Antibody and cytokine responses to the cilium-associated respiratory bacillus in BALB/c and C57BL/6 mice

The cilium-associated respiratory (CAR) bacillus is a gram-negative, gliding bacterium that causes persistent respiratory tract infections in rodents despite histologic and serologic evidence of a marked immune response. To assess humoral immunity and cytokine responses in CAR bacillus disease, 6-week-old female BALB/c and C57BL/6 mice were inoculated intratracheally with 10(5) CAR bacillus organisms. CAR bacillus-specific serum immunoglobulins (immunoglobulin M [IgM], IgG1, IgG2a, IgG2b, IgG3, and IgA) and local pulmonary cytokines (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], and interleukin-4 [IL-4]) were evaluated by enzyme-linked immunosorbent assay every 7 days for 49 days. BALB/c mice developed CAR bacillus-induced lesions early in the course of disease that became more severe with time. Correlating with increasing disease severity, BALB/c mice had elevations in all antibody isotypes tested, and elevations in pulmonary TNF-alpha, IFN-gamma, and IL-4. C57BL/6 mice developed mild lesions with mild increases in serum IgM, IgG1, IgG2b, and IgG3 levels and minimally detectable IgG2a and IgA. Cytokine perturbations were not detected in C57BL/6 mice. The persistence of infection in BALB/c mice with vigorous serum antibody responses and increased IFN-gamma and IL-4 responses suggests that humoral immunity and T-cell responses are ineffective at preventing CAR bacillus disease. Furthermore, the lackluster antibody responses and undetectable cytokine responses in C57BL/6 mice suggest that humoral immunity and T-cell responses are not critical in resistance to CAR bacillus-induced disease.     

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

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

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.     

Differential susceptibility of C57BL/6 and DBA/2 mice to ovalbumin-induced pulmonary eosinophilia regulated by Th1/Th2-type cytokines

To test the effect of genotype on immune response, C57BL/6 and DBA/2 mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) and challenged with aerosolized OVA. The serum immunoglobulin (Ig) E and IgG1 levels in C57BL/6 mice were higher than those in DBA/2 mice. In contrast, IgG2a levels in C57BL/6 mice were lower than that in DBA/2 mice. C57BL/6 mice were also much more susceptible than DBA/2 mice to OVA-induced pulmonary eosinophilia. Furthermore, patterns of cytokine generation in lung tissue were different between C57BL/6 and DBA/2 mice after OVA challenge. Th2-type cytokine interleukin (IL-) 4 and IL-5 generation in C57BL/6 mice was higher than that in DBA/2 mice, while Thl-type cytokine interferon-gamma (IFN-gamma) generation in C57BL/6 mice was lower than that in DBA/2 mice. Similar patterns of IL-4 and IL-5, and IFN-gamma production in splenocytes from both strains after OVA stimulation in vitro were also observed. The participation of IL-4 and IL-5, and IFN-gamma in the regulation of eosinophil infiltration into the lung was confirmed by injection of anti-IL-5, -IL-4 and -IFN-gamma monoclonal antibodies. These results indicate that C57BL/6 mice preferentially induce IL-4 and IL-5-mediated Th2-type response, while DBA/2 mice induce IFN-gamma-mediated Thl-type response. Thus, the genotype of laboratory strains partially determines whether Th1- or Th2-type immune responses are elicited.     

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.     

Antigen dose defines T helper 1 and T helper 2 responses in the lungs of C57BL/6 and BALB/c mice independently of splenic responses

