Distinct CD8 T cell functions mediate susceptibility to histoplasmosis during chronic viral infection.

It has long been recognized that some viral infections result in generalized immune suppression. In acute infections, this period of suppressed immunity is relatively short. However, chronic infections associated with a prolonged period of immune suppression present far greater risks. Here, we examined the role of CD8 T cell responses following viral infection in immunity to systemic histoplasmosis. Although wild-type mice with systemic histoplasmosis were able to control the infection, those simultaneously infected with lymphocytic choriomeningitis virus clone 13 showed reduced immunity with greater fungal burden and high mortality. The immune suppression was associated with loss of CD4 T cells and B cells, generalized splenic atrophy, and inability to mount a granulomatous response. Removing the anti-viral CD8 T cells in the coinfected mice enabled them to reduce the fungal burden and survive the infection. Their lymphoid organs were replenished with CD4 T and B cells. In contrast to wild-type mice, perforin-deficient mice infected with lymphocytic choriomeningitis virus clone 13 and Histoplasma showed an absence of immunopathology, but the animals still died. These results show that CD8 T cells can suppress immunity through different mechanisms; although immunopathology is perforin-dependent, lethality is perforin-independent.

Long-lived immune response to early secretory antigenic target 6 in individuals who had recovered from tuberculosis.

We sought to understand the persistence and relevance of the long-lived immune response to early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis in humans. ESAT-6 is recognized by memory cells involved in protection of animals against tuberculosis (TB). Recent reports also showed that ESAT-6 response can be recovered in patients with TB and in those soon after anti-TB therapy. We chose 18 individuals who had recovered from pulmonary TB (some in remission for >5 years), and 14 bacille Calmette-Guérin-vaccinated healthy individuals for this study. The results showed that peripheral blood mononuclear cells of 10 (55.6%) of 18 patients with TB remission responded to ESAT-6 with stimulation indices >3.0, whereas none of the healthy controls responded. Functional analysis showed that 13 (72.2%) of 18 patients with TB remission produced significant amounts of IFN-gamma in response to ESAT-6, whereas only 1 (7.1%) of the 14 healthy control subjects did so. It appears that responses to ESAT-6 can persist in individuals who had recovered from pulmonary TB.

Nitric oxide synthase expression in macrophages of Histoplasma capsulatum-infected mice is associated with splenocyte apoptosis and unresponsiveness.

Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of NG-monomethyl-L-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice.

Antihistoplasma effect of activated mouse splenic macrophages involves production of reactive nitrogen intermediates.

The mechanism by which recombinant murine gamma interferon (rMuIFN-gamma) and bacterial lipopolysaccharide (LPS) activate mouse resident splenic macrophages to inhibit the intracellular growth of the fungus Histoplasma capsulatum was examined. Growth inhibition depended on L-arginine metabolism. The growth inhibitory state normally induced by rMuIFN-gamma and LPS in resident splenic macrophages did not occur when the macrophages were cultured in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of L-arginine metabolism. Resident splenic macrophages treated with rMuIFN-gamma and LPS produced nitrite (NO2-), an end product of L-arginine metabolism. When macrophages were cultured in the presence of NG-monomethyl-L-arginine together with rMuIFN-gamma and LPS, only baseline levels of NO2- were detected. Spleen cells from H. capsulatum-infected mice produced high levels of NO2- in culture. The production of NO2- correlated with in vitro inhibition of the intracellular growth of H. capsulatum. Anti-tumor necrosis factor alpha antibody did not block NO2- production by the immigrant splenic macrophages and did not abolish the antihistoplasma activity.

Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity.

The antihistoplasma activity of recombinant murine gamma interferon (rMuIFN-gamma)-treated macrophages of the RAW 264.7 cell line depends on the generation of nitric oxide (NO.) from L-arginine. Macrophages of the P388D1 cell line treated with rMuIFN-gamma do not produce NO. or inhibit the intracellular growth of Histoplasma capsulatum. NO. is generated by the inducible enzyme nitric oxide synthase (iNOS) formed by stimulated macrophages. Northern (RNA) blot analysis of RAW 264.7 cells revealed the expression of iNOS mRNA after exposure to rMuIFN-gamma. In contrast, rMuIFN-gamma-treated P388D1 cells did not produce detectable levels of iNOS. These data suggest that the failure of P388D1 cells to generate NO. and to restrict the intracellular growth of H. capsulatum is due to a lack of expression of iNOS following treatment with rMuIFN-gamma.

Recombinant murine gamma interferon stimulates macrophages of the RAW cell line to inhibit intracellular growth of Histoplasma capsulatum.

Macrophages of the RAW 264.7 cell line, activated by pretreatment with recombinant murine gamma interferon, inhibit the intracellular growth of Histoplasma capsulatum. Growth inhibition occurred by a mechanism that was operative only when L-Arg metabolism was allowed to occur. When activated macrophages were cultured in the absence of L-Arg or in the presence of NG-monomethyl-L-Arg, a competitive inhibitor of L-Arg metabolism, activation to the antihistoplasma growth-inhibitory state did not occur. An increase in levels of NO2-, an end product of L-Arg metabolism, was detected only after activation of RAW 264.7 cells to the growth-inhibitory state. In contrast, only baseline levels of NO2- were detected when L-Arg was excluded or when NG-monomethyl-L-Arg was added to the culture medium. Nitric oxide (NO.), a reactive intermediate product of L-Arg metabolism, was implicated as the relevant antihistoplasma effector molecule. When H. capsulatum yeast cells were cultured for 24 to 28 h in a system designed to generate soluble NO., a dose-dependent cytotoxic effect was observed.

Cytokines in the host response to mycotic agents.

In summary, different approaches have been taken to understand cytokine responses to different fungal infections. Singer-Vermes and co-investigators indirectly examined cytokine responses to paracoccidioidomycosis by studying the types of cellular and humoral immune responses that were induced in resistant and susceptible mouse strains. Their results implicated Th1 cell responses in the resistant mouse strain and Th2 cell responses in the mouse strain susceptible to paracoccidioidomycosis. By measuring cytokine production and through cytokine depletion experiments, Wu-Hsieh showed that besides IFN gamma, TNF alpha was important in host defences against the intracellular pathogen, H. capsulatum. Both cytokines play important roles in the regulation of other cytokines. In histoplasmosis, the dynamics of the complex interactions amongst cytokines govern the efficiency of host clearance of the fungus from tissues. Ferrante and collaborators, examining TNF alpha and TNF alpha receptors on neutrophils presented data showing that TNF alpha plays an important role in the activation of neutrophils for anti-Candida activity. Through the detection of cytokine mRNAs with RT-PCR, Moser and co-workers found that cytokine mRNAs of macrophage origin were produced preferentially in the lungs of mice infected with Histoplasma or Blastomyces. A great challenge still lies ahead of us. It is well understood that the interactions of cytokines are extremely complex at the levels of the induction and expression of the immune responses as well as on effects on natural cellular defences. Work accomplished thus far has laid the ground work for future studies in the effort to dissect host cytokine responses and to understand the roles of cytokines in protection against fungal infections.

Gamma interferon cooperates with lipopolysaccharide to activate mouse splenic macrophages to an antihistoplasma state.

Inhibition of the intracellular growth of Histoplasma capsulatum by murine resident red pulp splenic macrophages was examined. Splenic macrophages, unlike resident peritoneal macrophages, required a prolonged preincubation (18 h) with recombinant murine gamma interferon (rMuIFN-gamma) for activation. To be fully activated, the splenic macrophages required incubation with rMuIFN-gamma in combination with 0.1 microgram of lipopolysaccharide (LPS) per ml. Splenic macrophages stimulated with rMuIFN-gamma, LPS, or rMuIFN-gamma and LPS produced tumor necrosis factor alpha (TNF-alpha), but recombinant murine TNF-alpha (rMuTNF-alpha) did not activate macrophages when used alone or as a second signal with rMuIFN-gamma. Anti-TNF-alpha antibody did not block IFN-gamma-LPS activation of splenic macrophages to any significant extent. One hundred micromolar ferrous sulfate antagonized IFN-gamma-LPS activation of splenic macrophages, indicating that iron was involved in the fungistatic activity of cytokine-stimulated phagocytes. Our results indicate that (i) splenic macrophages differ significantly from peritoneal macrophages in their requirements for activation and (ii) the mechanism by which splenic macrophages exert their antifungal effects involves iron.

Resistance mechanisms in murine experimental histoplasmosis.

The study by use of immunocytochemical methods shows that the spleen of mouse infected intravenously by Histoplasma capsulatum is heavily infiltrated by macrophages. The CD4+ and CD8+ T cells are diffused and sparsely distributed throughout the spleen. It appears that experimental histoplasmosis in animals presents as a disease of the mononuclear phagocyte systems. Macrophages are important cells in controlling replication of intracellular H. capsulatum. Factors that affect the infiltration and activation of macrophages are, thus, important in host defense against histoplasmosis. Depletions of endogenous TNF-alpha in animals infected with sublethal dose of H. capsulatum results in death of these animals. The fungus burden in these animals is high and macrophages are not capable of restricting proliferations of the fungus. However, the role of TNF-alpha in histoplasmosis is not a direct activation of macrophages and is still yet to be defined. IFN-gamma has been shown to fully activate mouse peritoneal macrophages and partially activate splenic macrophages for anti-histoplasma activity. The importance of IFN-gamma in host defense against histoplasmosis is studied by use of resistant A/J and susceptible C57BL/6 mouse strains. There is a good correlation of early production of IFN-gamma by spleen cells of infected mice with the ability of the animals to clear the infection. Spleen cells of resistant A/J mice are more efficient than susceptible C57BL/6 mice in production of IFN-gamma. Recombinant inbred progeny of A/J and C57BL/6 mice are used to locate the genes that control resistance to histoplasmosis. Preliminary studies show that the resistance phenotype is controlled not by a single gene but by multiple genes.

Early activation of splenic macrophages by tumor necrosis factor alpha is important in determining the outcome of experimental histoplasmosis in mice.

Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally. The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells. Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice. Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection. Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion. It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.

Intracellular growth inhibition of Histoplasma capsulatum induced in murine macrophages by recombinant gamma interferon is not due to a limitation of the supply of methionine or cysteine to the fungus.

Recombinant murine gamma interferon (rMuIFN-gamma) stimulates mouse peritoneal macrophages to inhibit the intracellular growth of the zoopathogenic fungus Histoplasma capsulatum. In some systems, the inhibition of growth of an intracellular parasite by rIFN-gamma has been related to nutritional constraints induced in the host cells by the lymphokine. Such an explanation might apply to H. capsulatum because the fungus is a functional methionine-cysteine (Met-Cys) auxotroph at 37 degrees C; its sulfite reductase is repressed at that temperature. For this reason, we set about to examine whether or not the antihistoplasma state induced in rMuIFN-gamma is due to a restriction in the availability of Met-Cys. Omission of Met-Cys from the medium in which macrophages were cultivated prevented H. capsulatum from growing within them. Addition of Met or Cys to the macrophage cultures did not antagonize the inhibitory effect induced in the cells by rMuIFN-gamma. Thus, there was no evidence from our work that rMuIFN-gamma evokes the antihistoplasma effect in mouse peritoneal macrophages by limiting the supply of Met-Cys to the fungus.

Iron limitation and the gamma interferon-mediated antihistoplasma state of murine macrophages.

The zoopathogenic fungus Histoplasma capsulatum requires iron for growth. Intracellular growth of the fungus within mouse peritoneal macrophages is inhibited by recombinant murine gamma interferon (IFN-gamma). Such treatment of mouse peritoneal macrophages induces a marked downshift in transferrin receptors. We tested whether the antihistoplasma effect of IFN-gamma-treated macrophages is the result of iron deprivation. Treatment of mouse peritoneal macrophages with the intracellular iron chelator deferoxamine inhibits the intracellular growth of H. capsulatum. Exposure of macrophages to holotransferrin antagonizes the effect of both recombinant murine IFN-gamma and deferoxamine treatments. These results suggest that iron restriction may be one of the bases for the IFN-gamma-induced antihistoplasma effect of mouse macrophages.

Inhibition of the intracellular growth of Histoplasma capsulatum by recombinant murine gamma interferon.

Recombinant murine gamma interferon as well as lymphokines prepared from immune splenocytes and concanavalin A-stimulated T-cell hybridoma activated normal mouse peritoneal macrophages to inhibit the intracellular growth of Histoplasma capsulatum. The activities of the lymphokine from immune splenocytes and of recombinant murine gamma interferon were neutralized by rabbit anti-murine gamma interferon antibody. The intracellular yeasts were not killed by the interaction even though growth was completely inhibited.