Interferon-gamma inhibits the efficacy of interleukin 1 to generate a Th2-cell biased immune response induced by Leishmania major.

Splenic adherent cells from L. major-infected resistant and susceptible mice were restimulated in vitro and analyzed for the expression of IL-1 activity. Three weeks or later after infection, cells from parasite infected susceptible BALB/c mice produced substantially more IL-1 activity than those from non-infected controls or from L. major-infected resistant C57BL/6 animals. More than 95% of the IL-1 bioactivity was mediated by IL-1 alpha, as determined by blocking experiments with an anti-IL-1 alpha antiserum. The strain-specific differences in IL-1 production correlated with different accumulation of IL-1 producing adherent cells in the spleens of infected animals, but also with different IL-1 producing capacity on a per cell basis. When adherent cells were mixed with syngeneic IFN-gamma producing CD4+ T lymphocytes from L. major-infected C57BL mice or from animals that had been pretreated with anti-CD4 monoclonal antibody prior to infection, the level of detectable IL-1 decreased depending on the number of T cells added. This inhibition could be blocked completely with an anti-IFN-gamma antibody. No such effect was seen, when CD4+ cells were used that were derived from parasite-infected BALB/c mice and did not produce IFN-gamma. In contrast to L. major, L. donovani antigen not only failed to induce IL-1 production, but also dose-dependently suppressed the IL-1 activity elaborated by L. major antigen. We conclude from these data that IFN-gamma effectively inhibits the efficacy to IL-1 to generate to Th2-cell biased immune response induced by L. major. A T cell independent and as yet unknown mechanism to inhibit the IL-1 response is used by L. donovani.     

The capacity to produce IFN-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to Leishmania donovani infection in mice.

The immune response against Leishmania donovani infection has been investigated in one resistant mouse strain (C3H/HeJ) and three susceptible mouse strains (C57BL/6, BALB/c, and B10D2/n). In order to correlate the strain-specific course of infection with the individual T cell response phenotype, the ex vivo cytokine secretion patterns of splenic lymphocytes were assessed by ELISA (interferon-y [IFN-gamma], interleukin-4 [IL-4], IL-10) or by bioassay (IL-2). The strain-dependent differences in the course of infection correlated closely with the potency of T cells to produce IFN-gamma. C3H/HeJ mice produced high amounts of IFN-gamma before and during infection, whereas susceptible mice produced low amounts of IFN-gamma early during L. donovani infection. However, C57BL/6 mice, which recovered from the infection rapidly after the acute stage, developed marked IFN-gamma response within the first 30 days of infection. In contrast, in BALB/c and B10D2/n mice, the IFN-gamma production diminished during the acute stage, and this was associated with a delay in recovery and with subsequent switching into the chronic stage. Interestingly, CD8+ T cells contributed significantly to IFN-gamma production during this phase. In contrast to IFN-y, the levels of IL-4 in response to antigen or mitogen ex vivo were always very low. Moreover, neutralization of endogenous IL-4 in vivo by treatment with soluble murine IL-4 receptor did not result in significant decreases in the parasite burdens in spleen and liver but did cause a decrease in the serum IgE level of L. donovani-infected BALB/c mice. These results confirm that in visceral leishmaniasis a Thl-dominated immune response is protective against the L. donovani parasites and, furthermore, that the capacity to produce IFN-gamma rather than the presence of IL-4 determines the efficacy of the immune response in susceptible mice. The data show that CD8+ T cells represent an important source of IFN-gamma during L. donovani infection in susceptible mice, implying a role for this cell type in healing and development of protective immunity.     

Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis.

Leishmaniae are protozoans which, depending upon both the host and parasite species, can cause either a healing or nonhealing infection. While C57BL/10 mice are able to heal following infection with Leishmania major, they fail to heal following infection with Leishmania amazonensis. In order to address the role of Th1 and Th2 cell responses in the outcome of these infections in C57BL/10 mice, gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production was assessed. While cells from L. major-infected C57BL/10 mice produced high levels of IFN-gamma, cells from L. amazonensis-infected animals produced little or no IFN-gamma. On the other hand, IL-4 was produced only by cells from L. amazonensis-infected C57BL/10 mice, but this production was restricted to the first few weeks of infection. Later in infection, when lesions were evident, no IL-4 was detected. Treatment of BALB/c mice with a monoclonal antibody (11B11) directed against IL-4 induced a dramatic reduction in L. amazonensis lesions. This reduction was associated with a decrease in IL-4 levels and an increase in IFN-gamma production. However, only a slight reduction in lesion sizes and parasite numbers was observed when anti-IL-4-treated C57BL/10 mice were infected with L. amazonensis. These results suggest that IL-4 may have an important role in mediating susceptibility to L. amazonensis in BALB/c mice, as previously demonstrated for L. major. More importantly, however, the data suggest that susceptibility to L. amazonensis in C57BL/10 mice is due to the absence of a Th1 cell response, rather than to the presence of a Th2 cell response.     

Generalized infection and lack of delayed hypersensitivity in BALB/c mice infected with Leishmania tropica major.

The susceptibility of a few strains of mice to a subcutaneous injection of Leishmania tropica major, the causative agent of cutaneous leishmaniasis in humans, was studied. The infection in six strains (CBA, AKR/J, AKR/cu, C57BL/6, A/J, and C3H) remained cutaneous, and the animals recovered within 3 to 4 months. In contast, the infection in BALB/c became generalized and killed 1005 of infected animals. Intraperitoneal injection of infected liver of BALB/c to A/J and syngeneic mice produced a lethal disease in BALB/c but no infection in A/J mice. Lower doses of the parasite produced a lethal infection in BALB/c but no apparent disease in A/J. Hence, the host rather than the parasite is responsible for the outcome of the disease. The peak antibody titer of BALB/c mice was not significantly higher than that of A/J mice. However, BALB/c failed to show any delayed hypersensitivity to leishmania tested by footpad reaction, whereas A/J mice showed a strong response.     

Ineffective elimination of Leishmania major by inflammatory (MRP14-positive) subtype of monocytic cells

Myeloid-related protein (MRP) 14, an intracellular protein involved in calcium-dependent activation of myeloid cells, presents a differentiation marker for a subtype of macrophages. In experimental leishmaniasis, BALB/c mice succumb to visceral dissemination after infection with L. major, due to a Th2 cell response, while C57Bl/6 mice develop protective immunity associated with a Th1 cell response. We have previously shown that resistance in (C57Bl/6 mice was also associated with a significantly lower percentage of MRP14-positive cells in the infiltrate than in susceptible BALB/c mice. In C57Bl/6 mice, weekly injections of bone marrow (BM) cells enriched with MRP14-positive cells (d1 of culture) did not reverse, but prolonged the course of infection, associated with increased local parasite spread. In BALB/c mice a single dose of an antiphlogistic agent (dexamethasone or lipoxygenase inhibitor) was associated with reduction of infiltrating MRP14-positive cells and also with a decrease of parasite loads in footpads, lymph nodes as well as spleens, and with delayed progression of disease, Double labeling experiments in vitro revealed that at least 43.1% of MRP14-positive mononuclear cells in BM cultures (8h) had phagocytosed parasites after 4 h of co-incubation. Activation by IFN-gamma (20 U/ml) for 24h and 48h did not significantly reduce parasite load in these cells. In contrast, 77.0% of F4/80-positive macrophages (6d of culture) were infected with L. major parasites and these cells responded to activation with IFN-gamma (20 U/ml) with significant reduction of parasite load (25.3%). The protein MRP14 did not have an effect on parasite survival in vitro. Thus, the impaired capability of MRP14-positive cells to kill L. major upon stimulation may be one reason for the adverse course of infection observed with their increased appearance.     

