In susceptible mice,Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1−

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-γ (IFN-γ) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-γ dependent. Treatment of resistant C57BL/6 mice with anti-IFN-γ allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in Vβ8, 7, 2^? CD4⁺ cells in BALB/c mice and NK1.1^? CD4⁺ cells in anti-IFN-γ treated C57BL/6 mice. These results show that the NK1.1⁺ CD4⁺ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.     

T cell response in murine Leishmanial mexicana amazonensis infection: production of interferon-γby CD8+ cells

The immune response to Leishmania major has been the subject of many investigations. However, Leishmania includes many species with different clinical manifestations. In this report, we studied the Tcell response to L. mexicana amazonensis, a New World species, in a murine model. We found that, similar to L. major, an Old World species, resistant C57BL/6 mice produced a high level of IFN-γ and a low level of IL-4. Conversely, susceptible BALB/c mice produced a much lower level of IFN-γ and higher level of IL-4. Although IFN-γ is one of the important lymphokines that mediate macrophage activation and thus the destruction of the intracellular parasites, which lymphocyte subsets are producing the IFN-γ is still a controversy. Much evidence including the isolation of protective, IFN-γ-producing, CD4⁺ cell lines have confirmed the participation of CD4⁺ Thl cells unequivocally. However, both CD4⁺ and CD8⁺ cells produced IFN-γ. Recently, an increasing body of evidence has appeared suggesting that CD8⁺ cells also play a role in the resolution of murine L. major infection. We found that in the L. m. amazonensis model, when CD8⁺ lymphocytes from resistant C57BL/6 mice were eliminated by anti-CD8 antibody and complement-mediated lysis, the IFN-γ production was reduced by 77%. This indicated that CD8⁺ cells produced a significant amount of the IFN-γ. However, our results also indicate that IFN-γ production by CD8⁺ cells was dependent on CD4⁺ cells.     

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4⁺ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/ 6 × 129 mice that were rendered T cell deficient (TCR β^–/– × TCR δ^–/–) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-γ production in draining lymphnodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-γ mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4⁺ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. majorinfection and raises the possibility of utilizing this approach for vaccination strategies.     

Interleukin-12 but not interferon-γ production correlates with induction of T helper type-1 phenotype in murine candidiasis

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-γ (IFN-γ), IL-4, and IL-10 in CD4⁺ and CD8⁺ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection (“healer mice”) or in a Th2-associated progressive disease (“nonhealer mice”). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-γ or IL-10 mRNA in CD4⁺ and CD8⁺ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4⁺ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4⁺ and CD8⁺ cells was assessed in vitro, while IFN-γ-producing cells were enumerated in CD4^? CD8^? cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-γ protein in vitro by CD4⁺ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4⁺ cells would expand in nonhealer mice in the face of high levels of circulating IFN-γ, likely released by CD4^? CD8^? lymphocytes; (iii) a finely regulated IFN-γ production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-γ-producing CD4⁺ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-γ by early CD4⁺ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-γ production may be an indicator of Th1 differentiation.     

T cells made deficient in interleukin-2 production by exposure to staphylococcal enterotoxin B in vivo are primed for interferon-γ and interleukin-10 secretion

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor Vβ domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-γ and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 μg SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-γ and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-γ levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4⁺ or CD8⁺ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4⁺ and CD8⁺ cells from SEB-pretreated mice were primed for IL-10 and IFN-γ production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-γ was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-γ monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-γ and IL-10 production, and that IFN-γ up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.     

Interleukin-4 transgenic mice of resistant background are susceptible to Leishmania major infection

The outcome of cutaneous leishmaniasis is dependent on the balance of Th1 and Th2 cells. In the murine model, Th1 cells are host-protective whereas the Th2 cells are disease-promoting. However, the in vivo role of interleukin-4 (IL-4), a signature product of Th2 cells, is uncertain. We compared the course of Leishmania major infection in the genetically resistant 129/Sv mice and the mutant 129/Sv mice transgenic for the murine IL-4 gene under the control of the immunoglobulin heavy chain enhancer and promoter. We report here that in contrast to their wild-type parents, the IL-4 transgenic mice are susceptible to L. major infection. This is associated with the development of inexorably progressive lesions and parasite loads. Spleen cells from infected transgenic mice produced significantly higher levels of IL-4 but lower amounts of interferon-γ when stimulated in vitro with leishmanial antigens compared to those from infected normal 129/Sv mice. Furthermore, sera from the infected transgenic mice contained higher levels of IL-4 and IgE than the sera of infected normal 129/Sv mice. These results, therefore, establish in a new animal model that IL-4 promotes disease development in murine cutaneous leishmaniasis.     

Diversion of CD4+ T cell development from regulatory T helper to effector T helper cells alters the contact hypersensitivity response

Cutaneous sensitization to reactive haptens and subsequent challenge results in a T cell-mediated response, contact hypersensitivity (CHS). Recent results from this laboratory have indicated that hapten sensitization induces two populations of reactive T cells: CD8⁺ T cells producing interferon (IFN)-γ which mediate the response and CD4⁺ T cells producing interleukin (IL)-4 and IL-10 which negatively regulate the magnitude and duration of the response. Since CD4⁺ T cell development to either IFN-γ- (Th1) or IL-4/IL-10- (Th2)-producing cells is dependent upon the cytokine environment during antigen priming, we hypothesized that CD4⁺ T cell induction in a Th1-promoting environment would not only alter the CD4⁺ T cell cytokine-producing phenotype but also the course of the CHS response. Administration of the Th1-promoting cytokine IL-12 during hapten sensitization resulted in a CHS response of greater magnitude following challenge and extended the duration of the response. In hapten-sensitized mice depleted of CD8⁺ T cells, treatment with IL-12 induced effector CD4⁺ T cells. Histological examination of challenged ear tissue from these mice indicated minimal edema and an acute mononuclear cell infiltration more typical of classical delayed-type hypersensitivity than CHS. Hapten-primed CD4⁺ T cells from IL-12 treated, sensitized mice produced IFN-γ, but not IL-4 in response to T cell receptor-mediated stimulation. Use of neutralizing anti-IFN-γ antibody indicated that IL-12 not only directly promoted Th1 development but also indirectly inhibited Th2 development through stimulation of IFN-γ production at the time of hapten sensitization. Overall, these results demonstrate that diversion of CD4⁺ T cell development to Th1 effector cells rather than to Th2 cells alters the efferent nature of CHS and removes a primary regulatory mechanism of the immune response.     

Reconstitution of C.B-17 scid mice with BALB/c T cells initiates a T helper type-1 response and renders them capable of healing Leishmania major infection

C.B-17 scid mice, which were found to be very susceptible to infection with Leishmania major, were reconstituted with various doses of T cells, T plus B cells or unfractionated spleen cells from nonhealer BALB/c mice. All reconstitution protocols, except for the transfer of very high numbers of BALB/c spleen cells, led to a spontaneously healing infection and resistance to reinfection, rather than the lethal, nonhealing infection typical of BALB/c mice. These healing responses were associated with a strong T helper 1 (Th1)-like response characterized by delayed-type hypersensitivity (DTH) responsiveness, but no elevation of serum IgE, and by the production of high levels of interferon-γ (IFN-γ), but no interleukin-4 (IL-4) by lymph node and spleen cells after restimulation with antigen in vitro. The development of this Th1 response from BALB/c Th cells requires IFN-γ during the initial infection period. Treatment of scid mice with a single injection of neutralizing anti-IFN-γ antibody prior to infection and reconstitution prevented healing and permitted the development of a Th-2 like response as indicated by elevated serum IgE, but no DTH, and by the production of IL-4, but very little IFN-γ, after antigen stimulation in vitro. As few as 10⁴ transferred T cells led to a Th1-like response, suggesting that the IFN-γ is of host rather than donor origin. The transfer of very high numbers (7.5 x 10⁷) of BALB/c spleen cells overcame the effects of the IFN-y and led to the nonhealing infection and cytokine pattern characteristic of BALB/c mice. The enrichment or depletion of B cells from the transferred T cells had no measureable effect upon the development of a healing response in reconstituted scid mice.     

Regulation of IFN-γ production in granulocyte-macrophage colony-stimulating factor-deficient mice

Granulocyte-macrophage colony-stimulating factor-deficient (GM-CSF−/−) mice produce far lower serum levels of IFN-γ in response to LPS than GM-CSF+/+ mice. CD4⁺ and CD8⁺ T cells from LPS-injected GM-CSF−/− mice showed a deficiency in IFN-γ production and proliferative activity in response to IL-2 and IL-12, whereas IFN-γ production by NK cells was not compromised. These defects of T cells were reversed by administration of GM-CSF in vivo, but not by supplementation with GM-CSF in vitro. GM-CSF−/− mice do not have an intrinsic defect in IFN-γ production, because IL-12 injection induces the same high levels of IFN-γ in GM-CSF−/− and GM-CSF+/+ mice. To investigate the inhibitory effect of LPS on GM-CSF−/− T cells and the indirect restorative activity of GM-CSF, we tested the action of supernatants from cultured dendritic cells (DC). A factor or factors in the DC supernatant normalized serum IFN-γ levels and T cell responses in LPS-injected GM-CSF−/− mice. IL-18 reproduced some but not all of these in vivo and in vitro effects of DC supernatants. Our results indicate that GM-CSF is important in protecting T cells from inhibitory signals generated during immunization or exposure to LPS, and that this effect of GM-CSF is indirect and mediated by factors produced by DC.     

Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major

Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires the development of a resistance-promoting CD4⁺ -mediated Th1 response. Epidermal Langerhans cells (LC) are critically involved in the induction of the primary immune response to Leishmania infection. They are able to ingest the parasites, to express MHC class II molecules with extraordinarily long half-life and to activate naive L. major -specific Th cells. Considering these unique properties, we studied the capacity of LC to mediate resistance to L. major in vivo. A single i.v. application of LC that had been pulsed with L. major antigen in vitro induced the protection in susceptible BALB/c mice against subsequent challenges with L. major parasites. Resistance could neither be induced by unpulsed LC, nor by L. major antigen alone or by L. major -pulsed macrophages. Development of resistance was paralleled by a reduced parasite burden and by a shift of the cytokine expression towards a Th1-like pattern. In contrast, control mice developed a Th2 response. In vitro exposure of LC to L. major antigen induced the expression of IL-12 (p40) mRNA. In conclusion, our data demonstrate that LC are able to serve as a natural adjuvant and to induce a protective immune response to L. major infection. This effect is based on the initiation of a Th1-like response that is likely to be mediated by IL-12.     

Early parasite containment is decisive for resistance to Leishmania major infection

We investigated the early spread of Leishmania major in various mouse strains. In BALB/c mice, which are extremely vulnerable to L. major infection, the parasites disseminated within 10-24 h from the site of subcutaneous footpad infection in to the popliteal lymph node, spleen, lung, liver and bone marrow. Application of recombinant (r)IL-12 prior to infection prevented the early dissemination of parasites into visceral organs and the animals healed the infection. In three mouse strains tested, C57BL/6, CBA/J and C3H/HeJ, which are all resistant to L. major infection, the parasites remained localized in the footpad and in the draining LN for 3 days without evidence of dissemination. In C57BL/6 mice, depletion of NK1.1⁺ cells or neutralization of interferon (IFN)-γ prior to infection led to rapid parasite spreading with kinetics similar to those seen in susceptible animals. Depletion of either CD4⁺ or CD8⁺ T cells in vivo prior to infection did not alter the kinetics of dissemination in any mouse strain tested. Experiments with severe-combined immunodeficient mice provided further evidence that parasite containment depends on natural killer cells and IFN-γ, but is independent of T cells. The finding that all resistant mouse strains restrict the spread of the parasites within the first 24 h after infection strongly suggests that early parasite containment is closely associated with a resistant phenotype. The data show that local restriction of parasites in the pre-T cell phase of the infection is mediated by the innate immune system and suggest that this function plays an important role in the development of a protective T cell response.     

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.     

Interferon-γ confers resistance to experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), T cells infiltrate the central nervous system (CNS) and induce inflammation. These CD4⁺ T cells secrete interferon (IFN)-γ, levels of which correlate with disease severity, and which is proposed to play a key role in disease induction. Many strains of mice are resistant to EAE. We have studied the effect of deletion of IFN-γ on the ability to induce EAE in resistant BALB/c-backcrossed mice. As expected, only 0–6 % of BALB/c or BALB/c-backcrossed mice developed EAE when immunized with myelin basic protein in adjuvant. Strikingly, abrogation of IFN-γ expression by targeted disruption of the IFN-γ gene (GKO mice) converted them to a susceptible phenotype. As many as 71 % of these IFN-γ-deficient mice developed EAE, a frequency comparable to that seen with the susceptible SJL/J strain. In addition, EAE was of unusually high severity in mice lacking IFN-γ. Immunological characteristics of disease in IFN-γ-deficient mice were comparable to those seen in susceptible (SJL/J) mice with EAE, including perivascular infiltration in the CNS and order-of-magnitude increases for both CD3 γ chain and TNF-α mRNA levels in the spinal cord. We thus demonstrate that lack of IFN-γ converts an otherwise EAE-resistant mouse strain to become susceptible to disease. Therefore, in BALB/c mice, IFN-γ confers resistance to EAE.     

Intestinal IFN-γ production is associated with protection from clinical signs,but not with elimination of worms,in Echinostoma caproni infected-mice

In the present paper, we assess the relationship between the expression of IFN-γ and the development of clinical signs in Echinostoma caproni-infected mice. For this purpose, we studied the course of the infection in three mouse strains: ICR (CD-1®) (a host of high compatibility with E. caproni), BALB/c (a prototypical Th2 strain), and BALB/c deficient for IFN-γ mice (IFN-γ^?/?). Infection in ICR mice is characterized by the elevated expression of IFN-γ and iNOS in the intestine concomitantly with the lack of clinical signs. In contrast, the infection was more virulent in BALB/c and IFN-γ-deficient mice that developed a severe form of the disease together with the absence of IFN-γ expression. The disease was more severe in IFNγ^?/? mice in which the disease was lethal during the few first weeks of the infection. The analysis of different parameters of the infection in each host strain showed that most of the features were similar in the three mouse strains, suggesting the IFN-γ plays a central role in that protection against severe disease. Thus, IFN-γ seems to play a dichotomous role in the infection facilitating the parasite establishment, but it may also benefit mice since it protects the mice from morbidity and mortality induced by the parasite.