Comparative roles of free fatty acids with reactive nitrogen intermediates and reactive oxygen intermediates in expression of the anti-microbial activity of macrophages against Mycobacterium tuberculosis

We assessed the role of free fatty acids (FFA) in the expression of the activity of macrophages against Mycobacterium tuberculosis in relation to the roles of two major anti-microbial effectors, reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). Intracellular growth of M. tuberculosis residing inside macrophages was accelerated by treatments of macrophages with either quinacrine (phospholipase A2 (PLA2) inhibitor), arachidonyl trifuloromethylketone (type IV cytosolic PLA2 inhibitor), NG-monomethyl-L-arginine (nitric oxide synthase inhibitor), and superoxide dismutase plus catalase (ROI scavengers). In addition, M. tuberculosis-infected macrophages produced and/or secreted these effectors sequentially in the order ROI (0-3 h), FFA (0-48 h), and RNI (3 to at least 72 h). Notably, membranous FFA (arachidonic acid) of macrophages translocated to M. tuberculosis residing in the phagosomes of macrophages in phagocytic ability- and PLA2-dependent fashions during cultivation after M. tuberculosis infection. FFA, RNI and H2O2-mediated halogenation system (H2O2-halogenation system) displayed strong activity against M. tuberculosis in cell-free systems, while ROI alone exerted no such effects. Combinations of 'FFA + RNI' and 'RNI + H2O2-halogenation system' exhibited synergistic and additive effects against M. tuberculosis, respectively, while 'FFA + H2O2-halogenation system' had an antagonistic effect. Moreover, a sequential attack of FFA followed by RNI exerted synergistic activity against M. tuberculosis. Since M. tuberculosis-infected macrophages showed simultaneous production of RNI with FFA secretion for relatively long periods (approx. 45 h) and prolonged RNI production was seen thereafter, RNI in combination with FFA appear to play critical roles in the manifestation of the activity of macrophages against M. tuberculosis.     

Differential potentiation of anti-mycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal macrophages activated by interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α)

The anti-mycobacterial activities of IFN-γ and TNF-α-treated murine peritoneal macrophages were determined. Resident macrophages pretreated with IFN-γ or TNF-α for 2 days were infected with test organisms and subsequently cultured for up to 7 days. First, the early-phase growth of Mycobacterium tuberculosis (days 0–3) was strongly suppressed in IFN-γ-treated macrophages, and progressive bacterial elimination was subsequently observed. Although TNF-α treatment of macrophages did not affect the early phase growth of organisms, bacterial killing was observed in the later phase of cultivation. Second, although IFN-γ-treated macrophages killed M. avium during the first 3 days of culture, regrowth of the intracellular organisms was subsequently observed. TNF-α treatment of macrophages did not influence the mode of intracellular growth of M. avium. Third, IFN-γ but not TNF-α enhanced production of reactive nitrogen intermediates (RNI) by macrophages infected with M. tuberculosis or M. avium, whereas both cytokines increased macrophage release of reactive oxygen intermediates (ROI). The present findings therefore show that IFN-γ and TNF-α potentiated the anti-mycobacterial activity of murine peritoneal macrophages in different fashions. They also suggest that RNI played more important roles than did ROI in the expression of macrophage anti-mycobacterial, particularly anti-M. avium, activity.     

Phospholipids and reactive nitrogen intermediates collaborate in expression of the T cell mitogenesis-inhibitory activity of immunosuppressive macrophages induced in mycobacterial infection

We studied the role of phospholipids and nitric oxide in expression of the suppressor activity of splenic macrophages induced by Mycobacterium avium-intracellulare complex infection (MAIC-induced macrophages) in mice against mitogenic response of concanavalin A (Con A)-stimulated splenocytes (SPC) as follows. First, phosphatidylserine (PS) and phosphatidylinositol were found to suppress Con A-induced mitogenesis of SPC via inhibition of IL-2 production and acquisition of IL-2 reactivity in Con A-stimulated T cells. The mitogenesis-inhibitory activity of PS was increased when SPC were cultured under mildly acidic condition (pH 6·3). When SPC were pretreated with PS for 24 h prior to Con A blastogenesis, their mitogenic response was irreversibly abrogated. Second, N^G-monomethyl-l-arginine, an inhibitor of nitric oxide (NO) synthase, was found to attenuate in part the expression of the suppressor activity of MAIC-induced macrophages. Third, reactive nitrogen intermediates (RNI) including NO generated from acidified NO2− exerted potent inhibitory activity against SPC mitogenic response, and the suppressive activity of RNI was significantly augmented by the combination with PS. These findings indicate that phospholipids and RNI play an important role in the expression of suppressor activity of MAIC-induced macrophages as the effector molecules.     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

