∆sIL-2Rα) was the best criterion for detecting transplant rejection /11/.

Treatment with anti-IL-2R antibodies with human-mouse chimeric monoclonal anti-CD25 antibodies (Basiliximab) or humanized monoclonal anti-CD-25 antibodies (Daclizumab) reduces the incidence of acute rejection by reducing the effect of IL-2. The antibodies influence the measurement of sIL-2Rα.

Lymphoproliferative disease /11/

Compared to healthy individuals, patients with lymphoproliferative diseases (Tab. 20.5-1 – Causes of elevated sIL-2Rα levels) have elevated concentrations of sIL-2Rα. The highest concentrations have been reported in patients with hairy cell leukemia (48,000 U/mL) and acute T-cell leukemia (69,000 U/mL). Patients with aggressive non-Hodgkin lymphoma also have 10-fold higher concentrations than healthy individuals. In 174 patients with Hodgkin’s lymphoma, the concentration was 1842 ± 129 U/mL compared to 420 ± 10 U/mL in healthy controls /12/.

The concentration of sIL-2Rα prior to treatment is a criterion of tumor burden; a progressive increase during the course of the disease is indicative of a poor prognosis and a decrease is a marker of response to treatment. According to one study /13/, the risk of relapse of Hodgkin’s lymphoma is 16.4% in adults with sIL-2Rα concentrations above 1500 U/mL, but only 1.5% in patients with lower concentrations.

Malignant tumor

Compared to lymphoproliferative diseases, in most solid tumors elevated concentrations of sIL-2Rα are seen only in advanced stages of the tumor /2/.

Autoimmune disease

In autoimmune diseases, sIL-2Rα can be elevated, depending on immunologic activity. As an example, the following concentrations have been reported: 1709 ± 855 U/mL in active systemic lupus erythematosus vs. 252 ± 66 U/mL in healthy controls /15/, approximately 2500 U/mL in Wegener’s granulomatosis vs. less than 500 U/mL in healthy controls /16/ and 946 U/mL in Graves’ ophthalmopathy vs. less than 650 U/mL in healthy controls /17/. Elevated levels are also seen in rheumatoid arthritis, although here their diagnostic value is questionable, since sIL-2Rα lacks sufficient specificity and sensitivity for assessing disease activity. In addition, the sIL-2Rα concentration does not correlate with disease activity following treatment with gold salts, methotrexate or sulfasalazine /18/.

Multiple sclerosis (MS)

In patients with active disease or in a relapse, the concentration of sIL-2Rα in plasma/serum is elevated whereas the findings in cerebrospinal fluid (CSF) are inconsistent. Some studies report elevated levels, even in the chronic progressive form, others do not /19/.

CNS involvement in acute lymphoblastic leukemia (ALL)

In ALL, involvement of the central nervous system is likely if the CSF concentration of sIL-2Rα is above 10 U/mL (diagnostic sensitivity of 89.5% at a specificity of 89.6%). In one study /3/, the CSF sIL2-R level of cell-containing samples averaged 162 ± 248 U/mL compared to cell-free samples with 11 ± 45 U/mL.

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. /4/.

y y y y y y y y y y GA B MAPK y P P13K y y y y y y y y y y y y y y y y y y y y y IL-6 JAK JAK STAT3 SHP2 SHP2 gp130 gp130 Y759 YxxQ Signalling to cell nucleus

Figure 20.1-2 Regulation of hematopoiesis by cytokines. Modified according to Ref. /1/. 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

IL-2IL-4IL-12 IL-4IL-5IL-6 IL-3IL-7 IL-3, IL-7,SCF Lymphoidstem cell Hematopoieticstem cell Self-renewalFit-3L, SCF,gp130 stimulation Myeloidstem cell IL-3, GM-CSF, EPO IL-3,IL-11, GM- CSF, EPO IL-3,GM-CSF IL-3,GM-CSF IL-3,GM-CSF,IL-6 CSF, GM-CSF G-CSF,GM-CSF IL-5GM-CSF IL-4,GM-CSF IL-9 EPO TPOEPOGM-CSIL-6 F B-progenitorcell T-progenitor cell Erythroidprogenitor cell Basophilprogenitor cell Eosinophil progenitor cell Granulocytemonocyteprogenitor cell Megakaryocyte Helper T cell B cell Cyto-toxic T cell Erythrocyte Thrombo-cytes Basophilgranulocyte Eosinophilgranulocyte Neutrophilgranulocyte Monocyte Mastcell IL-7

Figure 20.1-3 Function of cytokines in innate and adaptive immunity, modified according to Ref. /1/. 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.

Neutrophilgranulocyte Eosinophilgranulocyte Restinghelper T cell Fibroblasts Endothelial cells Makro-phage Activatedhelper T cell Mast cell Naturalkiller cell Resting B cell Antibodyproducingplasma cell Resting cytotoxic T cell Contact with antigen Contact with antigenpresenting cell Clonal propagationof helper T cell Activatedcytotoxic T cell IL-6 IL-2 IL-2IL-12IFN-γ IL-2IL-4IL-5IFN-γ IL-2 IL-12 IL-15 IL-8 TNF-α IL-3 IL-4 IL-10 IL-3 IL-5 IL-1TNF-α IL-1TNF-α IL-1, 6, 8,10, 12, 15,IFN-γ,IFN-α IL-3IL-4IL-10IL-13

Figure 20.1-4 Development of the subpopulations of T-helper cells (Th1 and Th2) under the influence of IL-4 and IL-12.

Antigenicstimulation Th0 IL-12 Th1 IFN-γIL-2 Th2 IL-4 IL-4IL-10IL-13

Figure 20.2-1 Increase and decline of IL-6 upon inflammatory stimulation by injection of lipopolysaccharide.

IL-6Half life time < 1 h 200018001600140012001,0008006004002000 IL-6 (ng/L) Time after Injection (h) 0 1 1.5 2 3 4 6 8 IL-61 h

Figure 20.2-2 Cytokine activated transformation of naive T-helper cells (Th0) into different CD4+T-cell subtypes /2/. 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.

Th1T-Bet Th2GATA-3 Th17RORγt TregFoxp3 IL-12 IL-4 TGF-β +IL-6 TGF-β IL-10TGF-β IL-17 IL-4 IFN-γ Th0 InfectionAutoimmunedisease AllergyInflammationAutoimmunediseaseSuppressionof immuneresponse

Figure 20.4-1 Synthesis of TNF-α and action with target cells. Modified with kind permission according to Ref. /5/. 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.

TNF-α binding cell TACE sTNF TNF-α target cell TNFR TNFR sTNF(mono-mers) tmTNF Stimulus Receptor Immature TNF-α DNA mRNA Ribosome Nucleus 5‘ 3‘ Nucleus Apoptosis Activation

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. /5/.

IL-2 Box 1 Box 2 338 357 355 IL-2 358 361 392 510 303 325 363 1 2 PROX(SH2-similar) α β γ c α β γ c JAK1 Lck/Fyn/Lyn Syk JAK3 A H
Goto top <