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 7alpha-hydroxy-DHEA (7alpha-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 7alpha-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 7alpha-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 7alpha-OH-DHEA with the use of high-performance liquid chromatography. RESULTS: The CYP7B enzyme product 7alpha-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1alpha (IL-1alpha), IL-1beta, 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 beta inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFalpha increased CYP7B activity in all cell lines tested. CONCLUSION: Exposure to the proinflammatory cytokines TNFalpha, IL-1alpha, IL-1beta, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7alpha-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.     

Basic FGF-induced activation of telomerase in rheumatoid synoviocytes

To investigate the mechanism of persistent proliferation of rheumatoid arthritis (RA) synoviocytes in situ, we examined the activity of telomerase enzyme and the expression of telomerase related factors in cultured synoviocytes. Cultured synoviocytes obtained from patients with rheumatoid arthritis (n=29), osteoarthritis (OA, n=18), and traumatic joint disease (TJD, n=4) were examined. Telomerase activity was detected by TRAP (telomeric repeat amplification protocol) assay, and 12 out of 29 samples of synoviocytes (41%) from RA patients showed a positive telomerase activity, whereas none of the samples from OA and TJD patients showed this activity. Results were confirmed by PCR-ELISA. The telomerase activity was enhanced by basic fibroblast growth factor (bFGF). The mRNA expression of telomerase related factors, such as hTERC, TRF2, and TEP-1, showed no difference between RA and OA synoviocytes. Our results suggest that telomerase is activated in rheumatoid synoviocytes, and that bFGF upregulates the activity of this enzyme in RA synoviocytes. Received: 15 June 1999 / Accepted: 10 January 2000     

RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells

Peptides containing the Arg-Gly-Asp (RGD) motif inhibit cell adhesion and exhibit a variety of other biologic effects including anticoagulant and antimetastatic activities. The aim of the present study was to examine the anchorage-independent effects of an RGD-containing peptide, Arg-Gly-Asp-Ser (RGDS), on human umbilical vein endothelial cells (HUVECs). Assays were performed on HUVECs seeded onto collagen IV; under these experimental conditions RGDS did not exert antiadhesive effects but significantly reduced FGF-2-dependent chemotaxis after 4 hours of treatment and reduced proliferation after 24 hours of treatment. Experiments carried out with caspase-specific inhibitors indicated that the observed antichemotactic effects required caspase 8 and caspase 9 activation. RGDS activated both caspase 8 and caspase 9 after 4 hours of treatment and caspase 3 after 24 hours of treatment, and markedly enhanced HUVEC apoptosis by transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)/Hoechst staining and fluorescence-activated cell sorting (FACS) analysis. Finally, confocal microscopy showed that RGDS localizes in the cytoplasm of live HUVECs within 4 hours and in vitro experiments showed that RGDS directly interacts with recombinant caspases 8 and 9 in a specific way. In summary, these results indicate that RGDS directly binds and activates caspases 8 and 9, inhibits chemotaxis, and induces apoptosis of HUVECs with a mechanism independent from its antiadhesive effect.     

ATM kinase activity modulates Fas sensitivity through the regulation of FLIP in lymphoid cells

Ataxia telangiectasia (A-T) is a rare cancer-predisposing genetic disease, caused by the lack of functional ATM kinase, a major actor of the double strand brakes (DSB) DNA-damage response. A-T patients show a broad and diverse phenotype, which includes an increased rate of lymphoma and leukemia development. Fas-induced apoptosis plays a fundamental role in the homeostasis of the immune system and its defects have been associated with autoimmunity and lymphoma development. We therefore investigated the role of ATM kinase in Fas-induced apoptosis. Using A-T lymphoid cells, we could show that ATM deficiency causes resistance to Fas-induced apoptosis. A-T cells up-regulate FLIP protein levels, a well-known inhibitor of Fas-induced apoptosis. Reconstitution of ATM kinase activity was sufficient to decrease FLIP levels and to restore Fas sensitivity. Conversely, genetic and pharmacologic ATM kinase inactivation resulted in FLIP protein up-regulation and Fas resistance. Both ATM and FLIP are aberrantly regulated in Hodgkin lymphoma. Importantly, we found that reconstitution of ATM kinase activity decreases FLIP protein levels and restores Fas sensitivity in Hodgkin lymphoma-derived cells. Overall, these data identify a novel molecular mechanism through which ATM kinase may regulate the immune system homeostasis and impair lymphoma development.     

