Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity.

Abstract

The anamnestic response to infection with Listeria monocytogenes is characterized by the rapid elimination of normally lethal doses of bacteria and accelerated granuloma formation. These phenomena are mediated by listeria-specific memory T cells within the first 24 h after reinfection. In order to elucidate the mechanisms operative during this decisive phase of infection, we conducted a comprehensive kinetic and quantitative analysis of cytokine gene expression in the livers of naive and immune mice. Organs were removed at 30 min, and 1, 2, 6, and 24 h after primary and secondary infections, and PCR3-assisted messenger RNA (mRNA) amplification was performed on matched samples using primers specific for IL-1 beta, IL-6, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, IL-10, IL-4, IL-2, IL-3 and I1-2Rp55. The cytokine pattern characteristic of secondarily infected animals differed qualitatively by the expression of mRNA for IL-2, IL-2Rp55, IL-3, and IL-4, demonstrating the accumulation and activation of specific T cells in the livers as early as 1 to 2 h after reinfection. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection almost completely abrogated the differentiated cytokine profile typical of the anamnestic response. Using competitive PCR for semiquantitative determination of mRNA levels, the amount of IL-1 beta and IL-6 mRNA was found to be very similar during primary and secondary infection, whereas TNF-alpha mRNA was found to be increased by approximately 10-fold 2 h and IFN-gamma mRNA by approximately 50 to 100-fold 6 h after reinfection when compared with a primary challenge. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection resulted in a substantial (approximately 10-fold) decrease in IFN-gamma mRNA expression. To correlate these findings with cytokine secretion, spleen cells from naive and immune as well as normal and CD4+ and CD8+ cell depleted mice infected 6 h previously were cultured for 48 h, and supernatants were analyzed for the amount of the above mentioned cytokines. Semiquantitative PCR-assisted mRNA amplification is demonstrated to be a superior tool in dissociating the mediators of innate resistance from those operative in protective immunity and granuloma formation.