In vitro immunity of rat peritoneal macrophages to Candida albicans

Phagocytic, germ tube inducing and candidacidal activities were investigated in monolayers of peritoneal macrophages of rats. The phagocytic activities observed in macrophages of the healthy rats in the presence of normal serum, those in the presence of immune serum and of immunized rats in the presence of normal serum were 40%, 45.3% and 44.8% respectively. The percent of macrophages in which intracellular Candida formed germ tubes in the above three situations were 10, 9.59 and 10.19, respectively and the percent of intracellular Candida that formed germ tubes were 6.6, 3.7 and 4.1, respectively. The candidacidal activity observed in the above three sets of macrophages were 5.33%, 22.66% and 19.88%, respectively. Induction of germ tube in C. albicans in supplemented tissue culture medium containing normal serum was 15 per cent. These observations indicate that immunisation/sensitisation of individuals with C. albicans organisms does provide some degree of cell mediated immunity by activating macrophages. This may partly be due to the appearance of specific antibodies. It is likely that this type of immunity can be produced by subclinical infections during invasion by the commensal organism thus preventing further invasion establishment of infection and keeping the organism (C. albicans) in a state of commensalism. However, the degree of immunity so produced is so low that predisposing factors suppress it and allow establishment of infection.     

Uptake of transferrin by rat peritoneal macrophages

S ummary . Activated rat peritoneal macrophages bind ¹²⁵I-apotransferrin in a time and temperature-dependent process, the amount of transferrin taken up at 4°C amounting to only about 15% of that bound at physiological temperatures. Binding is reversible, saturable, and largely abolished by prior treatment of the cells with Pronase. A single class of high affinity binding sites is evidenced by Scatchard analysis, each cell binding about 110 000 apotransferrin molecules with an apparent affinity constant of 1.4 x 10⁶ 1 mol^-1. Macrophages are also capable of binding about one-third as much iron-saturated transferrin as iron-free transferrin. Since binding of neither form of the protein is influenced by the presence of the other, separate and independent binding sites for apotransferrin and iron transferrin are presumed to exist on the macrophage.     

Aging affects lipopolysaccharide-induced upregulation of heme oxygenase-1 in the lungs and alveolar macrophages

Lung injuries are generally more serious and cause high mortality in aged humans and animals. Heme Oxygenase-1 (HO-1) is known to be readily inducible in alveolar macrophages (AMs) and airway epithelial cells to confer cytoprotection against oxidative stress. We thus investigated whether aging impairs the stress-induced upregulation of HO-1. In this study, we first quantified basal levels of HO-1 expression in lungs from male ICR mice of various ages. Second, young (9–11 weeks) and old (65–66 weeks) mice were subjected to intratracheal administration of lipopolysaccharide (LPS) and expression of HO-1 in the lungs was quantified at 2, 24 and 72 h. HO-1 expression in bronchiolar epithelial cells harvested by laser capture microdissection (LCM) was also specifically quantified in the two age groups. Third, we examined HO-1 expression in AMs lavaged from 22-week-old and 86–96-week-old male ICR mice in response to LPS for 24 h in vitro. We found that basal expression of HO-1 in the lungs did not differ with age. LPS-induced HO-1 upregulation was significantly impaired in the lungs of 65–66-week-old mice than in 9–11-week-old mice at 2 and 24 h, although there were no differences in the magnitude of HO-1 upregulation in bronchiolar epithelium at 2 h. LPS-induced upregulation of HO-1 was observed in AMs from 22-week-old mice (1.8-fold), but not in AMs from 86–96-week-old mice in vitro. In summary, we demonstrated age-related defects in HO-1 induction in the whole lungs and in AMs in response to LPS.     

Interaction of transferrin with iron-loaded rat peritoneal macrophages

Rat peritoneal macrophages are capable, in vitro, of processing and releasing iron derived from phagocytosed, immunosensitized red cells. From 20% to 60% of the red cell iron can be returned to the culture medium in 24 h, with resident macrophages more active than inflammatory, peptone-induced macrophages. When apotransferrin is present in the culture medium, from 39% to 72% of iron released from macrophages is bound to the protein, with most of the remainder in a ferritin-like form. No distinct preference of released iron for either site of transferrin could be observed. The absence of apotransferrin depresses iron release only slightly, with much of the iron then released in a form readily available to the protein in vitro. Pronase treatment of macrophages, which abolishes their ability to bind transferrin, depresses iron release no more than 10-15%. It appears, therefore, that binding of apotransferrin to macrophages may not be essential for iron excretion by the cells.     

