Sister chromatid exchange in murine alveolar macrophages,regenerating liver and bone marrow cells — a simultaneous multicellular in vivo assay

Differential labeling of sister chromatids was achieved simultaneously in murine alveolar macrophages, regenerating liver, and bone marrow cells of partially hepatectomized mice as well as in alveolar macrophages and bone marrow cells of nonhepatectomized mice. The mean frequency of SCE/cell ±S.D. and the percentage of second division cells for each cell type were determined. No significant differences in mean frequencies of SCE/cell were observed among the cell types or between hepatectomized (alveolar macrophages –3.6±2.2, bone marrow –3.4±2.2; regenerating liver –3.6±2.4) and nonhepatectomized (alveolar macrophages —3.4 ±1.9; bone marrow —2.9±1.8). Although the percentage of second division cells was dependent upon cell type, no significant differences were apparent between hepatectomized (alveolar macrophages —57±8%; bone marrow —37±6%; regenerating liver –65±6%) and nonhepatectomized mice (alveolar macrophages –53±6%; bone marrow –36±4%). Comparisons between BrdU treated and nontreated nonhepatectomized mice revealed no significant alteration in mitotic yields.     

GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.     

The expression of Fc gamma receptors on the surface of macrophages activated by a vaccinal strain of Yersinia pestis

The expression of Fc gamma R on the surface of macrophages in the process of antiplague immunity formation is analyzed. The stud is performed on the alveolar and peritoneal macrophages obtained from intact and immunized guinea pigs in different periods after vaccination (the 1st, 7th, 14th and 21st day). It is established that during the formation of the antiplague immunity there occurs activation of macrophages which is accompanied by an increase of the Fc gamma R expression on the outer surface of the membrane both of peritoneal and alveolar macrophages and the pattern of response of these cells to the interaction with the vaccine strain of the plague microbe changes. The Fc gamma R expression heterogeneity of certain macrophage populations is revealed both in an intact and in immune organism as well as different pattern of the intact alveolar and peritoneal macrophage response during the interaction with the vaccine strain of the antiplague microbe. These differences are levelled in the process of the antiplague immunity formation.     

Exogenous surfactant changes the phenotype of alveolar macrophages in mice

Alveolar macrophages are essential for the maintenance of surfactant homeostasis. We asked whether surfactant treatment would change alveolar macrophage number and whether the alveolar macrophage phenotype would become activated or apoptotic when challenged in vivo with exogenous surfactant. Surfactant pool size in mice was increased by repetitive surfactant treatments containing 120 mg/kg (110 micromol/kg) saturated phosphatidylcholine. The number of alveolar macrophages recovered by alveolar lavage decreased after the first dose by 49% and slightly increased after the second and third doses. Up to 28.5% of the macrophages became large and foamy, and their appearance normalized within 12 h. Surfactant treatment did not increase the percent of apoptotic or necrotic cells. The alveolar macrophages were not activated as indicated by no change in expression of CD14, CD16, CD54, CD95, and scavenger receptor class A types I and II after surfactant treatment. Surfactant treatment in healthy mice transiently changed the phenotype of alveolar macrophages to large and foamy without indications of changes in the surface markers characteristic of activation.     

Influenza virus-induced immune complexes suppress alveolar macrophage phagocytosis

Immune complexes in the lungs are capable of inducing adverse responses. Herein we have detailed the formation of immune complexes in the lungs of influenza virus-infected mice and examined their effect on alveolar macrophage defenses. On days 3, 7, 10, 15, and 30 after aerosol infection with influenza A/PR8/34 virus, the acellular pulmonary lavage fluid was tested for viral antigen, specific viral antibody, and immune complexes by immunoassays. Whereas peak viral antigen (day 3) diminished to undetectable levels by day 10, specific viral antibody remained at a low concentration until day 10, after which it rapidly increased. Immune complex concentrations increased through day 7, peaked at day 10, and gradually returned to the control level by day 30. These data demonstrate that immune complexes of detectable size are induced by influenza virus infection during the interface between antigen excess and antibody excess conditions. Since alveolar macrophages are the pivotal phagocytic defense cells in the lung, the ability of normal alveolar macrophages to ingest opsonized erythrocytes was quantitated in the presence of immune complexes from lavage fluid. Immune complexes from day 10 virus-infected lungs caused a dose-dependent suppression of antibody-mediated phagocytosis to 30% of control values. In contrast, although these immune complexes also markedly decreased the phagocytosis of antibody-coated yeast cells, they did not significantly impair the antibody-independent ingestion of unopsonized yeast cells by macrophages. the suppressive effects of immune complexes on alveolar macrophages may, in part, explain the phagocytic dysfunction that occurs 7 to 10 days after influenza virus pneumonia.     

