IFN-gamma-induced TNF-alpha is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFN-gamma

Dopamine is formed form L-tyrosine by tyrosine hydroxylase and aromatic L-amino acid decarboxylase. In addition to this pathway, however, the formation of catecholamines, including dopamine, from trace amines such as tyramine by hepatic microsomes has been demonstrated. In this study, we investigated the formation of dopamine from trace amines, using human hepatic microsomes and human cytochrome P450 (CYP) isoforms expressed in yeast. Among the 11 isoforms of human CYP expressed in yeast, CYP2D6 was the only isoform exhibiting strong ability to convert p-tyramine and m-tyramine to dopamine. In studies with human hepatic microsomes, the hydroxylation of tyramine to dopamine was inhibited by bufuralol, a typical substrate for CYP2D isoforms, and anti-CYP2D1 antiserum. This is the first report showing that CYP2D is capable of converting tyramine to dopamine. The Km values of CYP2D6, expressed in yeast, for p-tyramine and m-tyramine were 190.1 +/- 19.5 microM and 58.2 +/- 13.8 microM, respectively. Tyramine is an endogenous compound which exists in the brain as a trace amine but is also an exogenous compound which is found in foods such as cheese and wine. Our results suggest that dopamine is formed from endogenous and/or exogenous tyramine by this CYP2D isoform.     

Differential nitric oxide and TNF-alpha production of murine Kupffer cell subfractions upon priming with IFN-gamma and TNF-alpha

The effects of a nitric oxide (NO) donor on microcirculation and contractile function of reperfused skeletal muscle were studied. Rat cremaster muscles underwent 5 hours of ischemia and 90 minutes of reperfusion and were divided into two groups systemically infused with S-nitroso-N-acetylcysteine (SNAC, 100 nmol/min) and phosphate-buffered saline (PBS), respectively. The results showed that the vessels in the SNAC group had more rapid and complete recovery than that in controls. A significant difference was found from 10 to 40 minutes and at 90 minutes in 10-20-microm arterioles, from 10 to 90 minutes in 20-40-microm arterioles, and at 10 and 90 minutes in 40-70-microm arteries. When compared to controls, SNAC-treated muscles showed larger fluorescein filling areas at 15, 30, 60, and 90 minutes and greater isometric tetanic contractile forces in response to stimulation frequencies of 40, 70, 100, and 120 Hz. The data indicate that supplementation of exogenous NO could effectively improve microcirculation and contractile function of skeletal muscle during early reperfusion.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.     

Nitric oxide prevents IL-1beta and IFN-gamma-inducing factor (IL-18) release from macrophages by inhibiting caspase-1 (IL-1beta-converting enzyme)

Procytokine processing by caspase-1 is required for the maturation and release of IL-1beta and IFN-gamma-inducing factor (IGIF) (or IL-18) from activated macrophages (Mphi). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1beta-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1beta, but not TNF-alpha, in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine. Stimulated peritoneal Mphi from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1beta if exposed to N(G)-monomethyl-L-arginine, whereas Mphi from iNOS knockout mice (iNOS-/-) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1beta and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1beta and IFN-gamma than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1beta and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1beta and IGIF release.     

Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) stimulate the production of inducible nitric oxide synthase (iNOS) protein in RAW 264.7 macrophages

Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) are members of a family of superantigens produced by group A streptococci that appear to play a key role in the pathogenesis of streptococcal toxic shock syndrome. Since it is known that nitric oxide (NO) and tumor necrosis factor (TNF) are largely responsible for the shock and multiple organ dysfunction of Gram-negative sepsis, we hypothesized that SpeA and/or SpeC could trigger the production of inducible nitric oxide synthase (iNOS) and/or TNF by murine macrophages. We exposed RAW 264.7 macrophages to increasing concentrations of SpeA or SpeC alone and in combination with recombinant murine interferon-gamma (rIFN gamma) for 16-24 h. We found that both SpeA and SpeC triggered iNOS production in the presence of low concentrations of rIFN gamma, while neither provoked iNOS accumulation in the absence of rIFN gamma. Neither SpeA nor SpeC (with or without rIFN gamma) reproducibly induced TNF production by these murine macrophages. These data indicate that two streptococcal exotoxins up-regulate iNOS production by murine macrophages and suggest that nitric oxide production may play an important role in the pathogenesis of streptococcal toxic shock syndrome.     

