Cadherin 11 promotes invasive behavior of fibroblast‐like synoviocytes

Objective

To define the expression pattern of cadherin 11 in the destructive pannus tissue of patients with rheumatoid arthritis, and to determine whether cadherin 11 expression in fibroblast‐like synoviocytes controls their invasive capacity.

Methods

Cadherin 11 expression in rheumatoid synovial tissue was evaluated using immunohistochemistry. To examine the role of cadherin 11 in regulating the invasive behavior of fibroblast‐like synoviocytes, we generated L cell clones expressing wild‐type cadherin 11, mutant cadherin 11, and empty vector–transfected controls. The invasive capacity of L cell transfectants and cultured fibroblast‐like synoviocytes treated with a blocking cadherin 11–Fc fusion protein or control immunoglobulin was determined in Matrigel invasion assays.

Results

Immunohistochemical analysis revealed that cadherin 11 is abundantly expressed in cells at the cartilage–pannus junction in rheumatoid synovitis. Assays to determine invasion demonstrated a 2‐fold increased invasive capacity of cadherin 11–transfected L cells compared with L cells transfected with E‐cadherin or control vector. The invasive behavior of L cells stably transfected with a cadherin 11 construct that lacked the juxtamembrane cytoplasmic domain was diminished to the level of vector control L cells. Furthermore, treatment with the cadherin 11–Fc fusion protein diminished the invasive capacity of fibroblast‐like synoviocytes.

Conclusion

The results of these in vitro studies implicate a role for cadherin 11 in promoting cell invasion and contribute insight into the invasive nature of fibroblast‐like synoviocytes in chronic synovitis and rheumatoid arthritis.

     

PUMA regulation and proapoptotic effects in fibroblast‐like synoviocytes

Objective

Although p53 is overexpressed in rheumatoid arthritis (RA) synovial tissue (ST), few synoviocytes undergo apoptosis. This could be partly due to low expression of proapoptotic genes. Deficient p53 up‐regulated modulator of apoptosis (PUMA), which is a major effector of p53‐mediated cell death, could contribute to this phenomenon. To evaluate a method to induce apoptosis, the expression and function of PUMA was investigated in ST and cultured fibroblast‐like synoviocytes (FLS).

Methods

PUMA expression in ST was measured by immunohistochemistry, Western blot analysis, and quantitative polymerase chain reaction analysis. Ad‐p53 and plasmids encoding hemagglutinin‐tagged, full‐length PUMA expression vector (HA‐PUMA), PUMA lacking the Bcl‐2 homology 3 domain, or pCEP4 were used to transfect FLS. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation, and caspase 3 activation.

Results

PUMA protein was detected in RA ST, although most of the immunoreactive protein was localized to sublining cells rather than the intimal lining synoviocytes. Western blot analysis showed no difference between RA ST and osteoarthritis (OA) ST. PUMA messenger RNA was detected in RA and OA ST, although the amounts were markedly lower than in the spleen and FLS. To determine if PUMA was inducible, FLS were transduced with Ad‐p53. Even though p53 protein was produced and p21 expression was increased, PUMA expression was not enhanced. Consistent with this observation, Ad‐p53 did not induce apoptosis in FLS. However, HA‐PUMA transfection into FLS resulted in rapid apoptosis with the activation of caspase 3.

Conclusion

PUMA can induce apoptosis by FLS and represents a potential target in RA.

     

Specifically modified osteopontin in rheumatoid arthritis fibroblast‐like synoviocytes supports interaction with B cells and enhances production of interleukin‐6

Objective

Osteopontin (OPN) is expressed by fibroblast‐like synoviocytes (FLS) in rheumatoid arthritis (RA), but its pathologic role is still obscure. The present study was undertaken to analyze the role of OPN in RA by focusing on its effects on cell–cell interactions between FLS and B lymphocytes.

Methods

FLS obtained from 10 patients with RA and 10 non‐RA subjects and a B lymphocyte cell line were studied. The characteristics of OPN expression by FLS were analyzed by Western blotting, immunoprecipitation, and immunofluorescence studies. In cocultures of FLS and B lymphocytes, the effects of OPN on adhesion of B lymphocytes to FLS and the consequent production of interleukin‐6 (IL‐6) were analyzed in experiments involving overexpression and knockdown of OPN and inhibitory studies with an OPN‐blocking antibody. In vivo, the expression of OPN in RA synovium was examined by immunohistochemistry.

Results

A specifically modified 75‐kd form of OPN was predominantly expressed in RA FLS, and this was associated with expression of >200‐kd thrombin‐cleaved OPN that was crosslinked with fibronectin and localized on the surface of the FLS. In FLS–B lymphocyte cocultures, 75‐kd OPN–positive FLS produced a significantly higher amount of IL‐6 than did 75‐kd OPN–negative FLS. When the FLS were separated from B lymphocytes or cultured alone, the production of IL‐6 was low and was not significantly different between these 2 culture conditions. Moreover, OPN overexpression enhanced production of IL‐6 in 75‐kd OPN–positive FLS–B lymphocyte cocultures. Addition of the OPN‐blocking antibody inhibited the adhesion of B lymphocytes to FLS. Immunohistochemical analyses revealed that localization of IL‐6–positive cells coincided with the sites at which OPN and B lymphocytes were colocalized.

Conclusion

Specifically modified 75‐kd OPN was expressed by RA FLS. This form of OPN affected FLS–B lymphocyte interactions by supporting the adhesion of B lymphocytes to FLS and enhancing the production of IL‐6.

