Murine peritoneal macrophages activated with interferon (IFN)-gamma and lipopolysaccharide (LPS) produce high levels of nitric oxide (NO) and are efficient in killing the intracellular protozoan parasites Leishmania major in vitro. Earlier studies have shown that NO, whose synthesis in murine macrophages is catalyzed by an inducible enzyme NO synthase, plays a major effector role in the host resistance against microbial infection. We now shown that both the NO synthesis and the leishmanicidal activity can be inhibited by prior treatment of the cells with recombinant interleukin 4 (IL4). IL4 treatment had no effect on the binding of IFN-gamma to macrophages but prevented the induction of NO synthase in these cells activated with IFN-gamma and LPS. Since IFN-gamma is produced by murine T helper type-1 (Th1) cells, whereas IL4 is secreted by Th2 cells, these results suggest a novel pathway by which Th2 cells regulate an activity of Th1 cells, namely by inhibiting the induction of NO synthase. These results may also account for the mechanism by which the disease-promoting Th2 cells counteract the host-protective effect of Th1 cells in leishmaniasis and other intracellular parasitic diseases.

BALB/c mice are highly susceptible to Leishmania major infection. They develop a progressive fatal disseminating disease even with a minimum infecting dose. However, these mice are able to contain the disease if they are exposed to sublethal gamma-irradiation shortly before infection. Earlier studies demonstrated that CD4+ T cells from mice which had recovered from infection (Tr) can adoptively transfer resistance. In contrast, CD4+ cells from mice with progressive disease (Ts) not only failed to protect, but can reverse the protective effect of the Tr cells. Spleen cells from BALB/c mice which had recovered from L. major infection or which had progressive disease were cultured with leishmanial antigens in vitro. The culture supernatant from spleen cells of recovered mice (TrSN) contains high levels of macrophage-activating factor (MAF) activity which can activate peritoneal macrophages to kill 51Cr-labeled P815 cells and to eliminate intracellular parasites as measured by the reduction in [3H]thymidine uptake by residual parasites released from macrophages following sodium dodecyl sulfate treatment. The MAF activity of TrSN parallels that of recombinant interferon-gamma (IFN-gamma). In contrast, culture supernatant of spleen cells from mice with progressive disease (TsSN) contains no detectable MAF but it is able to neutralize the MAF activity of TrSN. The MAF-inhibiting function of TsSN appears to be mediated by interleukin (IL)3 and IL4, since the MAF activity of TrSN and rIFN-gamma also can be inhibited by the addition of rIL3 and rIL4 but not by rIL1 or rIL2. Furthermore, the MAF-inhibiting activity of TsSN can be partially reversed by the addition of specific anti-IL3 or anti-IL4, but completely reversed by the combination of the two antibodies in vitro. These findings provide a mechanism for the immune regulation in leishmaniasis and a means by which the two subsets of CD4+ T cells influence each other through their modulation of macrophage function.

Leishmaniasis provides a biologically relevant model to analyze the heterogeneity of CD4+ T cells and may lead to answering the major question of the mechanism for the preferential induction of T helper type 1 (Th1) and Th2 cells. Using synthetic peptides corresponding to the tandemly repeating regions of Leishmania proteins, we have identified an epitope that can preferentially induce the disease-exacerbating Th2 cells in susceptible BALB/c mice. Lymph node cells from BALB/c mice immunized subcutaneously with the octamer (p183) of the repeating 10-mer peptide EAEEAARLQA proliferated strongly against the peptide as well as the soluble antigen extract (SolAg) of Leishmania major. The proliferative T cells are CD4+, major histocompatibility complex class II restricted, and secrete interleukin 4 (IL-4) but little or no IL-2 and interferon gamma when stimulated with the peptide in vitro. T cells from BALB/c mice with progressive disease, but not from BALB/c mice cured of the infection, recognized this epitope. BALB/c mice injected subcutaneously with p183 developed significantly exacerbated disease when subsequently challenged with L. major. Furthermore, subcutaneous injection with p183 prevented the subsequent induction of resistance against L. major by intravenous immunization with soluble antigen. The T cell response to p183 is H-2d restricted. Immunization of the genetically resistant B10.D2 mice with p183 also produced strong T cell responses and exacerbated disease when challenged with L. major.

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Seeds of the leguminous plant Millettia thonningii were shown to possess promising molluscicidal activity against Bulinus trunctatus. The size of the snails was an important determinant of their susceptibility to the molluscicide; specimens with shell lengths of 2 to 3 mm being more susceptible than snails 5 to 6 mm long.