Polyamine-mediated apoptosis of alveolar macrophages during Pneumocystis pneumonia

The number of alveolar macrophages is decreased during Pneumocystis pneumonia (Pcp), partly because of activation of apoptosis in these cells. This apoptosis occurs in both rat and mouse models of Pcp. Bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals were found to contain high levels of polyamines, including spermidine, N1-acetylspermine, and N1-acetylspermidine. These BAL fluids and exogenous polyamines were able to induce apoptosis in alveolar macrophages. Apoptosis of alveolar macrophages during infection, after incubation with BAL fluids from Pneumocystis-infected animals, or after incubation with polyamines was marked by an increase in intracellular reactive oxygen species, activation of caspases-3 and -9, DNA fragmentation, and leakage of mitochondrial cytochrome c into the cytoplasm. When polyamines were depleted from the BAL fluids of infected animals, the ability of these BAL fluids to induce apoptosis was lost. Interestingly, the apoptosis inducing activity of the polyamine-depleted BAL fluids was restored when polyamines were added back. The results of this study suggested that Pneumocystis infection results in accumulation of high levels of polyamines in the lung. These polyamines activate apoptosis of alveolar macrophages, perhaps because of the ROS that are produced during polyamine metabolism.     

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.     

Factors influencing Pneumocystis infection in the immunocompromised rat.

A compromised immune system is the primary predisposing condition for Pneumocystis infection. Factors that contribute to this underlying state of immunosuppression are poorly understood. The presence of common rodent viruses and the role of anti-Pneumocystis antibodies on the progression of natural infection in the corticosteroid-treated rat model of Pneumocystis pneumonia were evaluated. The development and intensity of infection were not affected by the presence or absence of antibodies to these viruses or to major Pneumocystis antigens. A significant increase in survival of Pneumocystis-infected viral antibody-positive rats was observed when these rats were housed under barrier conditions.     

Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia

CD4+CD25+FoxP3+ regulatory T cells are decreased in patients infected with HIV and have been shown to be critical in mediating Ag tolerance in the lung. Because a subset of Pneumocystis-infected individuals develop substantial lung injury, which can be modeled in immune reconstituted scid mice, we used mouse models of Pneumocystis carinii to investigate the role of regulatory T cells in opportunistic infection and immune reconstitution. In this study, we show that CD4+CD25+FoxP3+ cells are part of the host response to Pneumocystis in CD4+ T cell-intact mice. Moreover, lung injury and proinflammatory Th1 and Th2 cytokine levels in the bronchoalveolar lavage fluid and lung homogenate were increased following CD4+CD25- immune reconstitution in Pneumocystis-infected SCID mice but not in CD4+CD25+ T cell-reconstituted animals. The ability of CD4+CD25+ T cells to control inflammation and injury during the course of Pneumocystis was confirmed by treatment of wild-type C57BL/6 mice with anti-CD25 mAb. These data show that CD4+CD25+ T cells control pulmonary inflammation and lung injury associated with Pneumocystis infection both in the setting of immune reconstitution as well as new acquisition of infection.     

Influence of Pneumocystis carinii on Nitrite Production by Rat Alveolar Macrophages1

ABSTRACT Nitrite production by rat alveolar macrophages was studied to determine the role of L-arginine oxidation in the interaction between these cells and Pneumocystis carinii. Alveolar macrophages from rats obtained from two different breeders were used: rats from Janvier breeder had latent P. carinii infection, while those from Charles River breeder were bred in a germ-free environment. Pneumocystis carinii increased in vitro nitrite generation by unstimulated alveolar macrophages from Janvier rats only, and this was blocked by N^G-monomethyl-L-arginine. Incubation of cells from Janvier and Charles River rats with lipopolysaccharide and/or interferon-gamma increased nitrite production to a similar extent. Pneumocystis carinii partially decreased nitrite release by activated alveolar macrophages, and this was still inhibited by N^G-monomethyl-L-arginine. In the presence of P. carinii, superoxide dismutase used as a superoxide anion scavenger had no effect on nitrite production by activated cells. These results show that prior exposure to P. carinii leads to nitric oxide production by rat alveolar macrophages. Although the magnitude of this production seems to be moderate, it is of biological significance since cells of P. curinii-naive rats do not generate nitrite whereas those of latently infected rats do.     