Role of Notch-1 intracellular domain in activation of rheumatoid synoviocytes

OBJECTIVE: Notch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch-1 and its role in the activation of RA synoviocytes. METHODS: The expression of Notch-1 protein was detected by a specific antibody raised against the Notch-1 intracellular domain. Notch-1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch-1 protein expression was confirmed by Western blotting with anti-Notch-1 antibody. To analyze the role of Notch-1 in synoviocyte proliferation, we examined the effects of antisense Notch-1 oligonucleotides (ODNs) and MW167, a gamma-secretase inhibitor. RESULTS: Notch-1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct approximately 63-kd protein detected by anti-Notch-1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch-1 intracellular domain (NICD). Furthermore, tumor necrosis factor alpha (TNFalpha) increased NICD nuclear translocation in a dose-dependent manner. Antisense Notch-1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFalpha-induced proliferation in both OA and RA synoviocytes. In addition, gamma-secretase inhibitor, which blocks the production of NICD, also inhibited TNFalpha-induced proliferation of RA synoviocytes. CONCLUSION: Our results demonstrate the expression of Notch-1 in synoviocytes and the presence of Notch-1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch-1 signaling in the TNFalpha-induced proliferation of RA synoviocytes.     

Mediation of the proinflammatory cytokine response in rheumatoid arthritis and spondylarthritis by interactions between fibroblast-like synoviocytes and natural killer cells

OBJECTIVE: Fibroblast-like synoviocytes (FLS) are potentially directly involved in the propagation of inflammation. We have previously shown evidence of an expanded activated population of natural killer (NK) cells in spondylarthritis (SpA) patients. In the present study, we sought to determine whether the interaction between NK cells and FLS from SpA patients results in a proinflammatory response. METHODS: Autologous NK cells and FLS were obtained from 6 patients with SpA, 4 patients with rheumatoid arthritis (RA), and 8 patients with osteoarthritis (OA). Physical interactions between NK cells and FLS were studied by time-lapse phase-contrast microscopy. Fluorescence-activated cell sorting was used to study the activation, proliferation, and survival of NK cells in contact with FLS. Cytokine and stromal factor production were measured by a multiple cytokine bead assay. RESULTS: NK cells both adhered to and migrated beneath the FLS monolayer (pseudoemperipolesis). FLS from SpA and RA patients supported increased pseudoemperipolesis, activation, cytokine production, and survival of NK cells. The production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, IL-1beta, and IL-15, was increased in cocultures of NK cells and FLS, particularly in those from RA and SpA patients. Production of interferon-gamma, RANTES, and matrix metalloproteinase 3 (MMP-3) by NK cell and FLS coculture was greatest in SpA patients. Surface expression of IL-15 on FLS was significantly increased in SpA and RA patients, but not OA patients. Blockade with an IL-15 monoclonal antibody resulted in increased apoptosis of NK cells. CONCLUSION: FLS promote the migration, activation, and survival of NK cells. The interaction of NK cells with FLS results in increased IL-15 expression by FLS and the production of proinflammatory chemokines, cytokines, and MMPs, which may contribute to joint inflammation. This response was much more marked in SpA and RA patients as compared with OA patients.     

Apomodulation as a novel therapeutic concept for the regulation of apoptosis in rheumatoid synoviocytes.

Fas-mediated apoptosis is observed in synoviocytes of patients with rheumatoid arthritis (RA). This process may be involved in the pathophysiology of RA. We have recently found that Fas-mediated apoptosis of RA synoviocytes is associated with activation of two signaling pathways, the c-Jun amino-terminal kinase (JNK)/activator protein-1 (AP-1) pathway, and the FADD (Fas-associated death domain protein)/Caspase-8/Caspase-3/PARP (poly(ADP-ribose)polymerase) pathway. The latter appears to be one of the major signaling pathways required for Fas-mediated apoptosis in RA synoviocytes. Interestingly, Fas-mediated apoptosis in synoviocytes may be induced at least in part by tumor necrosis factor-alpha. Paradoxically, tumor necrosis factor-alpha also causes proliferation of synoviocytes. Employing these molecular processes in the treatment of RA, we have recently shown that ex vivo gene transfer of human Fas ligand (hFasL) induced apoptosis of synoviocytes and infiltrated mononuclear cells of RA synovial tissue through cell-to-cell interaction via the Fas/FasL system. We believe that further understanding of the complex regulatory mechanisms of apoptosis in RA synoviocytes would uncover further aspects of the pathophysiologic mechanisms of RA and contribute to the development of new and effective therapies for RA.     

Role of notch‐1 intracellular domain in activation of rheumatoid synoviocytes

Objective

Notch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch‐1 and its role in the activation of RA synoviocytes.

Methods

The expression of Notch‐1 protein was detected by a specific antibody raised against the Notch‐1 intracellular domain. Notch‐1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch‐1 protein expression was confirmed by Western blotting with anti–Notch‐1 antibody. To analyze the role of Notch‐1 in synoviocyte proliferation, we examined the effects of antisense Notch‐1 oligonucleotides (ODNs) and MW167, a γ‐secretase inhibitor.

