Endogenous interleukin-12 is involved in resistance to Brucella abortus infection.

Protective immunity against Brucella abortus is mediated by acquired cellular resistance, with gamma interferon (IFN-gamma)-producing T cells playing a key role. Interleukin-12 (IL-12) is a cytokine that has a profound effect on the induction of IFN-gamma-producing Th1 and NK cells. Here we report that depletion of endogenous IL-12 before infection of mice significantly exacerbated brucella infection. IL-12-depleted mice also had reduced splenomegaly resulting from infection and showed a decrease in percentage and absolute numbers of macrophages compared with those in control infected mice. Furthermore, spleen cells from IL-12-depleted mice had a reduced ability to produce nitrite, a product of activated macrophages. This could be the result of the low production of IFN-gamma by splenic T cells observed in the IL-12-depleted mice. The mechanism whereby IL-12 controls antibacterial resistance is discussed.     

Endogenous interleukin-12 is involved in resistance of mice to Mycobacterium avium complex infection.

Acquired cellular resistance against Mycobacterium avium complex (MAC) infections involves the induction of Th1 type gamma interferon (IFN-gamma)-producing T cells. Interleukin-12 (IL-12) is a cytokine involved in the control of IFN-gamma production by T cells and NK cells. The role of IL-12 in the response to MAC infection was investigated. Depletion of endogenous IL-12 by injection of monoclonal antibody prior to and during intranasal infection with MAC resulted in an 150- to 550-fold increase in bacterial load in lung, spleen, and liver homogenates by 10 weeks postinfection. Depletion of IL-12 abrogated the ability of spleen cell cultures to produce IFN-gamma in response to stimulus with live MAC. IL-12-depleted mice showed a 75% decrease in the number of inflammatory cells entering the lungs following intranasal infection with MAC, with significant reductions in cytotoxic activity and nitric oxide production by lung cells. This work suggests that IL-12 plays a major role in the activation of IFN-gamma-producing cells during MAC infection.     

Role of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 in the pathogenesis of severe murine monocytotropic ehrlichiosis: increased resistance of TNF receptor p55- and p75-deficient mice to fatal ehrlichial infection

Intraperitoneal (i.p.) infection with a high dose of a highly virulent Ehrlichia strain (IOE) results in a toxic shock-like syndrome characterized by severe liver injury and systemic overproduction of tumor necrosis factor alpha (TNF-alpha) by CD8+ T cells. We examined the role of TNF-alpha and TNF receptors in high-dose-IOE-induced shock/liver injury. TNF receptor (TNFR) I/II-/- mice lacking both the p55 and p75 receptors for this cytokine were more resistant to IOE-induced liver injury than their wild-type background controls. TNFR I/II-/- mice survived longer, dying between 15 and 18 days, with evidence of mild liver necrosis/apoptosis. In contrast, wild-type mice were not rescued from the lethal effect of IOE by TNF-alpha neutralization. TNF-alpha-depleted mice developed severe liver injury and succumbed to disease between days 9 and 11 postinfection, similar to sham-treated, infected wild-type mice. Although IFN-gamma production in the spleens of IOE-infected TNFR I/II-/- and TNF-alpha-depleted mice was higher than that detected in wild-type controls, these mice had higher bacterial burdens than infected controls. Following high-dose IOE challenge, TNFR I/II-/- and TNF-alpha-depleted mice have an early increase in IL-10 levels in sera and spleens, which was produced mainly by adherent spleen cells. In contrast, a late burst of interleukin-10 (IL-10) was observed in control mice. Nonadherent spleen cells were the major source of IL-10 in IOE-infected wild-type mice. We conclude that TNFR I/II and TNF-alpha participate in Ehrlichia-induced shock and host defense by regulating liver injury and controlling ehrlichial burden. Our data suggest that fatal ehrlichiosis could be a multistep process, where TNF-alpha is not solely responsible for mortality.     

Cytokine production in the murine response to brucella infection or immunization with antigenic extracts.

