Faculty Bibliography

OBJECTIVE: To examine the effect of human interleukin-17 (IL-17) on nitric oxide (NO) production in human osteoarthritis (OA) cartilage under ex vivo conditions. METHODS: OA cartilage from patients undergoing knee replacement surgery was used in explant assays to assess the effect of IL-17. NO production was measured by estimating the stable NO metabolite, nitrite, in conditioned medium. RESULTS: IL-17 augmented the spontaneous production of nitric oxide. This augmentation was sensitive to cycloheximide and pyrrolidine dithiocarbamate, but not to dexamethasone or soluble IL-1 receptor. CONCLUSION: IL-17 augments nitric oxide production in OA cartilage via nuclear factor kappaB activation, but independently of IL-1beta signaling

Cartilage specimens from osteoarthritis (OA)-affected patients spontaneously released PGE2 at 48 h in ex vivo culture at levels at least 50-fold higher than in normal cartilage and 18-fold higher than in normal cartilage + cytokines + endotoxin. The superinduction of PGE2 production coincides with the upregulation of cyclooxygenase-2 (COX-2) in OA-affected cartilage. Production of both nitric oxide (NO) and PGE2 by OA cartilage explants is regulated at the level of transcription and translation. Dexamethasone inhibited only the spontaneously released PGE2 production, and not NO, in OA-affected cartilage. The NO synthase inhibitor HN(G)-monomethyl-L-arginine monoacetate inhibited OA cartilage NO production by > 90%, but augmented significantly (twofold) the spontaneous production of PGE2 in the same explants. Similarly, addition of exogenous NO donors to OA cartilage significantly inhibited PGE2 production. Cytokine + endotoxin stimulation of OA explants increased PGE2 production above the spontaneous release. Addition of L-NMMA further augmented cytokine-induced PGE2 production by at least fourfold. Inhibition of PGE2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE2 did not significantly affect the spontaneous NO production. These data indicate that human OA-affected cartilage in ex vivo conditions shows (a) superinduction of PGE2 due to upregulation of COX-2, and (b) spontaneous release of NO that acts as an autacoid to attenuate the production of the COX-2 products such as PGE2. These studies, together with others, also suggest that PGE2 may be differentially regulated in normal and OA-affected chondrocytes

Neutrophil aggregation is mediated by the beta2 integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]-labeled CD11b/CD18 was immunoprecipitated from purified neutrophil-specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The alpha chain, CD11b, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule-associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (pp1 > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA-treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane-associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule-associated phosphatase, probably distinct from ppl. Therefore adhesion-competent plasma membrane CD11b/CD18 and adhesion-incompetent specific granule CD11b/CD18 differ in their state of phosphorylation

Tetracyclines have recently been shown to have 'chondroprotective' effects in inflammatory arthritides in animal models. Since nitric oxide (NO) is spontaneously released from human cartilage affected by osteoarthritis (OA) or rheumatoid arthritis in quantities sufficient to cause cartilage damage, we evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent inducible NOS (iNOS). Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1 beta-induced OA-NOS activity in ex vivo conditions. Similarly, minocycline > or = doxycycline inhibited both lipopolysaccharide- and interferon-gamma-stimulated iNOS in RAW 264.7 cells in vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors of L-arginine that directly inhibit the specific activity of NOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for beta-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of protein of beta-actin and cyclooxygenase 2 expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inflammatory diseases, these observations suggest that tetracyclines should be evaluated as potential therapeutic modulators of NO for various pathological conditions

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