Immunohistochemical dynamics of leukotoxin (9.10-eooxv-12-octadecenoic acid) in lungs of rats

This paper investigates the immunohistochemical dynamics of leukotoxin (9,10-epoxy-12-octadecenoic acid, LTx) in the lungs of rats exposed to hyperoxia with or without paraquat. The rats were treated with 100% oxygen or ambient air for 24. 48, 72 and 96 h in the presence or absence of a low or high dose paraquat (1,1-di-methyl-4,4-bipyridinium, PQ) injection. Immunostaining for LTx demonstrated positive reactions in the neutrophils that showed a progressive increase in intensity of staining with time in all groups exposed to 100% oxygen and in the group with high dose PQ, but the positive findings were weak in the group injected with low dose PQ only. We found the positive immunostaining reaction not only in neutrophils but also in alveolar macrophages. This indicates that LTx is produced by alveolar macrophages as well as by neutrophils depending on the treatment period under hyperoxic conditions, suggesting that LTx is an important chemical mediator in pulmonary diseases.     

脾切除对小鼠腹腔巨噬细胞数量和功能的影响

目的：研究脾切除对小鼠腹腔巨噬细胞数量和功能活性的影响。方法：术后4周获取腹腔常居巨噬细胞和炎症巨噬细胞,检测其数量、吞噬功能和一氧化氨水平。结果：与正常对照组和假手术组比较,脾切除组小鼠的2种巨噬细胞不仅数量明显减少,而且它们的吞噬功能和一氧化氮水平呈明显下降。结论：提示脾切除造成的单核吞噬细胞继发性炎症反应的捐伤和巨噬细胞功能活性的降低,可能是脾切除后(凶险性)感染的重要因素。     

白细胞介素-1受体拮抗剂对博莱霉素致大鼠肺纤维化模型的影响

目的：探讨白细胞介素１受体拮抗剂（ｉｎｔｅｒｌｅｕｋｉｎ１ｒｅｃｅｐｔｏｒａｎｔａｇｏｎｉｓｔ,ＩＬ１ｒａ）治疗博莱霉素（ｂｌｅｏｍｙｃｉｎ,ＢＬＭ）致大鼠肺纤维化模型的作用机制。方法：利用ＭＴＴ法测定经ＩＬ１ｒａ作用前、后１周时该模型大鼠肺泡巨噬细胞产生肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα,ＴＮＦα）,及其培养上清促肺成纤维细胞增殖能力的变化;利用Ｎｏｒｔｈｅｒｎ杂交测定肺泡巨噬细胞ＴＮＦα、血小板衍化生长因子（ｐｌａｔｅｌｅｔｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ,ＰＤＧＦ）ｍＲＮＡ的表达。结果：ＢＬＭ致肺纤维化模型１周时,肺泡巨噬细胞ＴＮＦα的产生及该组细胞上清的促成纤维细胞增殖活性与正常对照组相比均明显增强,而ＩＬ１ｒａ（１０ｍｇ·Ｌ－１）对肺泡巨噬细胞ＴＮＦα的产生及该组细胞上清的促成纤维细胞增殖活性有明显的抑制作用。ＢＬＭ致肺纤维化模型１周时肺泡巨噬细胞ＴＮＦα、ＰＤＧＦｍＲＮＡ的表达较正常对照组增高,而ＩＬ１ｒａ对其表达无明显影响。结论：ＩＬ１ｒａ通过对肺泡巨噬细胞的ＴＮＦα产生及其对成纤维细胞增殖活性的抑制,可能对该模型的肺纤维化起到抑制作用     

