Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages

C14orf28 [alias dopamine receptor-interacting protein (DRIP1)] is belonging to the family of DRIPs. However, the function of C14orf28 in cancer remains unclear. Herein, we found that C14orf28 was upregulated in colorectal cancer tissues compared to the adjacent non-tumor tissues. Overexpression of C14orf28 promoted the cellular proliferation, migration, invasion of colorectal cancer cells. In addition, C14orf28 inhibited apoptosis and promoted the EMT process. To explore the mechanism of dysregulation, C14orf28 was identified to be a target of miR-519d by targeting its 3′UTR. Furthermore, in agreement, C14orf28 overexpression counteracted the inhibitory effect of miR-519d. Together, these results evidenced that C14orf28 downregulated by miR-519d contributes to tumorigenesis and might provide new potential targets for colorectal cancer therapy.     

Exogenous nitric oxide modulates cytokine production in human leukocytes

Exogenous nitric oxide was found to modify the pattern of cytokine secretion from human leukocytes, with similar outcome in 11 different healthy blood donors. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohaemagglutinin (PHA) in the presence of increasing amounts of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). The NO donor dose-dependently enhanced IL-4 secretion into the supernatant (p<0.01). In contrast, IFNgamma production was not affected while IL-10 levels were slightly decreased. Comparable changes were observed when analysing cytokine mRNA levels by semiquantitative RT-PCR. The differential effect of the NO donor on IL-4 versus IL-10 and IFNgamma gene expression suggests an immunomodulatory potential of NO, which may serve to limit inflammatory responses.     

Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in beta1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via beta1 integrins, suggesting that TNF and beta1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-kappaB. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.     

Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro

Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50?~?200?μg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.     

Immunomodulatory cytokines suppress epithelial nitric oxide production in inflammatory bowel disease by acting on mononuclear cells

Inducible nitric oxide synthase (iNOS) activity in colonic epithelial HT-29 cells is modulated by the T-cell-derived cytokines IL-4 and IL-13, but is not affected by IL-10 despite its effect in models of colitis. We studied the effects of these cytokines on nitric oxide (NO) production by colonic tissue. IL-10 and IL-4 but not IL-13 suppressed the NO production and iNOS expression by inflamed tissue and cytokine-stimulated noninflamed tissue from patients with ulcerative colitis, whereas the three cytokines suppressed NO production in cytokine-stimulated biopsies from controls. To examine why colonic biopsies and HT-29 cells respond differently to immunomodulatory cytokines, a coculture of mixed mononuclear monocytes (MMC) and HT-29 cells was studied. Treatment of HT-29 cells with conditioned medium from IFN-γ/LPS-stimulated MMC produced significant amounts of NO, which suggested the presence of an MMC-derived soluble factor modifying epithelial NO production. Pretreatment of IFN-γ/LPS-stimulated MMC with IL-10 and IL-4 but not IL-13 suppressed NO production by HT-29 cells. Interestingly, pretreatment of HT-29 cells with IL-1 receptor antagonist suppressed the IFN-γ/LPS-stimulated MMC-induced NO production. These results suggest that immunomodulatory cytokines might exert an inhibitory effect on NO up-regulation by colonic epithelium via the inhibition of MMC-derived soluble mediators, such as IL-1.     

Endogenous nitric oxide inhibits neutrophil adherence to lung epithelial cells to modulate interleukin-8 release

To investigate the effect of neutrophil adherence to epithelial cells on the release of interleukin 8 (IL-8), we measured neutrophil adherence in the presence or absence of IFN-gamma+TNF-alpha+IL-1beta (cytomix) stimulation on cultured A549 epithelial cells. The extent of neutrophil adherence to A549 epithelial cells was measured and the concomitant production of IL-8 and nitrite were assayed. The roles of adhesion molecules and nitrite in modulation of neutrophil adherence were examined by pretreatment with oversaturating ICAM-1 blocking antibody and L-NAME (1 mM), respectively. There was a time-dependent spontaneous and cytomix-induced release of IL-8 from epithelial cells, as well as a time-dependent increase in the magnitude of neutrophil adherence to epithelial cells. Stimulation of epithelial cells with cytomix induced a further increase in neutrophil adherence. Pretreatment with oversaturated ICAM-1 monoclonal antibody inhibited neutrophil adherence with or without cytomix stimulation. The inhibition of neutrophil adherence to epithelial cells with ICAM-1 monoclonal antibody or a semipermeable membrane downregulated the release of IL-8 with or without cytomix stimulation. Stimulation with cytomix decreased nitrite production. Both neutrophil adherence and L-NAME pretreatment significantly inhibited the production of nitrite. The inhibition of neutrophil adherence to epithelial cells with ICAM-1 monoclonal antibody or a semipermeable membrane upregulated nitrite production. Pretreatment with L-NAME failed to modify the spontaneous release of IL-8, but significantly enhanced the response to adherence and cytomix. In conclusion, endogenous nitric oxide may play a role in preventing neutrophil adherence to lung epithelial cells, thus modulating concomitant IL-8 release.     

