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Infect. Immun. doi:10.1128/IAI.00858-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Guinea pig neutrophils infected with Mycobacterium tuberculosis produce cytokines which activate alveolar macrophages in non-contact cultures

Department of Microbial and Molecular Pathogenesis, The Texas A&M University System Health Science Center, College Station, TX 77843-1114

^* To whom correspondence should be addressed. Email: Ksawant{at}medicine.tamhsc.edu.

	Abstract

The early influx of neutrophils to the site of infection may be an important step in host resistance against Mycobacterium tuberculosis. In this study, the effect of M. tuberculosis infection on the ability of guinea pig neutrophils to produce IL-8 (CXCL8) and TNF-, and to activate alveolar macrophages, was studied. Neutrophils and alveolar macrophages were isolated from naïve guinea pigs, cultured together or alone, and infected with virulent M. tuberculosis for 3, 12 and 24 h. IL-8 protein production in co-cultures, as measured by ELISA, was found to be additive at 24 h and significantly greater in M. tuberculosis-infected co-cultures as compared to uninfected co-cultures and the infected neutrophils or macrophages alone. The IL-8 mRNA levels, determined by real-time RT-PCR, were elevated at 24 h in infected co-cultures and infected cells alone. In order to elucidate the contribution of neutrophils and their soluble mediators to the activation of alveolar macrophages, neutrophils and alveolar macrophages were cultured in a contact-independent manner using a Transwell insert system. Neutrophils were infected with virulent M. tuberculosis in the upper well, and alveolar macrophages were cultured in the lower well. Release of hydrogen peroxide from alveolar macrophages exposed to soluble products from infected neutrophils was significantly increased compared to unexposed alveolar macrophages. Significant up-regulation of IL-1 and TNF- mRNA levels was observed in alveolar macrophages at 24 and 30 h respectively, as compared to cells not exposed to soluble neutrophils products. Treatment with anti-gpTNF- polyclonal antibody completely abrogated the response of alveolar macrophages to neutrophil products. This suggests that TNF- produced by infected neutrophils may be involved in activation of alveolar macrophages, and hence may contribute to the containment of M. tuberculosis infection during the early period of infection.