Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE2 and TGF-β production by macrophages, while parasite killing depended on neutrophil elastase and TNF-α production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.

Recent studies indicate important roles for CTLA-4 engagement in T cells, and for TGF-β production in the immunopathogenesis of murine kalaazar or visceral leishmaniasis, but a functional link between these two pathways in helping intracellular parasite growth is unknown. Here we report that Ag or anti-CD3 activation of splenic CD4+ T cells from visceral leishmaniasis leads to intense CTLA-4-mediated TGF-β1 production, as assessed either by CTLA-4 blockade or by direct CTLA-4 cross-linkage. Production of TGF-β1 accounted for the reciprocal regulation of IFN-γ production by CTLA-4 engagement. Following CD4+ T cell activation, intracellular growth of Leishmania chagasi in cocultured splenic macrophages required both CTLA-4 function and TGF-β1 secretion. Cross-linkage of CTLA-4 markedly increased L. chagasi replication in cocultures of infected macrophages and activated CD4+ T cells, and parasite growth could be completely blocked with neutralizing anti-TGF-β1 Ab. Exogenous addition of rTGF-β1 restored parasite growth in cultures protected from parasitism by CTLA-4 blockade. These results indicate that the negative costimulatory receptor CTLA-4 is critically involved in TGF-β production and in intracellular parasite replication seen in murine kalaazar.

Infection of BALB/c mice with Leishmania chagasi results in progressive increase of parasite burden in spleen, in spite of extensive T cell activation in situ. Explanted splenic CD4+ T cells showed decreased proliferation to anti-CD3, compared with controls, and no response to L. chagasi recombinant antigen Lcr1. Blockade of the negative costimulatory receptor CTLA-4 restored responses to anti-CD3 and induced vigorous responses to Lcr1. Blockade of B7-1, but not B7-2, also enhanced T cell responsiveness. CTLA-4 blockade completely restored activation-induced interleukin-2 secretion and increased interferon-gamma production. The effect, however, was not restricted to Th1 responses, since CTLA-4 blockade also enhanced antigen-induced interleukin-4 secretion. CTLA-4 blockade induced almost complete elimination of parasite burden in splenocyte cultures activated with anti-CD3 or Lcr1. These results indicate that CTLA-4 engagement by B7-1 plays an important role in maintaining unresponsiveness in CD4+ T cells in this model of chronic visceral leishmaniasis.

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