Interleukin 6 and Haemostasis (2001)

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 & Sagar, 1980), enhancement of proliferation of haemopoietic progenitors (Wong et al, 1988) and induction of acute-phase reactant release from hepatocytes (Gauldie et al, 1990) – a feature shared with other cytokines, collectively known as the IL-6 cytokine family. It is also this last action that leads to the production of C-reactive protein (CRP), a useful surrogate marker of IL-6 (Heinrich et al, 1990). IL-6 may also be involved in the pathogenesis of disease as has been demonstrated in myeloma (Kawano et al, 1988). Summary of some of the diverse stimuli, sites of production and effects of IL-6. The subject of this review, however, is the role of IL-6 in haemostasis, which is mediated through a series of differing effects on endothelial cells, leucocytes and hepatocytes with promotion of synthesis of coagulation factors such as fibrinogen, tissue factor and factor VIII (Amrani, 1990; 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 & Kruithof, 1991; 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 & Fuller, 1995; 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 & Fuller, 2000). IL-4, IL-10 and IL-13 have also been shown to down-regulate the biosynthesis of fibrinogen by inhibiting the effect of IL-6 on hepatocytes (Vasse et al, 1996). This illustrates the complicated and not yet fully understood interacting cytokine effects on haemostasis. More recently, the transcription factor serum amyloid A activating factor (SAF)-1 has also been demonstrated to induce expression of the γ-fibrinogen promoter in response to IL-6 (Ray, 2000). In the case of Aα-fibrinogen, a novel DNA binding protein is associated with the CTGGGA site when it is not stimulated by IL-6. IL-6 stimulation causes this protein to leave the CTGGGA site and bind 12 basepairs downstream. This protein then re-associates with the CTGGGA site 1–2 h later. Through this effect on the kinetics of the protein, IL-6 controls Aα-fibrinogen transcription (Liu & Fuller, 1995). Most fibrinogen is synthesized in the liver but other sources such as epithelial cells have also been identified and these are also IL-6 responsive (Haidaris, 1997). Peak levels of fibrinogen tend to occur in the morning, which has been associated with an increase in ischaemic events, and this may relate to the circadian rhythm of IL-6 levels (Kanabrocki et al, 1999). The relationship between IL-6, fibrinogen and thrombotic disease is further discussed below. Tissue factor is a potent promoter of thrombin generation through direct activation of factor X by the tissue factor–factor VIIa complex (Osterud & Rapaport, 1977). IL-6, in a concentration of 100 ng/l, has been shown to increase tissue factor mRNA 4·5-fold and also increase the surface expression of tissue factor on cultured monocytes (Neumann et al, 1997). Effects of IL-6 on tissue factor may also be mediated indirectly through CRP, an acute-phase protein released by hepatocytes after IL-6 stimulation (Heinrich et al, 1990). CRP at concentrations found during inflammation has been shown to increase tissue factor procoagulant activity 75-fold in studies using monocyte cultures (Cermak et al, 1993). This is an example of how IL-6 can act synergistically to influence haemostasis through two pathways. Moreover, other cytokines have been shown to contribute to tissue factor upregulation including IL-1 and TNF-α in endothelial cells and monocytes, and IFN-γ and IL-8 in monocytes (Dosquet et al, 1995; 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 & Brownlee, 1995; 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 & Perry, 2001). IL-6 contributes further to haemostasis by enhancing platelet production as elegantly reviewed by Burstein (1997). IL-6 has been shown to promote megakaryocyte maturation in the absence of other growth factors. Also, it possibly stimulates megakaryocyte proliferation. Administration of IL-6 has been shown to result in a significant increase in the platelet count. IL-6 also alters platelet function and enhances thrombin-induced platelet activation. Thus by stimulating the mechanisms of both primary and secondary haemostasis, IL-6 appears to be an important mediator of the development of a stable fibrin clot. Endothelial cells are central to haemostasis through a number of vasoregulatory, coagulation and thrombolytic mechanisms. In the healthy resting state, endothelial cells inhibit haemostasis through production of the anticoagulants AT, tissue-factor pathway inhibitor (TFPI), membrane-bound thrombomodulin (which converts thrombin from a pro- to an anticoagulant role) and through vasodilators such as nitric oxide (NO) and prostaglandins. Endothelial cells also produce tissue plasminogen activator (t-PA). Endothelial procoagulants include VWF and thrombospondin, which promote platelet adhesion and aggregation, and endothelin, which causes vasoconstriction. In addition, production of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells inhibits fibrinolysis (Pearson, 1999). Recent evidence suggests that factor