移植骨髓间充质干细胞减轻脂多糖诱导的小鼠急性肺损伤

目的 观察移植骨髓间充质干细胞 （MSCs） 对脂多糖 （LPS） 诱导小鼠急性肺损伤 （ALI）的治疗修复作用。方法 全骨髓培养法培养小鼠骨髓MSCs;细胞免疫化学染色鉴定MSCs特异表面标记;小鼠咽后壁吸入LPS制造小鼠肺损伤;尾静脉注射引入MSCs;称重计算肺水肿指数;肺组织切片HE染色观察组织病理改变;ELISA检测肺泡灌洗液和肺组织匀浆中IL-1β含量;Brdu（5-Bromo-2-Deoxyuridine）标记供体MSCs,免疫组织化学染色及双染色观察移植细胞的迁移和分化状态。结果 培养的MSCs细胞表面标记CD44阳性,而造血系表面标记CD34阴性。吸入LPS后,小鼠出现典型的肺损伤病理改变,肺水肿指数和肺组织匀浆IL-1β含量明显增加。标记的MSCs移植入同种异体的肺损伤小鼠,其肺部出现标记的MSCs,并表达上皮细胞标志抗原-细胞角蛋白（CK）。治疗后小鼠的肺水肿指数和肺组织匀浆IL-1β含量下降。结论 外源性MSCs移植到肺损伤小鼠体内,可迁移至肺损伤部位,并表达上皮细胞标志;减轻肺水肿程度,减少炎症因子释放。     

Expression of scavenger receptor class A and CD14 in lipopolysaccharide-induced lung injury

CD14 and macrophage scavenger receptor class A type I and II (MSR-A) are receptors for lipopolysaccharide (LPS). In this study, the expressions of both receptors in the lung after administration of LPS in aerosol to mice with a nebulizer were observed. Bronchiolar epithelial cells and alveolar macrophages immediately incorporated LPS and expressed CD14. CD14-positive neutrophils then appeared in the alveolar space followed by the appearance of MSR-A-expressing cells in the vascular lumen, pulmonary interstitium, and alveolar space. Numbers of apoptotic cells increased after 1 day, and MSR-A-expressing macrophages actively incorporated apoptotic bodies. Daily administration of macrophage colony stimulating factor (M-CSF) to the mice resulted in increased levels of MSR-A expression and reduced levels of CD14 as well as several cytokine expressions, leading to shortening of the inflammatory process. The numbers of apoptotic cells were reduced in M-CSF injected mice. These findings imply that CD14 acts as an immediate expressing receptor for LPS and MSR-A exerts a protective function by scavenging LPS and apoptotic cells in LPS-induced lung injury.     

Differential augmentation of in vivo natural killer cytotoxicity in normal primates with recombinant human interleukin-1 and granulocyte-macrophage colony-stimulating factor.

The effect of recombinant human interleukin-1 (IL-1) alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and combined factor therapy (CFT) on Rhesus monkey peripheral blood natural killer (NK) activity in vivo was compared. During a 14-day treatment period, IL-1-treated animals demonstrated a 170% increase in NK activity against K562 target cells by day 4, reaching maximal levels (300%) at day 16, and returning to baseline by day 30. NK activity of GM-CSF-treated monkeys increased slightly (60-100%) during days 4-12, as did saline-treated monkeys, but returned to baseline values by day 16. A delayed increase in NK activity resulted after GM-CSF treatment, reaching a peak (260%) on day 23 and remaining elevated through day 39. CFT resulted in a bimodal response pattern, with two peaks of NK activity: one at day 16 and a second at day 39. The first peak of activity (223%) was significantly less than the activity attained with IL-1 alone; the second peak (300%) was of greater duration and occurred later than the peak observed in GM-CSF-treated monkeys. Unlike IL-1, GM-CSF treatment did not lead to a immediate stimulation of NK activity; augmentation was delayed by more than 7 days post treatment. CFT results suggest that GM-CSF reduced the direct NK response to IL-1; while IL-1 led to an enhanced delayed NK response. Therefore, IL-1 and GM-CSF augment NK activity through different but interrelated pathways.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not granulocyte colony-stimulating factor (G-CSF) induces plasma membrane expression of proteinase 3 (PR3) on neutrophils in vitro