Intraepithelial lymphocytes coinduce nitric oxide synthase in intestinal epithelial cells

The study of mucosal immunity has revealed that complex reciprocal interactions occur between intestinal intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC). The present study focuses on the induction of inducible nitric oxide (NO) synthase in cocultures of freshly isolated rat IEL and the rat epithelial cell line IEC-18 after the addition of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, or lipopolysaccharide. When IEL and IEC were separated using Transwell chambers, NO synthesis was not induced, indicating that cell-cell contact was required. Culture of IEC-18 with IEL, even in the absence of inflammatory stimuli such as IL-1beta, resulted in upregulation of class I and II antigens on IEC-18, due to the interferon-gamma (IFN-gamma) that is constitutively produced by IEL. Addition of anti-IFN-gamma antibody to the NO-producing cocultures resulted in inhibition of NO synthesis as well as the upregulation of class I and II antigen expression. These data indicate that IFN-gamma production by IEL conditions IEC for the expression of other components of the inflammatory process.     

Characterization of basal nitric oxide production in living cells

Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.     

Effects of macrophage inducible nitric oxide synthase in murine septic lung injury

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS-/- AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 +/- 0.1 vs. 1.4 +/- 0.1 microg EB.g lung(-1).min(-1); P < 0.05) and MPO activity (37 +/- 4 vs. 67 +/- 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 +/- 0.3 microg EB.g lung(-1).min(-1)) but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.     

Toxoplasma gondii partially inhibits nitric oxide production of activated murine macrophages

Activated macrophages produce nitric oxide (NO) and as such are able to control the multiplication of Toxoplasma gondii. Until now, no reports have described a possible modulation of NO production of macrophages after T. gondii infection. To investigate this possibility, murine blood monocyte-derived and peritoneal macrophages were activated in vitro with interferon-gamma and lipopolysaccharide and infected with T. gondii and Trypanosoma cruzi, and NO production was evaluated. NO was produced by monocyte-derived macrophages only if cultured in the presence of macrophage-colony-stimulating factor. Monocyte-derived or peritoneal macrophages infected with T. gondii presented a significant reduction in NO production. NO production inhibition was not detected after T. cruzi infection. Macrophages infected with higher T. gondii/macrophage ratios presented lower NO production. Furthermore, only viable T. gondii could cause partial inhibition of NO production. In macrophages activated 24 h before the interaction, partial inhibition was detected after 3 h of infection and continued for 48 h. In macrophages activated immediately after the interaction, partial inhibition was not detected at 12 h, but was observed at 24 h. T. gondii-infected macrophages present lower inducible nitric oxide synthase expression as assayed by immunofluorescence. T. gondii did not develop in monocyte-derived macrophages producing NO, but were not totally eliminated. These results demonstrate that T. gondii infection partially inhibits NO production by murine macrophages, suggesting that a deactivating macrophage mechanism may be used for better survival into phagocytic cells.     

Indomethacin prevents the induction of inducible nitric oxide synthase in murine peritoneal macrophages and decreases their nitric oxide production

Indomethacin (0.14-.5 mM concentration) inhibits nitric oxide production in murine peritoneal macrophages. This was evidenced by measuring both nitrite production or 14C-L-citrulline formation. The inhibition was caused by the diminution of de novo inducible nitric oxide synthase production as demonstrated by Western blotting experiment. The effect of indomethacin after 4 h treatment was irreversible. NO synthase and arginase activities and the uptake of arginine were not directly affected by the drug. Indomethacin also decreased uridine incorporation in macrophages. The effect of indomethacin on the induction of other enzymes (i.e. arginase) was weaker.     

