FTS reduces bleomycin-induced cytokine and chemokine production and inhibits pulmonary fibrosis in mice.

Bleomycin (BLM), an antitumour drug, is known to cause interstitial pneumonia followed by pulmonary fibrosis, and has often been used to produce an animal model of pulmonary fibrosis. In the present study, we examined the effect of a nonapeptide thymic hormone, facteur thymique serique (FTS), on the murine lung fibrosis induced by intratracheal instillation of BLM. Treatment with FTS ameliorated BLM-induced fibrotic changes in a dose-dependent manner, as indicated by the reduced accumulation of hydroxyproline (HP). In addition, FTS suppressed BLM-induced cellular inflammatory response in the lungs, as evidenced by inhibition of increased lung weight, reduced accumulation of inflammatory leucocytes, including lymphocytes and neutrophils, but not macrophages, and less pronounced histopathological changes. Finally, BLM challenge increased the local synthesis of proinflammatory cytokines, TNF-alpha and IL-1beta and chemokines, MCP-1, MIP-1alpha RANTES, MIP-2 and KC, while administration of FTS suppressed the production of these cytokines, except for MCP-1. These effects of FTS were observed only when mice received intratracheal instillation with BLM. Considered collectively, our results indicated that FTS treatment ameliorated the cellular inflammatory responses and fibrotic changes in the lungs caused by BLM and such inhibition was well correlated with reduced synthesis of several fibrosis-related cytokines, and suggested that FTS may be potentially useful for the treatment of pulmonary fibrosis.

Mechanisms of the in vitro fungicidal effects of human neutrophils against Penicillium marneffei induced by granulocyte-macrophage colony-stimulating factor (GM-CSF).

We examined the in vitro fungicidal activity of human neutrophils against conidia and yeast cells of Penicillium marneffei. Neutrophils showed a small but significant anti-fungal effect against the yeast form of P. marneffei. Treatment of neutrophils with GM-CSF significantly augmented their anti-fungal activity. In contrast, the conidia form resisted killing even by stimulated neutrophils. Neutrophil fungicidal effect was not inhibited by superoxide dismutase (SOD), while the same treatment significantly suppressed the killing of Candida albicans by GM-CSF-stimulated neutrophils. For effective killing of P. marneffei yeast cells by GM-CSF-stimulated neutrophils, direct contact between the two was essential; interference in such interaction by separation using a 0. 45-microm-pored membrane prevented such an effect. Addition of colchicine attenuated GM-CSF-stimulated neutrophil fungicidal activity in a dose-dependent manner. This effect did not appear to be mediated by interference with neutrophil mobility toward yeast cells, because similar results were obtained when the cultures were set in round-bottomed wells which facilitate their direct contact. Finally, granular extracts derived from unstimulated neutrophils significantly suppressed the growth of microorganisms. Pretreatment of neutrophils with GM-CSF markedly enhanced this effect. The fungicidal activity of granular lysates was strongly, but not completely, reduced by heat treatment. Considered together, our results indicate that GM-CSF-stimulated neutrophils killed the yeast form of P. marneffei present in close proximity, probably in a superoxide anion-independent mechanism, but through exocytosis of granular enzymes which were largely heat-labile.

Cytokine-induced fungicidal activity of human polymorphonuclear leukocytes against Penicillium marneffei.

We investigated the fungicidal activity of human polymorphonuclear leukocytes (PMN) against Penicillium marneffei. The yeast cells were cocultured in vitro with PMN for 24 h. Microscopic examination was also performed to examine the germination of yeast cells and their transformation to hyphal form during culture. Unstimulated PMN inhibited fungal growth when used at a higher effector/target (E/T) ratio but inhibited germination at a lower E/T ratio. We also examined the effects of various PMN-activating cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF), G-CSF, interleukin (IL)-8, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha on the activity of PMN. Among these, GM-CSF, G-CSF and IFN-gamma enhanced PMN activity from being fungistatic to fungicidal. However, the other cytokines had little or no effect on PMN activity. In contrast, all tested cytokines enhanced PMN inhibitory effects on germination and morphological changes of P. marneffei. These antifungal activities were most strongly induced by GM-CSF. The combined use of any of the above cytokines failed to synergistically enhance antifungal PMN activity. Our results demonstrated that cytokine-activated PMN exert a significant antifungal activity, by suppressing the growth and germination of P. marneffei. Our results suggest that PMN may contribute to host resistance to infection against this fungal pathogen.