In order to induce acquired cellular resistance (ACR) to facultative intracellular bacterial pathogens, infection with live organisms is required. It is possible that different cytokine responses to live bacteria or their extracted antigens could account for their different abilities to induce ACR. Therefore, mice were infected with live attenuated Brucella abortus vaccine strain 19, and their ability to produce cytokines, both in vivo and in vitro, was investigated over 12 weeks of infection. This was compared with the response to injection of soluble brucella proteins (SBP). During infection, serum levels of interleukin-6 (IL-6) were markedly increased over a period of 4 weeks during the peak of infection. SBP plus adjuvant induced a transient increase in serum IL-6. IL-1 and tumour necrosis factor-alpha (TNF-alpha) remained undetectable in both instances. Spleen cells taken at intervals after infection and cultured with brucella antigens produced high titres of IL-6, IL-1 and TNF-alpha. Immunization with SBP was less efficient than live infection at inducing these cytokines. Of the characteristically T-cell-derived lymphokines, interferon-gamma (IFN-gamma) production rose 2 weeks after infection, peaking at 6 weeks, while IL-2 was not detected until 6 weeks post-infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was produced in substantial amounts, but IL-3 production was minimal. In contrast, spleen cells from mice immunized with SBP produced IL-2 but failed to produce IFN-gamma. The implications of these results for the induction of ACR are discussed.     

Neutrophil depletion exacerbates experimental Chagas' disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions

To elucidate the roles of neutrophils in experimental Chagas' disease, we depleted the peripheral neutrophils in BALB/c and C57BL/6 mice with a monoclonal antibody 1 day before Trypanosoma cruzi infection. Neutrophil depletion in BALB/ c mice resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines, including IL-2 and IFN-gamma, IL-12p40 and TNF-alpha in their spleens after the infection, while a Th2 cytokine, IL-10, increased especially 1 day after infection. Neutrophils from infected BALB / c mice expressed mRNA for IL-12p40, IFN-gamma, TNF-alpha and Th1 chemoattractive chemokines, monokine induced by IFN-gamma (MIG) and macrophage inflammatory protein-1alpha (MIP-1alpha ). In contrast, in C57BL/6 mice neutrophil depletion induced resistance to the disease and enhanced the expression of the above Th1 cytokines, although IL-10 mRNA in neutrophil-depleted C57BL/6 mice was also higher than in control mice. Neutrophils from C57BL/6 mice did not express IL-12p40, IFN-gamma and MIG but expressed TNF-alpha, MIP-1alpha and IL-10. Therefore, neutrophils may play opposite roles in these two strains of mice with respect to protection versus exacerbation of T. cruzi infection, possibly through modulating the Th1/Th2 dichotomy in different directions.     

Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes.

Host resistance to infection by Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the first week of infection in mice, NK cell-derived gamma interferon (IFN-gamma) is involved in controlling intracellular parasite replication, mainly through the induction of NO biosynthesis by activated macrophages. Interleukin-12 (IL-12) has been shown to be a powerful cytokine in inducing IFN-gamma synthesis by NK cells, as well as in mediating resistance to different intracellular protozoa. We have therefore studied the ability of T. cruzi to elicit IL-12 synthesis by macrophages and the role of this cytokine in controlling parasite replication during acute infection in mice. Our results show that macrophages cultured in the presence of live trypomastigote forms (but not epimastigotes) release IL-12 that can induce IFN-gamma production by normal spleen cells. IL-12 was detected in as little as 12 h after the addition of the trypomastigotes, and the level of IL-12 peaked at 48 h after the initial macrophage-parasite incubation. The addition of anti-IL-12 monoclonal antibody to macrophage-trypomastigote supernatants dose-dependently inhibited IFN-gamma production by naive splenocytes. Finally, the in vivo role of IL-12 in resistance to infection by T. cruzi was analyzed. Mice treated with anti-IL-12 monoclonal antibody had significantly increased parasitemia and mortality in comparison with those of control infected mice treated with control antibody. Together, these results suggest that macrophage-derived IL-12 plays a major role in controlling the parasitemia in T. cruzi-infected mice and that the animal's resistance during the acute phase of infection may, at least in part, be a consequence of postinfection levels of IL-12.     

Interleukin-12 modulates the protective immune response in SCID mice infected with Histoplasma capsulatum.