Curcumin modulates eukaryotic initiation factors in human lung adenocarcinoma epithelial cells

Curcumin, a polyphenolic compound, is the active component of Curcuma longa and has been extensively investigated as an anticancer drug that modulates multiple pathways. Eukaryotic initiation factors (eIFs) have been known to play important roles in translation initiation, which controls cell growth and proliferation. Little is known about the effects of curcumin on eIFs in lung cancer. The objective of this study was to exam the curcumin cytotoxic effect and modulation of two major rate-limiting translation initiation factors, including eIF2α and eIF4E protein expression levels in lung adenocarcinoma epithelial cell line A549. Cytotoxicity was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and protein changes were determined by Western blot. A549 cells were treated with 0–240 μM curcumin for 4–96 h. The inhibitory effects of curcumin on cytotoxicity were dose- and time-dependent (P < 0.001). The 50% inhibitory curcumin concentrations (IC50s) at 24, 48, 72, and 96 h were 93, 65, 40, and 24 μM, respectively. Protein expressions of eIF2α, eIF4E, Phospho-4E-BP1 were down-regulated, while Phospho-eIF2α and Phospho-eIF4E were up-regulated after A549 cells were treated with 20 and 40 μM curcumin for 24 h. In addition, the effects of curcumin on these protein expression changes followed a significant dose-response (P < 0.05, trend test). These findings suggest that curcumin could reduce cell viability through prohibiting the initiation of protein synthesis by modulating eIF2α and eIF4E.     

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Arginase activity in the frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate, arginase can affect the NO synthesis. In the present work, the properties of arginase from the frog Rana temporaria L. urinary bladder epithelial cells possessing the NO-synthase activity were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme had the temperature optimum in the range of 55-60 degrees C, K(m) for arginine 23 mM, and V(max) about 10 nmol urea/mg protein/min, and its activity was effictively inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10(-6) to 10(-4) M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in the isolated urinary bladder epithelial cells was 3 times higher than that in the intact urinary bladder. To evaluate the role of arginase in the regulation of NO production, epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO2-, the stable NO metabolite, was determined in the culture fluid after 18-20 h of cells incubation. The vast majority of the produced nitrites are associated with the NOS activity, as L-NAME, the NOS-inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in 199 medium. BEC (10(-4) M) increased the nitrite production by 18.0 % +/- 2.7 in the L-15 medium and by 24.2 +/- 3.5 in the 199 medium (p < 0.05). The obtained data indicate a relatively high arginase activity in the frog urinary bladder epithelium and its involvement in regulation of NO production by epithelial cells.     

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.     

Transition metals and nitric oxide production in human endothelial cells

The bioavailability of endothelial nitric oxide (NO) is regulated by transition metals but their mechanisms of action on NO synthesis and degradation are not clearly understood. Using differential pulse amperometry and NO microelectrodes, local NO concentration was measured at the surface of cultured human umbilical vein endothelial cells (HUVECs) stimulated by histamine or thrombin in the presence of transition metal chelators. The agonist-activated NO release required both extracellular Ca2+ and transition metals. In the presence of 1 mM external Ca2+, a low concentration of EGTA (5 microM) inhibited by 40% the NO release from stimulated HUVECs. In the presence of extracellular L-arginine, the inhibitory effect of EGTA was even more marked and, in its absence, it was suppressed by adding exogenous superoxide dismutase. The decrease in NO release induced by the copper chelators, cuprizone and DETC, suggests that extracellular traces of Cu2+ could regulate NO availability.     

