The Anti-inflammatory Effect Of Extracellular HSP70 On Rheumatoid Arthritis And Its Mechanisms

Rheumatoid arthritis (RA) is an autoimmune disease, characterized by chronic inflammation of synovial membranes and proliferation of the synovial lining, leading to cartilage damage and ultimately joint destruction. Although the pathogenesis of RA remains unclear, a variety of cytokines have been implicated in the development of the disease. Heat shock proteins (HSPs) are a superfamily of highly conserved proteins found in all eukaryotes and prokaryotes. Among them HSP70 is the most important subfamily which contains multiple members such as constitutive HSP70 (HSC70) and stress-inducible HSP70 (HSP70). Although HSP70 are traditionally regarded as intracellular proteins and their primary functions appear to be as molecular chaperones, involved in protein folding and transport, accumulating data suggest that HSP70 are also actively released and have important extracellular functions. It has been shown that HSP70 are released from a variety of cells in response to cellular stress. HSP70 may serve as a "danger signal" to the innate immune system and may also be relevant to the pathogenesis of autoimmune diseases. Although elevated HSP70 have been found in synovial fluid from RA patients, their sources and functions remain unclear.In present studies, the investigators observed the clinical significance of HSP70 alteration in serum or synovial fluid of RA patients, explored the effects of stress on HSP70 release and its mechanism, and further studied the effects of extracellular HSP70 on inflammatory response and its mechanisms from TNF-αstimulated fibroblast-like synoviocytes (FLSs) as well as a mouse model with collagen induced arthritis (CIA). The main results are as follows:1.The clinical significance of HSP70 alteration in serum or synovial fluid of RA patients. 1) Changes of HSP70 in serum of RA patients. The serum levels of HSP70 were significantly higher in RA patients than those in osteoarthritis (OA) or nomal control groups, and the serum levels of HSP70 were higher in active RA than those in inactive RA. The levels of HSP70 were correlated with the levels of TNF-αand IL-6 in serum of RA. Moreover, the levels of HSP70 in serum of RA were correlated with erythrocyte sedimentation rate (ESR), C-response protein (CRP) and rheumatoid factor (RF). 2) Changes of HSP70 in synovial fluid (SF) of RA patients. The SF levels of HSP70 were significantly higher in RA group than those in OA group, while the SF levels of HSP70 were higher in active RA than those in inactive RA. The levels of HSP70 were correlated with TNF-α, IL-6 and IL-10 in SF of RA. It suggests that extracellular HSP70 is correlated with the inflammatory response in RA, and the examination of HSP70 levels may be helpful in the evaluation of prognosis of RA.2. The effects of stress on HSP70 release in FLSs. Firtly, the effects of heat stress on HSP70 release were observed. Exposure of FLSs from RA patients to nonlethal HS resulted in a marked up-regulation of intracellular HSP70 and HSP70 release without significant cell death as indicated by LDH release. Then, the effects of inflammatory stress on HSP70 were studied. Treatment of FLSs with TNF-αdose- and time-dependently increased the expression of intracellular HSP70 and the release of HSP70 into the medium, without significant increase in cell death as indicated by LDH release. In addition, the investigators explored the role of lipid raft in HSP70 release. Treatment of FLSs with methyl-β-cyclodextrin (MβD), a lipid raft-disrupting reagent, inhibited nonlethal HS or TNF-αinduced HSP70 release from FLSs. It suggests that FLSs can actively release HSP70 when exposed to different kinds of stressors, and lipid raft may be involved in HSP70 release in FLSs.3. The effects of extracellular HSP70 on inflammatory cytokine secretion in FLSs and its mechanisms. 1) The effects of extracellular HSP70 on the secretion of inflammatory cytokine in FLSs. Recombinant human HSP70 inhibited the secretion of IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 induced by TNF-αstimulation in RA FLSs, although HSP70 alone had no effect on FLSs. HSP70 directly induced IL-10 secretion in dose-and time-dependent manners. Moreover, the effects of human HSP70 on cytokine secretion were completely abolished by the treatment of HSP70 with boiling but not with polymyxin B (PMB). 2) The analysis of receptor for extracellular HSP70 on cell surface of FLSs. By RT-PCR and Western blotting, it was shown that human RA FLSs could express TLR2 and TLR4. When the FLSs were preincubated with TLR2 or TLR4 antibody, and the effects of HSP70 on the secretion of IL-6, IL-8, MCP-1 and IL-10 were blocked by TLR4 antibody but not by TLR2 antibody. It suggests that the effects of human HSP70 on cytokine secretion in FLSs were mediated through TLR4, and TLR4 on cell surface of FLSs may be as the possible receptor of HSP70. 3) The effects of HSP70 on inflammatory signal pathways. Recombinant human HSP70 inhibited activation of MAPK signal pathways (p38, JNK, and ERK ) induced by TNF-α. HSP70 also inhibited the degradation of IκBαand nuclear translocation of NF-κB p65 induced by TNF-α. However, HSP70 directly induced the activation of STAT3 and expression of SOCS3. It suggests that the anti-inflammatory effect of extracellular HSP70 on human RA FLSs may be explained by its ability to suppress TNF-α-induced activation of MAPK and NF-κB, two vital pro-inflammatory signal pathways in RA, and promote activation of JAK-STAT3-SOCS3, a known anti-inflammatory signal pathway.4. The effect of HSP70 on collage induced arthritis (CIA). Recombinant human HSP70 significantly suppressed paw swelling of mice with CIA. Histological analysis confirmed the suppression of joint inflammation and bone destruction in mice with CIA after treatment with HSP70. Serum TNF-αand IL-6 levels were significantly reduced after treatment of HSP70. These results suggest that HSP70 is effective in suppressing inflammatory response and bone damage in CIA, and may have potential value in the treatment of rheumatoid arthritis.Taken together, it is suggested that 1) human HSP70 is up-regulated and actively released by RA FLSs exposed to stress; 2) Extracellular HSP70 inhibits the release of IL-6, IL-8 and MCP-1 induced by TNF-αand promotes the release of IL-10 in FLSs; 3) the anti-inflammatory role of extracellular HSP70 in human RA FLSs can be blocked by TLR4 antibody and recombinant human HSP70 inhibits the activation of MAPK and NF-κB signal pathways induced by TNF-α, but activates Jak-STAT3-SOCS3 pathway; 4) recombinant HSP70 suppresses systemic and joint inflammation and cartilage and bone destruction in mice with CIA, indicates a possible therapeutic effect of HSP70 on RA.