Infection with Histoplasma capsulatum results in a subclinical infection in immunocompetent hosts due to an effective cellular immune response. By contrast, immunodeficient individuals can have a severe disseminated and potentially fatal disease. In a previous study, it was demonstrated that normal mice infected intravenously with H. capsulatum and treated with interleukin-12 (IL-12) at the time of infection were protected from a fatal outcome. In this study, we examined the immunomodulatory effects of IL-12 on disseminated histoplasmosis in immunodeficient SCID mice. SCID mice infected with H. capsulatum and treated with IL-12 showed an increase in survival and a reduction in the colony counts of H. capsulatum in internal organs at 14 days after infection. The protective effect of IL-12 was abrogated if animals were also treated with a neutralizing antibody to gamma interferon (IFN-gamma). IL-12 treatment also resulted in an increase in mRNA expression and protein production for IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and nitric oxide from spleen cells. When IL-12 was combined with amphotericin B (AmB) treatment, there was a significant increase in survival compared with either modality alone. Moreover, combined treatment resulted in an increase in both IFN-gamma and TNF-alpha production, as well as in a substantial reduction in H. capsulatum burden at 35 and 90 days postinfection. This study demonstrates that IL-12 modulates the protective immune response to histoplasmosis in SCID mice and also suggests that IL-12 in combination with AmB may be useful as a treatment for H. capsulatum in immunodeficient hosts.     

Induction of interleukin-12 and gamma interferon requires tumor necrosis factor alpha for protective T1-cell-mediated immunity to pulmonary Cryptococcus neoformans infection

The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-alpha) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-alpha expression was detected in lavage sample cells at days 2, 3, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-alpha-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-alpha prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-alpha monoclonal antibody (day 0) diminished the lung levels of TNF-alpha, interleukin-12 (IL-12), and gamma interferon (IFN-gamma) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-alpha (day 0) also altered the IFN-gamma/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-alpha-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-alpha-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-gamma in response to the infection. These findings demonstrate that afferent-phase TNF-alpha production is essential for the induction of IL-12 and IFN-gamma and neutralization of early TNF-alpha results in a T2 shift of the T1/T2 balance of antifungal immunity.     

Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha.

Previous studies of mice have implicated natural killer (NK) cells as mediators of protective activity against Toxoplasma gondii through their production of gamma interferon (IFN-gamma). In the present study, we have compared NK-cell activity in infected and uninfected SCID mice. Our data reveal that infection results in increased levels of IFN-gamma in serum and elevated NK-cell activity but that these NK cells were not cytotoxic for T. gondii-infected P815 cells. Treatment with anti-IFN-gamma antibody abrogated the increase in NK-cell activity and resulted in earlier mortality of infected mice. In vivo treatment with anti-asialo GM1 antiserum reduced NK cell activity and levels of IFN-gamma in serum but did not alter time to death. Spleen cells from infected mice produced higher levels of IFN-gamma than those from uninfected mice when stimulated in vitro with live T. gondii or parasite antigen preparations. Further analysis revealed that interleukin 10 (IL-10) inhibited, whereas tumor necrosis factor alpha (TNF-alpha) and IL-12 enhanced, IFN-gamma production by spleen cells from infected or uninfected mice. The combination of IL-12 and TNF-alpha induced higher levels of IFN-gamma from whole spleen cells of infected mice than from those of uninfected mice. Depletion of the adherent cell population from the spleen cells of infected mice led to a significant reduction in the levels of IFN-gamma produced after stimulation with IL-12 plus TNF-alpha. Similar results did not occur with cells from uninfected mice. These data indicate that other cytokines produced by the adherent cell population from infected mice may be involved in maximal production of IFN-gamma by NK cells stimulated with IL-12 and TNF-alpha. To assess the importance of endogenous IL-12, a polyclonal anti-IL-12 was administered to infected SCID mice. This treatment led to earlier mortality, indicating that endogenous IL-12 mediates resistance to T. gondii.     

Roles of interleukin-12 and gamma interferon in murine Chlamydia pneumoniae infection

BALB/c and strain 129 mice infected intranasally with Chlamydia pneumoniae displayed a moderate-to-severe inflammation in the lungs and produced interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10, with peak levels on days 1 to 3 postinfection (p.i.), returning to basal levels by day 16 p.i. Anti-IL-12 treatment resulted in less-severe pathological changes but higher bacterial titers on days 3 and 7 p.i. By day 16 p.i., the inflammatory responses of control antibody-treated mice subsided. The bacterial titers of both anti-IL-12- and control antibody-treated mice decreased within 3 weeks to marginally detectable levels. Anti-IL-12 treatment significantly reduced lung IFN-gamma production and in vitro spleen cell IFN-gamma production in response to either C. pneumoniae or concanavalin A. In gamma-irradiated infected mice, cytokine production was delayed, and this delay correlated with high bacterial titers in the lungs. Following C. pneumoniae infection, 129 mice lacking the IFN-gamma receptor alpha chain gene (G129 mice) produced similar IL-12 levels and exhibited similarly severe pathological changes but had higher bacterial titers than 129 mice. However, by day 45 p.i., bacterial titers became undetectable in both wild-type 129 and G129 mice. Thus, during C. pneumoniae lung infection, IL-12, more than IFN-gamma, plays a role in pulmonary-cell infiltration. IFN-gamma and IL-12, acting mostly through its induction of IFN-gamma and Th1 responses, play an important role in controlling acute C. pneumoniae infection in the lungs, but eventually all mice control the infection to undetectable levels by IL-12- and IFN-gamma-independent mechanisms.     

Effect of 6-Hydroxydopamine on Host Resistance against Listeria monocytogenes Infection

Recent studies have shown that immunocompetent cells bear receptors of neuropeptides and neurotransmitters and that these ligands play roles in the immune response. In this study, the role of the sympathetic nervous system in host resistance against Listeria monocytogenes infection was investigated in mice pretreated with 6-hydroxydopamine (6-OHDA), which destroys sympathetic nerve termini. The norepinephrine contents of the plasma and spleens were significantly lower in 6-OHDA-treated mice than in vehicle-treated mice. The 50% lethal dose of L. monocytogenes was about 20 times higher for 6-OHDA-treated mice than for vehicle-treated mice. Chemical sympathectomy by 6-OHDA upregulated interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) production in enriched dendritic cell cultures and gamma interferon (IFN-gamma) and TNF-alpha production in spleen cell cultures, whereas chemical sympathectomy had no apparent effect on phagocytic activities, listericidal activities, and nitric oxide production in peritoneal exudate cells and splenic macrophages. Augmentation of host resistance against L. monocytogenes infection by 6-OHDA was abrogated in IFN-gamma(-/-) or TNF-alpha(-/-) mice, suggesting that upregulation of IFN-gamma, IL-12, and TNF-alpha production may be involved in 6-OHDA-mediated augmentation of antilisterial resistance. Furthermore, adoptive transfer of spleen cells immune to L. monocytogenes from 6-OHDA-treated mice resulted in untreated naive recipients that had a high level of resistance against L. monocytogenes infection. These results suggest that the sympathetic nervous system may modulate host resistance against L. monocytogenes infection through regulation of production of IFN-gamma, IL-12, and TNF-alpha, which are critical in antilisterial resistance.     

Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon.

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.     

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.     

Inflammatory response following intranasal infection with Mycobacterium avium complex: role of T-cell subsets and gamma interferon.

The role of CD4+ and CD8+ T cells in the response to intranasal infection with a Mycobacterium avium complex isolate (MAC) was investigated. Depletion of CD4+ T cells by injected antibody exacerbated infection in the lung, spleen, and liver. There were decreased numbers of inflammatory cells in the lungs of CD4-depleted mice and a significant decrease in lung cytotoxic activity. The neutrophil response was unaffected, and in CD4-depleted mice, unlike intact infected mice, these cells were found with large numbers of associated MAC. Purified CD4+ splenic T cells produced gamma interferon (IFN-gamma) in vitro in response to MAC antigen. IFN-gamma production by cultured spleen, lung, or mediastinal lymph node cells was markedly reduced in CD4-depleted mice. In contrast, CD8+ T cells did not produce IFN-gamma in vitro, and depletion of CD8+ T cells from infected mice had no effect on bacterial growth or lung cell activation. Depletion of IFN-gamma by injected monoclonal antibody had effects similar to those of CD4 depletion, namely, exacerbation of infection and decreased lung cell cytotoxicity. We conclude that CD4+ T cells are the main T cells involved in the lung response to MAC infection and that this response is at least partially dependent on the production of IFN-gamma.     

