Interleukin 6 and Haemostasis

Interleukin 6 (IL-6) is a 26 kDa protein produced by many cell types including activated monocytes and macrophages, endothelial cells, adipose cells and the Th-2 subset of T-helper cells (Aarden et al, 1987; Jirik et al, 1989; Mohamed-Ali et al, 1997; Laharrague et al, 2000). After interaction with a specific saturable receptor, present on a great variety of responsive cells, it promotes a range of activities (Fig 1 and Table I) including antiviral effects (Sehgal Cermak et al, 1993; Stirling et al, 1998), and also through stimulation of platelet production (Burstein, 1997). IL-6 was discovered almost simultaneously in 1986 by a number of groups who had been thought to be investigating different proteins – a factor related to its diverse activity (Hirano et al, 1986; Zilberstein et al, 1986). It consists of 212 amino acids with a hydrophilic signal sequence of 28 residues (Hirano et al, 1986). The gene for IL-6, mapped to chromosome 7p21 (Bowcock et al, 1988), consists of four introns and five exons, and has three transcriptional initiation sites (Zilberstein et al, 1986; Yasukawa et al, 1987). IL-6 expression in different cell types is regulated in response to a number of stimuli including endotoxins, IL-1, tumour necrosis factor-α (TNF-α), IL-4 and interferon-γ (IFN-γ) (Van Damme et al, 1987; Shalaby et al, 1989; Howells et al, 1991). Different cell types may respond differently to these stimuli. Indeed, IL-6 expression may be promoted by some cytokines in certain cell types, whereas the same cytokine may inhibit IL-6 expression in other cell types (Howells et al, 1991). The interleukin-6 receptor (IL6-R) is a specific receptor for IL-6 (Fig 2). It is a 468 amino acid protein that has three main regions (Yamasaki et al, 1988; Kishimoto et al, 1995). The first is an outer immunoglobulin-(Ig) like domain that is not required for IL-6 binding (Yawata et al, 1993). The remaining extracellular portion binds IL-6 and is composed of two fibrinonectin type III-like modules (Bazan, 1990). An intracytoplasmic domain, which is also not necessary for IL-6 binding, completes the protein (Yamasaki et al, 1988). Mechanisms of interaction of IL-6 with its receptor. (1) Cells with the IL-6R may bind IL-6 directly inducing homodimerization of gp130 leading to signal transduction. (2) Alternatively, the outer domain of the IL-6R may be shed after proteolytic cleavage by protein kinase C to form soluble IL-6R which may also bind IL-6. (3) The IL-6–soluble IL-6R complex can then cause homodimerization of gp130 and subsequent signal transduction in cells that lack the IL-6R (Kishimoto et al, 1995; Peters et al, 1998). As the IL-6R lacks a tyrosine kinase domain, an alternative method of signal transduction after IL-6 binding must occur. This led to the discovery of an accessory signal transducer, gp130, a 130 kb glycoprotein (Taga et al, 1989) that also acts as a signal transducer for other cytokines such as IL-11. Binding of the IL-6/IL−6R complex to gp130 induces its homodimerization and this is followed by the tyrosine-specific phosphorylation and subsequent activation of a variety of transcription factors (Kishimoto et al, 1995). Several examples of such transcription factors that are involved in fibrinogen gene expression are described below. Soluble IL-6R has subsequently been discovered, and it is formed as a result of cleavage by protein kinase C from the membrane-bound receptor protein (Mullberg et al, 1993). The IL-6R is only expressed on certain cell types, e.g. hepatocytes and B cells (Yamasaki et al, 1988), whereas not on others, e.g. endothelial and haemopoietic cells (Peters et al, 1997;Romano et al, 1997). In contrast, gp130 appears to be on all cells with relatively constant expression (Hibi et al, 1990). This allows cells lacking the IL-6R, which cannot be stimulated by IL-6 alone, to be stimulated by circulating IL-6–soluble IL-6R complex (Peters et al, 1998). Thus IL-6 alone will not stimulate endothelial cells directly, whereas in the presence of soluble IL-6R it will cause homodimerization of gp130 leading to cellular activation. Therefore, soluble IL-6R must be added for in vitro studies of endothelial cell response to IL-6 (Romano et al, 1997). IL-6 promotes coagulation without affecting fibrinolysis. It