Mitogen- and antigen-specific proliferation of T cells in murine toxoplasmosis is inhibited by reactive nitrogen intermediates.

Acute and chronic infections with Toxoplasma gondii result in a nonspecific suppression of immunologic function in mice and humans. Proliferation of spleen cells in response to concanavalin A (ConA) and toxoplasma lysate antigen (TLA) was studied during the course of infection in mice susceptible (CBA/Ca) and resistant (BALB/c) to development of toxoplasmic encephalitis to determine if reactive nitrogen intermediates (RNI) are involved in the suppression of the proliferative responses. Maximal suppression of proliferation of spleen cells in response to ConA and TLA was observed on days 7 and 14 after infection and correlated with elevated levels of nitrite in spleen cell culture supernatants. By day 68 postinfection in BALB/c mice, proliferative responses returned to normal levels, whereas in CBA/Ca mice, they remained suppressed. The addition of an inhibitor of production of RNI (NG-monomethyl-L-arginine) increased proliferation of spleen cells in response to both ConA and TLA at days 7, 14, and 21 after infection. Depletion of adherent cells from spleen cell preparations obtained from acutely infected mice followed by their repletion with adherent spleen cells from uninfected mice resulted in increased proliferation of spleen cells from infected mice and a significant decrease in nitrite in the cultures. These results indicate that production of RNI by macrophages contributes significantly to the suppression of the spleen cell proliferation observed in the acute stage of toxoplasmosis.     

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.     

Effect of interleukin 12 neutralization on host resistance and gamma interferon production in mouse typhoid.

Innately resistant (Ityr) A/J mice infected with the virulent Salmonella typhimurium C5 strain suppress the early exponential bacterial growth in the reticuloendothelial system toward the end of the first week of infection, with spleen and liver bacterial counts reaching a plateau phase. In vivo administration of neutralizing anti-interleukin-12 (IL-12) antibodies did not affect early bacterial growth in the tissues (days 1 to 3) but impaired the establishment of the plateau, with higher spleen and liver counts by day 7 of the infection in anti-IL-12 treated mice than in untreated controls. Gamma interferon (IFN-gamma) was detectable in the sera and spleen homogenates of both control and anti-IL-12-treated mice on days 3 and 7 of the infection. Noticeably, IFN-gamma levels were significantly lower in anti-IL-12 treated mice than in control animals. Splenocytes from uninfected A/J mice released IFN-gamma in response to concanavalin A (ConA) or to S. typhimurium C5. In vitro IL-12 neutralization dramatically impaired the IFN-gamma response to S. typhimurium but not to ConA. Splenocytes harvested from infected anti-IL-12 treated mice on day 7 of the infection produced significantly lower amounts of IFN-gamma upon in vitro stimulation with ConA and with a Salmonella protein-rich extract than did cells from similarly infected untreated control animals. Spleen cells from infected mice showed lower proliferative (mitogenic) responses to ConA and to a Salmonella soluble extract than did cells from uninfected mice. In vivo anti-IL-12 treatment significantly restored the ability of splenocytes from infected mice to proliferate in response to the antigens and ConA. In vivo neutralization of IL-12n in innately susceptible BALB/c mice ((ItyS)) immunized with a live attenuated aromatic-dependent Salmonella vaccine reduced host resistance to virulent oral challenge with S. typhimurium C5. Thus, in primary Salmonella infections, IL-12 mediates the suppression of growth of virulent salmonellae in the reticuloendothelial system, positively modulates IFN-gamma production, and is involved in the immunosuppression which accompanies the acute stages of the disease. IL-12 also contributes to host resistance to virulent organisms in secondary infections.     

