Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.     

Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF

We recently reported that mast cells stimulated via FcepsilonRI aggregation can enhance T cell activation by a TNF-dependent mechanism. However, the molecular mechanisms responsible for such IgE-, Ag- (Ag-), and mast cell-dependent enhancement of T cell activation remain unknown. In this study we showed that mouse bone marrow-derived cultured mast cells express various costimulatory molecules, including members of the B7 family (ICOS ligand (ICOSL), PD-L1, and PD-L2) and the TNF/TNFR families (OX40 ligand (OX40L), CD153, Fas, 4-1BB, and glucocorticoid-induced TNFR). ICOSL, PD-L1, PD-L2, and OX40L also are expressed on APCs such as dendritic cells and can modulate T cell function. We found that IgE- and Ag-dependent mast cell enhancement of T cell activation required secreted TNF; that TNF can increase the surface expression of OX40, ICOS, PD-1, and other costimulatory molecules on CD3(+) T cells; and that a neutralizing Ab to OX40L, but not neutralizing Abs to ICOSL or PD-L1, significantly reduced IgE/Ag-dependent mast cell-mediated enhancement of T cell activation. These results indicate that the secretion of soluble TNF and direct cell-cell interactions between mast cell OX40L and T cell OX40 contribute to the ability of IgE- and Ag-stimulated mouse mast cells to enhance T cell activation.     

Potentiation of tumor necrosis factor-alpha-mediated cytotoxicity of mast cells by their production of nitric oxide

Nitric oxide (NO or endothelium-derived relaxing factor) has many of biologic actions, including the maintenance of blood pressure, inhibition of platelet aggregation, and cytotoxicity by phagocytic cells. Several cell types produce NO from L-arginine. Given recent emphasis on mast cell (MC)-dependent TNF-alpha-mediated cytotoxicity, we investigated the role of NO in rat peritoneal MC (PMC)-and intestinal mucosal mast cell-mediated cytotoxicity. MC cytotoxicity against the TNF alpha-sensitive target, WEHI-164, was potentiated by L-arginine. The NO competitive inhibitors, N omega-nitro-L-arginine and NG-methyl-L-arginine, diminished the cytotoxicity of rat PMC by 27 and 17%, respectively. However, hemoglobin, which binds to NO, inhibited the cytotoxic activity of PMC by 49% in the presence of 1 mM L-arginine and by 24% in L-arginine-free medium. The latter suggests that PMC use intracellular stores of L-arginine to produce NO. Neither hemoglobin nor NO metabolites affected human rTNF-alpha cytotoxicity. Furthermore, sodium nitroprusside, with its free radical NO group, restored PMC cytotoxicity in L-arginine-free medium to the level observed in 1 mM L-arginine medium. Studies with a platelet aggregation bioassay and various NO inhibitors confirmed that PMC produce NO. In addition, increased levels of NO2- were observed in medium of A23187, TNF-alpha, or WEHI-164-stimulated PMC.     

Nitric oxide inhibits IFN-alpha production of human plasmacytoid dendritic cells partly via a guanosine 3',5'-cyclic monophosphate-dependent pathway

NO, a free radical gas, is known to be critically involved not only in vascular relaxation but also in host defense. Besides direct bactericidal effects, NO has been shown to inhibit Th1 responses and modulate immune responses in vivo, although the precise mechanism is unclear. In this study, we examined the effect of NO on human plasmacytoid dendritic cells (pDCs) to explore the possibility that NO might affect innate as well as adaptive immunity through pDCs. We found that NO suppressed IFN-alpha production of pDCs partly via a cGMP-dependent mechanism, which was accompanied by down-regulation of IFN regulatory factor 7 expression. Furthermore, treatment of pDCs with NO decreased production of IL-6 and TNF-alpha and up-regulated OX40 ligand expression. In accordance with these changes, pDCs treated with NO plus CpG-oligodeoxynucleotide AAC-30 promoted differentiation of naive CD4(+) T cells into a Th2 phenotype. Moreover, pDCs did not express inducible NO synthase even after treatment with AAC-30, LPS, and several cytokines. These results suggest that exogenous NO and its second messenger, cGMP, alter innate as well as adaptive immune response through modulating the functions of pDCs and may be involved in the pathogenesis of certain Th2-dominant allergic diseases.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.     

Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms

Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.     

IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells.

IL-1 is a proinflammatory cytokine that plays pleiotropic roles in host defense mechanisms. We investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Ab production against SRBC was significantly reduced in IL-1alpha/beta-deficient (IL-1(-/-)) mice and enhanced in IL-1R antagonist(-/-) mice. The intrinsic functions of T, B, and APCs were normal in IL-1(-/-) mice. However, we showed that IL-1(-/-) APCs did not fully activate DO11.10 T cells, while IL-1R antagonist (-/-) APCs enhanced the reaction, indicating that IL-1 promotes T cell priming through T-APC interaction. The function of IL-1 was CD28-CD80/CD86 independent. We found that CD40 ligand and OX40 expression on T cells was affected by the mutation, and the reduced Ag-specific B cell response in IL-1(-/-) mice was recovered by the treatment with agonistic anti-CD40 mAb both in vitro and in vivo. These observations indicate that IL-1 enhances T cell-dependent Ab production by augmenting CD40 ligand and OX40 expression on T cells.     

Regulation of gene expression of murine MD-1 regulates subsequent T cell activation and cytokine production

The immunoadhesin (OX2:Fc) comprising the extracellular domain of murine OX2 linked to IgG2aFc, inhibits production of IL-2 and IFN-gamma by activated T cells and increases allograft and xenograft survival in vivo. Increased expression of OX2 on dendritic cells (DC) in vivo following preimmunization via the portal vein is also associated with elevated expression of MD-1. We have used antisense oligodeoxynucleotides (ODNs) to MD-1 to investigate the effect of inhibition of expression of MD-1 by DC on their function as allostimulatory cells. We also investigated by FACS analysis the cell surface expression of OX2, CD80, and CD86 on DC incubated with ODN-1 blocking MD-1 expression. Blocking MD-1 gene expression inhibits surface expression of CD80 and CD86, but not of OX2. DC incubated with ODN-1 to MD-1 did not stimulate IL-2 or IFN-gamma production, but generated cells able to suppress, in a second culture of fresh DC plus allogeneic T cells, production of IL-2 and IFN-gamma. This inhibition was blocked by anti-OX2 mAb. Infusion of DC preincubated with ODN-1 prolonged renal allograft survival, an effect also reversed by anti-OX2 mAb. By FACS, incubation of DC with anti-MD-1 Ab to promote capping eliminated cell surface expression of MD-1 and CD14 without altering DEC205, DC26, CD80, CD86, or OX2 expression. Thus OX2 and MD-1 are independent surface molecules on DC that may reciprocally regulate T cell stimulation. MD-1 is linked to CD14, a "danger receptor complex," and activation of this complex can regulate cell surface expression of CD80/CD86, which signal T cells.     

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.     

Endothelium-dependent and -independent vasorelaxation by a theophylline derivative MCPT: roles of cyclic nucleotides, potassium channel opening and phosphodiesterase inhibition

The vasorelaxation activities of MCPT, a newly synthesized xanthine derivative, were investigated in this study. In phenylephrine (PE)-precontracted rat aortic rings with intact endothelium, MCPT caused a concentration-dependent relaxation, which was inhibited by endothelium removed. This relaxation was also reduced by the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine methylester (L-NAME, 100 microM), soluble guanylyl cyclase (sGC) inhibitors methylene blue (10 microM), 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one (ODQ, 1 microM), adenylyl cyclase (AC) blocker SQ 22536 (100 microM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (1 microM), a Ca2+ activated K+ channels blocker tetraethylammonium (TEA, 10 mM) and a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 microM). The vasorelaxant effects of MCPT together with IBMX (0.5 microM) had an additive action. In PE-preconstricted endothelium-denuded aortic rings, the vasorelaxant effects of MCPT were attenuated by pretreatments with glibenclamide (1 microM), SQ 22536 (100 microM) or ODQ (1 microM), respectively. MCPT enhanced cAMP-dependent vasodilator isoprenaline- and NO donor/cGMP-dependent vasodilator sodium nitroprusside-induced relaxation activities in endothelium-denuded aortic rings. In A-10 cell and washed human platelets, MCPT induced a concentration-dependent increase in intracellular cyclic GMP and cyclic AMP levels. In phosphodiesterase assay, MCPT displayed inhibition effects on PDE 3, PDE 4 and PDE 5. The inhibition % were 52 +/- 3.9, 32 +/- 2.6 and 8 +/- 1.1 respectively. The Western blot analysis on HUVEC indicated that MCPT increased the expression of eNOS. It is concluded that the vasorelaxation by MCPT may be mediated by the inhibition of phosphodiesterase, stimulation of NO/sGC/ cGMP and AC/cAMP pathways, and the opening of K+ channels.     

