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Destiny and Intracellular Survival of Leishmania amazonensis in Control and Dexamethasone-treated Glial Cultures : Protozoa-specific Glycoconjugate Tagging and TUNEL Staining

Wagner Baetas-da-Cruz, Roger M. Macedo-Silva, Alessandra Santos-Silva, Andrea Henriques-Pons, Maria F. Madeira, Suzana Corte-Real¹ and Leny A. Cavalcante¹

Departmento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil (WB-d-C,AH-P,SC-R); Laboratório de Protozoologia, Escola Nacional de Saúde Pública, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil (RMM-S,MFM); and Laboratório de Neurobiologia do Desenvolvimento, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (AS-S,LAC)

Correspondence to: Dr Leny A. Cavalcante, Laboratório de Neurobiologia do Desenvolvimento, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil. E-mail: Lacav{at}abc.org.br

Leishmania amazonensis, an obligatory intracellular parasite, survives internalization by macrophages, but no information is available on the involvement of microglia. We have investigated microglia–protozoa interactions in mixed glial cultures infected with promastigote forms of L. amazonensis after lipopolysaccharide (LPS) or dexamethasone (DM) treatment. After 2 hr of exposure to parasites in control cultures, there was a small number of infected microglia (1%). Preincubation with LPS or DM led to 14% or 60% of microglial cells with attached parasites, respectively. DM treatment resulted in 39% of microglial cells with internalized parasites (controls or LPS-treated cells had 1%). Scanning electron micrographs showed numerous filopodia in DM-treated cells, whereas these projections were rarely observed in LPS-treated or control cells. DM treatment also affected the intramicroglial survival of Leishmania. In control cultures, internalized parasites, tagged with an anti-lipophosphoglycan (anti-LPG) antibody, showed fragmented DNA [terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL+)] after 4 hr of interaction, but changes seemed slightly delayed in DM-treated cultures. After 12 hr, there were no LPG+/TUNEL+ profiles in controls, whereas rare LPG+ profiles still persisted in DM-treated cells. Our results suggest that microglia are highly effective in the elimination of Leishmania and that the process can be effectively studied by LPG/TUNEL double labeling. (J Histochem Cytochem 52:1047–1055, 2004)

Key Words: microglia–protozoa • interactions • lipophosphoglycan • microglial cytotoxicity • lipopolysaccharide

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