Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly.

OBJECTIVE: The migration of cells of chondrocyte lineage is believed to play a role in cartilage growth and repair. The present study examined 1) whether chondrocytes are capable of migration in vitro; and 2) the effects of nitric oxide (NO) on chondrocyte migration, adhesion, and cytoskeletal assembly. METHODS: Chondrocyte migration was evaluated by 2 assays: 1) "centrifugal" migration within a 3-dimensional collagen matrix (dot culture); and 2) directed migration under agarose in response to bone morphogenetic protein. To assess the effects of NO, chondrocytes were treated with either exogenous NO (S-nitrosoglutathione [SNO-GSH]) or a mixture of cytokines known to induce endogenous NO production. The effects of NO on chondrocyte adhesion to fibronectin-coated surfaces, as well as on actin polymerization (determined by indirect immunofluorescence), were also examined. RESULTS: The capacity of chondrocytes to migrate was demonstrated both by the dot culture and by agarose methods. Both SNO-GSH and endogenous NO induced by cytokines inhibited this migration. Exposure to NO also inhibited attachment of chondrocytes to fibronectin and disrupted assembly of actin filaments. These effects of SNO-GSH and cytokine-induced NO production were reversed in the presence of hemoglobin and the NO synthase inhibitor NG-monomethyl arginine, respectively. CONCLUSION: NO interferes with chondrocyte migration and attachment to fibronectin, an extracellular matrix protein, probably via effects on the actin cytoskeleton. These effects of NO may result in impairment of cartilage repair, by interfering with the extracellular matrix regulation of chondrocyte function.

Steady-state levels of mitochondrial messenger RNA species characterize a predominant pathway culminating in apoptosis and shedding of HT29 human colonic carcinoma cells.

A differentiated human colonic epithelial cell has undergone relatively stable molecular, biochemical, and cellular alterations resulting in the acquisition of structures, activities, and functions that characterize it as one of at least three mature phenotypes: a columnar absorptive, secretory, or enteroendocrine cell. We have shown previously that induction of HT29 cells with the short-chain fatty acid sodium butyrate elevates alkaline phosphatase activity, a marker of the absorptive cell phenotype, and increases mitochondrial gene expression. Furthermore, this induction is accompanied by subsequent apoptosis and cell shedding. In this report, we have investigated the effects of forskolin, a potent inducer of the MUC2 gene in HT29 cells, a marker of the secretory phenotype, and have shown that neither apoptosis nor mitochondrial gene expression are significantly stimulated. Thus, differentiation along the secretory cell lineage may not play a major role in apoptosis of colonic epithelial cells. Moreover, we have also investigated two polar solvents, DMSO and dimethylformamide, which have been reported to induce a more differentiated, but as yet not well characterized, phenotype in colonic carcinoma cells in culture. Although neither polar solvent induces alkaline phosphatase expression or MUC2 expression, both induce significant apoptosis, again associated with significant elevation of mitochondrial gene expression. Thus, elevation of mitochondrial gene expression appears to be an important pathway in the induction of apoptosis in colonic epithelial cells in culture, whether or not markers characteristic of differentiation along either the absorptive or secretory cell lineage are induced.

The inducible production of nitric oxide by articular cell types.

Nitric oxide (NO) may play a role in tissue remodeling associated with arthritis. The articular cell sources of human inducible NO synthesis, however, have not been defined. This study demonstrates that human articular chondrocytes in primary or organ culture, but not synovial fibroblasts, produce NO in response to catabolic cytokines such as interleukin-1 (IL-1). As measured by the accumulation of NO2- in culture medium, NO production by IL-1-stimulated chondrocytes was inhibited by the NO synthase inhibitor Ng-monomethyl-L-arginine (NMA) and dependent on the presence of exogenous L-arginine. Other inflammatory cytokines such as tumor necrosis factor, but not transforming growth factor-beta, induced chondrocyte NO synthesis. The stimulation of NO synthesis required both RNA and protein synthesis. Chondrocytes isolated from cartilage derived from osteoarthritic patients also produced large amounts of NO in response to IL-1. In beginning to define potential effects of NO on chondrocyte function, it is shown that IL-1 induced an increase in cyclic guanosine monophosphate (cGMP) which was inhibited by NMA. In summary, these results demonstrate that cytokine-induced production of NO is a response of human articular chondrocytes but not of synovial fibroblasts. A potential role of NO in cytokine-induced tissue remodeling in the joint is provided by the induction of cGMP.

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon-gamma and interleukin-1 alpha.

Articular chondrocytes from rheumatoid joints have been shown to express class II major histocompatibility (MHC) antigens that were correlated with the presence of interferon-gamma (IFN-gamma) in the inflamed joint. Chondrocytes expressing MHC antigens function as antigen presenting cells and thus stimulate lymphocyte proliferation. These responses suggest a powerful role for the IFN-gamma stimulation of chondrocytes. The present studies were designed to examine the functional role of chondrocytes exposed to IFN-gamma during cartilage degradation that occurs in synovial disease. Destruction of cartilage in arthritis is partially attributable to metalloproteinases released by the chondrocytes in response to interleukin-1 (IL-1). Bovine articular chondrocytes treated with interleukin-1 alpha (IL-1 alpha) produced enhanced levels of stromelysin mRNA, however, Northern blots could not determine the percentage of cells responding. Exposure of bovine articular chondrocytes to IFN-gamma induced the expression of bovine HLA-DR (boHLA-DR) antigen in 50% of the cells. Using a modified cell sorting technique, chondrocytes that expressed class II MHC antigens produced two fold greater stromelysin mRNA than chondrocytes that did not express this antigen. In contrast, collagen type II mRNA levels were similar in chondrocytes, regardless of the expression of class II MHC antigens. In situ hybridization studies showed that less than half of all cartilage chondrocytes were induced to synthesize stromelysin mRNA. These observations suggest that IFN-gamma stimulates specific subpopulations of chondrocytes to be functionally active in inflammation-induced metalloprotease secretion.

