Membrane perturbation of macrophages stimulated by bacterial lipopolysaccharide.

Macrophages recovered from regressing Moloney sarcomas lose their capacity to kill tumor cells when held 12–24 h in culture. Exposure of this population to low concentrations (ng/ml range) of bacterial lipopolysaccharide will cause a rapid resurgence of cytolytic activity, however. By using electron spin resonance techniques and either 5- or 12-doxyl stearate spin labels, we have shown that exposure of cultured tumor macrophages to lipopolysaccharide (100 ng/ml) is followed by plasma membrane perturbation. The change was most pronounced 3–4 h after stimulation, and had subsided within 5–6 h. The relationship of the described perturbation to the induction of killing cannot be determined from these studies; however, neither cytolytic activity nor the membrane change developed in other kinds of macrophages exposed to similar concentrations of lipopolysaccharide.     

Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

Syntheses of NAMDA derivatives inhibiting NO production in BV-2 cells stimulated with lipopolysaccharide

Sixteen derivatives of N-acetyl-3-O-methyldopamine (NAMDA), an inhibitor of BH4 synthesis, were designed and synthesized. The ability of these derivatives to inhibit NO and BH4 production by lipopolysaccharide-stimulated BV-2 microglial cells was determined. While NAMDA at 100 microM inhibited NO and BH4 production by only about 20%, its catecholamide 8, indole 23 derivative, 13, and N-acetyl tetrahydroisoquinoline 25 inhibited the NO production by >50% at the same concentration. In particular, 13 and 25 inhibited both NO and BH4 production to similar degrees, which suggested that these compounds might inhibit NO production by blocking BH4-dependent dimerization of the newly synthesized iNOS monomer.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Physiological and hypoxic O2 tensions rapidly regulate NO production by stimulated macrophages

Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O₂ availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O₂-mediated regulation of NO production, we used a novel forced convection cell culture system to rapidly (response time of 1.6 s) and accurately (±1 Torr) deliver specific O₂ tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFN-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O₂ availability rapidly (30 s) and reversibly decreased NO production with an apparent K_mO₂ of 22 (SD 6) Torr (31 µM) and a V_max of 4.9 (SD 0.4) nmol·min^–1·10^–6 cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O₂-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected (P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr (P = 0.008) and 157 Torr (P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O₂ and NO tensions and negligible at low O₂ and NO tensions. These results are consistent with O₂ substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O₂ tensions suggest that O₂ tension has the potential to regulate NO production in vivo. inducible nitric oxide synthase; substrate limitation; nitric oxide consumption     

Enhancement of in vivo adenovirus-mediated gene transfer and expression by prior depletion of tissue macrophages in the target organ.

Based on the hypothesis that tissue macrophages present an obstacle for adenovirus (Ad) vector-mediated gene transfer to internal organs, this study evaluated the consequences of transient depletion of Kupffer cells on subsequent transfer of the Ad vector genome and Ad vector-directed gene expression in the livers of experimental animals. Depletion of Kupffer cells in mice by intravenous administration of multilamellar liposomes containing dichloromethylene-bisphosphonate permitted subsequent intravenous administration of an Ad vector to provide a higher input of recombinant adenoviral DNA to the liver, an absolute increase in transgene expression, and a delayed clearance of the vector DNA and transgene expression. These observations suggest that the tissue macrophages pose a significant hurdle to Ad vector-mediated gene transfer and that strategies to transiently suppress macrophage defenses might be useful in enhancing the efficiency of this in vivo gene transfer system.     

SHP-1 inhibits LPS-mediated TNF and iNOS production in murine macrophages

Several lines of evidence have suggested that protein tyrosine phosphatases, including CD45 and SHP-1, regulate macrophage activation. Macrophages from mice lacking SHP-1 (motheaten mice) are hyper-responsive to many stimuli, suggesting that SHP-1 may negatively regulate macrophage activation. Herein we report that the repressible/inducible over-expression of wild-type SHP-1 in a subclone of RAW 264.7 macrophages (RAW-TT10 cells) inhibited both TNF secretion and iNOS protein accumulation in response to stimulation with lipopolysaccharide (LPS) and recombinant murine interferon-gamma and led to diminished LPS-mediated tyrosine phosphorylation of vav1. In contrast, expression of a truncated SHP-1 construct previously shown to interfere with endogenous SHP-1 function modestly augmented LPS-mediated TNF and iNOS production and did not inhibit vav1 tyrosine phosphorylation. Taken together, these data provide the first direct evidence that SHP-1 inhibits macrophage activation by LPS and suggest that this effect may be mediated in part by dephosphorylation of vav1.     