2R,4R-APDC,a Metabotropic Glutamate Receptor Agonist,Reduced Neuronal Apoptosis by Upregulating MicroRNA-128 in a Rat Model After Seizures

This study aimed to study the protective effect of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a selective metabotropic glutamate receptor agonist, against hippocampal neuronal apoptosis induced by seizures in a rat model of pilocarpine-induced epilepsy. The Morris water maze test was used to assess the spatial memory abilities of epileptic rats with or without 2R,4R-APDC treatment. TUNEL assay was performed to examine neuronal apoptosis in hippocampus. Western blot was conducted to evaluate changes in the levels of caspase-3 and caspase-9 in hippocampus. Real-time PCR was used to determine the levels of microRNA-128 (miR-128) in hippocampus. The results of the Morris water maze test showed that the 2R,4R-APDC treatment reduced the escape latencies and swimming lengths of rats after seizures. The TUNEL assay showed that 2R,4R-APDC significantly counteracted seizure-induced cell apoptosis. The western blot confirmed this finding, demonstrating that the levels of cleaved caspase-3 and cleaved caspase-9 were potently decreased by 2R,4R-APDC in rat hippocampus after seizures. In addition, 2R,4R-APDC upregulated miR-128 expression levels in the hippocampus. A miR-128 mimic or inhibitor decreased or increased the percentage of TUNEL-positive cells in rats after seizures and 2R,4R-APDC treatment, respectively. The levels of both cleaved caspase-3 and cleaved caspase-9 were decreased in hippocampus exposed to the miR-128 mimic, whereas they were markedly increased in miR-128 inhibitor-treated hippocampus. In conclusion, 2R,4R-APDC protected hippocampal cells from cell apoptosis after seizures, possibly by upregulating miR-128.     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Impaired recognition by Toll-like receptor 4 is responsible for exacerbated murine Pneumocystis pneumonia

We investigated the effect of Toll-like receptor 4 (TLR4) on the progression of murine Pneumocystis pneumonia. TLR4-mutant C3H/HeJ and wild-type C3H/HeN mice were infected with Pneumocystis after depletion of CD4 T cells. Mutant mice lost body weight more quickly and showed exacerbated pulmonary injury even though there was no difference in Pneumocystis organism burden in the lung. Mutant mice showed reduced levels of IL-10, IL-12p40 and MIP-2 accompanied by elevated levels of TNF-alpha and IL-6 in the bronchoalveolar lavage fluid compared with those of wild-type mice 8 weeks after the infection. In response to stimulation with Pneumocystis antigen, the production of IL-10, IL-12p40 and MIP-2 by alveolar macrophages was partially impaired in mutant mice, while that in wild-type mice was suppressed by the anti-TLR4/MD-2 mAb, MTS510. Unlike the response to lipopolysaccharide stimulation, TLR4-reconstituted HEK293 cells showed no elevated NF-kappaB activation after stimulation with Pneumocystis antigen. Taken together, these findings suggest that recognition of Pneumocystis by TLR4 helps to regulate the host inflammatory responses through cytokine and chemokine production by alveolar macrophages.     

Caspase-3 Mediates In Part Hippocampal Apoptosis in Sepsis

The brain is one of the first organs affected during sepsis development resulting in apoptosis for a short-term and cognitive impairment for a long-term. Despite its importance, the mechanisms of brain dysfunction during sepsis are not fully elucidated. Thus, we here, in an animal model of sepsis, evaluated apoptosis in the dentate gyrus cell layer of the hippocampus to document the involvement of caspase-3 in the pathogenesis of neuronal apoptosis. Wistar rats sham-operated or submitted to the cecal ligation and perforation (CLP) procedure were killed at 12, 24, 48 h, and 10 days after surgery for the determination of caspase-3 and apoptosis rate. In a separate cohort of animals, a caspase-3-specific inhibitor was administered and animals were killed at 12 h after sepsis. An increase in the number of apoptotic cells 12, 24, and 48 h by histopathological evaluations and an increase of caspase-3 apoptotic cells 12 and 24 h after sepsis induction were observed. The caspase-3 inhibitor decreases the number of apoptotic cells by histopathological evaluations but not by immunohistochemistry evaluations. Caspase-3 is involved in part in apoptosis in the dentate gyrus cell layer of the hippocampus in septic rats submitted by CLP.     

Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor

Caspases have been implicated in the effector process of apoptosis in several systems including the Fas-Fas ligand pathway. We previously demonstrated that excessive apoptosis of lung epithelial cells and the Fas-Fas ligand pathway were essential in the pathogenesis of bleomycin-induced pneumopathy in mice. Therefore, the purpose of this study was to investigate whether a caspase inhibitor could prevent the development of this model. The expression of caspase-1 and caspase-3 was upregulated on lung epithelial cells, alveolar macrophages, and infiltrating inflammatory cells in this model. We demonstrated that a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, decreased the caspase-1- and caspase-3-like activity, the number of apoptotic cells, the pathological grade of lung inflammation and fibrosis, and the hydroxyproline content in lung tissues in this model. We conclude that caspase inhibitors could be a new therapeutic approach against lung injury and pulmonary fibrosis.     

Detection of Fc receptors for IgA on rat alveolar and peritoneal macrophages.

The presence of Fc receptors for IgA on alveolar macrophages was determined by rosette assay and immunogold labeling. IgA-mediated phagocytosis by alveolar macrophages was observed. Results of these assays were compared between rats receiving no treatment and those receiving long-term cortisone administration. Sheep erythrocytes coated with dextran and an IgA monoclonal antibody specific for the alpha 1,3 linkages of dextran bound to 16% of alveolar macrophages. However, peritoneal macrophages did not form rosettes with dextran-IgA-coated erythrocytes. Immunogold labeling by transmission electron microscopy revealed that most Fc receptors for IgA were found on the membrane of pseudopodia of activated alveolar macrophages. Long-term cortisone administration diminished the phagocytosis and phagocytic index of alveolar macrophages, thereby contributing to decreased host resistance to infection (e.g., Pneumocystis carinii pneumonia).     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

The involvement of caspase-11 in TPEN-induced apoptosis

The depletion of intracellular zinc with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induces protein synthesis-dependent apoptosis. Here we examined the involvement of caspase induction in apoptosis. Among the examined caspases, only caspase-11 was increased by TPEN. Caspase-11 activity also increased, which resulted in caspase-3 activation. Cycloheximide or actinomycin D blocked caspase-11 induction, reduced caspase-11 and -3 activation, and attenuated TPEN-induced neuronal apoptosis. Blockade of caspase-11 by a chemical inhibitor or genetic deletion attenuated TPEN-induced apoptosis, indicating a critical role of caspase-11 in TPEN-induced apoptosis. Although mitochondria-mediated caspase-9/-3 activation also contributed to TPEN-induced apoptosis, caspase-11 is likely a key inducible apoptosis-inducing protein.     

Staurosporine-induced apoptosis in P388D1 macrophages involves both extrinsic and intrinsic pathways

Treatment of P388D1, a macrophage-like cell line, with staurosporine triggered apoptosis through the activation of caspase-9 and caspase-3. Unexpected effects of staurosporine on the induction of apoptosis were the activation of caspase-8, and an increase of the levels of TNF-α. The increased TNF-α levels led to activation of caspase-8 by an autocrine effect via the TNF receptor expressed by the P388D1 macrophages. In contrast, P388D1 macrophages that either had been exposed to UV light or treated with dexamethasone did not undergo apoptosis.     

Comparison of pulsed field gel electrophoresis karyotypes of Pneumocystis carinii derived from rat lung, cell culture, and ferret lung

Pulsed field gel electrophoretic karyotypes of Pneumocystis carinii derived from three sources were compared: immunosuppressed virus-free rats transtracheally inoculated with Pneumocystis-infected rat lung; WI-38 cell/Cytodex bead cell cultures inoculated with the same material; and immunosuppressed ferrets which reactivated latent Pneumocystis pneumonia. Karyotypes of DNA from Pneumocystis trophozoites or cysts from rat lung, and trophozoites from cell culture were identical. In contrast, ferret Pneumocystis DNA karyotypes were distinctly different. Rat Pneumocystis gene probes reacted with Southern- transferred rat Pneumocystis DNA but not with ferret Pneumocystis DNA. We concluded that neither the source nor life stage of rat Pneumocystis carinii influenced genomic karyotype, and that rat and ferret Pneumocystis are genetically diverse.