Alterations of intralesional and lymph node gene expression and cellular composition induced by IL-12 administration during leishmaniasis.

Changes in gene expression and cellular distribution in the lymph node and at the site of infection, the footpad, during Leishmania major infection and/or IL-12 administration were evaluated. Otherwise susceptible BALB/c mice given IL-12 are able to resolve infection. Interestingly, iNOS was not induced in the lymph node by IL-12, yet, nitric oxide is critical in the control of leishmaniasis. However, we observed an increase in iNOS at the lesion site in response to IL-12. These results reflect the importance of examining the primary site of infection. We observed no changes in inflammation at the lesion site; however, IL-12 promoted an early inflammatory response in the lymph nodes. IL-12 administration differentially affected both the local and systemic immune response to invading leishmanial parasites. IL-12 induced iNOS at the lesion site and an early granulomatous inflammation in the lymph node; therefore, we hypothesize that these are key events responsible for the resolution of disease in BALB/c mice treated with IL-12.

Expression of Th2 cytokines and the stable Th2 marker ST2L in the absence of IL-4 during Leishmania major infection.

In this study we characterized Th2 responses in the absence of IL-4. We show that ST2L, a stable Th2 marker, is expressed at similar levels in Leishmania major-infected IL-4-deficient (IL-4(-/-)) and wild-type BALB/c (IL-4(+/+)) mice. Th2 cytokines are secreted by in vivo differentiated lymphocytes in response to specific activation in the absence of IL-4. Although IL-13 is produced, its neutralization did not alter the outcome of infection. Thus, we demonstrate that Th2 differentiation as assessed by the expression of ST2L and the production of Th2 cytokines can occur in vivo in the absence of IL-4.

Interleukin-12 is capable of generating an antigen-specific Th1-type response in the presence of an ongoing infection-driven Th2-type response.

Previously we demonstrated that recombinant murine interleukin-12 (rmIL-12) administration can promote a primary Th1 response while suppressing the Th2 response in mice primed with 2,4, 6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). The present studies examined the capacity of rmIL-12 to drive a Th1 response to TNP-KLH in the presence of an ongoing Th2-mediated disease. To establish a distinct Th2 response, we used a murine model of leishmaniasis. Susceptible BALB/c mice produce a strong Th2 response when infected with Leishmania major and develop progressive visceral disease. On day 26 postinfection, when leishmaniasis was well established, groups of mice were immunized with TNP-KLH in the presence or absence of exogenous rmIL-12. Even in the presence of overt infection, TNP-KLH-plus-rmIL-12-immunized mice were still capable of generating KLH-specific gamma interferon (IFN-gamma) as well as corresponding TNP-specific immunoglobulin G2a (IgG2a) titers. In addition, the KLH-specific IL-4 was suppressed in infected mice immunized with rmIL-12. However, parasite-specific IL-4 and IgG1 production with a lack of parasite-specific IFN-gamma secretion were maintained in all infected groups of mice including those immunized with rmIL-12. These data show that despite the ongoing infection-driven Th2 response, rmIL-12 was capable of generating an antigen-specific Th1 response to an independent immunogen. Moreover, rmIL-12 administered with TNP-KLH late in infection did not alter the parasite-specific cytokine or antibody responses.

IL-13 is a key regulatory cytokine for Th2 cell-mediated pulmonary granuloma formation and IgE responses induced by Schistosoma mansoni eggs.

