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Originally published online as doi:10.1189/jlb.0608354 on June 29, 2009

Published online before print June 29, 2009
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(Journal of Leukocyte Biology. 2009;86:989-998.)
© 2009 Society for Leukocyte Biology

Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages

Célio X. C. Santos*,1, Beatriz S. Stolf{dagger}, Paulo V. A. Takemoto*, Angélica M. Amanso*, Lucia R. Lopes{ddagger}, Edna B. Souza§, Hiro Goto§,2 and Francisco R. M. Laurindo*,2

* Vascular Biology Laboratory, Heart Institute (InCor), and
§ Tropical Medicine Institute, School of Medicine, and Departments of
{dagger} Parasitology and
{ddagger} Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

1. Correspondence: Heart Institute (InCor), University of São Paulo School of Medicine, Vascular Biology Laboratory, Av. Eneas Carvalho Aguiar, 44-Annex II, 9th Floor, CEP 05403-000; São Paulo, Brazil. E-mail: celioxcs{at}yahoo.com.br

ABSTRACT

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss- or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi.

Key Words: thiols • chaperone • PDI • Nox • parasite • infection