Fas-mediated Apoptosis (Fas-mediat + apoptosi)

Distribution by Scientific Domains

Selected Abstracts

IFN-, induces apoptosis in mouse embryonic stem cells, a putative mechanism of its embryotoxicity

Gang-Ming Zou
It has been reported that interferon (IFN)-, should inhibit in vitro mouse embryo growth by direct cell toxicity. However, the mechanism involved has not been clearly established. In the present study, this question was addressed using the embryonic stem (ES) cell model. It was found that IFN-, induces a dose-dependent apoptosis in ES cells, as assessed by trypan-blue staining, by Annexin-V labeling and DNA analysis. Moreover, IFN-, treatment cooperates with Fas-mediated apoptosis, a phenomenon that has been recently reported. As Bcl-2 oncoprotein functions as a death repressor molecule in an evolutionarily conserved cell death pathway, its expression was analyzed by flow cytometry. It was demonstrated that Bcl-2 is expressed in ES cells. When compared to untreated ES cells, IFN-,-treated, apoptotic cells expressed a lower Bcl-2 level and a normal level of Fas, whereas surviving cells expressed a normal level of Bcl-2 but a lower Fas expression. Altogether, these data suggest that IFN-, may influence early mouse embryo development by promoting apoptosis, which may constitute a novel mechanism of IFN-, embryotoxicity. [source]

High-dose glucose-insulin-potassium treatment reduces myocardial apoptosis in patients with acute myocardial infarction

L. Zhang
Abstract Background, Several clinical trials have suggested that a metabolic cocktail of glucose-insulin-potassium (GIK) decreases mortality rates in patients with acute myocardial infarction (AMI). It has also been reported that Fas-mediated apoptosis plays an important role in ischaemic/reperfusion injury in the rat model. This study was designed to evaluate the interaction of ischaemic/reperfusion and reperfusion therapy coadministered with high-dose GIK treatment on soluble Fas/APO-1 (sFas) and Fas ligand (sFasL) plasma concentration in patients with AMI. Materials and methods, Seventy-four patients presenting with AMI who underwent reperfusion therapy were randomized into a GIK group (n = 35) receiving high-dose GIK for 24 h or a vehicle group (n = 39). Thirty-four control subjects were also enrolled in the present study. Strepavidin-biotin ELISA was used to determine the soluble sFas and sFasL plasma concentration at baseline, 24 h (h), 3 day (d), 7 d and 14 d. Results, Soluble Fas and sFas-L serum concentrations ([sFas] and [sFas-L]) of patients with AMI were significantly elevated at baseline as compared with normal controls (NCs; P < 0·01 vs. NC). The sFas in the GIK and vehicle groups markedly decreased 24 h after the GIK infusion (10·7,5·9 ng mL,1 and 9·7,6·5 ng mL,1; P < 0·01 vs. baseline) and then increased during the 3,7-d period (5·9,12·1 ng mL,1 and 6·5,11·1 ng mL,1; P < 0·01 vs. 24 h). The GIK group demonstrated reduced sFas (12·1,5·9 ng mL,1) at 14 d (P < 0·01 vs. 7 d), with no concomitant changes in the vehicle group. The sFas-L in the GIK and vehicle groups was not significant different during the 14-d period. Conclusions, These results indicate that the sFas and sFasL in patients with AMI increased significantly compared with NC. Owing to the cardioprotective effects reported here and by others, a high-dose GIK infusion co-administered with the timely re-establishment of nutritive perfusion should be strongly considered as a treatment of choice for AMI. Additionally, sFas may be a valuable marker of the physiological response to ischaemic/reperfusion injury and reperfusion associated with high-dose GIK treatment. [source]

