Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage.

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.

The effects of cyclosporin A on bone and cartilage.

Cyclosporin A (CyA) is a potent immunomodulatory agent with an increasing number of clinical applications. Although its precise mechanisms of action are yet to be elucidated, one of the most important known properties of CyA is its ability to inhibit the production of cytokines involved in the regulation of T cell activation. There is also evidence for direct effects on other cell types, such as B cells, macrophages, and bone and cartilage cells. The effects of CyA on T cells and on bone, cartilage and synovial cells, which can produce a range of cytokines, are of interest in the study of inflammatory diseases such as RA. It has been shown, for example, that in vitro CyA inhibits bone resorption induced by interleukin-1, 1,25-dihydroxy-vitamin D3, parathyroid hormone and prostaglandin E2. In vivo, it protects against adjuvant arthritis.

The effect of interleukin-1 on cytokine gene expression in cultured human articular chondrocytes analyzed by messenger RNA phenotyping.

OBJECTIVE: To investigate the pattern of cytokine gene expression in human articular chondrocytes in culture in response to interleukin-1 beta (IL-1 beta). The effect of serum and variations in culture conditions was also studied. METHODS: Messenger RNA was extracted from cells, reverse-transcribed to complementary DNA, and amplified by the polymerase chain reaction (PCR), using specific oligonucleotide primers. The PCR products were validated by restriction analysis with specific enzymes and by Southern blot analysis. RESULTS: In cultured articular chondrocytes, IL-1 beta, IL-1 alpha, granulocyte colony-stimulating factor (CSF), and granulocyte-macrophage CSF cytokine genes were expressed only after induction by IL-1 beta. However, IL-6, IL-8, and macrophage CSF genes were expressed constitutively. The expression of IL-1 beta was dose and time dependent. CONCLUSION: Using PCR, it was possible to demonstrate gene expression for several cytokines in human articular chondrocytes in culture. It was evident that some cytokine genes were expressed constitutively and some were inducible by IL-1 beta.

Mechanisms of action of cyclosporine and effects on connective tissues.

Cyclosporine is a potent immunomodulatory agent with an increasing number of clinical applications. Its major mode of action is inhibition of the production of cytokines involved in the regulation of T-cell activation. In particular, cyclosporine inhibits the transcription of interleukin 2. Although cyclosporine's major actions are on T cells, there is some evidence that it produces direct effects on other cell types. Its immunosuppressive action is closely linked to its binding of cyclophilin, a member of a family of high-affinity cyclosporine-binding proteins widely distributed in different cell types and in different species. The cyclophilins have been shown to have peptidyl-prolyl cis-trans isomerase enzyme activity that is blocked by cyclosporine. Although this may be a factor in cyclosporine's selective inhibition of cytokine gene transcription, it is still unclear whether inhibition of this activity is the mechanism through which cyclosporine exerts its effects on target cells. The ubiquitous presence of cyclophilins raises the question of why cyclosporine has major effects on T cells. Perhaps the critical proteins affected are transcriptional regulators restricted in their tissue distribution. The effects of cyclosporine on T cells and, directly or indirectly, on connective tissue cells, all of which can produce a range of cytokines, are of interest in relation to the tissue changes that occur in such inflammatory conditions as rheumatoid arthritis.

Cyclosporin A. Mode of action and effects on bone and joint tissues.

Cyclosporin A is an established immunomodulatory agent with an increasing number of clinical applications. Although its precise mechanisms of action remain elusive, one of the most important known properties of CyA is its ability to inhibit the production of cytokines involved in the regulation of T-cell activation. In particular, CyA inhibits de novo synthesis of interleukin 2(IL-2), the major cytokine involved in T-cell proliferation, as well as other cytokines, probably at the level of gene transcription, as shown by the suppression of mRNA levels in activated T-cells. Although the major actions of CyA are on T-cells, there is some evidence for possible direct effects on other cell types e.g. B-cells, macrophages and, from our own work, on bone and cartilage cells. Cyclosporin A is thought to enter cells and to bind to cyclophilins, which are members of a family of high-affinity cyclosporin A-binding proteins, now known as immunophilins. The binding of cyclosporins to such proteins appears to be closely linked to the immunosuppressive action of cyclosporins. The immunophilins possess enzyme activity, ie. peptidyl-prolyl cis-trans isomerase, also known as rotamase, which can regulate protein folding, and may therefore alter the functional state of many cell proteins. Cyclosporin A blocks peptidyl-prolyl cis-trans isomerase activity but it is not clear whether this plays a part in its selective inhibition of cytokine-gene transcription. Moreover, the ubiquitous presence of cyclophilins and immunophilins raises the question of why cyclosporin A has its apparent major effects only on T-cells. Recent proposals regarding the intracellular mode of action of CyA suggest that it interacts with cyclophilin and other regulatory proteins including calmodulin and calcineurin, which is a serine/threonine phosphatase, and thereby affects the functional state of key regulators of gene transcription in its target cells. The effects of CyA on T-cells and directly or indirectly on connective tissue cells, including bone, cartilage and synovial cells, which all can produce a range of cytokines, are of interest in relation to the tissue changes that occur in inflammatory diseases, such as rheumatoid arthritis. Thus, for example, cyclosporin A inhibits in vitro the bone resorbing activity of interleukin 1, 1,25-dihydroxy-vitamin D3, parathyroid hormone and prostaglandin E2 by apparently non-T-cell effects, while in vivo protects against bone and cartilage loss in adjuvant arthritis. More needs to be known about the direct and indirect modulation of cytokine production by cyclosporin A in connective tissues, in order to understand its potential value in clinical disorders.

Susceptibility to murine cutaneous leishmaniasis correlates with the capacity to generate interleukin 3 in response to leishmania antigen in vitro.

Spleen cells from genetically susceptible BALB/c mice infected with Leishmania major produced higher levels of IL-3 in response to leishmania antigens or concanavalin-A in vitro compared to that of genetically resistant CBA mice throughout the course of infection. The capacity to generate IL-3 in BALB/c mice increased with disease progression. The correlation between susceptibility to L. major infection and the capacity of spleen cells to produce IL-3 also extends to other mouse strains. Thus genetically highly resistant CBA and Biozzi Low mice are low IL-3 producers, whereas the highly susceptible BALB/c and Biozzi High mice are high IL-3 producers. The resistant C57BL/10 and C3H mice produce intermediate levels of IL-3. BALB/c mice recovered from L. major infection following a sublethal dose of gamma-irradiation are refractory to further infection. The capacity of the spleen cells from these cured mice to produce IL-3 upon a challenge infection was similar to those of the resistant CBA mice. The IL-3 generated by activated T cells was measured by IL-3 dependent cell lines, 32D and FDC-P2. The spleen cells from infected BALB/c mice also contain a population of IL-3 responding cells whose number increases as disease progresses. A similar population of IL-3 responding cells was barely detectable in the resistant CBA mice or BALB/c mice rendered immune by prior gamma-irradiation. These results therefore demonstrate a direct correlation between the susceptibility to L. major infection and the capacity of splenic T cells from infected mice to produce a continuous elevated level of IL-3. The possible role of IL-3 in the immune regulation of cutaneous leishmaniasis is discussed.