The relative efficacy of different strain combinations of lactic acid bacteria in the reduction of populations of Salmonella enterica Typhimurium in the livers and spleens of mice

Multispecies probiotics have been reported to be more effective than monostrain probiotics in health promoting for the host. In this study, 12 lactic acid bacteria (LAB) strains were selected based on the level of induction of tumor necrosis factor (TNF)-α in RAW 264.7 macrophage cells. Their adherence to Caco-2 cells and inhibitory effects on Salmonella invasion of Caco-2 cells were compared. Strains with different probiotic properties were then combined and BALB/c mice were fed with LAB strains for 63 days; then the mice were challenged with Salmonella on day 64. For Salmonella-unchallenged mice that received a multistrain combination of LAB strains that have greater TNF-α production in macrophages, greater adherence and inhibit Salmonella invasion of Caco-2 cells to a greater extent, their peritoneal macrophages had greater phagocytic activity. For Salmonella-challenged mice, a significant reduction of Salmonella cells in the livers and spleens of the mice was observed 8 days post challenge. The addition of 12% skim milk powder together with LAB strain combinations significantly enhanced the reduction of Salmonella cells in the mice livers and spleens. In conclusion, we have shown that LAB strain combinations with particular probiotic properties when fed to mice can inhibit Salmonella invasion of the liver and spleen.     

光,电镜观察小鼠呼吸道合胞病毒性肺炎的免疫病理改变

Ｂａｌｂ／ｃ小鼠经鼻吸入呼吸道合胞病毒（ＲＳＶ）悬液感染成ＲＳＶ肺炎。于感染第５天后连续隔日取肺,光镜与透射电镜检查。感染第５～７天,肺组织病理改变最严重,多数小鼠表现为间质淋巴细胞（ＬＣ）套状浸润,肺泡隔增宽;少数小鼠出现间质内大量ＬＣ浸润与肺泡内大量单个核细胞渗出的两种病理改变。病毒包涵体出现于肺泡上皮细胞内,细胞受感染后发生肿胀、坏死。Ⅰ型细胞核周胞质内有核衣壳复制,表面病毒芽生形成长短不等的丝状体。第９天,肺泡隔增宽与间质ＬＣ浸润逐渐减轻。第１２天,病毒包涵体明显减少。     

Mortality of Altitude-exposed Mice Infected with Pasturella tularensis

The influence of reduced barometric pressure equivalent to an altitude of 18,000 ft (5,486 m) on the susceptibility of mice to tularemia was investigated by exposing groups of animals to the test environment before, after, or before and after intraperitoneal inoculation of 225 colony-forming units of Pasteurella tularensis. Similarly infected control animals were not exposed to the experimental environment. Two measurements of mortality were employed: (i) the day on which 50% of the mice were dead; and (ii) the number of dead mice on the 8th day. Continuous altitude exposure for 14 days prior to infection had no effect on host susceptibility but exposure after infection significantly increased mortality (P < 0.001).     

VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice

To determine whether increased levels of VEGF disrupt postnatal lung formation or function, conditional transgenic mice in which VEGF 164 expression was enhanced in respiratory epithelial cells were produced. VEGF expression was induced in the lungs of VEGF transgenic pups with doxycycline from postnatal day 1 through 2 and 6 wk of age. VEGF levels were higher in bronchoalveolar lavage fluid (BALF) and lung homogenates of VEGF transgenic mice compared with endogenous VEGF levels in controls. Neonatal mortality was increased by 50% in VEGF transgenic mice. Total protein content in BALF was elevated in VEGF transgenic mice. Surfactant protein B protein expression was unaltered in VEGF transgenic mice. Although postnatal alveolar and vascular development were not disrupted by VEGF expression, VEGF transgenic mice developed pulmonary hemorrhage, alveolar remodeling, and macrophage accumulation as early as 2 wk of age. Electron microscopy demonstrated abnormal alveolar capillary endothelium in the VEGF transgenic mice. In many locations, the endothelium was discontinuous with segments of attenuated endothelial cells. Large numbers of hemosiderin-laden macrophages and varying degrees of emphysema were observed in adult VEGF transgenic mice. Overexpression of VEGF in the neonatal lung increased infant mortality and caused pulmonary hemorrhage, hemosiderosis, alveolar remodeling, and inflammation.     

Sequential analysis of cell differentials and IFN-gamma production of splenocytes from mice infected with Toxoplasma gondii

To assess the relationship between the changes of cellular components and the production of Th1 cytokine in the immune tissue, inbred C57BL/6 mice were orally infected with 40 cysts of 76K strain of Toxoplasma gondii. The sequential change of cell differentials and IFN-gamma production of splenocytes were analyzed by Diff-Quik stain and RT-PCR. There were no significant proportional changes of cellular components of splenocytes until day 4 postinfection (PI) as compared to those of day 0, and the relative percentage of macrophages and neutrophils/eosinophils increased significantly (p < 0.01) thereafter. The expression of IFN-gamma mRNA of CD3- cells was observed from day 1 PI at a low level. However, IFN-gamma production of CD3+ cells increased significantly from day 4 PI (p < 0.01) which progressively increased thereafter. These findings provide the relative percentages of granulocytes and macrophages were increased in conjunction with increase of total number of splenocytes after oral infection with T. gondii in the susceptible murine hosts, and lymphocytes were the major cellular components and the important source of IFN-gamma.     

Antigen-specific (p30) mouse CD8+ T cells are cytotoxic against Toxoplasma gondii-infected peritoneal macrophages.

The importance of CD8+ T cells in immunity against Toxoplasma gondii is now well recognized. The mechanism by which these CD8+ T cells are able to confer this immunity is not yet understood. To examine the Ag specificity of this response, immune splenocytes from mice immunized with p30, a major surface parasite Ag, were evaluated for their ability to lyse peritoneal macrophages infected with three different strains of T. gondii. Macrophages infected with either the RH or P wild-type strain tachyzoites were lysed at varying E:T ratios by nylon wool nonadherent immune splenocytes whereas macrophages infected with a p30-deficient mutant (B mutant) of the P strain were not. The gene encoding p30 for the wild type and B mutant were amplified by the polymerase chain reaction. This revealed a nonsense mutation in the B mutant such that its primary translation product is predicted to be about two-thirds the size of the wild-type p30 molecule. mAb depletion studies indicate that the cytotoxic effect of the immune splenocytes is mediated by the CD8+ T cell population. Peritoneal macrophages infected with the three different strains (RH, P wild type, B mutant) from mice genetically restricted were not lysed by the immune CD8+ effector cell population. A cloned line (C3) of p30 Ag-specific CD8+ T cells exhibited significant cytotoxicity against syngeneic peritoneal macrophages infected with either the RH or P strain tachyzoites. There was no macrophage lysis observed by these CD8+ effector cells of either syngeneic macrophages infected with the B mutant or nonsyngeneic macrophages infected with the three different tachyzoite strains.     