The role of Fas ligand as an effector molecule in corneal graft rejection

Abstract Previous studies have shown that the expression of Fas ligand (FasL; CD95L) by donor corneas is critical to their survival when placed on allogeneic recipients. Since there have been reports that the cornea expresses Fas, we tested the idea that FasL on lymphoid cells could be an effector molecule during rejection episodes. When FasL defective BALB/c- gld mice were engrafted with allogeneic corneas, significantly more of these corneas were accepted than by normal BALB/c mice. However, this was not due to impaired FasL-mediated effector function in these mice as the allogeneic corneas did not express detectable Fas by Western blot or RT-PCR analysis. Furthermore, donor corneas without Fas were given no survival advantage, but were rejected similar to wild-type donor allogeneic corneas. Examination of the T cell compartment in gld mice revealed that these cells express higher levels of Fas and are more susceptible to Fas-mediated death than wild-type cells. These results indicate that FasL is not an effector molecule in corneal graft rejection and that gld mice show reduced graft rejection due to greater susceptibility of their T cells to Fas-mediated apoptosis. [source]

Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injury

Wen Ru Yu
Abstract Activation of the Fas receptor has been recently linked to apoptotic cell death after spinal cord injury (SCI). Although it is generally considered that Fas activation mediates apoptosis predominantly through the extrinsic pathway, we hypothesized that intrinsic mitochondrial signaling could be involved in the underlying mechanism of Fas-induced apoptosis after SCI. In the present study, we utilized the FejotaTM clip compression model of SCI at T5,6 in C57BL/6 Fas-deficient (lpr) and wild-type mice. Complementary studies were conducted using an in vitro model of trauma or a Fas-activating antibody to induce apoptosis in primary neuronal,glial mixed spinal cord cultures. After in vivo SCI, lpr mice, in comparison with wild-type mice, exhibited reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells at the lesion, reduced expression of truncation of Bid (tBid), apoptosis-inducing factor, activated caspase-9 and activated caspase-3, and increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. After in vitro neurotrauma or the induction of Fas signaling by the Jo2 activating antibody, lpr spinal cord cultures showed an increased proportion of cells retaining mitochondrial membrane integrity and a reduction of tBid expression, caspase-9 and caspase-3 activation, and TUNEL-positive cells as compared to wild-type spinal cord cultures. The neutralization of Fas ligand (FasL) protected against traumatically induced or Fas-mediated caspase-3 activation and the loss of mitochondrial membrane potential and tBid expression in wild-type spinal cord cultures. However, in lpr spinal cord cultures, FasL neutralization had no protective effects. In summary, these data provide direct evidence for the induction of intrinsic mitochondrial signaling pathways following Fas activation after SCI. [source]

The adaptor molecule FADD from Xenopus laevis demonstrates evolutionary conservation of its pro-apoptotic activity

GENES TO CELLS, Issue 12 2004
Kazuhiro Sakamaki
FADD is an adaptor protein that transmits apoptotic signals from death receptors such as Fas to downstream initiator caspases in mammals. We have identified and characterized the Xenopus orthologue of mammalian FADD (xFADD). xFADD contains both a death effector domain (DED) and a death domain (DD) that are structurally homologous to those of mammalian FADD. We observed xFADD binding to Xenopus caspase-8 and caspase-10 as well as to human caspase-8 and Fas through interactions with their homophilic DED and DD domains. When over-expressed, xFADD was also able to induce apoptosis in wild-type mouse embryonic fibroblasts (MEF), but not in caspase-8-deficient MEF cells. In contrast, DED-deficient xFADD (xFADDdn) acted as a dominant-negative mutant and prevented Fas-mediated apoptosis in mammalian cell lines. These results indicate that xFADD transmits apoptotic signals from Fas to caspase-8. Furthermore, we found that transgenic animals expressing xFADD in the developing heart or eye under the control of tissue-specific promoters show abnormal phenotypes. Taken together, these results suggest that xFADD can substitute functionally for its mammalian homologue in death receptor-mediated apoptosis, and we suggest that xFADD functions as a pro-apoptotic adaptor molecule in frogs. Thus, the structural and functional similarities between xFADD and mammalian FADD provide evidence that the apoptotic pathways are evolutionally conserved across vertebrate species. [source]

Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes,

HEPATOLOGY, Issue 3 2007
Marta Casado
Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2,selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. Conclusion: These results demonstrate that expression of COX-2,dependent prostaglandins exerted a protection against liver apoptosis. (HEPATOLOGY 2007;45:631,638.) [source]