Structural significance of the benzoyl group at the C-3'-N position of paclitaxel for nitric oxide and tumor necrosis factor production by murine macrophages

The antitumor agent paclitaxel (Taxol) mimics the actions of lipopolysaccharide (LPS) on murine macrophages (M phi). Recently, we have shown that the benzoyl group at the C-3' position of paclitaxel is the most important site to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by C3H/HeN M phi (Biochem. Biophys. Res. Commun. 210, 678-686, 1996). In the present study, synthetic analogs of paclitaxel with replacement of the C-3'-N position were examined for their potencies to induce NO and TNF production by peritoneal M phi of LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice, by human blood cells and human M phi. In this structure-activity relationship study, we found that (i) the p-substitution of the benzoyl group definitely affects the activity to activate C3H/HeN M phi, (ii) the analogs having a methyl or chloro group at the p-position exhibit stronger activity than that of paclitaxel, (iii) there is good correlation between NO and TNF production by the M phi in response to compounds, (iv) the compounds tested do not induce either NO or TNF production by C3H/HeJ M phi or TNF production by human cells, (v) a previous treatment of C3H/HeN M phi with the inactive compounds can hardly affect either paclitaxel- or LPS-induced TNF production by the M phi, (vi) paclitaxel and its analogs marginally affect LPS-induced TNF production by human blood cells, and (vii) there is no correlation between the NO/TNF inducibility to C3H/HeN M phi and growth inhibitory activity against M phi-like J774.1 and J7.DEF3 cells.     

IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha

IL-17 is a novel T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated whether hapten-specific T cells isolated from patients with allergic contact dermatitis (ACD) to nickel produce IL-17 and the effects of IL-17 alone or in combination with IFN-gamma or TNF-alpha on the immune activation of keratinocytes. Skin affected with ACD to nickel and skin-derived, nickel-specific CD4+ T cell lines expressed IFN-gamma, TNF-alpha, and IL-17 mRNAs. Four of seven nickel-specific CD4+ T cell clones positive for the skin-homing receptor, cutaneous lymphocyte-associated Ag, were shown to corelease IL-17, IFN-gamma, and TNF-alpha. In contrast, two nickel-specific CD8+ T cell clones failed to synthesize IL-17. Normal human keratinocytes were found to express constitutively the IL-17 receptor gene. IL-17 specifically and dose-dependently augmented IFN-gamma-induced ICAM-1 expression on keratinocytes at both the mRNA and the protein level, whereas HLA-DR, MHC class I, and CD40 levels were not modulated by IL-17. On the other hand, IL-17 alone did not affect ICAM-1 or enhance TNF-alpha-induced ICAM-1. In addition, IL-17, both directly and in synergism with IFN-gamma and/or TNF-alpha, stimulated synthesis and release of IL-8 by keratinocytes. In contrast, IFN-gamma- and TNF-alpha-induced production of RANTES was markedly inhibited by IL-17, and the synthesis of macrophage chemotactic protein 1 was not changed. Taken together, the results suggest that IL-17 is an important player of T cell-mediated skin immune responses, with synergistic or antagonist effects on IFN-gamma- and TNF-alpha-stimulated keratinocyte activation.     

Antisense oligonucleotides targeting protein kinase C-alpha, -beta I, or -delta but not -eta inhibit lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages: involvement of a nuclear factor kappa B-dependent mechanism