Critical role of type 1 cytokines in controlling initial infection with Burkholderia mallei

Burkholderia mallei is a gram-negative bacterium which causes the potentially fatal disease glanders in humans; however, there is little information concerning cell-mediated immunity to this pathogen. The role of gamma interferon (IFN-gamma) during B. mallei infection was investigated using a disease model in which infected BALB/c mice normally die between 40 and 60 days postinfection. IFN-gamma knockout mice infected with B. mallei died within 2 to 3 days after infection, and there was uncontrolled bacterial replication in several organs, demonstrating the essential role of IFN-gamma in the innate immune response to this pathogen. Increased levels of IFN-gamma, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 were detected in the sera of immunocompetent mice in response to infection, and splenic mRNA expression of IFN-gamma, IL-6, IL-12p35, and IL-27 was elevated 24 h postinfection. The effects of IL-18, IL-27, and IL-12 on stimulation of the rapid IFN-gamma production were investigated in vitro by analyzing IFN-gamma production in the presence of heat-killed B. mallei. IL-12 was essential for IFN-gamma production in vitro; IL-18 was also involved in induction of IFN-gamma, but IL-27 was not required for IFN-gamma production in response to heat-killed B. mallei. The main cellular sources of IFN-gamma were identified in vitro as NK cells, CD8+ T cells, and TCRgammadelta T cells. Our data show that B. mallei is susceptible to cell-mediated immune responses which promote expression of type 1 cytokines. This suggests that development of effective vaccines against glanders should target the production of IFN-gamma.     

Tumor necrosis factor alpha-mediated toxic shock in Trypanosoma cruzi-infected interleukin 10-deficient mice

Using interleukin-10 (IL-10)-deficient (IL-10(-/-)) mice, previous studies revealed a pathological immune response after infection with Trypanosoma cruzi that is associated with CD4(+) T cells and overproduction of proinflammatory cytokines. In this study we further investigate the pathology and potential mediators for the mortality in infected animals. T. cruzi-infected IL-10(-/-) mice showed reduced parasitemia accompanied by increased systemic release of gamma interferon (IFN-gamma), IL-12, and reactive nitrogen intermediates and overproduction of tumor necrosis factor alpha (TNF-alpha). Despite this early resistance, IL-10(-/-) mice died within the third week of infection, whereas all control mice survived acute infection. The clinical manifestation with weight loss, hypothermia, hypoglycemia, hyperkalemia, and increased liver-derived enzymes in the blood together with hepatic necrosis and intravascular coagulation in moribund mice indicated a toxic shock-like syndrome, possibly mediated by the systemic TNF-alpha overproduction. Indeed, high production of systemic TNF-alpha significantly correlated with mortality, and moribund mice died with critically high TNF-alpha concentrations in the blood. Consequent treatment with anti-TNF-alpha antiserum attenuated pathological changes in T. cruzi-infected IL-10(-/-) mice and significantly prolonged survival; the mice died during the fourth week postinfection, again with a striking correlation between regaining high systemic TNF-alpha concentrations and the time of death. Since elevated serum IL-12 and IFN-gamma concentrations were not affected by the administration of antiserum, these studies suggest that TNF-alpha is the direct mediator of this toxic shock syndrome. In conclusion, induction of endogenous IL-10 during experimentally induced Chagas' disease seems to be crucial for counterregulating an overshooting proinflammatory cytokine response resulting in TNF-alpha-mediated toxic shock.     

Endogenous gamma interferon mediates resistance to Brucella abortus infection.

Depletion of endogenous gamma interferon (IFN-gamma) with anti-IFN-gamma monoclonal antibody resulted in increased numbers of Brucella abortus in the spleen and liver of infected CBA mice. This increase was accompanied by a decrease in splenomegaly and a lower proportion of macrophages in the spleen. Furthermore, treatment of recipient mice with anti-IFN-gamma antibody blocked the adoptive transfer of resistance with immune T cells. Together, the results indicated that endogenous IFN-gamma plays an important role in mediating resistance to primary and secondary Brucella infection.     

Sex-determined resistance to Toxoplasma gondii is associated with temporal differences in cytokine production.

Examination of a wide range of inbred mice of diverse genetic backgrounds and major histocompatibility complex haplotypes revealed a dramatic difference in the susceptibilities of males and females to Toxoplasma gondii infection. Female mice were found to be more susceptible to acute infection, as determined by higher mortality levels, than male mice, while those female mice surviving to have chronic infections harbored more cysts in their brains than did surviving males. This phenomenon was therefore investigated in greater depth immunologically in the BALB/K mouse, a strain showing moderate susceptibility to infection with T. gondii. Plasma tumor necrosis factor alpha (TNF-alpha) levels were elevated in both male and female BALB/K mice on days 8 and 10 postinfection, but not thereafter, with males producing significantly higher levels than females. However, it was not until day 12 postinfection that the first deaths occurred, and these were among female mice, indicating that TNF-alpha production was not responsible for mortality. In vitro examination of T. gondii-specific T-cell proliferative responses from day 15 postinfection onwards revealed significantly higher stimulation indices in male mice than in their female counterparts. This difference was most apparent in splenocyte cultures initiated at day 15 postinfection, where complete suppression of proliferation was noted in the splenocytes from female mice but not from male mice. Analysis of tissue culture supernatants from these cultures revealed distinct differences in the kinetics of production as well as the quantities of gamma interferon (IFN-gamma) and interleukin 10 (IL-10) produced. Spleen cells from male mice produced higher levels of IFN-gamma in the early stages of infection than those from female mice. IFN-gamma levels were highest in the supernatants from male splenocyte cultures initiated at day 15 postinfection. Similar levels of IFN-gamma were not obtained from the supernatants of female splenocyte cultures until day 22 postinfection. IL-10 production, on the other hand, peaked at maximal levels in the cell cultures from both sexes initiated at day 22 postinfection. These results suggest that, in male mice, a rapid response to infection with high levels of TNF-alpha and IFN-gamma helps to control parasite multiplication, after which IL-10 production may be important in down regulating these potentially harmful inflammatory mediators. The failure of female mice to respond quickly in terms of T-cell proliferation and IFN-gamma production compared with their male counterparts may account for their poor survival rates and higher cyst burdens.     

Perforin, a cytotoxic molecule which mediates cell necrosis, is not required for the early control of mycobacterial infection in mice.

Host defense against mycobacterial infection requires the participation of monocytes and T cells. Both CD4+ and CD8+ T cells have been shown to be important in resistance to mycobacterial infection in vivo. The main contribution of CD4+ T cells to the protective antituberculosis response involves the production of Th1-type cytokines, including interleukin-2 (IL-2) and gamma interferon (IFN-gamma). CD8+ T cells have been considered to be responsible primarily for cytotoxicity mediated by toxic molecules, including perforin. CD8+ T cells may also elaborate Th1-type cytokines, such as IFN-gamma, in response to the infection. To elucidate the contribution of perforin-mediated target cell death to the control of mycobacterial infection in vivo, mice with a disruption in the perforin gene (P-/-) were infected with Mycobacterium bovis BCG or M. tuberculosis Erdman for 5 and 13 weeks, respectively. At 1, 3, 5, and 13 weeks postinfection, the number of viable mycobacteria in the lungs, spleens, and livers of mice were determined by CFU assay. The infected tissues were examined histologically, and cytokine mRNA levels in the spleens of these mice were determined. Similar studies were carried out in Fas receptor-defective (CBA/lpr(cg)) mice to evaluate the contribution of this alternative cytotoxic pathway to the control of mycobacterial infection. The absence of either perforin gene function or Fas receptor gene function did not modify the course of experimental mycobacterial infection in these mice. In addition, both P-/- and Fas receptor-defective mice appeared to have a compensatory activation of cytokine genes, even in the absence of the experimental infection. P-/- mice had a mean 3.4- to 5-fold increase in mRNA levels for IL-10, IL-12p35, IL-6, and IFN-gamma. Similarly, Fas receptor-defective mice had a mean 3- to 3.6-fold increase in mRNA levels for IFN-gamma, IL-12p35, and IL-10. Our results indicate that both perforin-mediated cytotoxicity and Fas-mediated cytotoxicity do not appear to be necessary for the early control of mycobacterial infection in vivo.