We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.

BALB/c mice infected with Leishmania major develop fatal, progressive disease, despite an immune response characterized by expansion of CD4+ T cells in the draining lymph nodes. The immune response has been further characterized by a lack of IFN-gamma mRNA, but increased IL-4 mRNA in lymphoid tissues, and striking elevation of serum IgE. Treatment of infected BALB/c mice with rIFN-gamma at doses shown to be beneficial in other protozoan infections was insufficient to ameliorate L. major infection. In contrast, neutralization of IL-4 by six weekly injections of mAb 11B11 led to attenuation of disease in 100% of animals, and complete cure in 85%. Resolution of disease required the presence of T cells, and recovered mice remained resistant to reinfection at 12 wk. This immunity was adoptively transferable and was dependent on both CD4+ and CD8+ cells. Although administration of anti-IL-4 was associated with fourfold increase in IFN-gamma mRNA in lymph node cells draining the lesion, the coadministration of neutralizing R4 6A2 anti-IFN-gamma mAb had no effect on resistance to disease. This was in marked contrast to resolution of disease in both resistant C57BL/6- and GK1.5-pretreated BALB/c mice that was abrogated by in vivo treatment with anti-IFN-gamma. These data suggest a novel mechanism of cellular immunity established by interference with the development of Th2 cells during infection.

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Patients with acute kala azar are generally nonreactive in a number of immunologic assays, including T cell proliferation and generation of macrophage-activating cytokines, principally IFN-'y, in response to leishmania antigens in vitro. To test for potential immunosuppressive factors, a series of T cell lines and clones were established from patients with acute kala azar, from patients after chemotherapy for kala azar, and from skin test-positive adults from the same endemic region. Although CD4+ T cell lines and clones could be readily established from the skin test-positive adults, lines and clones from acute or treated patients were heavily biased in expression of CD8+. The CD8 + cells from acute patients did not themselves release cytokines in response to leishmania antigens in vitro, but markedly affected the cytokine profile of peripheral blood mononuclear cells isolated 1 yr later after recovery. Addition of the CD8 + cells caused inhibition of lymphoproliferation and IFN-y release, with augmentation of IL-6 and IL-10 release. The inhibitory effects of the CD8+ cells could be partially abrogated by antibodies to IL-10 but not by antibodies to IL4. Analysis of four patients with acute kala azar demonstrated release of IL-10 that could not be demonstrated in supernatants from asymptomatic skin test-positive individuals. Generation of IL-10 may contribute to the profound suppression of IFN--y release that occurs during kala azar due to Leishmania chagasi. (J. Clin. Invest. 1993. 92:2626-2632.) Key words: leishmania -interleukin-10 * kala azar * interferon-'y -immunosuppression

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Patients from across the spectrum of clinical manifestations of Leishmania chagasi infection were evaluated for in vitro correlates of immunity. Peripheral blood mononuclear cells were assayed for parasite-specific lymphoproliferation, cytokine generation, and the capacity to activate autologous macrophages to kill intracellular amastigotes. Patients with acute kala-azar were generally unreactive in each of these assays. Children with subclinical infection demonstrated relatively low levels of proliferation and interferon-gamma production, but none went on to develop overt kala-azar during the study. Patients evaluated after therapy for kala-azar demonstrated yet higher levels of lymphoproliferation and cytokine generation and produced low but significant levels of cytokines in vitro in response to parasite antigens, but not during the activation of infected macrophages. Finally, peripheral blood mononuclear cells from adults with positive delayed-type hypersensitivity responses and no history of kala-azar showed the broadest reactivity in vitro. These patients' cells generated the largest amounts of activating cytokines in vitro during the activation of autologous macrophages to a leishmanicidal state.

This study examined the role and source of endogenous interleukin-10 (IL) secretion in visceral leishmaniasis (VL). The amounts of endogenous and Leishmania specific IL-10 and interferon-gamma (IFN) secreted by peripheral blood mononuclear cells (PBMC) from VL patients were compared. The correlation coefficient between endogenous IL-10 secretion and Leishmania specific IFN-gamma was -0.77, suggesting a major role for endogenous IL-10 secretion in VL. The effects of CD4+ and CD8+ T cell clones, isolated from a treated VL patient, on IL-10 secretion were assayed by mixing the clones with autologous, inactivated PBMC. The CD8+ clones mediated increased levels of IL-10 secretion in the presence of PBMC alone suggesting that CD8+ T cells may mediate endogenous IL-10 secretion

The course of human Leishmania chagasi infections appears to be determined by the balance between type 1 (T1) CD4+ and CD8+ T suppressor (Ts)