| Rheumatoid arthritis (RA) is a globally prevalent autoimmune diseasecharacterized by chronic polyarticular synovial inflammation and progressive erosionof cartilage and bone. The pathogenesis of RA is still not clear, but it related withgenetic background, microbial infection and its induction of immune disorder, andalso related with auto-reactive T cells, B cells and the autoantibodies it produced.Inflammatory cytokines produced by multiple cells in joint of RA patients playimportant roles in RA pathogenesis.Animal models of autoimmune arthritis are indispensable tools for investigatingdetail pathogenic hypotheses and therapeutic strategies. Various animal models havebeen developed, among them, adjuvant induced arthritis (AIA), described in1956, isthe first and still most widely used experimental arthritis model. Originally, AIA wasinduced by a single intradermal injection with complete Freund’s adjuvant (CFA) atthe hind footpad in susceptible strains of rats, like Lewis and Wistar. Upon induction,rats developed typical manifestations of arthritis similar to human RA, characterizedby progression of an easily measurable polyarticular inflammation, marked infiltrationof lymphocytes, proliferation of synoviocytes and fibroblasts, pannus formation,cartilage destruction and bone erosion.CFA is composed of Bacille Calmette Guerin (BCG) and incomplete Freund’sadjuvant (IFA), a water-in-oil emulsion containing paraffin and lanolin. In themycobacteria, the mycobacterial heat-shock proteins (MHSPs) have been implicatedin autoimmune arthritis in human and animal models for molecular mimicry.Therefore, we studied whether E. coli expressed, BCG derived HSP65could replaceBCG in CFA to induce arthritis in rats. HSP65-IFA emulsion was prepared to observethe its effect on inducing paw swelling, histopathological lesions in ankle joint andproduction of anti-dsDNA antibody in male Wistar rats.Furthermore, the effect of inhibitory ODN on CFA and HSP65-IFA inducedarthritis was analyzed. 1. Effect of HSP65-IFA on inducing arthritis in ratsTo study whether BCG derived HSP65could replace BCG in CFA to inducearthritis with IFA in rats, we prepared HSP65-IFA emulsion by mixing equal volumesof HSP65and IFA with HSP65at5μg in100μl/rat. HSP65-IFA, CFA, IFA, HSP65(5μg/rat) and saline were intradermally injected in left footpads of male Wistar rats,respectively, on day0.4rats were tested in each group.We found HSP65-IFA and CFA injected rats developed the most severe pawswelling and arthritic lesions in ankle joints, who could not walk normally due to thepain in injected foot, while HSP65alone did not induce any paw swelling or arthriticlesion in ankle joints. Moreover, on day19, HSP65-IFA induced more severe pawswelling than CFA. In histopathological analysis, we found HSP65-IFA inducedtypical lesions in ankle joints, including cartilage erosion and synovial thickeningwith pannus formation. Double blinded scoring of histopathological lesions showedHISP65-IFA induced higher levels of lesions than CFA and IFA. X-ray analysisshowed HSP65-IFA and CFA induced soft tissue edama in injected side andosteoporosis in ankle joints. HSP65-IFA also inhibited body weight increase in rats.Next, we prepared three HSP65-IFA emulsions by mixing IFA and HSP65atdoses of1,5and25μg/rat, respectively, to test the dose-effect of HSP65for inducingarthritis in rats. The results showed IFA plus HSP65at5μg/rat induced the mostsevere paw swelling and histopathological lesions in ankle joints, slightly higher thanIFA plus HSP65at25μg/rat, though serologic analysis showed higher levels ofanti-HSP65/dsDNA antibody in IFA plus HSP65at25μg/rat treated rats.2. Effects of VP1/OVA-IFA on inducing arthritis in ratsTo eliminate the effects of6×his-tag and LPS, a possible contamination inE.coli expressed protein HSP65, we chose VP1which also contained6×his-tag andexpressed in E.coli, and ovalbumin (OVA) which was not expressed in E.coli and didnot contain6×his-tag, emulsified with IFA at final dose of5μg/rat and injected atleft hind footpads in male Wistar rats, to observe the effect of VP1/OVA-IFA on pawswelling and histopathological changes in ankle joints. We found VP1/OVA-IFA couldalso induce paw swelling, histopathological lesions in ankle joints and anti-dsDNAantibody production, and OVA-IFA induced significantly more severe swelling thanIFA. In histopathological analysis of ankle joints, we found VP1/OVA-IFA inducedarthritic lesions in certain extant, including synovium hyperplasia and cartilagedestruction. However, histopathological scoring showed no significant difference between VP1/OVA-IFA and IFA treated rats, thus it suggested that VP1or OVA couldnot effectively induce autoimmune arthritis with IFA as HSP65, and arthritis inducedby HSP65-IFA was related with the property of HSP65, but not with LPS or6×his-tag.3. Therapeutic effects of SAT05f on arthritis in ratsSince it had been reported that inhibitory ODN A151had therapeutic effects oncollagen and CpG ODN induced arthritis, we applied inhibitory ODN SAT05f ontherapy of HSP65-IFA and CFA induced arthritis. SAT05f was treated from day0today42in arbitrary intervals, and the first treatment was carried out on day0twohours before arthritis indction. We found SAT05f could ameliorate HSP65-IFAinduced paw swelling, histopathological lesions in ankle joints and anti-dsDNAantibody production, while it had no therapeutic effects on CFA induced arthritis.Therefore, we speculate CFA and HSP65-IFA induced arthritis in different pathways.In summary, we successfully set up a novel autoimmune arthritis modle in ratswith HSP65-IFA, which could be used in studies of RA pathogenesis and therapeuticdrugs. We also found inhibitory ODN SAT05f had therapeutic effects on HSP65-IFAinduced arthritis, indicating the perspective of SAT05f on RA treatment. |
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