Aging affects passive stiffness and spindle function of the rat soleus muscle

Aging affects many motor functions, notably the spinal stretch reflexes and muscle spindle sensitivity. Spindle activation also depends on the elastic properties of the structures linked to the proprioceptive receptors. We have calculated a spindle efficacy index, SEI, for old rats. This index relates the spindle sensitivity, deduced from electroneurograms recording (ENG), to the passive stiffness of the muscle. Spindle sensitivity and passive incremental stiffness were calculated during ramp and hold stretches imposed on pseudo-isolated soleus muscles of control rats (aged 4 months, n=12) and old rats (aged 24 months, n=16). SEI were calculated for the dynamic and static phases of ramp (1-80 mm/s) and for hold (0.5-2mm) stretches imposed at two reference lengths: length threshold for spindle afferents discharges, L(n) (neurogram length) and slack length, L(s). The passive incremental stiffness was calculated from the peak and steady values of passive tension, measured under the stretch conditions used for the ENG recordings, and taking into account the muscle cross-sectional area. The pseudo-isolated soleus muscles were also stretched to establish the stress-strain relationship and to calculate muscle stiffness constant. The contralateral muscle was used to count muscle spindles and spindle fibers (ATPase staining) and immunostained to identify MyHC isoforms. L(n) and L(s) lengths were not significantly different in the control group, while L(n) was significantly greater than L(s) in old muscles. Under dynamic conditions, the SEI of old muscles was the same as in controls at L(s), but it was significantly lower than in controls at L(n) due to increased passive incremental stiffness under the stretch conditions used to analyze the ENG. Under static conditions, the SEI of old muscles was significantly lower than control values at all the stretch amplitudes and threshold lengths tested, due to increased passive incremental stiffness and decreased spindle sensitivity at L(s). The muscle stiffness constant values were greater in old muscles than in controls, confirming the changes in elastic properties under passive conditions due to aging. Aging also altered the intrafusal fibers: it increased the mean number of intrafusal fibers and the contents in the slow, neonatal and developmental isoforms intrafusal of MyHC have been modified. These structural modifications do not seem great enough to counteract the loss of the spindle sensitivity or the spindle efficacy under passive conditions and after the nerve was severed. However, they may help to maintain the spindle afferent message under natural conditions and under fusimotor control.     

地塞米松对大鼠腹腔巨噬细胞抗弓形虫作用的影响

目的研究地塞米松(Dexamethasone,DXM)对大鼠腹腔巨噬细胞抗弓形虫感染的影响。方法采用瑞-姬染色观察弓形虫在与DXM共孵育大鼠腹腔巨噬细胞内的增殖;以半定量RT-PCR法检测与DXM共孵育大鼠腹腔巨噬细胞IFN-γ、TNF-α和IL-2mRNA的表达,以ELISA法检测细胞培养上清中IFN-γ、TNF-α和IL-2的含量。结果弓形虫在DXM孵育的腹腔巨噬细胞内大量增殖,其弓形虫密度0h为(37±7)个/100个细胞,而24h时为(173±32)个/100个细胞(P<0.01);与DXM共孵育大鼠腹腔巨噬细胞24h时的IL-2、IFN-γ和TNF-αmRNA及其蛋白的表达与对照组相比均明显降低(P<0.01),如:DXM孵育组的TNF-α(187.52±39.41pg/ml)与对照组(115.43±22.46pg/ml)相比差异显著(P<0.01)。结论 DXM可诱发腹腔巨噬细胞易感弓形虫,其机制可能与细胞因子IFN-γ、TNF-α和IL-2表达下调有关。     

Eicosanoid profile of healing colon anastomosis and peritoneal macrophages in the rat.

Because intraperitoneal administration of prostaglandin E2 (PGE2) has a negative influence on the healing of colonic anastomosis, the production of eicosanoid products in the healing rat colon after resection and anastomosis was studied using high performance liquid chromatography. Normal colonic tissue metabolizes small amounts of arachidonic acid into cyclo-oxygenase and lipoxygenase products. After construction of an anastomosis, however, there is increased production of lipoxygenase products, while cyclooxygenase activity remains low. Increased amounts of PGE2 and other cyclo-oxygenase products are not produced after anastomosis of the colon and probably do not play a major role in uncomplicated healing of the large intestine in the rat. During the first eight days of repair in the anastomosed colonic tissue, a statistically significant increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was found compared with control colon tissue (p = 0.001). At the same time peritoneal macrophages from these rats showed increased 12-HETE production. Eicosanoid synthesis of peritoneal macrophages resembled eicosanoid synthesis of anastomosed colon taken from the same rat indicating that 12-HETE, in particular, may be of macrophage origin.     

