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.     

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.     

Horseradish peroxidase and glycosylated BSA induce nitric oxide production in murine macrophages

The in vitro activation of murine macrophages by horseradish peroxidase (HRP) induced nitric oxide production in a dose-dependent manner, and increased the induction of NO-synthase by LPS. Nitrite production after HRP stimulation was inhibited by NG-monomethyl-L-arginine (NMMA), a specific inhibitor of NO-synthase. Equivalent amounts of nitrite were obtained with native and heat-inactivated HRP. High concentrations of mannose inhibited nitric oxide production, while the HRP inhibitor 3-aminotyrosine did not. Glycosylated serum albumin derivatives also induced murine macrophage NOS, probably by an interaction between carbohydrates and their specific cell membrane receptors. The inability of HRP apoprotein to stimulate NO production, and the specific inhibition of HRP-mediated activation of macrophages by hemin suggests that the heme moiety of this enzyme is involved in NO-synthase induction.     

LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.     

Effect of prolactin on nitric oxide and interleukin-1 production of murine peritoneal macrophages: role of Ca2+ and protein kinase C

The Psychiatric Hospital at the Municipal (General) Hospital in G?rlitz, Germany, was the only Department of Psychiatry in a non-University (general) hospital in the newly integrated German provinces who were originally part of the so-called "German Democratic Republic" before the re-unification of Germany, to participate in the German collective study on "Psychiatric and Psychosomatic Consultation and Liaison Service in German General Hospitals-A Multicentre Empirical Study to Assess and Evaluate Existing Structures and Services". This study is an independent part project that includes specific questions forming part of the European collective study on "Effectiveness of Mental Health Consultation and Liaison Service Delivery in the General Hospital".     

Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages

The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone. On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.     

An improved experimental model for the study of in vitro release of nitric oxide by murine peritoneal macrophages

This exposition analyses and contextualizes the complex problem of structural inequality in South African health care. Socio-economic conditions, racial divisions and geographical location are isolated as the main determinants of inequality in the provision, allocation and distribution of health care; the prevailing inequalities are attributed to a wide range of underlying causes, including the absence of a central, binding health policy, the prominent role of apartheid and white domination, the free market and the medical profession, as well as the unique sociocultural set-up of the country. The urgent need for deliberate strategies to equalize the prevailing disparities and discrepancies is posed.     

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.     

Stimulation of nitric oxide production in macrophages by Babesia bovis

Gamma interferon (IFN-gamma)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-gamma, B. bovis merozoites stimulated NO production, as indicated by the presence of increased L-arginine-dependent nitrite (NO2-) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-gamma per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-gamma or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-gamma, were also found to induce L-arginine-dependent NO2- production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.     

Nitric oxide production in mouse peritoneal macrophages enhanced with glycyrrhizin

The enhancement of nitric oxide (NO) production in glycyrrhizin (GL)-induced macrophages (M phi) in response to lipopolysaccharide (LPS) was investigated. No production in GL-induced macrophage culture supernatants was stimulated in response to LPS (10 micrograms/ml) for 24- or 48- h cultures, and these levels were compared three times with the levels in saline-induced peritoneal exudate cell cultures. Furthermore, M phi induced with proteose peptone (PP) containing GL could generate greater NO production than M phi induced with PP alone. However, no stimulation of NO production was observed by addition of GL in the cultures of M phi induced with thioglycollate or Bacillus Calmette Guerin. Moreover, GL-induced M phi showed cytostasis against such tumor target cells as L 1210 and P 388 lymphoma cell lines. These observations indicate that GL can activate the M phi in vivo system and stimulate NO production in response to LPS.     

The triterpenoid quinonemethide pristimerin inhibits induction of inducible nitric oxide synthase in murine macrophages

Inducible nitric oxide synthase dependent production of nitric oxide (NO) plays an important role in inflammation. We investigated whether pristimerin ((20alpha)-3-hydroxy-2-oxo-24-nor-friedela-1(10),3,5,7-te traen-carboxylic acid-(29)-methylester), an antitumoral, antimicrobial as well as anti-inflammatory plant compound, has an effect on the inducible NO synthase system in lipopolysaccharide-activated RAW 264.7 macrophages. Pristimerin dose dependently (IC50: 0.2-0.3 microM) reduces nitrite accumulation, a parameter for NO synthesis, in supernatants of lipopolysaccharide-stimulated (1 microg/ml, 20 h) macrophages. This effect correlates with a reduced inducible NO synthase enzyme activity measured by conversion of [3H]L-arginine to [3H]L-citrulline and significantly lower levels of enzyme protein (Western blotting) in homogenates of cells cotreated with lipopolysaccharide and pristimerin (12 h). Northern blot analysis and polymerase chain reaction (PCR) showed decreased inducible NO synthase mRNA levels in activated macrophages exposed to pristimerin (4 h). Electrophoretic mobility shift assay (EMSA) demonstrated a markedly reduced binding activity of nuclear factor-kappa B (NFkappaB) in nuclear extracts of pristimerin-treated cells. These results suggest that pristimerin inhibits the induction of inducible NO synthase by a mechanism which involves inhibition of NFkappaB activation. This feature of pristimerin is likely to contribute to its anti-inflammatory activity.     