Analysis of the Capacity to Produce IL-3 in Murine AIDS

Adult C57BL/6 mice infected with LP-BM5 murine leukaemia virus represent a model of murine AIDS (MAIDS). In this study we have analysed the capacity of CD4⁺ T cells from infected mice to produce IL-3 following stimulation with ConA for 24–72 h. In contrast to the position with IL-2, the production of which is markedly impaired during LP-BM5 infection, similar levels of IL-3 were measured in culture supernatants of splenocytes from infected and uninfected mice harvested at 24 h of stimulation. Forty eight and 72 h of ConA stimulation led to increasing levels of IL-3 being measured in cultures from uninfected mice, whilst in cultures from infected animals, IL-3 levels remained stagnant. Similar results were obtained 4, 8 and 13 weeks post-infection. In view of the fact that parallel experiments revealed markedly impaired proliferative responses to ConA during MAIDS, we conclude that IL-3 production is basically intact at the cellular level in T cells during MAIDS; but when in a situation requiring clonal expansion of the activated T cells, IL-3 production will be inhibited owing to the impaired capacity for proliferation.     

Interleukin-12 promotes pathologic liver changes and death in mice coinfected with Schistosoma mansoni and Toxoplasma gondii

We previously demonstrated that mice concurrently infected with Schistosoma mansoni and Toxoplasma gondii undergo accelerated mortality which is preceded by severe liver damage. Abnormally high levels of serum tumor necrosis factor alpha (TNF-alpha) in the dually infected mice suggested a role for this and related proinflammatory mediators in the pathologic alterations. In order to evaluate the factors involved in increased inflammatory-mediator production and mortality, interleukin-12(-/-) (IL-12(-/-)) mice were coinfected with S. mansoni and T. gondii, and survival and immune responses were monitored. These IL-12(-/-) mice displayed decreased liver damage and prolonged time to death relative to wild-type animals also coinfected with these parasites. Relative to the response of cells from the coinfected wild-type animals, levels of TNF-alpha, gamma interferon, and NO produced by splenocytes from coinfected IL-12(-/-) mice were reduced, and levels of IL-5 and IL-10 were increased, with the net result that the immune response of the dually infected IL-12(-/-) mice was similar to that of the wild-type mice infected with S. mansoni alone. While dually infected wild-type animals succumb in the absence of overt parasitemia, the delayed death in the absence of IL-12 is associated with relatively uncontrolled T. gondii replication. These data support the view that S. mansoni-infected mice are acutely sensitive to infection with T. gondii as a result of their increased hepatic sensitivity to high levels of proinflammatory cytokines; IL-12 and TNF-alpha are implicated in this process.     

Susceptibility of interleukin-2-deficient mice to Toxoplasma gondii is associated with a defect in the production of gamma interferon

Costimulation through the B7-CD28 interaction is an important second signal for T-cell activation, and previous studies have shown that CD28(-/-) mice infected with Toxoplasma gondii generate suboptimal CD4(+) T-cell responses, associated with a defect in production of the T-cell growth factor interleukin-2 (IL-2). To address the role of IL-2 in the expansion of T cells during toxoplasmosis, IL-2(-/-) mice were infected with T. gondii and their ability to generate a protective T-cell response was assessed. Although IL-2(-/-) mice produced normal levels of IL-12p40, they had reduced levels of gamma interferon (IFN-gamma) in serum, had an increased parasite burden, and succumbed to infection with T. gondii within 20 days. Fluorescence-activated cell sorter analysis revealed that, although uninfected IL-2(-/-) mice had an increased number of activated T cells compared with uninfected IL-2(+/+) mice, following infection they were unable to further upregulate this population. Examination of the ability of splenocytes from uninfected and infected mice to produce IFN-gamma revealed that IL-2(-/-) mice were hyporesponsive to stimulation with anti-CD3 or parasite antigen compared with wild-type mice, and the addition of IL-2 alone or in combination with IL-12 or stimulation with phorbol myristate acetate and ionomycin did not restore the production of IFN-gamma. Together, these studies reveal that IL-2(-/-) mice are unable to generate a protective IFN-gamma response following infection with T. gondii and suggest that IL-2(-/-) mice have an intrinsic defect in their ability to activate and expand IFN-gamma-producing T cells required for resistance to T. gondii.     

