EVIDENCE FOR A DITHIOL-ACTIVATED SIGNALING PATHWAY IN NATURAL-KILLER-CELL AVIDITY REGULATION OF LEUKOCYTE FUNCTION ANTIGEN-1 - STRUCTURAL REQUIREMENTS AND RELATIONSHIP TO PHORBOL ESTER-TRIGGERED AND CD16-TRIGGERED PATHWAYS

Dithiothreitol (DTT) activation of the adhesive function of several di fferent integrins suggests the existence of a common DTT-sensitive int egrin regulatory element. Ui11/E3, a natural killer (NK) cell-resistan t murine target cell line genetically engineered to constitutively exp ress human intercellular adhesion molecule-1 (ICAM-1; CD54) was used i n a flow cytometric experimental model to evaluate DTT effects on the NK cell integrin adhesion molecule, leukocyte function antigen-1 (LFA- 1;alpha L beta 2, CD11a/CD18). DTT and several structurally related di thiol compounds elicited a dramatic elevation in conjugate formation t hat was dependent on target cell ICAM-1 expression, was blocked by LFA -1 alpha L or beta 2 chain-specific antibodies, and occurred in the ab sence of Ui11/E3 target cell exposure to DTT or quantitative changes i n NK cell membrane LFA-1 expression. This avidity modulation of LFA-1 by DTT required actin polymerization, was abrogated by the protein kin ase C inhibitor calphostin C, involved activities of calyculin A- and okadaic acid-sensitive serine/threonine protein phosphatases PP-1 and/ or PP-2A but not geldanamycin-sensitive tyrosine kinases, and differed with respect to kinetics and enzyme inhibitor sensitivity from LFA-1 activation promoted by cross-linking of NK cell CD16 or phorbol ester treatment. A key structural feature of DTT was the presence of two thi ol groups, both reduced but not physically adjacent as in the nonstimu latory dithiol, 2,3-dimercaptopropanol. LFA-1 activation was not becau se of DTT chelation of Ca2+ or Zn2+. Immunoblotting studies identified multiple NK cell plasma membrane-associated proteins to be reduced by DTT under LFA-l-activating conditions, but similar effects were also promoted by reducing agent treatments that failed to alter adhesive fu nction. Direct chemical modification of LFA-1 seemed an unlikely basis of activation because (1) DTT activated LFA-1 in HSB2 T cells without detectable disulfide reduction in LFA-1 alpha L or beta 2 chains immu noprecipitated from these cells and (2) DTT treatment of NK cells did not hinder binding of KIM127 and KIM185, monoclonal antibodies that re cognize epitopes in the potentially DTT-susceptible cysteine-rich doma in of the beta 2 chain. Thus, these results extended the range of DTT- activatible integrins to include NK cell LFA-1 and characterized for t he first time signaling-associated enzymatic activities involved in DT T activation of NK cell LFA-1. Moreover, they suggested that structura l features of DTT, particularly SH group spatial positioning, are impo rtant in LFA-activation for reasons other than cation chelation or dis ulfide reduction. Despite apparent differences in some aspects of DTT activation of different integrins, the actin-dependent cytoskeleton or membrane skeleton was implicated as a likely candidate for a common l ocus of DTT action. (C) 1995 by The American Society of Hematology.