Roles of AKT and sphingosine kinase in the antiapoptotic effects of bile duct ligation in mouse liver,

HEPATOLOGY, Issue 6 2005
Yosuke Osawa
Tumor necrosis factor (TNF) receptor, and Fas-mediated apoptosis are major death processes of hepatocytes in liver disease. Although antiapoptotic effects in the injured liver promote chronic hepatitis and carcinogenesis, scant information is known about these mechanisms. To explore this issue, we compared acute liver injury after TNF-, or anti-Fas antibody (Jo2) between livers from sham-operated mice and chronic injured liver via bile duct ligation (BDL). BDL inhibited hepatocyte apoptosis induced by TNF-, but not by Jo2. On the other hand, BDL inhibited the massive hemorrhage seen in livers treated with either TNF-, or Jo2. Inactivation of AKT blocked the antiapoptotic effect of BDL. Sphingosine kinase knockout mice also lost the antihemorrhagic effect of BDL and attenuated the antiapoptotic effects of BDL. In bile duct,ligated livers, hepatic stellate cells (HSCs) were activated and produced tissue inhibitor of metalloproteinase 1 in a sphingosine kinase (SphK)-1,dependent mechanism. In conclusion, BDL exerts antiapoptotic effects that appear to require activation of AKT in hepatocytes and SphK in HSCs.(HEPATOLOGY 2005;42:1320,1328.) [source]

COX-2 inhibits Fas-mediated apoptosis in cholangiocarcinoma cells

HEPATOLOGY, Issue 3 2002
Ugochukwu C. Nzeako
Fas expression has been shown to negatively regulate the progression of cholangiocarcinoma cells in xenografts. However, many human cholangiocarcinomas express Fas, suggesting these cancers have developed mechanisms to inhibit Fas-mediated apoptosis. Cyclooxygenase-2 (COX-2), which generates prostanoids, is expressed by many cholangiocarcinomas. Therefore, our aim was to determine whether COX-2 expression inhibits death receptor,mediated apoptosis in KMBC cells, a cholangiocarcinoma cell line. These cells express messenger RNA for the death receptors Fas, tumor necrosis factor receptor 1 (TNF-R1), death receptor 4 (DR4), and DR5. Agonists for these death receptors, CH-11, TNF-,, and TRAIL all induced apoptosis. However, COX-2, whether induced by proinflammatory cytokines or transient transfection, only significantly inhibited Fas-mediated apoptosis. The COX-2 inhibitor NS-398 restored Fas-mediated apoptosis in COX-2 transfected cells. Prostaglandin E2 reduced apoptosis and mitochondrial depolarization after treatment with the Fas agonist CH-11. Of a variety of antiapoptotic proteins examined, COX-2/prostaglandin E2 only increased expression of Mcl-1, an antiapoptotic member of the Bcl-2 family. In conclusion, these data suggest that prostanoid generation by COX-2 specifically inhibits Fas-mediated apoptosis, likely by up-regulating Mcl-1 expression. Pharmacologic inhibition of COX-2 may be useful in augmenting Fas-mediated apoptosis of cholangiocarcinoma cells. [source]

Roles of proinflammatory cytokines and the Fas/Fas ligand interaction in the pathogenesis of inflammatory myopathies

