Role of inducible nitric oxide synthase-derived nitric oxide in lipopolysaccharide plus interferon-gamma-induced pulmonary inflammation

Exposure of mice to lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) increases nitric oxide (NO) production, which is proposed to play a role in the resulting pulmonary damage and inflammation. To determine the role of inducible nitric oxide synthase (iNOS)-induced NO in this lung reaction, the responses of inducible nitric oxide synthase knockout (iNOS KO) versus C57BL/6J wild-type (WT) mice to aspirated LPS + IFN-gamma were compared. Male mice (8-10 weeks) were exposed to LPS (1.2 mg/kg) + IFN-gamma (5000 U/mouse) or saline. At 24 or 72 h postexposure, lungs were lavaged with saline and the acellular fluid from the first bronchoalveolar lavage (BAL) was analyzed for total antioxidant capacity (TAC), lactate dehydrogenase (LDH) activity, albumin, tumor necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2). The cellular fraction of the total BAL was used to determine alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) counts, and AM zymosan-stimulated chemiluminescence (AM-CL). Pulmonary responses 24 h postexposure to LPS + IFN-gamma were characterized by significantly decreased TAC, increased BAL AMs and PMNs, LDH, albumin, TNF-alpha, and MIP-2, and enhanced AM-CL to the same extent in both WT and iNOS KO mice. Responses 72 h postexposure were similar; however, significant differences were found between WT and iNOS KO mice. iNOS KO mice demonstrated a greater decline in total antioxidant capacity, greater BAL PMNs, LDH, albumin, TNF-alpha, and MIP-2, and an enhanced AM-CL compared to the WT. These data suggest that the role of iNOS-derived NO in the pulmonary response to LPS + IFN-gamma is anti-inflammatory, and this becomes evident over time.     

Role of inducible nitric oxide synthase-derived nitric oxide in silica-induced pulmonary inflammation and fibrosis

Inhalation of crystalline silica can produce lung inflammation and fibrosis. Inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) is believed to be involved in silica-induced lung disease. To investigate the role of iNOS-derived NO in this disease, the responses of iNOS knockout (KO) versus C57Bl/6J wild-type (WT) mice to silica were compared. Male mice (8-10 wk old, mean body weight 24.0 g) were anesthetized and exposed, by aspiration, to silica (40 mg/kg) or saline. At 24 h and 42 d postexposure, lungs were lavaged with saline. The first bronchoalveolar lavage (BAL) fluid supernatant was analyzed for lactate dehydrogenase (LDH) activity, levels of albumin, tumor necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2), as well as total antioxidant capacity (TAC). The cellular fraction of the total BAL was used to determine alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) counts, and zymosanstimulated AM chemiluminescence (AM-CL). In separate mice, lung histopathological changes were evaluated 42 d postexposure. Acute (24-h) silica exposure decreased AMs, increased PMNs, increased LDH activity and levels of albumin, TNF-alpha, and MIP-2 in BAL fluid, and enhanced AM-CL in both iNOS KO and WT mice. However, iNOS KO mice exhibited less AM activation (defined as increased AM-CL and decreased AM yield) than WT. Furthermore, TAC following acute silica decreased in WT but was maintained in iNOS KO mice. Pulmonary reactions to subchronic (42 d) silica exposure were similar to acute. However, histopathological and BAL fluid indices of lung damage and inflammation, AM activation, and lung hydroxyproline levels were significantly less in iNOS KO compared to WT mice. These results suggest that iNOS-derived NO contributes to the pathogenesis of silica-induced lung disease in this mouse model.     

Resistance to endotoxic shock in mice lacking natriuretic peptide receptor-A

Background and purpose:

Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock.

Experimental approach:

Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated.

Key results:

LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NO_x] and cyclic GMP).

Conclusions and implications:

These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition.     