Schistosoma mansoni egg-induced pulmonary granuloma formation is a cell-mediated inflammatory response associated with dominant Th2-type cytokine expression, tissue eosinophilia, and high levels of serum IgE. In the present study, we show that in vivo blockade of the Th2 cytokine IL-13, using soluble IL-13R alpha2-Fc fusion protein, significantly reduced the size of pulmonary granulomas in unsensitized as well as egg-sensitized mice. Blocking IL-13 also significantly reduced total serum IgE levels. Interestingly, however, IL-13 blockade did not affect the evolving egg-induced Th2-type cytokine response. IL-4, IL-5, as well as IL-13 responses were indistinguishable in control-Fc- and soluble IL-13R alpha2-Fc fusion protein-treated animals. The smaller granulomas were also phenotypically like the control Fc-treated mice, displaying a similar eosinophil content. Additional studies in IL-4-deficient mice demonstrated that IL-13 was produced, but at much lower levels than in wild-type mice, while IL-4 expression was completely independent of IL-13. Moreover, while granuloma formation was partially reduced in IL-4-deficient mice, blocking IL-13 in these animals almost completely abrogated granuloma development and the pulmonary eosinophilia, while it simultaneously increased IFN-gamma production. Together, these data demonstrate that IL-13 serves as an important mediator of Th2-mediated inflammation and plays a role in eliciting IgE responses triggered by schistosome eggs.

Characterization of a relatively rare class B, type 2 trypanosome variant surface glycoprotein gene.

The variant surface glycoprotein (VSG) gene of Trypanosoma brucei rhodesiense LouTat 1.5, a defined African trypanosome variant antigenic type, was cloned and sequenced. Southern blot analysis revealed 2 DNA restriction fragments in both VSG 1.5 expressor and nonexpressor populations, suggesting that there are 2 genomic copies of the VSG 1.5 gene and no expression-linked copies. Pulsed-field gel electrophoresis followed by Southern blot analysis showed that each copy of the VSG 1.5 gene exists on a separate large chromosome in both the expressor (approximately 3.5- and 4-megabase (Mb) chromosomes) and nonexpressor (approximately 4- and 5.7-Mb chromosomes) populations. Thus, VSG genes may be present on larger chromosomes than previously reported. Sequence analysis and alignments revealed that the VSG 1.5 molecule is a class B VSG with 12 cysteine residues in the N-terminus and is classified as a type 2 VSG based on C-terminus motifs. This classification shows that the VSG 1.5 molecule represents a relatively rare VSG class and type. Taken together, these studies provide additional information on VSG genes and proteins and supply the foundation for structure-function analysis of the VSG 1.5 surface antigen expressed by trypanosomes of the LouTat 1 serodeme.

The role of recombinant murine IL-12 and IFN-gamma in the pathogenesis of a murine systemic Candida albicans infection.

Studies on murine candidiasis suggest that resistance to disease is linked to a Th1 response and production of IFN-gamma, while failure to elicit protection is associated with a Th2 response and production of IL-4 and IL-10. Experimental infection of C57BL/6 mice, IL-12 treatment of these mice, or both infection and IL-12 treatment resulted in a characteristic Th1 cytokine mRNA profile as measured by quantitative competitive PCR. Specifically, little or no IL-4 transcripts were detected, while IFN-gamma message was elevated, particularly with IL-12 treatment. Despite its role in driving increased IFN-gamma expression and production, IL-12 treatment, paradoxically, promoted disease progression in our model. Therefore, we examined the effect of IFN-gamma neutralization on IL-12-induced susceptibility to infection. None of the systemically infected mice receiving IL-12 alone survived, while IL-12- and anti-IFN-gamma-treated mice had a 70% survival rate, similar to that after infection alone. These results suggested that IFN-gamma induced by IL-12 treatment contributed to lethality. However, in separate studies, IFN-gamma knockout mice were more susceptible to infection than their wild-type counterparts, suggesting that IFN-gamma is required for resistance. Nonetheless, infected IFN-gamma knockout mice treated with recombinant murine IL-12 exhibited enhanced resistance, suggesting that the toxicities observed with IL-12 are directly attributable to IFN-gamma and that an optimal immune response to Candida infections necessitates a finely tuned balance of IFN-gamma production. Thus, we propose that although IFN-gamma can drive resistance, the overproduction of IFN-gamma during candidiasis, mediated by IL-12 administration, leads to enhanced susceptibility.

Interleukin-4-dependent immunoglobulin G1 isotype switch in the presence of a polarized antigen-specific Th1-cell response to the trypanosome variant surface glycoprotein.

