Interferon-gamma alone triggers the production of nitric oxide from serum-starved BNL CL.2, murine embryonic liver cells.

A previous study has demonstrated that both interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were needed to induce the production of nitric oxide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstrate that when BNL CL.2 cells were cultured with serum-free medium, they were induced to produce NO by the stimulation of IFN-gamma alone. BNL CL.2 cells were cultured with serum-free or serum-containing medium for 1-3 days and then stimulated to synthesize NO by IFN-gamma. Surprisingly, only serum-starved cells showed significant amount of nitrite accumulation and iNOS protein expression in response to IFN-gamma in dose- and time-dependent manners, but serum-supplied cells did not. When the cells were stimulated with IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), or LPS in combinations, only the combination of IFN-gamma and LPS produced more NO than that produced by IFN-gamma alone. The production of NO by the cells stimulated with IFN-gamma or IFN-gamma plus LPS was blocked by the addition of N(G)-monomethyl-L-arginine (N(G)MMA), a NO synthesis inhibitor. To address the intracellular signal pathway responsible for the production of NO by the cells stimulated with IFN-gamma aloneor IFN-gamma plus LPS, we examined the effects of several protein kinase inhibitors on the production of NO from the cells. The production of NO was significantly inhibited by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-gamma and the involvement of PTK signal transduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.     

NITRIC OXIDE PREVENTS THE IFN-γ/LPS-INDUCED HEPATOTOXICITY IN A PROTEIN KINASE G-INDEPENDENT MANNER

Although it has been well known that the role of LPS on hepatotoxicity is mediated through TNF-α, the direct cytotoxic effect of LPS on IFN-γ-primed hepatocytes has not yet been clearly demonstrated. Here, we demonstrate that the IFN-γ-mediated death of murine embryonic liver BNL CL2 cells is potentiated by LPS (0.5 μg/ml). In addition, an exogenous NO donor, sodium nitroprusside (SNP) significantly prevents cell death induced by IFN-γ alone or IFN-γ plus LPS (IFN-γ/LPS) in a dose-dependent manner over 25 μM. SNP significantly blocked the death of BNL CL2 cells only when it was added within 12 hr after treatment of IFN-γ and IFN-γ/LPS. The preventive effect of SNP occurred in parallel with the suppression of caspase 3-like protease activation. We have also demonstrated that a relatively high concentration as well as an appropriate period of exposure to NO may be critical to maintain cell viability from the cytotoxic effect of IFN-γ and IFN-γ/LPS. Furthermore, the preventive effect of SNP on IFN-γ/LPS-induced cell death is mediated by a protein kinase G (PKG)-independent manner.     

RHODIOLA SACHALINESIS INDUCES THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE BY MURINE FETAL HEPATOCYTES (BNL CL.2)

We have examined the effect of the aqueous extract of Rhodiola sachalinensis root (RSE), a traditional herbal medicine, on nitric oxide (NO) synthesis in murine fetal hepatocytes (BNL CL.2) by measuring the stable end-product nitrite and the mRNA of inducible NO synthase (iNOS). Interferon-gamma (IFN-γ) by itself failed to induce NO synthesis in BNL CL.2 cells. RSE also did not elicit NO synthesis at concentrations up to 1000 μg/ml, but dose- and time-dependently induced NO synthesis in the presence of IFN-γ in BNL CL.2 cells. Whereas RSE or IFN-γ failed to induce detectable levels of iNOS mRNA, a combination of RSE and IFN-γ markedly induced iNOS mRNA in BNL CL.2 cells. Thus, we found that RSE triggered IFN-γ-primed BNL CL.2 cells to synthesize NO by inducing iNOS gene expression. The capability of RSE to induce NO synthesis might be related to the therapeutic efficacy of RSE on the liver diseases.     

Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major

Murine macrophages can be activated to produce nitric oxide (NO) and superoxide and these two radicals can react to form peroxynitrite, a powerful oxidant which may be involved in parasite killing. We now show that murine macrophages activated with zymosan and interferon-γ (ZYM/IFN-γ) produced both superoxide (peaking 1–2 h after stimulation, then rapidly declining) and NO (barely detectable at 6 h, peaking by 24 h). Macrophages activated with ZYM alone produced only superoxide, while stimulation with lipopolysaccharide (LPS) and IFN-γ induced NO but not superoxide. Cells stimulated with ZYM/IFN-γ or LPS/IFN-γ killed Leishmania major to a similar degree, an effect that was completely blocked by the addition of N-iminoethyl-L -ornithine. However, macrophages stimulated with ZYM alone were unable to kill L. major. S-nitroso-acetyl-penicillamine, which release NO, was highly leishmanicidal when added directly to the parasites. 3-morpholino-sydnonimine hydrochloride which releases both NO and superoxide simultaneously, was also efficient at killing L. major and this cytotoxicity was greatly enhanced by the addition of superoxide dismutase. Finally, authentic peroxynitrite failed to induce any cytotoxic effect, even at a high concentration. Thus macrophages can produce either NO, superoxide or both, depending on the stimulus. However, the killing of L. major is dependent only on the production of NO.     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Nitric oxide prevents the IFN-gamma/LPS-induced hepatotoxicity in a protein kinase G-independent manner.

Although it has been well known that the role of LPS on hepatotoxicity is mediated through TNF-alpha, the direct cytotoxic effect of LPS on IFN-gamma-primed hepatocytes has not yet been clearly demonstrated. Here, we demonstrate that the IFN-gamma-mediated death of murine embryonic liver BNL CL2 cells is potentiated by LPS (0.5 microg/ml). In addition, an exogenous NO donor, sodium nitroprusside (SNP) significantly prevents cell death induced by IFN-gamma alone or IFN-gamma plus LPS (IFN-gamma/LPS) in a dose-dependent manner over 25 microM. SNP significantly blocked the death of BNL CL2 cells only when it was added within 12 hr after treatment of IFN-gamma and IFN-gamma/LPS. The preventive effect of SNP occurred in parallel with the suppression of caspase 3-like protease activation. We have also demonstrated that a relatively high concentration as well as an appropriate period of exposure to NO may be critical to maintain cell viability from the cytotoxic effect of IFN-gamma and IFN-gamma/LPS. Furthermore, the preventive effect of SNP on IFN-gamma/LPS-induced cell death is mediated by a protein kinase G (PKG)-independent manner.     

Different involvement of the MAPK family in inflammatory regulation in human pulmonary microvascular endothelial cells stimulated with LPS and IFN-γ

Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.     

红景天苷对小鼠腹腔巨噬细胞体外增殖、凋亡、吞噬、ROS和NO产生的影响

目的:研究红景天苷(Sal)对小鼠腹腔巨噬细胞体外增殖、凋亡、吞噬、胞内活性氧簇(ROS)及分泌一氧化氮(NO)的影响,初步探讨其对小鼠腹腔巨噬细胞的免疫调节作用。方法:无菌分离小鼠腹腔巨噬细胞,并制备单细胞悬液,以不同终浓度(80μmol/L、160μmol/L及320μmol/L)的Sal和巨噬细胞共培养4 h,再以脂多糖(LPS)和γ-干扰素(IFN-γ)进行共刺激。利用MTT比色法检测Sal对巨噬细胞体外增殖的影响。用放线菌酮(CHX)诱导巨噬细胞凋亡,用Sytox G reen染色结合荧光酶标仪检测Sal对CHX诱导巨噬细胞凋亡的影响。用流式细胞术(FCM)检测Sal对巨噬细胞吞噬功能的影响。用2-7-二氯氢化荧光素乙二脂(H2DCFDA)染色法结合荧光酶标仪检测Sal对胞内ROS产生的影响;用G riess反应检测Sal对巨噬细胞分泌NO的影响。结果:MTT比色法检测显示,终浓度为80、160、320μmol/L的Sal均可显著促进LPS+IFN-γ刺激巨噬细胞增殖(P<0.05)。荧光酶标仪检测Syto xG reen染色法的结果显示,160μmol/L的Sal可抑制CHX诱导的巨噬细胞凋亡(P<0.01)。FCM结果显示,各浓度的Sal均能促进单纯药物组和实验药物组LPS+IFN-γ刺激巨噬细胞的吞噬功能(P<0.05)。用荧光酶标仪检测DH2DCFDA染色结果表明,各浓度的Sal对LPS+IFN-γ刺激的巨噬细胞胞内ROS的产生均具有显著的抑制作用(P<0.01)。Griess反应检测NO含量的结果显示,各浓度的Sal对LPS+IFN-γ刺激巨噬细胞产生NO均具有促进作用(P<0.05)。结论:Sal对LPS和IFN-γ刺激的巨噬细胞增殖具有显著的促进作用,对CHX诱导的巨噬细胞凋亡具有显著的抑制作用,对静息态和活化态的巨噬细胞的吞噬功能均有增强作用,并能减少LPS和IFN-γ活化的巨噬细胞胞内ROS的产生;但能促进LPS和IFN-γ活化的巨噬细胞NO的分泌。     

转移程度不同的小鼠肝癌细胞株对腹腔巨噬细胞免疫功能的影响

目的 研究转移程度不同的小鼠肝癌细胞株对腹腔巨噬细胞功能的影响.方法 在两株转移程度不同的小鼠腹水型肝癌模型中,取荷瘤小鼠腹腔巨噬细胞,检测其在干扰素γ(IFN-γ)和脂多糖(LPS)刺激下产生一氧化碳(NO)和肿瘤坏死因子α(TNF-α)的水平,并检测其活化后的杀伤能力.进一步采用夹心酶联免疫吸附测定(ELISA)方法,研究不同荷瘤小鼠腹水中IFN-γ与转化生长因子β1(TGF-β1)的水平,并用相应抗体封闭TGF-β1后,检测活化巨噬细胞产生NO能力及杀伤活性.结果 经IFN-γ和LPS活化后,荷瘤小鼠腹腔巨噬细胞分泌NO和TNF-α能力明显低于正常巨噬细胞,杀伤能力下降.高转移性肝癌小鼠巨噬细胞分泌NO水平和杀伤能力均低于低转移性小鼠,但其分泌TNF-α量较高.此外,荷瘤小鼠腹水含较高水平IFN-γ与TGF-β1,不同转移程度荷瘤小鼠IFN-γ水平接近,但高转移性肝癌小鼠腹水含更多TGF-β1,而且TGF-β1的封闭可导致与肿瘤细胞共培养的巨噬细胞分泌NO的能力部分恢复.结论 肿瘤细胞可以通过分泌TGF-β1等抑制性因子下调巨噬细胞的活性和免疫功能.肿瘤的转移程度可能与其分泌免疫抑制因子的能力相关.     

Suppression of IL-12 production by phosphodiesterase inhibition in murine endotoxemia is IL-10 independent

Phosphodiesterase (PDE) inhibitors are potent regulators of various immune processes. Immune cells contain type IV and type III PDE. Here we studied in mice the effects of rolipram, a selective PDE IV inhibitor, and amrinone, a selective PDE III blocker, on plasma levels of IL-12 (p70), IFN-γ, IL-1, TNF-α, and nitric oxide (NO) induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (80 mg/kg). Pretreatment of BALB/c mice with both rolipram (1 – 25 mg/kg) and amrinone (10 – 100 mg/kg) decreased plasma IL-12 levels in a dose-dependent manner. Similarly, LPS-elicited plasma IFN-γ concentrations were suppressed by both rolipram and amrinone. However, LPS-induced plasma IL-1α levels were not affected by either of these compounds. In addition, rolipram inhibited IL-12, IFN-γ, TNF-α and nitrite/nitrate (breakdown products of NO) production in C57BL/6 IL-10^+/+ mice as well as in their IL-10-deficient counterparts (C57BL/6 IL-10^−/−). Our results suggest that rolipram and amrinone decrease the immune activation in endotoxemia through inhibition of the production of pro-inflammatory mediators IL-12, IFN-γ, TNF-α and NO. These effects are not the consequences of the increase in IL-10 production by PDE inhibition.     

Tumour Growth Causes Suppression of Autoreactive T-Cell Proliferation by Disrupting Macrophage Responsiveness to Interferon-γ

Normal immune homeostasis is regulated partly by a small population of CD4⁺ T cells that react to autologous major histocompatibility complex class-II molecules on self-cells. Decreased autoreactive T-cell responses are associated with cancer. Tumour growth causes syngeneic macrophages (Mø) to suppress autoreactive T-cell proliferation by decreasing Mø class-II expression and increasing Mø production of the suppressor molecule prostaglandin E₂ (PGE₂). Because interferon-γ (IFN-γ) is a potent Mø activation molecule which regulates both Mø PGE₂ and class-II expression, the effects of IFN-γ on tumour-induced suppression of autoreactive T-cell proliferation were investigated. Exogenous IFN-γ increased normal host (NH) CD4⁺ autoreactive T-cell proliferation stimulated by syngeneic NHMø but decreased proliferation stimulated by tumour-bearing host (TBH) Mø. Antibody (Ab) neutralization of endogenous IFN-γ activity reduced TBH Mø-mediated suppression. Kinetic studies showed that endogenous IFN-γ suppressor activity was not exclusive during T-cell activation. Indomelhacin treatment blocked IFN-γ-induced suppression in TBH Mø-T cell cultures. TBH Mø-T cell cultures contained significantly more PGE₂ than those containing NH Mø. Exogenous IFN-γ increased early PGE₂ production in TBH Mø cultures but decreased production in NHMø cultures. The Ab-mediated neutralization of endogenous transforming growth factor-β or tumour necrosis factor-x reduced TBH Mμ-mediated suppression and blocked IFN-γ-induced suppression. Short-term treatment of Mμ with IFN-γ before their addition to T cells caused TBH Mμ to stimulate T-cell proliferation, which suggests that early suppressor molecule production by TBHMø inhibits synthesis or activity of IFN-γ-induced stimulatory monokines. These results show that tumour growth causes Mø to suppress autoreactive T-cell responses by allowing IFN-γ to induce Mø suppressor molecules, which block production or activity of stimulatory monokines.     

Interferon-γ and interleukin-4 reciprocally regulate the production of monocytes/macrophages and neutrophils through a direct effect on committed monopotential bone marrow progenitor cells

We studied the direct effects of interferon-γ (IFN-γ) in single cell colony assays of CD34⁺HLA-DR++ bone marrow progenitor cells stimulated by either granu-locyte-colony-stimulating factor (G-CSF), interleukin(IL)-3, granulocyte/macro-phage-colony-stimulating factor (GM-CSF), combinations of these CSF or medium conditioned by the 5637 human bladder carcinoma cell line. In this culture system IFN-γ stimulated monocytic colonies (CFU-M) no matter which CSF or CSF combination was used to support them and inhibited granulocytic colonies (CFU-G) if they were generated in the presence of G-CSF. IL-4 antagonized the myelopoietic effects of IFN-γ: the IFN-γ induced suppression of G-CSF-supported CFU-G, as well as the stimulation of CFU-M, were reversed by IL-4. In all cultures, IFN-γ had a limited, but statistically non-significant, inhibitory effect on CFU-GM, which was not affected by the presence of IL-4. These data show that IFN-γ and IL-4 reciprocally regulate the generation of myeloid cells involved in humoral (neutrophils) and cellular (macrophages) immune responses through a direct effect on monopotential myeloid progenitor cells.     

Induction of nitric oxide production by bovine alveolar macrophages in response toPasteurella haemolyticaA1

We assessed the kinetics of inducible nitric oxide synthase (iNOS) mRNA expression and production of nitric oxide (NO) in bovine alveolar macrophages (AMs) stimulated with purified lipopolysaccharide (LPS) fromPasteurella haemolyticastrain 12296. The effect of LPS on iNOS gene expression was dose-dependent and was expressed maximally at 24 h after stimulation with 10 μg/ml of LPS. Production of NO measured as secreted nitrite in supernatants took place in a time and dose-dependent manner with peak production at 24 h after LPS stimulation. Recombinant bovine gamma interferon (rbγIFN) augmented the LPS-induced iNOS gene expression and production of NO. The ability of LPS to induce iNOS gene expression and NO production either alone or in combination with rbγIFN was significantly abrogated by polymyxin B. In addition, the iNOS inhibitor N^G-monomethyl-Larginine (L-NMMA) significantly inhibited LPS and rbγIFN+LPS induced NO production. Our results also demonstrated that NO produced from an exogenous NO donor sodium nitroprusside (SNP), and NO generated from LPS-stimulated AMs (endogenous) caused cytotoxic injury to bovine pulmonary artery endothelial cells in a dose-dependent manner. The cytotoxic injury caused by NO generated from LPS stimulated AMs was inhibited by polymyxin B or L-NMMA. There was a markedly increased concentration of nitrite in the lung lavage fluids of calves followingP. haemolyticainfection. These findings support a role for NO in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.