     

CYP7B expression and activity in fibroblast‐like synoviocytes from patients with rheumatoid arthritis: Regulation by proinflammatory cytokines

Objective

The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7α‐hydroxy‐DHEA (7α‐OH‐DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collagen‐induced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7α‐OH‐DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast‐like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).

Methods

The presence of 7α‐OH‐DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7α‐OH‐DHEA with the use of high‐performance liquid chromatography.

Results

The CYP7B enzyme product 7α‐OH‐DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin‐1α (IL‐1α), IL‐1β, and IL‐17 up‐regulated CYP7B activity in an FLS cell line 2–10‐fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor β inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFα increased CYP7B activity in all cell lines tested.

Conclusion

Exposure to the proinflammatory cytokines TNFα, IL‐1α, IL‐1β, and IL‐17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7α‐OH‐DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.

     

Lysophosphatidic acid receptor 1 suppression sensitizes rheumatoid fibroblast‐like synoviocytes to tumor necrosis factor–induced apoptosis

Objective

To investigate the role of lysophosphatidic acid (LPA) receptors in the proliferation and apoptosis of fibroblast‐like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).

Methods

Expression of LPA receptors 1–3 was analyzed by real‐time polymerase chain reaction (PCR). LPAR1 and LPAR2 were suppressed in RA FLS by small interfering RNA (siRNA) transfection. Proliferation of RA FLS after tumor necrosis factor (TNF) and LPA stimulation was determined with a luminescent cell viability assay. Apoptosis was analyzed by quantification of nucleosome release and measurement of activated caspase 3/7. Genes involved in the apoptotic response were identified with a human apoptosis PCR array and validated with Western blot assays. The requirement of these genes for apoptosis sensitization was assessed by siRNA transfection. Secretion of mediators of inflammation was analyzed by enzyme‐linked immunosorbent assay.

Results

Only LPAR1 and LPAR2 were expressed by RA FLS, and their levels were higher than those in osteoarthritis (OA) FLS. Suppression of LPAR1 abrogated TNF‐induced proliferation and sensitized the RA FLS, but not the OA FLS, to TNF‐induced apoptosis. These changes occurred despite an increased early inflammatory response to TNF. Sensitization to apoptosis was associated with changes in expression of multiple apoptosis‐related genes. Three of the up‐regulated proapoptotic genes were further studied to confirm their involvement. In contrast, suppression of LPAR2 showed no effect in any of these analyses.

Conclusion

LPA₁ is an important receptor in RA FLS. Its suppression is accompanied by a global increase in the response to TNF that is ultimately dominated by sensitization to apoptosis.

     

Rheumatoid arthritis fibroblast‐like synoviocytes express BCMA and are stimulated by APRIL

Objective

Fibroblast‐like synoviocytes (FLS) are among the principal effector cells in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to examine the variety of stimulating effects of APRIL and its specific effect on FLS in the affected RA synovium.

Methods

Synovium and serum samples were obtained from patients with RA, patients with osteoarthritis (OA), and healthy subjects. Soluble APRIL proteins were assayed by enzyme‐linked immunosorbent assay. The relative gene expression of APRIL, BCMA, interleukin‐6 (IL‐6), tumor necrosis factor α (TNFα), IL‐1β, and RANKL was assessed in RA and OA FLS by polymerase chain reaction. Effects of APRIL on the production of proinflammatory cytokines and RANKL in RA FLS were investigated by flow cytometry and with the use of a BCMA‐Fc fusion protein.

Results

A significantly higher level of soluble APRIL was detected in RA serum compared with normal serum. Among the 3 receptors of APRIL tested, RA FLS expressed only BCMA, whereas OA FLS expressed none of the receptors. APRIL stimulated RA FLS, but not OA FLS, to produce IL‐6, TNFα, IL‐1β, and APRIL itself. In addition, APRIL increased RA FLS expression of RANKL and also enhanced progression of the cell cycle of RA FLS. Neutralization of APRIL by the BCMA‐Fc fusion protein attenuated all of these stimulating effects of APRIL on RA FLS.

Conclusion

RA FLS are stimulated by APRIL and express the APRIL receptor BCMA. These results provide evidence that APRIL is one of the main regulators in the pathogenesis of RA.

     

Modulation of interleukin‐6 and matrix metalloproteinase 2 expression in human fibroblast‐like synoviocytes by functional ionotropic glutamate receptors

Objective

Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology.

Methods

Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast‐like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin‐6 (IL‐6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP‐2] and MMP‐9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP‐1] and TIMP‐2). IL‐6 concentrations were determined by enzyme‐linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL‐6 or MMP‐2.

Results

Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N‐methyl‐D ‐aspartate (NMDA) receptors in RA FLS increased proMMP‐2 release, whereas non‐NMDA ionotropic glutamate receptor antagonists reduced IL‐6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors.

Conclusion

Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL‐6 expression.

     

Presentation of arthritogenic peptide to antigen‐specific T cells by fibroblast‐like synoviocytes

Objective

To assess the ability of rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) to function as antigen‐presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues.

Methods

Human class II major histocompatibility complex (MHC)–typed FLS were used as APCs for murine class II MHC–restricted CD4 T cell hybridomas. Interferon‐γ (IFNγ)–treated, antigen‐loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp‐39 (HC gp‐39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme‐linked immunosorbent assay of culture supernatants for interleukin‐2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2.

Results

Human FLS could present peptides from the autoantigens HC gp‐39 and human CII to antigen‐specific MHC‐restricted T cell hybridomas. This response required pretreatment of FLS with IFNγ, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp‐39 and human CII T cell hybridomas in an IFNγ‐dependent and MHC‐restricted manner.

Conclusion

RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.