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.

Different susceptibilities of yeasts and conidia of Penicillium marneffei to nitric oxide (NO)-mediated fungicidal activity of murine macrophages.

Penicillium marneffei is an important opportunistic fungal pathogen. Host defence mechanisms against P. marneffei are not fully understood. We investigated the fungicidal activity of murine peritoneal macrophages against two forms of P. marneffei, conidia and yeast cells, and the involvement of the NO-mediated killing system. Peritoneal macrophages suppressed the intracellular growth of P. marneffei yeast cells and conidia. The number of live yeast cells within macrophages was significantly reduced by activation of macrophages by interferon-gamma (IFN-gamma), while a similar response was not observed with conidia. IFN-gamma-induced macrophage fungicidal activity against yeast cells was mediated by NO and was almost completely inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthesis, while N(G)-monomethyl-D-arginine (D-NMMA), an optical isomer of L-NMMA, did not show any influence. NO production by macrophages stimulated with IFN-gamma was significantly enhanced when these macrophages were cultured with P. marneffei yeast cells, while conidia did not enhance macrophage NO production. Furthermore, yeast cells were more susceptible to the killing effect of chemically generated NO than conidia. Our results indicate that the yeast form of P. marneffei is more sensitive to the fungicidal activity of IFN-gamma-stimulated macrophages than conidia, and suggest that the different effects of two forms of P. marneffei on macrophage NO production and their different susceptibilities to NO may be reasons for the present findings.

CD4+ T cell-mediated fatal hyperinflammatory reactions in mice infected with Penicillium marneffei.

In the present study, we demonstrate that all mice survived when a low dose of Penicillium marneffei was instilled intratracheally, while severe infection induced by instillation of 1 x 10(8) and 1 x 10(7) colony-forming units (CFU) killed all and 50% of mice, respectively, within 14 days, although the number of live microorganisms in the lungs of these animals was found to decrease with time. The cellular inflammatory responses in the lungs were much more striking in mice with severe infection than in animals infected with a low dose of microorganisms. The number of leucocytes, macrophages, neutrophils and lymphocytes in the lungs increased progressively during infection, and were more than 50 times higher on day 11 than in the control count. CD4+ T cells were the predominant cells in the lungs, and played an important role in hyperinflammatory host reactions, because neutralizing anti-CD4 MoAb increased the survival rate in infected mice despite the presence of a high number of live microorganisms in the lungs. Our results indicate that severe infection with P. marneffei induces fatal hyperinflammatory host reactions, mediated to a large extent by CD4+ T cells, although the infection is well controlled. However, the contribution of endogenous tumour necrosis factor-alpha (TNF-alpha) remains unsubstantiated, since administration of neutralizing anti-TNF-alpha MoAb in the present study failed to prolong survival in infected mice.

Different susceptibility of three clinically isolated strains of Cryptococcus neoformans to the fungicidal effects of reactive nitrogen and oxygen intermediates: possible relationships with virulence.

We investigated the susceptibility of three clinically isolated strains of Cryptococcus neoformans with different virulences to reactive nitrogen and oxygen intermediates (RNI and ROI, respectively), representing two important mediators of macrophage microbicidal activity. All mice infected with the highly virulent strain of C. neoformans, YC-11, died within 3 to 6 weeks because of rapid multiplication of the organism in the lungs and dissemination to the brain. In contrast, a weakly virulent strain, YC-13, was almost completely eradicated from the lungs and did not disseminate to the brain, leading to survival of all infected animals during the period of observation (15 weeks). The virulence of the third strain, YC-5, was intermediate between the other two strains. To examine the susceptibility of C. neoformans to the fungicidal effect of nitric oxide (NO) and superoxide anions (O2-), the organisms were exposed to these oxidants, which were chemically generated in a cell-free system. Interestingly, the number of live YC-13 yeast cells was markedly reduced after exposure to NO and O2-. In contrast, YC-11 was almost completely resistant to the killing effect of these oxidants. YC-5 showed an intermediate susceptibility. Our results demonstrate that the resistance of C. neoformans to the fungicidal effects of RNI and ROI is related to virulence, and suggest that the resistance to nitrogen- and oxygen-derived oxidants may be one of the factors to determine the outcome of infection with C. neoformans.

