Differential Regulation of T Cell Receptor-Mediated Th1 Cell IFN- γ Production and Proliferation by Divergent cAMP-Mediated Redox Pathways

Abstract

Culture of an H-2^s-restricted, bovine myelin basic protein (BMBP)-specific murine Th1 clone with the adenyl cyclase agonist forskolin (FSK) or isobutylmethylxanthine (IBMX), an inhibitor of cAMP catabolism, before culture with anti-CD3 or BMBP and antigen-presenting cells (APC) suppressed antigen or anti-CD3-induced proliferation and production of interferon-γ (IFN-γ). Other H-2^s-derived or H-2^b-derived clones specific for BMBP or keyhole limpet hemocyanin (KLH) were similarly affected. FSK did not affect the expression of CD4 or the T cell receptor(TCR) but did diminish levels of the phosphorylated (activated) mitogen-activated protein (MAP) kinases early response kinase-1 (ERK-1) and ERK-2. Immunoblotting of lysates from an FSK-treated Th1 clone with antibodies to a carboxy-terminal epitope of p56 ^lck , a signal transduction enzyme upstream from ERK-1 and ERK2, did not detect p56 ^lck unless the lysates were reduced prior to electrophoresis. Immunoblotting of nonreduced lysates with antibodies to an amino-terminal epitope demonstrated p56 ^lck with a lower apparent molecular weight, characteristic of oxidized proteins. Reduction restored the detection of p56 ^lck by anticarboxy-terminal p56 ^lck and to mobilities indistinguishable from controls detected by the antiamino-terminal p56 ^lck . N-acetylcysteine or catalase prevented FSK-induced suppression of antigen-induced proliferation and the loss of carboxy-terminal epitopes of p56 ^lck . An inhibitor of cAMP-dependent protein kinase A (PKA) or nitric oxide synthase (NOS) did not affect FSK-induced inhibition of antigen-induced proliferation. In contrast, inhibitors of PKA or NOS, but not catalase, prevented FSK-induced suppression of IFN-γ production. Moreover, immunoblots of lysates precipitated with anti-p56 ^lck , phosphotyrosine, or CD4 demonstrated that in FSK-treated, anti-CD3-stimulated cells, p56 ^lck is not associated with CD4 ζ chain, nor is p56 ^lck or ζ chain phosphorylated. In vitro kinase assays demonstrated that p56 ^lck from FSK-treated cells does not have kinase activity. Taken together, the results suggest that an elevation of intracellular cAMP (in the absence of antigen) creates an oxidative environment that oxidizes and inactivates p56 ^lck by an H₂O₂-dependent, PKA-independent mechanism and inhibits the production of IFN-γ by an NO, PKA-dependent mechanism. Thus, antigen-induced proliferation and IFN-γ production in a Th1 clone are controlled separately by different cAMP-dependent, redox-based mechanisms.