Alveolar macrophages contribute to alveolar barrier dysfunction in ventilator-induced lung injury

In patients requiring mechanical ventilation for acute lung injury or acute respiratory distress syndrome (ARDS), tidal volume reduction decreases mortality, but the mechanisms of the protective effect have not been fully explored. To test the hypothesis that alveolar macrophage activation is an early and critical event in the initiation of ventilator-induced lung injury (VILI), rats were ventilated with high tidal volume (HV(T)) for 10 min to 4 h. Alveolar macrophage counts in bronchoalveolar lavage (BAL) fluid decreased 45% by 20 min of HV(T) (P < 0.05) consistent with activation-associated adhesion. Depletion of alveolar macrophages in vivo with liposomal clodronate significantly decreased permeability and pulmonary edema following 4 h of HV(T) (P < 0.05). BAL fluid from rats exposed to 20 min of HV(T) increased nitric oxide synthase activity nearly threefold in na?ve primary alveolar macrophages (P < 0.05) indicating that soluble factors present in the air spaces contribute to macrophage activation in VILI. Media from cocultures of alveolar epithelial cell monolayers and alveolar macrophages exposed to 30 min of stretch in vitro also significantly increased nitrite production in na?ve macrophages (P < 0.05), but media from stretched alveolar epithelial cells or primary alveolar macrophages alone did not, suggesting alveolar epithelial cell-macrophage interaction was required for the subsequent macrophage activation observed. These data demonstrate that injurious mechanical ventilation rapidly activates alveolar macrophages and that alveolar macrophages play an important role in the initial pathogenesis of VILI.     

GM-CSF regulates protein and lipid catabolism by alveolar macrophages

Metabolism of surfactant protein (SP) A and dipalmitoylphosphatidylcholine (DPPC) was assessed in alveolar macrophages isolated from granulocyte-macrophage colony-stimulated factor (GM-CSF) gene-targeted [GM(-/-)] mice, wild-type mice, and GM(-/-) mice expressing GM-CSF under control of the SP-C promoter element (SP-C-GM). Although binding and uptake of (125)I-SP-A were significantly increased in alveolar macrophages from GM(-/-) compared with wild type or SP-C-GM mice, catabolism of (125)I-SP-A was markedly decreased in GM(-/-) mice. Association of [(3)H]DPPC with alveolar macrophages from GM(-/-), wild-type, and SP-C-GM mice was similar; however, catabolism of DPPC was markedly reduced in cells from GM(-/-) mice. Fluorescence-activated cell sorter analysis demonstrated decreased catabolism of rhodamine-labeled dipalmitoylphosphatidylethanolamine by alveolar macrophages from GM(-/-) mice. GM-CSF deficiency was associated with increased SP-A uptake by alveolar macrophages but with impaired surfactant lipid and SP-A degradation. These findings demonstrate the important role of GM-CSF in the regulation of alveolar macrophage lipid and SP-A catabolism.     

CD4+ T cells are required for HSP65 expression in host macrophages and for protection of mice infected with Plasmodium yoelii

We have reported that macrophages expressing heat-shock protein 65 play an essential role in protection of mice infected with Plasmodium yoelii. In this study, we investigated the function and expression mechanism of HSP65 in macrophages of mice infected with P. yoelii. C57BL/6 (B6) mice are susceptible to infection with the lethal (L) strain but resistant to infection with the non-lethal (NL) strain of P. yoelii. The percentage of apoptotic macrophages in mice infected with the L strain was higher than that in mice infected with the NL strain. However, the percentage was low in L strain infected mice if they acquired resistance to the infection by primary infection with the NL strain. That apoptosis was reversely correlated with HSP65 expression in splenic macrophages from mice infected with P. yoelii suggests HSP65 may contribute to protective immunity by preventing apoptosis of macrophages in malarial infection. Cell depletion/transfer experiments showed that CD4+ T cells, but not CD8+ T cells, gammadelta T cells, NK cells or NK T cells, were required for HSP65 expression in macrophages as well as for protection of mice infected with P. yoelii. In conclusion, HSP65 may play a role in preventing apoptosis of macrophages in mice infected with P. yoelii. CD4+ T cells are required for HSP65 expression and for protective immunity against P. yoelii infection.     

