CO-STIMULATION OF HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS WITH IL-2 AND ANTI-CD3 MONOCLONAL-ANTIBODIES INDUCES PHOSPHORYLATION OF CREB

Phosphorylation of the cAMP-response element binding protein CREB with in 1 h of CD2 but not CD3 cross-linking of human PBMC was recently dem onstrated. The absence of P-CREB following CD3 cross-linking was unexp ected, as other laboratories reported increased phosphorylation of CRE B following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in p rimary lymphocytes. Therefore, P-CREB was evaluated following co-stimu lation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 d id not further augment phosphorylation of CREB following CD2 cross-lin king. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of I L-2/CD3 co-stimulation was observed. Phosphorylation was not associate d with the induction of cAMP, and inhibition of PKA signaling had no e ffect on P-CREB. Consistent with signal transduction through p56(lck) or p59(fyn), inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further incr eased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulate d cells not pretreated with a PKC inhibitor. In contrast to previous s tudies performed in the absence of exogenous IL-2, no increase in bind ing of CREB to a P-32-labeled oligonucleotide probe was observed by el ectrophoretic mobility shift assay. These data suggest that the IL-2 a nd CD3 signaling pathways provide a necessary and co-operative stimulu s promoting phosphorylation of CREB following receptor cross-linking. (C) 1998 Elsevier Science B.V. All rights reserved.