Stimulation of rat peritoneal mast cells enhances uptake of low density lipoproteins by rat peritoneal macrophages in vivo

The effect of mast cell stimulation on the uptake of low density lipoproteins (LDL) by macrophages was tested in vivo in the peritoneal cavity of the rat, a site known to contain both macrophages and mast cells. The concentration of LDL in the peritoneal cavity was raised by injecting [14C]sucrose-labeled LDL ([14C]sucrose-LDL). In the treated rats, in the absence of mast cell stimulation, the uptake of LDL by the peritoneal macrophages was low. But when the peritoneal mast cells were concomitantly stimulated by intraperitoneal administration of compound 48/80, an agent known to induce mast cell degranulation, the rate of uptake of labeled LDL by macrophages rose by 7-24-fold. The reason for this rise was that exocytosed mast cell granules bound LDL and carried it into macrophages when phagocytosed. Thus, cyclohexanedione treatment of LDL, or injection of avidin along with LDL, 2 measures known to inhibit binding of LDL to mast cell granules, totally prevented the mast cell-dependent uptake of LDL. Furthermore transmission electron microscopic studies with gold-labeled LDL disclosed phagocytosis of LDL-bearing granules by the peritoneal macrophages. This is the first demonstration of a natural proteoglycan being able to enhance the rate of LDL uptake by macrophages in vivo. These observations on the relation between stimulation of mast cells and uptake of LDL by macrophages in vivo may have relevance in other sites where mast cells and macrophages coexist, such as the arterial intima.     

In vitro activation of tumoricidal properties of rat peritoneal macrophages using lipopolysaccharide

The peritoneal macrophages from normal Lewis rats were characterized by their capacity to phagocytose, by the presence of nonspecific esterase and Fc receptors. In vitro, these macrophages were maintained in culture 7 and/or 21 days, respectively, and for the last 24 h (activation period) were cultured with 0.1-4.0 micrograms/ml of lipopolysaccharide (LPS) and were found tumoricidal against different rat fibrosarcomas, at a ratio of 50:1 (BP6-Tu2, MC-1 and B77). Macrophage-mediated tumor cytolysis was determined using 51Cr-release assay. The sensitivity of used tumors to macrophage-mediated cyto-toxicity was different. In vivo the transfer of the activated macrophages together with the mentioned tumor cells to rats inhibited the growth of tumors. The adoptive transfer of macrophages activated with "activators" might lead to a new kind of immunotherapy of neoplastic diseases.     

Phenotypic and functional differences between rat alveolar, pleural, and peritoneal macrophages

Tissue macrophages (M phi) play a central and essential role in modulating the initiation and perpetuation of the inflammatory response. Phenotypical and functional differences among alveolar M phi (AM) and peritoneal M phi (PM) have been reported, but less is known about pleural M phi (PLM) and their ability and capacity to release biologically active substances. Therefore, the aim of this study was to determine the production of superoxide anion, nitric oxide (NO), and tumor necrosis factor alpha (TNF-alpha) by PLM in comparison to AM and PM in vitro. M phi from rats were isolated by lavage of the respective body compartment and characterized by evaluating the expression of the surface antigens MHC class II molecules, CD11b, and ED2-like antigen. Upon activation, AM produced significantly higher amounts of superoxide anion, NO, and TNF-alpha compared to PM and PLM. Taken together, the findings of this study demonstrate that rat PLM resemble PM more than AM in terms of production of key inflammatory mediators.     

