[No Title Available]

Abstract

Leishmaniasis are worldwide spread syndromes affecting approximately 12 million people in at least 88 countries. Among several distinct clinical manifestations, diffuse cutaneous leishmaniasis (DCL) is of pivotal importance due to its frequent unresponsiveness to chemotherapy. Its pathology is characterized by non-ulcerated nodular lesions affecting most part of the body surface due to signaling defects in the skin immune system during early infection which promote a specific anergy and the exacerbation of the disease. It has been shown that serine¤threonine-asssociated signaling pathways are altered in infected macrophages which in turn fail to overcome infection (reviewed in Reiner 1994, Olivier 1986. Protein tyrosine kinase (PTK) activation in parasitic diseases has been largely overlooked despite its outstanding importance in phagocytosis (Greenberg 1995) and microbicidal processes (e.g. Dong et al. 1993). The infection of mononuclear phagocytes by Leishmania plays an important role in the pathophysiology of leishmaniasis, since this cell type not only sustains the parasite but also takes part in the immune response. Alterations in macrophage signaling may lead to defective immune regulation (Tapia et al. 1996). Several receptor-ligand systems have been implicated in the macrophage invasion by Leishmania sp. (Mosser & Rosenthal 1993), but little is known about the cellular responses triggered by the parasite binding and their relative role in its uptake and intracellular survival, as well as how parasites manage to subvert signaling cascades and thus impair effective immune responses.Upon L. amazonensis binding to macrophage receptors an early and transient tyrosine phosphorylation of target proteins is triggered underneath the host cell plasma membrane. This response is restricted to contact areas between the promastigote flagellum and macrophage surface and participates in parasite uptake. Inhibition of phagocyte PTK activity by selective antagonists decreases both binding and uptake of parasites in a dose-dependent fashion (Martiny et al. 1996). The observations that virulent promastigotes induce a lower PTK activity than avirulent ones and that PTK inhibition diminishes NO release abrogating parasite killing strongly indicate that virulence factors might affect host cell PTK-mediated microbicidal mechanisms. Therefore this conceivable escape mechanism in leishmanial parasites was investigated in amastigote infection.Internalization of amastigotes was inhibited neither by the tyrosine kinase antagonists nor by staurosporine, a broad spectrum protein kinase inhibitor and no immunolabeling using the 4G10 anti-phosphotyrosine monoclonal antibody could be observed at the parasite attachment areas, unless they were fixed with paraformaldehyde or pretreated with sodium orthovanadate. The reversion of tyrosine phosphorylation was assessed by Western blotting, in macrophages challenged with amastigotes for 60 min. Kinetic studies revealed that tyrosine dephosphorylation took place within 45 min and was completed...