Studies on the synergy between collagen and adjuvant arthritis in rats

Intravenous administration of subarthritogenic doses of anticollagen IgG and adjuvant-sensitized spleen cells to syngeneic naive rats induces an erosive arthritis in recipients. The onset of the clinical disease in recipients is rapid and the disease is severe when compared to those recipients receiving cells alone. Immunocytochemical analysis of the knee synovium indicates the accumulation in the adipose tissue of Ia+ (ED1+)macrophages, OX-19+ T lymphocytes, and neutrophils. A large proportion of the lining cells of the proliferative synovium are Ia+. The knee synovium is extremely edematous and contains fibrin. If recipient rats are decomplemented, clinical disease is delayed and the number of mononuclear and polymorphonuclear cells accumulating in the synovium is decreased. Similar results are observed if recipient rats are treated with anti-Ia+ antibody. However, anti-Ia+ treatment does not induce depletion of serum complement.     

The cellular basis of adjuvant arthritis. I. Enhancement of cell-mediated passive transfer by concanavalin A and by immunosuppressive pretreatment of the recipient

Two reliable systems for the cell-mediated passive transfer of adjuvant arthritis have been developed. Donor rats were sensitized with Mycobacterium butyricum in mineral oil. In the first system, intravenous injection of adjuvant-sensitized donor lymph node or spleen cells into adult-thymectomized, lethally irradiated, bone marrow cell-reconstituted syngeneic rats induced arthritis in the recipients. In the second system, adjuvant-sensitized donor lymph node or spleen cells were cultured in vitro with concanavalin A; these cells induced arthritis in normal recipients as well as in thymectomized, irradiated, bone marrow cell-reconstituted recipients. The passively transferred disease in both systems resembled classical adjuvant-induced arthritis clinically, radiographically, and histologically. Neither irradiated, adjuvant-sensitized donor cells nor cells from donors not injected with complete adjuvant could passively transfer arthritis.     

Interleukin-34 as a promising clinical biomarker and therapeutic target for inflammatory arthritis

Interleukin-34 (IL-34), recently identified as a novel inflammatory cytokine and the second ligand for colony-stimulating factor-1 receptor, is known to play regulatory roles in the development, maintenance, and function of mononuclear phagocyte lineage cells – especially osteoclasts. Regarding its primary effect on osteoclasts, IL-34 has been shown to stimulate formation and activation of osteoclasts, which in turn magnifies osteoclasts-resorbing activity. In addition to its role in osteoclastogenesis, IL-34 has been implicated in inflammation of synovium via augmenting production of inflammatory mediators, in which altered IL-34 expression is regulated by pro-inflammatory cytokines responsible for cartilage degradation. Indeed, IL-34 has been documented to be highly expressed in inflamed synovium of rheumatoid arthritis (RA) and knee osteoarthritis (OA) patients, which are recognized as inflammatory arthritis. Furthermore, a number of clinical studies demonstrated that IL-34 levels were significantly increased in the circulation and synovial fluid of patients with RA and knee OA. Its levels were also found to be positively associated with disease severity – especially radiographic severity of both RA and knee OA patients. Interestingly, emerging evidence has accumulated that functional blockage of IL-34 with specific antibody can alleviate the severity of inflammatory arthritis. It is therefore reasonable to speculate that IL-34 may be developed as a potential biomarker and a new therapeutic candidate for inflammatory arthritis. To date, there are numerous studies showing IL-34 involvement and association with many aspects of inflammatory arthritis. Herein, this review aimed to summarize the recent findings regarding regulatory role of IL-34 in synovial inflammation-mediated cartilage destruction and update the current comprehensive knowledge on usefulness of IL-34-based treatment in inflammatory arthritis – particularly RA and knee OA.     

BMP-7 inhibits cartilage degeneration through suppression of inflammation in rat zymosan-induced arthritis

Bone morphogenetic protein-7 (BMP-7) regulates cartilage metabolism and promotes matrix synthesis. However, the effect of BMP-7 on inflammatory arthritis remains unknown. We investigated the effect and mechanism of exogenous BMP-7 on cartilage and synovium in vivo in rat zymosan-induced arthritis. Zymosan was injected into the left knees of Wistar rats. Phosphate-buffered saline or BMP-7 at 10, 100, or 1000 ng per joint was injected into the left knee every 2 days. Normal joints acted as normal controls. The knee joints were analyzed histologically and immunohistologically at 14 days. Joint swelling was evaluated by measuring the transverse diameter of the knee joints. Synovial lysates were collected, and the concentrations of interleukin-1β (IL-1β), IL-6, and IL-10 were measured by enzyme-linked immunosorbent assay. Intra-articular injection of zymosan resulted in acute inflammation and was followed by cartilage degeneration. Local administrations of BMP-7 inhibited this loss of cartilage matrix in a dose-dependent manner. Immunohistochemical analysis demonstrated enhanced type II collagen levels in cartilage and enhanced BMP-7 levels in cartilage and synovium after exogenous BMP-7 treatment. Joint swelling and cell infiltration into synovium were significantly reduced by BMP-7 injections. Administration of BMP-7 decreased IL-1β production significantly and increased IL-10 production in the synovium. Thus, intra-articular injections of BMP-7 had a protective effect on cartilage degeneration in the inflammatory arthritis model by enhancing levels of BMP-7 in cartilage and suppressing the production of IL-1β in synovium.     

