Anna Osiecka-Iwan

Influence of LPS, TNF, TGF-ß1 and IL-4 on the expression of MMPs, TIMPs and selected cytokines in rat synovial membranes incubated in vitro

Synovial membranes are formed by four main types of cells, i.e. fibroblasts, macrophages, epitheliocytes and adipocytes. To study the combined effect of various factors on these cell populations, synovial membranes dissected from rat knee joints were incubated in control medium or medium with lipopolysaccharide (LPS), TNF, TGF-ß1 or IL-4 for 12 h. LPS stimulated TNF secretion and both agents stimulated secretion of IL-6. TGF-ß1 slightly increased IL-6 secretion. LPS increased the mRNA levels of IL-6, IL-1ß, TGF-ß1, MMP1a, MMP1b, MMP3, MMP9, MMP13, MMP14, TIMP1 and TIMP3 while the mRNA levels of MMP2, TIMP2 and TIMP4 were significantly decreased. Expression of IL-1ß, MMP1a, MMP1b, MMP3, MMP9, MMP13 and TIMP1 increased after TNF treatment, while mRNA levels of MMP2, MMP14, TIMP2, TIMP3 and TIMP4 were decreased. TGF-ß1 decreased the mRNA levels of IL-1ß, all MMPs, TIMP1, TIMP2, TIMP4 and increased mRNA levels of itself and TIMP3. IL-4 decreased mRNA levels of IL-1ß, TGF-ß1, MMP2, MMP9, MMP13 and all TIMPs. Only LPS decreased the amount and activity of MMP2. The effect of LPS and cytokines on most of the MMPs and TIMPs produced by whole synovial membrane was in good agreement with previous studies on their action on similar types of cells as those present in synovial membranes, but originating from other tissues. All tested agents decreased MMP2 mRNA expression levels and in the case of LPS also the protein level and its activity determined by zymography, contrary to previous observations on isolated cell populations. This indicates that the response of the organized tissue is an interplay of all components and cannot be deduced from the individual reactions.

Transplants of rat chondrocytes evoke strong humoral response against chondrocyte-associated antigen in rabbits.

Rat chondrocytes transplanted intramuscularly in rabbits produced cartilage. In 1-day-old transplants chondrocytes remained viable. After 1 week peripheral chondrocytes of the transplant were dead and the cartilage was surrounded and resorbed by macrophages. In 2-week-old transplants cartilage deteriorated and was invaded by fibroblast-like cells and macrophages. Sera of rabbits that received two or three consecutive transplants of rat chondrocytes with 2-week intervals contained high titer of antichondrocyte cytotoxic antibodies. A part of the cytotoxic activity could be removed by absorption with rat splenocytes. Western blot analysis of lysates from fresh or 24-h cultured chondrocytes with absorbed sera detected antigen with Mr of ~74 and ~23 kDa. Only the latter remained after reduction in 2-mercaptoethanol. In lysates of fibroblasts and endotheliocytes the 23-kDa antigen was not found but the serum reacted with Mr 39-kDa antigen. In lysates of thymocytes a weak band corresponding to Mr of 35 kDa was present. Serum from rabbits receiving transplants of living chondrocytes followed by chondrocytes suspended in complete Freunds adjuvant contained antibodies directed against components of crude collagenase used for cell isolation. Such antibodies could not be detected in sera of rabbits receiving transplants of living chondrocytes only. Molecular weight of detected antigen differs from that of collagen type II, core of aggrecan, link proteins, and several other macromolecules of cartilage matrix. It could represent either a component of chondrocyte membrane or a membrane-bound substance resistant to enzymes used for isolation. Availability of antibodies against presumably chondrocyte-specific antigen produced during transplant rejection may help to characterize it more precisely and to ascertain whether its presence may influence results of autogenous chondrocyte transplants in humans.

Immunosuppression and rejection of cartilage formed by allogeneic chondrocytes in rats.