The theoretical risk of triggering vasculitis resulting from administration of G-CSF and GM-CSF to patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), such as Wegener's granulomatosis (WG), who develop agranulocytosis due to cytotoxic therapy, is unknown. Since there is strong evidence that activation of polymorphonuclear neutrophils (PMN) induced by binding of ANCA to PR3 or myeloperoxidase (MPO) expressed on their plasma membrane is involved in the pathogenesis of systemic vasculitides (SV), we studied the surface expression of PR3 and MPO on PMN from healthy donors in response to G-CSF and GM-CSF in vitro by flow cytometric analysis. Increasing doses of G-CSF did not alter PR3 expression on either untreated or tumour necrosis factor-alpha (TNF-alpha)-primed donor PMN significantly. In contrast, GM-CSF significantly increased PR3 membrane expression on both intact PMN and neutrophils primed with TNF-alpha. MPO expression was not significantly altered by either G-CSF or GM-CSF. In summary, these data demonstrate that GM-CSF, but not G-CSF, induces plasma membrane expression of PR3 on PMN in vitro. Since in AAV accessibility of the antigen (PR3 or MPO) to the antibody (ANCA) on the plasma membrane of PMN is thought to be essential for neutrophil activation by ANCA, the results of the present study suggest that administration of GM-CSF to patients with WG with neutropenia implies a definite theoretical risk of deterioration of vasculitis via this mechanism.     

Activation process of macrophages after in vitro treatment of mouse lymphocytes with dodecylglycerol.

Alkylglycerols, inflammation products of cancerous membrane lipids, efficiently activate macrophages. A brief in vitro treatment (30 min) of peritoneal cells (mixture of non-adherent and adherent cells) with a small amount (50 ng/ml) of synthetic dodecylglycerol (DDG) resulted in greatly enhanced Fc-receptor-mediated ingestion activity of macrophages. However, treatment of adherent cells (macrophages) alone with DDG produced no significant enhancement of macrophage ingestion activity, implying that macrophage activation requires a contribution of non-adherent cells. DDG-treated non-adherent cells were found to generate a macrophage-activating signal factor. Studies with a serum free-0.1% egg albumin-supplemented RPMI 1640 medium revealed that a serum factor is essential for macrophage activation process. Time course analysis of stepwise transfers of conditioned media of DDG-treated or untreated B cells and T cells revealed that DDG-treated B cells rapidly transmit a factor to untreated T cells which yield the ultimate macrophage-activating factor. This signal transmission among these cells for the macrophage activation process is too rapid to allow time for synthesis of inducible gene products. Thus, we hypothesized that a serum factor is modified by the pre-existing function of DDG-treated B cells and further modified by the pre-existing function of untreated T cells to yield macrophage-activating factor. This hypothesis was confirmed by the demonstration that DDG-treated splenic non-adherent cell ghosts modify a serum factor to yield macrophage-activating factor.     

Inhibition of in vitro differentiation of human monocytes to macrophages by lipopolysaccharides (LPS): phenotypic and functional analysis.

Blood monocytes (MO) undergo maturation into macrophages (MAC) upon migration from the capillary bed to tissue sites of inflammation where they are exposed to environmental signals. Functional competence and phenotype heterogeneity is the result of both differentiation-inducing and -activating events. In vitro, MO to MAC maturation is induced by serum factors, can be followed by the expression of specific maturation-associated antigens and is accompanied by a characteristic change in the secretory repertoire of MAC in comparison to MO. Here we report that bacterial lipopolysaccharides (LPS) at subnanogram quantities very effectively inhibited the serum-induced maturation of human MO in vitro. At the same time LPS induced the up-regulation of CD14 antigens. The lipid A moiety was shown to be responsible for this novel biological activity of the LPS molecule. Inhibition of maturation was not due to secondary LPS-induced signals like interleukin (IL)-1, IL-6, tumor-necrosis factor (TNF)-alpha or interferon (IFN)-alpha--even though the latter by itself suppressed MAC maturation in vitro. The inhibitory activity of IFN-alpha could be abolished by neutralizing anti-IFN-alpha antibodies whereas these antibodies had no effect on LPS-induced suppression of MAC maturation. Functional analysis of LPS-treated MO long-term cultures showed that the pattern of secretory products released was similar to that of freshly-isolated immature blood MO: compared with mature MAC, LPS-treated MO released high amounts of IL-6 but significantly less TNF-alpha, neopterin, lysozyme and beta-2-microglobulin. At the same time, in LPS-treated MO cultures the MAC maturation-associated molecules alpha-2-macroglobulin and fibronectin could be detected only in trace amounts. The ability to secrete IL-1, however, was lost both in control as well as in LPS-treated MO cultures. The results indicate that endotoxins may influence the biology of the MO/MAC system distinctively: they not only induce a functional activation but also interfere with the ontogeny of this cell family.     

