Role of Integrin A4я7/A4яp in Lymphocyte Adherence To

Role ofIntegrin a4ß7/a4ßP in Lymphocyte Adherence to Fibronectin and VCAM4 and in Homotypic Cell Clustering Curzio Rüegg, Antonio A. Pbstigo,t Elizabeth E. Sikorski,* Eugene C. Butcher,* Robert Pytela, and David J. Erle The Lung Biology Center, Department ofMedicine, University of California, San Francisco, California 94143; * Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School ofMedicine, Stanford, California 94305 ; the Center for Molecular Biology in Medicine, Veterans Administration Medical Center, Palo Alto, California 94305 ; and Cervizio de Immunologia, Hospital de la Princesa, Universidad Autonoma de Madrid, Madrid, Spain Abstract. Integrins are heterodimeric cell surface pro- fragment containing the RGD sequence. Adhesion to teins that mediate both cell-cell and cell-extracellular fibronectin was inhibited by antibodies to a4, suggest- matrix interactions . We and others recently identified ing that a4ß7 is a fibronectin receptor. We confirmed cDNAs encoding a novel integrin ß subunit, 07, in that a4ß7 binds to the CS-1 region of fibronectin lymphocytes. We have now detected 07 mRNA in using affinity chromatography. TK-1 cell adhesion to mouse TK-1 T lymphoma cells, which are known to the vascular cell adhesion molecule VCAM-1 was also express the putative Peyer's patch homing receptor inhibited by antibodies to a4, implying that a4ß7 also a4ßP. We used an anti-peptide antiserum and a novel plays a role in the adherence of lymphocytes to endo- mAb against the 07 subunit to show that TK-1 cells thelial cells. TK-1 cell binding to fibronectin and express 07 as the only subunit associated with a4. We VCAM-1 is markedly increased by brief PMA stimula- conclude that 07 and ßP are identical. We also show tion. We also found that mAbs against a4 and 07 in- that activated peripheral blood T cells express a4ß7. duce homotypic clustering of TK-1 cells. Taken to- We studied the function of a4ß7/a4ßP in TK-1 cells, gether these results suggest that a4ß7/a4ßP recognizes which do not express very late antigen (VLA)-4 (a4ß1) . some or all of the same widely distributed ligands rec- Cells adhered to intact fibronectin and to a fibronectin ognized by VLA-4 (a4ß1) and that the role of a4ß7/ fragment containing the CS-1 region, but not to a a4ßP may not be restricted to lymphocyte homing. HE ability to interact with a variety of cells or with a4ß1 (VLA-4) has been shown to be involved in at least three proteins of the extracellular matrix is crucial for a different kinds ofadhesive interactions . First, a4ß1 mediates variety of leukocyte functions, including cell activa- cell adhesion to fibronectin by binding to the alternatively Ttion, phagocytosis, recruitment to sites of inflammation, re- spliced C9-1 region of fibronectin (Wayner et al., 1989; circulation, and homing (for reviews see Stoolman, 1989; Mould et al., 1990; Guan and Hynes, 1990). Second, a4ß1 Butcher, 1990; Springer, 1990). Many adhesion molecules mediates T cell adhesion to endothelium by binding to the expressed on T lymphocytes belong to the integrin family. vascular cell adhesion molecule VCAM-1 (Elices et al ., Integrins are noncovalently linked heterodimers consisting 1990) . T cell activation causes rapid enhancement of c4ß1- of an a and a ß subunit that mediate cell-extracellular matrix mediated binding to both fibronectin and VCAM-1 (Shimizu as well as cell-cell interactions (Hynes, 1987; Ruoslahti, et al., 1990a,ó). Third, certain antibodies directed against 1991) . The a and ß subunits each have a large extracellular the a4 subunit of a4ß1 induce homotypic lymphocyte domain, a short transmembrane region, and a cytoplasmic clustering (Campanero et al., 1990 ; Bednarczyk and McIn- tail. At least 13 different a and eight different ß subunits have tyre, 1990; Pulido et al., 1991) . This clustering is dependent been identified to date; these combine to produce at least 18 upon divalent cations. Clustering can be abolished by other different integrin heterodimers . Both a and ß subunits par- antibodies directed against a4 and by some anti-01 antibod- ticipate in the determination ofligand specificity. The tripep- ies, but not by antibodies against other integrins or VCAM-1. tide RGD (Arg-Gly-Asp) is the recognition site for many of It is possible that binding of anti-ce4 antibodies induces a the integrins that bind to extracellular matrix proteins. conformational change in a4ß1 that allows a4ß1 to bind to Members of the 01 integrin subfamily (a1ß1 to a6ß1), also an unidentified counterreceptor on adjacent cells. Alterna- referred to as the very late antigens (VLA-1 to VLA-6), bind tively, binding of anti-c«4 antibodies might cause clustering and mediate adhesion to several extracellular matrix proteins by triggering an a4ß1-mediated signal that activates other such as laminin, collagens, and fibronectin (Hemler, 1990). adhesion pathways. © The Rockefeller University Press, 0021-9525/92/04/179/11 $2 .00 The Journal of Cell Biology, Volume 117, Number 1, April 