IL-12 and IFN-gamma production, and NK cell activity, in acute and chronic experimental Trypanosoma cruzi infections

Resistance to acute Trypanosoma cruzi infection is mainly associated with a Th1 immune response, characterized by gamma-interferon (IFN-gamma) production and activation of macrophages. The outcome of the Th1 response in the spleen and serum of BALB/c and C3H mice infected with T. cruzi, Tulahuén strain was studied. The levels of interleukin-12 p40 (IL-12 p40) and IFN-gamma, as well as natural killer (NK) cell cytotoxicity were determined at different time-points during the acute phase, and the production of cytokines was also studied in the chronic infection. At 2 days post-infection (pi), spleen cells from C3H mice increased their NK cell activity and the ex vivo spontaneous release of both IL-12 p40 and IFN-gamma. On the other hand, BALB/c mice reached low levels of NK cell cytotoxicity and no IFN-gamma production was detected at this time pi, but the cytokine was released at high amounts in the second week of the infection. Seric IL-12 p40 concentrations showed a 3-fold increase in both mouse strains on the second day pi and remained high throughout the acute phase. However, seric IFN-gamma levels increased during the late acute infection and were higher in BALB/c than in C3H mice. In chronically infected mice IL-12 p40 was as high as in the acute phase in the serum of both strains, but only BALB/c mice still produced IFN-gamma. To the authors' knowledge this is the first report showing the protein levels of IL-12 p40 determined in vivo in acute and chronic T. cruzi infections. The results reveal differences between both mouse strains in the mechanisms controlling the onset and fate of the Th1 response triggered by the parasite and a long lasting pro-inflammatory stimuli.     

An avirulent lipophosphoglycan-deficient Leishmania major clone induces CD4+ T cells which protect susceptible BALB/c mice against infection with virulent L. major.

An avirulent clone of Leishmania major was used to immunize susceptible BALB/c mice against challenge with virulent L. major. By using the immunized animals as a source of cells, CD4+ parasite-specific T-cell lines could be generated in vitro which, when adoptively transferred to naive BALB/c recipients, conferred marked protection against challenge with virulent L. major. Compared with CD4+ parasite-specific T-cell lines generated from nonimmunized BALB/c mice infected with L. major, the protective T-cell lines generated from immunized mice produced substantially less interleukin-4 and substantially more tumor necrosis factor and interleukin-2. Interestingly, the protective CD4+ T cells did not mediate L. major-specific delayed-type hypersensitivity in vivo and proliferated in vitro only in response to living L. major and not to frozen-and-thawed antigen preparations of the parasite. Finally, the avirulent clone of L. major was found to express the major surface glycolipid of L. major, lipophosphoglycan, at a level that was sixfold less than expression of this molecule by virulent L. major. In addition, lipophosphoglycan of the avirulent parasite failed to mature into the larger, or metacyclic, form of the molecule.     

Identification and characterization of host-protective T-cell epitopes of a major surface glycoprotein (gp63) from Leishmania major

By using a series of overlapping synthetic peptides that cover more than 75% of the amino acid sequence of the major surface glycoprotein (gp63) from Leishmania major, 11 T-cell epitopes in CBA and BALB/c mice have been identified. Six of the peptides were recognized by T cells of CBA mice recovered from L. major infection, while one was recognized by the T cells from BALB/c mice recovered from the infection following sublethal doses of gamma-irradiation. Lymph node cells from mice immunized with the peptides also responded to a number of the same peptides (seven in CBA and one in BALB/c). Peptide p10-28 induced proliferative T-cell responses in both CBA and BALB/c mice. Five of the peptides (p10-28, p22-40, p289-309, p459-471 and p467-482) induced vigorous T-cell response in CBA mice but were not recognized by T cells from recovered mice. Four other peptides (p321-336, p364-476, p372-385 and p378-396) were recognized by T cells from recovered CBA mice but could not induce a T-cell response in normal CBA mice. Three peptides (p146-171, p289-309 and p395-414) were both able to induce a T-cell response and were recognized by T cells from recovered mice. However, only two peptides (p146-171 and p467-482) were able to activate T cells, which also recognized epitopes expressed by antigen-presenting cells infected with promastigotes. T cells induced by p146-171 and p467-171 or a mixture of these two peptides were mainly CD4+ and produced interleukin (IL-2) and interferon-gamma (IFN-gamma) but not IL-4 upon antigen stimulation in vitro. These two peptides also induced a classical delayed type hypersensitivity (DTH) response in CBA mice. Furthermore, CBA mice immunized with a mixture of the two peptides in Coryne parvum or entrapped in liposomes induced significant resistance against L. major infection. The implications of these results in terms of a synthetic vaccine against leishmaniasis and the mechanism of the induction of Th1 and Th2 cells are discussed.     