Up-regulation of microglial CD11b expression by nitric oxide

Increased expression of CD11b, the beta-integrin marker of microglia, represents microglial activation during neurodegenerative inflammation. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) induced the production of NO and increased the expression of CD11b in mouse BV-2 microglial cells and primary microglia. Either a scavenger of NO (PTIO) or an inhibitor of inducible nitric-oxide synthase (L-NIL) blocked this increase in microglial CD11b expression. Furthermore, co-microinjection of PTIO with LPS was also able to suppress LPS-mediated expression of CD11b and loss of dopaminergic neuronal fibers and neurotransmitters in striatum in vivo. Similarly, other inducers of NO production such as interferon-gamma, interleukin-1beta, human immunodeficiency virus type-1 gp120, and double-stranded RNA (poly(IC)) also increased the expression of CD11b in microglia through NO. The role of NO in the expression of CD11b was corroborated further by the expression of microglial CD11b by GSNO, an NO donor. Because NO transduces many intracellular signals via guanylate cyclase (GC), we investigated the role of GC, cyclic GMP (cGMP), and cGMP-activated protein kinase (PKG) in microglial expression of CD11b. Inhibition of LPS- and GSNO-mediated up-regulation of CD11b either by NS2028 (a specific inhibitor of GC) or by KT5823 and Rp-8-bromo-cGMP (specific inhibitors of PKG), and increase in CD11b expression either by 8-bromo-cGMP or by MY-5445 (a specific inhibitor of cGMP phosphodiesterase) alone suggest that NO increases microglial expression of CD11b via GC-cGMP-PKG. In addition, GSNO induced the activation of cAMP response element-binding protein (CREB) via PKG that was involved in the up-regulation of CD11b. This study illustrates a novel biological role of NO in regulating the expression of CD11b in microglia through GC-cGMP-PKG-CREB pathway that may participate in the pathogenesis of devastating neurodegenerative disorders.     

Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMP-dependent modulation of NF-κB activity

Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (≤10μM) and decreased at higher (>100μM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-κB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-κB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway.     

NO regulates LPS-stimulated cyclooxygenase gene expression and activity in pulmonary artery endothelium

We examined whether nitric oxide (NO) inhibits prostanoid synthesis through actions on cyclooxygenase (COX) gene expression and activity. Bovine pulmonary artery endothelial cells were pretreated for 30 min with the NO donors 1 mM S-nitroso-N-acetylpenicillamine (SNAP), 0.5 mM sodium nitroprusside (SNP), or 0.2 microM spermine NONOate; controls included cells pretreated with either 1 mM N-acetyl-D-penicillamine or the NO synthase (NOS) inhibitor 1 mM N(G)-nitro-L-arginine methyl ester with and without addition of lipopolysaccharide (LPS; 0.1 microg/ml) for 8 h. COX-1 and COX-2 gene and protein expression were examined by RT-PCR and Western analysis, respectively; prostanoid measurements were made by gas chromatography-mass spectrometry, and COX activity was studied after a 30-min incubation with 30 microM arachidonic acid. LPS induced COX-2 gene and protein expression and caused an increase in COX activity and an eightfold increase in 6-keto-PGF(1alpha) release. LPS-stimulated COX-2 gene expression was decreased by approximately 50% by the NO donors. In contrast, LPS caused a significant reduction in COX-1 gene expression and treatment with NO donors had little effect. SNAP, SNP, and NONOate significantly suppressed LPS-stimulated COX activity and 6-keto-PGF(1alpha) release. Our data indicate that increased generation of NO attenuates LPS-stimulated COX-2 gene expression and activity, whereas inhibition of endogenous NOS has little effect.     

Nitric oxide: a major determinant of mast cell phenotype and function

Mast cells (MC) are important in the numerous physiological processes of homeostasis and disease. Most notably, MC are critical effectors in the development and exacerbation of allergic disorders. Nitric oxide (NO) is a diatomic radical produced by nitric oxide synthase (NOS), and has pluripotent cell signaling and cytotoxic properties. NO can influence many MC functions. Recent evidence shows the source of this NO can be from the mast cell itself. Governing the production of this endogenous NO, through alterations in the expression of tetrahydrobiopterin (BH4), a NOS cofactor, has stabilizing effects on MC degranulation. Furthermore, NO regulates the synthesis and secretion of de novo generated mediators, including leukotrienes and chemokines. These novel observations add to the growing body of knowledge surrounding the role of NO in the MC.     

Nitric oxide regulation of human peripheral blood mononuclear cells: critical time dependence and selectivity for cytokine versus chemokine expression

NO is antiproliferative for T cells and other immune cells, but there is debate over whether it influences cytokine expression and if so whether it shows cytokine selectivity. Furthermore, the NO effect may depend on exposure time. To address these issues, we precultured human PBMC with the NO donors S-nitrosoglutathione (a natural storage form of NO) or S-nitroso-N-acetyl-D-penicillamine for up to 48 h before cell activation and then monitored proliferation and cytokine and chemokine expression. S-nitrosoglutathione or S-nitroso-N-acetyl-D-penicillamine, but not their non-NO-releasing analogues, inhibited proliferation induced by PHA or IL-2, the effect declining progressively from 48 to 0 h pre-exposure to the mitogen. This was accompanied by reduced PHA-induced IL-2 release and reduced IL-2, IFN-gamma, and IL-13 mRNA expression. In contrast, NO did not influence PHA-induced expression of mRNA for the chemokines lymphotactin, RANTES, IFN-gamma-inducible protein, macrophage-inhibitory protein-1alpha, macrophage-inhibitory protein-1beta, macrophage chemoattractant protein-1, and IL-8 or release of RANTES or IL-8. The NO effects were not toxic and were not accompanied by changes in PHA-induced CD25 expression. We conclude that exposure time to NO is critical to altered PBMC responsiveness and that NO inhibits expression of both Th1 and Th2 cytokines but not chemokines.     

Nitric oxide inhibits gastric acid secretion by increasing intraparietal cell levels of cGMP in isolated human gastric glands

We have previously identified cells containing the enzyme nitric oxide (NO) synthase (NOS) in the human gastric mucosa. Moreover, we have demonstrated that endogenous and exogenous NO has been shown to decrease histamine-stimulated acid secretion in isolated human gastric glands. The present investigation aimed to further determine whether this action of NO was mediated by the activation of guanylyl cyclase (GC) and subsequent production of cGMP. Isolated gastric glands were obtained after enzymatic digestion of biopsies taken from the oxyntic mucosa of healthy volunteers. Acid secretion was assessed by measuring [(14)C]aminopyrine accumulation, and the concentration of cGMP was determined by radioimmunoassay. In addition, immunohistochemistry was used to examine the localization of cGMP in mucosal preparations after stimulation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). SNAP (0.1 mM) was shown to decrease acid secretion stimulated by histamine (50 microM); this effect was accompanied by an increase in cGMP production, which was histologically localized to parietal cells. The membrane-permeable cGMP analog dibuturyl-cGMP (db-cGMP; 0.1-1 mM) dose dependently inhibited acid secretion. Additionally, the effect of SNAP was prevented by preincubating the glands with the GC inhibitor 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (10 microM). We therefore suggest that NO in the human gastric mucosa is of physiological importance in regulating acid secretion. Furthermore, the results show that NO-induced inhibition of gastric acid secretion is a cGMP-dependent mechanism in the parietal cell involving the activation of GC.     

Regulation of A1 by OX40 Contributes to CD8+ T Cell Survival and Anti-Tumor Activity

The TNFR family member OX40 (CD134) is critical for optimal clonal expansion and survival of T cells. However, the intracellular targets of OX40 in CD8 T cells are not fully understood. Here we show that A1, a Bcl-2 family protein, is regulated by OX40 in effector CD8 T cells. In contrast to wild-type T cells, OX40-deficient CD8 T cells failed to maintain A1 expression driven by antigen. Conversely, enforced OX40 stimulation promoted A1 expression. In both situations, the expression of A1 directly correlated with CD8 T cell survival. In addition, exogenous expression of A1 in OX40-deficient CD8 T cells reversed their survival defect in vitro and in vivo. Moreover, forced expression of A1 in CD8 T cells from OX40-deficient mice restored the ability of these T cells to suppress tumor growth in a murine model. These results indicate that OX40 signals regulate CD8 T cell survival at least in part through maintaining expression of the anti-apoptotic molecule A1, and provide new insight into the mechanism by which OX40 may impact anti-tumor immunity.     

Regulation of the nitric oxide system in human adipose tissue

Nitric oxide (NO) is involved in adipose tissue biology by influencing adipogenesis, insulin-stimulated glucose uptake, and lipolysis. The enzymes responsible for NO formation in adipose cells are endothelial NO synthase (eNOS) and inducible NO synthase (iNOS), whereas neuronal NO synthase (bNOS) is not expressed in adipocytes. We characterized the expression pattern and the influence of adipogenesis, obesity, and weight loss on genes belonging to the NO system in human subcutaneous adipose cells by combining in vivo and in vitro studies. Expression of most of the genes known to belong to the NO system (eNOS, iNOS, subunits of the soluble guanylate cyclase, and both genes encoding cGMP-dependent protein kinases) in human adipose tissue and isolated human adipocytes was detected. In vitro adipogenic differentiation increased the expression level of iNOS significantly, whereas eNOS expression levels were not influenced. The genes encoding eNOS, iNOS, and cGMP-dependent protein kinase 1 were expressed at higher levels in obese women. Expression of these genes, however, was not influenced by 5% weight loss. Insulin and angiotensin II (Ang II) increased NO production by human preadipocytes in vitro. Increased eNOS and iNOS expression in adipocytes and local effects of insulin and Ang II may increase adipose tissue production of NO in obesity.