We explored the possibility that pulsed antigen-presenting cells (APC) provide a model vector system for site-specific delivery of immunosuppressive proteins during collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. Thus, mice were treated with either B cells or macrophages engineered to secrete IL-4 and loaded (or not) with type II collagen (CII). Systemic injection of an IL-4-producing B cell hybridoma resulted in a reduction of arthritis severity which was further improved when APC were incubated with CII before their transfer. Unmanipulated B cells loaded with CII also exerted a potent suppressive effect. Likely, clinical amelioration was observed in mice given at priming syngeneic bone marrow-derived macrophages producing IL-4 and pulsed with CII in comparison to the other groups. When the same dose of cells was transferred at disease onset, a moderate beneficial effect was observed. Whatever the APC inoculated, the beneficial effect did not rely upon an IL-4-driven shift towards Th2 phenotype. Systemic administration of fluorescent dye labeled macrophages to arthritic mice has shown that some of these cells rapidly migrate to joints. Moreover, IL-4 transfected macrophages retained their potent capacity to present CII peptides to T cells. These findings validate the use of CII peptide-loaded engineered APC as therapeutic vector cells in CIA and allow consideration of this strategy for the administration of various anti-inflammatory proteins. (+info)
Frizzled-7 and limb mesenchymal chondrogenesis: effect of misexpression and involvement of N-cadherin.
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Products of the Frizzled family of tissue polarity genes have been identified as putative receptors for the Wnt family of signaling molecules. Wnt-signaling is implicated in the regulation of limb mesenchymal chondrogenesis, and our recent study indicates that N-cadherin and related activities are functionally involved in Wnt-7a-mediated inhibition of chondrogenesis. By using an in vitro high-density micromass culture system of chick limb mesenchymal cells, we have analyzed the spatiotemporal expression patterns and the effects on chondrogenesis of RCAS retroviral-mediated misexpression of Chfz-1 and Chfz-7, two Frizzled genes implicated in chondrogenic regulation. Chfz-1 expression was localized at areas surrounding the cartilaginous nodules at all time points examined, whereas Chfz-7 expression was limited to cellular aggregates during initial mesenchymal condensation, and subsequently was down-regulated from the centers toward the periphery of cartilage nodules at the time of chondrogenic differentiation, resembling the pattern of N-cadherin expression. Chondrogenesis in vitro was inhibited and limited to a smaller area of the culture upon misexpression of Chfz-7, but not affected by Chfz-1 misexpression. Analyses of cellular condensation and chondrogenic differentiation showed that the inhibitory action of Chfz-7 is unlikely to be at the chondrogenic differentiation step, but instead affects the earlier precartilage aggregate formation event. At 24 hr, expression of N-cadherin, a key component of the cellular condensation phase of chondrogenesis, was delayed/suppressed in Chfz-7 misexpressing cultures, and was limited to a significantly smaller cellular condensation area within the entire culture at 48 hr, when compared with control cultures. Chfz-1 misexpressing cultures appeared similar to control cultures at all time points. However, neither Chfz-1 nor Chfz-7 misexpression affected mesenchymal cell proliferation in vitro. These results suggest that Chfz-7 is active in regulating N-cadherin expression during the process of limb mesenchymal chondrogenesis and that Chfz-1 and Chfz-7 are involved in different Wnt-signaling pathways. (+info)
A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness.
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T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases. (+info)
Comparison of collagenase-cleaved articular cartilage collagen in mice in the naturally occurring STR/ort model of osteoarthritis and in collagen-induced arthritis.
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OBJECTIVE: The STR/ort mouse develops a naturally occurring osteoarthritis of the femorotibial joint that provides a model with which to establish the time course of biochemical changes taking place in articular cartilage in the disease. Our objective was to define the onset, location and progression of type II collagen cleavage by collagenase in the tibial cartilage of the STR/ort mouse. For comparison, cartilage collagen cleavage was also studied in collagen-induced arthritis in DBA mice. DESIGN: STR and control CBA mice aged 6-45 weeks were examined. DBA/1 mice were studied 2 and 3 weeks after initiating collagen-induced arthritis. Collagen cleavage was detected by immunolocalization using the antibody COL2-3/4Cshort which recognizes a carboxy terminal neoepitope created by collagenase cleavage of type I and II collagens. RESULTS: No COL 2-3/4Cshort immunostaining was observed in the intact cartilage of healthy young or old mice. The earliest detectable collagen degradation occurred at the cartilage surface coincident with the appearance of surface roughening. As fibrillations developed, further collagen degradation was evident around the edge of the lesion and in adjacent extracellular matrix. In contrast, staining was observed throughout the cartilage matrix in type II collagen-induced arthritis prior to the development of histopathological lesions. CONCLUSION: No evidence was found for collagen cleavage in intact/pre-lesional cartilage from STR/ort mice. Local collagen cleavage was, however, clearly associated with very early histopathological lesions and immunostaining with COL 2-3/4Cshort increased with progression of the latter. In contrast, type II collagen cleavage occurs throughout the articular cartilage at an early stage in collagen-induced arthritis. (+info)
Control of extracellular matrix homeostasis of normal cartilage by a TGFbeta autocrine pathway. Validation of flow cytometry as a tool to study chondrocyte metabolism in vitro.
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OBJECTIVE: To validate flow cytometry as an experimental technique for the study of the homeostasis of the extracellular matrix (ECM) of human articular cartilage. METHODS: Given the established insights in the relation between the transforming growth factor (TGF)-beta type II Receptor (TGF-betaRII)/TGF-beta auto/paracrine pathway, the intracellular levels of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), and the accumulation of ECM molecules in the ECM of articular cartilage, this metabolic pathway was used as a reference model to fulfill the objective. Chondrocytes were liberated from visually intact femoral condyle cartilage and cultured in gelled agarose to maintain their differentiated phenotype. After 2 weeks of culture, the chondrocytes were isolated from the agarose and flow cytometry was used to analyse the expression of TGF-betaRII on the plasmamembrane, the expression of TGFbeta1, MMP-1, MMP-3, TIMP-1 and TIMP-3 inside the cells, as well as the amounts of aggrecan, type II collagen and hyaluronan in the cell-associated matrix (CAM). The expression of the different substances was analysed with flow cytometry and reported as mean fluorescence intensity (MFI), which is due to the binding of FITC-labeled antibodies to their specific antigens. In addition, the effects of exogenous TGFbeta1 on the expression of these proteins was investigated on chondrocytes cultured in serum-free media. Enzyme Linked Immunosorbent Assay (ELISA) was performed to evaluate the MMP-1, MMP-3, TIMP-1 and MMP-1/TIMP-1 complex in the culture medium collected after the last 3 days of the culture period. The correlations between the data were analysed with the Spearman's test. RESULTS: Exogenous TGF-beta1 increased the accumulation of aggrecan and hyaluronan in the CAM of chondrocytes and down-regulated the intracellular levels of MMP-1 and -3. TIMP-1 and -3 were increased after exposure to TGF-beta1. Baseline expression of TGF-betaRII on the plasmamembrane of normal human articular chondrocytes significantly correlated with the intracellular levels of TGFbeta1, TIMP-1 and TIMP-3. TGFbeta1 was correlated with TIMP-1, TIMP-3 and MMP-1. Aggrecan in the CAM was inversely correlated with the ratio of MMP-1 to TIMPs. In addition, there were correlations between TIMP-1 and TIMP-3, aggrecan and hyaluronan. ELISA also revealed the correlation between MMP-1 and TIMP-1 secreted by the chondrocytes into the nutrient medium. MMP-1/TIMP-1 complex was hardly found in the medium. CONCLUSIONS: Some aspects of ECM metabolism of normal cartilage were evaluated by flow cytometry. Chondrocytes from normal human cartilage, when cultured in gelled agarose, showed correlations between the expression of TGF-betaRII/TGF-beta1 and the intracellular levels of TIMPs, indicating that TGF-beta autocrine pathway may contribute to homeostasis of the ECM in the normal cartilage. The relations between MMPs, TIMPs and the ECM molecules support that a physiological balance between MMPs and TIMPs results in a well-controlled matrix turnover in normal cartilage. (+info)
Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen.
