A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii

Long-term resistance to Toxoplasma gondii is dependent on the development of parasite-specific T cells that produce IFN-gamma. CD28 is a costimulatory molecule important for optimal activation of T cells, but CD28(-/-) mice are resistant to T. gondii, demonstrating that CD28-independent mechanisms regulate T cell responses during toxoplasmosis. The identification of the B7-related protein 1/inducible costimulator protein (ICOS) pathway and its ability to regulate the production of IFN-gamma suggested that this pathway may be involved in the CD28-independent activation of T cells required for resistance to T. gondii. In support of this hypothesis, infection of wild-type or CD28(-/-) mice with T. gondii resulted in the increased expression of ICOS by activated CD4(+) and CD8(+) T cells. In addition, both costimulatory pathways contributed to the in vitro production of IFN-gamma by parasite-specific T cells and when both pathways were blocked, there was an additive effect that resulted in almost complete inhibition of IFN-gamma production. Although in vivo blockade of the ICOS costimulatory pathway did not result in the early mortality of wild-type mice infected with T. gondii, it did lead to increased susceptibility of CD28(-/-) mice to T. gondi associated with reduced serum levels of IFN-gamma, increased parasite burden, and increased mortality compared with the control group. Together, these results identify a critical role for ICOS in the protective Th1-type response required for resistance to T. gondii and suggest that ICOS and CD28 are parallel costimulatory pathways, either of which is sufficient to mediate resistance to this intracellular pathogen.     

Blockade of T cell costimulation by CTLA4-Ig inhibits lung inflammation in murine hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is characterized by an influx of activated T cells in the lungs. The CD28/B7 system provides costimulatory signals essential for complete T cell activation and differentiation. We have previously demonstrated that alveolar macrophages from patients with HP have an up-regulated expression of B7 molecules. In the present study, we investigated the effect of i. p. administration of CTLA4-Ig, a CD28/B7 antagonist, on the lung inflammation of mice inoculated with Saccharoplyspora rectivirgula (SR), a major causative agent of HP. Five groups of C57BL/6 mice were intranasally instilled with SR or saline for 3 consecutive days per wk during 3 wk. CTLA4-Ig was administered starting either after 1 wk of SR challenge or 6 h before the first antigenic exposure and continued during the whole period of sensitization. A control-IgG was given similarly during the 3 wk of SR exposure. The groups included: 1, saline; 2, SR; 3, SR + control-Ig; 4, SR + CTLA4-Ig for the last 2 wk; and 5, SR + CTLA4-Ig for 3 wk. CTLA4-Ig treatment markedly decreased lung inflammation as shown by significantly fewer inflammatory cells in the bronchoalveolar lavage and in lung tissue and reduced SR-specific serum and bronchoalveolar lavage Ig levels. Production of IL-4, IL-10, and IFN-gamma by IL-2-stimulated pulmonary T cells was also decreased by CTLA4-Ig. Administration of CTLA4-Ig did not affect the SR-induced up-regulation of B7-2 expression. These results show that blockade of CD28/B7 interactions by CTLA4-Ig inhibits SR-induced lung inflammation and immune response to SR Ag in mice and may provide a novel approach in the treatment of HP.     

Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis

Toxoplasma gondii infects astrocytes, neurons and microglia cells in the CNS and, after acute encephalitis, persists within neurons. Robust astrocyte activation is a hallmark of Toxoplasma encephalitis (TE); however, the in vivo function of astrocytes is largely unknown. To study their role in TE we generated C57BL/6 GFAP-Cre gp130(fl/fl) mice (where GFAP is glial fibrillary acid protein), which lack gp130, the signal-transducing receptor for IL-6 family cytokines, in their astrocytes. In the TE of wild-type mice, the gp130 ligands IL-6, IL-11, IL-27, LIF, oncostatin M, ciliary neurotrophic factor, B cell stimulating factor, and cardiotrophin-1 were up-regulated. In addition, GFAP(+) astrocytes of gp130(fl/fl) control mice were activated, increased in number, and efficiently restricted inflammatory lesions and parasites, thereby contributing to survival from TE. In contrast, T. gondii- infected GFAP-Cre gp130(fl/fl) mice lost GFAP(+) astrocytes in inflammatory lesions resulting in an inefficient containment of inflammatory lesions, impaired parasite control, and, ultimately, a lethal necrotizing TE. Production of IFN-gamma and the IFN-gamma-induced GTPase (IGTP), which mediate parasite control in astrocytes, was even increased in GFAP-Cre gp130(fl/fl) mice, indicating that instead of the direct antiparasitic effect the immunoregulatory function of GFAP-Cre gp130(fl/fl) astrocytes was disturbed. Correspondingly, in vitro infected GFAP-Cre gp130(fl/fl) astrocytes inhibited the growth of T. gondii efficiently after stimulation with IFN-gamma, whereas neighboring noninfected and TNF-stimulated GFAP-Cre gp130(fl/fl) astrocytes became apoptotic. Collectively, these are the first experiments demonstrating a crucial function of astrocytes in CNS infection.     