An increase in intracellular cyclic AMP modulates nitric oxide production in IFN-gamma-treated macrophages

Macrophages treated with IFN-gamma alone are stimulated to produce nitric oxide. The level of nitric oxide production can be enhanced significantly when IFN-gamma treatment is combined with other agents (e.g., LPS, TNF-alpha, IL-2, etc.). We tested the hypothesis that cAMP plays a role in the IFN-gamma-induced activation of macrophages. Our experiments indicate that factors that increase the concentration of cAMP in the murine macrophage cell line ANA-1 can also enhance IFN-gamma-induced production of nitric oxide. PGE2 and cholera toxin increased the production of nitrite (an indicator of nitric oxide production) in IFN-gamma-treated ANA-1 macrophages by at least twofold. These factors produced no increase in nitric oxide production in the absence of IFN-gamma treatment. The increase in nitric oxide production corresponded to an increase in the accumulation of nitric oxide synthase mRNA without a change in stability of mRNA. Dibutyryl cAMP and Sp-cAMPs (a selective activator of cAMP-dependent protein kinase I and II) also increased nitric oxide production in IFN-gamma-treated macrophages. However, at very high concentrations (i.e., >100 microM), the stimulatory effect was decreased. These studies indicate that elevation of intracellular cAMP causes a dose-dependent, biphasic alteration of IFN-gamma-induced nitric oxide production in murine macrophages. Moreover, they suggest that agents that affect nitric oxide synthesis may do so via modulation of the cAMP second messenger system.     

Metal-induced modulation of nitric oxide production in vitro by murine macrophages: lead, nickel, and cobalt utilize different mechanisms

Macrophages (M phi) can be induced to produce nitric oxide (NO), which has been suggested to be important for macrophages to exercise various functions. We have previously reported that an environmental toxicant, lead (Pb), can significantly inhibit NO production by murine splenic M phis. Herein, eight additional metal ions, gold (Au), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), and zinc (Zn), were assessed. In addition to Pb, Hg and Cd significantly suppressed NO production by cytokine (interferon-gamma and tumor necrosis factor-alpha)-stimulated murine M phis. Au and Cu also were inhibitory, but less than Pb, Hg, and Cd. In contrast, Cr and Zn were not modulatory, and Ni and Co significantly enhanced NO production by cytokine-stimulated M phis. The enhancement by Ni and Co was inhibited by the arginine analog N-monomethylarginine. The metals showed different activating/inhibiting profiles when added to a cell-free (activated M phi lysate) NO-producing-system in which inducible NO synthase (iNOS) is already expressed. Cr, Cu, Pb, and Zn moderately suppressed iNOS, which suggests that they may directly modify enzyme or cofactor activity. Cd, Hg, Mg, Ni, or Co did not produce any significant effect on NO production by the cell-free system. Inhibition of NO production by Pb-exposed M phis was not due to decreased expression of iNOS nor limited to its modest direct inhibition of iNOS; thus, other mechanism(s) must be accountable for the efficient Pb-induced inhibition of NO production by M phi. Ni or Co did induce a substantial increase of iNOS protein. Overall, these observations provide additional insight into the means by which metals via inhibition or enhancement of NO production may be pathogenic, by suppression of defense mechanisms or induction of hypersensitivity, respectively.     

Scavenger receptors may regulate nitric oxide production from macrophages stimulated by LPS

A new crystal form of papain from the latex of Carica papaya, complexed with an inhibitor (Z-Arg-Leu-Val-Gly-CHN2) was obtained by the vapor-diffusion method using a methanol/ethanol mixture as a precipitant. The slat-like crystals are monoclinic, space group P2(1), with unit cell parameters a = 42.6 A, b = 49.8 A, c = 50.5 A, A = 111.9 degrees, and contain one molecule in the asymmetric unit. The crystals are stable in the X-ray beam and diffract beyond 1.8 A. A molecular model has been placed in the unit cell by molecular replacement.