Inhibition of interleukin-12 p40 transcription and NF-kappaB activation by nitric oxide in murine macrophages and dendritic cells

Nitric oxide (NO), an important effector molecule of the innate immune system, can also regulate adaptive immunity. In this study, the molecular effects of NO on the toll-like receptor signaling pathway were determined using interleukin-12 (IL-12) as an immunologically relevant target gene. The principal conclusion of these experiments is that NO inhibits IL-1 receptor-associated kinase (IRAK) activity and attenuates the molecular interaction between tumor necrosis factor receptor-associated factor-6 and IRAK. As a consequence, the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibits lipopolysaccharide (LPS)-induced IL-12 p40 mRNA expression, protein production, and promoter activity in murine macrophages, dendritic cells, and the murine macrophage cell line RAW 264.7. Splenocytes from inducible nitric-oxide synthase-deficient mice demonstrate markedly increased IL-12 p40 protein and mRNA expression compared with wild type splenocytes. The inhibitory action of NO on IL-12 p40 is independent of the cytokine IL-10. The effects of NO can be directly attributed to inhibition of NF-kappaB activation through IRAK-dependent pathways. Accordingly, SNAP strongly reduces LPS-induced NF-kappaB DNA binding to the p40 promoter and inhibits LPS-induced IkappaB phosphorylation. Similarly, NO attenuates IL-1beta-induced NF-kappaB activation. These experiments provide another example of how an innate immune molecule may have a profound effect on adaptive immunity.     

Following direct CD40 activation, human primary microglial cells produce IL-12 p40 but not bioactive IL-12 p70

There is accumulating evidence that interleukin 12 (IL-12) is involved in the pathogenesis of multiple sclerosis. In the periphery, this cytokine is produced by antigen-presenting cells (APCs) following interaction with activated T cells. CD40 ligation plays a crucial role in this production. Microglial cells are thought to play a major role in antigen presentation in the central nervous system. In this work, we examined IL-12 production by human primary microglial cells after CD40 ligation. These cells expressed CD40 and MHC class II following interferon-gamma activation. IL-12 p40 mRNA and protein, but not bioactive IL-12 p70, were detected in response to direct CD40 activation. Microglial cells co-cultured with activated allogenic CD4+ T lymphocytes also produced IL-12 p40 but not IL-12 p70. This IL-12 p40 production was inhibited by anti-CD40 ligand. Altogether, these results suggest that CD40-CD40-ligand interaction provides a signal that triggers IL-12 p40 expression. However, other interaction(s) may be required during antigen presentation for bioactive heterodimeric IL-12 p70 to be produced by microglial cells.     

Induction of tumor necrosis factor-alpha (TNF-alpha) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

The present study was undertaken to explore the role of interleukin-12 (IL-12) p40 in the expression of TNF-alpha in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-alpha and the expression of TNF-alpha mRNA in BV-2 microglial cells. In addition to BV-2 microglial cells, p70, p402 and p40 also induced the production of TNF-alpha in mouse primary microglia and peritoneal macrophages. As the activation of both NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) is important for the expression of TNF-alpha in microglial cells, we investigated the effect of p40 on the activation of NF-kappaB as well as C/EBPbeta. Activation of NF-kappaB as well as C/EBPbeta by p40 and inhibition of p40-induced expression of TNF-alpha by Deltap65, a dominant-negative mutant of p65, and DeltaC/EBPbeta, a dominant-negative mutant of C/EBPbeta, suggests that p40 induces the expression of TNF-alpha through the activation of NF-kappaB and C/EBPbeta. In addition, we show that p40 induced the activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Interestingly, PD98059, an inhibitor of ERK, inhibited p40-induced expression of TNF-alpha through the inhibition of C/EBPbeta, but not that of NF-kappaB, whereas SB203580, an inhibitor of p38 MAPK, inhibited p40-induced expression of TNF-alpha through the inhibition of both NF-kappaB and C/EBPbeta. This study delineates a novel biological function of p40 in inducing TNF-alpha in microglia and macrophages.     

Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.     

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.     

The role of tetrahydrobiopterin in the activation of oxygen by nitric-oxide synthase

We have studied the reaction of reduced nitric-oxide synthase (NOS) with molecular oxygen at -30 degrees C. In the first reaction cycle (from L-Arg to hydroxy-L-Arg), an oxygen adduct complex formed rapidly. Experiments in the absence of the reductase domain demonstrated that this complex was then further reduced by one electron stemming from the cofactor tetrahydrobiopterin (BH4). Spectral evidence suggested an iron(IV) porphyrin pi-cation radical as an intermediate. The nature of the oxidized BH4 was identified by EPR as a BH3* radical. Within the second cycle (from hydroxy-L-Arg to citrulline and NO), an iron(III)-NO complex could be identified clearly by its spectral characteristics. The strict requirement of BH4 for its formation suggests that BH4 plays a redox role, although transient, also in the second reaction cycle.     

Lysophosphatidic acid and receptor-mediated activation of endothelial nitric-oxide synthase

Both lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are platelet-derived phospholipids that elicit diverse biological responses. In endothelial cells, S1P stimulates the EDG-1 receptor-mediated activation of the endothelial isoform of nitric oxide synthase (eNOS), but the role of LPA in eNOS regulation is less well understood. We now report that LPA treatment of bovine aortic endothelial cells (BAEC) activates eNOS enzyme activity in a pathway that involves phosphorylation of eNOS on serine 1179 by protein kinase Akt. In contrast to the cellular responses elicited by S1P in COS-7 cells, LPA can stimulate the activation of eNOS and Akt independently of EDG-1 receptor transfection. LPA-stimulated enzyme activation was significantly attenuated in an eNOS mutant lacking the site that is phosphorylated by kinase Akt (eNOS S1179A). In BAEC, activation of eNOS by LPA is completely blocked by pertussis toxin, by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy) ethane-N,N,N',N'-tetraacetic acid), and by the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, but is unaffected by U0126, an inhibitor of mitogen-activated protein (MAP) kinase pathways. Analysis of the LPA dose response for eNOS activation reveals an EC(50) of approximately 40 nM, a concentration well below the potency of LPA at the EDG-1 receptor. Taken together, these results indicate that LPA potently activates eNOS in BAEC in a pathway distinct from the EDG-1 receptor, but mediated by a similar receptor-mediated pathway dependent on pertussis toxin-sensitive G proteins and involving activation of the PI3-K/Akt pathway. These studies have identified a role for the phospholipid LPA in eNOS activation, and point out the complementary role of distinct platelet-derived lipids in endothelial signaling pathways.     

Induction of endothelial nitric-oxide synthase in rat brain astrocytes by systemic lipopolysaccharide treatment

In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection.     

Salicylanilides: selective inhibitors of interleukin-12p40 production

Interleukin (IL)-12p40, a subunit component of both IL-12 and IL-23, is being widely studied for its role in inflammatory disease. As part of an effort to profile cellular signaling pathways across different cell types, we report salicylanilide inhibitors of IL-12p40 production in stimulated dendritic cells. Based on a hypothesis that a desirable therapeutic profile is one that could block IL-12p40 but not IL-6 production, we engaged in directed analoging. This resulted in salicylanilides with similar IL-12p40 related potency but enhanced selectivity relative to IL-6 production.