The effect of a bacterial extract on synovial cells in tissue culture.

The studies reported here were undertaken to determine if Erysipelothrix crude extract (CE) could elicit consistent, detectable alterations in synovial cells in vitro. Synovial cells of rabbits were cultured in vitro and data about normal synovial cells in vitro were presented. Such cells were consistently and characteristically affected by exposure to small amounts of CE; an effect that was neutralized in the presence of specific and anti-CE anti-serum.With an increase in the amount of CE added to the cells in culture, a decreasing number of cells was formed but protein content remained relatively constant.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

Autocrine regulation of rheumatoid arthritis synovial cell growth in vitro

Rheumatoid arthritis (RA), and not osteoarthritis (OA) synovial cells proliferate in serum-free medium, a finding that suggests that, in vitro, RA synovial cells may be stimulated to grow by the continuous autocrine production of at least one polypeptide growth factor. Adding monoclonal antibody 1D11.16, or rabbit polyclonal anti-tumor growth factor beta (anti-TGF-beta) antibodies (both neutralizing antibodies to TGF-beta 1 and TGF-beta 2) to RA synovial cells, in culture, caused a significant reduction in cell growth, an effect not seen when other growth factor antibodies (platelet-derived growth factor [PDGF], epidermal growth factor [EGF], or EGF receptor) were added to the culture medium. Taken together, these data are consistent with the concept that RA synovial cell growth in vitro is driven endogenous TGF-beta. Moreover, when EGF was added to the culture medium, this caused the numbers of RA, and not OA, synovial cells to increase significantly. This finding suggests that RA synovial cells are in G1 phase of the cell cycle; an effect that could be mediated by endogenous TGF-beta.     

Inhibition of in vitro T cell activation by corneal endothelial cells.

Cells and tissues of the anterior uvea and aqueous humor express activities which inhibit immune responses. These activities include soluble factors such as TGF-beta and uncharacterized cell surface interactions. Relatively little is known regarding the immunologic activities of corneal endothelium, despite its potentially important role in contributing to the immune privilege of the anterior chamber and the high success rate of corneal transplantation. In this report, in vitro studies of cultured rat corneal endothelial (CE) cells were done using S-antigen-specific LEW rat T cell lines, or S-antigen-specific T cell hybridomas, to examine the immunologic capabilities of CE cells. Monolayers of LEW rat CE cells were unable to present antigen or a mitogen, Con A, to T cell lines or hybridomas as assessed by the lack of a proliferative response or IL-2 secretion. Furthermore, the CE cells exerted a potent inhibitory effect when added to in vitro proliferation assays of T cell lines stimulated with antigen or Con A. When T cells were preactivated on conventional antigen presenting cells and then transferred to wells containing CE cells, their proliferation was not inhibited. Although CE cells inhibited activation of T cell lines and hybridomas, they did not inhibit the growth of T cell hybridomas or CTLL cells, nor did the CE cells adversely affect the viability of resting T cells cultured on CE monolayers. The inhibitory effect was reversible as preincubation of T cells on CE cells for up to 6 days followed by washes restored T cell responsiveness when assayed on splenocytes. The inability to stimulate proliferative responses was not affected by preincubation of the CE cells with lymphokines which increase MHC antigen expression. The inhibition observed in these assays was not MHC-restricted as CE cells from both LEW and BN rats were equally inhibitory. CE cells from rabbits and cats were also potent inhibitors of T cell activation, suggesting that the mechanism is evolutionarily conserved. The mechanism of inhibition of CE cells is unknown at this time.     

Transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. Studies on secretion by synovial fibroblast-like cells and immunohistologic localization

The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.     

Association of cathepsin E with tumor growth arrest through angiogenesis inhibition and enhanced immune responses

Cathepsin E (CE) is an intracellular aspartic proteinase implicated in various physiological and pathological processes, yet its actual roles in vivo remain elusive. To assess the physiological significance of CE expression in tumor cells, human CE was stably expressed in human prostate carcinoma ALVA101 cells expressing very little CE activity. Tumor growth in nude mice with xenografted ALVA101/hCE cells was slower than with control ALVA101/mock cells. Angiogenesis antibody array and ELISA assay showed that this was partly due to the increased expression of some antiangiogenic molecules including interleukin 12 and endostatin in tumors induced by CE expression. In vitro studies also demonstrated that, among the cathepsins tested, CE most efficiently generated endostatin from the non-collagenous fragment of human collagen XVIII at mild acidic pH. Histological examination revealed that tumors formed by ALVA101/hCE cells were partitioned by well-developed membranous structures and covered with thickened, well-stratified hypodermal tissues. In addition, both the number and extent of activation of tumor-infiltrating macrophages were more profound in ALVA101/hCE compared to ALVA101/mock tumors. The chemotactic response of macrophages to ALVA101/hCE cells was also higher than that to ALVA/mock cells. These results thus indicate that CE expression in tumor cells induces tumor growth arrest via inhibition of angiogenesis and enhanced immune responses.     

