title: 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.
abstract: 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)
authors.affiliation: Institute for Basic and Applied Medical Research, Lovelace Institutes, Albuquerque, NM 87108, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Edwards
authors.forename: M S
authors.initials: MS
authors.lastname: Curry
authors.forename: E A
authors.initials: EA
authors.lastname: Southon
authors.forename: A S
authors.initials: AS
authors.lastname: Chong
authors.forename: L H
authors.initials: LH
authors.lastname: Graf
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D004229:Dithiothreitol / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D007694:Killer Cells, Natural / Q000187:drug effects / Q000502:physiology*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000096:biosynthesis* / Q000235:genetics; D008387:Marine Toxins; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000378:metabolism; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010084:Oxidation-Reduction; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017452:Receptors, IgG / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000199:Actins; D002413:Cations, Divalent; D004988:Ethers, Cyclic; D016169:Lymphocyte Function-Associated Antigen-1; D008387:Marine Toxins; D008565:Membrane Proteins; D009281:Naphthalenes; D009363:Neoplasm Proteins; D010080:Oxazoles; D017452:Receptors, IgG; D013439:Sulfhydryl Reagents; D018799:Intercellular Adhesion Molecule-1; D019319:Okadaic Acid; C059041:calyculin A; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D004229:Dithiothreitol