支气管哮喘患者肺泡巨噬细胞释放内皮素-1的研究

为研究支气管哮喘患者肺泡巨噬细胞（Ａｍ）所释放的内皮素１（ＥＴ１）在哮喘发病中的作用,对发作期哮喘患者２０例,对照组８例,经纤支镜行支气管肺泡灌洗术获得Ａｍ,调整Ａｍ至１×１０６／ｍｌ后分３孔体外培养（１ｍｌ／孔）;生理盐水对照孔、氟美松孔（１μｇ／ｍｌ）、氨茶碱孔（１５μｇ／ｍｌ）,培养６ｈ后取上清液,采用放免法测定ＥＴ１。显示哮喘组血浆ＥＴ１明显高于对照组,并于第１秒用力肺活量（ＦＥＶ１．０）呈显著负相关。哮喘组Ａｍ体外释放ＥＴ１与对照组无显著差异,与ＦＥＶ１．０无相关性。氟美松孔Ａｍ释放ＥＴ１明显低于生理盐水对照孔;氨茶碱孔与生理盐水孔或氟美松孔无差异。提示哮喘Ａｍ所释放ＥＴ１在哮喘发病中意义不大     

The role of VCAM-l/VLA-4 in the activation of allogenic T cells by murine macrophages

Vascular cell adhesion molecule 1 (VCAM-1) is a member of immunoglobulin superfamily. The principal ligand for VCAM-1 is integrin α4β/VLA-4 (very late antigen 4). It was reported that VCAM-1 was expressed on macrophages and dendritic cells, but little is known about its function on these professional antigen presenting cells (APC). The present study was performed to investigate the expression of VCAM-1 on macrophages and the role of VCAM-l/VLA-4 in the activation of allogenic T cells by murine macrophages. We analyzed VCAM-1 expression on peritoneal macrophages and macrophage cell line J774A.1 by fluorescence-activated cell sorting (FACS). Using neutralizing antibodies, we further analyzed the role of VCAM-l/VLA-4 interaction in macrophage and allogenic T cell mixed lymphocyte reaction (MLR). We found that VCAM-1 was constitutively expressed on macrophages and its expression level was upregulated by soluble tumor associated antigen (freeze-thaw lysates of FBL-3 leukemia cells) and TNF-a. In MLR assays, we observed that blocking VCAM-l/VLA-4 interaction with anti-VCAM-1 or anti-VLA-4 mAbs caused significant inhibition of the proliferative response and IL-2 production. These results suggest that VCAM-lon macrophages not only facilitates the cell-to-cell contact through adhesive interaction but also plays a role in the costimulation of T cells via its interaction with VLA-4 on the T cells. This work was supported by grants from the National Natural Science Foundation of China.No. (39730420). This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997).     

Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A(2).

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A(2), and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H(2)O(2)) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 microg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcgamma, complement, mannose and beta-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and beta-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H(2)O(2) by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H(2)O(2) only at a concentration of 3.2 microg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5-2.5 microg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages up regulating phagocytosis, mainly via mannose and beta-glucan receptors, and the respiratory burst.     

Microcystin-LR promote intestinal secretion of water and electrolytes in rats.

We showed previously that exposure to microcystin-LR causes renal toxic effects in isolated perfused rat kidney, and that inflammatory mediators from supernatants of macrophages stimulated by microcystin-LR are involved in this process. The aim of this research was to examine water and electrolytes secretion in vivo, induced by microcystin-LR and supernatant of macrophages stimulated for this toxin (SUP.MphiS + MCLR), using perfused rat ileal segment and ligated intestinal loop models. We found microcystin-LR at 1 microg/ml (0.09 +/- 0.003* vs. control 0.07 +/- 0.001 g of secretion/2 cm of loop; P < 0.05*) and the SUP.MphiS + MCLR after 18 h postinoculation (0.10 +/- 0.003 vs. control 0.03 +/- 0.002 g/cm) caused intestinal secretion. In addition, microcystin-LR caused significant sodium secretion (-2.18 +/- 0.72* vs. control 2.18 +/- 0.50 microEq g(-1) min(-1)), potassium (-0.26 +/- 0.04* vs. control 0.32 +/- 0.03 microEq g(-1) min(-1)), chloride (MCLR = -3.29 +/- 1.93* vs. control 0.88 +/- 1.25 microEq g(-1) min(-1)) and water (-0.012 +/- 0.004* vs. control 0.002 +/- 0.002 ml g(-1) min(-1)). We also demonstrated SUP.MphiS + MCLR to induce intestinal secretion of electrolytes (sodium, potassium, chloride) and water. These findings suggested that microcystin-LR and lamina propria macrophages-derived mediators are able to induce intestinal secretion in vivo, probably via inhibition of protein phosphatase.     

