Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex.

Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.

Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage. Influence of nitric oxide.

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.

Inducible nitric oxide synthase from human articular chondrocytes: cDNA cloning and analysis of mRNA expression.

Human articular chondrocytes can be induced by IL-1 beta, TNF-alpha or LPS to release high levels of nitric oxide. Using degenerate PCR primers based on homologous regions from previously cloned NOS enzymes, a 1.9 kb cDNA fragment was amplified from IL-1 beta stimulated but not from resting chondrocytes. Screening of a lambda gt11 cDNA library, which was prepared from RNA of IL-1 beta activated chondrocytes, resulted in the isolation of the complete cDNA, encoding a protein of 1153 amino acids. Comparison of the cDNA sequence identified human chondrocyte iNOS to be almost identical to the sequence recently reported for the hepatocyte enzyme, differing in 12 amino acids. Northern blot analysis revealed, that stimulated chondrocytes express a single 4.5 kb iNOS mRNA species. IL-1 beta induction of iNOS mRNA was detectable by 6 h and continued to be elevated throughout a 72 h culture period. Screening of a human bone cDNA library identified this inducible NOS to be also expressed by bone cells.

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.

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.

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.

Identification of a splice variant of neutrophil collagenase (MMP-8).

We have identified a splice variant of human neutrophil collagenase (MMP-8) transcript (MMP-8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP-8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP-8, the translation product of MMP-8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP-8alt protein may lead to uncover novel roles it plays in cellular physiology.

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.

Expression of nitric oxide synthase in human peripheral blood mononuclear cells and neutrophils.

It has been clearly demonstrated in rodents that nitric oxide (NO) plays an important role in host defense and immunity. However, evidence that human leukocytes express inducible nitric oxide synthase (iNOS) or its products has been inconclusive and a source of controversy. We report that iNOS could not be detected in human monocytes, HL-60 cells, neutrophils, and T cells by Western blotting analysis (< or = 10 pg) or by radiolabeled L-arginine-to-L-citrulline conversion (< or = 20 pmol L-citrulline) under conditions sufficient to induce iNOS in the rodent system and in human hepatocytes, which include activation with cytokines, endotoxins, and/or chemoattractants. However, sensitive methods such as RT-PCR and Northern blot analysis show "constitutively expressed" iNOS mRNA from human monocytes, neutrophils, Jurkat cells, and HL-60 cells. This iNOS mRNA is 4.4 kb and is similar to that seen in human hepatocytes and rodent macrophages. In spite of the constitutive expression of mRNA in neutrophils and the lack of detectable NOS activity (based on Western blotting and L-arginine-to-L-citrulline conversion assay), stimulation of human neutrophils unit FMLP in vitro induced the ADP-ribosylation of an intracellular NO target, glyceraldehyde-3-PO4 dehydrogenase (GAPDH), in a NO-dependent manner. These studies indicate that low levels of NOS protein are expressed in neutrophils (and perhaps T cells and monocytes) and produce NO following stimulation. The data indicate that, in addition to its phagocytic and tumoricidal activity. NO may also function as an autacoid signaling molecule within the cells.

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.

Human monocytes respond to leukotriene B4 with a transient increase in cytosolic calcium.

To define the intracellular activation events stimulated by leukotriene B4 (LTB4) in human monocytes, we investigated the transient increase in cytosolic free calcium levels ([Ca2+]i) elicited by this lipid mediator. Using elutriated human monocytes, LTB4 caused a dose-dependent increase in [Ca2+]i with an ED50 of 1.0 nM. The LTB4-induced [Ca2+]i response exhibited ligand selectivity, with both the diastereomer 5S-12S-diHETE and the 20-COOH LTB4 inactive at 500 nM, while 20-OH LTB4 was a partial agonist with an approximate ED50 of 10 nM. This response demonstrated stimulus-specific deactivation and was inhibited by the specific LTB4 receptor antagonist LY-223982. These results suggest that LTB4 stimulated an increase in [Ca2+]i via interaction with a defined LTB4 receptor. The inhibitory effects of pertussis toxin and PMA on the LTB4-induced [Ca2+]i suggest that a receptor-linked guanine nucleotide-binding protein and protein kinase C are involved in the regulation of the LTB4-elicited increase in [Ca2+]i. The LTB4-induced cell activation event may play a key role in the functional responses elicited by LTB4 in human monocytes.

Substrate-dependent differences in growth and biological properties of fibroblasts and epithelial cells grown in microcarrier culture.

