Interleukin 6 (IL-6)

IL-6 is a cytokine that is produced by a variety of cells, including macrophages, marrow stromal cells, endothelial cells and fibroblasts (Kishimoto et a l, 1995). In vivo studies in both mice and primates have shown that IL-6 promotes megakaryocytic maturation, ploidy, and platelet production (Asano et a l, 1990; Ishibashi et a l, 1989).

1.5.1 The structure o f IL-6 and its gene

Human lL-6 is a variably glycosylated, 22-27 kDa secreted glycoprotein that serves as a prototype for several other cytokines including leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) and IL-11. Although all molecules possess a similar helical structure, their association is due to their functional redundancy and receptor interactions. lL-6 is translated as a 212 amino-acid molecule, with a 28 amino-acid signal sequence and a 184 amino-acid mature segment. It contains four cysteines and two potential N- linked gylcosylation sites with its primary structure showing limited homology to G- CSF. An alternate splice variant of lL-6 was identified in monocytes and lymphocytes. This form is 17 kDa and 148 amino-acids long and appears to lack a binding site for the lL-6 signal transducing molecule gpl30. Mouse and rat lL-6 also have been cloned and are approximately 40% identical to human lL-6 at the amino- acid level (Chiu et a l, 1988). Unlike human lL-6, mouse and rat lL-6 lack potential N-linked gylcosylation sites, but may be 0-glycosylated (van Snick et a l, 1988). The presence or absence of gylcosylation, however, has no effect on bioactivity.

1.5.2 Genetic polymorphisms

Several polymorphisms in the lL-6 gene have been described although only one of these has shown to have a functional correlate (Fishman et a l, 1998). This polymorphism involves a change of a single base, from guanine to cytosine at position -174 in the 5’ flanking region of the interleukin-6 gene. The G allele is associated with higher lL-6 production than the C allele. This has been demonstrated both in vitro and in vivo. In vitro studies have shown that HeLa cells transfected with the G allele produce a much greater response to stimulation with lipopolysaccharide or lL-1, than cells transfected with the C-allele. In vivo studies have shown that basal

IL-6 levels are twice as high in volunteers with the G allele than those homozygous for the C allele.

1.5.3 The production and regulation o f IL-6

IL-6 production is generally correlated with cell activation and is induced by TNF-a, IL-1 and LPS. Circulating IL-6 can be found in the blood of normal individuals in the 1 pg/mL range (Yamamura et a i, 1998), with slight elevations during the menstrual cycle (Angstwurm et a l, 1997), modest elevations in certain cancers (melanoma) (10 pg/mL) (Mouawad et a l, 1996) and large elevations after surgery (30-430 pg/mL) (Sakamoto et a l, 1994). Consideration of the action of IL-6 involves an understanding of the interaction of the cytokine with its receptor. The functional receptor for IL-6 is a complex of two transmembrane glycoproteins (gpl30 and IL-6 receptor - IL-6R) that are members of the Class I cytokine receptor superfamily. IL- 6 binds only to IL-6R that then interacts with gpl30 - the signal transducing mechanism that is also used by the other cytokines mentioned above. It is suggested that IL-6R is also upregulated in response to stimuli that produce the release of IL-6 thereby conferring IL-6 responsiveness on a cell (Peters et a l, 1996). This interaction allows the recruitment of ligands to the cell surface that activate the signalling pathways via gpl30 (Taga & Kishimoto, 1997). Added to the control mechanism for IL-6 is the finding of soluble IL-6R in human serum. Soluble IL-6R binds circulating IL-6 extending its half-life, and, on the surface of cells expressing gpl30, forms a signal transducing complex (Kishimoto et a l, 1995). Cells known to express IL-6R include CD4^ and CD8^ T cells (Wognum et a l, 1993), hepatocytes (Geisterfer et a l, 1993), CD34^ stem cells (Tajima et a l, 1996), neurons (Schobitz et a l, 1993), neutrophils (Modur et a l, 1997), monocytes (Mullberg et a l, 1993) and osteoblasts (Udagawa et a l, 1995).

1.5.4 Biological activities o f IL-6

1.5.4.1 7h vitro activity of IL-6

Biological properties attributed to EL-6 include its ability to induce B-cell differentiation, haematopoietic stem cell differentiation, and plasmacytoma and myeloma cell growth. Additionally, IL-6 possesses immunomodulating properties involving enhancement of NK cell activity and induction of cytotoxic T-cell activity (Hirano et a l, 1990; Kishimoto, 1989; Sehgal, 1990).

EL-6 is capable of promoting MK maturation in the absence of other added growth factors. Its effects on thrombopoiesis are mediated through effects on MK differentiation, with a relatively small effect on circulating haematopoietic progenitors (Clarke et a l, 1996). This process would, however, appear to be mediated by the soluble IL-6 receptor that in turn stimulates the gpl30 receptor (Sui

et a l, 1999). MKs themselves produce both IL-6 and IL-6R (Navarro et a l, 1991). Although the significance of this is unclear it has been suggested that this may be part of a self-regulatory mechanism in which the conditions for megakaryocytopoiesis are optimised (Wickenhauser et a l, 1995)

1.5.4.2 In vivo activity of EL-6

Recombinant human IL-6 has been shown to increase platelet counts in rodents (Nagasawa et a l, 1990), primates (Mayer et a l, 1991) and in patients with advanced cancer participating in phase I clinical trials (Weber et a l, 1993). IL-6 induced thrombocytosis appears to be a result of accelerated megakaryocytopoiesis since circulating IL-6 levels appear to correlate with increased MK size and ploidy (Mei & Burstein, 1991). High dose IL-6 administration to primates however, results in aberrant megakaryocytic development including membrane hyperplasia, megakaryocytic cell death, and the presence of giant platelets (Stahl et a l, 1991). Studies in IL-6 treated dogs show increased platelet activation, as assessed by P- selectin expression, in response to thrombin or platelet activating factor (FAF) (Peng

et a l, 1994a). In contrast to human studies, EL-6 did not directly stimulate canine platelets. The increase in platelet function observed in animals treated with IL-6 was therefore attributed to changes occurring during platelet production.

However, as far as human platelets are concerned IL-6 renders platelets more sensitive to activation by thrombin and PAF (Asano et a l, 1990; Ishibashi et a l,

1989) as well as increasing platelet production and MK maturation. The sensitisation of platelets by EL-6 has been shown to be a direct effect of IL-6 on the platelets via a mechanism involving arachidonic acid metabolism (Oleksowicz et a l, 1994; Oleksowicz et a l, 1995).