Induction of tumor necrosis factor-alpha and inducible nitric oxide synthase fails to prevent toxoplasmic encephalitis in the absence of interferon-gamma in genetically resistant BALB/c mice

Following infection with Toxoplasma gondii, certain strains of mice, such as BALB/c, are genetically resistant to development of toxoplasmic encephalitis (TE) and establish a latent chronic infection as do humans. Thus, these animals appear to be a suitable model to analyze the mechanism of resistance to TE. Since the mechanism for their genetic resistance is unknown, we examined the role of interferon-gamma (IFN-gamma) tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) in the resistance using BALB/c-background IFN-gamma-deficient (IFN-gamma(-/-)) mice. IFN-gamma(-/-) and control mice were infected with the ME49 strain of T. gondii and treated with sulfadiazine to establish chronic infection. After discontinuing sulfadiazine, the IFN-gamma(-/-) mice all died, whereas the control mice all survived. Histological studies revealed remarkable inflammatory changes associated with large numbers of tachyzoites in brains of the IFN-gamma(-/-) mice but not in the control mice after discontinuation of sulfadiazine. Large amounts of mRNA for tachyzoite-specific SAG1 were detected in brains of only the IFN-gamma(-/-) mice. IFN-gamma mRNA was detected in brains of only the control mice, whereas mRNA for TNF-alpha and iNOS were detected in brains of both strains of mice. The amounts of the mRNA for TNF-alpha and iNOS did not differ between these mice. Treatment of IFN-gamma(-/-) mice with recombinant IFN-gamma prevented development of TE. These results demonstrate that IFN-gamma is crucial for genetic resistance of BALB/c mice against TE and that TNF-alpha and iNOS are insufficient to prevent TE in the absence of IFN-gamma.     

TCR V beta 8+ T cells prevent development of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease

BALB/c are genetically resistant to development of toxoplasmic encephalitis (TE) when infected with Toxoplasma gondii, whereas CBA/Ca mice are susceptible. We compared TCR Vbeta chain usage in lymphocytes infiltrated into brains between these animals following infection. TCR Vbeta8(+) cells were the most frequent T cell population in brains of infected, resistant BALB/c mice, whereas TCR Vbeta6(+) T cells were more prevalent than Vbeta8(+) T cells in brains of infected, susceptible CBA/Ca mice. Adoptive transfer of Vbeta8(+) immune T cells, obtained from infected BALB/c mice, prevented development of TE and mortality in infected athymic nude mice that lack T cells. In contrast, adoptive transfer of Vbeta6(+) immune T cells did not prevent development of TE or mortality in the nude mice. The protective activity of Vbeta8(+) immune T cells was greater than that of the total Vbeta8(-) population. In addition, Vbeta8(+) immune T cells produced markedly greater amounts of IFN-gamma than did the Vbeta8(-) population after stimulation with tachyzoite lysate Ags in vitro. Thus, Vbeta8(+) T cells appear to play a crucial role in the genetic resistance of BALB/c mice against development of TE.     

Role of CD28 in the generation of effector and memory responses required for resistance to Toxoplasma gondii.

CD28 deficient (CD28-/-) mice were used to study the role of costimulation in the T cell-mediated, IFN-gamma-dependent mechanism of resistance to Toxoplasma gondii. These mice were resistant to infection with the ME49 strain of T. gondii. Analysis of the immune response of acutely infected CD28-/- mice revealed that IL-12 was required for T cell production of IFN-gamma and this was independent of the CD40/CD40 ligand interaction. A similar mechanism of IL-12-dependent, CD28/B7 independent production of IFN-gamma by T cells was also observed in wild-type mice. Interestingly, although chronically infected wild-type mice were resistant to rechallenge with the virulent RH strain of T. gondii, chronically infected CD28-/- mice were susceptible to rechallenge with the RH strain. This deficiency in the protective memory response by CD28-/- mice correlated with a lack of IL-2 and IFN-gamma in recall responses and reduced numbers of CD4+ T cells expressing a memory phenotype. Together, our findings demonstrate that CD28 is not required for the development of a protective T cell response to T. gondii, but CD28 is required for an optimal secondary immune response.     

