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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- and CD16-triggered pathways
BS Edwards, MS Curry, EA Southon, AS Chong and LH Graf
Institute for Basic and Applied Medical Research, Lovelace Institutes,
Albuquerque, NM 87108, USA.
Dithiothreitol (DTT) activation of the adhesive function of several
different integrins suggests the existence of a common DTT-sensitive
integrin regulatory element. Ui11/E3, a natural killer (NK) cell- resistant
murine target cell line genetically engineered to constitutively express
human intercellular adhesion molecule-1 (ICAM-1; CD54) was used in 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 dithiol compounds
elicited a dramatic elevation in conjugate formation that 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 absence of Ui11/E3 target
cell exposure to DTT or quantitative changes in NK cell membrane LFA-1
expression. This avidity modulation of LFA-1 by DTT required actin
polymerization, was abrogated by the protein kinase 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 thiol groups, both reduced but not
physically adjacent as in the nonstimulatory dithiol, 2,3-
dimercaptopropanol. LFA-1 activation was not because of DTT chelation of
Ca2+ or Zn2+. Immunoblotting studies identified multiple NK cell plasma
membrane-associated proteins to be reduced by DTT under LFA-1- activating
conditions, but similar effects were also promoted by reducing agent
treatments that failed to alter adhesive function. 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 immunoprecipitated from these cells and
(2) DTT treatment of NK cells did not hinder binding of KIM127 and KIM185,
monoclonal antibodies that recognize epitopes in the potentially
DTT-susceptible cysteine-rich domain of the beta 2 chain. Thus, these
results extended the range of DTT-activatible integrins to include NK cell
LFA-1 and characterized for the first time signaling-associated enzymatic
activities involved in DTT activation of NK cell LFA-1. Moreover, they
suggested that structural features of DTT, particularly SH group spatial
positioning, are important in LFA-activation for reasons other than cation
chelation or disulfide reduction.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 86,
Issue 6,
pp. 2288-2301,
09/15/1995
Copyright © 1995 by The American Society of Hematology
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