has been shown that activation of the coagulation cascade, as measured by thrombin–antithrombin (TAT) complex levels increases after infusion of IL-6 to patients with renal carcinoma (Stouthard et al, 1996). In contrast, levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and plasmin-α2–antiplasmin (PAP) complexes are unaffected suggesting that IL-6 does not affect fibrinolysis (Bergonzelli Stouthard et al, 1996). IL-6 promotes haemostasis through a number of pathways (summarized in 3, 4) and the individual mechanisms are described below. Pathways by which IL-6 contributes towards haemostasis – an overview. IL-6 increases platelet production and enhances platelet activation. IL-6 promotes the coagulation cascade through a number of effects which are summarized in Fig 4. Vessel wall injury or inflammation stimulate IL-6 synthesis by endothelial cells. Endothelial cells also respond to IL-6 through the soluble IL-6 receptor. Thus, through these cumulative mechanisms IL-6 promotes haemostasis. Pathways by which IL-6 influences the coagulation cascade. Broken arrows indicate direct action of IL-6. Negative effects are accompanied by the (–) symbol; all other effects are positive. IL-6 increases tissue factor production by monocytes and factor VIII transcription in hepatocytes. Serum levels of VWF rose and protein S fell after administration of IL-6 in a canine model. IL-6 increases antithrombin production by hepatocytes and increases fibrinogen production by a variety of cells. Fibrinogen is a large dimeric protein, each half consisting of three polypeptides – Aα, Bβ and γ which are encoded by separate genes. The transcription of fibrinogen can be significantly upregulated by IL-6 (Amrani, 1990). Hexanucleotide CTGGGA clusters in the promoter region of the fibrinogen Aα− and γ-chain genes have been recognized as being IL-6-responsive elements (Liu Zhang et al, 1995). Studies of the fibrinogen γ-chain gene show that the signal transducer and activator of transcription, STAT 3, associates with the CTGGGA hexanucleotide promoter (Zhang et al, 1995). As its name suggests, STAT 3 carries out the dual functions of signal transduction and transcription activation (Zhong et al, 1994). It may become activated through phosphorylation in response to IL-6 or other stimuli such as epidermal growth factor (EGF). Interestingly, IL-1β which stimulates IL-6 synthesis (Van Damme et al, 1987) has also been shown to inhibit IL-6-mediated rat γ-fibrinogen gene expression by blocking STAT 3 binding (Zhang Mulder et al, 1996; Osnes et al, 1996; Neumann et al, 1997; ten Cate et al, 1997). Other cytokines act to oppose such effects. IL-4, IL-10 and IL-13 have been shown to inhibit IL-1-induced tissue factor synthesis (Osnes et al, 1996) and IL-10 has also been shown to inhibit IFN-γ and TNF-α production during experimental endotoxaemia (Marchant et al, 1994). This illustrates a small part of the complexity of cytokine interactions and their effects on haemostatic balance. We have reported that transcription of the factor VIII gene is also promoted by IL-6 (Stirling et al, 1998). Levels of mRNA increased six- to ninefold basal values in liver cell lines after stimulation with IL-6. In contrast, other cytokines studied (IL-1 and IL-2) did not significantly affect factor VIII gene transcription. The transcription factors NFκB and C/EBP have been shown using mutational analysis to be necessary for increased factor VIII mRNA transcription in the acute-phase response as stimulated by lipopolysaccharide (LPS) (Begbie et al, 2000). Further study is required to elucidate whether these transcription factors are involved in the IL-6-mediated response and/or response to other cytokines associated with the acute-phase response such as IL-1 and TNF-α. Indeed, many protein binding sites have been identified within the factor VIII gene promoter region (Figueiredo McGlynn et al, 1996). Thus, several proteins may act/interact with involvement of a variety of transcription factors to regulate factor VIII synthesis. In a canine model, IL-6 caused an increase in von Willebrand factor (VWF) and a decrease in protein S, the co-factor for protein C inactivation of factors V and VIII (Burstein et al, 1996). We have also shown in man that after infusion of desmopressin (1-desamino-8-arginine