Exacerbated susceptibility to infection-stimulated immunopathology in CD1d-deficient mice

Mice lacking functional CD1d genes were used to study mechanisms of resistance to the protozoan parasite Toxoplasma gondii. Wild-type (WT) BALB/c mice, CD1d-deficient BALB/c mice, and WT C57BL/6 mice all survived an acute oral infection with a low dose of mildly virulent strain ME49 T. gondii cysts. In contrast, most CD1d-deficient C57BL/6 mice died within 2 wk of infection. Despite having parasite burdens that were only slightly higher than WT mice, CD1d-deficient C57BL/6 mice displayed greater weight loss and intestinal pathology. In C57BL/6 mice, CD4(+) cells can cause intestinal pathology during T. gondii infection. Compared with WT mice, infected CD1d-deficient C57BL/6 mice had higher frequencies and numbers of activated (CD44(high)) CD4(+) cells in mesenteric lymph nodes. Depletion of CD4(+) cells from CD1d-deficient mice reduced weight loss and prolonged survival, demonstrating a functional role for CD4(+) cells in their increased susceptibility to T. gondii infection. CD1d-deficient mice are deficient in Valpha14(+) T cells, a major population of NKT cells. Involvement of these cells in resistance to T. gondii was investigated using gene-targeted Jalpha18-deficient C57BL/6 mice, which are deficient in Valpha14(+) T cells. These mice did not succumb to acute infection, but experienced greater weight loss and more deaths than B6 mice during chronic infection, indicating that Valpha14(+) cells contribute to resistance to T. gondii. The data identify CD4(+) cells as a significant component of the marked susceptibility to T. gondii infection observed in CD1d-deficient C57BL/6 mice, and establish T. gondii as a valuable tool for deciphering CD1d-dependent protective mechanisms.     

Microglia and macrophages as innate producers of interferon-gamma in the brain following infection with Toxoplasma gondii

We previously reported the requirement of interferon-gamma (IFN-gamma) expression by cells other than T and natural killer (NK) cells in the brain, in addition to T cells, for prevention of toxoplasmic encephalitis following infection with Toxoplasma gondii. In the present study, we analysed the identity of the IFN-gamma-producing non-T, non-NK cells in the brain using infected athymic nude and SCID mice that lack T cells but express IFN-gamma in their brains. Intracellular staining for IFN-gamma followed by flow cytometry revealed that approximately 45-60% of the cells expressing IFN-gamma in their brains were positive for CD11b or F4/80 on their surfaces. Smaller portions of the cells were positive for pan-NK marker. Further smaller portions were positive for CD11c, and these cells were less than 5% of the IFN-gamma-expressing cells in brains of infected SCID mice. In addition to IFN-gamma proteins, large amounts of mRNA for IFN-gamma were detected in CD11b+ cells purified from brains of infected mice, but it was not the case in the cells obtained from uninfected animals. In infected SCID mice depleted of NK cells by treatment with anti-asialo-GM1 antibody, cells expressing IFN-gamma in their brains were all positive for CD11b, and the IFN-gamma-producing cells were detected in both CD45low and CD45high populations. These results suggest that CD11b+ CD45low microglia and CD11b+ CD45high blood-derived macrophages are the major non-T, non-NK cells which express IFN-gamma in the brain of mice infected with T. gondii.     