Rat syngeneic and allogeneic chondrocytes were transplanted intramuscularly or into defects prepared in articular cartilage (intracartilaginous transplants). Recipients of allogeneic transplants received cyclosporin A (CsA), cladribine (2-chlorodeoxyadenosine, 2-CdA), or both drugs in combination. Transplants were taken for examination after 5 weeks. Cartilage formed intramuscularly by syngeneic chondrocytes was ossified. Allogeneic cartilage was resorbed by infiltrating cells. CsA or 2-CdA partially suppressed, and both these agents in combination strongly suppressed, formation of infiltrations. Both syngeneic and allogeneic chondrocytes formed cartilage in joint surface defects but only allogeneic cartilage was attacked by infiltrating cells. CsA + 2-CdA treatment slightly decreased intensity of infiltrations but did not prevent cartilage resorption. Antichondrocyte response was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte–chondrocyte cultures and by detection of antichondrocyte cytotoxic antibodies. SMC stimulation index (SI) was calculated separately for syngeneic and allogeneic chondrocytes. Comparison of SMC SI for syngeneic and allogeneic chondrocytes indicated lack of stimulation of SMC from control or syngeneic transplant recipients and significant stimulation of SMC from recipients of allogeneic transplants. SMC from animals treated with CsA + 2-CdA were not stimulated. Additional experiments aiming at an explanation of the lack of stimulation of SMC from intact animals by syngeneic chondrocytes reported in this work and contrary to other findings disclosed that it was caused by the use of collagenase solution containing Nα-p-tosyl-l-lysine chloromethyl ketone for chondrocyte isolation. Spontaneous antichondrocyte cytotoxic antibody activity was found in intact rats raised only in sera from recipients of allogeneic intramuscular transplants without immunosuppression. Thus, strong immunosuppressive treatment of rats with allogeneic chondrocyte transplants was more effective in relation to the general immunological response than to the local reaction.

Chondrocyte-associated antigen and matrix components in a 2- and 3-dimensional culture of rat chondrocytes

The goal of this study was to compare expression of chondrocyte-associated antigen (CAA) and cartilage matrix molecules in 2-D (monolayer) and 3-D (Matrigel) culture. Chondrocytes isolated from the cartilage of 3-day-old rats were expanded in monolayer culture for 28 days. CAA expression gradually decreased and was not detected beyond the 96th hour of monolayer culture. Collagen type II and aggrecan mRNA levels decreased during culture. The collagen type I mRNA level increased during the first week and remained high. The increase in the versican mRNA level was less pronounced during the first week and declined slightly after further cultivation. Freshly isolated chondrocytes introduced into Matrigel still expressed CAA after 7 days. Moreover, CAA expression returned in chondrocytes re-cultured in Matrigel after 7 days in monolayer. Similarly, the increase in the mRNA levels of collagen type II and aggrecan in Matrigel was limited to freshly isolated chondrocytes and to those that remained in monolayer for 1 week. Collagen type I mRNA in monolayer and Matrigel cultures of freshly isolated chondrocytes was at a similar level. The introduction of freshly isolated or 7-day monolayer-cultured chondrocytes into Matrigel caused a decrease in the versican mRNA level in comparison with 7- and 14-day 2-D cultures, respectively. On the other hand, chondrocytes seeded in Matrigel after 14 days of monolayer culture did not express CAA, showed decreased levels of collagen type II and aggrecan mRNA and an increase in versican mRNA. In conclusion, it appears that changes in the expression of CAA in 2- and 3-D cultures occur in parallel to changes in typical cartilage matrix molecule expression.

Cartilage produced after transplantation of syngeneic chondrocytes is rejected in rats presensitized with allogeneic chondrocytes.