1992 179-189 179 In contrast to other integrin a subunits, the 150-kD a4 RNA Extraction, cDNA Synthesis, Homology PCR, subunit can be cleaved ata site close tothe middle ofthe mol- and Northern Blotting Analysis ecule, resulting in variable amounts of 80- and 70-kD a4 Total RNA extraction, cDNA synthesis, and homology polymerase chain fragments (Sanchez-Madrid et al., 1986; Hemler et al., reaction (PCR) amplification of integrin ß subunits was performed as previ- 1987; Takada et al., 1989). Both the intact and the cleaved ously described (Erle et al., 1991b) . Briefly, total RNA was prepared from forms of cA can associate with 01 on the cell surface to form TK-1 cells using the LiCl-urea method and reverse transcribed to cDNA. Integrin ß subunit partial cDNAs were amplified using the primer mixtures functionally active integrins. The relative expression of the BIAF and B2AR that recognize two regions that are conserved in all known intact and cleaved forms greatly varies between different cell 0 subunit sequences (Erle et al ., 1991b) . Amplified cDNAs were cloned lines and is affected by T cell activation. (using the pBluescript plasmid) and sequenced . RNA gel electrophoresis In some murine lymphocytes, a4 can associate with a sec- and Northern blotting analysis were performed as previously described with ond ß subunit, ßP (Holzmann et al., 1989; Holzmann and a probe made from a partial 07 cDNA clone containing nucleotides 857-2761 of the ß7 cDNA using the random primer method (Erie et al., Weissman, 1989) . The molecular cloning and sequence of 1991x) . ßP has not yet been reported . Antibodies directed against a4ßP block adhesion of lymphocytes to Peyer's patch high Antisera andAntibodies endothelial venules (HEV)', but not peripheral lymph node lympho- A peptide modeled after the carboxyl-terminal sequence of 07 (Erle et al., HEV, suggesting that a4ßP may be a Peyer's patch 1991x) plus an amino-terminal cysteine (CQDSNPLYKSAITTTINPRF- cyte homing receptor. The ligand(s) of a4ßP have not been QAEDSPTL, kindly provided by Dr. L. Reichardt, University of Califor- previously identified. nia, San Francisco, CA) was coupled to keyhole limpet hemocyanin (Cal- We and others have recently reported the cloning and se- biochem Corp., San Diego, CA) using m-Maleimidobenzoyl-N-hydroxy- of cDNAs coding for a novel human integrin ß sulfosuccinimide (Pierce Chemical Co., Rockford, IL) and glutaraldehyde quencing (Fisher Scientific Co ., Pittsburgh, PA) (Harlow andLane, 1988). A 1:1 mix- subunit, 07, expressed in lymphocytes (Yuan et al., 1990; ture of the two conjugates was used to immunize a rabbit (CALTAG Labora- Erle et al., 1991x). It has recently been shown that the 07 tories, South San Francisco, CA). The antiserum was affinity purifiedusing subunit is part of the murine M290 antigen complex (Kil- the same peptide coupled to Sepharose . Antisera against other integrin shaw and Murant, 1990; Yuan et al., 1991) . M290 and its subunits were also obtained by immunizing rabbits with peptides modeled after the carboxyl termini and were generously provided to us by other putative human homolog HML-1 are highly expressed on investigators (a4 antiserum, Drs . M. Johannson and E. Ruoslahti, La nearly all intraepithelial lymphocytes of the intestine (Cerf- Jolla Cancer Research Foundation (La Jolla, CA) ; /31 antiserum, Dr. L. Bensussan et al ., 1987; Kilshaw and Murant, 1990) . How- Reichardt ; ß2 antiserum, Dr. E. Small, University of California, San ever, expression ofHML-1 was reported on peripheral blood Francisco, CA) . T cells after in vitro activation (Schieferdecker et al ., 1990) . The mAb LS722 was obtained by immunizing a rat with supernatants from chymotrypsin-treatedmesenteric lymphocytes andboosting with TK-1 The M290/HML-1 a subunits have not yet been identified, cells (Sikorski, E. E., and E. C. Butcher, manuscript in preparation) . Hy- but these subunits do not appear to be related to any of the bridomas were selected by testing culture supernatant for their ability to previously described a subunits. The functions of M290/ specifically stain TK-1 cells but not thymocytes. Rat anti-mouse a4 mAbs HML-1 remain to be determined . were generous gifts of Dr. I. Weissman (Rl-2) (Holzmann et al., 1989) and Dr. P. Kincade (Oklahoma Medical Research Foundation, Oklahoma City, We now report the detection of the 07 protein using a ß7- OK) (PS/2) (Miyake et al., 1991b) . The mAb M290 was a generous gift specific antiserum and a mAb. We present evidence that a4 of Dr. S. Mutant and Dr. P. Kilshaw (Institute for Animal Physiology and associates with 07 on mouse TK-1 lymphoma cells, that Genetic Research, Babraham, Cambridge) (Kilshaw and Murant, 1990). a4ß7 and a4ßP are identical, and that a4ß7 is involved in The rat anti-mouse hybridoma FD441, anti-LFA1 a subunit, was ob- VCAM-1 and in homotypic T cell tained from the American Type Culture Collection (Rockville, MD) (ATCC adhesion to fibronectin and TIB213) (Sarmiento et al ., 1982).

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