B-Cell Homotypic Adhesion Through Exon-A Restricted Epitopes of CD45 Involves LFA-l/ICAM-1, ICAM-3 Interactions, and Induces Coclustering of CD45 and LFA-1 zyxw By zyxwvutsrqp Juan M. Zapata, Miguel R. Campanero, Monica Marazuela, Francisco Sanchez-Madrid, and Manuel 0. de Landazuri Lymphocyte interactions with other leukocytes and other cell types, as well as with components of the extracellular matrix, are one of the key steps in the immune response. Three novel monoclonal antibodies (MoAbs) have been pro- duced and selected for their ability t o induce intercellular adhesion in B cells. These three MoAbs immunoprecipitated a polypeptide of zyxwvutsrq 220 kD, displaying specific phosphotyrosine phosphatase activity that has been identified as CD45. These MoAbs recognize epitopes located on the alternative spliced exon-A+ncoded region of CD45. These epitopes are of poly- peptidic nature, but they can be masked by additionof car- bohydrate during CD45 biosynthesis. Interestingly enough, CD45 epitopes recognized by these MoAbs appeared to be selectively expressed on both peripheral blood and tonsillar B lymphocytes as well as on peripheral blood natural killer (NK) cells. CD45-mediated intercellular adhesion was abro- gated upon incubation with anti-leukocyte function-associ- zyxwvuts D45 IS A MEMBRANE glycoprotein with intrinsic phosphotyrosine phosphatase activity that is expressed specifically on nucleated hematopoietic cells."4 Several iso- forms of this molecule have been described that are mainly generated by the alternative splicing of exons 4, zyxwvuts 5, and 6 (also named exons A, B, and C, respectively) of a transcript produced by a single gene. Additional antigenic variability of the CD45 molecules is achieved by glycosylation events. There are many potential sites for both 0- and N-glycosyla- tion located on the extracellular region of CD45 that are differentially This complex antigenic variability of the CD45 molecules is of great importance because of the existence of a tight regulation of the expression of the differ- ent CD45 isoforms during leukocyte development and lym- phocyte activation."3.'" CD45 functions as a lymphocyte membrane receptor capa- ble of transducing signals via their phosphotyrosine phospha- tase domains.'-4 In this regard, evidence suggesting that CD45RO is a ligand for CD22 has been reported." Neverthe- less, although colateral associations between CD45 and a number of T-cell accessory molecules such as T-cell receptor (TCR), Thy-l, CD2, CD4, and CD8 have been described,' no additional evidence of other putative ligands for this or other CD45 isoforms has been obtained. Recently, it has been shown that specific CD45 isoforms could differentially regulate TCR signaling." However, although evidence has been described indicating that tyrosine phosphorylation of CD45 by p5OCsk kinase is involved in the activation of CD45 phosphatase activity,13 the identity of the mechanism in- volved in the regulation of the phosphatase activity of CD45 remains Several studies on the function of CD45 in T lymphocytes support a crucial role of CD45 in T-cell activation and devel- opment.2.~.~n.~4 Recent evidence also suggests that CD45 acts as a regulatory molecule that controls B-cell f ~ n c t i o n . ~ In this regard, CD45 has been involved in the regulation of B- cell proliferation triggered by anti-IgM or anti-CD40 mono- clonal antibody (MoAb).""' Moreover, a recent report'" C zyxw Blood, Vol 86, No 5 (September l), 1995: pp 1861-1872 ated antigen 1 (anti-LFA-l), intercellular cell adhesion mole- cule 1 (ICAM-l), and ICAMJ MoAbs, thus indicating that this phenomenon involved both LFA-l/ICAM-l and LFA-l/ICAM- z 3 cell adhesion pathways. Moreover, CD45-mediated cell ag- gregation was also inhibited by preincubation with some conventional anti-CD45 MoAbs. Interestingly, the triggering of cell aggregation through CD45 induced membrane surface relocation of CD45 and LFA-1 molecules, with both of them colocalizing at cell-cell contact areas of B-cell aggregates. Studies with inhibitors of both phosphotyrosine phospha- tase and tyrosine kinase activities suggest that CD45 phos- photyrosine phosphatase activity could be involved in CD45- mediated cell aggregation. Taken together, theseresults support the notion that CD45 is a key molecule in the regula- tion of LFA-l-mediated cell-cell interactions. zyx 0 1995 zyxwv by The American Society of Hematology. shows that CD45 phosphatase activity is elevated during mitosis, thus supporting the notion that CD45 could regulate cell division. CD45 appears also to be involved in the regula- tion of the B-cell antigen receptor function."," Interestingly, using mice in which exon C6 has been targeted by homolo- gous recombination, Kishihara et a]' showed that, despite the apparently normal B-cell development in these mice, signaling through the B-cell antigen receptor was abrogated. Nevertheless, evidence indicating that CD45 is implicated in the regulation of isotype switching has been ~ b t a i n e d . ' ~ ~ ~ ~ A recent report" pointed out that in vivo administration of MoAb against a B-cell-restricted CD45 epitope abrogated the B-cell response to a T-dependent antigen, thus further supporting the important role of CD45 in B-cell function. One of the key steps in the immune response is the regula- tion of leukocyte interactions. The correct establishment of cell-cell and cell-extracellular matrix interactions is essential for the proper development and function of leukocyte^.'^ We report herein that CD45 is implicated in the regulation of B- cell-cell interactions. We found that CD45-mediated B-cell homotypic adhesion was dependent on the leukocyte func- From the Servicio de Inmunologia and Servicio de Endocrino- logia, Hospital de la Princesa, Universidad Autcinoma de Madrid, Madrid, Spain. Submitted November 15, 1995; accepted April 21, 1995. Supported by grants from INSALUD (Fondo de Investigaciones Sanitarias 94/1695) and Comunidad Autbnoma de Madrid (CAM 028/92). J.M.Z. is the recipient of a postdoctoral fellowship from the Comunidad Autrinoma de Madrid. Address reprint requests to Manuel 0. de Landdzuri, MD, Servicio de Inmunologia, Hospital de la Princesa, C/Diego de Lebn 62, Ma- drid 28006, Spain. The publication costsof this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with zyxw 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/8605-0001$3.00/0 1861 Downloaded from http://ashpublications.org/blood/article-pdf/86/5/1861/617011/1861.pdf by guest on 08 December 2021