Role of surfactant protein-A (SP-A) in lung injury in response to acute ozone exposure of SP-A deficient mice

Millions are exposed to ozone levels above recommended limits, impairing lung function, causing epithelial damage and inflammation, and predisposing some individuals to pneumonia, asthma, and other lung conditions. Surfactant protein-A (SP-A) plays a role in host defense, the regulation of inflammation, and repair of tissue damage. We tested the hypothesis that the lungs of SP-A(-/-) (KO) mice are more susceptible to ozone-induced damage. We compared the effects of ozone on KO and wild type (WT) mice on the C57BL/6 genetic background by exposing them to 2 parts/million of ozone for 3 or 6 h and sacrificing them 0, 4, and 24 h later. Lungs were subject to bronchoalveolar lavage (BAL) or used to measure endpoints of oxidative stress and inflammation. Despite more total protein in BAL of KO mice after a 3 h ozone exposure, WT mice had increased oxidation of protein and had oxidized SP-A dimers. In KO mice there was epithelial damage as assessed by increased LDH activity and there was increased phospholipid content. In WT mice there were more BAL PMNs and elevated macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1. Changes in MIP-2 and MCP-1 were observed in both KO and WT, however mRNA levels differed. In KO mice MIP-2 mRNA levels changed little with ozone, but in WT levels they were significantly increased. In summary, several aspects of the inflammatory response differ between WT and KO mice. These in vivo findings appear to implicate SP-A in regulating inflammation and limiting epithelial damage in response to ozone exposure.     

Palmitoleic acid reduces the inflammation in LPS‐stimulated macrophages by inhibition of NFκB,independently of PPARs

Palmitoleic acid (PM , 16:1n‐7) has anti‐inflammatory properties that could be linked to higher expression of PPAR α, an inhibitor of NF κB. Macrophages play a major role in the pathogenesis of chronic inflammation, however, the effects of PM on macrophages are underexplored. Thus, we aimed to investigate the effects of PM in activated macrophages as well the role of PPAR α. Primary macrophages were isolated from C57BL /6 wild type (WT ) and PPAR α knockout (KO ) mice, cultured under standard conditions and exposed to lipopolysaccharides LPS (2.5 μg/ml ) and PM 600 μmol/L conjugated with albumin for 24 hours. The stimulation with LPS increased the production of interleukin (IL )‐6 and IL ‐1β while PM decreased the production of IL ‐6 in WT macrophages. In KO macrophages, LPS increased the production of tumour necrosis factor (TNF )‐α and IL ‐6 and PM decreased the production of TNF α. The expression of inflammatory markers such NF κB and IL 1β were increased by LPS and decreased by PM in both WT and KO macrophages. PM reduced the expression of MyD88 and caspase‐1 in KO macrophages, and the expression of TLR 4 and HIF ‐1α in both WT and KO macrophages, although LPS had no effect. CD 86, an inflammatory macrophage marker, was reduced by PM independently of genotype. PM increased PPAR γ and reduced PPAR β gene expression in macrophages of both genotypes, and increased ACOX ‐1 expression in KO macrophages. In conclusion, PM promotes anti‐inflammatory effects in macrophages exposed to LPS through inhibition of inflammasome pathway, which was independent of PPAR α, PPAR ? and AMPK , thus the molecular mechanisms of anti‐inflammatory response caused by PM is still unclear.     

Beneficial effect of hyperbaric oxygen pretreatment on lipopolysaccharide-induced shock in rats

1. We investigated the effect of hyperbaric oxygenation (HBO2) pretreatment on the production of exhaled nitric oxide (ENO) and the expression of lung inducible nitric oxide synthase (iNOS) by Escherichia coli lipopolysaccharide (LPS)-induced shock in an experimental rat model. 2. Rats were randomized into four groups, anaesthetized, mechanically ventilated with room air and infused with normal saline (2 mL/h) through the jugular vein for 5 h. Group 1 (NS) received only normal saline. Group 2 (HBO2-NS) was pretreated with HBO2 at 2.8 absolute atmospheres for 2 h and then received normal saline. Group 3 (LPS) received LPS, 20 mg/kg, i.v., bolus. Group 4 (HBO2-LPS) was pretreated with HBO2 for 2 h, followed by LPS. 3. Arterial blood gases, blood pressure, blood pH and ENO production were measured every 30 min. Plasma nitrite/nitrate (NOx) concentrations were assessed at the beginning (baseline) and at the end of the study. Lung myeloperoxidase (MPO) activity, iNOS expression and histological scores were measured for the evaluation of lung injury. 4. Administration of LPS was associated with decreased blood pressure and pH, increased ENO production, plasma NOx concentrations, lung iNOS expression and MPO activity. 5. Pretreatment with HBO2 significantly alleviated the LPS-induced hypotension, acidosis and decreased ENO production, plasma NOx concentrations, lung MPO activity and expression of iNOS. Hyperbaric O2 had no effect on control rats. 6. Our data show that HBO2 pretreatment has beneficial haemodynamic effects in rats with endotoxin shock. The beneficial effects of HBO2 may be partially mediated by decreased ENO production via reduced LPS-induced lung iNOS expression.     

Hemodynamic and renal involvement of B1 and B2 kinin receptors during the acute phase of endotoxin shock in mice

B1 kinin receptor (B1R) is up-regulated by endotoxins and thus may represent a therapeutic target in sepsis. We investigated the expression and role of B1R and B2R in the acute phase of lipopolysaccharide (LPS)-induced endotoxin shock in C57BL/6 mice (WT) and B1R and B2R knock out mice (B1KO, B2KO). B1R mRNA was enhanced from 6 to 48 h after LPS while B2R mRNA was further increased in B1KO. Maximal hypotension was found 24 h after LPS, and was more pronounced in B1KO, but was reduced in B2KO. Glomerular filtration rate was more reduced by LPS in B1KO than in WT and B2KO. Glycemia was reduced by LPS and particularly in B1KO and B2KO mice. Mortality was increased by LPS in B1KO. These data suggest that the up-regulated B1R plays, at least transiently, a significant beneficial role in acute LPS-induced hypotension. Conversely, supra activation of B2R could be also involved in the increased mortality observed in B1KO mice.     

Angiotensin II stimulates superoxide production via both angiotensin AT1A and AT1B receptors in mouse aorta and heart

The present study was conducted to determine the roles of angiotensin AT(1A) and AT(1B) receptors in angiotensin II-induced superoxide anion production in mouse aorta and heart. Superoxide anion production in aorta was determined by the lucigenin chemiluminescence method, and thiobarbituric acid reactive substances in heart tissues were measured by biochemical assay. The basal production rate of superoxide anion in aorta of wild type (WT) mice was significantly higher than in angiotensin AT(1A) receptor knockout (AT(1A) KO) mice. Angiotensin II (2.8 mg/kg/day, s.c. for 13 days) significantly increased superoxide anion production in aorta of both AT(1A) KO and WT mice. However, the superoxide anion production rate in aorta of angiotensin II-infused AT(1A) KO mice was significantly lower than in angiotensin II-infused WT mice. Valsartan (40 mg/kg/day in drinking water) prevented angiotensin II-induced superoxide anion production in aorta of WT and AT(1A) KO mice. Similarly, thiobarbituric acid reactive substances levels in heart tissues of angiotensin II-treated WT and AT(1A) KO mice were significantly higher than those in vehicle-infused WT and AT(1A) KO mice, respectively. Valsartan prevented angiotensin II-induced increases of thiobarbituric acid reactive substances levels in heart tissue of both WT and AT(1A) KO mice. These results indicate that angiotensin II stimulates superoxide anion production via both angiotensin AT(1A) and AT(1B) receptors, and that angiotensin AT(1A) receptors appear to play a predominant role in angiotensin II-induced superoxide anion production in mouse aorta and heart.     

Induction of chemokines by low-dose intratracheal silica is reduced in TNFR I (p55) null mice.

Previous studies suggest that tumor necrosis factor alpha (TNF-alpha) and the TNFRI (p55) and TNFRRII (p75) receptors mediate the pulmonary fibrotic response to silica. In order to further define the role of the TNFRI (p55) receptor in induction of profibrotic chemokines by low-dose silica/crystalline silica (50 micro g/50 micro l/mouse) or control diluent saline was instilled into the trachea of TNFRI gene ablated ((-/-)) and C57BL/6 (WT) control mice. Lung tissue was harvested and bronchoalveolar lavage (BAL) performed 24 h and 28 days following silica administration. Selected profibrotic chemokine mRNAs were quantified by ribonuclease protection assay, normalized to ribosomal protein L32 mRNA content and expressed relative to saline control treated lungs. Induction of MIP-1beta, MIP-1alpha, MIP-2, IP-10, and MCP-1 mRNAs was attenuated in the TNFRI(-/-) mice, in comparison to WT mice, particularly at 28 days after exposure. ELISA assays for MIP-1alpha and MIP-2 in homogenized lung tissue similarly demonstrated marked induction of both chemokines 24 h after silica treatment, which was persistent at 28 days in WT but not in TNFRI(-/-) mice. The percentage of BAL cells that was neutrophils was comparably increased in WT and RI(-/-) lungs at 24 h (49 +/- 12% vs. 46 +/- 10%) and 28 days (6.2 +/- 1.5% vs. 4.5 +/- 1%). The increase in total lavagable cells and BAL protein was also independent of strain. Histology revealed mild alveolitis without granuloma formation in both strains, slightly decreased in TNFRI(-/-). This study demonstrates an increase in pro-fibrotic chemokines in response to a single intratracheal exposure to crystalline silica that was sustained at 28 days after treatment in WT but not in TNFRI(-/-) mice. Silica dependent recruitment of neutrophils to the alveolar space and alveolar protein leak were, however, not altered by the absence of the TNF receptor.     

Dopamine D2 receptor modulates sodium handling via local production of dopamine in the kidney

We have recently demonstrated that a deletion of the dopamine D2 receptor gene caused suppression of urinary sodium excretion and salt-sensitive elevation of blood pressure in mice. In order to understand the mechanisms underlying this impaired sodium excretion, we studied renal dopamine production and dopamine-induced sodium excretion in 20- to 30-week-old male D2-receptor knockout (D2KO) mice and age- and sex-matched wildtype (WT) mice. Renal local dopamine synthesis, examined by 24-h urine free dopamine excretion (UDAV), was significantly (p < 0.05) reduced in D2KO mice compared to that in WT mice (D2KO versus WT: 1.06 +/- 0.2 versus 1.5 +/- 0.3 ng/mg creatinine). Such a difference between D2KO and WT mice was also observed after oral administration of 3,4-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine, at 5 mg/kg per day for 24 h. Furthermore, activity of aromatic 1-amino acid decarboxylase, a dopamine synthetase, was significantly suppressed in D2KO mice. Next, we examined changes in 24-h urine flow (UV) and 24-h sodium excretion (UNaV) during chronic infusion of dopamine at sub-pressor doses (3-4 microg/kg per min, sq.) or a vehicle via an osmotic pump. Urine flow in 24 h and UNaV were significantly (p < 0.05) smaller in D2KO mice infused with vehicle than in WT mice infused with vehicle (UV: 210 +/- 43 versus 650 +/- 163 microl/day; UNaV: 20.6 +/- 13.2 versus 44.4 +/- 21.6 microEq/day). After administration of dopamine, UV and UNaV in D2KO mice were restored to a level similar to that in WT mice. These results indicate that D2-dopamine receptors play a significant role in renal local dopamine synthesis and that a shortage of dopamine was, at least in part, responsible for the suppression of UV and UNaV in D2KO mice. However, we could not conclude from the present study whether renal tubular sodium reabsorption is intact in D2KO mice because the baseline dopamine contents in kidneys of D2KO mice and WT mice may be different.     

Inhibitory effect of trimidox on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages

We examined the effect of trimidox (3,4,5-trihydroxybenzamidoxime) on the production of nitric oxide (NO) by lipopolysaccharide (LPS) in mouse RAW 264.7 macrophages. Trimidox (50 - 300 microM) concentration-dependently inhibited NO production by LPS (0.01, 0.1, or 1 microg/ml) after incubation for 24 h. LPS-induced expression of inducible NO synthase (iNOS) and degradation of IkappaBalpha were prevented by trimidox. The protective effect against NO production by LPS was not only observed in prior incubation but also later incubation with trimidox until iNOS was activated by LPS. These results suggest that trimidox has a predominantly protective effect against LPS-induced production of NO via iNOS expression.     

