Cytokines as cellular communicators

Cytokines and their receptors are involved in the pathophysiology of many diseases. Here we present a detailed review on cytokines, receptors and signalling routes, and show that one important lesson from cytokine biology is the complex and diverse regulation of cytokine activity. The activity of cytokines is controlled at the level of transcription, translation, storage, processing, posttranslational modification, trapping, binding by soluble proteins, and receptor number and/or function. Translation of this diverse regulation in strategies aimed at the control of cytokine activity will result in the development of more specific and selective drugs to treat diseases.


Introduction
For over two decades, it has been recognized that immune-competent cells produce peptide mediators, now termed cytokines. Cytokines act as chemical communicators between cells, but mostly not as effector molecules in their own right. The biological function of many cytokines is mediated through their ability to regulate gene expression in target cells. Recent developments have made it clear that cytokines play crucial roles in the regulation of normal growth and development, protection against inflammation and infection, neoplastic transformation, wound-healing and angiogenesis. For example, inflammatory mediators are involved in the pathophysiology of septic shock and they are often associated with disease severity. In such diseases the balance between pro-inflammatory cytokines (interleukin-1 (IL-1), IL-6, IL-8, tumour necrosis factor-alpha (TNF<z)) and anti-inflammatory compounds (IL-1 receptor antagonist (IL-lra), IL-IO, soluble IL-1 receptor (slL-1R), sTNFR) determines to a large extent the final outcome. Also, in many diseases it was shown that production levels or activity of important cytokines is perturbed. Understanding the control of cytokine activity is therefore important as it may provide new potential strategies to treat diseases.
In general, cytokines are not expressed constitutively, but are rapidly produced after stimu-lation and act locally to restore homeostasis. Cytokines bind with high affinity to specific receptors on the surface of target cells. When cytokine receptors are expressed on the cytokine-producing cell, autocrine cellular activation may be the result. If the receptor is expressed on a neighbouring cell, binding may result in paracrine cytokine activation. These features could explain the sometimes high local concentration and profound local effects of cytokines. A characteristic feature of cytokines is their functional pleiotropy (cytokines result in diverse effects upon binding to different target cells) and redundancy (several cytokines can take over each other's effects), which can be largely explained at the molecular level of receptor systems (see below).  IL-6, IL-11, LIF, OM, CNTF, CT-1  IL-3, IL-5, GM-CSF  IL-2, IL-4, IL-7, IL-9, IL-13, IL-15   IL-10, IFN-, IFN-I, IFN-y   M-CSF   IL-lcz, IL-113, IL-lra, FGF-(z, FGF-I   TNF-ot, TNF-I, LT-I   TGF-(z, EGF   TGF-II, TGF-132, TGF- growth factor (PDGF), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) (cysteine knots); and chemotactic cytokines (chemokines) (triple-stranded, anti-parallel ]3-sheets in Greek key motiO. [2][3][4] The large family of chemokines can be subdivided into at least cchemokines (the two most amino-proximal cysteine residues are separated by an intervening amino acid: CXC) and [3-chemokines (CC), which are functionally distinct, as they predominantly attract neutrophils and monocytes, 5 respectively. It is intriguing that different structural domains of cytokines (e.g. IL-I[3) may display different or even antagonistic cytokine activities.6

Families of Cytokine Receptors
Cytokine receptors are classified into several superfamilies based on common homology regions. The main superfamilies recognized today are the cytokine receptor superfamilies class I and class II, the immunoglobulin superfamily, the TNF receptor superfamily, the protein tyrosine kinase receptor superfamily, and the chemokine receptor superfamily. Figure 1 illustrates schematically these cytokine receptor superfamilies. It should be noted that the superfamilies are not restricted to cytokine receptors (for example, several hormone receptors and leukocyte membrane polypeptides also belong to cytokine receptor superfamilies) and several cytokine receptors belong to more than one 418 Mediators of Inflammation Vol 5 1996 superfamily (e.g. IL-6 receptor belongs to both the class I receptor and immunoglobulin superfamilies). All cytokine receptors are single transmembrane proteins, except for the chemokine receptors which contain seven transmembrane domains. The cytokine receptor superfamily class I receptors are characterized by the presence of combinations of cytokine and fibronectin III domains in the extracellular regions. The amino terminal cytokine domain(s) contains four conserved cysteine residues, and (one of) the membrane proximal fibronectin III domains contains the Trp-Ser-X-Trp-Ser (WSXWS) motif required for ligand binding. 7 Functional class I receptors consist of a complex of two or three receptor chains. These multisubunit receptors can be grouped according to the identity of common chains which are required for high affinity ligand binding and signal transduction. Three subgroups of class I receptors were identified sharing either gpl30 (a glycoprotein with a relative molecular mass (Mr) of 130 kDa, cluster of differentiation (CD) 130), the common -chain (Mr: 120 kDa, KH97) or the common ,-chain (Mr: 64-kDa) (see Table 1 and Fig.   1). The gp130 chain is shared by the receptors.
for IL-6, IL-11, ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OM) and cardiotrophin-1 (CT-1). 9 Ligand binding by this group of receptors induces homodimerization of gpl30 ( gp130 and LIFR (in case of CNTFR). LIE OM and CT-1 are believed to directly induce heterodimerization of gpl30 and LIFR (OM and perhaps CT-1 also induce heterodimerization of gpl30 and the ligand-specific receptor). The LIFR, granulocyte-colony stimulating factor (G-CSF) R and IL-12R are highly homologous with gpl30 and belong to the so-called gp130 family.
Recent evidence suggests that the cytoplasmic parts of these signal transducing chains harbour two functional domains: a membrane-proximal region which is essential for proliferation signals (c-myc induction), and a membrane-distal region which is essential for differentiation signals (c-101 los and c-jun induction).' The signalling routes which result from activation of the common chains are discussed below. The cytokine receptor superfamily class II receptors (interferon (IFN) R and IL-IOR) contain one or two extracellular fibronectin III domains, which lack the WSXWS motif charac-  Recent studies have demonstrated that the cytoplasmic domains of cytokine receptors are cytokine genes distinct combinations of these critical for signal transduction. Ligand-induced transcription factors determine the transcripreceptor di-or oligomerization typically results tional activity of these loci. Activation of the in activation of (receptor-associated) protein individual cytokine genes requires binding of tyrosine kinases (such as members of the SRC distinct sets of transcription factors. The data kinases OAK)) which are associated with the on the involvement of the JAK/STAT pathway cytoplasmic tails of cytokine receptors. Subse-do not preclude that the activation of these quently, activated JAK1 are thought to phos-genes may be secondary to the activation of the phorylate several substrates (such as the above mentioned combination of transcription receptors themselves, members of the RASfactors. mitogen activated protein (MAP) kinase cascades, and transcription factors). Phosphorylation of tyrosine-based motifs in the cytoplasmic Soluble Cytokine Receptors domains of the receptors is followed by specific docking and activation of members of the STAT It seems that essentially all single transmemfamily of proteins. 15'16 Activated STATs form brane cytokine receptors exist as soluble homodimers, translocate into the nucleus, and forms. 19 Soluble receptors can be translated bind to specific responsive elements to initiate from differentially spliced pre-mRNA molecules gene transcription. This JAK-STAT pathway relacking the transmembrane domain (e.g. IL-4R, suits in rapid and specific transcriptional IL-5R, IL-6R, IL-7R, IL-9R, GM-CSFR, G-CSFR, activation of target genes, and may, at least in TGF-cR). However, the presence of mRNA for part, contribute to cytokine-specific responses soluble receptors not always seems to represent through selective activation of individual JAK protein synthesis. Soluble receptors can also be kinases (JAK1, JAK2, JAK3 and TYK2)and STAT generated by proteolysis of cell surface recepfactors (STAT1 through STAT6). 7 '18 Table 2 tors (e.g. IL-1R, IL-2Rcz, IL-6R, gpl30, PDGFR, presents more details on receptor-mediated NGFR, TNFR, TGF-R) and by phospholipase activation of induced JAK kinases and STAT action on the glycosyl-phosphatidylinositol factors for various cytokine receptors. Besides (GPI) anchor of CNTFR. Proteolytic shedding this tyrosine kinase pathway, also the serine may be controlled by protein kinase C and may kinase pathway is involved in cytokine-receptor involve a metalloprotease, since phorbol myrismediated signalling. In activated T-cells the tate acetate (PMA)-induced shedding of IL-6R, activation of transcription factors, like activating TNFR type I and II as well as membrane TNF-(z protein-1 (AP-1) (fos/jun dimers), members of is blocked by a metalloprotease inhibitor, which the nuclear factor in activated T cells (NF-AT) was first identified as TNF-0t protease inhibitor family and NF-KB, play distinct roles in the (TAPI). 2'2] Soluble cytokine receptors are found induced differentiation. In the promotors of in appreciable amounts in body fluids, although 420 Mediators of Inflammation Vol 5 1996 the exact cellular source and function of these proteins is not completely clear.

