Lymphocytes induce resorption of cartilage by producing catabolin.
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ABSTRACT:
SUBMITTER: Saklatvala J
PROVIDER: S-EPMC1158104 | biostudies-other | 1982 Jan
REPOSITORIES: biostudies-other
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Project description:Catabolin, a protein that causes proteoglycan resorption in explants of living cartilage, was purified to homogeneity from culture medium conditioned by culturing buffy-coat leucocytes from 60 litres of pig blood in the presence of concanavalin A. The purification steps were (1) gel filtration of concentrated medium, (2) chromatofocusing, (3) hydroxyapatite chromatography, (4) anion-exchange chromatography (Mono Q), (5) reversed-phase high-pressure liquid chromatography (h.p.l.c.) (Zorbax ODS). These achieved approx. 9000-fold purification from the starting material. The purified protein when reduced ran as a single band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis with Mr 21000. On isoelectric focusing its pI was 4.8-5.0, and there was evidence of micro-heterogeneity. The protein co-migrated with active material on h.p.l.c., isoelectric focusing and SDS gels (15 and 12.5% acrylamide) under both reducing and non-reducing conditions. The pure protein caused proteoglycan release from cultured bovine nasal cartilage at 20pM. Its possible identity with interleukin 1 is discussed.
| S-EPMC1152407 | biostudies-other
Project description:1. Pig synovium in organ culture produces material which induces living cartilage to resorb its proteoglycan in vitro. 2. The bioassay for this material was to measure glycosaminoglycan released from explants of bovine nasal-septal cartilage cultured for 8 days. The performance of the assay was greatly improved by adding cortisol succinate (0.1mug/ml). This decreased the release of glycosaminoglycan from unstimulated cartilage without inhibiting its response to catabolic factors from the synovium. 3. By using this improved assay it was shown that 90% of the active materials in synovial culture medium were retained by dialysis membrane. 4. An active protein was partially purified from synovial culture medium by (NH(4))(2)SO(4) precipitation, ion-exchange chromatography, gel filtration and preparative isoelectric focusing. 5. This protein, called catabolin, had mol.wt. 17000 and pI4.6. 6. Synovial culture medium concentrated in dialysis tubing was subjected to gel chromatography and found to contain one major active component, which was eluted at the same position as the partially purified catabolin. 7. The synovial culture medium was not inactivated by heating (70 degrees C for 10min), nor were diluted preparations of partially purified catabolin, but concentrated crude preparations were thermolabile. 8. These results suggest that catabolin is the major substance produced by the synovial tissue in culture which induces resorption of proteoglycan of living cartilage in vitro. 9. Other cultured soft connective tissues produced catabolin-like activity. The example of sclera is shown, and production was inhibited by cortisol succinate (0.1mug/ml). 10. It is suggested that catabolin may be a general product of soft connective tissues in culture, and its physiological function may be to induce resorption of connective-tissue matrix after injury.
| S-EPMC1163428 | biostudies-other
Project description:Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.
| S-EPMC1154099 | biostudies-other
Project description:ObjectiveAutoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption.MethodsAnti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption.ResultsNET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts.ConclusionIgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies.
| S-EPMC8281931 | biostudies-literature
Project description:BACKGROUND:Osteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. The osteoclast resorption processes required to degrade subchondral bone and cartilage-the remodeling of which is important in the osteoarthritic disease process-have not been previously described, although cathepsin K has been indicated to participate. In this study we profile osteoclast-mediated degradation of bovine knee joint compartments in a novel in vitro model using biomarkers of extracellular matrix (ECM) degradation to assess the potential of osteoclast-derived resorption processes to degrade different knee joint compartments. METHODS:Mature human osteoclasts were cultured on ECMs isolated from bovine knees-articular cartilage, cortical bone, and osteochondral junction ECM (a subchondral bone-calcified cartilage mixture)-in the presence of inhibitors: the cystein protease inhibitor E-64, the matrix metalloproteinase (MMP) inhibitor GM6001, or the vacuolar-type H+-ATPase (V-ATPase) inhibitor diphyllin. Biomarkers of bone (calcium and C-terminal type I collagen (CTX-I)) and cartilage (C2M) degradation were measured in the culture supernatants. Cultures without osteoclasts were used as background samples. Background-subtracted biomarker levels were normalized to the vehicle condition and were analyzed using analysis of variance with Tukey or Dunnett's T3 post hoc test, as applicable. RESULTS:Osteochondral CTX-I release was inhibited by E-64 (19% of vehicle, p = 0.0008), GM6001 (51% of vehicle, p = 0.013), and E-64/GM6001 combined (4% of vehicle, p = 0.0007)-similarly to bone CTX-I release. Diphyllin also inhibited osteochondral CTX-I release (48% of vehicle, p = 0.014), albeit less than on bone (4% of vehicle, p <?0.0001). Osteochondral C2M release was only inhibited by E-64 (49% of vehicle, p = 0.07) and GM6001 (14% of vehicle, p = 0.006), with complete abrogation when combined (0% of vehicle, p = 0.004). Cartilage C2M release was non-significantly inhibited by E-64 (69% of vehicle, p = 0.98) and was completely abrogated by GM6001 (0% of vehicle, p = 0.16). CONCLUSIONS:Our study supports that osteoclasts can resorb non-calcified and calcified cartilage independently of acidification. We demonstrated both MMP-mediated and cysteine protease-mediated resorption of calcified cartilage. Osteoclast functionality was highly dependent on the resorbed substrate, as different ECMs required different osteoclast processes for degradation. Our novel culture system has potential to facilitate drug and biomarker development aimed at rheumatic diseases, e.g. osteoarthritis, where pathological osteoclast processes in specific joint compartments may contribute to the disease process.
