title: 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.
authors.affiliation: Institute für Medizinsche Mikrobiologie und Infektionsimmunologie, Freie Universität Berlin, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Ehlers
authors.forename: M E
authors.initials: ME
authors.lastname: Mielke
authors.forename: T
authors.initials: T
authors.lastname: Blankenstein
authors.forename: H
authors.initials: H
authors.lastname: Hahn
mesh_terms: D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D007156:Immunologic Memory; D007371:Interferon-gamma / Q000096:biosynthesis; D007376:Interleukin-2; D007378:Interleukins / Q000096:biosynthesis; D008088:Listeriosis / Q000276:immunology*; D008099:Liver / Q000378:metabolism*; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014611:Vaccination
chemical_list: D016207:Cytokines; D007376:Interleukin-2; D007378:Interleukins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D015375:Receptors, Interleukin-2; D014409:Tumor Necrosis Factor-alpha; D016173:Macrophage Colony-Stimulating Factor; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor