The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated

Richard S. Larson, Margaret L. Hibbs, Timothy A. Springer

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Abstract

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 αβ heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the α and β subunit cDNAs. Immunoprecipitation studies demonstrated that the α and β subunits were associated in heterodimers. The α or β subunit was expressed at lower levels after transfection with the α or β subunit cDNA alone. Cotransfection of the α and β subunit cDNAs, but not transfection of α or β 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.

Original languageEnglish
Pages (from-to)359-367
Number of pages9
JournalMolecular Biology of the Cell
Volume1
Issue number4
DOIs
Publication statusPublished - 1 Jan 1990

Cite this

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title = "The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated",
abstract = "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 αβ heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the α and β subunit cDNAs. Immunoprecipitation studies demonstrated that the α and β subunits were associated in heterodimers. The α or β subunit was expressed at lower levels after transfection with the α or β subunit cDNA alone. Cotransfection of the α and β subunit cDNAs, but not transfection of α or β 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.",
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The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated. / Larson, Richard S.; Hibbs, Margaret L.; Springer, Timothy A.

In: Molecular Biology of the Cell, Vol. 1, No. 4, 01.01.1990, p. 359-367.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated

AU - Larson, Richard S.

AU - Hibbs, Margaret L.

AU - Springer, Timothy A.

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N2 - 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 αβ heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the α and β subunit cDNAs. Immunoprecipitation studies demonstrated that the α and β subunits were associated in heterodimers. The α or β subunit was expressed at lower levels after transfection with the α or β subunit cDNA alone. Cotransfection of the α and β subunit cDNAs, but not transfection of α or β 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.

AB - 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 αβ heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the α and β subunit cDNAs. Immunoprecipitation studies demonstrated that the α and β subunits were associated in heterodimers. The α or β subunit was expressed at lower levels after transfection with the α or β subunit cDNA alone. Cotransfection of the α and β subunit cDNAs, but not transfection of α or β 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.

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