IL-33敲除促进弓形虫感染小鼠腹腔巨噬细胞的M1偏移

目的 研究IL-33敲除(IL-33^-/-)对小鼠急性弓形虫感染腹腔巨噬细胞(pMφ)极化的影响,探讨IL-33^-/-在弓形虫感染免疫应答中的作用。方法 收集C57BL/6 IL-33^-/-小鼠和野生型(WT)小鼠pMφ,各分为弓形虫感染组和未感染组,比较各组pMφ 感染率、细胞因子及表面分子表达水平的变化。结果 IL-33^-/-小鼠pMφ 弓形虫感染30 min后的感染率低于WT小鼠(t=-2.49,P<0.05);IL-33^-/-小鼠感染组pMφ M1型标志物NO(t=29.71,P<0.05)、MHCⅡ(t=19.05,P<0.05)、TLR4(t=8.34,P<0.05)表达高于WT小鼠感染组,而M2型标志物CD206表达低于WT小鼠感染组(t=-3.34,P<0.05);感染组小鼠pMφ NO、IL-10、TNF-α、MHCⅡ类分子及CD86的表达高于各自未感染对照组;IL-33敲除和弓形虫感染对IL-33^-/-与WT小鼠pMφ MHCⅡ(F=5.25,P<0.05)、TLR2(F=14.88,P<0.05)分子的表达有交互作用。结论 在急性弓形虫感染早期,IL-33敲除驱动小鼠pMφ 向M1方向极化,促进pMφ 抗感染。     

腱糖蛋白-C对大鼠腹腔巨噬细胞活性及其分泌表达基质金属蛋白酶-2,9的影响

目的 探讨腱糖蛋白(tenascin-C)对大鼠腹腔巨噬细胞活性及分泌表达基质金属蛋白(MMPs)的影响。方法 分别在tenascin-C、纤维连接蛋白(fibronectin)和tenascin—C fibronectin包被的培养板中，采用MTT、Zymography、Westem和Northem blot法检测大鼠腹腔巨噬细胞的活性，以及MMP-2，9活性、蛋白和mRNA水平的表达。结果Tenascin-C不但使巨噬细胞活性增加，而且可上调其MMP-9蛋白和mRNA的表达，并显著增加MMP9的活性，但对MMP-2无明显影响；而浓度相同的fibronectin，对MMP-2，9活性及蛋白和mRNA的表达都无明显影响。tenascin-C fibronectin组，对MMP-9表达及活性的上调要明显高于tenasein-C组，说明tenascin-C和fibronectin具有协同作用。结论 tenascin-C可能具有与其他细胞外基质糖蛋白不同的作用，推测动脉粥样硬化病变中tenascin—C表达的上调，可能会通过增加巨噬细胞的活性并上调MMP-9的表达削弱斑块的稳定性。     

Synthesis of thymosin beta 4 by peritoneal macrophages and adherent spleen cells.

Thymosin beta 4, previously identified as a component of thymosin fraction 5 isolated from calf thymus, is synthesized by peritoneal macrophages derived from rats or mice, including cells from nu/nu mice. Identification of thymosin beta 4 containing [35S]methionine was based on its retention time in reverse-phase HPLC and the recovery of radioactivity in peptides generated by mild acid hydrolysis or by digestion with trypsin. Thymosin beta 4 is also synthesized by rat and mouse spleen cells, and evidence is presented for its release from these cells.     

白细胞介素-4对巨噬细胞缺氧诱导促有丝分裂因子分泌的影响

目的：探讨白细胞介素-4（IL-4）对巨噬细胞缺氧诱导促有丝分裂因子（HIMF）分泌及HIMF对血管平滑肌细胞增殖的影响。方法：用不同剂量的重组IL-4刺激培养的巨噬细胞，以逆转录聚合酶链反应（RTPCR）检测巨噬细胞HIMFmRNA表达，荧光免疫组化激光共聚焦显微镜检测巨噬细胞HIMF蛋白表达；用终浓度分别为3×10^-4mmol／L，9×10^-4mmol／L，2．7×10^-5 mmol／L的重组HIMF刺激培养的平滑肌细胞，以^3 H-胸腺嘧啶核苷（^3 H—TdR）掺入法测定HIMF对平滑肌细胞增殖的影响。结果：Th2型细胞因子IL-4刺激巨噬细胞，HIMFmRNA及其蛋白明显表达（P〈0．01）；浓度为3×10^-6 mmol／L，9×10^-4～mmol／L，2．7×10^-5mmol／L的重组HIMF明显促进平滑肌细胞增殖（与对照组比较P〈0．01），且随着HIMF剂量的增加，促增殖作用增强。结论：Th2型细胞因子IL-4能刺激巨噬细胞表达HIMF mRNA及其蛋白，HIMF能促进体外培养的血管平滑肌细胞增殖。     

Molecular organization of the cytokine gene cluster, involving the human IL-3, IL-4, IL-5, and GM-CSF genes, on human chromosome 5

