Faculty Bibliography

The phagocytic macrophage plays a critical role in host immune responses to microbial infection, and represents a major source of inflammatory and growth cytokines. Intramacrophage infection by the protozoan parasite Leishmania donovani results in increased viability of the host cell in the absence of exogenous growth factor. We demonstrate that infection of bone marrow-derived macrophages (BMMs) by L. donovani promastigotes or treatment of BMMs with lipophosphoglycan LPG, the major surface molecule of the promastigote, inhibits apoptosis in the macrophage induced by the removal of macrophage (M)-CSF. This effect was also achieved by supernatants collected from L. donovani-infected macrophages, implicating the elaboration of a soluble factor by infected cells as the mediator of this inhibition. To identify candidate factors, reverse transcription PCR was employed to characterize the mRNA cytokine profile of infected macrophages. L. donovani infection of BMMs was found to induce gene expression for granulocyte-macrophage CSF, TNF-alpha, TGF-beta, and IL-6, but not M-CSF or IL-1 beta. Of the cytokines induced by L. donovani, rTNF-alpha and recombinant granulocyte-macrophage CSF were shown to inhibit apoptosis of BMMs induced by the removal of M-CSF. The amount of these cytokines in L. donovani-infected cell supernatants was quantified by ELISA. The mechanism by which L. donovani may inhibit apoptosis is discussed.

Bone marrow-derived macrophages rapidly die in the absence of macrophage growth factor (M-CSF). However, as demonstrated here, bone marrow-derived macrophages infected with Leishmania donovani exhibit increased viability in the absence of exogenous growth factor. Forty-eight hours after inoculation with promastigotes or amastigotes, infected cell cultures contained 180 and 95% more cells, respectively, than control cultures. This effect was specific to Leishmania infection, as uptake of latex beads or avirulent promastigotes by macrophages did not enhance cell viability. L. donovani-infected macrophages also displayed increased phagocytic capacity, as compared with control macrophages and macrophages grown continuously in M-CSF-containing medium. Supernatants collected from infected cells elaborated a factor(s) that enhanced macrophage viability but did not stimulate macrophage DNA synthesis. This activity of L. donovani-infected cell-conditioned medium could be abrogated by preincubation of macrophages with cycloheximide before inoculation with the parasite, implying that macrophage protein synthesis is required for the elaboration of this factor(s).

Leishmania donovani is an obligate intracellular protozoan which residues and multiples in macrophages. The molecular basis for this host-parasite interaction is poorly understood. Targeting a signal transduction pathway in the macrophage would allow this parasite to manipulate cellular gene expression, and this may aid in ensuring its survival. We demonstrate that in macrophages infected with L. donovani for 18 h, c-fos gene expression mediated through protein kinase A was unaffected under conditions where there was an impairment of protein kinase C (PKC)-mediated c-fos gene expression. This selective impairment of PKC-mediated c-fos gene expression was substantially augmented in macrophages put in contact with L. donovani promastigotes or amastigotes for only 1 h. Treatment of macrophages with L. donovani-conditioned media was not sufficient to significantly impair signal transduction. These data revealed that L. donovani selectively impaired the transmission of information from the cell surface to the nucleus and that this effect is induced very soon after macrophage-parasite contact. The biologic significance of this altered signal transduction in the macrophage with respect to infection with L. donovani was then examined by treating macrophages with various protein kinase inhibitors prior to infection with amastigotes. Macrophages that were treated with PKC inhibitors demonstrated an increase in the initial uptake of the parasite and carried heavier infection levels than did controls. In contrast, treatment of macrophages with an inhibitor of calmodulin-dependent protein kinase (CaM-PK) did not show significant differences in the initial uptake of parasite, but prolonged impairment of CaM-PK resulted in a decrease in the level of macrophage infection. Further experiments revealed that promastigote proliferation was severely impaired by the CaM-PK inhibitor but not any of the other inhibitors.