Divergent regulation of cell surface protease expression in HL-60 cells differentiated into macrophages with granulocyte macrophage colony stimulating factor or neutrophils with retinoic acid

Taking advantage of the recently demonstrated presence of N-aminopeptidasesand the serine protease dipeptidyi aminopeptidase IV (DPP IV)at the surface of human myeloblastic HL-60 cells, the regulationof these protease activities in HL-60 cell differentiation hasbeen assessed using combined spectrophotometric and flow cytometricassays. Addition of human recombinant granulocyte macrophagecolony stimulating factor (rHu-GM-CSF) to HL-60 cells to inducedifferentiation into macrophages led to a time and dose-dependentincrease in both cell surface N-aminopeptidase and DPP IV activities.Protease up-regulation was due to an enhancement in cell surfaceprotease number, associated with a slight rise in apparent affinitiesof the enzymes for their substrates. In contrast, in HL-60 cellsinduced to differentiate into neutrophils in the presenceofretinoic acid, expression of cell surface N-amlnopeptidaseswas almost completely abolished in a time-and dose-dependentfashion, and this down-regulation was accompanied by a weakbut significant decrease in affinity. However, no noticeabledifference was seen in serine DPP IV expression between retinoicacid-treated and untreated HL-60 cells. Retinoic acid treatmentalso reduced soluble protease activity in vitro indicating thatdown-regulation of membrane aminopeptldases was not due to theirproteolytic clip. No modulation in the activity of any of theenzymes tested was seen with human recombinant tumor necrosisfactor- or retinol which do not induce HL-60 cell differentiation.The up-regulation of cell surface protease expression in HL-60cells differentiated into macrophages was similar to that observedin monocytes isolated from peripheral blood: both DPP IV andN-aminopeptidase activities strictly increased on cells thatundergo macrophage maturation (up to 5-fold) and independentlyof the nature of the differentiation inducer. Thus, the distinctivepatterns of N-aminopeptidase and DPP IV expression that areseen in differentiating neutrophils and macrophages appear tobe relatedto differences in stage of myeloid maturation. Becausecell surface proteases are crucially involved in leukocyte functions,the data presented suggest that alterations in cell surfaceprotease expression are associated with events controlling thedifferentiation of immature cells.     

Mechanisms involved in mycobacterial growth inhibition by gamma interferon-activated bone marrow macrophages: role of reactive nitrogen intermediates.

Murine bone marrow-derived macrophages are able to inhibit the growth of Mycobacterium bovis after stimulation with recombinant gamma interferon. This antimycobacterial activity was inhibited by NG-monomethyl-L-arginine, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Furthermore, there was a complete lack of mycobacterial growth inhibition in a medium deficient in L-arginine. Nitrite is generated by gamma interferon-activated bone marrow-derived macrophages after infection with M. bovis, and a correlation between mycobacterial growth inhibition and nitrite production was observed. These results indicate that reactive nitrogen intermediates derived from L-arginine are crucially involved in macrophage antimycobacterial activity.     

Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro

Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.     

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.     

Antiretroviral treatment reverses HIV-induced reduction in the expression of surface antigens on alveolar macrophages in AIDS patients.

