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Cytokine Profiles in Primary and Secondary Pulmonary Granulomas of Guinea Pigs with Tuberculosis

¹ Department of Microbial and Molecular Pathogenesis, Texas A&M University System Health Science Center, College Station, Texas; and ² Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas

Correspondence and requests for reprints should be addressed to Lan H. Ly, Texas A&M Health Science Center, Microbial and Molecular Pathogenesis, 463 Reynolds Medical Building, College Station, TX 77843. E-mail: lhly{at}medicine.tamhsc.edu

The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-β, with some IFN- and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF- mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-β mRNA with some IL-12p40 and IFN- mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally "vaccinated" by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF- mRNA in unimmunized animals, and mostly TGF-β mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.

Key Words: guinea pig • vaccine • tuberculosis • granuloma • cytokine

CLINICAL RELEVANCE The studies presented here extend our knowledge of granuloma formation and hematogenous reseeding in a highly relevant animal model, as well as add to our knowledge base of the mechanisms by which the bacillus Calmette-Guérin vaccine exerts its protective effects.