Interleukin-12 is critical for induction of nitric oxide-mediated immunosuppression following vaccination of mice with attenuated Salmonella typhimurium.

Studies from our laboratory have shown that infection of mice with an attenuated strain of Salmonella typhimurium causes a marked suppression in the capacity of splenocytes to generate an in vitro plaque-forming cell (PFC) response to sheep erythrocytes. The suppression has been shown to be mediated by mature, adherent macrophages (Mphis) and nonadherent, precursor Mphis. Nitric oxide has been identified as the suppressor factor. The present study investigated the role of interleukin-12 (IL-12) in the generation of nitric oxide-mediated immunosuppression in this model. Salmonella inoculation resulted in marked suppression of PFC responses and high levels of nitrite production. When mice were treated with anti-IL-12 prior to inoculation, nitrite levels in splenocyte cultures were reduced by 75% and the suppression of PFC responses was prevented. The nonadherent splenocyte fraction from Salmonella-inoculated mice, which contains precursor Mphis and is weakly immunosuppressive, was treated with IL-12 in vitro. IL-12 augmented the capacity of this fraction to suppress PFC responses by normal splenocytes in a coculture system. Additionally, IL-12 induced nitrite and gamma interferon (IFN-gamma) production in a dose-dependent manner. Treatment with anti-IFN-gamma blocked nitrite production and suppression, indicating that IFN-gamma is an important intermediary in the pathway of IL-12-induced immunosuppression. These results indicate that IL-12 is critical for the induction of nitric oxide-mediated immunosuppression following S. typhimurium inoculation and, through its ability to stimulate IFN-gamma production, can induce nitric oxide-producing suppressor Mphis.     

HFRSV感染乳鼠的脾细胞增殖及IL-2产生能力的变化

本文研究了HFRSV感染3～5日龄BALB/C新生乳鼠脾脏的病理变化、脾细胞对丝裂原或/和细胞因子诱导的增殖效应及IL-2产生能力的变化。结果表明:感染组鼠较正常组鼠(1)脾脏体积明显缩小,脾细胞数量显著减少;(2)脾细胞在无刺激原条件下培养,~3H-TdR掺入cpm值明显升高;(3)脾细胞对ConA或/和rHuTNF-α、rHu IL-2诱导的增殖反应显著降低,但是rHu IL-2、rHuTNF-α对ConA诱导的感染鼠脾细胞增殖反应仍有明显的促进作用,且这两种细胞因子有协同效应;(4)脾细胞经ConA诱导后的IL-2产生能力显著降低。此结果对于进一步了解HFRSV感染过程中细胞免疫功能变化有重要意义,同时也为临床应用细胞因子进行免疫治疗提供了有价值的实验依据。     

Attenuated Salmonella vaccine-induced suppression of murine spleen cell responses to mitogen is mediated by macrophage nitric oxide: quantitative aspects.