Borrelia burgdorferi downregulates ICAM-1 on human synovial cells in vitro.

Lyme arthritis following infection with Borrelia burgdorferi (B. burgdorferi) is associated with the presence of bacteria in the joint, but the mechanism of persistent infection in the presence of specific antibodies and lymphocytes remains unknown. To investigate how an infection with B. burgdorferi might influence the local immune response in the joint, we examined the expression of cell adhesion molecules, human leucocyte antigens and inducible nitric oxide synthase (iNOS)-1 and -2 in human synovial cells after infection with B. burgdorferi in vitro. Synovial cells are known to influence the function of local immunologic effector cells and play a key role in the pannus formation of erosive arthritis. It has been shown previously that B. burgdorferi can persist in the cytosol of human synovial cells. The expression of the surface molecules ICAM-1, VCAM-1, HLA-class-I and -class-II and the cytosolic production of iNOS-1 and -2 in synovial cells was measured by flow cytometry for up to 5 days after infection with B. burgdorferi. A significant, lasting downregulation of surface ICAM-1 could be demonstrated on synovial cells, whereas no significant changes were seen in the expression of VCAM-1, HLA-class-I and -II, and of iNOS-1 and -2. To determine the biological significance of this downregulation an in vitro adhesion assay using peripheral blood mononuclear cells was developed. After infection with B. burgdorferi a significantly smaller number of mononuclear cells was adhering to the synovial cell monolayer. Adhesion of peripheral mononuclear cells was shown to be in part mediated by ICAM-1 by using a blocking mononuclear antibody against ICAM-1. Downregulation of ICAM-1 on synovial cells due to infection with B. burgdorferi might suppress the local immunosurveillance and might help the bacteria to persist in joint cells in vivo.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

The small cells of coelomic fluid and coelomic epithelium isolated from starfish <Emphasis Type="Italic">Asterias rubens</Emphasis> and <Emphasis Type="Italic">Asterias amurensis</Emphasis> (Echinodermata: Asteroidea): Comparative analysis of cell morphology and proliferative activity in vivo and in vitro

Earlier we revealed the probable candidates for the role of Asteroidea stem cells in the starfish Asterias rubens L., small coelomic epithelial cells (SECs-1) with a high nuclear–cytoplasmic ratio that were able to proliferate in vivo and in vitro. To check the existence of a similar cell type in other members of Asteroidea, the small cells in suspensions of coelomic fluid (CF) and coelomic epithelium (CE) of A. amurensis were analyzed with respect to their morphology and proportion in the total cell pool. The morphology of proliferating cells and the proliferative activity of CF and CE cells in vivo and in vitro were studied. The small cells with parameters identical to those of A. rubens SECs-1, were found both in CF and CE of related species. The subpopulation of weakly attached CE cells, highly enriched with SECs-1, was detected. These cells were able to migrate from CE and to proliferate in vivo and in vitro. Additionally, large proliferating cells were described in both starfish. The dynamics of proliferative activity in primary cell cultures of these starfish had some distinctions. Moreover, for the first time, the formation of “crystals”, the potential centres of spiculogenesis, was observed in primary culture of A. amurensis CE cells. The data prove that SECs may fulfil the common functions in two members of Asteroidea.     

Persistent induction of the chemokine receptor CXCR4 by TGF-beta 1 on synovial T cells contributes to their accumulation within the rheumatoid synovium

Chemokines and their receptors determine the distribution of leukocytes within tissues in health and disease. We have studied the role of the constitutive chemokine receptor CXCR4 and its ligand, stromal-derived factor-1 (SDF-1) in the perivascular accumulation of T cells in rheumatoid arthritis. We show that synovial T cells, which are primed CD45RO+CD45RBdull cells and consequently not expected to express constitutive chemokine receptors, have high levels of the chemokine receptor CXCR4. Sustained expression of CXCR4 was maintained on synovial T cells by specific factors present within the synovial microenvironment. Extensive screening revealed that TGF-beta isoforms induce the expression of CXCR4 on CD4 T cells in vitro. Depletion studies using synovial fluid confirmed an important role for TGF-beta1 in the induction of CXCR4 expression in vivo. The only known ligand for CXCR4 is SDF-1. We found SDF-1 on synovial endothelial cells and showed that SDF-1 was able to induce strong integrin-mediated adhesion of synovial fluid T cells to fibronectin and ICAM-1, confirming that CXCR4 expressed on synovial T cells was functional. These results suggest that the persistent induction of CXCR4 on synovial T cells by TGF-beta1 leads to their active, SDF-1-mediated retention in a perivascular distribution within the rheumatoid synovium.     

Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts

Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.     

The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome

Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (kcat/Km) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.     

Possible role for intracellular cholesteryl ester transfer protein in adipocyte lipid metabolism and storage

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride (TG) between lipoproteins in plasma. However, short term suppression of CETP biosynthesis in cells alters cellular cholesterol homeostasis, demonstrating an intracellular role for CETP as well. The consequences of chronic CETP deficiency in lipid-storing cells normally expressing CETP have not been reported. Here, SW872 adipocytes stably expressing antisense CETP cDNA and synthesizing 20% of normal CETP were created. CETP-deficient cells had 4-fold more CE but an approximately 3-fold decrease in cholesterol biosynthesis. This phenotype of cholesterol overload is consistent with the observed 45% reduction in low density lipoprotein receptor and 2.5-fold increase in ABCA1 levels. However, cholesterol mass in CETP-deficient adipocytes was actually reduced. Strikingly, CETP-deficient adipocytes stored <50% of normal TG, principally reflecting reduced synthesis. The hydrolysis of cellular CE and TG in CETP-deficient cells was reduced by >50%, although hydrolase/lipase activity was increased 3-fold. Notably, the incorporation of recently synthesized CE and TG into lipid storage droplets in CETP-deficient cells was just 40% of control, suggesting that these lipids are inefficiently transported to droplets where the hydrolase/lipase resides. The capacity of cellular CETP to transport CE and TG into storage droplets was directly demonstrated in vitro. Overall, chronic CETP deficiency disrupts lipid homeostasis and compromises the TG storage function of adipocytes. Inefficient CETP-mediated translocation of CE and TG from the endoplasmic reticulum to their site of storage may partially explain these defects. These studies in adipocytic cells strongly support a novel role for CETP in intracellular lipid transport and storage.     

Effects of synovial fluid on the respiratory burst of granulocytes in rheumatoid arthritis

Neutrophil infiltration in the synovia is an important feature of the local inflammatory process associated with rheumatoid arthritis. The present study is focused on the effects exerted in vitro by the synovial fluid versus serum on the respiratory burst of granulocytes isolated either from blood or synovial fluid of rheumatoid arthritis patients. The respiratory burst was evaluated as superoxide anion release, by lucigenin-amplified chemiluminescence. Our data show that the respiratory burst of granulocytes isolated from rheumatoid arthritis patients might trigger a significant oxidative stress both in periphery and the inflamed joint. These cells show no pathological pattern when activated in vitro by the chemotactic peptide fMLP, heterologous synovial fluid or serum. Acellular synovial fluid amplifies the superoxide anion release induced by fMLP more than the corresponding serum, indicating that a bacterian infection in the joint might enhance the oxidative damage in the inflamed synovium.     

Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.     

Crisscross CTL induction by SYT-SSX junction peptide and its HLA-A*2402 anchor substitute

To investigate the effects of anchor substitutions in SYT-SSX junction peptide, an HLA-A24 anchor residue (position 9) of the SYT-SSX B peptide (GYDQIMPKK) was substituted to more favorable residues according to the HLA-A24-binding motif. Among four substitutes constructed, a substitute with isoleucine (termed K9I peptide) most apparently enhanced the affinity for HLA-A24 molecule. Subsequent in vitro CTL induction analysis using PBMCs of 15 HLA-A24(+) synovial sarcoma patients revealed that the original B peptide allowed to induce synovial sarcoma-specific CTLs from 7 patients (47%), whereas such CTLs were inducible from 12 patients (80%) with K9I peptide. Moreover, the extent of cytotoxicity against HLA-A24(+) synovial sarcoma cell lines was higher in K9I peptide-induced CTLs than B peptide-induced CTLs. Influence of anchor substitution on peptide/TCR interaction was evaluated by cytotoxicity assays against autologous cells and tetramer analysis. CTLs induced from a synovial sarcoma patient using K9I peptide did not lyse autologous PHA blasts or EBV-infected B cells. In vitro stimulations of PBMCs from 5 HLA-A24(+) synovial sarcoma patients with K9I peptide increased the frequency of T cells reacting with both HLA-A24/K9I peptide tetramer and HLA-A24/B peptide tetramer. In contrast, the frequency of T cells reacting with HLA/HIV-derived peptide tetramer remained low. These findings support the validity in design of anchor residue substitution in SYT-SSX fusion gene-derived peptide, and provide a potential clue to the current stagnation in vaccination trials of fusion gene-derived natural junction peptides.     