Cell-mediated immunity in host resistance against infection caused by Penicillium marneffei.

Penicillium marneffei is one of the most important opportunistic infectious pathogens in AIDS patients in Thailand and Southeast Asia. However, very little is known about the host defence mechanisms against P. marneffei infection. In the present study, we established the first experimental murine model of chronic pulmonary and disseminated infection using P. marneffei, and examined the immunological response to such infection in euthymic and athymic mice. In this model, micro-organisms inoculated intratracheally multiplied progressively in the lungs and disseminated to the liver and spleen. However, the number of organisms decreased gradually in these organs. In contrast, congenitally athymic mice developed severe pulmonary and disseminated systemic mycosis. Pulmonary penicilliosis marneffei was associated with a marked cellular inflammatory response as evident by histological abnormalities and increased intraparenchymal leucocyte count. To confirm the importance of cell-mediated immunity in host resistance to P. marneffei infection, we transferred nylon wool non-adherent spleen cells into the athymic mice. Such treatment significantly reduced the number of yeasts in the organs of athymic mice. Taken together, our results demonstrate that the cell-mediated immunity play a central role in a host defence mechanism against infection with P. marneffei, and suggest that our new model may be a useful approach for studying the pathogenesis of this fungal disease.

Contribution of interferon-gamma in protecting mice during pulmonary and disseminated infection with Cryptococcus neoformans.

In the present study, the role of interferon-gamma (IFN-gamma) in the host resistance against Cryptococcus neoformans was examined using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with live yeast cells, and the histological changes in the lungs and the number of microorganisms in the lung and brain were compared in mice treated and untreated with anti-IFN-gamma monoclonal antibody (mAb) to define the contribution of endogenously synthesized IFN-gamma in the natural course of infection. Administration of this mAb reduced the accumulation of inflammatory cells in the alveolar septa, peribronchial and perivascular areas, and promoted the expansive growth of microorganisms in the alveoli and destruction of alveolar structure. The neutralization of endogenous IFN-gamma by mAb increased the number of microorganisms in the lung and brain, and significantly shortened the survival time of infected mice. On the other hand, administration of IFN-gamma decreased the number of microorganisms in these organs, and significantly extended their survival time. Considered together, our results suggest that endogenous IFN-gamma protects mice from infection with C. neoformans by inducing a cellular inflammatory response, potentiating the clearance of microorganism from the lungs and preventing its dissemination into the central nervous system.

Mac1 discriminates unusual CD4-CD8- double-negative T cells bearing alpha beta antigen receptor from conventional ones with either CD4 or CD8 in murine lung.

Pulmonary intraparenchymal leukocytes were purified from normal mice. By flow cytometry, 20-30% of the lymphocytes were positive for the expression of Mac1, a cell-surface antigen largely restricted to macrophages, neutrophils and natural killer (NK) cells. Sorted Mac1+ lung lymphocytes were large and had abundant cytoplasm with few azurophilic granules. Because Mac1+ lymphocytes did not contain any asiallo GM1+ cells, they are not likely to be NK cells. By a two-color flow cytometric analysis, Mac1+ lymphocytes were demonstrated to be TCR-alpha beta intermediate+, TCR-gamma delta-, CD3intermediate+, CD4-, CD8-, Thy1-, CD5-, and B220-. These Mac1+ alpha beta T cells were not found in other organs such as spleen, thymus, liver, bone marrow and intestine of mice uninfected and infected with Mycobacterium bovis BCG. There was a considerable population of this unusual subset of alpha beta T cells in the lungs of congenitally athymic nude mice. In the Mac1+ alpha beta T-cell population, the proportions of V beta 8+ T cells and of forbidden T-cell clones expressing V beta 6 TCR were not much different from that in the conventional T-cell population. These results indicated that extrathymically developed alpha beta T cells reside in considerable proportions in the lung and that Mac1 clearly discriminates these cells from conventional ones. Interestingly, the proportion of these cells increased in the lungs of mice infected with M. bovis BCG, which raises a possibility that these cells may play some role in the host defense against mycobacterial infection.

T cell-dependent activation of macrophages and enhancement of their phagocytic activity in the lungs of mice inoculated with heat-killed Cryptococcus neoformans: involvement of IFN-gamma and its protective effect against cryptococcal infection.

Previous investigations have demonstrated that macrophages play a critical role in the first-line cellular defense mechanism against infection with Cryptococcus neoformans. In the present study, to elucidate the way in which anticryptococcal activity of macrophages is regulated at the site of infection, pulmonary intraparenchymal macrophages were directly analyzed for expression of their surface molecules and their phagocytic activities against the organism, and the effects of depletion of T cells and endogenous IFN-gamma in vivo on these parameters were examined. In the lungs of mice intratracheally inoculated with heat-killed C. neoformans, macrophages were activated, as indicated by augmented expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1) and Fc receptor (FcR), and about two-thirds of macrophages were found to have ingested an average of 3.77 +/- 0.12 yeast cells per macrophage. In mice depleted of both CD4+ and CD8+ T cells by injecting the specific monoclonal antibodies (mAbs) or anti-IFN-gamma mAb, not only augmentation of the expression of macrophage activation markers but also phagocytosis of C. neoformans was significantly reduced. These results suggest that anticryptococcal activity of macrophages is regulated by IFN-gamma endogenously produced by T cells. Additionally, treatment with IFN-gamma were shown to significantly prolong the survival time of mice infected with viable C. neoformans. Additionally, preimmunization with heat-killed C. neoformans significantly prolonged the survival time of mice which received the following infection.

[A therapeutic trial of experimental tuberculosis with gamma-interferon in an immunocompromised mouse model].

It has been well documented that IFN-gamma plays an important role in the host defense against Mycobacterium tuberculosis infection through activating macrophages to kill the organism. In the present study, we studied the effects of in vivo injection of monoclonal antibody against IFN-gamma (anti-IFN-gamma mAb) on the mycobacterial infection to confirm the role of this cytokine. The injection of anti-IFN-gamma mAb suppressed the enhanced expression of MHC class II and ICAM-1 on pulmonary parenchymal macrophages induced by intravenous injection of Mycobacterium bovis BCG. The number of bacilli recovered from lung of mice treated with anti-IFN-gamma mAb and injected with Mycobacterium tuberculosis H37Rv was significantly larger than that of the control infected mice. From these results, it was indicated that anti-IFN-gamma mAb blocked the activities of endogenously synthesized IFN-gamma, thus inhibited the activation of macrophages to kill the bacilli. Next, CD4+ T cell-depleted mice were prepared by injecting anti-CD4 mAb and used as immunocompromised animal. When infected with M. tuberculosis, the multiplication of the bacilli within the lungs of such immunocompromised mice was much more enhanced in comparison with the control mice with intact CD4+ T cells. Administration of IFN-gamma significantly reduced the number of the bacilli in lung. Further, in an in vitro study with human lung macrophages, IFN-gamma enhanced the killing activity of macrophages against M. tuberculosis in a dose dependent manner, and suboptimal dose of 1 alpha, 25-dihydroxyvitamin D3 synergistically augmented the effect of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of acetaminophen-induced pneumonitis].

A case of acetaminophen-induced pneumonitis is described. A 63-year-old woman took 2350 mg of acetaminophen for fever. She subsequently developed a non-productive cough and dyspnea. Chest X-ray revealed diffuse reticulonodular shadows in bilateral lung field and she was admitted to our hospital. PaO2 was 45.0 Torr and PaCO2 35.7 Torr while breathing room air. Bronchoalveolar lavage examination showed an increase in the percentage of lymphocytes and a decrease of CD4/CD8 ratio. Microscopic examination of a transbronchial lung biopsy specimen showed alveolar septal thickening. The lymphocyte stimulation test was positive for acetaminophen, with a stimulation index of 237%, while other drugs used were negative. Because drug-induced pneumonitis was suspected, all drugs were stopped and she was administered methylprednisolone. Consequently her symptoms, laboratory data and chest X-ray findings improved. Based on these findings, we diagnosed this case as acetaminophen-induced pneumonitis. To our knowledge, there has been only one previously reported case of acetaminophen-induced pneumonitis.