Nicotinamide inhibits inducible nitric oxide synthase enzyme activity in macrophages by allowing nitric oxide to inhibit its own formation

Nitric oxide (NO) production by macrophages is mainly regulated by induction of nitric oxide synthase (iNOS) by cytokines and microbial products. Nicotinamide (NIC) inhibits NO production by activated macrophages in a dose dependent manner. NIC also inhibits NOS enzyme activity in extracts from activated macrophages. The inhibition was noncompetitive with L-arginine (Ki 13.37 +/- 4.40 mM, n=3), uncompetitive versus NADPH (Ki 3.06 +/- 0.17 mM, n=3) and tetrahydrobiopterin. Finally, the inhibition by nicotinamide was fully reversed by scavenging NO with hemoglobin. We suggest that NIC acts by allowing NO to inhibit its own formation.     

Phlebotomus papatasi saliva inhibits protein phosphatase activity and nitric oxide production by murine macrophages

Leishmania parasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand fly Phlebotomus papatasi is the vector of Leishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show that P. papatasi saliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate that P. papatasi saliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that the P. papatasi salivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by which P. papatasi saliva exacerbates cutaneous leishmaniasis.     

Nitric oxide superoxide and peroxynitrite modulate osteoclast activity

PURPOSE: To assess whether magnetic resonance (MR) spectroscopic imaging with MR imaging can improve prostate cancer localization in postbiopsy hemorrhage cases. MATERIALS AND METHODS: Records of 175 patients with prostate cancer were retrospectively reviewed; 42 patients (135 hemorrhagic sites) had spatially correlated biopsy data. Patients underwent both phased-array coil-endorectal coil MR imaging and three-dimensional MR spectroscopic imaging within 180 days after transrectal ultrasound (US)-guided biopsy. High-signal-intensity hemorrhage on T1-weighted images and corresponding high- or low-signal-intensity areas on T2-weighted images and the metabolic ratio (choline + creatine)/citrate were recorded. Cancer was identified as a low-signal-intensity area at T2-weighted MR imaging or a metabolite ratio greater than 3 standard deviations above normal at MR spectroscopic imaging. MR imaging, spectroscopic, and biopsy findings were compared. RESULTS: Forty-nine patients had postbiopsy hemorrhage. On T2-weighted images, a higher (P < .01) percentage of hemorrhagic sites demonstrated low signal intensity (80% [108 of 135 sites]), which is similar to the signal intensity seen with cancer. The addition of MR spectroscopic imaging to MR imaging resulted in a significant increase (P < .01) in the accuracy (52% to 75%) and specificity (26% to 66%) of tumor detection. CONCLUSION: The addition of MR spectroscopic imaging to MR imaging significantly improves the ability to determine the presence of prostate cancer and spatial extent when postbiopsy changes hinder interpretation with MR imaging alone.     

Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption

Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.     

Inhibition of nitric oxide synthesis inhibits rat sleep

Previous findings indicate that nitric oxide (NO) may play a role in the regulation of sleep-wake activity. In rabbits, blocking the production of endogenous NO by a nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NAME) suppresses spontaneous sleep and interferes the somnogenic actions of interleukin 1. In the present experiments we extended our earlier work by studying the long-term effects of L-NAME treatment on sleep-wake activity including power spectra analyses of the electroencephalogram (EEG) in rats. Rats implanted with EEG electrodes, brain thermistor, and intracerebroventricular (i.c.v.) guide cannula were injected i.c.v. with vehicle or 0.2, 1, or 5 mg L-NAME at light onset. In separate experiments, rats were injected intraperitoneally (i.p.) with L-NAME three times (50, 50, 100 mg/kg), 12-12 h apart. Both i.c.v. and i.p. injections of L-NAME elicited decreases in time spent in NREMS and REMS. After i.c.v. injection of 5 mg L-NAME the sleep responses were long-lasting; NREMS did not return to baseline even 72 h after injection. EEG delta-wave activity during NREMS (slow wave activity) was also suppressed after 0.2 and 5 mg L-NAME. Brain temperature was slightly increased after the two lower doses of L-NAME, whereas there was a transient decrease in Tbr after 5 mg L-NAME. Acute i.p. injection of 50 mg/kg L-NAME elicited an immediate decrease in NREMS which lasted for approximately 2 h. The second injection of 50 mg/kg L-NAME and the following injection of 100 mg/kg L-NAME induced biphasic decreases in NREMS but not REMS.     

Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms

Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis, and here we report that NO prevents hepatocyte apoptosis initiated by the removal of growth factors or exposure to TNFalpha or anti-Fas antibody. We postulated that the mechanism of the inhibition of apoptosis by NO would include an effect on caspase-3-like protease activity. Caspase-3-like activity increased coincident with apoptosis due to all three stimuli, and treatment with the caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde inhibited both proteolytic activity and apoptosis. Endogenous or exogenous sources of NO prevented the increase in caspase-3-like activity in hepatocytes. Exposure of purified recombinant caspase-3 to an NO or NO+ donor inhibited proteolytic activity. Dithiothreitol (DTT), but not glutathione, reversed the inhibition of recombinant caspase-3 by NO. When lysates from cells stimulated to express inducible NO synthase or cells exposed to NO donors were incubated in DTT, caspase-3-like activity increased to about 55% of cells not exposed to a source of NO. Similarly, administration of an NO donor to rats treated with TNFalpha and D-galactosamine also prevented the increase in caspase-3-like activity as measured in liver homogenates. The effect of the NO donor was reversed by about 50% if the homogenate was incubated with DTT. TNFalpha-induced apoptosis and caspase-3-like activity were also reduced in cultured hepatocytes exposed to 8-bromo-cGMP, and both effects were inhibited by the cGMP-dependent kinase inhibitor KT5823. The suppression in caspase-3-like activity in hepatocytes exposed to an NO donor was partially blocked by an inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one, (ODQ), while the incubation of these lysates in DTT almost completely restored caspase-3-like activity to the level of TNFalpha-treated controls. These data indicate that NO prevents apoptosis in hepatocytes by either directly or indirectly inhibiting caspase-3-like activation via a cGMP-dependent mechanism and by direct inhibition of caspase-3-like activity through protein S-nitrosylation.     

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.     

Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity

The effects of substrate, L-Arg and cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4B) and calmodulin (CaM), on chiral discrimination by rat neuronal nitric oxide synthase (nNOS) for binding the enantiomers of 1-(1-naphthyl)ethylamine (ligand I), 1-cyclohexylethylamine (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied under anaerobic conditions by optical absorption spectroscopy. The ratio of the dissociation constant (Kd) values for the S- and R-enantiomers of ligand I (S/R) was 30, while the S/R ratio for ligand II and the R/S ratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 microM H4B. However, in the presence of 1 mM L-Arg, the S/R ratio of the Kd values for ligand I was decreased down to 5.9. In the presence of both 1 mM L-Arg and 0.1 mM H4B, the S/R ratios for ligands I and II and the R/S ratio for ligand III were enormously increased up to 29, > 80, and 60, respectively. These and other spectral observations strongly suggest that strict chiral recognition at the active site of nNOS during catalysis is exhibited only in the presence of the active effector.     

Oncostatin-M: a new bone active cytokine that activates osteoblasts and inhibits bone resorption

Osteoblasts and their precursors respond to specific cytokines, growth factors, and hormones. One facet of this response includes the secretion of additional cytokines, some of which are part of the circuitry involved in the regulation of osteoblast and osteoclast function. Therefore, understanding which cytokines are able to activate osteoblastic cells and the consequences of that activation are central to understanding normal and pathologic bone remodeling. Oncostatin M (OSM) is a glycoprotein belonging to a new subfamily of cytokines related by sequence and structural homology and the use of the signal transducing receptor component gp130. Osteoblastic cells secrete and respond to leukemia-inhibiting factor (LIF) both in vitro and in vivo, suggesting that LIF is an autocrine regulatory factor. OSM is closely related to LIF, and therefore we hypothesized that OSM should regulate the function of cells in the osteoblastic lineage. Primary neonatal murine or fetal rat calvarial osteoblastic cultures were treated with OSM or LIF and a series of biochemical and biological parameters were determined. In these cultures, OSM induced proliferation, collagen synthesis, and interleukin-6 secretion, whereas it inhibited alkaline phosphatase activity. Bone resorption was also inhibited by OSM. These data represent the first report of OSM's effects on bone cell function and indicate that, like some other members of the LIF/interleukin-6 subfamily, OSM has potent bone regulatory activity.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Expression of nitric oxide synthase isoforms in bone and bone cell cultures

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.     

Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models

Alendronate is an aminobisphosphonate that acts as a potent inhibitor of osteoclastic bone resorption. To understand the mechanism of action of alendronate in vivo, in this study we investigated the relationship between distribution of [14C]-alendronate in rat bone and its effects on bone resorption in vitro or in rat hypercalcemic models. A single IV dose of 0.05 approximately 1.25 mg/kg inhibited the increase in plasma calcium level induced by bovine PTH or 1 alpha(OH)D3. The minimal effective dose of pamidronate (1.25 mg/kg) and etidronate (over 31.25 mg/kg) were at least 5 times and 25 times, respectively, higher than the dose of alendronate in the rat hypercalcemic model prepared by 1 alpha(OH)D3. The relative potencies of compounds in the hypercalcemic rat models reflected those of inhibitory effects on bone resorption in vitro. We conducted the ivory-slice assay under two conditions: (a) addition of a given bisphosphonate after adherence of the osteoclasts; and (b) preincubation of the ivory slices with a given bisphosphonate. The inhibitory IC50 values of alendronate under condition (b) were similar to those under condition (a). To evaluate the interaction between osteoclasts and alendronate in bone, we investigated the localization of [14C]-alendronate in the tibia of growing rats (4-day-old rats). Alendronate did not distribute uniformly in the tibia. At 1 day after injection (0.05 mg SC), dense labeling was seen primarily under osteoclasts. We injected 0.05 mg/kg of [14C]-alendronate (single i.v.) into rats [14C]-alendronate was rapidly eliminated from plasma, and mainly distributed to the bone in rats. These data suggest that alendronate which distributed on bone surface mainly contributed to the antihypercalcemic action in vivo.     

Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase

Physiological levels of shear stress reduce endothelial cell turnover and exert a potent antiatherosclerotic effect. Here we demonstrate that oxidative stress-induced apoptosis of human endothelial cells was inhibited by shear stress exposure (15 dynes/cm2). Incubation with H2O2 (200 mumol/L) for 18 hours induced apoptosis of human umbilical venous endothelial cells as demonstrated by an enzyme-linked immunosorbent assay specific for histone-associated DNA fragments and visual analysis of fluorescence-stained nuclei. Shear stress-mediated inhibition of apoptosis was partially prevented by pharmacological inhibition of glutathione (GSH) biosynthesis with buthionine sulfoximine (BSO) or nitric oxide (NO) synthase with NG-monomethyl-L-arginine (LNMA), whereas inhibition of catalase by aminotriazol did not affect the inhibitory action of shear stress. Combined inhibition of NO synthase and GSH biosynthesis completely reversed the protective effect of shear stress, suggesting that both NO synthase and the GSH redox cycle system are involved in the apoptosis-suppressing effect of shear stress. Similar results were obtained when apoptosis was stimulated by tumor necrosis factor alpha (TNF alpha). To gain further insights into the interference of shear stress with apoptosis signal transduction, we measured caspase-3-like activity, a cysteine protease that has been shown to play a predominant role in the cell death effector pathway. Indeed, shear stress prevented the activation of caspase-3-like activity induced by H202 or TNF alpha. The inhibitory effect of shear stress was prevented by LNMA and BSO, suggesting that the reduction of oxidative flux by shear stress prevents the activation of caspase-like proteases and thereby inhibits apoptotic cell death in human endothelial cells.     

Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis

OBJECTIVE: Chondrocytes produce nitric oxide (NO) and undergo apoptosis in response to exogenous NO. This study sought to examine the relationship between NO synthesis, chondrocyte apoptosis, and the development of cartilage degradation during experimental osteoarthritis (OA). METHODS: OA was induced in rabbits by anterior cruciate ligament transection (ACLT). Knees were harvested after 4 weeks and assessed for OA severity and chondrocyte apoptosis. Conditioned media from cultured cartilage explants were analyzed for nitrite content. Cartilage sections were analyzed by immunohistochemistry for the presence of nitrotyrosine. RESULTS: All ACLT knees demonstrated osteoarthritic changes. Conditioned media from ACLT cartilage organ cultures contained higher levels of nitrite as compared with cartilage samples from the nonoperated side or from rabbits that had not received ACLT. Cultures of specific areas of cartilage from ACLT knees showed high levels of NO production in the medial femoral and medial tibial cartilage. Approximately 28.7% of chondrocytes isolated from ACLT cartilage and 6.7% of chondrocytes from cartilage of the nonoperated side underwent apoptosis. In situ staining demonstrated apoptotic cells in the superficial and middle zones of ACLT cartilage. A high number of apoptotic cells was present at the pannus-cartilage junction. In control cartilage, the superficial zone contained a small number of cells in apoptosis. The prevalence of apoptotic cells was significantly correlated with the levels of nitrite production and OA grade. CONCLUSION: These observations suggest that, during the early phases of OA, NO production may lead to chondrocyte apoptosis, and that both events contribute to the pathogenesis of cartilage degradation. Inhibitors of NO synthesis and chondrocyte apoptosis may therefore be of therapeutic value after cartilage injury and in patients with OA.     