Increased HO-1 expression and decreased iNOS expression in the hippocampus from adult spontaneously hypertensive rats

Brain expression of heme oxygenase (HO) and nitric oxide synthase (NOS) in hypertension may participate in the pathogenesis of hypertension-related neuronal disorders, such as vascular dementia. In the present study, expression levels of HO and NOS in spontaneously hypertensive rats (SHR) were investigated using Western immunoblotting assay. Expression level of inducible HO-1 in hippocampus of 4-wk prehypertensive SHR was about twofold of that in age-matched Sprague-Dawley (SD) rats (p<0.01). In 23-wk SHR with fully developed hypertension, hippocampal HO-1 level was significantly greater than that of age-matched SD rats (p<0.05), but not different from 4-wk SHR. There was no difference in expression levels of hippocampal HO-2 between SHR and SD rats at different ages. Total enzymatic activity of hippocampal HO was significantly greater in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Although hippocampal expression of nNOS protein was relatively unchanged, iNOS expression in 23-wk SHR was about fourfold lower than that in age-matched SD rats and 4-wk SD/SHR (p<0.01). Total enzymatic activity of hippocampal NOS was significantly lower in 23-wk SHR than in age-matched SD rats or 4-wk SD/SHR (p<0.01). Significantly suppressed Morris water maze performance was found in 23-wk SHR in comparison with age-matched SD rats. Because SHR has been used as a model of vascular dementia and hippocampus is essential for spatial learning and memory, understanding of altered HO/CO and NOS/NO systems in the hippocampus of adult SHR may shed light on the pathogenic development of memory deficits associated with vascular dementia.     

Immune interferon enhances the production of interleukin 1 by human endothelial cells stimulated with lipopolysaccharide

Interferon-gamma (IFN-gamma) is a macrophage-activating factor that has also been shown to act on endothelial cells (EC). Interleukin 1 (IL 1), first described as a monocyte product, is also produced by EC after stimulation by lipopolysaccharide (LPS). In this study, the effect of IFN-gamma on the release of IL 1 by EC stimulated with LPS has been investigated. Although IFN-gamma did not stimulate the release of IL 1 or increase the apparent intracellular pool of IL 1 when incubated with EC, there was an increase in the amount of IL 1 released when cells preincubated with IFN-gamma were stimulated with LPS. The effect of IFN-gamma increased with concentration (1 to 1000 U/ml) and with duration of preincubation (24 to 96 hr). The presence of IFN-gamma was not required during the stimulation with LPS. When EC were cultured without IFN-gamma for increasing time periods up to 96 hr, the amount of IL 1 released by EC on subsequent stimulation with LPS progressively decreased. Addition of as little as 1 U/ml of IFN-gamma, however, prevented the loss in capacity of EC to secrete IL 1 when stimulated with LPS. In vivo, EC are involved in the emigration of mononuclear cells from the blood to inflammatory sites. Because IL 1 is chemotactic for lymphocytes and also increases the binding of lymphocytes to EC, activation of EC by T cell-derived factors such as IFN-gamma may augment lymphocyte emigration by increasing the release of IL 1 at the blood-tissue interface.     

Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines.

Catecholamines are elaborated in stress responses to mediate vasoconstriction, and elevate systemic vascular resistance and blood pressure. They are elaborated in disorders such as sepsis, cocaine abuse, and cardiovascular disease. The aim of the study was to determine whether catecholamines affect nitric oxide (NO) production, as NO is a vasodilator and counteracts the harmful effects of catecholamines. RAW264.7 macrophage cells were cultured with lipopolysaccharide (LPS)+/-epinephrine, norepinephrine, and dopamine at 5x10(-6)M concentrations for 24h. Supernatants were harvested for measuring NO by spectrophotometry using the Greiss reagent and cells were harvested for detecting inducible NO synthase (iNOS) by Western blot. NO production in RAW 264.7 macrophages was increased significantly by addition of LPS (0.5-10ng/ml) in a dose-dependent fashion. The NO production induced by LPS was further enhanced by epinephrine and norepinephrine, and to a lesser extent by dopamine. These increases in NO correlated with expression of iNOS protein in these cells. The enhancing effect of iNOS synthesis by epinephrine and norepinephrine on LPS-induced macrophages was down regulated by beta-adrenoceptor antagonist, propranolol, and dexamethasone. The results suggest that catecholamines have a synergic effect on LPS in induction of iNOS synthesis and NO production, and this may mediate some of the vascular effects of infection. These data support a novel role for catecholamines in disorders such as septic shock and cocaine use, and indicate that beta-adrenoceptor antagonists and glucocorticoids may be used therapeutically for modulation of the catecholamine-NO axis in disease states.     

High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production

In this study we investigated the role of nitric oxide (NO) in monocyte fungicidal activity against Paracoccidioides brasiliensis. We found that cells primed with IFN-γ, TNF-α or GM-CSF and challenged with a high-(Pb18) or low-virulence (Pb265) strain of the fungus increase their fungicidal activity. Expression of iNOS mRNA was increased after priming cells with each cytokine, and tended to be inhibited by Pb18. Despite up-regulation of iNOS mRNA expression by Pb265, an equivalent increase in NO production was not detected, as metabolite levels were similar in all cultures. The results indicated that high expression of human monocyte iNOS mRNA induced by P. brasiliensis is not correlated with NO concentrations produced.     