Previous reports from our laboratory have shown that 7 days after infection of C3HeB/FeJ mice with an attenuated strain of Salmonella typhimurium, there is profound suppression of responses to B- and T-cell mitogens and suppression of the capacity of spleen cells to mount a primary, in vitro plaque-forming-cell (PFC) response to sheep erythrocytes. Inhibition of the PFC response was shown to be mediated by nitric oxide (NO), as NG-monomethyl-L-arginine (NMMA) gave complete reversal of suppression. The experiments reported here examined the role of NO in suppression of the response to the mitogen concanavalin A (ConA). In contrast to the PFC system, it was found that addition of NMMA to ConA-stimulated immune spleen cells resulted in less than 20% reversal of suppression. However, addition to NMMA resulted in a 50% reversal of suppression in cocultures of immune and normal spleen cells at a ratio of 1:4. A complete restoration of ConA-induced responses was achieved in cocultures incubated in medium containing a reduced concentration of L-arginine plus 1.25 mM NMMA. Investigation of why NMMA alone was not 100% effective in reversing suppression showed that addition of ConA significantly augmented production of nitrite and gamma interferon (IFN-gamma) in cocultures containing immune cells. Addition of anti-IFN-gamma reduced nitrite levels in the cultures, although results with the combination of anti-IFN-gamma and NMMA were not significantly better than results with NMMA alone. These findings suggest that suppression in cultures stimulated with ConA is difficult to reverse completely with NMMA alone because of an overproduction of NO, which can be offset by either reducing the L-arginine concentration or blocking IFN-gamma. The quantitative relationship between nitrite levels and suppression in cocultures was examined. It was found that suppression did not correlate directly with the nitrite concentration but rather with the log10 of the nitrite concentration. Nitrite levels above 15 microM gave almost complete suppression, and levels between 1 and 10 microM gave a wide range of suppression. These results strongly support NO as the suppressor factor in Salmonella-induced immunosuppression of responses to ConA and, by inference, suppression of responses to mitogens induced by other microbes. The results show that involvement of NO cannot always be demonstrated by simple addition of NMMA to suppressed mitogen-stimulated spleen cell cultures.     

The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis

Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-gamma), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-gamma when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-gamma or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L(-/-) mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-gamma but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L(-/-) mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-gamma, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.     

Gamma Interferon Modulates CD95 (Fas) and CD95 Ligand (Fas-L) Expression and Nitric Oxide-Induced Apoptosis during the Acute Phase of Trypanosoma cruzi Infection: a Possible Role in Immune Response Control

We have previously shown that splenocytes from mice acutely infected with Trypanosoma cruzi exhibit high levels of nitric oxide (NO)-mediated apoptosis. In the present study, we used the gamma interferon (IFN-gamma)-knockout (IFN-gamma(-/-)) mice to investigate the role of IFN-gamma in modulating apoptosis induction and host protection during T. cruzi infection in mice. IFN-gamma(-/-) mice were highly susceptible to infection and exhibited significant reduction of NO production and apoptosis levels in splenocytes but normal lymphoproliferative response compared to the infected wild-type (WT) mice. Furthermore, IFN-gamma modulates an enhancement of Fas and Fas-L expression after infection, since the infected IFN-gamma(-/-) mice showed significantly lower levels of Fas and Fas-L expression. The addition of recombinant murine IFN-gamma to spleen cells cultures from infected IFN-gamma(-/-) mice increased apoptosis levels, Fas expression, and NO production. In the presence of IFN-gamma and absence of NO, although Fas expression was maintained, apoptosis levels were significantly reduced but still higher than those found in splenocytes from uninfected mice, suggesting that Fas-Fas-L interaction could also play a role in apoptosis induction in T. cruzi-infected mice. Moreover, in vivo, the treatment of infected WT mice with the inducible nitric oxide synthase inhibitor aminoguanidine also led to decreased NO and apoptosis levels but not Fas expression, suggesting that IFN-gamma modulates apoptosis induction by two independent and distinct mechanisms: induction of NO production and of Fas and Fas-L expression. We suggest that besides being of crucial importance in mediating resistance to experimental T. cruzi infection, IFN-gamma could participate in the immune response control through apoptosis modulation.     

CD4+ T cells of schistosomiasis naturally resistant individuals living in an endemic area produce interferon-gamma and tumour necrosis factor-alpha in response to the recombinant 14KDA Schistosoma mansoni fatty acid-binding protein