IMMUNOLOGY, Issue 1pt2 2009
Masahiro Kondo
Summary Within the lesions of inflammatory myopathies, muscle fibres and invading mononuclear cells express Fas and Fas ligand (FasL), respectively. However, the roles of the Fas/FasL interaction in the pathogenesis of inflammatory myopathies are not fully understood. In the present study, we investigated the roles of proinflammatory cytokines and the Fas/FasL system in the pathogenesis of inflammatory myopathies. In vitro culturing of muscle cells with the proinflammatory cytokines interferon-,, tumour necrosis factor-,, and interleukin (IL)-1, synergistically increased Fas expression, susceptibility to Fas-mediated apoptosis, and the expression of cytoplasmic caspases 8 and 3. In addition, culturing of muscle cells with activated CD4+ T cells induced muscle cell apoptosis, which was partially inhibited by anti-FasL antibody. We also tested the possibility that T helper (Th) 17, which is an IL-17-producing helper T-cell subset that plays crucial roles in autoimmune and inflammatory responses, participates in the pathogenesis of inflammatory myopathies. Interestingly, in vitro culturing of dendritic cells with anti-Fas immunoglobulin M (IgM) or activated CD4+ T cells induced the expression of mRNA for IL-23p19, but not for IL-12p35, in addition to proinflammatory cytokines. Furthermore, IL-23p19 and IL-17 mRNAs were detected in the majority of biopsy samples from patients with inflammatory myopathies. Taken together, these results suggest that proinflammatory cytokines enhance Fas-mediated apoptosis of muscle cells, and that the Fas/FasL interaction between invading dendritic cells and CD4+ T cells induces local production of IL-23 and proinflammatory cytokines, which can promote the proliferation of Th17 cells and enhance Fas-mediated apoptosis of muscle cells, respectively. [source]

FAP-1-mediated activation of NF-,B induces resistance of head and neck cancer to fas-induced apoptosis

Eva Wieckowski
Abstract Molecular mechanisms responsible for tumor resistance to apoptosis often involve the Fas/FasL pathway. While squamous cell carcinomas of the head and neck (SCCHN) express both Fas and FasL, their resistance to self-induced apoptosis or apoptosis mediated by Fas agonistic antibody (CH-11Ab) was independent of the level of Fas surface expression or the presence of soluble Fas in supernatants of primary or metastatic SCCHN cell lines. By in vitro immunoselection, using PCI-15A cell line treated with successive cycles of CH-11 Ab, Fas-resistant sublines with the parental genotype were selected. Such sublines failed to cleave caspase-8 upon Fas engagement and were resistant to CH-11 Ab, although they remained sensitive to VP-16 or staurosporin. In the presence of cycloheximide, the selected SCCHN sublines become susceptible to CH-11 Ab, and showed cleavage of caspase-8, suggesting that apoptosis resistance was mediated by an inhibitory protein(s) acting upstream of caspase-8. Overexpression of Fas-associated phosphatase 1 (FAP-1), but not cellular FLICE-inhibitory protein (cFLIP) in SCCHN sublines was documented by Western blots and RT-PCR analyses. The FAP-1+ selected sublines also downregulated cell surface Fas. A high phosphorylation level of I,B,, NF,B activation and upregulation of Bcl-2 expression were observed in the FAP-1+ sublines. Treatment with the phosphatase inhibitor, orthovanadate, or silencing of FAP-1 with siRNA abolished their resistance to apoptosis, suggesting that FAP-1 phosphatase activity could be responsible for NF-,B activation and resistance of SCCHN cells to Fas-mediated apoptosis. J. Cell. Biochem. 100: 16,28, 2007. © 2006 Wiley-Liss, Inc. [source]

Contribution of death receptor and mitochondrial pathways to Fas-mediated apoptosis in the prostatic carcinoma cell line PC3