Characterization of the binding of Bolton-Hunter labeled [125I]C5a to human neutrophil, monocyte and U-937 cell membranes.

The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.

Platelet activating factor stimulates intracellular calcium transients in human neutrophils: involvement of endogenous 5-lipoxygenase products.

Stimulation of human neutrophils with platelet activating factor (PAF) resulted in a transient elevation of free cytosolic calcium. Neutrophils exhibited a two-component calcium response observed as a double peak when stimulated with greater than 5 nM PAF. In contrast, leukotriene B4 (LTB4), C5a, or formylmethionyl-leucyl-phenylalanine stimulated only a single-peak calcium response. The double-peak calcium response was not elicited in human monocytes or differentiated U937 cells, which demonstrated a single peak. Pretreatment of neutrophils with a 5-lipoxygenase inhibitor or a specific LTB4-receptor antagonist selectively blocked the second calcium peak. These results suggest that PAF-mediated activation of human neutrophils results in the activation of the 5-lipoxygenase and the subsequent generation of LTB4. This LTB4 in turn elicits a secondary rise in calcium, which contributes to the overall response of neutrophils of PAF. These results demonstrate how LTB4 participates in the cellular responses elicited by PAF in human neutrophils.

A simple and rapid radioimmunoassay for human interleukin-6.

Studies were undertaken to develop a sensitive radioimmunoassay for human IL-6 using commercially available reagents. The assay utilized a polyclonal rabbit anti-IL-6 binding to iodinated IL-6; the reaction was carried out in solution and the immune complexes were precipitated using anti-rabbit IgG coupled to magnetic particles. Using a format where sample IL-6 was added in advance of the radiolabelled IL-6, the working range of the assay was found to fall between 0.1 and 10 ng/ml (IC50 around 1 ng/ml) for a 50 microliters sample volume, and was run overnight. However, the assay could be completed in 2 h, using a direct competitive format when less sensitivity is required (working range 1.5-25 ng/ml, IC50 12 ng/ml). The RIA correlated directly with a standard functional assay for IL-6 (proliferation of the mouse hybridoma B9.9) for both recombinant IL-6 and natural IL-6 from human monocytes. The total assay variance was less than 12% and no reactivity with interleukins-1-5 was found. Using the RIA, IL-6 produced in culture by human monocytes in response to various stimuli (LPS, IL-1, dibutyryl cAMP) was measured.

Fluorometer based multi-parameter analysis of phagocytic cell activation.

Simultaneous measurements of the calcium rise, membrane potential change, and 90 degrees light scatter (shape change) responses exhibited by neutrophils upon activation, can be obtained with identical result as that obtained when independently performing each measurement. The putative intracellular mediator diacylglycerol depolarizes membrane potential and causes a decrease in light scatter. Leukotriene B4 causes a rise in calcium and a decrease in light scatter. The chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine, causes a depolarization of membrane potential, a calcium rise, and a decrease in light scatter. The fura 2 measurements of intracellular free calcium indicate that the calcium concentration of unstimulated cells is much lower than previously thought based on quin 2 studies.

Effect of prostaglandins on complement production by tissue macrophages.

Tissue macrophages produce several proteins of the complement system. The mechanisms that regulate this process are poorly understood. The established ability of certain prostaglandins to influence macrophage secretory activity suggests that these lipid mediators may also modulate complement production (CP). Using the guinea pig peritoneal macrophages, we determined the effects of selected prostaglandins on in vitro CP and found that PGE2 inhibited production of complement proteins but not lysozyme; the response of elicited and resident peritoneal cells to PGE2 was identical; and PGE1, PGF2 alpha, and PGI2 had no detectable effect. PGE2 may contribute to regulation of CP in vivo.

Arachidonic acid-mediated and serum-opsonized-zymosan-mediated inhibition of complement production by macrophages. Lack of requirement for endogenous arachidonic acid metabolites.

The ability of exogenous prostaglandins to inhibit complement production (CP) by monocytes and macrophages (M phi) suggests that endogenous arachidonic acid metabolites produced by these cells may also regulate their rate of CP. We assessed the regulatory influence of endogenous metabolites on CP by M phi utilizing exogenous arachidonic acid and serum-opsonized zymosan as stimulators of production of cyclooxygenase and lipoxygenase metabolites. The results of this study show that (i) the inhibition of CP caused by both agents is is independent of arachidonic acid metabolites, suggesting that endogenously produced metabolites do not influence CP, and (ii) arachidonic acid and serum-opsonized zymosan inhibit production by independent mechanisms.