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.     

Response of scid mice to establishment of Leishmania major infection.

The initiation of Leishmania major infection in susceptible BALB/c mice is regulated by interferon-gamma (IFN-gamma). To examine further the mechanisms of IFN-gamma-dependent regulation of the establishment of L. major, we studied the characteristics of the infection in severe combined immunodeficient (scid) mice. In the first 2 weeks of infection, we observed a delay in the development of the lesions in the footpads and lower numbers of parasites in scid compared with BALB/c mice. By week 5 after infection, the size of the leishmanial lesion was similar in both strains of mice, but the number of parasites in scid mice was 100-fold higher than in BALB/c. Treatment with anti-IFN-gamma during the establishment of L. major did not alter the course of infection in scid mice, while it exacerbated lesion development in BALB/c mice. Macrophages from scid mice were unable to kill L. major when stimulated with IFN-gamma in vitro, and produced lower levels of nitric oxide compared with macrophages from susceptible BALB/c or the resistant C57Bl/6 mice. We examined whether delayed lesion development in scid mice was due to their inability to mount appropriate inflammatory responses. While significantly fewer nucleated cells were present in the footpads of scid mice compared with BALB/c, 2 and 3 weeks after infection, no difference in inflammatory response between scid and BALB/c mice was observed in response to L. major antigen in the footpads. In contrast, there was a dramatic increase in the number of cells in the popliteal lymph nodes of BALB/c mice. Decreased inflammatory responses of scid mice in the footpad (at the site of infection) may contribute to slower development of leishmanial lesions during the first 2 weeks of infection.     

In the absence of endogenous gamma interferon, mice acutely infected with Neospora caninum succumb to a lethal immune response characterized by inactivation of peritoneal macrophages.

Following infection with Neospora caninum, BALB/c mice were shown to be resistant to an acute infection but developed a latent chronic infection. However, BALB/c background gamma interferon (IFN-gamma)-deficient mice were sensitive to the acute infection. Since the immune response in IFN-gamma-deficient mice is scantly known, we examined the function of macrophages, major histocompatibility complex (MHC) class II expression, T-cell responses, and serum cytokine levels in the mice. All IFN-gamma-deficient mice died within 9 days of infection with N. caninum, whereas those treated with exogenous IFN-gamma lived longer. Although N. caninum invaded various organs in both types of mice at the early stage of infection, the parasite was not detected in the brains of resistant hosts until 21 days postinfection (dpi). Peritoneal macrophages from IFN-gamma-deficient mice were activated by exogenous IFN-gamma associated with inhibition of parasite growth and nitric oxide production as were those from BALB/c mice. IFN-gamma-deficient mice failed to increase MHC class II expression on macrophages. Moreover, BALB/c mice induced T-cell proliferation while IFN-gamma-deficient mice did not. However, in vivo treatment with exogenous IFN-gamma induced up-regulated MHC class II expression in IFN-gamma-deficient mice. BALB/c mice treated with an antibody to CD4 showed an increase in morbidity and mortality after parasite infection. In serum, significant levels of IFN-gamma and interleukin-4 (IL-4) were detected in resistant hosts, whereas IL-10 was detected in IFN-gamma-deficient mice. The levels of IL-12 in IFN-gamma-deficient mice were higher than those in BALB/c mice at 7 dpi. The present study indicates that early IFN-gamma production has a crucial role in the activation of peritoneal macrophages for the induction of protective immune responses against N. caninum.     

Role of gamma interferon in natural clearance of Bordetella pertussis infection.

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-gamma) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4+ and produced IFN-gamma but no detectable interleukin-4. Analyses of IFN-gamma mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-gamma mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-gamma demonstrated that infection of adult BALB/c mice depleted of IFN-gamma in vivo with anti-IFN-gamma monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-gamma-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-gamma gene resulted in bacterial clearance with a time course similar to those seen with IFN-gamma-depleted mice. These results indicate that IFN-gamma plays a role in controlling B. pertussis infection.     

Early IFN-gamma production is related to the presence of interleukin (IL)-18 and the absence of IL-13 in experimental Trypanosoma cruzi infections

Gamma-interferon (IFN-gamma) production, the hallmark of the Th1 immune response, has been shown to play a central role in the resistance to Trypanosoma cruzi infections, in particular when produced in the very early acute infection. BALB/c mice infected with T. cruzi, Tulahuén strain, reach high parasitemias during the acute phase, and their spleen cells release IFN-gamma in the second week of the infection, while those of the resistant C3H strain produce the cytokine earlier, at 2 days post-infection (pi). We studied in the spleen cells supernatants of infected BALB/c and C3H mice, the spontaneous production of cytokines involved in the induction, interleukin (IL)-18 and IL-12 p70, as well as in the downregulation, IL-13 and IL-10, of the Th1 immune response. We found that, at 2 days pi, only C3H mice produced IL-18, while IL-12 p70 was detected in both mouse strains. Moreover, at this time pi splenocytes from BALB/c mice spontaneously produced high amounts of IL-13. At 14 days pi, despite the increased levels of IL-13 and IL-10 detected in C3H mice, they still showed high concentrations of IL-18 and IL-12 p70. In contrast, spleen cells from BALB/c mice did not secrete IL-18, IL-12 p70 and IL-13 at this time pi, but produced higher amounts of IL-10 than C3H mice. Non of these cytokines was found increased in the cell supernatants of chronically infected mice. The addition of lipopolysaccharide (LPS) or Concanavalin A (Con A) to the cell cultures did not enhance the production of IL-18 and IL-12 at the time points tested. On the other hand, at 21 days pi, when parasitemia peaked, an inhibition of both the LPS induced IL-10 release and the IL-13 production upon Con A stimulation was observed in C3H, but not in BALB/c mice. We did not find an increase of IL-18, IL-10, or IL-12 p70 in the serum of the infected mice, despite the high seric IL-12 p40 concentrations reached during the infection. The data show that the different kinetics of the production of these cytokines in the spleen of both mouse strains could have a key role in the in vivo regulation of IFN-gamma production. In these experimental models, early IFN-gamma release and thus resistance to T. cruzi infection, could be related to the combined effect of both IL-18 and IL-12p70 in the absence of IL-13.     

