Leishmania major induces differential expression of costimulatory molecules on mouse epidermal cells.

Levels of expression of costimulatory molecules have been proposed to influence the outcome of antigen-specific T cell priming. We found that Leishmania major selectively modulated the expression of costimulatory molecules on various populations of epidermal cells. B7.2 expression was down-regulated on Thy1.2+ epidermal cells (keratinocytes) from disease-resistant C3H mice, but not from disease-susceptible BALB/c mice. In addition, epidermal cells from BALB/c mice showed a down-regulation of B7.1 expression on NLDC 145+ Langerhans cells. In vitro T cell priming experiments, using syngeneic epidermal cells as antigen-presenting cells (APC), showed that the production of IFN-gamma was inhibited when either B7.1 or B7.2 signaling pathways were blocked. Blockade of B7.2, but not B7.1, significantly inhibited the ability of epidermal cells to induce IL-4 production from CD4+ T cells. In addition, C3H CD4+ T cells, which were unable to secrete detectable levels of IL-4 in cultures with syngeneic APC, were now able to secrete IL-4 following presentation of L. major antigens by congenic BALB/K epidermal cells. Conversely, C3H epidermal cells supported the priming of BALB/K CD4+ T cells for IL-4 production in vitro. Thus, the differential expression of B7 molecules on epidermal cells may not represent the sole factor governing the polarization of L. major-specific CD4+ T cells in vitro.

Interleukin-6 deficiency influences cytokine expression in susceptible BALB mice infected with Leishmania major but does not alter the outcome of disease.

Since interleukin-6 (IL-6) may promote Th2 responses, we infected BALB IL-6-deficient (IL-6(-/-)) mice with Leishmania major. There was not a significant difference between the courses of infection (lesion size and parasite burden) in IL-6(-/-) and wild-type mice, but IL-6(-/-) mice expressed lower levels of Th2- and Th1-associated cytokines.

Influence of costimulatory molecules on immune response to Leishmania major by human cells in vitro.

The importance of CD40, CD80, and CD86 costimulatory molecules in anti-Leishmania immune responses has been established in murine models. A role for these costimulatory molecules in human anti-Leishmania immune responses was investigated in this study. Autologous macrophages and peripheral blood leukocytes (PBL) were prepared from peripheral blood mononuclear cells of Leishmania-naive donors and cultured with or without Leishmania major in various combinations. After 7 days of culture, high levels of CD40 and CD86 were expressed on macrophages in the presence or absence of L. major. When macrophages were cultured for an additional 7 days with PBL, expression of all three costimulatory molecules was detected. When L. major was present in these cultures, the expression of CD80, and to a lesser extent CD40, on macrophages was enhanced. Blockade of CD80, CD86, or both molecules (in the order of greatest effect) in cultures containing macrophages, PBL, and L. major significantly inhibited the production of gamma interferon, interleukin-5 (IL-5), and IL-12. Blockade of CD40-CD154 interactions also significantly inhibited production of these cytokines in response to L. major. Production of IL-10 was unaltered by the blockade of these costimulatory molecules. Thus, these data suggest that CD40, CD80, and CD86 expression and regulation may significantly impact anti-Leishmania immune responses in humans.

A method for the isolation and analysis of leucocytic cells from Leishmanial ear lesions in mice.

The standard model of experimental cutaneous leishmaniasis involves infection of mice with Leishmania major in a single footpad or the rump, and analysis of the subsequent immune response in draining lymph nodes. Relatively few studies have examined the lesion directly. Here, we describe a method for the isolation of cells from established leishmanial lesions in mouse ears. After physical disruption of lesion tissue and isolation of cells on density gradients, a variety of leucocytic cell phenotypes were identified by flow cytometry and cytology. The phenotypes of the viable cells obtained were similar, in proportion, to those observed in histologic sections of ear lesions. This technique may be useful for studying lesion-specific cell function within the first weeks after infection with Leishmania parasites.

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice.

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.

Analysis of the immune responses of mice to infection with Leishmania braziliensis.

Leishmania major and Leishmania braziliensis both cause cutaneous leishmaniasis, but the former kills BALB/c mice while the latter is killed by the mice. This killing of L. braziliensis occurred by a gamma interferon-dependent mechanism, potentially made possible by the observed lack of high interleukin-4 production.

Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant and prototypic ligand for the aryl hydrocarbon receptor, is a potent immunotoxicant. To understand the underlying mechanisms of TCDD immunotoxicity, we have characterized the time course of changes in CTL, alloantibody, and cytokine responses to the P815 tumor allograft in C57B1/6 mice treated with 0 or 15 microg TCDD/kg. Suppression of CTL activity by TCDD directly correlated with reduced numbers of splenic CTL effector cells identified by their CD8+CD44 high CD45RB low phenotype, while suppression of the alloantibody response correlated with a lack of expansion of the B220+ splenocyte population. Cytokine production was differentially modulated following TCDD treatment. Although type 1 cytokine production (IFN-gamma, IL-2, and TNF) was initially induced in TCDD-treated mice, production failed to increase normally after day 5. In contrast, the production of IL-1 beta, IL-4, and IL-6 was mostly unaffected by TCDD exposure. This differential effect of TCDD on cytokine production was reflected in the degree of suppression of specific alloantibody isotypes. TCDD abrogated the production of IgG2a (promoted by IFN-gamma), but had much less effect on the level of IgG1 (promoted by IL-4). IgM Ab titers were also highly suppressed. CD8+ cells were the exclusive producers of IFN-gamma and IL-2 when spleen cells from P815-injected mice were cultured in vitro on days 4 to 7 after P815 injection. However, CD4+ cells were shown to play a crucial role in the generation of both CTL and alloantibody responses, since their depletion in vivo abolished both responses. Based on similar temporal effects produced by TCDD and anti-CD4 Ab on alloimmune responses, we postulate that TCDD interferes with the initial activation of CD4+ T cells, which leads to downstream inhibition of the activation and/or differentiation of CD8+ T cells and B cells. In addition, since delayed treatment with either anti-CD4 Ab or TCDD suppressed the alloantibody but not the CTL response, TCDD may also affect later CD4+ T helper-B cell interactions.

Effects of exogenous corticosterone treatment on alloantigen-specific cytotoxic T lymphocyte activity in mice.

The intent of this study was to examine the effects of stress-like plasma corticosterone (CS) elevation on the generation of alloantigen-specific cytotoxic T lymphocyte (CTL) activity in mice. Elevation of plasma CS was achieved by infusion of exogenous CS via osmotic pumps. CS infusion at 16 mg/kg/day on days -4 through 10 relative to alloantigen challenge led to slight, but significant, suppression of CTL activity on day 10 but no elevation of plasma CS levels. Infusion of lower CS doses (1, 2, 4 or 8 mg/kg/day) had no effect on CTL activity. Serial sampling of mice infused with CS at 0.09, 0.9 or 9 mg/kg/day over a 14-day period indicated that only the 9 mg/kg/day infusion rate caused significant plasma CS elevation. Peak CS levels (approximately 500 ng/ml) were observed 1 day after the start of CS infusion, but CS levels fell to below 200 ng/ml by day 7 and were approximately 50 ng/ml on day 12 indicating that elevated plasma CS levels could not be maintained for extended periods by CS infusion. An attempt to define the windows of CS sensitivity during CTL development was made by infusing mice with CS at doses of 10-16 mg/kg/day on days 0-3, 3-6, 4-7, 5-8 and 6-9, relative to alloantigen challenge; however, CS infusion had no effect on CTL activity. In contrast, dexamethasone infusion (9.4 mg/kg/day) on days 0 to 3 suppressed CTL activity by approximately 90% indicating that the generation of CTL activity is sensitive to high dose GC treatment, but is refractory to stress-like CS elevation. In mixed lymphocyte-tumor cell cultures, CTL activity was suppressed by CS (2.5 x 10(-8) M) if added on the first day of culture but not if added on subsequent days. These results suggest that CTL are most sensitive to CS-induced suppression if exposed near to the time of alloantigen challenge.

Phenotypic analysis of spleen, thymus, and peripheral blood cells in aged C57B1/6 mice following long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

A mouse model was used to identify potential biomarkers of exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Female C57B1/6 mice were treated weekly with 0.2 microgram TCDD/kg body weight or vehicle for 14-15 months. Phenotypic analysis by flow cytometry identified the major cell subpopulations in the spleen, thymus, and peripheral blood as defined by the expression of CD4, CD8, B220, and Mac-1 molecules. These subpopulations were further characterized for the expression of I-A, Pgp-1, CD45RB, and/or T cell receptor antigens (CD3, alpha beta, gamma delta). A group of young (4 months old) mice was evaluated concurrently to document immunophenotype alterations associated with aging. Results showed several age-related changes in phenotype distribution in the spleen and blood, but not in the thymus, despite significant age-dependent thymic involution. The age-dependent changes in splenic phenotypes included a decreased frequency of CD4+ cells and a major shift in the frequency distribution from naive T cells to effector and memory T cells as defined by Pgp-1 and CD45RB expression. These phenotypic changes in the spleen due to aging correlated with similar changes in the blood, providing preliminary support for the use of spleen cells as surrogates for blood in the development of biomarkers of immunotoxicity. In comparison to the effects of aging, TCDD treatment produced relatively subtle changes in immunophenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)