Rheumatoid arthritis (RA) is an inflammatory autoimmune condition principally causing synovial joint inflammation and cartilage erosion. The pathogenesis encompasses intricate cellular and humoural manifestations, and vascular reactions that result in the infiltration of the synovium by white blood cells, of which release inflammatory mediators, inclusive of Interleukin-6 (IL-6). IL-6 serum levels of RA patients as shown by Capell et al. (1993) displayed a median of 55 IU/ml, as compared to that of healthy controls of 10 IU/ml.
With a wide-ranging pleiotropy endorsed by both a membrane-bound (IL-6R) and soluble (sIL-6R) receptor, and by the relative omnipresence of the trans-membrane protein gp130, IL-6 endorses a pro-inflammatory effect via its influence on numerous cell types and signalling-pathways. As a result, heightened levels of IL-6 aids in the promotion of osteitis, sequential joint damage, pain/discomfort and impaired function in RA patients.
Pleiotropy of IL-6
Interleukin 6 exerts effects on numerous pathways contributing to the pathophysiology of RA. IL-6 as it is called today has been known by several names that exemplify its pleiotropy for example, hepatocyte-stimulating factor known to cause the induction of C-reactive protein (CRP); due to IL-6 association with synovial fibril aggregation has been known as Amyloid protein; a thrombopoietin; both B-cell differentiation and stimulating factor 2; plasmacytoma growth factor; and cytotoxic T-cell differentiation factor. It also causes the differentiation of Th17 cells; is a causative factor in adhesion molecule expression on the surface of endothelial cells, and is involved in the differentiation to mature from precursor osteoclasts cells (REF!!). IL-6
IL-6 implements its influence via a protein complex primarily comprised of a membrane bound IL-6R and a glycoprotein comprised of two intra-cytoplasmic transducer sub-units, gp130. When IL-6 binds to membrane bound IL-6R (mIL-6R) it causes homo-dimerisation of the gp130 sub-units, of which triggers intra-cytoplasmic signal transduction. Whilst expression of gp130 is relatively omnipresent upon the surface of the body’s cells (Akil, et al., 2008), IL-6R is most prominently located on hepatocytes, macrophages, monocytes, neutrophils and select lymphocytes. However, (sIL-6R) of which is systemic also binds IL-6, and just as mIL-6R, can also engage with gp130 for sIL-6-gp130 trans-signalling (REF!).
Synoviocytes, for example do not express mIL-6R but do express gp130. Raised levels of IL-6 in the synovium is a characteristic biomarker of RA (Attar, et al., 2010), and Kim, et al. (1996) states enhanced IL-6/sIL-6R in synovial fluid increased the risk of joint destruction, as IL-6 stimulates endothelial cells to express adhesion cytokines and other molecules of which attract inflammatory cells to synovial membrane (Romano, et al., 1997) thus could contribute to exemplifying the significance of sIL-6R in RA pathophysiology. sIL-6R is formed by either an incomplete proteolytic enzymic dissection of mIL-6R or alternative splicing of mRNA (REF!!).
With the aforementioned ubiquitous nature of gp130, mIL-6R and systemic sIL-6R, increasing evidence REFERENCES SEE NOTES!! shows that a non-membrane bound, systemic, soluble gp130 (sgp130) found in higher circulatory concentrations than that of sIL-6, also binds IL-6/sIL-6R, thus functioning as a redundency factor inhibiting the cytoplasmic signal-transducing potential of mgp130 REFERENCES SEE NOTES!!, (IL-6/IL-6 receptor system and its role…) thus serving as a natural inhibitor of IL-6 signalling (IL-6/IL-6 receptor system and its role…).
gp130 dimerisation brings Janus Kinases (JAKS), a receptor-associated protein complex, into close proximity causing a trans-activation of the two molecule types. Auto-phosphorylation of JAKS occurs, of which causes intracellular signal transduction by recruiting signal-transducers and activators of transcriptions (STAT) that form either hetro or homo dimers and migrate to cell nucleus effecting target gene transcription of various physiological processes (REF). IL-6 can be detrimental to human physiology (REF!), thus expression of proteins known as the suppressors of cytokine signalling (SOCS) function as a negative-feedback system, and are activated by STAT’s. The regulation of the JAK-STAT signalling pathway by SOCS is more specifically down-regulated by SOCS 3 (REF!). SOCS 3 binds JAK’s causing negative-regulation thus functioning as an auto-regulatory mechanism, by inhibiting JAK activity.