Virokines
The immunological relevance of cytokines and (soluble) cytokine receptors is clearly illustrated by the observation that several pathogenic virus strains, particularly pox viruses, encode proteins which counteract the anti-viral cytokine activity during the host response. 22 Examples of these so-called virokines include a homologue of the host suppressive cytokine IL-10 encoded by Epstein-Barr virus 2 and several poxviral homologues of soluble IL-1R (sIL-1R), sTNFR and sIFNR, which prevent interaction of the ligands with cellular receptors. 2z'z4 In addition, herpes viruses and cytomegalovirus encode chemokine receptors which are expressed on the membrane of infected cells, but probably display an altered function. 2 Furthermore, intra-cellular viral proteins have been described that interfere with cytokine maturation and cytokine-mediated responses. 22 Cytokine Biology The current view on cytokine biology is that of a network of cytokines with additive, synergistic and opposite effects, and inducers and inhibitors of the expression of cytokines and/or their receptors, which combine to give an overall biological (or clinical) response. 26 Cytokines are potent molecules, of which the activity is regulated at multiple levels. Second, the extent of cytokine protein production is limited, since the consensus sequence UUAUUUAU in the 3' end of most cytokine mRNAs promotes message instability. 5 Translation of cytokine mRNA transcripts may, in addition to the first signal, require an additional signal (e.g. in the case of IL-I). 31 Third, several cytokines are not released through the classic secretory pathway. These cytokines are produced as larger biologically inactive precursor molecules, which are stored within the cytoplasm (e.g. IL-1, IL-8, GM-CSF, TGF-[3, PDGF) or expressed on the plasma membrane (e.g. TGF-c, IL-lc, TNF-cO, and need to be proteolytically cleaved to release the mature molecule (e.g. TGF-{x, IL-Iz, IL-I, TNFcz). [2][3][4] Extracellular enzymes may also contribute to the processing of precursor molecules (e.g. IL-I, TGF-). 5,6 Fourth, post-translational modifications (e.g. cleavage-site of leader peptide, glycosylation, phosphorylation and/or multimerization) affect the biological activity of mature cytokines (e.g. It-6, It-8). [37][38][39] Fifth, once secreted, several cytokines are trapped by cell surface binding proteins (e.g. TGF-), extracellular matrix (e.g. LIE IL-1, FGF), stromal cells (e.g. IL-3, IL-5, GM-CSF) and endothelial cells (e.g. IL-8). 3'4 It should be noted that the intracellular, membrane and extracellular pools of cytokines are available for rapid maturation and/or release in response to stimulation.
Sixth, soluble proteins which bind to cytokines can potentiate or antagonize cytokine activity. The proteins bind to cytokines either nonspecifically such as 2-macroglobulin 41 or specifically such as autoantibodies to cytokines (e.g. IL-I, IL-6, IL-8, IL-IO, monocyte chemotactic protein-1 (MCP-1), IFN-x) 42,4 and soluble cytokine receptors. 19 So far all known soluble forms of cytokine receptors were found to retain their ligand binding capacity. On the one hand, these soluble proteins may agonize cytokine activity by acting as chaperons, increasing 4,44 the cytokine's persistence.' Moreover, in case of slL-6R and sCNTFR, the soluble proteins form a biologically active binary complex with their ligands (IL-6msIL-6R, CNTFmsCNTFR) which may act on cells only expressing gp 130 on their cell surface, but not IL-6R or CNTFR (normally anticipated as cells being unresponsive to IL-6 or CNTF). 19 Note that in line with this 'trans-signalling pathway' is the fact that gp130 is present essentially on all cell types, whereas IL-6R and CNTFR are not expressed ubiquitously. On the other hand, soluble proteins may antagonize cytokine activity by acting as scavengers of cytokines (by competing with membrane receptors), by neutralizing their activity, by enhancing antibody-dependent cell-mediated cytotoxicity, by preventing extravascular escape and by promoting excretion. 4 Whether soluble proteins will act as agonists or antagonists of cytokine activity is, at least in part, determined by the ratio between membrane receptors and soluble proteins: the predominance of membrane receptors favours agonistic activity, whereas the predominance of soluble proteins favours antagonistic activity. 44 Finally, cytokine activity can be regulated by modulating the number and/or function of membrane receptors. Receptor number may be modulated by controlling gene expression, internalization or the generation of soluble receptors. Receptor affinity and/or function may be modulated by affecting receptor phosphorylation and/or glycosylation, by competition for common receptor chains or signal transduction molecules 16'41 or by binding cytokine receptor antagonists. These latter molecules bind to the receptor but do not cause signal transduction (for example, IL-lra, IL-462 and (p40)2 antagonize IL-1, IL-4 and IL-12 functions, respectively). [45][46][47] Strategies aiming at controlling cytokine activity take this into account and are schematically illustrated in Fig. 2.