Cellular immune responses to recombinant (r) Sm14 were examined in chronic, treated patients and uninfected individuals living in an endemic area for schistosomiasis. The lymphocyte proliferative responses and cytokine profile to this antigen were evaluated. Peripheral blood mononuclear cells (PBMC) of all groups studied proliferated to rSm14. However, the highest proliferation index to rSm14 was detected in uninfected endemic normal (EN) individuals who are naturally resistant to schistosomiasis. Regarding the cytokines produced, the levels of interleukin (IL)-5 and IL-10, known as Th2 cytokines, were not statistically different among all groups studied. In contrast, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were produced in significantly higher amounts by PBMC of EN individuals following rSm14 stimulation. Additionally, we have determined by flow cytometry that CD4+ T cells from these individuals are the main lymphocyte subpopulation producing IFN-gamma and TNF-alpha. Moreover, we have used rIL-10 or rIFN-gamma, or monoclonal antibodies (MoAb) against these two cytokines to determine their role on cellular reactivity to rSm14. Exogenous IL-10 suppressed T-cell proliferation and neutralization of endogenous IL-10 restored lymphocyte activation and enhanced IFN-gamma and TNF-alpha production in chronically infected patients. In contrast, the addition of anti-IFN-gamma totally abrogated the PBMC proliferation within the EN group. This study demonstrated that IL-10 is an important cytokine down-regulating T-cell responses in chronic schistosomiasis, whereas lymphocyte proliferation in the uninfected resistant group is dependent on IFN-gamma. Taken together these results suggest that Th1 type of immune response induced in EN individuals to a specific schistosome antigen might be associated with resistance to infection and also highlighted the importance of Sm14 as a potential vaccine candidate.     

The initial immune response during experimental cysticercosis is of the mixed Th1/Th2 type

The immunological events that occur during the initial stages of experimental cysticercosis are not known. The studies presented here examined the cytokines produced by peritoneal exudate cells (PECs), splenocytes and mesenteric lymph node (MLN) cells during the first week of infection with larval Taenia crassiceps in BALB/cJ mice. Proliferation assays determined that the earliest time when antigen-specific responses could be measured was 5 days post-infection. Concanavalin A (ConA) stimulation of host cells elicited an initial burst of IL-4 production at 24 h of infection and ConA-stimulated Th2-type cytokine production is predominant by 7 days post-infection. Thus, there are responses at day 1 of infection that seem to promote a Th2-type response. Stimulation of MLN cells, splenocytes and PECs with larval antigens supported previous reports of mixed Th1/Th2-type cytokine production with increases in interleukin (IL)-4, IL-10 and interferon (IFN)-gamma. Ex vivo IFN-gamma production by PECs from infected mice was increased at 3, 5 and 7 days post-infection, whereas at these times reduced ex vivo IL-10 production was observed. This ex vivo IFN-gamma response preceded an increasing IL-10 production by PECs between 3 and 7 days post-infection in parasite-specific and ConA-induced proliferation assays. Thus, infection with larval T. crassiceps results in an initial response mediated by IFN-gamma that is quickly followed by an increase in IL-10 production and subsequent reduction in the amount of IFN-gamma being produced.     

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity

Interleukin-18 (IL-18) has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response. Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18. This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18. IFN-gamma production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody. Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCG-mIL-18-induced IFN-gamma production. Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG. Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-gamma and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-gamma-secreting cells. rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dose-dependent manner. Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-alpha) as well as IFN-gamma severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process. Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-gamma and TNF-alpha in rBCG-mIL-18 cultures than in control BCG cultures. Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization.     

Interferon-gamma and tumor necrosis factor-alpha determine resistance to Paracoccidioides brasiliensis infection in mice

To investigate the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-gamma (GKO) or TNF-alpha receptor p55 (p55KO) were infected with the parasite. GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively. Typical inflammatory granulomas were found only in WT mice. In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice. Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs. Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-alpha levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-alpha but higher levels of IFN-gamma. Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO) resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice. On the contrary, the addition of IFN-gamma to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation. Taken together, these findings suggests that endogenous TNF-alpha, acting through the p55 receptor, and IFN-gamma mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness.     

Impairment of the cellular immune response in acute murine toxoplasmosis: regulation of interleukin 2 production and macrophage-mediated inhibitory effects.