IL-6 and the Adaptive Immune System
The trans-signalling of IL-6 is known to cause the induction of pre-B-cell-colony-stimulating factor (PBEF) in fibroblast cells of the synovium (Bryant, et al., 2006), and since PBEF upon its discovery was considered a B-cell differentiation cytokine(McNiece et al., 1994), it could be considered plausible that IL-6-induced PBEF and the IL-6 maturation of B-cells, collectively contribute to RA. It has been found the effect of IL-6 on plasmablasts indirectly induces the production of B-cell antibodies by assisting elements of CD4+ T-cells (of which act upon activated B-cells) due to elevated IL-21 production (Bond, et al., 2009), thus IL-6 may potentially be a co-adjuvant to humoural immunity enhancement (Bond, et al., 2009). Enhanced levels of the RA associated Rheumatoid Factor are located in the IgA, IgG and IgM isotypes (Ahmed, et al., 2010), and citruline antibodies located in serum and joints, can be linked to the plasmablast-induced antibody production of IL-6 (Ahmed, et al., 2010).
IL-6 enhances T-cell proliferation where they have been mitogen stimulated (Mihara, et al., 2002). IL-6 also impacts T-cell development (Mihara, et al., 2002). Along with transforming growth factor (TGF)- β, IL-6 contributes to Th 17 differentiation, an effector T cell with pro-inflammatory elements, and is further compounded by Th 17 production of the pro-inflammatory cytokine IL-17 (Bettelli, et al., 2007). Interestingly, without IL-6, TGF- β induces Treg production, of which are Th 17 cell suppressors (Hirota, et al., 2008). CD4+ Th cells have been considered Th 1 and Th 2 based upon their cytokine-producing characteristics (Diehl and Rincon, 2002).
Th 1 and Th 2 produce IFN-ᵧ and IL-13 respectively, of which are both pro-inflammatory molecules, however whereas IL-6 bolsters IL-4 induced differentiation of Th 2, it causes the inhibition of IL-12 induced differentiation of Th 1 (Diehl and Rincon, 2002). Acosta-Rodriguez, et al. (2007) found that in vitro levels of IL-1β – induced Th 17 polarisation of naïve human CD4+ T-cells were heightened by IL-6 involvement. More research however, is required to make clear the full extent of IL-6 role in human Th 17 cell development, in vivo.
During inflammation, neutrophils of which are essential inflammatory mediators, systemically increase substantially, resulting in relative neutrophilia. Endothelial cells, macrophages and monocytes all emit IL-6. Neutrophils are directly affected by IL-6 due to the expression of IL-6R. Filer, et al. (2005) found that co-cultured endothelial cells and fibroblasts extracted from synovial fluid of RA patients caused an increase in IL-6 and neutrophil recruitment. Adhesion molecules, of which it has been shown in the work of Woodfin et al. (2010) to be required in the transmigration of neutrophils, are augmented by IL-6 such as vascular cell adhesion molecule – 1 (VCAM) and intracellular adhesion molecule – 1 (ICAM) of which produce chemokine production.
Thus neutrophils being the most numerous and systemic of leucocytes have a strong initial synovial presence and is bolstered by the amplification of the inflammatory cascade, contributing to the inflammatory escalation during acute-phase response; and findings from animal and human studies revealed that the blockade of IL-6 caused a reduction in neutrophil levels at inflamed sites (Hashizume, et al., 2008), as well as a reduction in systemic neutrophil counts in RA patients (Deguchi, et al., 2003 and Broll, et al., 2006),thus indicating a prominent role for IL-6 in neutrophilia. IL-6: Acute to Chronic
Farnarier, et al. (2003) suggests the transition from acute to chronic inflammation as emphasised by a shift of biomarker from neutrophil to monocyte, is influenced by IL-6. It was found that if stimulated for a number of hours by inflammatory cytokines, neutrophils switched from the production of IL-8 and transitioned to monocyte chemoattractant protein-1 (MCP-1) (Yamashiro, et al., 1999). sIL-6R is released from neutrophils of which in turn causes the chemokine release of endothelial cells, thus Romano, et al. (1997) suggests the IL-6 – sIL6R complex contributes to the release of MCP-1 from endothelial cells.