This study examines B-cell immunoglobulin (Ig) class-switching events in the context of parasite antigen-specific Th-cell responses in experimental African trypanosomiasis. Inbred mice were infected with Trypanosoma brucei rhodesiense, and the coordinate stimulation of Th-cell cytokine responses and B-cell responses to the trypanosome variant surface glycoprotein (VSG) was measured. The cytokines produced by T cells in response to VSG, at both the transcript and protein levels, were gamma interferon and interleukin-2 (IL-2) but not IL-4 or IL-5. Isotype profiles of antibodies specific for VSG showed that IgG1, IgG2a, and IgG3 switch responses predominated; no VSG-specific IgE responses were detected. To determine whether cryptic IL-4 responses played a role in promoting the unexpected IgG1 switch response, IL-4 knockout mice were infected; the cytokine responses and Ig isotype profiles of IL-4 knockout mice were identical to those of the wild-type control mice except for dramatically reduced IgG1 levels in response to VSG. Thus, these results revealed an IL-4-dependent component of the VSG-driven B-cell Cmu-to-Cgamma1 switch. We speculate that an IL-4 response is mediated primarily by cells other than T lymphocytes since IL-4-secreting but parasite antigen-unresponsive, "background" cells were detected in all infected mice and since infected nude mice also displayed a detectable IgG1 switch response. Overall, our results suggest that B-cell clonal stimulation, maturation, and Ig class switching in African trypanosomiasis may be partially regulated by unusual mechanisms that do not include antigen-specific Th1 or Th2 cells.

Characterization of T helper cell responses to the trypanosome variant surface glycoprotein.

T cell responses to the variant surface glycoprotein (VSG) previously have not been detected in animals infected with the African trypanosomes despite the fact that such animals make strong T-dependent B cell responses to VSG molecules displayed by the parasites. In the present study, we have examined B 10.BR mice for VSG-specific Th cell responses at different times after infection with Trypanosoma brucei rhodesiense clone LouTat 1. T cell populations derived from different tissues were tested for their ability to proliferate and secrete cytokines when stimulated with purified LouTat 1 VSG. Furthermore, VSG-specific T cell lines and clones were derived from immunized mice and examined for their phenotypic and functional profiles in comparison with T cell responses of infected mice. The results of this study show that VSG-specific T cells were not consistently detected in the peripheral lymphoid tissues such as spleen or lymph nodes of infected animals. In contrast, VSG Ag-specific T cells were detectable principally in the peritoneal T cell populations of infected mice. Peritoneal T cells did not proliferate in response to VSG, yet produced substantial cytokine responses when stimulated; the cytokines produced were IFN-gamma and IL-2, without detectable IL-4. The cellular phenotype of VSG-responsive T cells was that of classical Th cells in that all cells were CD4-positive and expressed the CD3 alpha/beta TCR membrane complex. Thus, the VSG appears to preferentially stimulate a Th1 cell subset response during infection. Intrinsic molecular characteristics of the VSG molecule did not induce mice to make this response, however, since VSG-specific T cell lines derived from VSG-immunized mice displayed cytokine profiles characteristic of both Th1 and Th2 cells. Isolation of Th1 clones from selected lines demonstrated that these cells displayed the same membrane-phenotypic characteristics and cytokine profiles as the T cells from infected mice. Furthermore, all Th clones were VSG type-specific, APC-dependent, and I-Ak-restricted in their responses. In summary, these experiments provide the first direct evidence for VSG-specific responses at the T cell level. T cell responses to the VSG molecule during infection appear to be anatomically compartmentalized and exhibit evidence of clonal maturation (cytokine production) but not clonal expansion (proliferation) after antigenic stimulation. The cellular phenotype and cytokine profiles predict that infection predisposes the animals to mount Th1 cell subset responses to VSG. The results of this study, including the T clones generated, provide an experimental basis for examining the regulation of VSG-specific immune responses during infection.