信号传导与转录激活子4 信号通路在脂肪酶/Toll 样受体4调控M1/M2 型巨噬细胞分化的作用及机制

目的探讨信号传导与转录激活子4(STAT4)信号通路在脂肪酶(LPS)/Toll样受体4(TLR4)调控M1/M2型巨噬细胞分化的作用及机制。方法用野生型和STAT4-KO小鼠建立动脉内膜拉伤-增生模型,在术后1、3、7和14 d用流式细胞术分析免疫细胞M1/M2型巨噬细胞在外周血内的百分比变化。用免疫荧光双染检测M1,M2型巨噬细胞的表达。用实时荧光定量PCR(q-RT PCR)检测血管组织中STAT4和TLR4表达;酶联免疫吸附试验(ELISA)检测血管生长因子VEGF、PDGF-BB变化。从STAT4KO和蛋白质印迹法小鼠骨髓分离CD11b+细胞,用集落刺激因子GM-CSF和LPS处理,细胞实验分为WT con组、WT+LPS刺激组、STAT4KO con组、STAT4KO+LPS刺激组。观察巨噬细胞的分化情况。结果与野生组相比,STAT4-KO组在术后1、3、7和14天时M1 型巨噬细胞F4/80+iNOS+,F4/80+IL-12的细胞百分比降低(P<0.05),而M2型巨噬细胞F4/80+Arg-1+和F4/80+CD206+ 的细胞百分比明显升高(P<0.05)。STAT4-KO组的M1,M2型巨噬细胞阳性表达率(9.42±0.41)%、(89.48±10.43)%与野生组(11.14±1.49)%、(48.73±5.89)%比较,差异有统计学意义(P<0.05)。STAT4 和TLR4 在STAT4-KO 组［(0.23±0.04)、(0.47±0.06)］中的表达量明显较野生组［(1.31±0.07)、(0.89±0.08)］低(P<0.05)。VEGF、PDGF-BB 在STAT4-KO 组［(9.28±1.02)、(10.56±1.62)］中的表达量明显较野生组［(3.14±0.91)、(4.23±0.84)］高(P<0.05)。M2型巨噬细胞的诱导因子(IL-4)及分泌因子(IL-10)在STAT4KO+LPS 刺激组、WT+LPS 刺激组、STAT4KO con 组中的表达量明显较WT con 组高(F=13.412,F=15.012,P<0.05),其中STAT4KO+LPS刺激组表达量最为显著。结论STAT4信号通路可以促进抑制M1型巨噬细胞和增强M2型巨噬细胞分化。其作用机制可能是通过LPS/TLR4的结合。     

Inhibition of inducible nitric oxide synthase by beta-lapachone in rat alveolar macrophages and aorta

Beta-lapachone, a plant product, has been shown to be a novel inhibitor of DNA topoisomerase. In this study, we performed experiments to examine the effects of beta-lapachone on lipopolysaccharide (LPS)-induced inducible nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages and aortic rings. In alveolar macrophages, incubation with LPS (10 microg ml(-1)) for various time intervals resulted in a significant increase in nitrite production and iNOS protein synthesis, that was inhibited by coincubation with beta-lapachone (1-4.5 microM) without any cytotoxic effects. However, addition of beta-lapachone after induction of NO synthase by LPS failed to affect the nitrite production. Treatment with LPS (10 microg ml(-1)) for 6 h resulted in significant expression of mRNA for iNOS which was significantly inhibited in the presence of beta-lapachone (3 microM) in alveolar macrophages. In endothelium-intact rings of thoracic aorta, beta-lapachone (1 and 3 microM) markedly inhibited the hypocontractility to phenylephrine in aortic rings treated with LPS (10 microg ml(-1)) for 4 h. When beta-lapachone was added 3 h after LPS into the medium, the contractions evoked by phenylephrine were not significantly different in the presence or absence of beta-lapachone. Treatment with LPS (10 microg ml(-1)) for 4 h resulted in a significant increase in iNOS protein synthesis which was inhibited in the presence of beta-lapachone (3 microM), but did not affect the constitutive (endothelial and neuronal) NOS forms in aortic rings. These results indicate that beta-lapachone is capable of inhibiting expression and function of iNOS in rat alveolar macrophages and aortic rings. It is considered that beta-lapachone can be developed as a potential anti-inflammatory agent in the future.     