Normal diploid human fibroblasts and first passage monkey kidney epithelial cells were examined for growth and metabolic activity on microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells cm2 of surface area) on the glass microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells/cm2 of surface area) on the glass microcarriers than they did on the DEAE-dextran microcarriers and morphological differences were observed between the cells growing on the two substrates. On the DEAE-dextran microcarriers, the cells were much more resistant to protease-mediated detachment than were the cells on the glass microcarriers. In these respects, the cells grown on the glass microcarriers were similar to cells grown in conventional monolayer culture. Interestingly, the cells grown on the DEAE-dextran microcarriers expressed higher levels of proteolytic enzyme activity than the cells grown on the glass microcarriers. Substrate-dependent differences in prostaglandin production also occurred--both in unstimulated cells and in cells stimulated with 12-0-tetradecanoyl phorbol acetate. The unstimulated cells on the glass microcarriers produced slightly higher levels of three different prostaglandins than did the cells on the DEAE-dextran microcarriers. However, after stimulation the levels were much higher in the DEAE-dextran microcarrier cultures than in the glass microcarrier cultures. In contrast to these results, there was no significant, substrate-dependent difference in the production of infectious herpes simplex virus. Taken together, these findings suggest that when commercially-useful cells such as normal fibroblasts and epithelial cells are grown in large quantities on microcarriers, the nature of the substrate may have a profound effect on the growth and physiology of the cells. They also suggest that when microcarriers are used, unexpected results based on preliminary work in conventional monolayer culture may be obtained.

Binding of receptor for advanced glycation end products (RAGE) ligands is not sufficient to induce inflammatory signals: lack of activity of endotoxin-free albumin-derived advanced glycation end products.

AIMS/HYPOTHESIS: Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers cellular responses implicated in the pathogenesis of diabetes, such as increasing vascular cell adhesion molecule-1 (VCAM-1) expression on vascular endothelial cells and inducing TNF-alpha secretion by mononuclear cells. The objective of this study was to evaluate whether RAGE binding affinity of AGE-BSAs and cellular activation correlate. METHODS: To produce AGEs with varying glycation, bovine albumin AGEs were prepared with 500 mmol/l of glucose, fructose or ribose at times of incubation from 1 to 12 weeks. In addition, AGE-BSA was generated using either glyoxylic acid or glycolaldehyde. Cellular binding of the AGE-BSAs and the effect on endothelial cell VCAM-1 expression were studied in RAGE-expressing human microvascular endothelial cell line-4 cells. Induction of TNF-alpha secretion was assessed using RAGE-expressing human peripheral blood mononuclear cells (PBMCs). RESULTS: Cellular binding of the different AGE preparations correlated well with RAGE affinity. Interestingly, we found that the AGE preparations, which were essentially endotoxin free (< or =0.2 ng/mg protein), were incapable of inducing VCAM-1 or TNF-alpha secretion regardless of RAGE binding affinity, AGE concentration or incubation time. In contrast, the reported RAGE ligand S100b was confirmed to induce VCAM-1 expression on endothelial cells and TNF-alpha secretion by PBMCs after 24 h of treatment. CONCLUSIONS/INTERPRETATION: The results of this study suggest that AGE modification and high RAGE binding affinity are not sufficient to generate pro-inflammatory signalling molecules. Thus, RAGE binding affinity of AGE-BSAs does not seem to correlate with cellular activation. Our findings using AGEs with strong RAGE-binding properties indicate that AGEs may not uniformly play a role in cellular activation.

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.

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.

Nitric oxide attenuates cellular hexose monophosphate shunt response to oxidants in articular chondrocytes and acts to promote oxidant injury.

Nitric oxide (NO) has been implicated in both cartilage degradation and cell survival. Importantly, NO has been shown, in a cell-type-dependent manner, to directly cause cell death or indirectly promote cell death by compromising the ability of cells to detoxify intra- or extracellular oxidants. In this study we examined the role of NO in the survival of bovine chondrocytes exposed to catabolic cytokines (interleukin-1 (IL-1); tumor necrosis factor [TNF]) with or without the addition of an exogenous oxidant stress (e.g., H2O2, HOOCl, etc.). The exposure of chondrocytes to a mixture of IL-1 and TNF (IL-1/TNF) results in the release of NO but did not alter cell viability. However, there was evidence of NO-dependent oxidative responses in the IL-1/TNF group, as we observed an increased level of intracellular oxidants as well as the appearance of a 55 kD nitrated protein which reflects the formation of peroxynitrite. We next analyzed viability with H2O2. The LD50 for IL-1/TNF-treated cells was 0.1 mM (vs. 1 mM for control). The enhanced sensitivity was completely reversed when cells were incubated with the NO synthase inhibitor 1-n5-1-iminoethylornithine (NIO). To test whether cell death was caused by compromising the ability of cells to detoxify extracellular oxidants, we examined the hexose monophosphate shunt (HMPS) response in cells given H2O2. Treatment of control cells with H2O2 resulted in a fourfold increase in HMPS activity. In contrast, IL-1/TNF cells exhibited no increase in HMPS activity. The attenuation of stimulated HMPS activity was reversed by the coaddition of NIO. Thus, these data indicate that 1) endogenous NO mediates cytokine-dependent susceptibility to oxidant injury and 2) this effect is in part due to impaired activation of the HMPS. In inflamed joints replete with cytokines and oxidants, NO may contribute to chondrocyte death and progressive joint destruction.

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.

Activation of stress-activated protein kinase in osteoarthritic cartilage: evidence for nitric oxide dependence.

OBJECTIVE: We have demonstrated in bovine chondrocytes that nitric oxide (NO) mediates IL1 dependent apoptosis under conditions of oxidant stress. This process is accompanied by activation of c-Jun NH2-terminal kinase (JNK; also called stress-activated protein kinase). In these studies we examined activation of JNK in explant cultures of human osteoarthritic cartilage obtained at joint replacement surgery and we characterized the role of peroxynitrite to act as an upstream trigger. DESIGN: A novel technique to isolate chondrocyte proteins (<10% of total cartilage protein) from cartilage specimens was developed. It was used to analyse JNK activation by a western blot technique. To examine the hypothesis that chondrocyte JNK activation is a result of increased peroxynitrite, in vitro experiments were performed in which cultured chondrocytes were incubated with this oxidant. RESULTS: Activated JNK was detected in the cytoplasm of osteoarthritis (OA) affected chondrocytes but not in that of controls. In vitro, chondrocytes produce NO and superoxide anion. IL-1 (48 h), which induces nitric oxide synthase, resulted in an activation of JNK; this effect was reversed by N-monomethylarginine (NMA). TNFalpha treated chondrocytes at 48 h produce superoxide anion (EPR method). Exposure of cells to peroxynitrite led to an accumulation of intracellular oxidants, in association with JNK activation and cell death by apoptosis. CONCLUSION: We suggest that JNK activation is among the IL-1 elicited responses that injure articular chondrocytes and this activation of JNK is dependent on intracellular oxidant formation (including NO peroxynitrite). In addition, the extraction technique here described is a novel method that permits the quantitation and study of proteins such as JNK involved in the signaling pathways of chondrocytes within osteoarthritic cartilage.

Increased nitric oxide production accompanied by the up-regulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus.

OBJECTIVE: To investigate whether systemic lupus erythematosus (SLE) is accompanied by increased serum nitrite levels, whether active compared with inactive disease is associated with greater nitric oxide (NO) production, and whether endothelial cells or keratinocytes serve as cellular sources of NO by virtue of their increased expression of either constitutive nitric oxide synthase (cNOS) or inducible NOS (iNOS). METHODS: Fifty-one serum samples (46 from patients with SLE) were analyzed for NO production by measuring nitrite levels in a calorimetric assay. Skin biopsy samples from 21 SLE patients and 11 healthy volunteers were evaluated immunohistochemically, using monoclonal antibodies, for endothelial cell and keratinocyte cNOS and iNOS expression. RESULTS: Serum nitrite levels were significantly elevated in the 46 patients with SLE (mean +/- SEM 37 +/- 6 microM/liter) compared with controls (15 +/- 7 microM/liter; P < 0.01), and were elevated in patients with active SLE compared with those with inactive disease (46 +/- 7 microM/liter versus 30 +/- 7 microM/liter; P < 0.01). Serum nitrite levels correlated with disease activity (r = 0.47, P = 0.04) and with levels of antibodies to double-stranded DNA (r = 0.35, P = 0.02). Endothelial cell expression of iNOS in SLE patients (mean +/- SEM score 1.5 +/- 0.2) was significantly greater compared with controls (0.6 +/- 0.2; P < 0.01), and higher in patients with active disease compared with those with inactive SLE (1.7 +/- 0.2 versus 1.2 +/- 0.2; P < 0.01). Keratinocyte expression of iNOS was also significantly elevated in SLE patients (0.9 +/- 0.1) compared with controls (0.4 +/- 0.1; P < 0.001). With regard to expression of cNOS, there were no differences between patients with active SLE, those with inactive SLE, and normal controls in either the vascular endothelium or the keratinocytes. CONCLUSION: NO production is increased in patients with SLE, and 2 potential sources of excessive NO are activated endothelial cells and keratinocytes via up-regulated iNOS.

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.