CROSS-LINKING OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II MOLECULES BY STAPHYLOCOCCAL-ENTEROTOXIN-A SUPERANTIGEN IS A REQUIREMENT FOR INFLAMMATORY CYTOKINE GENE-EXPRESSION

Staphylococcal enterotoxin A (SEA) has two distinct binding sites for major histocompatibility complex (MHC) class II molecules. The asparti c acid located at position 227 (D227) in the COOH terminus of SEA is o ne of the three residues involved in its interaction with the DR beta chain, whereas the phenylalanine 47 (F47) of the NH2 terminus is criti cal for its binding to the DR alpha chain. Upon interaction with MHC c lass II molecules, SEA triggers several cellular events leading to cyt okine gene expression. In the present study, we have demonstrated that , contrary to wild-type SEA, stimulation of the THP1 monocytic cell li ne with SEA mutated at position 47 (SEA(F47A)) or at position 227 (SEA (D227A)) failed to induce interleukin 1 beta and tumor necrosis factor -or messenger RNA expression. Pretreatment of the cells with a 10-fold excess of either SEA(F47A) or SEA(D227A) prevented the increase in cy tokine messenger RNA induced by wild-type SEA. However, cross-linking of SEA(F47A) or SEA(D227A) bound to MHC class II molecules with F(ab') (2) anti-SEA mAb leads to cytokine gene expression, whereas cross-link ing with F(ab) fragments had no effect. Taken together, these results indicate that cross-linking of two MHC class II molecules by one singl e SEA molecule is a requirement for cytokine gene expression.