Evidence that the granulocyte colony-stimulating factor (G-CSF) receptor plays a role in the pharmacokinetics of G-CSF and PegG-CSF using a G-CSF-R KO model.

The covalent attachment of polyethylene glycol to filgrastim results in a new molecule pegfilgrastim, which has a significantly longer half-life than filgrastim. It is likely that the clearance of both filgrastim and pegfilgrastim involves granulocyte colony simulating factor (G-CSF) receptor binding, but the pharmacokinetics of these drugs have not been compared in mice with and without a functional G-CSF receptor. We sought to clarify the role of receptor-mediated clearance of filgrastim and pegfilgrastim using wild-type (WT) mice or mice with a non-functional G-CSF-R (knockout, KO). We administered single doses of filgrastim or pegfilgrastim (10 or 100 microg kg(-1)) intravenously to WT and KO mice. Plasma levels of protein were measured by enzyme-linked immunosorbent assay (ELISA) at preset time points, and AUC, MRT, CL, V(d), and T(1/2) were calculated. When compared with WT mice, the G-CSF-R KO mice had significantly greater AUC, longer MRT, longer T(1/2), and lower clearance. This was the case whether animals received 10 or 100 microg kg(-1) and whether they received filgrastim or pegfilgrastim. The volume of protein distribution was identical among WT and KO mice. However, the V(d) was larger after pegfilgrastim dosing than after filgrastim dosing. In both WT and KO mice, increasing the dose of figrastim or pegfilgrastim resulted in a proportional increase in the AUC. A functional G-CSF-R is an important mechanism in the plasma clearance of both filgrastim and pegfilgrastim.     

Involvement of NADPH oxidase and iNOS in rodent pulmonary cytokine responses to urban air and mineral particles

We have investigated the potential of two complex mineral particles (feldspar and mylonite), quartz (Min-U-Sil), and suspended particulate matter (SRM-1648) (SPM) from urban air to induce inflammatory cytokine responses in primary rat alveolar type 2 cells and alveolar macrophages, and the involvement of cellular formation of free radicals in these responses. All particle types induced an increased release of interleukin (IL)-6 and macrophage inflammatory protein (MIP)-2 from type 2 cells. Diphenyleneiodonium chloride (DPI), a selective inhibitor of NADPH-oxidase, reduced the IL-6 and MIP-2 responses to quartz, SPM and mylonite. N-(3-[Aminomethyl] benzyl) acetamidine (1400W), a selective inhibitor of inducible nitric oxide synthase (iNOS), significantly reduced the Il-6 response to SPM and feldspar in the type 2 cells. The macrophages displayed significantly increased TNF-alpha and MIP-2 release upon exposure to quartz or SPM. Here, DPI significantly reduced the tumor necrosis factor (TNF)-alpha and MIP-2 responses to quartz, and the MIP-2 response to SPM. No significant effect of 1400 W was detected in the alveolar macrophages. The role of particle-induced cellular generation of free radicals in lung cytokine responses was further elucidated in mice that lacked either NADPH-oxidase or iNOS as well as in wild-type (wt) mice. All particles were able to elicit increased cytokine levels in the bronchoalveolar lavage (BAL) fluid of the mice, although the levels depended on particle type. The NADPH-oxidase knockout (KO) mice demonstrated a significantly lower IL-6 and MIP-2 responses to SPM compared to their respective wt mice. The iNOS KO mice displayed significantly reduced IL-6, TNF-alpha, and MIP-2 responses to SPM. The overall results indicate the involvement of cellular free-radical formation in the pulmonary cytokine responses to particles of varying composition.     

Protective and anti-inflammatory effect of a traditional Chinese medicine, Xia-Bai-San, by modulating lung local cytokine in a murine model of acute lung injury

We investigated the effects of Xia-Bai-San (XBS) on acute lung inflammation induced by LPS in vivo. Mice were challenged with intratracheal lipopolysaccharide (100 microg) 30 min before administering XBS (1 mg/kg oral administration). Bronchoalveolar lavage fluid (BALF) was obtained after 4 and 24 h to measure proinflammatory cytokine (TNF-alpha, IL-1beta, IL-6), anti-inflammatory cytokines (IL-10), chemokines (KC, MCP-1 and MIP-2), total cell counts, nitric oxide production, and proteins. The results indicated that XBS down-regulated the LPS-induced expression of TNF-alpha, IL-1beta, IL-6, KC, MIP-2, and MCP-1. Furthermore, it also enhanced the production of IL-10, which had increased 24 h after LPS challenge. In addition, total leukocyte counts, nitric oxide production, iNOS expression, and BALF's proteins had significantly decreased 24 h after LPS challenge. XBS was also believes to have reduced the acute inflammation by attenuating the activation of NF-kappaB. In conclusion, XBS seem to suppress lipopolysaccharide-induced lung inflammation by stimulating the production of anti-inflammatory cytokines in lung. These results suggest that XBS could be a useful adjunct in the treatment of acute respiratory distress syndrome.     

Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase expression in glomerular mesangial cells

Nitric oxide (NO) that is produced by inducible nitric oxide synthase (iNOS) is associated with the pathophysiology of glomerulonephritis. Numerous studies have focused on the regulation of NO production by iNOS to reduce NO-mediated cytotoxicity. In the present study, we demonstrated the differential effects of two phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, on lipopolysaccharide- (LPS) and interferon (IFN)-γ-induced NO production in a glomerular mesangial cell line, MES-13 cells. At dosages without affecting cell viability of MES-13 cells, 5μM LY294002 showed a more-significant inhibitory effect on LPS/IFN-γ-induced NO production, and iNOS protein and gene expressions than did 1μM wortmannin. Akt phosphorylation in MES-13 cells declined upon the addition of wortmannin, but not upon treatment with LY294002. Suppression of PI3K expression by small interfering (si)RNA exhibited no effect on LPS/IFN-γ-stimulated NO production or iNOS protein expression in MES-13 cells. Neither LY294002 nor wortmannin reduced IFN-γ-induced STAT-1α phosphorylation. LY294002 exhibited a more-significant inhibitory effect on NF-κB luciferase activities than wortmannin in LPS/IFN-γ-stimulated MES-13 cells. Moreover, LY294002, but not wortmannin, accelerated iNOS protein degradation and reduced the iNOS dimer/monomer ratio in MES-13 cells. Although both LY294002 and wortmannin are known as PI3K inhibitors, their differential effects on iNOS expression in MES-13 cells indicate that the effects of LY294002 on inhibiting NF-κB activation and accelerating iNOS protein degradation are through a mechanism independent of PI3K.     

Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide

1. Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS-induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS-induced plasma leakage using iNOS deficient mice. 2. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100 - 400 microg site(-1)) produced a dose-related increase in dye leakage in both iNOS deficient and wild-type mice with about 40% less dye leakage in iNOS deficient mice. 3. Indomethacin (5 mg kg(-1)), N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide (NS-398) (1 mg kg(-1)), diphenhydramine (10 mg kg(-1)) and anti-TNF-alpha antibody (dilution 1 : 400, 10 ml kg(-1)) inhibited the LPS-induced dye leakage in both iNOS deficient and wild-type mice, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) or aminoguanidine (10 mg kg(-1)) inhibited that in wild-type but not in iNOS deficient mice. 4. Pretreatment with LPS (0.15 mg kg(-1) i.p.) 4 h before decreased the LPS-induced dye leakage in wild-type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild-type but not in iNOS deficient mice. 5. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF-alpha. The tolerance against LPS-induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.