As earlier discussed, neutrophil-count was found to be directly associated with the blockade of IL-6R?!; and endothelial cells expressing the gp130 but not the IL-6R, thus reliant upon the IL-6 – sIL6Rα complex for induction of MCP-1 release, i.e. the release of monocyte, not neutrophil specific chemo-attractants (Gres, et al., 2001), it would appear IL-6 tran-signalling plays an integral role in the transition from acute to chronic inflammation via neutrophil and endothelial cells. *CHART SHOWING CELLS THAT RELEASE IL-6!!*
IL-6 induces a disintigrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinase (MMP) proteinases of which are pivotal in the degradation of extracellular matrix. IL-6 more specifically, has been shown to induce ADAMTS-4 and MMP’s 1, 2 and 13 production in cells lining the synovium and chondrocytes (Hashizume and Mihara, 2009; Hashizume, et al., 2010; 2012). However, it has been suggested the IL-6 – sIL-6R complex has bearing on the extracellular matrix turnover, as it causes generation of tissue inhibitors of MMP’s (TIMP’s) in synovium fibroblasts and chondrocytes (Dayer, et al., 1998 and Hashizume, et al., 2012). The drug tocilizumab (TCZ), an IL-6 inhibitor reduces MMP-3 blood serum levels of RA patients (Garnero, et al., 2010), and has been show to restore biomarkers associated with cartilage turnover (Dayer, et al., 1998).
Thus, in RA patients the reduction of IL-6 activity appears to be a mediatory factor in sustainment of healthy joint cartilage. Angiogenesis is a key process in the local inflammatory process. Neovascularisation of the synovium and other angiogenic processes such as hyperplasia of synovial cells and permeation by inflammatory cells are characteristic processes in pannus development and RA pathology (Ballara et al., 2001). In addition to notable constituents of the inflammatory process such as monocytes and T cells, both of which as stated previously have affiliations with IL-6 expression (CITATION OF A FEW), levels of a key angiogenic specific growth factor Vascular Endothelial Growth Factor (VEGF) has also been associated with systemic levels of IL-6, as demonstrated in the work of Hasizume, et al., 2009 and Hagihara, et al., 2003 revealing IL-6 induces VEGF production from synovial cells.
VEGF induces proteins that contribute to the breakdown of endothelial basement membrane, including MMP’s, of which increases the permeability of blood vessels, thus allowing enhanced infiltration of inflammatory constituents (Delisle, et al., 2010). VEGF levels accordingly, have been shown to coincide with the articular severity of RA (Hagihana, et al., 2003), and has shown a reduction in response to TCZ (Hagihana, et al., 2003), of which correlates with findings of a semi-quantitative assessment by Akoi, et al. (2011) using ultrasonography that found TCZ responsible for a marked reduction in RA neovascularisation.
Bone remodelling is a highly regulated process in which mature bone tissue is removed by osteoclasts and formation by osteoblasts. The pathogenesis of RA favours bone loss (resorption) via the excessive production of osteoclasts. Anecdotal evidence by Kazuto et al. (1996) revealed synovial fluid highly +ve for IL-6 –sIL6R complex from RA patients, stimulated increased formation of osteoclasts in mouse co-culture of osteoblast and bone marrow cells. This study coincides with research by Balena, et al. (1994) of which indicated that mice deficient in IL-6 displayed no significant changes in gross or trabecular bone structure.
In human studies, biopsies of RA patients revealed that peri-articular bone loss was found to correlate with local excessive presence of IL-6 (Sugiyama, 2001), and work by Garnero et al. (2009) showed the administration of TCZ to RA patients in a multi-centre double-blind placebo-controlled study yielded an increase in bone-formation markers, with a decrease in bone-resorption markers. Collectively, research suggests IL-6 has a negative effect on bone mass.
