The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated.
The functional activity of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes can be regulated by T-cell receptor (TCR) stimulation and pharmacologic agents. It was of interest to determine if functionally active LFA-1 could be reconstituted on a nonhematopoietic, LFA-1-negative cell line. We report the expression of LFA-1 and diethylaminoethyl (DEAE) Mac-1 alpha beta heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the alpha and beta subunit cDNAs. Immunoprecipitation studies demonstrated that the alpha and beta subunit was expressed in heterodimers. The alpha or beta subunit was expressed at lower levels after transfection with the alpha or beta subunit cDNA alone. Cotransfection of the alpha and beta subunit cDNAs, but not transfection of alpha or beta alone, was sufficient to reconstitute intercellular adhesion molecule-1 (ICAM-1) binding activity. Consistent with this observation, LFA-1 on the fibroblastoid cells possesses the activation epitope defined by the L16 monoclonal antibody (mAb). This epitope marks the conversion of LFA-1 from the low to high avidity state on peripheral blood T lymphocytes (PBLs) and is constitutively present on activated cell lines. In contrast to LFA-1 on leukocytes, the functional activity of LFA-1 on fibroblastoid cells was not influenced by phorbol ester treatment. Furthermore, the use of agents that interfere with intracellular signaling, a protein kinase C inhibitor, cAMP analogue, or the combination of a phosphodiesterase inhibitor and adenyl cyclase activator, did not affect the binding of COS cells expressing LFA-1 to purified ICAM-1.
Larson
R S
RS
Center for Blood Research, Harvard Medical School, Boston, MA 02115.
Hibbs
M L
ML
Springer
T A
TA
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D005347:Fibroblasts / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1; D016169:Lymphocyte Function-Associated Antigen-1 / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D000470:Alkaloids; D015815:Cell Adhesion Molecules; D000939:Epitopes; D016023:Integrins; D016169:Lymphocyte Function-Associated Antigen-1; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D018799:Intercellular Adhesion Molecule-1; D003994:Bucladesine; D004247:DNA; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate