Interactions Between Cytokines

Individually, cytokines are potent molecules which, in vitro, can cause changes in cell proliferation, differentiation and movement at nanomolar to picomolar concentrations. Injection of cytokines into animals and humans either systemically or locally can also have profound effects on leukocyte migration and function, haematopoietic cell numbers, temperature regulation, acute phase responses, tissue remodelling and cell survival. The individual entries in this book describe the specific properties of each cytokine, but delineation of the mechanisms by which cytokines cause these effects is complicated by the tendency of cytokines to affect the expression of other cytokines and/or their receptors. In addition, it is clear that there are no circumstances in vivo in which cytokines are produced individually. Rather they are produced together with other cytokines in patterns characteristic of the particular stimulus or disease.

The potency of cytokines, and the potential for amplification and damage which excessive cytokine production carries, has resulted in elaborate controls on cytokine production and action. The current view of cytokine biology reflecting these concepts is of a network of positive and negative cytokines, and cytokine inhibitors and inducers, which combine to give an overall biological or clinical response'-6.

From experiments using cytokine proteins as agonists, or by blocking cytokine production or action with drugs or monoclonal antibodies, the contributions which many individual cytokines make to particular aspects of immunity, inflammation and haematopoiesis have been delineated. In addition, the use of transgenic mice which overexpress certain cytokines, or mice deficient in cytokines, their receptors or signalling proteins activated by them, have allowed investigators to determine more precisely the role of many individual cytokines7. What emerges is a highly complex network of interacting cytokines, and for the first time investigators are attempting to provide complex mathemathical models to predict cytokine function. Apparent redundancy in the action of cytokines and the production of inhibitory cytokines or soluble receptors contribute to a chaotic network of interactions*.

Cytokines act in cascades and networks

The effect of a cytokine in vivo will depend on the immediate cytokine environment. This is best illustrated in the context of inflammation; at an inflamed site, many different cytokines and their inhibitors can be detected. Apparent redundancy in the actions of cytokines would imply that blocking a given cytokine would have only a marginal effect. This is not the case, however, as is best illustrated by the profound anti-inflammatory effect of anti-TNF strategies, which currently comprise neutralizing antibodies or soluble receptors9. At the same time, anticytokine measures will be induced, such as increased production of the IL-1 receptor antagonist (IL-1 Ra) and the inhibitory cytokine IL-10. It is the balance of activating and inhibitory cytokines that will determine outcome6. Recently, it has been suggested that certain polymorphisms in the promoter regions of cytokine genes might lead to imbalances in cytokine networks. For example, individuals with a polymorphism in the gene for IL-ip, which gives rise to the overproduction of IL-1 in response to Helicobacter pylori, show a higher incidence of gastric cancer because of extensive gastritis70. It is likely that many more imbalances in the cytokine network will be described because of polymorphisms, and will be found to be important in the pathogenesis of many diseases.

Thl/Th2 polarization

A particularly informative paradigm with regard to cytokine networks has been the description of Thl/Th2 cytokine patterns. Work by Mossman and colleagues identified two groups of cytokines produced by different T helper (Th) lymphocytes which favour either cell-mediated and inflammatory immunity (Thl) or antibody-mediated humoral immunity (Th2)"-". The Th2 cytokines, such as 1L-4, IL-5, IL-6, IL-10 and IL-13, generally promote B-cell function, specifically favouring IgE and IgA responses. A major role here appears to be in host defence against gastrointestinal nematodes. The Thl cytokines, such as IFNy and IL-2, can also have some antibody-promoting effects, but favour an IgG2a response. The main role of Thl cells is actually to clear intracellular bacteria by promoting cellular immunity. These two cytokine groups are also antagonistic in that IFNy inhibits Th2 cytokine production, whereas IL-10, IL-4 and IL-13 inhibit Thl cytokine production, probably via effects on antigen-presenting cells. The initiating factors which predispose towards Thl or Th2 responses are not fully understood. Likely determinants include pathogen-derived molecules, the nature of the peptide being presented by antigen-presenting cells and the nature of costimulatory molecules, all in the context of genetic background7''.

Along with their roles in host defence, polarized Thl and Th2 responses can also contribute to the pathogenesis of immune-mediated diseases. Thl responses are involved in organ-specific autoimmune diseases while Th2 responses are implicated in allergic disorders in genetically susceptible individuals. A key question has therefore concerned the molecular basis for polarization. Both Thl and Th2 cells are derived from a common precursor termed ThO. The issue of polarization therefore lies at the heart of cellular differentiation, and discoveries in this area are proving informative to other areas of biology. The cytokine IL-12, which is produced by antigen-presenting cells, promotes differentiation into Thl cells, whilst IL-4 produced by ThO cells promotes their differentiation into Th2 cells". CD28 is expressed on the surface of Thl and Th2 cells and is an important costimulus to promote activation of both cell types, while a homologue of CD28, termed ICOS, may selectively promote activation of Th2 cells'5-'* Both are activated by different molecules on the surface of antigen-presenting cells, with B7 activating CD28 and B7h activating ICOS79 Different populations of dendritic cells may be involved in this process. In humans, lymphoid dendritic cells promote Th2 cell development while myeloid cells activate Thl cells20.

Transcriptional control is critical since it is the specific transcription of distinct genes that governs polarization. The transcription factors STAT6, GATA3, c-Maf, JunB and NFATc drive Th2 cytokines, while STAT4, T-bet and NFkB are implicated in Thl cytokines'"*-'15. Specific signals that activate these transcription factors have been studied, with Jun N-terminal kinase and p38 MAP kinase being essential for Thl cell function'5. Most recently, it has become clear that some transcription factors, such as T-bet, alter chromatin structure, allowing a Th cell to express specific genes'6. Detailed molecular understanding of these processes may provide opportunities for therapeutic intervention. Inhibitors of specific cell surface proteins, such as T1/ST2 or the chemokine receptor CCR3 on Th2 cells, or IL-18a or CCR5 on Thl cells may also provide possible means to control specific populations. Table 1 summarizes current knowledge on molecules specific for Thl and Th2 cells"-'9

REGULATION OF CYTOKINE PRODUCTION AND ACTION Gene expression

There is some evidence for the constitutive expression of haematopoietic cytokines such as M-CSF, G-CSF, SCF, IL-6 and Epo, which are necessary to maintain steady state

Table 3.1 Thl and Th2 cells

Roles

Inflammation and organ-specific autoimmunity

Humoral immunity and allergy

Cytokines produced IL-2, IFN-y, IL-18, LT

IL-4, ICOS

IFN-y

Activators

Inhibitors

Cell surface molecules

Transcripiton factors

Signals

JNK, p38 MAPK

haematopoiesis. Moreover, several cytokines are presynthesized, and stored either in cytoplasmic granules, e.g. GM-CSF, TGFfS, PF-4, PDGF2'; as membrane proteins, e.g. TNFa, IL-1 ¡3, EGF, TGFa22; or complexed with cell surface binding proteins or extracellular matrix, e.g. TGFp, MIP-ip, IL-823'2''. These pools of cytokine protein are available for rapid release in response to stimulation. Most cytokines, however, are not constitutively expressed in adult animals, but are rapidly produced in response to stimulation. The stimuli for gene expression are well characterized, particularly for the haematopoietic cells. In general, infectious agents such as bacteria, viruses, fungi and parasites, as well as mechanical injury and toxic stimuli are potent cytokine inducers.

In addition to classical antigens, infectious agents also express many nonspecific cytokine-inducing molecules, e.g. endotoxins25. It has recently emerged that Toll-like receptors (TLRs), which are members of the IL-1 receptor superfamily, are receptors for such pathogen-derived molecules. TLR-4 appears to be the receptor for LPS, TLR-2 is responsive to products from gram-positive bacteria (e.g. peptidoglycan)26 and TLR-9 responds to CpG DNA from bacteria27. The so-called Toll/IL-IR (TIR) domain in the cytosolic portions of these receptors signals cytokine induction. TLRs may therefore be the key receptors important for adjuvancy in the immune response. Two signals activated are the transcription factor NFkB and p38 MAP kinase. NFkB is critical for the induction of many cytokine genes, including IL-2, IL-6, IL-8, MCP-1, TNF and GM-CSF. The role of p38 MAP kinase is to stabilize cytokine mRNAs, which are unstable because of multiple AU repeats in their 3' ends. How p38 stabilizes these mRNAs is not known. Inhibition of both signals will block the production of many cytokines.

For many cells, cytokines are themselves potent cytokine inducers. Some, such as IL-1, TNF and IFNy, are particularly potent inducers of cytokine gene expression, and are referred to as proinflammatory cytokines2*. Others, like IL-1 and TNF, are even capable of stimulating their own production2', again acting via signals such as NFkB and p38 MAP kinase. As stated above, specific transcription factors play a key role in the induction of specific cytokines from Thl and Th2 cells.

In addition to the many positive stimuli, several mediators act to limit or prevent cytokine gene expression, or to limit cytokine action. The classical inhibitors of cytokine gene expression are the glucocorticoid hormones and the synthetic steroids which are widely used as immunosuppressants and anti-inflammatory drugs30. They are thought to act as part of an intracellular glucocorticoid receptor complex which binds to glucocorticoid-response elements present in the IL-1, IL-2, IL-3, IL-6, IL-8 and IFNy genes. Many glucocorticoid-sensitive genes do not have glucocorticoid-response elements however, and the steroid/ receptor complex may instead compete with activated transcription factors such as NFkB, for basal transcription factors such as p300/CBP, which are required for induced gene expression. Agents such as cyclosporin A. FK.506 and rapamycin also act via cytokine pathways'5/~''',. Prostaglandins are also known to inhibit the production of cytokines. As described above, many cytokines act to inhibit cytokine production. The antagonistic Thl and Th2 cytokines are the best-studied examples of this. Other examples include TGF(3, which is a broad-spectrum inhibitor of cytokine production, and IL-10, which is a potent inhibitor of TNF and IL-1 production by monocytes'"'^. The mechanism of action of IL-10 is still not fully understood.

Processing

Control of cytokine function can also be achieved by regulating the processing of precursor forms. Many cytokines are initially produced as biologically active integral membrane proteins which need to be proteolytically cleaved to release the active molecule. Examples in this category include EGF, TGFcx, IL-1 (3, IL-1 ex, TNFoe22. Alternatively, cytokines such as TGF(3 are produced as secreted but biologically inactive precursors, which are enzymatically processed to the active forms'i6. Progress has been made in the identification of enzymes involved in processing. IL-ip-converting enzyme is the founder of the caspase family of cysteine proteases, and is now termed caspase-1. It is required for processing of both pro-I Lip and pro-IL-18. TNFa-converting enzyme (TACE) is the processing enzyme for TNF, cleaving the membrane-bound form of TNF. It is a member of the ADAMs family of metalloproteinsasesJ7.

Sequestration

Some growth factors such as TGFp, FGF, LIF and IL-1 are sequestered on extracellular matrix in connective tissues, skin and bone2'. This serves as a sink of active cytokine that can be rapidly released when the matrix is broken down during injury or tissue repair. Other cytokines, such as GM-CSF, IL-3 and SCF. are localized to stromal cell layers in bone marrow where they stimulate haematopoiesis'W. Others, including MIP-ip, and IL-8, bind to endothelial cells at sites of inflammation where they promote leukocyte extravasation2''. In general, these cytokines are sequestered onto glycosaminoglycans such as heparin, decorin or CD44-like molecules.

Soluble binding proteins

Several binding proteins for cytokines are found in blood and tissue fluids. Some of these are secreted forms of the specific cell membrane receptors such as for TNF, IL-1, IL-2 and IL-6, whereas others are less specific such as aj-macroglobulin''9"". These binding proteins may serve as passive carriers of the cytokines, either extending their half-lives or promoting their excretion, or they may act as circulating inhibitors, limiting systemic effects of the cytokines. The soluble form of the IL-6 receptor is unusual in that it complexes with IL-6 to form a biologically active molecule which binds directly to the IL-6 receptor signalling chain*2.