Platelet-neutrophil-smooth muscle cell interactions: lipoxygenase-derived mono- and dihydroxy acids activate cholesteryl ester hydrolysis by the cyclic AMP dependent protein kinase cascade

Fluid-phase interactions between hematologic cells and those of the vessel wall were studied in order to define a role for lipoxygenase products as cell signals in the control of vascular cholesterol metabolism. A functional parameter for hydroxy acids in this system has not been previously demonstrated. We report herein for the first time a biochemical effect of lipoxygenase-derived eicosanoids in the modulation of cholesterol metabolism in smooth muscle cells. Products of platelet-neutrophil interactions served as cell signals in vitro to modulate cholesterol metabolism. We demonstrate that 12-HETE, 12,20-DiHETE, and 12-HETE-1,20-dioic acid activate both lysosomal and cytoplasmic cholesteryl ester (CE) hydrolytic activities, although no effect was observed on CE synthetic (ACAT) activity. The platelet lipoxygenase product, 12-HETE, was the most effective stimulator of CE hydrolysis in the smooth muscle cell, and its conversion to 12,20-DiHETE and the dioic acid derivative by the neutrophils was not necessary for the activation of CE hydrolase. A 2-fold enhancement on CE hydrolysis occurred and was independent of any "cross-activation" by hydroxy acids on production of cyclooxygenase or other lipoxygenase products. The activation of cytoplasmic CE hydrolysis had a lesser cofactor dependence on bile salts in the presence of 12-HETE. This suggested a reduced requirement for surface-active agents in an enzyme-substrate interaction where enzymes are hydrolyzing insoluble lipid substrates. Moreover, 12-HETE induced an additive effect with several lipolytic hormones in the activation of CE catabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of latent rheumatoid synovial collagenase by human mast cell tryptase

The functional role of mast cells in rheumatoid synovium was investigated by assessing the ability of mast cell tryptase to activate latent collagenase derived from rheumatoid synoviocytes. Tryptase, a mast cell neutral protease, was demonstrated in situ to reside in rheumatoid synovial mast cells, by an immunoperoxidase technique using a mouse mAb against tryptase, and in vitro to be released by dispersed synovial mast cells after both immunologic and nonimmunologic challenge. Each rheumatoid synovial mast cell contains an average of 6.2 pg of immunoreactive tryptase and the percent release values of this protease correlated with those of histamine (r = 0.58, p less than 0.01). The ability of purified tryptase to promote collagenolysis was demonstrated in a dose-dependent fashion using latent collagenase derived from rheumatoid synovium, synovial fluid, IL-1-stimulated cultured synoviocytes, and partially purified latent collagenase derived from conditioned media, with between 10 and 92% of the collagen substrate degraded. [3H] Collagen, treated with tryptase-activated latent collagenase, was subjected to electrophoresis on SDS polyacrylamide gels and autoradiography showed the collagen degradation pattern (A, B) characteristically produced by collagenase. Mast cell lysates also activated synovial latent collagenase yielding 24% digestion of collagen substrate. This activator in mast cell lysates could be inhibited by diisopropylflurophosphate or by immunoadsorption of tryptase. Thus, mast cells may activate metalloproteinases and play a role in the catabolism of collagen that occurs in rheumatoid synovium.     

Influence of synovial cells on cartilage in vitro: induction of breakdown and inhibition of synthesis.

Organoid or high density cultures of: (1) synovial cells from patients with rheumatoid arthritis, and (2) prechondrogenic mesenchymal cells from limb buds of 12-day-old mouse embryos, were co-cultured for 7-10 days using the Trowell culture system. Depending on the time of commencing co-cultivation, chondrogenesis was inhibited (co-cultivation from the start) or the cartilaginous matrix was partly degraded (co-cultivation after formation of embryonic cartilage, i.e. on day 4). These effects were obtained with cells from synovial fluid as well as from synovial tissue. Matrix degradation and the behaviour of the different cell types could be demonstrated well by electron microscopy under the in vitro conditions applied.