| S-EPMC5894194 | biostudies-literature
Project description:Recent studies have shown that tumor cells can release autophagosomes, which transport a broad array of biologically active molecules with potential modulatory effects on immune cell functions. In this study, we aimed to investigate the role of tumor cells-released autophagosomes (i.e. TRAP) in regulating B cell differentiation and function. TRAPs from murine tumor cell lines were found to induce splenic B cells to differentiate into IL-10-producing regulatory B cells (Bregs) with a distinct phenotype of CD1d(+) CD5(+), which could potently inhibit CD8(+) and CD4(+) T cell responses in IL-10-depedent manner both in vitro and in vivo. Notably, adoptive transfer of TRAP-induced Bregs abrogated the immune response and antitumor effect induced by OVA-loaded DC vaccinations in E.G7-OVA-bearing mouse model. Mechanistic studies revealed that membrane-bound high-mobility group B1 (HMGB1) on the intact TRAPs was crucial for inducing Breg differentiation via the activation of TLR2-MyD88-NF-κB signal pathway in B cells. Moreover, TRAPs enriched from malignant effusions of cancer patients could induce human B cells to differentiate into IL-10-producing B cells with immunoregulatory functions, the frequency of which were positively correlated with the HMGB1 levels on TRAPs. Together, our findings have demonstrated that TRAPs promote the generation of IL-10(+) Bregs, which may contribute to the suppression of antitumor immunity.
| S-EPMC5006924 | biostudies-literature
Project description:ObjectiveDemonstrate the impact of IL-10 producing T lymphocytes on mediating dermal scarring.Summary background dataWe demonstrated that CD4+ cells are essential to improving postinjury wound healing and preventing fibrosis. CD4+ subsets secrete differential cytokine and growth factor profiles, though their role in fibrosis is not known. IL-10, a key anti-inflammatory cytokine shown to promote regenerative wound healing, is secreted by some CD4+ subsets. We, therefore, hypothesize that IL-10 producing CD4+ T lymphocyte subsets selectively attenuate dermal wound fibrosis.MethodsIL-10-/- and wild-type murine splenocytes were enriched for CD4+ lymphocytes and adoptively transferred into severe combined immunodeficient (SCID) mice that received full-thickness wounds which were analyzed at days 7 and 28 for inflammation and collagen content. We then sorted CD4+CD44int/lowFoxP3-CD62L+ T cells (Tnaive) or CD4+CD44HiFoxP3- type 1 regulatory (Tr1) T cell subsets from 10BiT murine splenocytes, activated them, and transferred them into wounds. In vitro, dermal fibroblasts were cocultured with Tnaive or Tr1 and the effect on extracellular matrix (ECM) regulation was analyzed.ResultsThe anti-inflammatory and antifibrotic effects of CD4+ cells on SCID wounds were lost with cells from IL-10-/- mice. Adoptive transfer of Tr1 into SCID mice resulted in accelerated wound closure at d7 with reduced fibrosis at d28, with Tr1 favoring hyaluronan production by fibroblasts, an ECM molecule implicated in IL-10-induced regenerative healing.ConclusionsIL-10 producing T-lymphocytes, specifically Tr1, regulate inflammatory cell cytokine expression to promote HA-rich ECM deposition and attenuate fibrosis. Promoting IL-10 producing lymphocytes in wounds may be a therapeutic target to promote regenerative wound healing.
| S-EPMC8428868 | biostudies-literature
Project description:A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.
| S-EPMC1144916 | biostudies-other
Project description:Thiopurines are extensively used immunosuppressants for the treatment of inflammatory bowel disease (IBD). The polymorphism of thiopurine S-methyltransferase (TPMT) influences thiopurine metabolism and therapy outcome. We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome. We identified thirteen proteins with altered expression and nine proteins with altered phosphorylation signals. Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells. Furthermore, reactive oxygen species (ROS) assay results were consistent with a significant induction of oxidative stress by both TPMT knockdown and thiopurine treatments. Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown. Collectively, increased oxidative stress might be a mechanism involved in thiopurine induced cytotoxicity and adverse effects (i.e., hepatotoxicity) and an antioxidant cotherapy might help to combat this. Results highlight the significance of oxidative stress in thiopurines' actions and could have important implications for the treatment of IBD patients.
| S-EPMC4385670 | biostudies-literature
Project description:Pig synovial fibroblasts were shown to produce a protein that caused live cartilage to resorb its proteoglycan matrix in vitro. Fibroblasts were obtained either from synovial tissue digest or by allowing them to grow out of explants. The population derived from the digests was homogeneous and free of macrophage-like cells after two passages, but was still producing the cartilage-resorbing protein after seven passages. The active protein was found to have Mr 20,000 on gell filtration, and pI 4.8 on isoelectric focussing in polyacrylamide gel. It was indistinguishable from a protein with the same activity from pig mononuclear leucocytes, which has been called catabolin. Production of the protein was increased if the synovial fibroblasts were cultured with the tumour promoter phorbol 12-myristate 13-acetate. Fibroblasts from other sources (joint capsule and peritoneum) also apparently made the protein. The possibility that catabolin is the same as interleukin-1 is discussed: if they are, then the results suggest that fibroblasts can make an interleukin-1-life protein.
| S-EPMC1152527 | biostudies-other