The Contributions of Reactive Oxygen Intermediates and Reactive Nitrogen Intermediates to Listericidal Mechanisms Differ in Macrophages Activated Pre- and Postinfection

The contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the killing of Listeria monocytogenes by macrophages activated by addition of spleen cells from listeria-immune mice plus specific antigen was examined. When macrophages were infected with L. monocytogenes and then spleen cells were added, there was not as big a difference in listericidal activity between macrophages cultured with normal spleen cells and those cultured with immune spleen cells as expected. In this culture system, RNI was mainly involved in the macrophage intracellular killing. In macrophages first activated and then infected, a significant level of enhanced killing was observed. Blockade of ROI production drastically affected the enhanced killing ability, while inhibition of RNI production had a negligible effect. Thus, the contributions of ROI and RNI to listericidal mechanisms of macrophages were different between macrophages activated at pre- and postinfection stages.     

Bacteriostatic Activity of BCG/PPD-Activated Macrophages Against Mycobacterium fortuitum Does Not Involve Reactive Nitrogen or Oxygen Intermediates

Mycobacteria preferentially reside in resident macrophages whereas activated macrophages are presumed to eliminate the bacteria effectively, The aim of the present study was to determine the antibacterial activities of resident and activated murine peritoneal macrophages against Mycobacterium fortuitum and the intracellular mechanisms involved. After phagocytosis M. fortuitum could not be killed by either BCG/PPD-activated and IFN-γ-activated macrophages and resident macrophages. The mycobacteria did not multiply in BCG/PPD-activated macrophages and the rate of proliferation of M. fortuitum in IFN-γ-activated macrophages was only slightly inhibited compared to that in resident macrophages. Experiments with selective inhibitors of the production of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) demonstrated that these factors are not essential for the mycobacteriostatic activity of BCG/PPD-activated macrophages. After phagocytosis of M. fortuitum , BCG/PPD-activated and IFN-γ-activated macrophages produced substantial amounts of both RNI and ROI. No correlation was found between the levels of these intermediates and the proliferation of M. fortuitum in the macrophages. In conclusion, BCG/PPD-activated macrophages are bacteriostatic, but not bacteriocidal for M. fortuitum and the former does not involve reactive nitrogen and oxygen intermediates.     

Phagocytic Activity of Peritoneal and Omental Macrophages of Athymic Nude Mice

Phagocytosis of synthetic microspheres was determined by peritoneal macrophages and omental dendritic cells of nu/nu and nu/+ BALB/c and C57BL/10. LP mice during ontogenetic development. The macrophages of nu/nu mice phagocytized comparably to those of the nu/+ littermates till day 10 of their life. On day 25, the phagocytic activity of nu/nu mice sharply decreased but returned to normal and higher values at the age of 2 months. Omental dendritic cells showed similar ontogenetic behaviour in their ability to engulf microspheres as was found for free peritoneal macrophages.     

Phagocytic Activity of Peritoneal and Omental Macrophages of Athymic Nude Mice

Phagocytosis of synthetic microspheres was determined by peritoneal macrophages and omental dendritic cells of nu/nu and nu/+ BALB/c and C57BL/10. LP mice during ontogenetic development. The macrophages of nu/nu mice phagocytized comparably to those of the nu/+ littermates till day 10 of their life. On day 25, the phagocytic activity of nu/nu mice sharply decreased but returned to normal and higher values at the age of 2 months. Omental dendritic cells showed similar ontogenetic behaviour in their ability to engulf microspheres as was found for free peritoneal macrophages.     

Hydrocortisone Treatment of BCG-Infected Mice Impairs the Activation and Enhancement of Antimicrobial Activity of Peritoneal Macrophages

The present study concerns the effect of hydrocortisone (HC) on the effector functions of Bacillus Calmette Guerin-purified protein derivative (BCG-PPD)-activated macrophages. Such activated macrophages release greater amounts of H2O2 and NO2-, inhibit the intracellular proliferation of T. gondii and kill L. monocytogenes more efficiently than resident macrophages. This activation was not fully expressed by macrophages from BCG-activated mice that had received a subcutaneous injection of HC 2 days before intraperitoneal injection of PPD, since the inhibition of the intracellular proliferation of T. gondii, the release of NO2- and the rate of intracellular killing of L. monocytogenes were lower than in macrophages from BCG-PPD-activated mice. However, treatment with HC did not impair the release of H2O2 by BCG-PPD-activated macrophages. The results show that the treatment of infected mice with HC inhibits their ability to develop adequate intracellular microbicidal mechanisms.     

The Global Regulator ArcA Controls Resistance to Reactive Nitrogen and Oxygen Intermediates in Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis is a major cause of food-borne diseases associated with consumption of shell eggs. Clinical isolates of S. enterica serovar Enteritidis exhibit a wide spectrum of virulence in mice. A highly virulent isolate (SE2472) was previously shown to be more resistant in vitro than other clinical isolates to acidified sodium nitrite (ASN), a generator of reactive nitrogen and oxygen intermediates (RNI/ROI). SE2472 is also more resistant to S-nitrosoglutathione (GSNO) and hydrogen peroxide (H(2)O(2)) than an ASN-susceptible isolate of S. enterica serovar Enteritidis (SE8743). To investigate the molecular basis for the RNI/ROI resistance of S. enterica serovar Enteritidis, we transformed a genomic DNA library of SE2472 into SE8743. A plasmid clone conferred upon SE8743 enhanced resistance to ASN, GSNO, and H(2)O(2). The DNA insert in the clone encoded ArcA, a global regulator. An arcA mutant of SE2472 was constructed and was found to be more susceptible to GSNO and hydrogen peroxide but not more susceptible to ASN than wild-type SE2472. The susceptibility of the arcA mutant to GSNO and H(2)O(2) was complemented by a plasmid harboring arcA. The coding sequence of the arcA gene in SE2472 and the coding sequence of the arcA gene in SE8743 were identical, suggesting that the difference in resistance to RNI/ROI maybe due to the activity of genes regulated by ArcA. No significant difference in virulence between the wild type and the arcA mutant of SE2472 was observed in mice. These observations show that arcA is essential for resistance of S. enterica serovar Enteritidis to nitrosative and oxidative stress. However, additional genetic loci may contribute to the resistance to RNI/ROI and unusually high virulence for mice of SE2472.     

外周血单核细胞SOCS-1表达与MODS患者预后关系的研究

目的了解外周血单核细胞SOCS-1表达与多器官功能障碍综合征（MODS）患者预后的关系及临床意义。方法收集24例MODS患者，并采集其外周静脉血，采用淋巴细胞分离液密度梯度离心法分离外周血单核细胞（PBMCs），分别以RT-PCR法及Western-blot法检测PBMCs中SOCS-1的基因及蛋白表达，分析其与预后及MODS评分的关系。结果MODS组中死亡患者PBMCs中的SOCS-1mRNA表达量（0.4938±0.0273）显著低于存活患者（0.5475±0.0289）（P〈0.05），SOCS-1蛋白表达量（0.7924±0.0284）显著低于存活患者（0.8406±0.0407）（P〈0.05）。MODS患者的PBMCs中的SOCS-1mRNA表达量与MODS评分呈显著的负相关关系（r=-0.723,P〈0.01），SOCS-1蛋白表达量与MODS评分呈显著的负相关关系（r=-0.534，P〈0.01）。结论在MODS中，SOCS-1的表达可能起到保护组织避免损伤的作用,SOCS-1表达的减少可能提示患者的预后不良     

SOCS-1和TNF-α在有机磷中毒致MODS鼠肝脾中表达的研究

目的探讨SOCS-1和TNF-α在急性有机磷农药（AOPP）致多器官功能障碍综合症（MODS）中的发病机制，从而为MODS的防治提供新的策略。方法健康小鼠随机分成敌敌畏组、生理盐水组、正常对照组，分别于染毒后2、6、12、24h取小鼠的肝脾，以逆转录聚合酶链反应（RT-PCR）测定SOCS-1和TNF-α的mRNA表达水平。用SPSS统计软件进行分析。结果AOPP小鼠肝脾SOCS-1和TNF-α的mRNA水平均升高．与对照组比较差异有统计学意义（P均〈0．05）。结论AOPP可导致细胞因子TNF-α失调，可能参与了AOPP后SIRS向MODS的发病过程；TNF-α可诱导SOCS-1在肝脾中表达，考虑到SOCS的“负反馈”调控作用。若能抑制炎症反应的失控，则对中毒MODS的治疗将有新的突破。     

维生素E对家兔多器官功能障碍血管内皮细胞的保护作用

利用家免多器官功能障碍综合征（MODS）模型观察维生素E（VitE）对血管内皮细胞的保护作用。采用内皮细胞形态观察并同步检测血液MDA、SOD、CAT、TXB2、6－K－PGFIα以及应用VitE后对其影响。结果示MODS组动物血中MDA和TXB2含量随MODS的发生发展显著升高（P＜0．01），而SOD、CAT及6－K－PGFIα含量降低（P＜0．01）。利用扫描和透射电镜观察MODS血管内皮细胞发生显著损伤，失去正常形态；而补充VitE组前述各项指标均得到不同程度的改善．观察结果提示，MODS动物体内脂质过氧化损伤明显增强，而抗氧化能力明尼降低。这种变化可能在MODS发病中具有重要的病理生理意义。早期应用VitE对MODS的防治是有益的。     

MARS治疗重症恙虫病合并多脏器功能障碍综合征的疗效

目的评价分子吸附再循环系统（MARS）治疗重症恙虫病合并多脏器功能障碍综合征（MODS）的效果。方法60例重症恙虫病合并MODS患者，随机分为MARS治疗组、连续性肾脏替代（CRRT）治疗组和常规综合治疗组。观察并比较组间血流动力学、肝肾功能、凝血功能、呼吸功能、炎症介质以及Marshall评分等在治疗前后的变化，比较3组患者60d内的生存曲线的差异。结果MARS治疗后平均心率减慢，平均动脉压升高，总胆红素明显降低，白细胞计数、D-二聚体水平明显降低，血小板计数明显升高，一氧化氮、TNF-α、IL-6、IL-8、IL-10、IL-13和LBP水平明显降低，PaO2和氧合指数水平均显著提高，气道峰压明显降低。Marshall值降低，均优于常规综合治疗组和CRRT组（P〈0．05）。MARS治疗后呼吸频率和肌酐水平降低、SaO2和白蛋白水平升高，优于常规综合治疗组（P〈0．05）。60d的生存概率分析，MARS组存活率90％（18／20），优于常规综合治疗组存活率45％（9／20）（P=0．001），优于CRRT组60％（12／20）（P=0．031）。结论MARS治疗重症恙虫病合并MODS疗效良好，优于常规综合治疗和CRRT治疗。     

Role of Liver NK Cells and Peritoneal Macrophages in Gamma Interferon and Interleukin-10 Production in Experimental Bacterial Peritonitis in Mice

Gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) production by liver, spleen, lung, peripheral blood mononuclear cells (MNC), and peritoneal exudate cells (PEC) in experimental bacterial peritonitis was examined by cecum ligation and puncture (CLP) (with an 18-gauge needle) of BALB/c mice. MNC of organs were cultured for 18 h, and cytokine levels in supernatants were examined. Cytokines contained in peritoneal lavage fluid were regarded as those produced by PEC. Only liver MNC and PEC produced substantial amounts of IFN-γ, and PEC were the main source of IL-10, especially 12 h after CLP. As reflected by the cytokine production by liver MNC and PEC, serum IFN-γ and IL-10 levels were elevated after CLP. C57BL/6 (B6) mice and BALB/c nude mice showed a similar pattern of cytokine production. TNF-α levels in culture supernatants, peritoneal lavage fluid, and sera were not significantly elevated compared to those of sham-operated mice. In vivo depletion of NK cells of B6 mice with anti-asialo GM1 or anti-NK1.1 antibody greatly decreased IFN-γ levels in liver MNC culture supernatants and sera, suggesting that liver NK cells are IFN-γ producers. On the other hand, plastic-adherent PEC macrophages are the major IL-10 producers. Mice subjected to a cecum ligation and cut procedure (which have a more severe peritonitis) showed much higher IFN-γ and IL-10 levels than those subjected to CLP, while mice subjected to CLP with a smaller (22-gauge) needle showed low levels of these cytokines. These findings show that liver NK cells and PEC macrophages are important for the production of proinflammatory and anti-inflammatory cytokines in bacterial peritonitis.     

肠血管活性多肽对多器官功能障碍综合征大鼠肠淋巴细胞归巢的影响

为观察肠血管活性多肽 (VIP )对大鼠肠缺血再灌注致多器官功能障碍综合征 (MODS )时 ,肠淋巴细胞归巢至肠相关淋巴细胞 (GALT )的调节以及对MODS转归的影响。本研究用微型无创动脉夹夹住大鼠肠系膜动脉根部 4 5min ,松夹后再灌注6h ,制备MODS模型。将VIP分别从股静脉输入和从腹腔注入大鼠体内。收集肠淋巴液 ,计数淋巴细胞总数及T、B淋巴细胞比例 ,了解淋巴细胞进入血循环状况。体外用51Cr标记肠淋巴细胞 ,回输入大鼠体内 ,用γ计数器检测各器官组织内的51Cr 肠淋巴细胞 ,了解肠淋巴细胞归巢。检测血及肠淋巴TNF α、内毒素 ;测定血D 乳酸、谷丙转氨酶 (ALT )、肌肝 (Cr)、血氧分压 ;观察重要器官组织学改变 ,评价VIP对MODS时各脏器形态及功能改变。结果显示 ,VIP使MODS大鼠肠粘膜迁移至血循环肠淋巴细胞总数 [(0 4 2± 0 18)× 10 7/h]较未予处理的MODS组 [(0 2 8± 0 15 )× 10 7/h]显著增加 (P <0 0 5 ) ,以T细胞数上升明显。VIP减少了MODS大鼠归巢至肠粘膜的肠淋巴细胞 ,Peyer淋巴结及小肠分布的51Cr 细胞量分别占总51Cr量的 2 14 %± 1 4 9%、 1 5 8%± 0 4 2 % ,显著低于未予处理的MODS组 (5 0 4 %± 1 2 3%和 3 2 3%± 1 6 9% ,P <0 0 1及P <0 0 5 )。外源性给予MODS大鼠VIP后 ,肠淋巴     

牛至挥发油对小鼠腹腔巨噬细胞活性及免疫器官重量的影响

一定浓度的牛至挥发油(OVLVO)能显著提高Balb/c小鼠腹腔巨噬细胞(Mφ)形成EA和YC花环的能力,显著增强腹腔Mφ的吞噬作用,有明显的增加小鼠脾脏重量的作用,而对胸腺的重量则无显著的影响。表明OVLVO在一定浓度下不引起免疫器官萎缩并能显著提高小鼠腹腔Mφ的活性。提示OVLVO的抗感染作用与增强Mφ的功能有关。     

The Morphology of Echinoid Phagocytes and Mouse Peritoneal Macrophages During Phagocytosis in Vitro

The morphology of mouse peritoneal macrophages and echinoid phagocytes during phagocytosis in vitro was studied. A striking similarity in the function of the foreign surface receptor is found in the two systems. Glutaraldehyde-treated erythrocytes attached randomly over the entire surface of the cells and were internalized without circumferential attachment between the particles and the phagocyte membrane. The particles seemed to sink directly into the cytoplasm of the cells. Tannin-treated erythrocytes were phagocytosed by the echinoid cells in a similar mode. The complement-coated erythrocytes were attached only in the perinuclear area of the echinoid phagocyte's membrane, but the morphology of their internalization was similar to that mediated by the foreign surface receptor. A circumferential attachment between the particles and the phagocyte membrane did not seem necessary. This is also the case for mouse peritoneal macrophages.     

Effects of Reactive Oxygen and Nitrogen Metabolites on RANTES- and IL-5-Induced Eosinophil Chemotactic Activity in Vitro

Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis.     

Control of the Production of Oxygen Intermediates of Human Polymorphonuclear Leukocytes and Monocytes by B-Adrenergic Receptors

Abstract

The control by R-adrenergic receptors of the production of oxygen radicals by zymosan-stimulated human polymorphonuclear leukocytes (PMN) and monocytes (Mψ) was studied in vitro by means of chemiluminescence. In addition we asked whether PMN Mpsi; exhibit differential sensitivity to β-adrenergic stimulation. Eor β-adrenergic stimulation we applied fenoterol ranging from 10^?9 to 10 M × 2.7. We found a dose-dependent suppression of the production of oxygen radicals, the ID50 being approximately 10^?6 M both for PMN and Mpsi;. By assessment of lactic dehydrogenase release a cytotoxic effect of the drug could be ruled out. When incubated together with the β-adrenergic antagonist propranolol at 10^?6 and 10^?7 M the suppressive effect of fenoterol could be reversed in dose-dependency. Preincubation with fenoterol revealed that the inhibitory action on Mpsi; persisted, in contrast, no such suppression could be verified with PMN. Our findings indicate the control of the production of oxygen intermediates of human PMN and Mpsi; by β-adrenergic stimulation. Furthermore, selective functional modulation of resting PMN and Mpsi; by β-adrenoceptors is suggested. These effects may be of importance in vivo, in particular since fenoterol was applied in pharmacological doses.     

Control of the Production of Oxygen Intermediates of Human Polymorphonuclear Leukocytes and Monocytes by B-Adrenergic Receptors

The control by R-adrenergic receptors of the production of oxygen radicals by zymosan-stimulated human polymorphonuclear leukocytes (PMN) and monocytes (Mψ) was studied in vitro by means of chemiluminescence. In addition we asked whether PMN Mpsi; exhibit differential sensitivity to β-adrenergic stimulation. Eor β-adrenergic stimulation we applied fenoterol ranging from 10^-9 to 10 M × 2.7. We found a dose-dependent suppression of the production of oxygen radicals, the ID50 being approximately 10^-6 M both for PMN and Mpsi;. By assessment of lactic dehydrogenase release a cytotoxic effect of the drug could be ruled out. When incubated together with the β-adrenergic antagonist propranolol at 10^-6 and 10^-7 M the suppressive effect of fenoterol could be reversed in dose-dependency. Preincubation with fenoterol revealed that the inhibitory action on Mpsi; persisted, in contrast, no such suppression could be verified with PMN. Our findings indicate the control of the production of oxygen intermediates of human PMN and Mpsi; by β-adrenergic stimulation. Furthermore, selective functional modulation of resting PMN and Mpsi; by β-adrenoceptors is suggested. These effects may be of importance in vivo, in particular since fenoterol was applied in pharmacological doses.