Masaki Tohyama

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-γ 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‐γ 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‐γ 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‐γ endogenously produced by T cells. Additionally, treatment with IFN‐γ 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.

Enhanced Gamma Interferon Production through Activation of Vα14 + Natural Killer T Cells by α-Galactosylceramide in Interleukin-18-Deficient Mice with Systemic Cryptococcosis

ABSTRACT We showed recently that activation of Vα14+ natural killer T cells (NKT cells) by α-galactosylceramide (α-GalCer) resulted in increased gamma interferon (IFN-γ) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to α-GalCer-induced IFN-γ synthesis. Here we examined the role of IL-18 in α-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-γ in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-γ correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-γ synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-γ in WT mice. Finally, the enhanced production of IFN-γ in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in α-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-γ synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Vα14+ NKT cells.

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

Abstract 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- αβ intermediate+ , TCR- γδ − , CD3 intermediate+ , CD4 − , CD8 − , Thy1 − , CD5 − , and B220 − . These Mac1 + αβ 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 αβ T cells in the lungs of congenitally athymic nude mice. In the Mac1 + αβ T-cell population, the proportions of Vβ8 + T cells and of forbidden T-cell clones expressing Vβ6 TCR were not much different from that in the conventional T-cell population. These results indicated that extrathymically developed αβ 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.

Treatment of Murine Pulmonary Cryptococcosis with a Combination of Fluconazole and Interleukin-12

Interleukin-12 (IL-12) is known to protect the host from various pathogens by inducing cell-mediated immunity. In a murine model of pulmonary infection withCryptococcus neoformans, the daily administration of 0.1 μg/day of IL-12 for 7 day failed to protect mice from infection when treatment commenced 7 days after intratracheal instillation of the pathogen. However, IL-12 administered prophylactically or on the day of infection was effective in combating the infection. Because the number of yeast cells increase by 10-fold in the lungs within a short period of time, and in an attempt to control the growth of the fungus, a combination therapy of an antifungal agent, fluconazole (FCZ), and IL-12 was initiated 7 days after infection. Treatment with FCZ significantly prolonged survival time, and when combined with IL-12, there was a further prolongation of survival time. In addition, administration of both agents significantly reduced the number of live microorganisms isolated from the lung tissue. Our results suggest that the combined use of FCZ and IL-12 may represent a new therapeutic strategy for cryptococcal infections in patients with impaired cell-mediated immunity.