Depression of the cellular immune response to Toxoplasma gondii has been reported in both mice and humans. The present study was undertaken to determine the kinetics and mechanism of the observed downregulation of interleukin 2 (IL-2) production during experimental murine toxoplasmosis. For these investigations, the cell-mediated immune response to the wild type (PTg) was compared with that to the less-virulent mutant parasite (PTgB), which is deficient in the major surface antigen, p30 (SAG-1). Spleen cells from infected A/J mice failed to proliferate in response to Toxoplasma antigens during the first week of infection. Both PTg- and PTgB-infected A/J mice exhibited a significant reduction in the concanavalin A (Con A)-induced lymphoproliferative response. Further, the response of splenocytes from mice infected with the wild-type parasite was significantly diminished compared with that of mice infected with PTgB. The lymphoproliferative response to Con A reached its nadir at day 7 and remained below control levels for at least 14 days postinfection. By day 21 postinfection, the response to Con A and to Toxoplasma antigens was restored to the level observed prior to day 7. Con A-stimulated culture supernatants of spleen cells from mice on day 7 postinfection contained significantly less IL-2 than normal mice. There was no significant difference in the numbers of binding sites or capacity of high-affinity IL-2 receptors between infected and normal mouse splenocytes as determined by Scatchard analysis. Exogenous IL-2 at different concentrations failed to restore the proliferative response of lymphocytes from infected mice to Con A. Adherent macrophages from 7-day-infected mice were able to suppress IL-2 production by normal splenocytes following stimulation with Con A. The inhibitory activity mediated by infected cells was reversed by the antibody to IL-10 but not transforming growth factor beta. There were insignificant levels of nitric oxide production in both infected and normal splenocytes. These results indicate that during acute murine toxoplasmosis, there is a well-defined period (day 7) during which both the T-cell mitogen and parasite antigen-associated lymphoproliferative response are reduced. Further, there is a reduction in the production of IL-2 and an increase in IL-10, which appear to mediate, in part, the observed downregulation of immunity to T. gondii.     

Intestinal intraepithelial lymphocytes prevent pathogen-driven inflammation and regulate the Smad/T-bet pathway of lamina propria CD4+ T cells

Intraepithelial lymphocytes (IEL) play a key role in gut homeostasis and are critical effector cells preventing the inflammatory intestinal lesions induced in mice following oral infection with Toxoplasma gondii. In this intestinal inflammatory model, CD4(+) T lymphocytes from the lamina propria (LP) synergize with the infected enterocytes to secrete pro-inflammatory chemokines and cytokines. In this study, we assessed the mechanisms accounting for the ability of IEL to modulate the inflammatory activity of these cells. Adoptive transfer of IEL purified from wild-type mice, or CD154-,CD95L- or IL-10-deficient mice infected with T. gondii completely impairs the development of the lethal ileitis in recipient mice orally infected with T. gondii.Compared with unprimed IEL isolated from naive mice, the CD8 alpha beta TCR alpha beta subset of primed IEL, isolated from T. gondii-infected mice, secretes increased amount of TGF-beta. IEL interact with the LP CD4(+) T lymphocytes, down-regulate their production of inflammatory cytokines such as IFN-gamma and reduce their proliferative activity. These effects are linked to the secretion of TGF-beta and are correlated with a shift in the balance between Smad7/T-bet down-regulation and Smad2/Smad3 up-regulation in LP CD4(+) T lymphocytes.     

Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii.