VIII may also be synthesized by endothelial cells (Do et al, 1999). Although this remains controversial, it is supported by the observation that endothelial cells synthesize and release factor V (Cerveny et al, 1984), which has a major degree of strucutural homology with factor VIII, and which also has a very similar co-function in enzyme reactions on the platelet surface. After activation of endothelial cells, and in some disease states, there is increased production of pro-coagulants and decreased production of anticoagulants. As a result, the balance of endothelial cell activity moves to favour haemostasis (Fig 5) (Gross & Aird, 2000). Cytokines play a major role in the control of endothelial cell effects on haemostasis. After stimulation with endotoxins, TNF-α and IL-1, endothelial cells respond by producing a number of cytokines and growth factors including IL-1, IL-5, IL-6, IL-8, IL-11, IL-15, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF) and GM-CSF. These in turn not only change the balance in haemostatic factors as above, but also activate platelets and leucocytes. IL-1 and IL-6 appear to be particularly responsive to the local microenvironment (Bierhaus et al, 2000). IL-6 is not only produced by endothelial cells, they may also respond to this cytokine, for example, by upregulating protein S synthesis (Hooper et al, 1997). It has been shown recently that elevated levels of IL-6 are independently linked to markers of endothelial cell activation such as tPA, thrombomodulin and VWF (Yudkin et al, 1999). Heterogeneity in endothelial cell function in different tissues cannot be discerned in these in vivo studies, and it is only possible to speculate about their relationship with local thrombotic disease such as myocardial infarction. However, in vitro studies of endothelial cells from different tissues are limited by the need for soluble IL-6 receptor and also the removal of these cells from their normal microenvironment through which many interactions may occur. The effects of endothelial cell activation. Following activation the balance of endothelial cell activity moves to favour haemostasis. IL-6 is both by endothelial cells and endothelial cells. factor pathway – – nitric – – tissue VWF – von Willebrand – – A number of other related cytokines which have for the gp130 receptor and which promote the synthesis of acute-phase proteins have been described and with IL-6 are known as the IL-6 cytokine (Taga & 1997). These include factor factor and IL-11. These cytokines may also act to promote haemostasis. example, has been shown to induce fibrinogen biosynthesis and endothelial cell of and VWF et al, 1998). In administration of has been shown to significantly increase VWF and factor VIII, which appears to be mediated through an effect on transcription et al, 2001). However, other of cytokines of the IL-6 may be to IL-6. example, and inhibit whereas these cells are an of IL-6 et al, 1991). Serum IL-6 levels increase with as reviewed by & In contrast, no effect of is for the cytokines and IL-1β et al, 1998). An increase in IL-6 is particularly found in after the et al, et al, 1995). In IL-6 levels rise significantly in the and there is a further rise with the of et al, 1993). and are both factors for thrombotic The rise in serum IL-6 may be an factor to the factors that are in the pathogenesis of thrombotic disease in these The possible role of IL-6 in thrombotic particularly myocardial has a great of recently and there are several on this subject et al, 2000). IL-6 has other in to haemostasis and endothelial cell which may contribute to the development of example, IL-6 circulating concentration and has been shown to cause an increase in in the of a et al, 1999). In addition, IL-6 increases CRP which then binds to leading to and this may contribute to et al, 1999). IL-6 is also associated with (Yudkin et al, 1999). known factors for ischaemic disease may be linked to IL-6. tissue has been shown to contribute as as of the circulating IL-6, and it has been that may a may also affect IL-6 levels as may stimulate IL-6 release from adipose tissue (Yudkin et al, 1999). A to at in the IL-6 gene promoter has been discovered in patients with with the show IL-6 expression after stimulation with or IL-1 and basal levels of IL-6 et al, 1998). These may be from thrombotic Several studies have demonstrated an between IL-6 and/or CRP and ischaemic disease These are summarized and in Table In the IL-6 concentrations in the are associated with a of from both and all causes of that for concentrations in the et al, 1999). This study also demonstrated a of for CRP in the the of the normal both IL-6 and CRP are in the the of to IL-6 has also been shown to significantly with factors for fibrinogen, cell plasma and and et al, 1999). CRP in the of the normal range has been shown to be associated with disease and disease factors – increased increased and and in et al, 1996; 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 normal 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, need 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