Cartilage produced in 2-week-old intramuscular transplants of syngeneic chondrocytes in rats did not display any signs of rejection. Cartilage produced by similar transplants in animals presensitized with intramuscular transplants of allogeneic chondrocytes was surrounded by infiltrations composed mainly of lymphocytes and was partially resorbed. Spleen mononuclear cells (SMC) from recipients of syngeneic transplants alone were not stimulated in mixed splenocyte–chondrocyte cultures by syngeneic or allogeneic chondrocytes. SMC from recipients of allogeneic and subsequent syngeneic transplants were strongly stimulated by both syngeneic and allogeneic chondrocytes, although stimulation by the latter was significantly more pronounced. Sera from naive rats usually contained cytotoxic antichondrocyte antibodies but their level varied considerably in various individuals. In rats chosen as transplant recipients on the basis of low antichondrocyte cytotoxicity of their sera, this toxicity was markedly raised after sensitization with allo- and syngeneic chondrocytes. Absorption with thymocytes or fibroblasts decreased but did not abrogate cytotoxicity. These observations support previous reports suggesting expression of tissue-specific antigen(s) by chondrocytes.

Mechanical barrier as a protection against rejection of allogeneic cartilage formed in joint surface defects in rats.

Cartilage formed in transplants of allogeneic chondrocytes into joint cartilage defects in rats was infiltrated by immune cells migrating from the bone marrow while the surface on the side of the joint cavity remained free of infiltrations. This suggested that immunization occurred via bone marrow and not via joint cavity. Because articular cartilage is nourished exclusively by the synovial fluid, we have attempted to prevent cartilage rejection by protecting transplants from the contact with bone marrow. Defects in articular surface were filled with bone cement and chondrocytes were transplanted into a cavity prepared within the bone cement plug. Cartilage formed within the cement shell remained free of infiltrations and did not evoke systemic immunological response. However, distribution of glycosaminoglycans in the matrix of protected transplants was irregular. Cultures of chondrocytes growing in vitro on cement contained less glycosaminoglycans than the controls. This suggests that some factor(s) released from the cement unfavorably influenced chondrocytes and matrix production in protected transplants.

Chondrocyte-specific phenotype confers susceptibility of rat chondrocytes to lysis by NK cells

Normal chondrocytes are targets for natural killer (NK) cells. Since the mechanism of this phenomenon remains unknown, the present study was aimed at testing whether it is associated with chondrocyte-specific phenotype defined as ability of cartilage cells to produce sulfated glycosaminoglycans (GAG) and express collagen II and aggrecan mRNA. Lysis of rat epiphyseal chondrocytes by syngeneic spleen mononuclear cells (SMCs) was evaluated by (51)Cr-release assay. Loss of chondrocyte phenotype following long-term culture resulted in their decreased susceptibility to lysis. Similar effect was also observed after suppression of chondrocyte phenotype by TNF. On the other hand, stimulation of cartilage-specific matrix component synthesis by IGF-1 resulted in increased chondrocyte killing and exogenous chondroitin sulfate A stimulated NK cell-mediated cytotoxicity against chondrocytes and human K562 cells. This suggests that chondrocyte susceptibility to lysis by NK cells depends on chondrocyte-specific phenotype, especially sulfated GAG production.

A Role of NKR-P1A (CD161) and Lectin-like Transcript 1 in Natural Cytotoxicity against Human Articular Chondrocytes

Normal cartilage cells are susceptible to lysis by NK cells. This phenomenon may play a role in immune cartilage destruction; however, the mechanisms of chondrocyte recognition by NK cells remain poorly understood. Therefore, the aim of this study was to reveal a possible role of NKR-P1A/lectin-like transcript 1 (LLT1) interaction in NK cell–mediated cytotoxicity against normal human articular chondrocytes. Chondrocytes were isolated from articular cartilage obtained during talonavicular joint surgery. PBMC or polyclonal NK cells isolated from normal donors served as effector cells. Cell-mediated cytotoxicity against chondrocytes was evaluated by means of 18-h 51Cr-release assay. Specific mRNA expression was evaluated by classical and quantitative RT-PCR, and proteins were detected by Western blot analysis. We found that lysis of articular chondrocytes by PBMC or polyclonal NK cells was potentiated by stimulation with IL-2. Stimulation of effector cells with IL-2 downregulated mRNA expression of inhibitory NKR-P1A NK cell receptor, and blocking of NKR-P1A with specific mAbs resulted in increased chondrocyte killing. Chondrocytes constitutively expressed LLT1, a ligand of NKR-P1A. LLT1 expression by chondrocytes could be upregulated by IL-1α and TNF. Chondrocyte treatment with IL-1α resulted in their increased resistance to killing by natural cytotoxic cells. This could be reversed by blocking of NKR-P1A. These results show that susceptibility of normal articular chondrocytes to lysis by NK cells is modulated by NKR-P1A/LLT1 interactions. Thus, NKR-P1A/LLT1 interaction might provide some novel target for therapeutic interventions in the course of pathological cartilage injury.

