Elsevier

Molecular Immunology

Volume 45, Issue 6, March 2008, Pages 1703-1711
Molecular Immunology

LFA-1-mediated leukocyte adhesion regulated by interaction of CD43 with LFA-1 and CD147

https://doi.org/10.1016/j.molimm.2007.09.032Get rights and content

Abstract

The activity of the lymphocyte-function associated antigen 1 (LFA-1; CD11a/CD18) must be tightly controlled during the onset of cellular immunity. It is well known that the sialoglycoprotein CD43 can influence LFA-1-mediated cell adhesion in an either anti- or pro-adhesive manner through mechanisms not well understood. By using a yeast-2-hybrid screen and co-immunoprecipitation we identified physical association of CD43 with two novel partners, LFA-1 itself and the Ig-family member CD147 (EMMPRIN, basigin), and characterized how these interactions are involved in LFA-1-mediated cell adhesion. Monoclonal antibodies (mAbs) to both CD43 and CD147 induced similar homotypic cell aggregation and adhesion of Jurkat T cells and U937 myeloid cells. Both CD43 and CD147 mAbs induced dynamic co-capping of LFA-1 together with the CD43 and the CD147 molecule to cell contact zones. However, in contrast to CD43, we were not able to co-immunoprecipitate LFA-1 with CD147, which indicates that CD43 interacts with CD147 and LFA-1 in two distinct but similarly reorganized complexes. Co-transfection of CD43 interfered with the CD147-induced cell adhesion and aggregation, and siRNA-mediated knock down of CD43 in human T cells resulted in enhanced LFA-1 activation induced via CD147 and also the T cell antigen receptor. These results indicate that triggering CD43 and the underlying signaling pathways enhance LFA-1 adhesiveness while CD43 also negatively regulates LFA-1 induction via other receptors by dynamic interaction with either LFA-1 or CD147.

Introduction

The lymphocyte-function associated antigen 1 (LFA-1; CD11a/CD18) is essential for the action of human leukocytes, including lymphocyte recirculation, migration into inflammatory sites and recognition of foreign antigens (Pribila et al., 2004). LFA-1 mediates a variety of homotypic and heterotypic cell adhesion events between leukocytes, antigen presenting cells (APCs), endothelial cells within blood vessels and other cells within tissues. The major ligands for LFA-1 are the intercellular adhesion molecules (ICAM)-1-3, with ICAM-1 (CD54) having the highest affinity (de Fougerolles et al., 1994). The LFA-1-mediated cellular contacts are highly dynamic and in most cases transient, therefore they are proposed to be a subject of tight regulation. Integrin avidity depends on modulation of integrin affinity and valency via “inside-out”-signals initiated by G protein-coupled receptor, cytokine and T cell receptor-mediated signals (Dustin et al., 2004). While all of these signals can rapidly enhance LFA-1 avidity, there is also the need for coordinated de-adhesion of immune cells. Deactivation of LFA-1 and disassembly of LFA-1-mediated cell contacts seem to be vital for the generation of normal immune responses (Semmrich et al., 2005).

CD43, a major leukocyte cell surface sialoglycoprotein, is one of the potential mediators of LFA-1 de-adhesion. Its high level of glycosylation and net negative charge leads to an extended conformation of the extracellular domain enabling repulsion of cell contacts (Ostberg et al., 1998). Indeed, cells of CD43 knock out mice showed a substantial enhancement in both homotypic adhesion and ability to bind different ligands, including fibronectin and ICAM-1 (Manjunath et al., 1993, Manjunath et al., 1995). Furthermore, CD43 was shown to exert a negative regulatory effect on T cell trafficking by interfering with L-selectin-mediated adhesion (Stockton et al., 1998). Recently, it was shown that CD43 exerts its negative regulatory function on cell adhesion also in mast cells (Drew et al., 2005). However, the understanding of the physiological role of CD43 has been complicated by numerous reports showing rather pro-adhesive functions for CD43. Interestingly, CD43 itself was described to bind ICAM-1 (Rosenstein et al., 1991), which might be important for the initial interaction of T cells with other cells. Others and we demonstrated the induction of cell aggregation upon treatment with CD43 monoclonal antibodies (mAbs) (Nong et al., 1989, Rosenkranz et al., 1993, Sanchez-Mateos et al., 1995). On the other hand, a number of CD43 mAbs could block cellular interactions, like T cell conjugate formation with certain cell types (Stöckl et al., 1996), or T cell binding to lymph node and Peyer's patch high endothelial venules (McEvoy et al., 1997). We hypothesized that the dynamic interactions of CD43 with additional membrane partner molecules could explain these contradictory and puzzling anti- and pro-adhesive properties of CD43 towards LFA-1. Indeed, there is evidence for several counter-receptors and ligands of CD43 including MHC class I (Stöckl et al., 1996) and sialoadhesin (van den Berg et al., 2001).

To find such potential regulators, we performed a yeast-2-hybrid screen (Y2H), co-immunoprecipitations and confocal microscopy. As a result of these experiments, we can show here that CD43 interacts directly with LFA-1 as well as with CD147, a human Ig-family member (Kasinrerk et al., 1992). During the last couple of years, CD147 appeared to be a pleiotropic molecule involved in cell metabolism, cell adhesion and cell migration (Berditchevski et al., 1997, Cho et al., 2001, Kasinrerk et al., 1999, Kirk et al., 2000, Pushkarsky et al., 2001, Sun and Hemler, 2001, Wilson et al., 2002, Xu and Hemler, 2005, Yurchenko et al., 2001, Yurchenko et al., 2002). Treatment of cells with both CD43 and CD147 mAbs induces a dynamic re-arrangement of CD43, CD147 and LFA-1 to the areas of cell–cell contact. Moreover, the CD147-mediated adhesiveness is blunted by CD43 co-expression. Consistent with the latter finding, CD43 knock down by siRNA enhances CD147-mediated cell adhesion of human T cells to the LFA-1 ligand ICAM-1. Taken together, these data indicate that CD147 is an important co-receptor of CD43 in regulation of LFA-1-mediated cell adhesion.

Section snippets

Cells

The human monocytic cell line U937, the human T cell line Jurkat and the mouse thymoma cell line BW5147 were maintained in RPMI-1640 medium (Sigma, St. Louise, MO) supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine and 10% heat inactivated FCS (PAA, Linz, Austria). All cells were grown at 37 °C and 5% CO2 in a humidified atmosphere. Cells were passaged three times a week.

Antibodies

The mAbs used in this study were produced in our laboratories and described earlier with the

CD43 interacts with CD147 in human leukocytes

To find potential partners of CD43, we screened a human Jurkat T cell Y2H cDNA library using the intracellular domain of CD43 (amino acids 277–400) as bait. With the exception of Daxx, which was recently described by some of us as a part of the apoptosis pathway via CD43 (Cermak et al., 2002), the only specifically interacting clone was the intracellular part plus two amino acids of the putative transmembrane region of the transmembrane protein CD147 (amino acids 227-269; see Table 1). To

Discussion

The pro-adhesive and anti-adhesive role of CD43 in the regulation of cell adhesion is the matter of a long-term debate (Ostberg et al., 1998, Woodman et al., 1998). A possible mechanism mediating the negative regulatory function of CD43 for leukocyte adhesion is supposed to be steric hindrance of cell interactions via its extended conformation and its negatively charged sialic acid residues (Ostberg et al., 1998; Allenspach et al., 2001). Our finding that CD43 co-immunoprecipitated with LFA-1

Acknowledgements

This work was supported by the GEN-AU program of the Austrian Federal Ministry of Education, Science and Culture; the North-South-Dialogue-Scholarship Program, Department of Development Cooperation, Austrian Federal Ministry of Foreign Affairs; and the Competence Centre for Biomolecular Therapeutics. L.C., L.A. and V.H. were supported from the Center of Molecular and Cellular Immunology 1M0506. We are thankful to Dr. Waldemar Kolanus, University of Bonn, Germany, for providing the ICAM-1Rg.

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    P.K. and H.B.S. contributed equally to this work.

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