The Journal of Immunology, Vol 146, Issue 2 648-655, Copyright © 1991 by American Association of Immunologists

ARTICLES

Leukocyte integrin P150,95 (CD11c/CD18) functions as an adhesion molecule binding to a counter-receptor on stimulated endothelium

SA Stacker and TA Springer
Department of Pathology, Harvard Medical School, Boston, MA 02115.

p150,95 is a member of the beta 2 family of integrins, which includes both LFA-1 and Mac-1. These molecules are known to play a role in the adhesion of lymphocytes, granulocytes, and monocytes to various cell types including vascular endothelium. p150,95 is presumed to have an adhesive function because of its structural relationship to the other beta 2 integrins and the ability of anti-p150,95 mAb to inhibit some myeloid cell interactions with tumor cells, endothelial cells, and other substrates. In an endeavor to demonstrate directly that p150,95 can act as an adhesion molecule, we raised a mAb (CBRp150/4G1) to the alpha subunit of p150,95, which allows for the purification of functional intact p150,95 heterodimers. The antibody was selected by using a high pH elution ELISA. The assay was designed to select for antibodies directed to the alpha-chain of p150,95, which could be readily dissociated from p150,95 under conditions of high pH and 2 mM MgCl2. p150,95 purified under these conditions with CBRp150/4G1- Sepharose could be immunoprecipitated by using antibodies to the alpha- and beta-chains of p150,95 indicating that the structural integrity of the heterodimer was preserved during purification and elution. Elution in the absence of divalent cations yielded primarily dissociated alpha and beta subunits. Other antibodies previously made to p150,95 alpha- chain such as SHCL3 were greatly reduced in their efficiency of yielding intact heterodimer under these conditions. Mapping of the epitopes by using chimeric molecules of p150,95/Mac-1 revealed that antibodies that react with the divalent cation sites of p150,95 are inferior for the purification of intact p150,95. The adhesive capacity of p150,95 was demonstrated by the specific binding of 18-h rIL-1 beta or LPS-stimulated endothelial cells to purified p150,95 absorbed to plastic microtiter plates. These results indicate that p150,95 can function independently as an adhesion molecule and that it can interact with a counter-receptor on stimulated endothelium.

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