Human neutrophil-expressed CD28 interacts with macrophage B7 to induce phosphatidylinositol 3-kinase-dependent IFN-gamma secretion and restriction of Leishmania growth

We previously showed that CD28 is expressed on human peripheral blood neutrophils and plays an important role in CXCR-1 expression and IL-8-induced neutrophil migration. In this work we demonstrate that Leishmania major infection of macrophages results in parasite dose-dependent IL-8 secretion in vitro and in IL-8-directed neutrophil migration, as blocked by both anti-IL-8 and anti-IL-8R Abs, toward the L. major-infected macrophages. In the neutrophil-macrophage cocultures, both CTLA4-Ig, a fusion protein that blocks CD28-CD80/CD86 interaction, and a neutralizing anti-IFN-gamma Ab inhibit the anti-leishmanial function of neutrophils, suggesting that the neutrophil-macrophage interaction via CD28-CD80/CD86 plays an important role in the IFN-gamma-dependent restriction of the parasite growth. Cross-linking of neutrophil-expressed CD28 by monoclonal anti-CD28 Ab or B7.1-Ig or B7.2-Ig results in phosphatidylinositol 3-kinase association with CD28 and in wortmannin-sensitive but cyclosporin A-resistant induction and secretion of IFN-gamma. Whereas the neutrophils secrete IFN-gamma with CD28 signal alone, the T cells do not secrete the cytokine in detectable amounts with the same signal. Thus, neutrophil-expressed CD28 modulates not only the granulocyte migration but also induction and secretion of IFN-gamma at the site of infection where it migrates from the circulation.     

Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does not reactivate the latent disease in T. gondii-resistant BALB/c mice

Infection of C57BL/6 mice with Toxoplasma gondii leads to progressive and ultimately fatal chronic Toxoplasma encephalitis (TE). Genetic deletion or inhibition of inducible nitric oxide synthase (iNOS) from the beginning of infection increased the number of T. gondii cysts in the brain and markedly reduced the time-to-death in this mouse strain. In the present study, we addressed whether iNOS also contributes to the control of intracerebral parasites in a clinically stable latent infection that develops in T. gondii-resistant BALB/c mice after resolution of the acute phase of TE. iNOS was expressed in the inflammatory cerebral infiltrates of latently infected BALB/c mice, but the number of iNOS+ cells was significantly lower than in the brains of chronically infected T. gondii-susceptible C57BL/6 mice. In BALB/c mice with latent TE (> 30 days of infection), treatment with the iNOS inhibitors L-N6-iminoethyl-lysine or L-nitroarginine-methylester for < or = 40 days did not result in an increase of the intracerebral parasitic load and a reactivation of the disease, despite the presence of iNOS-suppressive inhibitor levels in the brain. However, L-nitroarginine-methylester treatment had remarkably toxic effects and induced a severe wasting syndrome with high mortality. In contrast to BALB/c mice, L-N6-iminoethyl-lysine treatment rapidly exacerbated the already established chronic TE of C57BL/6 mice. Thus, the containment of latent toxoplasms in T. gondii-resistant BALB/c mice is independent of iNOS, whereas the temporary control of intracerebral parasites in T. gondii-susceptible C57BL/6 mice with chronic TE requires iNOS activity.     

Augmentation of the CD8+ T cell response by IFN-gamma in IL-12-deficient mice during Toxoplasma gondii infection

The importance of IFN-gamma in regulating the host CD8+ T cell response during microbial infection has not been delineated. Mice deficient for the p40 chain of the IL-12 heterodimer have impaired IFN-gamma production and are susceptible to infection with the intracellular parasite Toxoplasma gondii. The administration of exogenous IFN-gamma to parasite-infected p40-/- mice increases survival and up-regulates the depressed CD8+ T cell response following infection. CD8+ T cells isolated from cytokine-treated p40-/- mice exhibit an increase in both precursor CTL frequency and IFN-gamma production compared with untreated controls. The enhancement of the CD8+ T cell response is independent of CD4+ T cell help. These CD8+ T cells induce protective immunity against a lethal challenge when adoptively transferred into naive p40-/- and IFN-gamma-/- mice. These observations indicate that IFN-gamma can regulate the CD8+ T cell response during T. gondii infection.     

Immune responses of different mouse strains after challenge with equivalent lethal doses of Toxoplasma gondii