THE PROSTATE, Issue 4 2002
Natalya V. Guseva
Abstract BACKGROUND Two main pathways of apoptosis in mammalian cells have been described: the death receptor pathway and the mitochondrial pathway. Two different cell types have been identified for Fas-mediated apoptosis, each using almost exclusively one of two different signaling pathways. Human prostatic carcinoma cell line, PC3 is sensitive to Fas-mediated apoptosis, but relation of receptor and mitochondrial pathways is not clear. METHODS Cell viability was estimated by calcein assay. Apoptosis was determined by preparation of DNA ladder. Expression of Fas-associated death domain-dominant negative (FADD-DN) and Bcl-2, activation of caspases, PARP, DFF45, Bid cleavage, and cytochrome c release were assessed using Western blotting techniques. [35S] Methionine-labeled caspase-3 was transcribed in vitro and translated using the TNT kit (Promega). A vector containing caspase-3 was prepared by the ligation of EcoR I/BamHI flanked PCR fragment of full size caspase-3 cDNA into pBlusckript II SK(+/,) (Stratagen). RESULTS Overexpression of both FADD-DN and Bcl-2 genes prevent Fas-mediated apoptosis in PC3. As predicted, overexpression of FADD-DN prevented activation of caspase-8 and Bid cleavage and attenuated the release of cytochrome c and activation of caspases -2, -7, and -9. Bcl-2 overexpression did not affect caspase-8 activation and cleavage of Bid but blocked the release of cytochrome c and activation of mitochondria localized caspases -2, -7, and,9. Overexpression of FADD-DN and Bcl-2 affected the activation of caspase-3 and PARP cleavage differently: FADD-DN attenuated the activation of caspase-3 and PARP cleavage whereas Bcl-2 overexpression prevented caspase-3 activation and completely blocked cleavage of PARP. CONCLUSIONS These data suggest that activation of caspase-8 is necessary but not sufficient to complete Fas-mediated apoptosis in PC3 cells without activation of the mitochondrial pathway. In addition, caspase-3 activation after Fas-receptor ligation involves two steps and is dependent on mitochondrial activation. Prostate 51: 231,240, 2002. © 2002 Wiley-Liss, Inc. [source]

Increased expression of CD40 on bone marrow CD34+ hematopoietic progenitor cells in patients with systemic lupus erythematosus: Contribution to Fas-mediated apoptosis

Katerina Pyrovolaki
Objective Patients with systemic lupus erythematosus (SLE) display increased apoptosis of bone marrow (BM) CD34+ hematopoietic progenitor cells. This study was undertaken to evaluate the expression of CD40 and CD40L in the BM of SLE patients, and to explore the possible involvement of these molecules in apoptosis of CD34+ cells. Methods The proportion and survival characteristics of CD40+ cells within the BM CD34+ fraction from SLE patients and healthy controls were evaluated by flow cytometry. The production of CD40L by BM stromal cells was assessed using long-term BM cultures, and the effect of CD40L on the survival characteristics and clonogenic potential of CD34+ cells was evaluated ex vivo by flow cytometry and clonogenic assays. Results SLE patients displayed an increased proportion of CD40+ cells within the CD34+ fraction as compared with controls. The CD34+CD40+ subpopulation contained an increased proportion of apoptotic cells compared with the CD34+CD40, fraction in patients and controls, suggesting that CD40 is involved in the apoptosis of CD34+ cells. Stimulation of patients' CD34+ cells with CD40L increased the proportion of apoptotic cells and decreased the proportion of colony-forming cells as compared with untreated cultures. The CD40L-mediated effects were amplified following treatment with recombinant Fas ligand, suggesting that the effects of these ligands are synergistic. CD40L levels were significantly increased in long-term BM culture supernatants and adherent layers of BM cells from SLE patients as compared with controls. Conclusion These data reveal a novel role for the CD40/CD40L dyad in SLE by demonstrating that up-regulation and induction of CD40 on BM CD34+ cells from patients with SLE contribute to the amplification of Fas-mediated apoptosis of progenitor cells. [source]

Crystallization and preliminary X-ray crystallographic analysis of human FAF1 UBX domain

Wonchull Kang
Fas-associated factor 1 (FAF1) is a multifunctional pro-apoptotic protein that is involved in Fas-mediated apoptosis, NF-,B signalling and the ubiquitin,proteasome pathway. In the ubiquitin,proteasome pathway, FAF1 binds to the N domain of p97/VCP, a molecular chaperone that acts in complex with the proteasome, through its C-terminal UBX domain and inhibits the proteasomal protein-degradation process. In an effort to elucidate the structural basis of the function of FAF1 in modulating p97/VCP activity related to proteasomal protein degradation, crystallographic analysis of the FAF1 UBX domain and the p97/VCP N domain was initiated. Following the recently reported crystallization of the FAF1 UBX domain bound to the p97/VCP N domain, the unbound FAF1 UBX domain was also crystallized for purposes of structural comparison. X-ray data were collected to 3.00,Ĺ resolution and the crystals belonged to space group F4132, with unit-cell parameters a = b = c = 176.40,Ĺ. The Matthews coefficient and solvent content were estimated to be 3.04,Ĺ3,Da,1 and 59.5%, respectively, assuming that the asymmetric unit contained two molecules of the UBX domain, which was subsequently confirmed by molecular-replacement calculations. [source]