vasopressin) there is an increase in the plasma concentration of IL-6, and that this occurs after the increase in VWF. This rise in IL-6 is not secondary to the VWF rise, as in patients with severe VWD the IL-6 response is retained although there is no increase in VWF (Newby et al, 2000). The synthesis of the potent inhibitor of coagulation, antithrombin (AT) may also be influenced by IL-6. AT production has been shown to be inhibited by both IL-6 and IL-1 in cultured hepatocytes (Niessen et al, 1997) and acquired AT deficiency may occur in sepsis as part of the acute-phase response (White et al, 1999). CRP in the was also associated with increased of myocardial and ischaemic but not in a study of healthy et al, 1997). This study also to be in with We have shown a significant of IL-6, CRP and soluble adhesion in with et al, 2001). is expressed on activated to of leucocytes to the site of injury and its upregulation may be the influence of IL-6. in this small a relationship to was not identified for of these at 3 review, IL-6 and CRP levels had but levels may be an marker to IL-6 for of in The has shown CRP to be associated with disease in patients of other factors for first An elevated CRP was a particularly marker in et al, 1996). with who have a CRP in the of have been found to have a increase in of a with in the first four et al, 1997). An increase in of disease has also been found to be associated with an elevated CRP for first et al, 1998). As discussed IL-6 increases transcription of fibrinogen and factor VIII, both of which are associated with an increased of A fibrinogen in the is associated with an increased of disease disease and at and a factor VIII in the is associated with an increased of disease and et al, 1987; et al, 1999). has also been in a between such as IL-6 and disease 1996; 1996). and endothelial cells with have been shown to have increased IL-6 which appears to be mediated through upregulation of the transcription factor et al, et al, 2000). Tissue factor and also found to be increased in cells. of in of the markers IL-1, IL-6, CRP and TNF-α et al, 1999). Further studies are required to elucidate whether this in a in Other and have also been shown to induce expression of pro-coagulants et al, 1997; et al, 1998). Although et did not an between IL-6 and an has been using a model. After elevated IL-6 levels associated with fibrin measured using fibrinogen and using et al, 1993). Although it cannot be from this study whether of IL-6 was the cause or effect of of no significant was found with for the other cytokines IL-8 and TNF-α. A further study has shown IL-6 levels to be to in after and that elevated levels associated with increased including et al, 1994). not found in this study for other acute-phase IL-1 and TNF-α. As described factor VIII transcription is promoted by IL-6. VIII levels have been recently as a major factor for primary and et al, et al, 2000). These studies have to effect of the acute-phase response during which IL-6 levels thrombotic may be As IL-6 has been shown to be central to haemostasis, to its effects may have include IL-6 to coagulation factors in and This may the of factor as has been demonstrated with desmopressin 2000). Alternatively, synthesis or blocking the function of IL-6 may be useful in thrombotic However, of these is without significant of the of function of IL-6, in its levels will also affect and acute-phase Thus, to be specific – for example, blocking the effect of IL-6 on fibrinogen and factor VIII transcription. We have shown desmopressin to cause a rise in plasma IL-6 which with a rise in VWF and factor VIII (Newby et al, 2000). This rise occurs for IL-6-mediated transcription, and other may be IL-6 synthesis may be by and 1999). This may the role associated with these may have an as as an role in the of myocardial infarction. with CRP in the shown to have a in of myocardial in a control of with a of only in with CRP in the et al, 1997). IL-6 induces a by expression of tissue fibrinogen, factor VIII and activation of endothelial cells and platelet production and by the levels of of haemostasis such as antithrombin and protein Although limited to for of IL-6 may become a useful in thrombotic Mechanisms to IL-6, or its the for novel to the of and inhibit the of We are very to for