The signaling pathway for protein kinase C (PKC) activation and the role of PKC isoforms in LPS-induced nitric oxide (NO) release were studied in RAW 264.7 macrophages. The tyrosine kinase inhibitor genestein attenuated LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression, as did the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 and the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609. LPS stimulated phosphatidylinositol (PI) hydrolysis and PKC activity in RAW cells; both were inhibited by genestein. The PKC inhibitors (staurosporine, calphostin C, Ro 31-8220, or Go 6976) or long-term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment also resulted in inhibition of LPS-induced NO release and iNOS expression. Western blot analysis showed expression of PKC-alpha, -betaI, -delta, -eta, and -zeta in RAW cells; down-regulation of PKC-alpha, -betaI, and -delta, but not -eta, was seen after long-term TPA treatment, indicating the possible involvement of one or all of PKC-alpha, -betaI, and -delta, but not -eta, in LPS-mediated effects. Treatment with antisense oligonucleotides for these isoforms further demonstrated the involvement of PKC-alpha, -betaI, and delta, but not -eta, in LPS responses. Stimulation of cells with LPS for 1 h caused activation of NF-kappaB in the nuclei by detection of NF-kappaB-specific DNA-protein binding; this was inhibited by genestein, U73122, D609, calphostin C, or antisense oligonucleotides for PKC-alpha, -betaI, and -delta, but not -eta. These data suggest that LPS activates PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, resulting in the stimulation of NF-kappaB DNA-protein binding, then initiated the expression of iNOS and NO release. PKC isoforms alpha, betaI, and delta were shown to be involved in the regulation of these LPS-induced events.     

Defect in the induction of nitric oxide synthase by lipopolysaccharide (LPS) in an LPS-resistant mutant of a murine macrophage-like cell line, J774.1

OBJECTIVE: To investigate the issue of systematic bias in self-reported weight and height, and produce a simple procedure which can be used to correct reporting bias. DESIGN: Cross-sectional, with self-reported questionnaires. SUBJECTS: A sub-sample (n = 143) of secondary school students in Siena, Italy, taken from the Food Behaviour Survey (sample size, n = 779). RESULTS: In the teenage sub-sample, both males and females under-reported their weight and over-reported their height, such that underestimation of the overweight prevalence was in the order of about 8% for both genders. For both weight and height, the correlations between self-reported and measured values were over 0.90. Conversion factors were derived to correct the reported body mass index (BMI) distribution by adjusting the percentages of erroneously classified subjects in the four BMI categories. CONCLUSION: High correlation coefficients (r > or = 0.75), showing a systematic tendency for erroneous self-reporting of a 'slim-body shape', justify the use of conversion factors (measured/self-reported) to correct BMI distributions calculated from self-reported values.     

Immunomodulation by TYB-2285 of Dermatophagoides farinae (Df) antigen-induced IFN-gamma and IL-4 production in lymphocytes from children with bronchial asthma

Effect of TYB-2285 and its metabolites on immune responses by peripheral blood mononuclear cells (PBMC) from children with bronchial asthma was investigated. TYB-2285 and its metabolites have immunosuppressive activity for the proliferation by Df-stimulated patients' lymphocytes. Concanavalin A (Con A)-activated peripheral blood mononuclear cells (PBMC) from the patients were not affected by the same treatment. The results indicate that TYB-2285 and its metabolites are capable of suppressing antigen-induced 3H-thymidine (3H-TdR) uptake but not the response induced by Con A. Interferon-gamma (IFN-gamma) production by Df-stimulated PBMC from patients with active asthma, which was lower than that of normal lymphocytes, were reversed beyond the levels of that in normal subjects. Thus reduced production of IFN-gamma by Df-stimulated patients' lymphocytes was increased by TYB-2285 and its metabolites in a dose-dependent manner. This phenomenon was not observed in lymphocytes from normal subjects. Furthermore, TYB-2285 inhibited IL-4 production induced by Df antigen in asthmatic patients' lymphocytes. Taken, together, TYB-2285 could work as a weak immunosuppressant to modify lymphocytes' responses to allergen in patients with bronchial asthma. These data underscore the potential benefit for the patients with bronchial asthma.     

Engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) induces transforming growth factor beta (TGF-beta) production by murine CD4(+) T cells

Evidence indicates that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor beta (TGF-beta) production by murine CD4(+) T cells. CD4(+) T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-beta after antibody cross-linking of CTLA-4, indicating that induction of TGF-beta by CTLA-4 signaling represents a ubiquitous feature of murine CD4(+) T cells. Stimulation of the CD3-T cell antigen receptor complex does not independently induce TGF-beta, but is required for optimal CTLA-4-mediated TGF-beta production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon gamma (Th1) and IL-4 (Th2). Moreover, addition of anti-TGF-beta partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-beta1 gene-deleted (TGF-beta1(-/-)) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4(+) T cell production of TGF-beta, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-beta, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4(+) T cell activation.     

Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype

Activated murine macrophages metabolize L-arginine via two main pathways that are catalyzed by the inducible enzymes nitric oxide synthase (iNOS) and arginase. We have previously shown that CD4+ T cell-derived cytokines regulate a competitive balance in the expression of both enzymes in macrophages; Thl-type cytokines induce iNOS while they inhibit arginase, whereas the reverse is the case for Th2-type cytokines. Here we addressed the regulation of both metabolic pathways by CD4+ T cells directly. Macrophages were used as APCs for established Th1 and Th2 T cell clones as well as for in vitro polarized Th1 or Th2 T cells of transgenic mice bearing an MHC class II-restricted TCR. Both systems revealed a similar dichotomy in the macrophages; Th1 T cells led to an exclusive induction of iNOS, whereas Th2 T cells up-regulated arginase without inducing iNOS. Arginase levels induced by Th2 T cells far exceeded those inducible by individual Th2 cytokines. Similarly, high arginase levels could be induced by supernatants of Th2 cells stimulated in various ways. Ab blocking experiments revealed the critical importance of IL-4 and IL-10 for arginase up-regulation. Finally, strong synergistic effects between IL-4/IL-13 and IL-10 were observed, sufficient to account for the extraordinarily high arginase activity induced by Th2 cells. Our results suggest that the iNOS/arginase balance in macrophages is competitively regulated in the context of Th1- vs Th2-driven immune reactions, most likely by cytokines without the requirement for direct cell interaction.     

Molecular cloning of cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its induction with nitric oxide synthase in a murine macrophage-like cell line

Arginase exists in two isoforms. Liver-type arginase (arginase I) is expressed almost exclusively in the liver and catalyzes the last step of urea synthesis, whereas the nonhepatic type (arginase II) is expressed in extrahepatic tissues. Arginase II has been proposed to play a role in down-regulation of nitric oxide synthesis. A cDNA for human arginase II was isolated. A polypeptide of 354 amino acid residues including the putative NH2-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. The arginase II precursor synthesized in vitro was imported into isolated mitochondria and proteolytically processed. mRNA for human arginase II was present in the kidney and other tissues, but was not detected in the liver. Arginase II mRNA was coinduced with nitric oxide synthase mRNA in murine macrophage-like RAW 264.7 cells by lipopolysaccharide. This induction was enhanced by dexamethasone and dibutyryl cAMP, and was prevented by interferon-gamma. Possible roles of arginase II in NO synthesis are discussed.     

Effective use of ribonucleotide reductase inhibitors (Didox and Trimidox) alone or in combination with didanosine (ddI) to suppress disease progression and increase survival in murine acquired immunodeficiency syndrome (MAIDS)

Ribonucleotide reductase inhibitors (RRIs) have been recently shown to inhibit retroviral replication. We examined a new series of RRIs, 3,4-dihydroxybenzohydroxamic acid (Didox) and 3,4,5-trihydroxybenzohydroxamidoxime (Trimidox) for their ability to alter disease progression in murine acquired immunodeficiency syndrome (MAIDS), both alone and in combination with 2',3'-dideoxyinosine (ddI). MAIDS disease was induced by inoculation of female C57BL/6 mice with the LP-BM5 murine leukemia virus (MuLV) and disease progression characterized by extensive peripheral lymphadenopathy and splenomegaly. Efficacy of treatment with these drugs was based upon their ability to influence survival and disease pathophysiology by monitoring the development of splenomegaly. Toxicity was determined by changes in body weight, total peripheral white blood cell count and hematocrit. Didox or trimidox monotherapy was associated with increased survival and decreased disease pathophysiology, with no apparent toxicity. Combined with ddI, their ability to reduce development of viral induced splenomegaly was enhanced compared to trimidox, didox or ddI alone. These results demonstrate RRIs have potent activity in reversing the disease manifestations characteristic of MAIDS. Further studies are warranted to determine human clinical efficacy.