Adenostemmoic acid B suppresses NO production by downregulating the expression and inhibiting the enzymatic activity of iNOS

Adenostemmoic acid B (AB) is a major compound found in Adenostemma lavenia; it shows anti-melanogenic, anti-inflammatory, and cytotoxic activities. By modifying the 19th position (carboxy: involved in the avoidance of cytotoxicity) of AB, we succeed to separate these activities. Short-chain alkylation of the carboxy group enhanced anti-melanogenic activity, while long-chain alkylation (hydrophobic) resulted in the suppression of nitric oxide (NO) production and inducible NO synthase (iNOS) expression without anti-melanogenic activity. Re-modification of hydrophilic properties in these long-chain derivatives restored anti-melanogenic activity but did not suppress NO production. Unexpectedly, AB and derivatives with long chains linked by an anhydride bond were new iNOS inhibitors. These results suggest that AB modulates multiple physiological activities by regulating different targets, including iNOS.     

Cardioprotection of H2S by downregulating iNOS and upregulating HO-1 expression in mice with CVB3-induced myocarditis

Aims

To explore the effects and potential mechanisms of hydrogen sulfide (H₂S) in CVB3-induced mice with myocarditis.

Main methods

A total of 75 six-week-old inbred male Balb/c mice were divided randomly into four groups (N, C, P and S). Group N was the negative control. The others were inoculated intraperitoneally (i.p.) with CVB3. Subsequently, groups P and S were injected i.p. once a day with DL-Proparglygylcine (PAG) and NaHS respectively. Group C was the positive control. Inducible nitric oxide synthase (iNOS) and heme oxygenase-1(HO-1) expression on cardiac tissues were evaluated by histopathological examination, immunohistochemistry, RT-PCR and Western blot.

Key findings

The heart-weight to body-weight (HW/BW) ratio, the histologic scores and the iNOS mRNA and protein expression levels were higher, and the HO-1 mRNA and protein expression levels were lower in mice treated with PAG than those mice solely inoculated with CVB3. Mice in group S had a significant decreased in the HW/BW ratio, the histologic scores and the iNOS mRNA and protein expression levels, and had a significant increased in the HO-1 mRNA and protein expression levels compared to the mice in group C. H₂S can attenuate inflammatory cell infiltration, alleviate cardiac edema, and limit myocardial lesions.

Significance

Our data support that H₂S can inhibit iNOS overexpression and induce HO-1 expression, both of which contribute to the cardioprotection of H₂S in CVB3-induced mice myocarditis.     

Nimesulide inhibits lipopolysaccharide-induced production of superoxide anions and nitric oxide and iNOS expression in alveolar macrophages

The study was designed to investigate the effect of nimesulide on lipopolysaccharide (LPS)-induced proinflammatory oxidants production by rat alveolar macrophages (AMs). Effects of LPS and nimesulide on antioxidant defense and the expression of inducible nitric oxide synthase (iNOS) were also studied. It was found that nimesulide could scavenge superoxide anions (O2*-), nitric oxide (NO*) and total oxidant burden induced by LPS in AMs in vitro. Approximately 850 nmoles of nimesulide had activity equivalent to one IU of superoxide dismutase (SOD). Further, to confirm the in vitro observation, Male Wistar rats were orally administered with nimesulide (9 mg/kg b. wt. twice daily) for one week followed by intratracheal instillation of 2 microg LPS to stimulate lung inflammation. AMs from bronchoalveolar lavage fluid were collected 18 h after instillation of LPS. Nimesulide pretreatment could inhibit O2*-, NO() and lipid peroxidation in AMs. Nimesulide also suppressed LPS-induced iNOS expression in AMs in vivo and in vitro. Nimesulide could also normalize LPS-induced changes in the levels of superoxide dismutase (SOD), glutathione reductase (GR) and reduced glutathione (GSH) in AMs. Inhibition in production of oxidants in LPS-challenged AMs by nimesulide could be one of the pathways for its anti-inflammatory action.     

Semecarpus anacardium L, nuts inhibit lipopolysaccharide induced NO production in rat macrophages along with its hypolipidemic property

Traditionally S. anacardium is used for rejuvenation, rheumatoid arthritis, fever and neurological disorders. In the present study it was observed that a fraction of S. anacacrdium at dose of 1 mg/100 g body wt, significantly reduced serum cholesterol from 378.87 mg/dl in the rats fed with atherogenic diet (AD) to 197.99 mg/dl (45-52%) in the rats fed with AD diet and increased serum HDL-cholesterol (33-37%). The same fraction also inhibited LPS induced NO production in the culture activated rat peritoneal macrophages in the dose dependent manner with IC50 value at 50 ng/ml of the culture medium. The drug in the above doses was completely safe and non-toxic, (no change in the enzymes), to liver and kidney functions.