Macrophage dysfunction and susceptibility to pulmonary Pseudomonas aeruginosa infection in surfactant protein C-deficient mice

To determine the role of surfactant protein C (SP-C) in host defense, SP-C-deficient (Sftpc-/-) mice were infected with the pulmonary pathogen Pseudomonas aeruginosa by intratracheal injection. Survival of young, postnatal day 14 Sftpc-/- mice was decreased in comparison to Sftpc+/+ mice. The sensitivity to Pseudomonas bacteria was specific to the 129S6 strain of Sftpc-/- mice, a strain that spontaneously develops interstitial lung disease-like lung pathology with age. Pulmonary bacterial load and leukocyte infiltration were increased in the lungs of Sftpc-/- mice 24 h after infection. Early influx of polymorphonuclear leukocytes in the lungs of uninfected newborn Sftpc-/- mice relative to Sftpc+/+ mice indicate that the lack of SP-C promotes proinflammatory responses in the lung. Mucin expression, as indicated by Alcian blue staining, was increased in the airways of Sftpc-/- mice following infection. Phagocytic activity of alveolar macrophages from Sftpc-/- mice was reduced. The uptake of fluorescent beads in vitro and the number of bacteria phagocytosed by alveolar macrophages in vivo was decreased in the Sftpc-/- mice. Alveolar macrophages from Sftpc-/- mice expressed markers of alternative activation that are associated with diminished pathogen response and advancing pulmonary fibrosis. These findings implicate SP-C as a modifier of alveolar homeostasis. SP-C plays an important role in innate host defense of the lung, enhancing macrophage-mediated Pseudomonas phagocytosis, clearance and limiting pulmonary inflammatory responses.     

Fas-mediated acute lung injury requires fas expression on nonmyeloid cells of the lung

Fas (CD95) is a membrane surface receptor, which, in the lungs, is expressed in macrophages, neutrophils, and epithelial cells. In mice, Fas activation leads to a form of lung injury characterized by increased alveolar permeability. We investigated whether Fas-mediated lung injury occurs primarily as a result of Fas activation in myeloid cells (such as macrophages) or in nonmyeloid cells (such as epithelial cells). Chimeric mice lacking Fas in either myeloid or nonmyeloid cells were generated by transplanting marrow cells from lpr mice (which lack Fas) into lethally irradiated C57BL/6 mice (MyFas(-) group) or vice versa (MyFas(+) group). Additional mice transplanted with marrow cells from their same strain served as controls (Fas(+) ctr and Fas(-) ctr groups). Sixty days after transplantation, the mice received intratracheal instillations of the Fas-activating mAb Jo2 (n = 10/group), or an isotype control Ab (n = 10/group), and were euthanized 24-h later. Only animals expressing Fas in nonmyeloid cells (Fas(+) ctr and MyFas(-)) showed significant increases in lung neutrophil content and in alveolar permeability. These same mice showed tissue evidence of lung injury and caspase-3 activation in cells of the alveolar walls. Despite differences in the neutrophilic response and lung injury, there was no statistical difference in the lung cytokine concentrations (KC and MIP-2) among groups. We conclude that Fas-mediated lung injury requires expression of Fas on nonmyeloid cells of the lungs. These findings suggest that the alveolar epithelium is the primary target of Fas-mediated acute lung injury, and demonstrate that apoptotic processes may be associated with neutrophilic inflammation.     

Granulocyte-macrophage colony-stimulating factor promotes the proliferation of human alveolar macrophages in vitro

The effects of granulocyte-macrophage (GM-CSF) or macrophage-CSF on in vitro proliferation of human alveolar macrophages (AM) were evaluated. AM of healthy volunteers incubated with recombinant human GM-CSF revealed incorporation of [3H]thymidine in vitro. The maximum incorporation was observed at 20 U/ml of GM-CSF on day 3. The proportion of proliferating cells incubated with 20 U/ml of GM-CSF from day 3 to day 4 was 8 to 11% of the total, whereas 3 to 5% of cells proliferated without GM-CSF. The number of cell nuclei increased 1.30- to 1.68-fold in the initial 7 days during incubation with 20 U/ml of GM-CSF, whereas there was a 1.07- to 1.13-fold increase without GM-CSF. Conditioned media obtained by the incubation with human lung tissue exhibited similar effects as recombinant human GM-CSF on macrophages. The effects were completely abrogated by antibody against human GM-CSF. Immunohistochemically, GM-CSF was detected in lung cells including AM, alveolar epithelium, alveolar interstitial cells, and endothelial cells. In contrast, recombinant macrophage-CSF did not induce the proliferation of human AM, although it has been known to promote the proliferation of murine AM. These observations suggest that GM-CSF plays an important role among the regulatory factors that locally support the population of AM in human lungs.     