Most immunological studies that utilize different strains of inbred mice following T. gondii infection fail to compensate for differences in host susceptibility to the size of the parasite innoculum. To address this concern, susceptible C57BL/6 and resistant CBA/J mice were orally infected with either an equivalent 50% lethal dose (LD50) of brain cysts of the 76K strain of T. gondii (15 cysts in C57BL/6, 400 cysts in CBA/J) or the same dose of parasites in each mouse strain. C57BL/6 mice receiving 400 cysts (LD50 of CBA/J mice) died post infection, whereas CBA/J mice that received 15 cysts (LD50 of C57BL/6 mice) survived. Parasite loads in the brains and serum Toxoplasma-specific IgG1 titers of LD50-infected C57BL/6 mice were significantly higher than those in LD50- or 15 cysts-infected CBA/J mice, whereas splenocyte proliferation to Toxoplasma antigen and the percentage of CD8 alpha+ T cells were reduced in LD50-infected C57BL/6 mice. In contrast, serum IgG2a and IgM titers, the percentage of gamma delta T cells and IFN-gamma expression of spleen of LD50-infected CBA/J mice were higher than those of either 15 cysts-infected CBA/J mice or LD50-infected C57BL/6 mice. These observations demonstrate that the immune response between LD50-infected C57BL/6 and CBA/J mice was more prominent when compared to C57BL/6 or CBA/J mice receiving the same parasite inoculum. These observations would suggest that caution must be excersized in the planning and interpretation of data when the size of the parasite inoculum has not been adjusted for mouse strain.     

Induction of tumor regression by administration of B7-Ig fusion proteins: mediation by type 2 CD8+ T cells and dependence on IL-4 production

CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8(+) effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4(-/-) and STAT6(-/-) mice. However, it was elicited in IFN-gamma(-/-) and STAT4(-/-) mice. By contrast, IL-12-induced tumor regression occurred in IL-4(-/-) and STAT6(-/-) mice, but not in IFN-gamma(-/-) and STAT4(-/-) mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8(+) T cells, although the induction of these effector cells required CD4(+) T cells. Tumor regression induced by CD8(+) T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8(+) T cells (Tc2) leading to Tc2-mediated tumor regression.     

Inhibition of NF-kappa B activity in T and NK cells results in defective effector cell expansion and production of IFN-gamma required for resistance to Toxoplasma gondii

To define the role of NF-kappa B in the development of T cell responses required for resistance to Toxoplasma gondii, mice in which T cells are transgenic for a degradation-resistant (Delta N) form of I kappa B alpha, an inhibitor of NF-kappa B, were challenged with T. gondii and their response to infection compared with control mice. I kappa B alpha(Delta N)-transgenic (Tg) mice succumbed to T. gondii infection between days 12 and 35, and death was associated with an increased parasite burden compared with wild-type (Wt) controls. Analysis of the responses of infected mice revealed that IL-12 responses were comparable between strains, but Tg mice had a marked reduction in systemic levels of IFN-gamma, the major mediator of resistance to T. gondii. In addition, the infection-induced increase in NK cell activity observed in Wt mice was absent from Tg mice and this correlated with NK cell expression of the transgene. Infection-induced activation of CD4(+) T cells was similar in Wt and Tg mice, but expansion of activated CD4(+)T cells was markedly reduced in the Tg mice. This difference in T cell numbers correlated with a reduced capacity of these cells to proliferate after stimulation and was associated with a major defect in the ability of CD4(+) T cells from infected mice to produce IFN-gamma. Together, these studies reveal that inhibition of NF-kappa B activity in T and NK cells results in defective effector cell expansion and production of IFN-gamma required for resistance to T. gondii.     

Differential requirement for CD80 and CD80/CD86-dependent costimulation in the lung immune response to an influenza virus infection

The CD28 costimulatory pathway is critical to T cell activation. Blockade of the interaction of CD28 with its ligands CD80 and CD86 using CTLA4-Ig has been proposed as a therapy for a number of immune-based disorders. We have used a murine model of influenza virus infection to study the role of CD28-dependent costimulation in the development of antiviral immune responses. In vivo treatment with CTLA4-Ig to block the interaction of CD28 with CD80 and CD86 reduced virus-specific cytotoxicity and IFN-gamma production by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro. It also resulted in decreased numbers of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid, lung, and spleen and lowered virus-specific Ab titers. Mice treated with CTLA4-Ig were able to control and clear the virus infection, but this was delayed compared with controls. Treatment with Y100F-Ig, a mutant form of CTLA4-Ig which selectively binds to CD80 and blocks the CD28-CD80 interaction leaving CD28-CD86 binding intact, did not affect Ab production, spleen cytotoxic precursors, or clearance of virus. However, Y100F-Ig treatment had a clear effect on lung effector cell function. Secretion of IFN-gamma by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro was decreased, and the number of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid and lungs of infected mice was reduced. These results indicate that CD28-dependent costimulation is important in the antiviral immune response to an influenza virus infection. The individual CD28 ligand, CD80, is important for some lung immune responses and cannot always be compensated for by CD86.     

Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii.

C57BL/6 mice chronically infected with an avirulent strain (ME-49) of Toxoplasma gondii were used to study the mechanisms by which T lymphocytes and IFN-gamma prevent reactivation of latent infection. Infected animals were treated with mAb, either anti-CD8, anti-CD4, anti-CD4 plus anti-CD8, anti-IFN-gamma, or anti-CD4 plus anti-IFN-gamma and the mice followed for survival, histopathology, cyst numbers, and spleen cell cytokine responses. In agreement with previously published findings, treatment with anti-IFN-gamma antibodies fully reactivated the asymptomatic infection, inducing massive necrotic areas in the brain with the appearance of free tachyzoites and death of all animals within 2 wk. Mice treated with the combination of anti-CD4 plus anti-CD8 antibodies showed augmented pathology and mortality nearly identical to the anti-IFN-gamma- treated animals. In contrast, treatment with anti-CD4 or anti-CD8 mAb alone failed to result in significantly enhanced brain pathology or mortality. In additional experiments, full reactivation of infection was observed in mice treated with anti-CD4 plus anti-IFN-gamma indicating that CD4+ lymphocytes are not required for the pathology resulting from IFN-gamma neutralization. Cytokine measurements on parasite Ag-stimulated spleen cells from mAb-treated mice indicated that both CD4+ and CD8+ cells produce IFN-gamma whereas only CD4+ cells contribute to parasite Ag-induced IL-2 synthesis. Together, these results suggest that CD4+ and CD8+ lymphocytes act additively or synergistically to prevent reactivation of chronic T. gondii infection probably through the production of IFN-gamma.     

Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-gamma, TNF-alpha, and inducible nitric oxide synthase

The role of B cells in resistance against Toxoplasma gondii was studied using B cell-deficient (muMT) mice. Following peroral infection with 10 cysts of the ME49 strain, all muMT mice survived the acute stage of the infection but died between 3 and 4 wk after infection. In contrast, all control mice were alive at 8 wk after infection. At the stage during which muMT animals succumbed to the infection, parasite replication and pathology were most evident in their brains; small numbers of tachyzoites were also detectable in their lungs. Significantly greater numbers of T. gondii cysts and areas of inflammation associated with tachyzoites were observed in brains of muMT than in control mice. Large areas of necrosis associated with numerous tachyzoites were observed only in brains of muMT mice. Anti-T. gondii IgG Abs were detected only in sera of control mice, whereas similar levels of IFN-gamma were detected in sera of both strains of mice. Amounts of mRNA for IFN-gamma, IL-10, and inducible NO synthase in the brain did not differ between infected muMT and control mice. Expression of mRNA for TNF-alpha was increased in brains of muMT mice. Administration of polyclonal rabbit anti-T. gondii IgG Ab prevented early mortality and pathology associated with tachyzoites in the brain in the infected muMT mice. These results indicate that B cells play an important role, most likely through their production of specific Abs, in resistance to persistent active (tachyzoite) infection with T. gondii in mice, especially in the brain and lung.     

Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice

The importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis was studied in mice chronically infected with Toxoplasma gondii by using a mAb to this lymphokine. Control mice chronically infected with the ME49 strain that received saline or normal IgG had slight inflammation in their brains whereas those that received the mAb developed severe encephalitis. In contrast to control mice, the mAb-treated mice had many areas of acute focal inflammation and infiltration of large numbers of inflammatory cells in the meninges and parenchyma of their brains. In the areas of acute focal inflammation, tachyzoites and Toxoplasma Ag were demonstrated by immunoperoxidase staining with the use of rabbit anti-Toxoplasma antibody, suggesting that the focal inflammation was induced by Toxoplasma organisms. Acute inflammation was also observed around cysts of Toxoplasma. Immunohistologic staining revealed tachyzoites and Toxoplasma Ag surrounding the periphery of these cysts suggesting cyst disruption had occurred. Mice treated with mAb against IFN-gamma had five times the numbers of cysts in their brains as did control mice. These results clearly indicate that endogenous IFN-gamma plays a significant and important role in prevention of encephalitis in mice chronically infected with Toxoplasma. The mAb-treated mice had the same Toxoplasma antibody titers and the same degree of macrophage killing of Toxoplasma as did untreated controls. These results suggest that IFN-gamma may have a direct role in preventing cyst rupture and toxoplasmic encephalitis.