Acute-phase response is an innate immune reaction in which IL-6 is notably involved via the stimulation of hepatocytes, and is a key inducer of the acute-phase protein CRP. CRP is considered a dependable biomarker of inflammation and RA activity as serum half-life remains constant due to its inflammatory-induced, hepatic-stimulated production being the exclusive systemic determinant and due to its noted increase in RA serum levels (Hirshfield and Pepys, 2003).
Approximately 1-in-4 RA patients will suffer symptoms of anaemia within the first year (Figenschau, Nikolaisen and Nossent, 2008). The hormone hepcidin, produced in the liver and integral in stemming the metabolism of iron, has been shown in vitro to increase in presence due to IL-6 stimulation of hepatoma cells (Ganz, 2003). This study correlates with Gabayan et al. (2004) in which IL-6 induced patients experienced a 7.5 fold increase in hepcidin production. Anaemia in RA patients, as a result of a hepcidin-IL-6 axis, has limited but supported credence. Osteoporosis has also shown a correlation with IL-6.
As mentioned earlier, healthy bone metabolism requires adequate regulation of osteoclasts and osteoblasts, in which excessive IL-6 expression and the Th17 derived IL-17 (Gillespie, et al., 1999) both contribute to excessive osteoclast formation (Campbell et al., 2005). This has also been found to be the case in transgenic mice (De et al., 2006; and Choy and Dayer, 2009) in which bone formation was reduced, and negative ossification was reported. As shown, there are comparable cross-over aspects of IL-6 pathology between RA and osteoporosis.
As highlighted throughout this review, IL-6 plays a pleiotropic pathophysiological role in RA, thus would make an ideal therapeutic target. The most promising and note-worthy of treatments at present, TCZ interrupts IL-6 induced trans-signalling. Numerous studies have shown promising results including a phase III clinical trial in 2005 using the ACR (American College of Rheumatology)* RA activity measure, which had improvement measures of 89, 70 and 47% at ACR’s of 20, 50 and 70 respectively at 52 weeks, in patients treated with TCZ. This multicentre, double-blind placebo-controlled trial by Hashimoto et al. (2004) showed bone resorption and joint destruction could potentially be completely prevented (Kishimoto, 2010), as exemplified when a culture of patient synovial and peripheral mononuclear cells, extracted from the same patients showed that osteoclast generation was completely prevented (Hashimoto et al., 2004; Kishimoto, 2010).
At 6 weeks within this study, it was also found that IgG, CRP and serum amyloid A levels all normalised. It has also been suggested that TCZ aids in Th17 differentiation, hence IL-17 production, thus aids in the improvement of RA symptoms (Kimura and Kishimoto, 2010). Other notable studies such as the SAMURAI (Study of Active controlled Monotherapy Used for RA, an IL-6 Inhibitor) and LITHE (TociLIzumab safety and THE prevention of structural joint damage trial) served to enhance conscensus. This humanised, monoclonal antibody has now been approved in many countries^^. 286 With a wide-ranging pleitropy, IL-6 has the most profuse SF and systemic cytokine presence in RA pathophysiology, and coincides with cartilage erosion and disease activity.
It has a role in elements of B-cell differentiation and increasing evidence suggests a definite yet ambiguous role in Th17 differentiation. IL-6 has several key positions in immune and inflammatory processes (recruitment, permeation and adhesion of inflammatory elements), and bone and joint degradation, of which over-expression causes adversity, such as release of hepcidin and CRP from the liver contributing to anaemia and inflammation, osteoporosis and pannus development.
It plays a key role in the cross-over from acute-to-chronic disease. TCZ has shown much promise in RA therapy by inhibiting IL-6 expression. *ACR 20, 50 and 70 = improvements of 20%, 50% and 70% in disease activity, respectively. ^Disease Modifying Anti Rheumatic Drugs. ^^Japan , April 2008, Europe, January 2009 and USA, January 2010. PUT IN ABSTRACT: The IL-6R, sIL-6R and gp130 system is known as IL-6 trans-signalling
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