Aldose reductase mediates endotoxin-induced production of nitric oxide and cytotoxicity in murine macrophages

Aldose reductase (AR) is a ubiquitously expressed protein with pleiotrophic roles as an efficient catalyst for the reduction of toxic lipid aldehydes and mediator of hyperglycemia, cytokine, and growth factor-induced redox-sensitive signals that cause secondary diabetic complications. Although AR inhibition has been shown to be protective against oxidative stress signals, the role of AR in regulating nitric oxide (NO) synthesis and NO-mediated apoptosis has not been elucidated to date. We therefore investigated the role of AR in regulating lipopolysaccharide (LPS)-induced NO synthesis and apoptosis in RAW 264.7 macrophages. Inhibition or RNA interference ablation of AR suppressed LPS-stimulated production of NO and overexpression of iNOS mRNA. Inhibition or ablation of AR also prevented the LPS-induced apoptosis, cell cycle arrest, activation of caspase-3, p38-MAPK, JNK, NF-kappaB, and AP1. In addition, AR inhibition prevented the LPS-induced down-regulation of Bcl-xl and up-regulation of Bax and Bak in macrophages. L-Arginine increased and L-NAME decreased the severity of cell death caused by LPS and AR inhibitors prevented it. Furthermore, inhibition of AR prevents cell death caused by HNE and GS-HNE, but not GS-DHN. Our findings for the first time suggest that AR-catalyzed lipid aldehyde-glutathione conjugates regulate the LPS-induced production of inflammatory marker NO and cytotoxicity in RAW 264.7 cells. Inhibition or ablation of AR activity may be a potential therapeutic target in endotoximia and other inflammatory diseases.     

Adiponectin stimulates production of nitric oxide in vascular endothelial cells

Adiponectin is secreted by adipose cells and mimics many metabolic actions of insulin. However, mechanisms by which adiponectin acts are poorly understood. The vascular action of insulin to stimulate endothelial production of nitric oxide (NO), leading to vasodilation and increased blood flow is an important component of insulin-stimulated whole body glucose utilization. Therefore, we hypothesized that adiponectin may also stimulate production of NO in endothelium. Bovine aortic endothelial cells in primary culture loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) were treated with lysophosphatidic acid (LPA) (a calcium-releasing agonist) or adiponectin (10 microg/ml bacterially produced full-length adiponectin). LPA treatment increased production of NO by approximately 4-fold. Interestingly, adiponectin treatment significantly increased production of NO by approximately 3-fold. Preincubation of cells with wortmannin (phosphatidylinositol 3-kinase inhibitor) blocked only adiponectin- but not LPA-mediated production of NO. Using phospho-specific antibodies, we observed that either adiponectin or insulin treatment (but not LPA treatment) caused phosphorylation of both Akt at Ser473 and endothelial nitric-oxide synthase (eNOS) at Ser1179 that was inhibitable by wortmannin. We next transfected bovine aortic endothelial cells with dominant-inhibitory mutants of Akt (Akt-AAA) or AMP-activated protein kinase (AMPK) (AMPKK45R). Neither mutant affected production of NO in response to LPA treatment. Importantly, only AMPKK45R, but not Akt-AAA, caused a significant partial inhibition of NO production in response to adiponectin. Moreover, AMPK-K45R inhibited phosphorylation of eNOS at Ser1179 in response to adiponectin but not in response to insulin. We conclude that adiponectin has novel vascular actions to directly stimulate production of NO in endothelial cells using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK. Thus, the effects of adiponectin to augment metabolic actions of insulin in vivo may be due, in part, to vasodilator actions of adiponectin.     

Protein SV-IV promotes nitric oxide production not associated with apoptosis in murine macrophages

SV-IV (seminal vesicle protein no. 4) is a potent immunomodulatory and anti-inflammatory secretory protein (Mr 9758) produced in large amounts by the rat seminal vesicle epithelium. Here we show that this protein possesses the ability to upregulate in J774 macrophages the expression of the gene coding for the inducible nitric oxide synthase (iNOS). The increase in NO production consequent on the marked enhancement of iNOS activity was not associated with apoptotic damage of the SV-IV-treated cells. In the same experimental model, however, LPS induced upregulation of iNOS coupled with an increase in NO production and marked apoptotic death. Differences in the ability of SV-IV and LPS to control the life/ death signal balance in target cells via trans-membrane activation of apoptotic (mediated by TNF-alpha and NO/iNOS system) and anti-apoptotic (mediated by bcl-2, c-myc, etc.) pathways are suggested to be the basis of the apoptotic fate of the experimentally treated cells. In addition, considering the important role played by NO in the process of mammalian reproduction, SV-IV may be involved in the fine tuning of NO concentration in the female genital tract mucosa via an SV-IV-mediated control of iNOS gene expression in local macrophages.