Recombinant Guinea Pig Tumor Necrosis Factor Alpha Stimulates the Expression of Interleukin-12 and the Inhibition of Mycobacterium tuberculosis Growth in Macrophages

Abstract

ABSTRACT Tumor necrosis factor alpha (TNF-α) plays an important role in the host immune response to infection with the intracellular pathogen Mycobacterium tuberculosis . It is essential for the formation of protective tuberculous granulomas and regulates the expression of other cytokines which contribute to a protective immune response. Interleukin-12 (IL-12) is known to promote a Th1 response, which is essential for antimycobacterial resistance. Recombinant guinea pig TNF-α (rgpTNF-α) protein (17 kDa) was purified, and its bioactivity was confirmed by its cytotoxicity for L929 fibroblasts. High titers of polyclonal anti-gpTNF-α antibody were obtained by immunization of rabbits. Resident alveolar and peritoneal macrophages were isolated from guinea pigs and infected with either the H37Ra or H37Rv strain of M. tuberculosis . The mRNA levels for TNF-α and IL-12 p40 were measured using real-time PCR. IL-12 p40 mRNA was up-regulated in a dose-dependent manner by rgpTNF-α alone. In infected macrophages, a lower dose of rgpTNF-α intensified the mRNA levels of TNF-α and IL-12 p40. However, higher doses of rgpTNF-α suppressed TNF-α and IL-12 p40 mRNA. The antimycobacterial activity of macrophages was assessed by metabolic labeling of M. tuberculosis with [ 3 H]uracil. Resident alveolar and peritoneal macrophages treated with anti-gpTNF-α antibody to block endogenous TNF-α exhibited increased intracellular mycobacterial growth. These data suggest that the dose of TNF-α is crucial to the stimulation of optimal expression of protective cytokines and that TNF-α contributes to the control of mycobacterial replication to promote host resistance against M. tuberculosis .