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Early steps in interferon-gamma production: possible involvement of Ca2+-calmodulin-dependent enzymes
G Antonelli1, V Ruggiero, A Cefaro
1Institute of Virology, University of Rome La Sapienza, Italy.
Abstract:
A23187 in combination with phorbol myristate acetate (PMA) strongly induces production of interferon-gamma (IFN-gamma) by human peripheral blood mononuclear cells (PBMC) and even by murine PBMC, which respond poorly to A23187 alone. Macrophage depletion of PBMC strongly reduces IFN-gamma production induced by several mitogens, but does not affect IFN-gamma production induced by A23187 and PMA. In addition the same stimuli are able in combination to induce strong amounts of IFN-gamma, even in the Jurkat T cell line. The protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and the calmodulin antagonist N-(6-aminoehexyl)-5-chloro-1-naphthalenesulfonamide (W-7) were examined for their ability to inhibit IFN-gamma production induced by PMA and A23187. At concentrations near the Ki for protein kinase C, H-7 failed to inhibit PMA- and A23187-induced IFN-gamma production. In contrast, W-7 at low concentrations inhibited IFN-gamma production induced by the same stimuli. In addition OAG, which is known to directly activate protein kinase C, failed to act synergistically with A23187 in the induction of IFN-gamma. On the basis of these results we propose that A23187 and PMA may mimic the early steps of lymphocyte activation, without the requirement of macrophage, bypassing antigen-, or lectin-induced signal. Our results suggest that Ca2+-calmodulin-dependent reactions other than protein kinase C activation may be essential for IFN-gamma production, at least at level of the producing cells.
Insights
The combination of A23187 and PMA strongly induces interferon-gamma (IFN-gamma) production in human and murine cells, independent of macrophages. This suggests calcium-calmodulin pathways, not protein kinase C, are crucial for IFN-gamma production.
Area of Science:
- Immunology
- Cellular Biology
Background:
- Interferon-gamma (IFN-gamma) is a critical cytokine in immune responses.
- Understanding the signaling pathways that induce IFN-gamma production is essential for immune modulation.
Purpose of the Study:
- To investigate the signaling mechanisms underlying IFN-gamma production induced by A23187 and phorbol myristate acetate (PMA).
- To determine the role of protein kinase C and Ca2+-calmodulin pathways in this process.
Main Methods:
- Stimulation of human peripheral blood mononuclear cells (PBMC) and Jurkat T cells with A23187 and PMA.
- Macrophage depletion experiments.
- Inhibition studies using protein kinase C inhibitor H-7 and calmodulin antagonist W-7.
- Assessment of IFN-gamma production.
Main Results:
- A23187 and PMA robustly induced IFN-gamma in human and murine PBMC, and in Jurkat T cells.
- Macrophage depletion did not affect A23187/PMA-induced IFN-gamma production.
- The calmodulin antagonist W-7 inhibited IFN-gamma production, while the protein kinase C inhibitor H-7 did not.
- Direct activation of protein kinase C with OAG did not synergize with A23187.
Conclusions:
- A23187 and PMA can mimic early lymphocyte activation signals, bypassing the need for macrophages or antigen/lectin.
- Ca2+-calmodulin-dependent pathways, distinct from protein kinase C activation, appear essential for IFN-gamma production in the producing cells.