Anti-atherosclerotic action of GW9508 – Free fatty acid receptors activator – In apoE-knockout mice

BackgroundIn the past two decades, enhanced understanding of the biology of G-protein-coupled receptors (GPRs) has led to the identification of several such receptors as novel targets for free fatty acids (FFAs). Two GPRs, FFAR1 and FFAR4, have received special attention in the context of chronic inflammatory diseases, thanks to their anti-inflammatory activities.MethodsThe present study investigates the influence of prolonged treatment with GW9508 – agonist of FFAR1 and FFAR4 – on the development of atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods.ResultsGW9508 administration has led to the reduction of atheroscletoric plaque size in an apoE-knockout mice model. Moreover, a FFAR1/FFAR4 agonist reduced the content of macrophages by almost 20%, attributed by immunohistochemical phenotyping to the pro-inflammatory M1-like activation state macrophages.ConclusionsProlonged administration of GW9508 resulted in significant amelioration of atherogenesis, providing evidence that the strategy based on macrophage phenotype switching toward an M2-like activation state via stimulation of FFAR1/FFAR4 receptors holds promise for a new approach to the prevention or treatment of atherosclerosis.     

Outer membrane protein A (OmpA) from Shigella flexneri 2a: A promising subunit vaccine candidate

Shigellosis is the leading cause of childhood mortality and morbidity. Despite many years of extensive research a practical vaccine is not yet available against the disease. Recent studies illustrate that bacterial outer membrane proteins are budding target as vaccine antigen. Outer membrane proteins A (OmpA) are among the most immunodominant antigens in the outer membrane of gram negative bacteria and possess many characteristics desired of a vaccine candidate. We observe that OmpA of Shigella flexneri 2a is crossreactive and common antigen among Shigella spp. and the epitope is widely exposed on the cell surface as well as capable of evoking protective immunity in mice. The protective immunity involves participation of both the humoral and cellular immune responses, since OmpA boosts rapid induction of IgG and IgA in both the systemic and mucosal compartments and also activates Th1 cells. The immunopotentiating activity of OmpA is mediated by its ability to bind and stimulate macrophages and up-regulate the surface expression of MHCII, CD80 and CD40, leading to activation of CD4⁺ T cells to secrete cytokines and express chemokine receptor and IL-12Rβ2, thereby orchestrating the bridge between innate and adaptive immune responses. This ability is dependent on Toll-like receptor 2 (TLR2), as demonstrated by lack of response by TLR2 knockdown macrophages to OmpA. Hence this property of OmpA to link innate and adaptive immunity via TLR2 offers a novel vista to develop vaccine against shigellosis.     

Silymarin Inhibits Morphological Changes in LPS-Stimulated Macrophages by Blocking NF-κB Pathway

The present study showed that silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), inhibited lipopolysaccharide (LPS)-induced morphological changes in the mouse RAW264.7 macrophage cell line. We also showed that silymarin inhibited the nuclear translocation and transactivation activities of nuclear factor-kappa B (NF-κB), which is important for macrophage activation-associated changes in cell morphology and gene expression of inflammatory cytokines. BAY-11-7085, an NF-κB inhibitor, abrogated LPS-induced morphological changes and NO production, similar to silymarin. Treatment of RAW264.7 cells with silymarin also inhibited LPS-stimulated activation of mitogen-activated protein kinases (MAPKs). Collectively, these experiments demonstrated that silymarin inhibited LPS-induced morphological changes in the RAW264.7 mouse macrophage cell line. Our findings indicated that the most likely mechanism underlying this biological effect involved inhibition of the MAPK pathway and NF-κB activity. Inhibition of these activities by silymarin is a potentially useful strategy for the treatment of inflammation because of the critical roles played by MAPK and NF-κB in mediating inflammatory responses in macrophages.