To investigate the effect of antigen dose on immune response, C57BL/6 and BALB/c mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) then challenged with aerosolized OVA. Low-dose sensitization (less than 8 microg of OVA) elicited T helper 2 (Th2)-type immunoglobulins (Igs) secretion from C57BL/6 mice, including high levels of serum IgE, IgG1 and low levels of IgG2a, while BALB/c mice secreted T helper 1 (Th1)-type Igs, including low levels of IgE, IgG1 and high levels of IgG2a. In contrast, high-dose sensitization (more than 50 microgram) elicited Th1-type Igs secretion in C57BL/6mice, while BALB/c mice exhibited Th2-type Igs secretion. Furthermore, the number of eosinophils infiltrating into the lungs of low-dose OVA-sensitized C57BL/6 mice was significantly greater than in BALB/c mice sensitized with the same amount of OVA. Only a very high dose of OVA (1 mg) could induce greater eosinophil infiltration into the lungs of BALB/c mice compared with C57BL/6 mice. Additionally, low-dose sensitization generated Th2-type cytokines, including high levels of interleukin (IL) -4, IL-5 and a low level of interferon-gamma (IFN-gamma) in the lungs of C57BL/6 mice, while BALB/c mice generated Th1-type cytokines in their lungs, including low levels of IL-4, IL-5 and a high level of IFN-gamma. In contrast, high-dose sensitization elicited Th1-type cytokines production in the lungs of C57BL/6 mice, while BALB/c mice generated Th2-type cytokines in their lungs. Interestingly, splenocyte cultures from C57BL/6 mice produced Th1-type cytokines, while cultures from BALB/c mice produced Th2-type cytokines regardless of OVA sensitization dose (100 ng-1 mg). These results indicate that C57BL/6 and BALB/c mice have different susceptibilities to OVA-sensitization and OVA-induced pulmonary eosinophilia regulated by Th1- and Th2-type cytokines, independent of splenic Th1- and Th2-type cytokines production.     

C57BL/6 mice are more susceptible to antigen-induced pulmonary eosinophilia than BALB/c mice, irrespective of systemic T helper 1/T helper 2 responses

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)-type cytokines interleukin (IL)-4 and IL-5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL-4 and IL-5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti-IL-4 and anti-IL-5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL-4 and IL-5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2-type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)-type responses in the lungs, despite considerable production of IL-4 and IL-5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background.     

Mycobacterium avium infection in mice is associated with time-related expression of Th1 and Th2 CD4+ T-lymphocyte response.

Disseminated infection caused by organisms of Mycobacterium avium complex is common in acquired immune deficiency syndrome (AIDS) patients. M. avium is an intracellular bacterium that multiplies within macrophages. We examined the effect of M. avium infection on the T-helper cell response in C57/BL/6 black mice. At weekly intervals, CD4+ T-cells were isolated from spleens and lines were created. T-cell lines were exposed to sonicated M. avium in the presence of feeder cells and macrophages and the supernatant were collected to measure the concentrations of interferon-gamma (IFN-gamma and interleukin-10 (IL-10). Production of IFN-gamma in CD4+ T-cells obtained from uninfected mice did not vary significantly during the 5 weeks. Levels of IFN-gamma produced by T-cell lines of infected mice were similar to the control mice during the first 2 weeks but significantly reduced (approximately 30 ng/ml) thereafter. In contrast, production of IL-10 by T-cell lines of infected mice was in a range of 190 to 342 pg/ml in weeks 1, 2 and 3, but increased to an average of 1300 pg/ml at weeks 4 and 5. Pre-immunized mice, when infected with M. avium strain 101, showed a different profile of T-cell cytokines, with high IFN-gamma and low IL-10 production. Proteins purified from a number of disease-associated (D-A) and non-D-A strains of M. avium were tested for the ability to induce IL-10. 65,000 MW and 60,000 MW proteins of M. avium induced significantly more IL-10 than 45,000 MW, 33,000 MW and 27,000 MW proteins. These results showed that M. avium predominantly stimulates either Th1 or Th2 T-helper cells according to the phase of the infection.     

Regulation of anaphylactic IgG1 antibody production by IL-4 and IL-10

BACKGROUND: Different cytokines have been implicated in the regulation of isotype expression in primary and secondary antibody responses. The aim of this study was to assess the regulation of anaphylactic IgG1 and IgE antibodies by IL-4, IL-10 and IFN-gamma at different time points of the antibody response against PI, an immunosuppressive fraction of Ascaris suum extract, and ovalbumin (OVA). METHODS: Wild-type or cytokine-deficient C57BL/6 or BALB/c mice were immunized with PI or OVA in different adjuvants. Twenty days later, they were boosted with the respective antigen. IgG1 and IgE antibodies produced during primary and secondary responses were measured by passive cutaneous anaphylaxis. RESULTS: PI induced low levels of anaphylactic IgG1 antibodies in the primary response and moderate levels after the antigenic booster, which were IL-4-dependent. In the absence of IL-10 and IFN-gamma, PI-specific IgG1 and IgE enhanced significantly, indicating that these cytokines downregulated antibody production in primary and secondary responses. The IgG1 response to OVA in aluminium hydroxide or complete Freund's adjuvant was IL-4-dependent in the beginning of the primary response. Later on, it became only partially regulated by IL-4 in C57BL/6 mice and IL-4-independent in Th2-prone BALB/c mice. In contrast, IgE antibodies depended exclusively upon IL-4 during the entire time course. CONCLUSIONS: These results indicate, first, that the IL-4 dependency of anaphylactic IgG1 antibody production, mainly in the secondary response, varies among mouse strains, and, second, that the nature of the antigen determines whether IL-10 and IFN-gamma limit the potential to make large amounts of anaphylactic IgG1 and IgE.     

Lack of Th1 or Th2 polarization of CD4+ T cell response induced by particulate antigen targeted to phagocytic cells

Several factors are involved in the selective activation of Th1 or Th2 subset of CD4+ T cells, such as the type of antigen-presenting cells, the dose of antigen, the route of immunization, etc. To analyze the influence of accessory cells on Th1/Th2 cell differentiation, we used a particulate antigen prepared by covalent linkage of hemocyanin (LH) to 1 microns synthetic microspheres. This particulate antigen was efficiently presented to T cells by macrophages but not by B lymphocytes. BALB/c mice immunized either with soluble LH in alum or with particulate LH without adjuvant produced both Th1 (IL-2 and IFN- gamma) and Th2 (IL-4 and IL-5) cytokines. Moreover, mice primed either with soluble or particulate LH secreted higher levels of IgG1- than of IgG2a-specific antibodies. The induction of this cytokine profile response was independent of the route of administration of the antigen, and was observed both in BALB/c and C57BL/6 mice. In contrast, immunization of mice with particulate LH in the presence of poly(I):(C) or of IL-12 induced a strong activation of Th1 cells, as shown by an up- regulated IFN-gamma production, and by decreased IL-4 and IL-5 levels associated to a greatly enhanced IgG2a antibody response. These results therefore demonstrate that targeting the antigen to phagocytic cells is not sufficient to stimulate a polarized Th response and that environmental cytokines play the major role in the selective activation of Th1 cells. This study provides important conclusions for the development of new vaccines and shows that particulate antigen associated with appropriate cofactor can selectively activate Th1 cells.      

Genetically programmed biases in Th1 and Th2 immune responses modulate atherogenesis

Atherosclerotic lesions contain T lymphocytes, which orchestrate adaptive immunity and regulate many innate immune pathways. This study examined the influence of Th1 and Th2 helper cell subsets on atherogenesis in two ApoE(-/-) mouse strains, C57BL/6 and BALB/c, which display opposite T-cell subset polarizations. ApoE(-/-) BL/6 mice showed predominant Th1-like immune responses on polyclonal stimulation of splenic CD4(+) T cells and had IgG2a antibodies to oxidized low-density lipoprotein (a disease-related antigen) whereas ApoE(-/-) BALB/c mice displayed predominant Th2 responses by CD4(+) T cells and an IgG1 isotype response toward oxidized low-density lipoprotein. ApoE(-/-) BL/6 and BALB/c mice consumed a high-cholesterol diet for 10, 16, and 24 weeks with equivalent cholesterolemic responses. The Th1-slanted BL/6 mice developed significantly more atherosclerosis in the aortic root and abdominal aorta at all time points compared with BALB/c mice, supporting a proatherogenic role for Th1 response. Progression of atherosclerosis was associated with increased levels of interleukin (IL)-6 in mouse serum and CD4(+) T-cell culture supernatants and increased levels of the acute-phase protein, serum amyloid A (SAA). Both IL-6 and SAA levels rose significantly in ApoE(-/-) BL/6 mice compared with BALB/c mice. The circulating cytokine milieu (IL-6) and acute phase reactants such as SAA may reflect alterations in the Th1/Th2 balance. The results presented here illustrate how genetically determined modifiers of both immune and inflammatory responses can modulate atherogenesis independently of lipid levels.     

Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis.