The human genes for the hematopoietic growth factors interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been mapped to 5q23-31. We present in situ hybridization evidence that the human IL-4 gene is located at 5q23.3-31.2, suggesting that the four cytokine genes may be closely linked. We used pulsed-field gel electrophoresis to prepare subchromosomal restriction maps surrounding these genes to define this possible linkage more precisely. The IL-4 and IL-5 genes are tightly linked, being 90 to 240 kilobases (kb) apart, as has been shown for the IL-3 and GM-CSF genes, which are only 9 kb apart. Possible overlap of the map containing the IL-4 and IL-5 genes with restriction sites 5' to the IL-3 gene suggests that the four cytokine genes may be localized within 500 kb of each other. The endothelial cell growth factor gene (ECGF), which has also been localized to the 5q31 region, did not appear to be close to the cytokine genes. Linkage of the IL-3, IL-4, IL-5, and GM-CSF genes has important implications in the evolutionary origin and regulation of expression of these genes. The four cytokine genes are located in the region of the long arm of chromosome 5, which is deleted in the 5q- anomaly. The present study provides a basis for further investigations of this disorder.     

The correlation between levels of IL-7Ralpha expression and responsiveness to IL-7 is lost in CD4 lymphocytes from HIV-infected patients

Measurements of Bcl-2 and CD25 expression suggested that IL-7R function is modified in CD4 lymphocytes of untreated viraemic patients. The extent of IL-7R function restoration post-HAART was analysed. A positive linear relationship was demonstrated between IL-7Ralpha expression and the magnitude of IL-7-induced responses in healthy individuals, whereas this relationship is lost in HIV-infected patients, suggesting that viraemic patients suffer a receptor signaling transduction defect in IL-7R function. IL-7 responsiveness is only partly restored by HAART.     

Cytoprotective and anti-inflammatory effects of kernel extract from Adenanthera pavonina on lipopolysaccharide-stimulated rat peritoneal macrophages

Objective:To investigate mechanism of anti-inflammatory activity of Adenanthera pavonina(A.pavonina) extracts.Methods:Rat peritoneal macrophages were treated with different concentrations of lipopolysaccharide and H_2O_2 in the presence and absence of kernel extract from A.pavonina.Nitric oxide,superoxide anion generation,cell viability and nuclear fragmentation were investigated.Results:The pre-treatment of kernel extract from A.pavonina suppressed nitric oxide,superoxide anion,cell death,nuclear fragmentation in lipopolysaccharide and H_2O_2stimulated or induced macrophages,respectively.Conclusions:These results suggest that A.pavonina extract suppresses the intra cellular peroxide production.     

Toll样受体4与大鼠肺泡巨噬细胞内毒素耐受性的实验研究

目的　观察大鼠肺泡巨噬细胞对内毒素 (LPS)重复刺激的耐受性与其Toll样受体 4(TLR4)表达的变化 ,研究两者之间的联系。方法　将 30只Wistar雄性大鼠分离所得肺泡巨噬细胞 ,用随机数字表法分为正常对照组 (A组 ) ,LPS单次刺激组 (B组 )和LPS 2次重复刺激组 (C组 )。用酶联免疫吸附测定 (ELISA)法和逆转录 聚合酶链反应 (RT PCR)法分别检测各组大鼠肺泡巨噬细胞分泌肿瘤坏死因子α(TNF α)及TLR4、白细胞介素 1 0 (IL 1 0 )、IL 1 8mRNA表达的变化 ,WesternBlot检测TLR4蛋白表达的变化。结果　大鼠肺泡巨噬细胞TNF α分泌和TLR4、IL 1 0、IL 1 8mRNA表达及TLR4的蛋白表达水平 ,A组分别为 (0 4 5 0± 0 0 1 0 ) μg/L、1 1 6± 0 0 4、0 97± 0 0 3、1 32 0± 0 0 2 0、5 8 1± 0 4 ;B组分别为 (0 76 0± 0 0 30 ) μg/L、2 1 8± 0 0 9、1 83± 0 0 7、2 0 6 0± 0 0 6 0、1 4 8.3± 1 4 ;B组与A组比较差异有显著性 (P <0 0 1 ) ;C组分别为 (0 4 90± 0 0 5 0 ) μg/L、1 2 3± 0 0 3、1 1 5± 0 0 5、1 1 70± 0 0 4 0、96 5±0 7;C组与B组比较差异也有显著性 (P <0 0 5或 <0 0 1 )。结论　LPS重复刺激可使大鼠肺泡巨噬细胞对LPS产生耐受性 ;LPS耐受性的产生与TLR4表达     

Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta-chain bound to an antagonist

Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)