Role of lung macrophages on susceptibility to respiratory infection following short-term moderate exercise training

Moderate exercise training is associated with a decreased risk for upper respiratory tract infection in human and animal studies, but the mechanisms have not been elucidated. Lung macrophages play an important role in resistance to respiratory infection, and moderate exercise can enhance macrophage antiviral resistance, but no studies have directly tested the role of lung macrophages in this response. This study tested the effect of lung macrophage depletion on susceptibility to infection following short-term moderate exercise training. Mice were assigned to one of four groups: exercise (Ex) and resting controls (Con) with and without clodronate encapsulated liposomes (CL(2)MDP-lip). Ex mice ran for 1 h on a treadmill for 6 days at 36 m/min, 8% grade. Fifteen minutes following exercise or rest on the last day of training, mice were intranasally inoculated with a standardized dose of herpes simplex virus type 1. Clodronate (Ex-CL(2)MDP-lip and Con-CL(2)MDP-lip) or PBS liposomes (Ex-PBS-lip and Con-PBS-lip) (100 microl) were intranasally administered following exercise or rest on the 4th day of training and again on the 4th day postinfection. Morbidity, mortality, and symptom severity were monitored for 21 days. Exercise decreased morbidity by 36%, mortality by 61%, and symptom severity score on days 5-7 (P < 0.05). Depletion of lung macrophages negated the beneficial effects of moderate exercise. This was indicated by no differences between Ex-CL(2)MDP-lip and Con-PBS-lip in morbidity (89 vs. 95%), mortality (79 vs. 95%), or symptom severity. Results indicate that lung macrophages play an important role in mediating the beneficial effects of moderate exercise on susceptibility to respiratory infection.     

Temporal relationship between immune cell influx and the expression of inducible nitric oxide synthase, interleukin-4 and interferon-gamma in pancreatic islets of NOD mice following adoptive transfer of diabetic spleen cells

Beta cell destruction in NOD mice can be accelerated by adoptive transfer of diabetic spleen cells into irradiated adult NOD mice. Here mice receiving diabetic spleen cells were examined at days 0, 7, 14, 21 and at onset of diabetes for the resulting insulitis and the number of intra-islet CD4 and CD8 cells and macrophages. The progression of insulitis and the number of intra-islet CD4 and CD8 cells and macrophages were correlated with the expression and co-localization of inducible nitric oxide synthase, interferon- and interleukin-4 by dual-label light and confocal immunofluorescence microscopy. Diabetes developed in 7/8 mice by 27 days following cell transfer. The insulitis score increased slightly by day 7 but rose sharply at day 14 (p=0.001) and was maintained until diabetes. The mean number of intra-islet CD4 and CD8 cells and macrophages showed a similar trend to the insulitis scores and were present in almost equal numbers within the islets. Immunolabelling for inducible nitric oxide synthase was observed at day 7 in only some cells of a few islets but increased sharply from day 14. It was restricted to islets with insulitis and was co-localized in selective macrophages. Weak intra-islet interleukin-4 labelling was observed at days 7 and 14 but became more pronounced at day 21 and at onset of diabetes, being present in selective CD4 cells. Intra-islet labelling for interferon- was first observed at day 21, but became more intense at onset of diabetes and was co-localized in a proportion of macrophages. Both cytokines were expressed in islets with advanced insulitis. Interferon- staining was also observed within endothelial cells located in the exocrine pancreas. We conclude that transfer of diabetic spleen cells results in a rapid influx of CD4 and CD8 cells and macrophages within the pancreas of recipient mice. During the period of heightened insulitis, selective immune cells begin to express inducible nitric oxide synthase and the opposing cytokines, interferon- and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.