Regulation of cyclooxygenase activity in airway epithelium by endogenous nitric oxide

The role of nitric oxide (NO) in the activity of cyclooxygenase (COX) in cultured canine tracheal epithelium was studied. Tracheal epithelium spontaneously released prostaglandin E2 (PGE2), which is a product of COX. The release of PGE2 was increased by bradykinin and was decreased by two NO synthase inhibitors: NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. That decrease was reversed in the presence of L-arginine. Chrolpromadin, but not aminoguanidine, inhibited PGE2 production, which suggests that constitutive NO synthase is involved. Two stable NO donors, sodium nitroprusside and S-nitroso-N-acetyl DL-penicillamine, also increased the production of PGE2. These effects were abolished by coincubation with hemoglobin, which binds and inactivates NO, but not by methylene blue, an inhibitor of soluble guanylate cyclase. NADPH diaphorase histochemistry of cultured tracheal cells revealed activity in the periphery of the cytoplasm. These results suggest that, in cultured canine tracheal epithelium, NO directly interacts with COX to regulate PGE2 production.     

Cytokine-induced inhibition of bone matrix proteins is not mediated by prostaglandins

Interleukin-1 (IL-1) and tumor necrosis factor (TNF), two pleiotropic cytokines produced in inflammatory processes, inhibit bone matrix biosynthesis and stimulate prostanoid formation in osteoblasts. In the present study, the importance of prostaglandin formation in IL-1 and TNF-induced inhibition of osteocalcin and type I collagen formation has been examined. In the human osteoblastic cell line MG-63, IL-1 alpha (10-1000 pg/ml), IL-1 beta (3-300 pg/ml) and TNF-alpha (1-30 ng/ml) stimulated prostaglandin E2 (PGE2) formation and inhibited 1,25(OH)2-vitamin D3-induced osteocalcin biosynthesis as well as basal production of type I collagen. Addition of PGE2 or increasing the endogenous formation of PGE2 by treating the cells with arachidonic acid, bradykinin, Lys-bradykinin or des-Arg9-bradykinin, did not affect osteocalcin and type I collagen formation in unstimulated or 1,25(OH)2-vitamin D3-stimulated osteoblasts. Four non-steroidal antiinflammatory drugs, indomethacin, flurbiprofen, naproxen and meclofenamic acid, inhibited basal, IL-1 beta- and TNF-alpha-stimulated PGE2 formation in the MG-63 cells without affecting IL-1 beta- or TNF-alpha-induced inhibition of osteocalcin and type I collagen formation. In isolated, non-transformed, human osteoblast-like cells, IL-1 beta and TNF-alpha stimulated PGE2 formation and concomitantly inhibited 1,25(OH)2-vitamin D3-stimulated osteocalcin biosynthesis, without affecting type I collagen formation. In these cells, indomethacin and flurbiprofen abolished the effects of IL-1 beta and TNF-alpha on prostaglandin formation without affecting the inhibitory effects of the cytokines on osteocalcin biosynthesis. These data show that IL-1 and TNF inhibit osteocalcin and type I collagen formation in osteoblasts independently of prostaglandin biosynthesis and that non-steroidal antiinflammatory drugs do not affect the effects of IL-1 and TNF on bone matrix biosynthesis.     

Interleukin-10 gene transfer inhibits murine mammary tumors and elevates nitric oxide

Transfection of cDNA for IL-10 into line 66.1 murine mammary tumor cells results in marked suppression of tumor growth and metastasis. Others have reported that nitric oxide has potent antitumor activity and IL-10 is known to regulate the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We identified nitric oxide production in mammary tumors as indicated by electron paramagnetic resonance detection of nitric oxide-hemoglobin (NO-Hb). IL-10 expression resulted in elevated levels of NO-Hb in mammary tumors. Immunohistochemical examination of mammary tumors for iNOS protein revealed few positively staining cells in parental or control neo-transfected tumors but strong iNOS staining in all IL-10 transfected tumors, consistent with the NO-Hb data. To determine if mammary epithelial tumor cells themselves, express nitric oxide synthase activity, cultured tumor cells were treated with pro-inflammatory cytokines and nitrite accumulation was assessed in the conditioned medium. All IL-10 producing cell lines accumulated uM concentrations of nitrite in response to short term (24 hr) cytokine stimulation. Cells not expressing IL-10 (parental and neo-transfectants) accumulated no nitrite under similar culture conditions. After longer stimulation (48 hr), parental and 66-neo cells accumulated lower amounts of nitrite. IL-10 gene transfer is associated with increased iNOS protein expression and enzymatic activity detected both in vitro and in vivo. Our findings suggest that the antimetastatic and antitumor activity of IL-10 is related to enhanced production of nitric oxide.     

L-arginine depletion inhibits glomerular nitric oxide synthesis and exacerbates rat nephrotoxic nephritis

Nitric oxide (NO) synthesis is induced in glomeruli in glomerulonephritis; its role in the pathogenesis of glomerular injury is unknown. Interpretation of its role using the currently available analogues of L-arginine as in vivo inhibitors of NO is complicated by their lack of specificity for inducible NO synthase (iNOS). As NO synthesis by iNOS depends on extracellular L-arginine, we have here examined effects of L-arginine depletion on glomerular NO synthesis and the course of accelerated nephrotoxic nephritis (NTN). Arginase, which converts L-arginine to urea and L-ornithine, was used to achieve L-arginine depletion. A single dose of i.v. arginase produced complete depletion of plasma arginine for four hours. Two forms of NTN were induced in preimmunised rats by nephrotoxic globulin: (1) the systemic form of the model by intravenous nephrotoxic globulin; or (2) the unilateral form of model by left kidney perfusion with nephrotoxic globulin, which avoids the complications of systemic administration of nephrotoxic globulin. Arginase reduced plasma arginine levels and the synthesis of nitrite (the stable end-product of NO) by NTN glomeruli (95% inhibition). Proteinuria was exacerbated. There was no effect on early (24 hr) leukocyte infiltration. In the systemic form of the model arginine depletion by i.v. arginase increased glomerular thrombosis at 24 hours, and the severity of histological changes at four days, accompanied by systemic hypertension. In the unilateral form of the model, where i.v. arginase did not induce hypertension, there was no increase in thrombosis or histological severity of nephritis. These results show that arginine depletion, which inhibits glomerular NO synthesis in NTN, leads to increased proteinuria. Where injury is severe, or accompanied by systemic hypertension, the disease is further exacerbated by glomerular thrombosis. These results suggest that NO has an important role in limiting acute glomerular injury.     

Osteopontin expression by osteoclast and osteoblast progenitors in the murine bone marrow: demonstration of its requirement for osteoclastogenesis and its increase after ovariectomy

Osteoclast development requires cell-to-cell contact between hematopoietic osteoclast progenitors and bone marrow stromal/osteoblastic support cells. Based on this, we hypothesized that osteopontin, an adhesion protein produced by osteoclasts and osteoblasts, plays a role in osteoclastogenesis. Using in situ hybridization, we demonstrate that cells expressing the osteopontin messenger RNA (mRNA) appear after 3 days of culturing murine bone marrow cells. The number of these cells increases thereafter, reaching a peak on day 5. In the same cultures, cells expressing alkaline phosphatase (AP) or tartrate resistant acid phosphatase (TRAP), phenotypic markers for osteoblastic and osteoclast-like cells, respectively, appeared subsequent to the appearance of the osteopontin-positive cells. By means of a combination of in situ hybridization and histostaining, it was shown that the osteopontin mRNA was localized in 30-50% of the AP-positive or the TRAP-positive, as well as in nonspecific esterase (NSE)-positive, cells. The number of cells expressing both the osteopontin mRNA and either one of the three phenotypic markers was significantly increased in bone marrow cultures from estrogen-deficient mice, as compared with controls. Conversely, the number of all three populations of double positive cells was decreased in cultures treated with a specific antimouse rabbit osteopontin antibody or an RGD peptide. These findings indicate that osteopontin is expressed during the early stages of the differentiation of osteoclast and osteoblast progenitors in the bone marrow and that its cell adhesion properties are required for osteoclastogenesis.