In search of chondrocyte-specific antigen

The purpose of this work was to establish, whether rat chondrocyte associated antigen, transmembrane Tmp21 protein belonging to the p24 protein family may immunize rats and thus be included into the panel of immunogens potentially involved in cartilage pathology. For immunization of rats extract from cultured chondrocytes containing surface chondrocyte proteins suspended in incomplete Freund’s adjuvant was used. Control animals were injected with incomplete Freund’s adjuvant without chondrocyte extract. Morphological observations indicated that both in control and experimental animals occurred subperiosteal resorption of bone, suggesting that it arised as the response to adjuvant. In trachea, however, resorption of cartilage and inflammatory changes in the respiratory epithelium and lamina propria were present only in animals exposed to antigen. Unexpectedly, sera from immunized rats strongly reacted with other antigen, which we were able to identify by Western blot and protein sequencing as cartilage oligomeric matrix protein (COMP). COMP is attached to chondrocyte membrane by integrins and its presence in chondrocyte extract is not surprising. Antibody response to COMP raises a question whether the observed changes in tracheal cartilage and epithelium represent anti-COMP reaction or were caused by some other, no specified factors. COMP is used as the marker of osteoarthritis progression, but its role in polychondritis, cartilage pathology involving i.a. tracheal cartilage resorption remains unknown. Thus, our observations may serve as the starting point for future studies in this direction.

Antigenic and immunogenic properties of chondrocytes. Implications for chondrocyte therapeutic transplantation and pathogenesis of inflammatory and degenerative joint diseases

In physiological conditions chondrocytes are protected from contact with immunocompetent cells by the extracellular matrix, and transplanted fragments of allogeneic cartilage are not rejected. Cartilage produced by allogeneic chondrocytes, however, evokes the immune response of the recipient and is gradually destroyed. Immunisation by allogeneic chondrocytes is induced by the contact of their surface molecules with cells of the immune system. Chondrocytes constitutively express class I and, in some species, class II major histocompatibility complex (MHC) molecules. Expression of MHC class II molecules is induced in vitro by pro-inflammatory cytokines and in vivo in the course of the rejection of transplanted allogeneic cartilage. Low level of MHC class II molecules is found on the surface of human articular chondrocytes in patients with rheumatoid arthritis and osteoarthritis. Cartilage produced by transplanted allogeneic chondrocytes is destroyed by monocytes/macrophages and cytotoxic T and natural killer (NK) cells. NK cells show spontaneous cytotoxic reactivity against isolated chondrocytes and participate in the rejection of transplanted isolated chondrocytes. Chondrocytes express molecules that can serve as potential antigens in inflammatory joint diseases. Chondrocytes express cartilage-specific membrane antigen (CH65), human cartilage glycoprotein-39 (HC gp-39), hyaluronan binding adhesion molecule CD44, thymocyte antigen-1 (Thy-1) – CD90, signal transducer – CD24, lymphocyte function-associated antigen-3 (LFA-3) – CD58, and type I transmembrane protein Tmp21. On the other hand, although chondrocytes express major histocompatibility complex (MHC) class I and class II molecules, they can also exert immunosuppressive and immunomodulatory effects on immunocompetent cells. Isolated chondrocytes do not trigger an efficient allogeneic immune response in vitro and suppress, in a contact-dependent manner, proliferation of activated T cells. This suppression is associated with the expression by chondrocytes of multiple negative regulators of immune response. Chondrocytes express programmed death-ligand (PD-L), chondromodulin-I and indoleamine 2,3-dioxygenase (IDO), molecules that promote self-tolerance and suppress the immune system.