In experimental Leishmania donovani infection in BALB/c mice, initial susceptibility gives way to T-cell-dependent acquired resistance and eventual control over visceral infection. Since various cytokines appear to underlie the host response to Leishmania infection, we examined infected liver tissue for gene expression of cytokines associated with Th1 (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]) and Th2 cells (IL-4 and IL-10). By Northern (RNA) blot analysis, only IFN-gamma mRNA expression was detected in livers of infected euthymic mice. To determine whether activation of Th1 cells develops selectively in this model, qualitative PCR analysis was used. These results indicated that mRNAs for IFN-gamma, IL-2, IL-4, and IL-10 were all induced by L. donovani infection. The potentially negative Th2 cell-associated response did not appear to play a functional role, however, since resistance was acquired, anti-IL-4 monoclonal antibody treatment did not accelerate control over visceral infection, and serum immunoglobulin E levels remained low. As judged by PCR analysis, IL-4 and IL-10 mRNAs were also expressed under three other conditions without apparent effect: in naive euthymic mice treated with IL-2, which induces leishmanicidal activity; in rechallenged immune mice, which resist reinfection; and in nude mice, which fail to control L. donovani. These results suggest that, like other Leishmania species, L. donovani infection may trigger a potentially suppressive Th2 cell-associated cytokine response. However, in T-cell-intact mice able to control L. donovani, this response either is insufficient to influence outcome or more likely is overshadowed by the Th1 cell response.     

Maintenance of IL-12-responsive CD4+ T cells during a Th2 response in Leishmania major-infected mice

BALB/c and anti-IL-12-treated C3H mice infected with Leishmania major develop a Th2 cell response. However, in contrast to BALB/c mice, C3H mice treated transiently with an anti-IL-12 monoclonal antibody switch from a Th2 to a Th1 response and resolve their lesions once treatment is terminated. We report here that the critical difference in the Th2 response between BALB/c and C3H mice is in their ability to respond to IL-12. Thus, C3H mice with a Th2 response maintain a CD4+ T cell population that expresses IL-12 receptor beta1 and beta2 mRNA and produces IFN-gamma after exposure to IL-12. These results indicate that Th2 cell populations from different genetic backgrounds differ in their stability, and that this difference can be related to differential regulation of the IL-12 receptor.     

Both Th1 and Th2 Cytokines Affect the Ability of Monoclonal Antibodies To Protect Mice against Cryptococcus neoformans

Variable-region-identical mouse immunoglobulin G1 (IgG1), IgG2b, and IgG2a monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans prolong the lives of mice infected with this fungus, while IgG3 is either not protective or enhances infection. CD4+ T cells are required for IgG1-mediated protection, and CD8+ T cells are required for IgG3-mediated enhancement. Gamma interferon is required for both effects. These findings revealed that T cells and cytokines play a role in the modulation of cryptococcal infection by antibodies and suggested that it was important to more fully define the cytokine requirements of each of the antibody isotypes. We therefore investigated the efficacy of passively administered variable-region-identical IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies against intravenous infection with C. neoformans in mice genetically deficient in interleukin-12 (IL-12), IL-6, IL-4, or IL-10, as well as in the parental C57BL/6J strain. The relative inherent susceptibilities of these mouse strains to C. neoformans were as follows: IL-12(-/-) > IL-6(-/-) > C57BL/6J approximately IL-4(-/-) > IL-10(-/-). This is consistent with the notion that a Th1 response is necessary for natural immunity against cryptococcal infection. However, none of the IgG isotypes prolonged survival in IL-12(-/-), IL-6(-/-), or IL-4(-/-) mice, and all isotypes significantly enhanced infection in IL-10(-/-) mice. These results indicate that passive antibody-mediated protection against C. neoformans requires both Th1- and Th2-associated cytokines and reveal the complexity of the mechanisms through which antibodies modulate infection with this organism.     

A central role for IL-2 in fate determination of mature T cells--I: role in determining the Th1/Th2 profile in primary T cell cultures.