Interleukin-10 (IL-10) is a cytokine which can inhibit T-cell and natural killer (NK) cell functions associated with cell-mediated immunity to intracellular infections. The production of IL-10 by mice infected with Toxoplasma gondii has been implicated in the suppression of lymphocyte proliferation observed during acute toxoplasmosis, as well as susceptibility to infection with this parasite. We have used C57BL/6 mice which lack a functional IL-10 gene (IL-10(-/-) mice) to investigate the role of IL-10 in acute toxoplasmosis. Intraperitoneal infection of IL-10(-/-) mice with T. gondii resulted in 100% mortality by day 13, whereas wild-type C57BL/6 (WT) mice survived acute infection. IL-10(-/-) mice infected with T. gondii had significantly higher serum levels of IL-12 and gamma interferon (IFN-gamma) than WT mice. Early mortality of infected IL-10(-/-) mice was prevented by treatment with IL-10 and significantly delayed by neutralizing antibodies to IL-12 and IFN-gamma. Further studies revealed that SCID/IL-10(-/-) mice infected with T. gondii had delayed time to death compared to IL-10(-/-) mice, indicating that lymphocytes contributed to death of IL-10(-/-) mice. In addition, infected SCID/IL-10(-/-) mice survived longer than infected SCID mice. These latter data indicate that in mice lacking lymphocytes, endogenous IL-10 is associated with increased susceptibility to T. gondii. However, the lack of IL-10 does not alter the infection-induced suppression of T cell and NK cell functions. Our experiments reveal that IL-10 is associated with protection or increased susceptibility to infection with T. gondii, depending on whether mice possess lymphocytes, and demonstrate the important roles of IL-12 and IFN-gamma in the early infection-induced mortality observed in the IL-10(-/-) mice.     

Role of Gamma Interferon in the Pathogenesis of Severe Schistosomiasis in Interleukin-4-Deficient Mice

In the absence of interleukin-4 (IL-4), infection with Schistosoma mansoni leads to a severe fatal disease rather than the chronic survivable condition that occurs in wild-type (WT) mice. Because the sustained production of NO most closely correlates to weight loss and fatality in infected IL-4(-/-) mice and because gamma interferon (IFN-gamma) is an important inducer of inducible NO synthase, infected IL-4(-/-) mice were treated with anti-IFN-gamma antibodies to determine the role of IFN-gamma during schistosomiasis in WT and IL-4(-/-) animals. When IFN-gamma was neutralized, Th2 responses were enhanced and NO production was reduced in both WT and IL-4(-/-) mice. The decreased NO production correlated with a rescue of proliferation in splenocytes from infected IL-4(-/-) mice. Furthermore, the neutralization of IFN-gamma in vivo improved the gross appearance of the liver and led to a reduction in granuloma size in infected IL-4(-/-) but not WT mice. However, the neutralization of IFN-gamma in vivo did not affect the development of severe disease in infected IL-4(-/-) mice. These results suggest that while the increased production of IFN-gamma does lead to some of the pathology observed in infected IL-4(-/-) mice, it is not ultimately responsible for cachexia and death.     

Cytokine production of CD8+ immune T cells but not of CD4+ T cells from Toxoplasma gondii-infected mice is polarized to a type 1 response following stimulation with tachyzoite-infected macrophages.

To examine whether cytokine production of CD4(+)immune T cells and CD8(+)immune T cells in Toxoplasma gondii-infected mice differ in their responses to infected cells and to soluble antigens of the parasite, we compared the production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-10 by the immune T cell populations following in vitro stimulation with tachyzoite-infected macrophages and tachyzoite lysate antigens (TLA). Both CD4(+)and CD8(+)immune T cells produced large amounts of IFN-gamma in response to either infected macrophages or TLA, but the CD4(+)T cells produced greater amounts of the cytokine than did the CD8(+)T cells with both stimulations. Both T cell populations also produced IL-2 after stimulation with infected macrophages, whereas only CD4(+)T cells did when stimulated with TLA. CD4(+)immune T cells also produced large amounts of IL-4 and IL-10 after stimulation with infected macrophages, but CD8(+)T cells did not. These results indicate that CD4(+)immune T cells produce IFN-gamma, IL-2, IL-4, and IL-10 in response to infected macrophages, whereas CD8(+)immune T cells produce predominantly IFN-gamma and IL-2. Since IL-4 and IL-10 could suppress IFN-gamma-mediated protective mechanisms against the parasite, the production of these cytokines by CD4(+)immune T cells in response to infected cells could negatively affect their protective activity in vivo.