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Decorin is a small leucine-rich chondroitin/dermatan sulfate proteoglycan reported to interact with fibrillar collagens through its protein core and to localize at d and e bands of the collagen fibril banding pattern. Using a solid-phase assay, we have determined the interaction of peptides derived by CNBr cleavage of type I and type II collagen with decorin extracted from bovine tendon and its protein core and with a recombinant decorin preparation. At least five peptides have been found to interact with all three decorin samples. The interaction of peptides with tendon decorin has a dissociation constant in the nanomolar range. The triple helical conformation of the peptide trimeric species is a necessary requisite for the binding. All positive peptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N-acetylation and N-methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in mild conditions do not significantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N-acetylation eliminates both the positive charge and the binding to decorin, whereas N-methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II collagen and that some collagen Lys/Hyl residues are essential for the binding. (+info)
Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis.
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During acute inflammation, leukocytes release proteolytic enzymes including matrix metalloproteinases (MMPs), but the physiopathological mechanisms and consequences of this process are not yet fully understood. Neutrophils, the predominant leukocyte type, produce neutrophil collagenase (MMP-8) and gelatinase B (MMP-9) but not the tissue inhibitors of MMPs. After stimulation, these cells also activate MMPs chemically. In arthritic diseases, neutrophils undergo great chemoattraction to the synovium, are activated by interleukin-8, and are stimulated to release gelatinase B in vivo. Production levels and net activities of gelatinase B were found to be absent in degenerative osteoarthritis but significantly increased in rheumatoid arthritis. The cleavage sites in cartilage type II collagen by gelatinase B were determined by a combination of reverse phase high-performance liquid chromatography, Edman degradation, and mass spectrometry analysis. The analysis revealed the site specificity of proline and lysine hydroxylations and O-linked glycosylation, the cleavage specificities by gelatinase B, and the preferential absence and presence of post-translational modifications at P2' and P5', respectively. Furthermore, gelatinase B leaves the immunodominant peptides intact, which are known from studies with (autoreactive) T cells. Lysine hydroxylation was detected at a critical position for T-cell activation. These data lend support to the thesis that extracellular proteolysis and other post-translational modifications of antigenic peptides may be critical in the establishment and perpetuation of autoimmune processes. (+info)
Catechins from green tea (Camellia sinensis) inhibit bovine and human cartilage proteoglycan and type II collagen degradation in vitro.
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Polyphenolic compounds from green tea have been shown to reduce inflammation in a murine model of inflammatory arthritis, but no studies have been undertaken to investigate whether these compounds are protective to joint tissues. We therefore investigated the effects of catechins found in green tea on cartilage extracellular matrix components using in vitro model systems. Bovine nasal and metacarpophalangeal cartilage as well as human nondiseased, osteoarthritic and rheumatoid cartilage were cultured with and without reagents known to accelerate cartilage matrix breakdown. Individual catechins were added to the cultures and the amount of released proteoglycan and type II collagen was measured by metachromatic assay and inhibition ELISA, respectively. Possible nonspecific or toxic effects of the catechins were assessed by lactate output and proteoglycan synthesis. Catechins, particularly those containing a gallate ester, were effective at micromolar concentrations at inhibiting proteoglycan and type II collagen breakdown. No toxic effects of the catechins were evident. We conclude that some green tea catechins are chondroprotective and that consumption of green tea may be prophylactic for arthritis and may benefit the arthritis patient by reducing inflammation and slowing cartilage breakdown. Further studies will be required to determine whether these compounds access the joint space in sufficient concentration and in a form capable of providing efficacy in vivo. (+info)