IL-2 signaling appears to play a significant role in enabling the synthesis of T(h)2 cytokines in an in vitro system for studying primary T cell responses. When T cells from C57BL/6J or BALB/c strains of mice were activated in vitro and re-stimulated through their TCR complex 48 h later, CD4(+) T cells producing the T(h)2 cytokines IL-4 and IL-10 were found only when IL-2 was present. IL-2 also enhanced IFN-gamma synthesis in C57BL/6J cells but not in BALB/c cells. By up-regulating production of anti-inflammatory T(h)2 cytokines during a primary response, IL-2 may play a critical role in limiting T(h)1-mediated responses.     

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4+ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0 x 10(7) cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-gamma, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4+ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c x C57BL/6 F1 mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-gamma than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-gamma-producing ability but rather derived from their poor responsiveness to IFN-gamma.     

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.     

TSH receptor-adenovirus-induced Graves' hyperthyroidism is attenuated in both interferon-gamma and interleukin-4 knockout mice; implications for the Th1/Th2 paradigm

The role of the Th1/Th2 balance in the pathogenesis of murine Graves' hyperthyroidism is controversial. In BALB/c mice injected with adenovirus expressing TSH receptor (TSHR-adeno model), we found that suppression of TSHR-specific Th1 immune responses by exogenous interleukin-4 (IL-4), alpha-galactosylceramide or helminth (Schistosoma mansoni) infection was associated with inhibition of hyperthyroidism, indicating the critical role for Th1 cytokines. In contrast, BALB/c IL-4 knockout (KO), but not interferon-gamma (IFN-gamma) KO mice failed to develop Graves' hyperthyroidism when injected with TSHR-expressing M12 B lymphoma cells (TSHR-M12 model), suggesting the importance of Th2 cytokine IL-4. To reconcile differences in these two models, we used IL-4 KO and IFN-gamma KO BALB/c mice in the TSHR-adeno model. Unlike wild-type (wt) BALB/c mice in which 60% developed hyperthyroidism, only 13 and 7% of IL-4 KO and IFN-gamma KO mice, respectively, became hyperthyroid. Thyroid stimulating antibodies were positive in most hyperthyroid mice. TSHR antibody titres determined by TSH binding inhibition and ELISA were comparable in all three groups. IgG1 and IgG2a TSHR antibody titres were similar in IFN-gamma KO and wt mice, whereas IgG1 TSHR antibody titres and TSHR-specific splenocyte IFN-gamma secretion were lower in IL-4 KO than in IFN-gamma KO and wt mice, respectively. Our results clearly implicate both IFN-gamma and IL-4 in development of hyperthyroidism in the TSHR-adeno model. These data, together with the previous report, also indicate different cytokine requirements in these two Graves' models, with IFN-gamma being more important in the TSHR-adeno than the TSHR-M12 model. Moreover, our previous and present observations indicate a difference in the role of exogenous versus endogenous IL-4 in TSHR-adenovirus induced Graves' hyperthyroidism.     

Anti-interleukin-4 modulation of the Th2 polarized response to the parasitic nematode Brugia pahangi.

Lymphatic filariasis is a chronic disease characterized by a pronounced Th2 bias in the immune response and impaired antigen (Ag)-specific Th1 responses. We have used a mouse model of filariasis to investigate the role of the infective form (the third-stage larvae [L3]) in modulating the immune response. Subcutaneous infection of BALB/c mice with L3 of Brugia pahangi has a profound effect on Th cell function. By day 12 post-infection, spleen cells from these mice exhibited a dramatic reduction in concanavalin A-driven proliferation and interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion in comparison with uninfected controls. However, exposure to L3 did not render the mice completely unresponsive; these animals mounted a strong Th2 response to the parasite, characterized by elevated levels of IL-4, IL-5, and IL-10 and parasite-specific serum immunoglobulin G (IgG), IgG1, and IgE. Treatment of spleen cells from L3-infected mice with neutralizing anti-IL-4 or recombinant IL-2 resulted in a dramatic increase in concanavalin A-induced proliferation and IL-2 and IFN-gamma production. Despite their defective polyclonal Th1 response, cells from L3 infected mice proliferated when stimulated with Ag, and this response was blocked by anti-IL-4. However, anti-IL-4 treatment failed to induce Ag-specific IL-2 or IFN-gamma production, indicating that B.pahangi-primed Th1 cells do not appear to be present or are still unable to respond even in the absence of IL-4.