Receptor antagonists

To date the only naturally occurring cytokine receptor antagonist that has been identified is IL-lRa. It is structurally related to IL-1 a and IL-1(3 and binds to the IL-1 receptors, but does not cause signal transduction, thereby acting as a specific receptor antagonist4''-^.

Receptor modulation

Control of cytokine function can also be achieved by modulating receptor number through controlling gene expression, internalization or receptor shedding. Modulation of receptor affinity or function can also occur by control of receptor phosphorylation, or by competition for shared receptor chains or signal transduction molecules'"'''6.

Viruses and the cytokine network

A major area of interest over the past 5 years has been the mechanisms used by viruses to avoid elimination by the immune system. Viruses use an array of strategies here, including inhibition of the humoral response, inhibition of apoptosis, modulation of MHC function and evasion of cytotoxic T lymphocytes''7. A key mechanism has concerned subversion of the cytokine network. Viruses achieve this in a number of ways. Poxviruses can make a range of soluble cytokine-binding proteins, including those for TNF, IL-1, interferons, IL-18 and chemokines. In some cases these are hijacked from the host genome but in other cases (e.g. chemokine-binding proteins) the virus may have coevolved the gene with the host. Other viruses which make various cytokine-binding proteins are Herpes virus 8, Cytomegalovirus and Shope sarcoma virus. Some viruses make homologues of cytokines (termed virokines) which the virus uses either as an antagonist (e.g. viral MIPs from Herpes virus 8) or an agonist (viral IL-10 from Epstein-Barr virus). Viral proteins can also inhibit cytokine signalling, There are many examples of viral proteins that can inhibit interferon signalling, including El A from adenovirus which blocks STAT1 function and Tat from HIV, which blocks PKR activity (a key signal for responses to viral double-stranded RNA). Two vaccinia proteins, A46R and A52R, block IL-1, IL-18 and TLR-4 signalling''*. African swine fever virus makes a homologue of the NFkB inhibitor 1-kB. Poxviruses make an inhibitor of caspase-1 termed CrmA, which blocks IL-1 p production.

Viruses have therefore acquired a range of strategies to manipulate the cytokine network, attesting to its importance for antiviral immunity. Viruses are in fact teaching us the key components of immunity, and in particular, which cytokines are critical for antiviral host defense. Table 2 lists the major strategies used by particular viruses to manipulate the cytokine network.

Table 3.2 Viruses and the cytokine network

Strategy

Used by

Function

Cytokine-binding proteins vIFNa/ßR vIFNyR vIL-18BP vIL-lRII vCKBP vCKR Virokines vCK

vGF vIL-10

vIL-6 Interference with signal transduction STAT inhibition PKR inhibition Inhibition of TNF signalling I-kB

homologue Inhibition of Toll/IL-IR signalling Inhibition of cytokine production Caspase-1 inhibition

Vaccinia virus (B18R) Vaccinia virus (B8R) Molluscum contagiosum virus Vaccinia virus (B15R) Myxoma virus (M-T7) Human herpes virus (ORF-74)

Human herpes virus (vMIP-II) Murine cytomegalovirus (MCK-1)

Vaccinia virus (CI 1R) Epstein-Barr virus (BCRF-1)

Human herpes virus (K.2)

Adenovirus (El A)

HIV (Tat) Adenovirus (E3)

African swine fever virus (A238L)

Vaccinia (A46R, A52R)

Sequester IFNa/p Sequester IFNy Sequester 1L-18 Sequester IL-lfS Sequester chemokines Sequester chemokines

Chemokine antagonist Chemokine agonist

EGF homologue: replication IL-10 homologue: inhibit Thl cells

IL-6 homologue: angiogenic

Blocks IFN signalling

Blocks dsRNA-dependent PKR Prevent TNF cytotoxicity

Inhibition of NFkB

Inhibition of NFkB

Cow pox virus (CrmA)

Inhibits processing of IL-lß and IL-18

Note: For additional examples of viral proteins which use these strategies see ref. 47.

References

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4 Cytokine Receptor Superfamilies

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