Results

Notch‐1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti–Notch‐1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct ∼63‐kd protein detected by anti–Notch‐1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch‐1 intracellular domain (NICD). Furthermore, tumor necrosis factor α (TNFα) increased NICD nuclear translocation in a dose‐dependent manner. Antisense Notch‐1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFα‐induced proliferation in both OA and RA synoviocytes. In addition, γ‐secretase inhibitor, which blocks the production of NICD, also inhibited TNFα‐induced proliferation of RA synoviocytes.

Conclusion

Our results demonstrate the expression of Notch‐1 in synoviocytes and the presence of Notch‐1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch‐1 signaling in the TNFα‐induced proliferation of RA synoviocytes.

     

Caspase-8 has an essential role in resveratrol-induced apoptosis of rheumatoid fibroblast-like synoviocytes

Objective. Resveratrol is a naturally occurring polyphenol,which possesses chemotherapeutic potential through its abilityto trigger apoptosis. The objective of this study was to investigatethe major determinant for the apoptotic cell death inductionby resveratrol in fibroblast-like synoviocytes (FLS) derivedfrom patients with RA. Methods. The effect of resveratrol on apoptotic cell death wasquantified in a population of subG1 in RA FLS by flow cytometry.The underlying signalling mechanism for apoptotic death wasexamined by analysing mitochondrial membrane potential, activationof the caspase cascade and translocation of Bid. Results. We show that activation of caspase-8 is essential fortriggering resveratrol-induced apoptotic signalling via theinvolvement of the mitochondrial pathway in RA FLS. Our findingsalso suggest that this enhanced apoptosis caused by resveratroloccurred in RA FLS irrespective of p53 status. Exposure to resveratrolcaused extensive apoptotic cell death, along with a caspase-dependent(activation of caspase-9 and -3, poly ADPribose polymerase (PARP)cleavage and mitochondrial cytochrome c release) or caspase-independent[translocation of apoptosis-inducing factor (AIF) to the nucleus]signalling pathway. Analysis of upstream signalling events affectedby resveratrol revealed that the activated caspase-8 triggeredmitochondrial apoptotic events by inducing Bid cleavage withoutany alteration in the levels of Bax, Bcl-xL or Bcl2. The caspase-8inhibitor or over-expression of crmA abrogated cell death inducedby resveratrol and prevented processing of the downstream cascade. Conclusion. The results suggest that resveratrol causes activationof caspase-8, which in turn results in modulation of mitochondrialapoptotic machinery to promote apoptosis of RA FLS. KEY WORDS: Resveratrol, Fibroblast-like synoviocytes, Apoptosis, Caspase-8, Mitochondria membrane potential     

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.

     

Cyclopentenone prostaglandins induce caspase activation and apoptosis in dendritic cells by a PPAR-gamma-independent mechanism: regulation by inflammatory and T cell-derived stimuli

OBJECTIVE: Dendritic cells (DC) are professional antigen-presenting cells playing a pivotal role in the induction of immunological responses. There is evidence that DC survival during ongoing immune responses is finite. However, little is known about the mechanisms regulating apoptosis in these cells. Here, we have investigated the effects of the anti-inflammatory cyclopentenone prostaglandins on human monocyte-derived DC. MATERIALS AND METHODS: Phenotype of DC was determined by flow cytometry and their allostimulatory potential in mixed leukocyte reaction. Induction of apoptosis in DC was monitored by staining with annexin-V-FITC and propidium iodide, propidium iodide staining of cell nuclei, and fluorimetric assay of caspase activity. Induction of maturation in DC was obtained by stimulation with TNF-alpha, LPS, IFN-gamma, CD40-ligand, or different combinations of these stimuli. PPAR-gamma expression in DC was determined by RT-PCR. RESULTS: Exposure of immature DC to cyclopentenone prostaglandins blunted their allostimulatory capacity and skewed their phenotype by downregulating CD1a and costimulatory molecules. These effects were due to activation of caspases and induction of apoptotic cell death in DC by cyclopentenone prostaglandins. Mature DC showed enhanced susceptibility to apoptosis via cyclopentenone prostaglandins as compared with immature DC. Although DC express PPAR-gamma, the corresponding receptor for some of these metabolites, PPAR-gamma activation by a synthetic high-affinity agonist failed to impair DC viability. CONCLUSIONS: Cyclopentenone prostaglandins induce apoptosis of human DC by a PPAR-gamma-independent mechanism. Since these compounds are released during an inflammatory event and show anti-inflammatory properties, they may contribute to the downregulation of DC function through apoptotic cell death.