Biological Characteristics Of Fibroblast-like Synoviocytes And Its Pathogenesis In Inflammatory Arthritis

Fibroblast-like synoviocytes(FLSs) have emerged as a pivotal effector cell in the inflamed joint,based on its ability to degrade the extracellular matrix and to provide chemotactic and activation signals to resident parenchymal cells and infiltrating immunocytes,and have the ability of non T cell-mediated damage to cartilage and bone. In vitro studies have demonstrated that cultured FLSs display unique biological characteristics that set them apart from fibroblasts isolated from non-synovial anatomic sites.These cells,most importantly,release an impressive array of cytokines and growth factors,which have the capacity to stimulate and,in some cases,dampen the inflammatory response.Moreover,the role of FLSs is increasingly concerned about its effect in pathogenesis of inflammatory arthritis.The aim of this study was to investigate the morphological characteristics of cultured FLSs in vitro from the patients with rheumatoid arthritis(RA),osteoarthritis(OA) or ankylosing spondylitis(AS),and to evaluate the cell proliferative curve and migration rate in resting or lipopolysaccharide(LPS) stimulating conditions;Further more cellular immune phenotype expression and proportion on FLSs were analyzed by flow cytometry; Finally,mRNA expression of inflammatory cytokines was determined by real-time polymerase chain reaction(PCR),and the levels of secreted inflammatory cytokines in culture supernatant were detected by Cytometric Bead Array(CBA),in comparion with the treatment by dexamethasone(Dex) or cyclosporin A(CsA).The results might provide us a clue to understand the mechanism between the pathogenesis of arthritis and FLSs.The thesis was divided into the following three parts:PART ONE The morphological characteristics of FLSs from different type of arthritis and the comparison with the proliferative curve and migration rate in resting or stimulated conditionObjective:To investigate the morphological characteristics of FLSs primary and subcultured in vitro from the patients with different type of arthritis and compare with the proliferation of cell growth and the cell migration capacity both in their resting and stimulated conditions.Methods:FLSs were separated from tissues in joint replacement surgery or extracted synovial fluid FLSs from 13 osteoarthritis,6 rheumatoid arthritis and 2 ankylosing spondylitis,then FLSs were cultured and subcultured.The morphological characterization of the cells were observed under the phase contrast microscopy;The proliferation to LPS was measured by WST-1 assay;The cell migration capacity was evaluated in resting and LPS stimulated by transwell chambers.Results:OA,RA and AS FLSs were no significantly different in morphology:The adherent cells in primary and early passage were shown for the spindle,stellate, polygonal and dendritic-shaped morphological diversity;After the passage following the intensive growth,the FLS cells gradually formed a single purification,and uniform size for the spindle,with kinds of parallel or whirlpool-like arrangement;The cells in synovial fluid were major round or oval-shaped adherent cells.RA and OA FLSs entered the logarithmic phase on the fourth day after passage,then they reached the growth plateau phase on the seventh day.The LPS stimulated group obviously proliferated on the second day,and researched the maximum on the third day;But on the fifth day maintaining the LPS concentration,the cell count dropped lower than the level of the unstimulated group.The OD values were significantly different between cultured RA and OA FLSs on the third day(1.48±0.07 vs 0.808±0.12,P＜0.001).AS FLSs from synovial fluid grew slowly and showed non obvious response to LPS stimulation.RA and OA FLSs cell migration in stimulated were significantly different with the control group(55.58±4.09 vs 30.25±1.79;36.10±3.50 vs 22.00±2.22,P value＜0.01);The number of RA migrated FLS cells in stimulated was also increased significantly compared with OA(25.33±3.86 vs 14.11±3.26,P＜0.005).Conclusion:There are no obvious morphological differences among the cultured FLSs in vitro from different type of inflammatory artirtis by the light microscope;FLSs in vitro have more highly capacity in proliferation and migration in RA comepared with OA.PART TWO Immunocytochemical-associated membrane molecule expression on FLSs and intracellular factors TNFαand IFNγdetected by flow cytometryObjective:To screen immunocytochemical characterization of membrane molecules on FLSs and detect intracellular factors TNFα,IFNγor other cytokines by flow cytometry;To determined the changes of phenotype and frequency of the cell subsets when stimulated by LPS.Methods:The cases chosen was the same as the first part;The membrane dye reagents for antibody were CD3-FITC,CD4-PE,CD8-APC,CD45-Pacific Orange, CD45RO-APC,CD45RA-PE,TCRαβ-PE,TCRγδ-PE;CD19-PE-Cy5,CD14-APC, CD68-FITC,CD11c-PE,HLA-DR-APC;CD2-PE,CD7-FITC,CD16-PE,CD56-PE, CD57-FITC,CD116-Pacific Blue;CD 13-PE,CD33-PE,CD34 -PE-Cy7, CD38-Percp-Cy5.5,CD44-Pacific Blue,CD69-PE-Cy5,CD90-APC and CD 117-PE;The intracellular cytokine staining for antibody were TNF-α-PacificBlue,IFNγ-PercpCy5.5, IL-4-FITC and IL- 17-PE.Results:FLSs were highly expressing CD44,CD90,CD13(98.05±2.18%,96.7±4.8%,88.83±8.2%,respectively),and CD56,CD34,HLA-DR expression were in low levels,but formed there independent subsets(8.36±2.76%;18.75±13.74%;3.28±2.34%,respectively) in the screening of 27 kinds of phenotype,and other 21 antibodies' positive ratio was＜0.5%.The frequency of CD34,CD56 and HLA-DR of FLSs subsets in RA(n = 6) were significant differences in comparison with OA(n = 13),in which the positive proportion for CD56 and HLA-DR of RA FLSs were higher than OA (respectively 33.65±6.12vs16.80±4.36;7.13±1.85 vs 3.75±1.24,P＜0.0001),and the frequency for CD34 of RA was lower than OA(4.34±1.63vs8.89±2.60,P＜0.01).After stimulated by LPS two hours,RA FLSs(n = 6) were more highly expressing CD56 and HLA-DR frequency significantly than unstimulated(47.61±8.35vs33.65±6.12,P＜0.05; 26.68±9.47vs7.13±1.85,P＜0.01,respectively),while the proportion of the CD34 positive FLSs subset was decreased significantly than unstimulated(1.93±0.92vs4.34±1.63,P＜0.01).A little TNFαand IFNγexpression was shown in two cases of OA FLSs and one case of RA FLSs when stimulated by LPS,and IL-4 and IL-17 were negative expression in each test.Conclusion:CD90,CD44 and CD13 are the main molecular markers on FLSs; CD56~+/CD34~+/HLA-DR~+ FLSs subsets may be associated with the controls of synovitis; A little TNFαand IFNγcan be produced by FLSs after stimulated.PART THREE The expression of IL-6,IL-1βand IL-10 mRNA produced by FLSs and the level of inflammatory cytokines secreted in culture supernatant in different experimental conditionObjective:To determined the expression of IL-6,IL- 1βand IL- 10 mRNA produced by FLSs and evaluate the changes of their expression when treated with dexamethasone or cyclosporin A at 2h after LPS stimulating in transcriptional levels;To detect IL-6, IL-8,IL-1β,IL-10,IL-12p70,TNFαand other major inflammatory factor expression in cell culture supernatant in protein levels when treated with Dex or CsA at 24h after LPS stimulating.Methods:The total RNA were extracted from FLSs and synthesized first strand cDNA,then madeβ-actin as a housekeeping gene to analysize IL-6,IL-1βand IL-10mRNA relative concentration by real-time PCR with or without the effect on Dex and CsA before and after LPS stimulating;Finally,the levels of secreted IL-6,IL-8, IL-1βand other inflammatory cytokines in culture supernatant were detected by CBA assay with or without Dex and CsA.Results:IL-6,IL-10 and IL-1βmRNA expression were significantly increased both in OA and RA FLSs after stimulated,in which IL-1βmRNA was the highest,and IL-6 mRNA the next;After addition of Dex,these cytokine mRNA expression was in significant low levels.However,addition of CsA had no obvious impact on these cytokines;RA and OA FLSs in resting can secret a low level of IL-6(1.12±1.05;3.53±3.99 ng/ml,respectively) and IL-8(0.15±0.08;0.15±0.07 ng/ml,respectively).When stimulated by LPS,IL-6 levels(42.78±12.49;10.18±3.32 ng/ml,respectively)and IL-8 levels(23.13±7.25;19.07±9.34 ng/ml,respectively) in RA and OA are increased,and RA FLSs were more obviously increased(P＜0.001).Dex could inhibit the secretion of IL-6 significantly,and the inhibition to IL-8 was relatively weak,and CsA were only played part of the inhibitory effect.And the levels of other cytokines such as IL-1β, IL-10,IL-12p70 and TNFαwere measured trace or not.Conclusion:IL-6 and IL-8 are the major inflammatory cytokines secreted by FLSs; IL-1βand TNFαmainly secreted by macrophages and T cells are rarely expressed on FLSs;Dex play stronger role than CsA on preventing the pathogenesis of inflammatory joints by FLSs.