Perforin expression in human peripheral blood mononuclear cells. Definition of an IL-2-independent pathway of perforin induction in CD8+ T cells.
Perforin gene expression upon in vitro stimulation was studied at the mRNA level in normal human PBMC and in subpopulations. Freshly isolated PBMC express low levels of perforin mRNA. Increased perforin expression is rapidly induced by the calcium ionophore A23187 and by rIL-2. Phorbolesters (PMA), by comparison, are poor inducers of perforin RNA. Perforin induction by Ca-ionophore, unlike granzyme 2 and IL-2 induction, did not synergize with phorbolesters in PBMC or in purified T cells. Instead, perforin mRNA induction by A23187 in purified T cells requires the presence of adherent cells. Ca-ionophore plus adherent cell-induced perforin occurred in CD8+ T cells and was abolished by depletion of CD8+ T cells but not by depletion of CD4+ T cells. Adherent cells alone did not express perforin under any condition. Perforin mRNA induction by both A23187 and by rIL-2 is independent of de novo protein synthesis. The half-life of perforin mRNA induced by either stimulus is approximately 100 min. Cyclosporin A completely abrogates perforin induction by A23187 but only slightly inhibits the effect of rIL-2 on perforin mRNA expression. These data show that A23187 activates perforin gene expression in CD8+ cells by an IL-2-independent pathway and that the molecular mechanism of perforin expression may be different from the one induced by IL-2. Granzyme 2 (human leukocyte protease-HLP, homologous to murine granzyme B) mRNA expression was studied in comparison to perforin. Granzyme 2 in contrast to perforin responds to the synergistic action of phorbolester and Ca-ionophore in PBMC. In addition, the kinetics of the induction of granzyme and perforin mRNA, by various signals are different. Our data suggest that situations in vivo may exist that allow perforin expression in CD8+ cells in the absence of cytokines by a combination of Ca signals and accessory receptor ligation. The same signals may not be sufficient for granzyme 2 expression in any T cell subpopulation.
Lu
P
P
Department of Microbiology and Immunology, University of Miami School of Medicine, FL 33101.
Garcia-Sanz
J A
JA
Lichtenheld
M G
MG
Podack
E R
ER
D001483:Base Sequence; D016827:CD8 Antigens / Q000032:analysis; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002448:Cell Adhesion; D016572:Cyclosporine / Q000494:pharmacology; D004640:Emetine / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D053804:Granzymes; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000502:physiology*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics; D016176:T-Lymphocyte Subsets / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D016827:CD8 Antigens; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger; D054353:Perforin; D000001:Calcimycin; D016572:Cyclosporine; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D004640:Emetine