The TGF-beta response to Leishmania chagasi in the absence of IL-12

Cure of leishmaniasis requires a type 1 immune response characterized by IFN-gamma production. Leishmania major infection leads to a type 2 response suppressing cure of susceptible BALB/c mice, and L. major causes an exacerbated type 2 response in mouse strains with a gene knockout (KO) such that they lack IL-12p40 (IL-12KO mice). In contrast, type 1 responses are inhibited by TGF-beta without Th2 cell expansion in BALB/c mice infected with L. chagasi. We questioned whether the type 2 or the TGF-beta response would dominate during L. chagasi infection of IL-12KO mice. C57BL/6 mice developed self-resolving L. chagasi infection with abundant IFN-gamma. In contrast, L. chagasi disease was exacerbated and IFN-gamma was low in IL-12KO mice. Total TGF-beta was significantly higher in IL-12KO than control C57BL/6 mice, but IL-4 and IL-10 levels were similar. TGF-beta was further augmented in IL-12/IFN-gamma double-KO mice. Thus, in contrast to L. major, the TGF-beta response was exacerbated whereas type 2 cells were not expanded during L. chagasi infection of IL-12KO mice. We conclude that L. chagasi has an inherent propensity to elicit a prominent TGF-beta response that either suppresses, or is suppressed by, a type 1 response. We propose this be termed a "type 3" immune response, which can antagonize a type 1 response.     

A recombinant Leishmania chagasi antigen that stimulates cellular immune responses in infected mice.

Cellular immune mechanisms resulting in gamma interferon production are critical for protection against visceral leishmaniasis. Antigens stimulating T-cell responses are likely present in the intracellular amastigote form of the parasite, since this is the form found in a mammalian host. To identify T-cell antigens of Leishmania chagasi, the parasite causing South American visceral leishmaniasis, we used a double antibody-T-cell technique to screen an amastigote cDNA library. One cDNA selected (Lcr1) encodes an antigen that stimulated proliferation of splenic T lymphocytes from infected mice that were either resistant (C3H.HeJ) or susceptible (BALB/c) to L. chagasi infection. The Lcr1 cDNA contains four highly divergent 201-bp repeats homologous to the 204-bp repeat of a Trypanosoma cruzi flagellar antigen gene. Results are consistent with a single copy of the Lcr1 gene producing an mRNA of > 10 kb and a protein of > 200 kDa. Recombinant Lcr1, cloned adjacent to polyhistidine and purified on a nickel affinity column, stimulated gamma interferon but not interleukin-4 (IL-4), IL-5, or IL-10 secretion by T-cell-enriched splenocytes from either susceptible or resistant mice during L. chagasi infection. Immunization with Lcr1 partially protected BALB/c mice against challenge with L. chagasi, indicating the utility of the double screening approach in selecting relevant T-cell antigens.     

DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice

C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-gamma) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-gamma. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.     

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.     

T-helper-cell cytokines in the early evolution of murine Lyme arthritis.

Genetic susceptibility to murine Lyme arthritis has been correlated with the dominance of T-helper (Th1)- or Th2-cell-associated cytokines. To determine when commitment of the Th cell phenotype occurs, we examined the kinetics of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production by lymph node T cells of disease-susceptible C3H/HeN and disease-resistant BALB/c mice from days 2 through 30 of infection, a period encompassing the evolution of disease and early regression. BALB/c mice produced more IFN-gamma on day 2 of infection than did C3H/HeN mice, whereas IL-4 was first detected on day 14. In contrast, only IFN-gamma could be detected in C3H/HeN mice, and the levels steadily increased from day 2 to surpass those seen in BALB/c mice by day 14 of infection. Despite the difference in cytokine profiles, both BALB/c and C3H/HeN mice developed comparable arthritis assessed at 14 days of infection. Arthritis regressed by day 30 in BALB/c mice but persisted in C3H/HeN mice. These studies are the first to demonstrate that the Th2 response to Borrelia burgdorferi infection of BALB/c mice is preceded by a Th1 cytokine response. Moreover, the timing of the appearance of IL-4 suggests that its primary effect is not in preventing disease, as suggested by others, but, rather, in hastening the resolution of inflammation. The implications of these findings for the orchestration of host defense against B. burgdorferi infection are discussed.     

Pretreatment with recombinant Flt3 ligand partially protects against progressive cutaneous leishmaniasis in susceptible BALB/c mice

Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-gamma)-producing Th1-type CD4(+) T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneous Leishmania major infection. Pretreatment of disease-susceptible BALB/c mice with 10 microg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alter L. major-induced IFN-gamma and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection with L. major reduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.