A novel angiogenesis inhibitor suppresses rat adjuvant arthritis

Rat adjuvant arthritis (AA) is an animal model of rheumatoid arthritis in which pannus formation and destruction of joints occur after immunization with complete Freund's adjuvant. Neovascularization is present within the synovium and may be critical for pannus growth. In this study the effects of a novel angiogenesis inhibitor, AGM-1470, on AA were evaluated. Lewis rats were immunized with CFA to induce arthritis. AGM-1470 treatment was initiated prior to arthritis onset (preventative protocol) or administered to rats with established disease (suppressive protocol). The severity of synovitis and the immunologic status of all rats were then evaluated. Using clinical and radiographic criteria, AGM-1470 significantly reduced arthritis incidence (preventative protocol) (P < 0.01) and disease severity (both protocols, P < 0.001, compared to controls) without affecting T cell function in vitro or phenotype in vivo. Additionally, histologic sections from control rats revealed marked pannus formation, destruction of bone/cartilage, and neovascularization. These findings were absent in AGM-1470-treated rats. AGM-1470 may offer a new treatment option for rheumatoid arthritis and other angiogenesis-dependent diseases.     

Blockade of chemokine receptor CXCR3 inhibits T cell recruitment to inflamed joints and decreases the severity of adjuvant arthritis

T lymphocytes expressing the chemokine receptors, CCR2, CCR5, CXCR3, and CXCR6 are increased in inflamed tissues in rheumatoid arthritis. The role of CXCR3 in autoimmune arthritis induced in Lewis rats was investigated. CXCR3+ T cells migrated 2- to 3-fold more than CXCR3- T cells to inflamed joints in arthritic animals. CXCR3-expressing in vivo Ag-activated T lymphoblasts and in vitro-activated lymph node cells from arthritic animals were strongly recruited to the arthritic joints, and treatment with anti-CXCR3 mAb significantly inhibited this T cell recruitment by 40-60%. Immune T cells from the spleen and lymph nodes of actively immunized arthritic donors adoptively transferred arthritis to naive rats. Treatment with anti-CXCR3 mAb delayed the onset of arthritis and significantly reduced the severity of joint inflammation with a >50% decrease in the clinical arthritis score. Blockade of CXCR3 also significantly reduced the weight loss in the arthritic animals and inhibited neutrophil accumulation in the joints by 50-60%. There was a marked reduction in the leukocyte infiltration of the synovium in the presence of CXCR3 blockade and a decrease in the loss of articular cartilage of the joints. In conclusion, CXCR3 on T cells has an essential role in T cell recruitment to inflamed joints and the development of joint inflammation in adjuvant arthritis.     

The regulatory C-terminal determinants within mycobacterial heat shock protein 65 are cryptic and cross-reactive with the dominant self homologs: implications for the pathogenesis of autoimmune arthritis

The 65-kDa mycobacterial heat shock protein (Bhsp65) has been invoked in the pathogenesis of both adjuvant arthritis (AA) in the Lewis rat (RT.1(l)) and human rheumatoid arthritis. Arthritic Lewis rats in the late phase of AA show diversification of the T cell response to Bhsp65 C-terminal determinants (BCTD), and pretreatment of naive Lewis rats with a mixture of peptides representing these neoepitopes affords protection against AA. However, the fine specificity and physiologic significance of the BCTD-directed T cell repertoire, and the role of homologous self (rat) hsp65 (Rhsp65), if any, in spreading of the T cell response to Bhsp65 have not yet been examined. We observed that T cells primed by peptides comprising BCTD can adoptively transfer protection against AA to the recipient Lewis rats. However, these T cells can be activated by preprocessed (peptide) form of BCTD, but not native Bhsp65, showing that BCTD are cryptic epitopes. The BCTD-reactive T cells can be activated by the naturally generated (dominant) C-terminal epitopes of both exogenous and endogenous Rhsp65 and vice versa. Furthermore, certain individual peptides constituting BCTD and their self homologs can also induce protection against AA. These results support a model for the diversification of T cell response to Bhsp65 during the course of AA involving up-regulation of the display of cryptic BCTD coupled with spontaneous induction of T cell response to the cross-reactive dominant C-terminal epitopes of Rhsp65. The identification of disease-regulating cryptic determinants in Ags implicated in arthritis provides a novel approach for immunotherapy of rheumatoid arthritis.     

Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.     

Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.     

Arthritis induced immunologically with cationic amidated bovine serum albumin in the Guinea pig

Arthritis was induced by injecting cationic amidated bovine serum albumin (aBSA) (pI approximately 9.2) into the knee joint of immunized guinea pigs and the mechanisms of articular cartilage destruction were studied morphologically and biochemically. Marked synovitis associated with polymorphonuclear leukocyte (PML) infiltration occurred within 1 day of the challenge. Articular cartilage infiltrated by PMLs was almost completely destroyed after 2 weeks. During the initial destructive process, proteoglycans were depleted from the cartilage and later collagen fibers disappeared. Granulation tissue growing in the inflamed synovium and bone marrow replaced the destroyed cartilage and joint cavity and formed fibrous scar tissue (fibrous ankylosis) by 8 weeks. Subsequently, the knee joints developed cartilagenous ankylosis by 12 weeks and finally bony ankylosis at 28 weeks. Autoradiography using 125I-aBSA and immunofluorescence studies for immunoglobulin (IgG) and complement (C3) demonstrated that the antigen is trapped in all zones of the articular cartilage and serves as a trigger for immune complex formation. Significantly increased neutral proteinase activities against substrates of proteoglycan subunits, [3H]carboxymethylated transferrin and L-pyroglutamyl-L-prolyl-L-valine-paranitroanilide were detected in homogenates of the synovium and cartilage from arthritic knee joints 1 and 2 weeks after induction. Inhibitor studies and pH curves suggested that the proteinase is leukocyte elastase. Measurable amounts of gelatinolytic activity, detected by activation with 4-aminophenylmercuric acetate and inhibited with EDTA, were also present in the same samples, but there was no detectable collagenase activity. The data on SDS-gelatin substrate gel showed that the proteinase is gelatinase derived from PMLs. These results suggest that in aBSA-induced arthritis, elastase and gelatinase from PMLs invading articular cartilage may play important roles in cartilage destruction.     

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1beta. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.     

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1β. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.     

Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation

During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcγ receptors (FcγRs) (mainly FcγRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcγRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced.     

MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis

Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovial, and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.     

Immunohistochemical demonstration of collagenase and tissue inhibitor of metalloproteinases (TIMP) in synovial lining cells of rheumatoid synovium

Degradation of fibrillar collagens is a central process in joint destruction in rheumatoid arthritis. Collagenase responsible for the collagenolysis has been immunolocalized on the extracellular matrix components at the cartilage/pannus junction in the rheumatoid joint, but very little is known about cellular source of the proteinase. In this paper monospecific antibodies against collagenase and tissue inhibitor of metalloproteinases (TIMP) were applied to rheumatoid and normal synovium to identify cells synthesizing and secreting the enzyme and its inhibitor. By treating the specimens with the monovalent ionophore, monensin, both collagenase and TIMP could be immunolocalized in hyperplastic synovial lining cells in rheumatoid synovium, but not in the cells of normal synovium. Dual immunolocalization studies demonstrated that the majority of the lining cells (approximately 64%) produce both collagenase and TIMP, while approximately 3% of the cells were positive only for collagenase, and 11% only for TIMP. Neither collagenase nor TIMP was immunolocalized on the extracellular matrix components in the synovia examined. These data suggest that synovial lining cells in rheumatoid arthritis secrete both collagenase and TIMP into the joint cavity. The role of collagenase in joint destruction in rheumatoid arthritis is discussed with reference to the regulation of the activity by TIMP.     

Pristane, a non-antigenic adjuvant, induces MHC class II-restricted, arthritogenic T cells in the rat

Pristane-induced arthritis (PIA) in rats, a model for rheumatoid arthritis (RA), is a T cell-dependent disease. However, pristane itself is a lipid and unable to form a stable complex with a MHC class II molecule. Therefore, the specificity and function of the T cells in PIA are as unclear as in rheumatoid arthritis. In this study, we show that activated CD4+ alphabetaT cells, which target peripheral joints, transfer PIA. The pristane-primed T cells are of oligo or polyclonal origin as determined by their arthritogenicity after stimulation with several mitogenic anti-TCRVbeta and anti-TCRValpha mAbs. Arthritogenic cells secreted IFN-gamma and TNF-alpha (but not IL-4) when stimulated with Con A in vitro, and pretreatments of recipient rats with either anti-IFN-gamma or a recombinant TNF-alpha receptor before transfer ameliorated arthritis development. Most importantly, we show that these T cells are MHC class II restricted, because treatment with Abs against either DQ or DR molecules ameliorates arthritis development. The MHC class II restriction was confirmed by transferring donor T cells to irradiated recipients that were syngenic, semiallogenic, or allogenic to MHC class II molecules, in which only syngenic and semiallogenic recipients developed arthritis. These data suggest that the in vivo administration of a non-antigenic adjuvant, like pristane, activates CD4+ alphabetaT cells that are MHC class II restricted and arthritogenic.     

Developments in the synovial biology field 2006

Synovial pathophysiology is a complex and synergistic interplay of different cell populations with tissue components, mediated by a variety of signaling mechanisms. All of these mechanisms drive the affected joint into inflammation and drive the subsequent destruction of cartilage and bone. Each cell type contributes significantly to the initiation and perpetuation of this deleterious concert, especially in rheumatoid arthritis. Rheumatoid arthritis synovial fibroblasts and macrophages, both cell types with pivotal roles in inflammation and destruction, but also T cells and B cells are crucial for complex network in the inflamed synovium. An even more complex cellular crosstalk between these key players maintains a process of chronic inflammation. As outlined in the present review, in the past year substantial progress has been made to elucidate further details of the rich pathophysiology of rheumatoid arthritis, which may also facilitate the identification of novel targets for future therapeutic strategies.     

佐剂诱导关节炎大鼠模型的建立及成纤维样滑膜细胞的分离

目的建立类风湿性关节炎（RA）动物模型;从炎性关节滑膜中分离成纤维样滑膜细胞（FLS）。方法应用热灭活结核杆菌H37Ra菌株与矿物油混合制备改良的佐剂,尾根部皮内注射Lewis大鼠诱导关节炎;剪取成功诱导关节炎大鼠的病变踝关节,从中剥离滑膜组织,充分剪碎后采用胶原酶消化法分离成纤维样滑膜细胞。结果成功诱导了大鼠关节炎,发病率为100%,发病时间有规律,组织学表现与RA相似;成功在体外培养了FLS并对其进行了鉴定,掌握了其生长形态和特征。结论成功制备RA动物模型并获得FLS,为今后RA发病机制探索和药物评价提供了良好的体内动物模型和体外细胞模型。     

Differential roles of VLA-4(CD49d/CD29) and LFA-1(CD11a/CD18) integrins and E- and P-selectin during developing and established active or adoptively transferred adjuvant arthritis in the rat

The role of the integrins VLA-4 and LFA-1 and of the selectin adhesion molecules in autoimmune arthritis was investigated. Adjuvant arthritis was induced in Lewis rats by active immunization (s.c.) with Mycobacterium butyricum or by adoptive transfer of immune T cells. With active adjuvant arthritis, Lewis rats develop maximal polyarticular joint inflammation and migration of radiolabelled (111In and 51Cr) blood neutrophils and monocytes to the joints 14 days post Mycobacterium butyricum immunization. Using blocking monoclonal antibodies we osbserved that at this stage monocyte recruitment was dependent (85%) on P-selectin plus VLA-4 (alpha4B1) and neutrophil recruitment depended (> 80%) on P-selectin plus LFA-1 (CD11a/CD18). E-selectin played a minimal role in inflammatory cell recruitment to the already inflamed joint. In contrast, during the development of active adjuvant arthritis, blockade of P-selectin beginning at day 5 post-immunization had no effect on subsequent arthritis. However, E-selectin blockade at this stage reduced arthritic scores by 70% (P < 0.01) and combined E-selectin plus VLA-4 blockade prevented development of arthritis. Either treatment nearly abolished neutrophil and monocyte recruitment to joints at day 14 and prevented cartilage damage. VLA-4 blockade alone was less effective. Adoptive T-cell transfer of adjuvant arthritis to naive rats employed spleen/lymph node lymphocytes from Mycobacterium butyricum immunized rats stimulated with Concanavalin A in vitro (48 h). E-selectin +/- P-selectin blockade had no effect on the development of adoptive arthritis. However, VLA-4 integrin blockade inhibited adoptive arthritis severity by 55% (P < 0.01). LFA-1 blockade had no effect. In adoptive adjuvant arthritis, inhibition of arthritis clinically and by histology was essentially complete (> 90%) when E- and P-selectin blockade was combined with VLA-4 blockade. Thus, in the development of actively induced arthritis E-selectin plays an important role, likely mediating early antigen reactive T-cell recruitment to joints. In contrast, VLA-4 and multiple selectin mechanisms are involved in arthritis induction by ex vivo restimulated arthritogenic T cells. Furthermore, in actively induced adjuvant arthritis, P- and E-selectin and VLA-4 are differently important in the initiation of arthritis, and at the time of fully developed joint inflammation.