Sodium butyrate induces P53-independent, Fas-mediated apoptosis in MCF-7 human breast cancer cells

Valérie Chopin
This study was performed to determine the effect and action mechanisms of sodium butyrate (NaB) on the growth of breast cancer cells. Butyrate inhibited the growth of all breast cancer cell lines analysed. It induced cell cycle arrest in G1 and apoptosis in MCF-7, MCF-7ras, T47-D, and BT-20 cells, as well as arrest in G2/M in MDA-MB-231 cells. Transient transfection of MCF-7 and T47-D cells with wild-type and antisense p53 did not modify butyrate-induced apoptosis. Pifithrin-,, which inhibits the transcriptional activity of P53, did not modify cell growth or apoptosis of MCF-7 and T47-D cells treated with butyrate. These results indicate that P53 was not involved in butyrate-induced growth inhibition of breast cancer cells. Treatment of MCF-7 cells with anti-Fas agonist antibody induced cell death, indicating that Fas was functional in these cells. Moreover, butyrate potentiated Fas-induced apoptosis, as massive apoptosis was observed rapidly when MCF-7 cells were treated with butyrate and anti-Fas agonist antibody. In addition, butyrate-induced apoptosis in MCF-7 cells was considerably reduced by anti-Fas antagonist antibody. Western blot analysis showed that butyrate increased Fas and Fas ligand levels (Fas L), indicating that butyrate-induced apoptosis may be mediated by Fas signalling. These results demonstrate that butyrate inhibited the growth of breast cancer cells in a P53-independent manner. Moreover, it induced apoptosis via the Fas/Fas L system and potentiated Fas-triggered apoptosis in MCF-7 cells. These findings may open interesting perspectives in human breast cancer treatment strategy. British Journal of Pharmacology (2002) 135, 79,86; doi:10.1038/sj.bjp.0704456 [source]

Hydroxychloroquine potentiates Fas-mediated apoptosis of rheumatoid synoviocytes

W.-U. Kim
Summary Inadequate apoptosis may contribute to the synovial hyperplasia associated with rheumatoid arthritis (RA). The Fas-associated death domain protein (FADD)-like interleukin (IL)-1,-converting enzyme (FLICE)-inhibitory protein (FLIP), which is an apoptotic inhibitor, has been implicated in the resistance to Fas-mediated apoptosis of synoviocytes. This study investigated whether hydroxychloroquine (HCQ), an anti-rheumatic drug, induces the apoptosis of rheumatoid synoviocytes, and modulates the expression of FLIP. Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and were cultured with various concentrations of HCQ in the presence or absence of the IgM anti-Fas monoclonal antibodies (mAb) (CH11). Treatment with HCQ, ranging from 1 to 100 µM, induced the apoptosis of FLS in a dose- and time-dependent manner. The increase in synoviocytes apoptosis by HCQ was associated with caspase-3 activation. A combined treatment of HCQ and anti-Fas mAb increased FLS apoptosis and caspase-3 activity synergistically, compared with either anti-Fas mAb or HCQ alone. The Fas expression level in the FLS was not increased by the HCQ treatment, while the FLIP mRNA and protein levels were decreased rapidly by the HCQ treatment. Moreover, time kinetics analysis revealed that the decreased expression of FLIP by HCQ preceded the apoptotic event that was triggered by HCQ plus anti-Fas mAb. Taken together, HCQ increases the apoptosis of rheumatoid synoviocytes by activating caspase-3, and also sensitizes rheumatoid synoviocytes to Fas-mediated apoptosis. Our data suggest that HCQ may exert its anti-rheumatic effect in rheumatoid joints through these mechanisms. [source]