MoAbs and immunoperoxidase methods were used to identify antigen-presenting and phagocytic cells and to assess expression of HLA-DR molecules on cells obtained by bronchoalveolar lavage (BAL) from 33 AIDS patients and nine normal volunteers. In 17 patients, not receiving antiretroviral therapy, the expression of HLA-DR molecules (MoAb RFDR1) as well as the percentages of cells expressing RFD1 marker for antigen-presenting cells and RFD7 marker for mature phagocytes were significantly reduced. However, in BAL obtained after commencing treatment with zidovudine (AZT) in 21 patients or with 2',3'-dideoxyinosine (DDI) in five patients, the expression of the markers studied was found to have returned to levels of expression seen in normal lavages. The changes observed were clearly associated with antiretroviral treatment and did not correlate with applications of other drugs, blood CD4 counts or presence of infectious organisms in BAL fluid. As the alterations in the expression of HLA-DR molecules and RFD1 marker on macrophages have been shown to be associated with functional capacities of these cells, the reversal of impaired expression of phenotypic markers on alveolar macrophages in AIDS patients by AZT and DDI signifies an important ability of these drugs to modify immune reactivity and emphasizes the need to monitor such functions in HIV disease.     

Comparative studies on the roles of mediator molecules in expression of the suppressor activity of Mycobacterium avium complex-induced immunosuppressive macrophages against T cell and B cell mitogenic responses

Mycobacterium avium complex-induced immunosuppressive macrophages (MAC-MPhis) exhibit suppressor activity against concanavalin A-induced T cell mitogenesis (T cell Con A mitogenesis). We examined the profiles of the MAC-MPhi-mediated suppression of lipopolysaccharide-induced B cell mitogenesis (B cell LPS mitogenesis) and found the following. First, although N(G)-monomethyl-L-arginine and carboxy-PTIO effectively blocked the MAC-MPhi's suppressor activity against T cell Con A mitogenesis, MAC-MPhi's action against B cell LPS mitogenesis was only weakly affected by these NO-reducing agents. Second, B cell LPS mitogenesis was remarkably more susceptible to MAC-MPhi-derived reactive oxygen intermediates than T cell Con A mitogenesis. Third, B cell LPS mitogenesis was less susceptible to the inhibitory effects of the other MAC-MPhi-derived suppressor mediators, including free fatty acids, TGF-beta and prostaglandin E(2), than T cell Con A mitogenesis. Fourth, MAC-MPhi's suppressor activity was strongly dependent on B7-1 like molecule-mediated cell contact with target cells only in the case of T cell Con A mitogenesis. Therefore, there are significant differences in the modes of suppressor action of MAC-MPhis against T cell and B cell mitogenesis.     

Further study on the roles of the effector molecules of immunosuppressive macrophages induced by mycobacterial infection in expression of their suppressor function against mitogen-stimulated T cell proliferation

Previously, we found that phospholipids and reactive nitrogen intermediates (RNI) collaborated in expression of the T cell mitogenesis-inhibitory activity of immunosuppressive macrophages induced by Mycobacterium avium-intracellularecomplex (MAIC) infection. In this study, we examined the roles of free fatty acids (FFA) and prostaglandins (PG) as effectors of MAIC-induced macrophages, and moreover, their collaborating effects with RNI. First, treatment of MAIC-induced macrophages with quinacrine (phospholipase A₂ (PLA₂) inhibitor), dexamethasone (inhibitor of PLA₂ and PG synthesis) or indomethacin (PG synthesis inhibitor) attenuated their suppressor activity against concanavalin A (Con A)-induced mitogenesis of splenocytes (SPC), indicating important roles of FFA liberated from membrane phospholipids and PG, as effectors. Second, oleic acid, PGE₂, RNI generated from NOR 4 (a new nitric oxide (NO) donor), and phosphatidylserine (PS) exhibited suppressor activity against SPC mitogenesis without showing significant cytotoxicity, in an irreversible manner. Third, the suppressor activities of RNI and PGE₂ were potentiated by combined use with oleic acid in a synergistic manner. Fourth, a dual-chamber experiment in which target SPC were separated from MAIC-induced macrophages by a Millipore filter revealed a requirement for cell-to-cell contact for expression of the suppressor function of MAIC-induced macrophages. These findings indicate that RNI, FFA, PG, and phospholipids (presumably PS) and their collaboration play central roles in expression of the T cell mitogenesis-inhibitory function of MAIC-induced suppressor macrophages.     

Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression

Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11c-positive cells were generated after 6-7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class II molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b^int/hi cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b^dull cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines.