Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1

Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS August 15, 1991 Pages 1498-l 504 EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF A SOLUBLE FORM OF VASCULAR CELL ADHESION MOLECULE 1 Roy Lobb, Gloria Chi-Rosso, Diane Leone, Margaret Rosa, Barbara Newman, Stefan Luhowskyj, Lauralee Osborn, Susan Schiffer, Christopher Benjamin, Irene Dougas, Catherine Hession, and Pingchang Chow Biogen Inc., 14 Cambridge Center, Cambridge, MA 02142 Received July 10, 1991 Vascular cell adhesion molecule 1 (VCAMl) is a leukocyte adhesion molecule induced on human endothelium in vitro and u w by inflammatory stimuli. A truncated cDNA for VCAMl was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAMl (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4- expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAMl during inflammatory and immune responses in vivo. 0 1991 Academic Press. Inc. VCAMl is a member of the immunoglobulin (Ig) superfamily which is expressed on human umbilical vein endothelial cells (HuvECS) stimulated in vitro by proinflammatory cytokines (l-3). VCAMl binds to the integrin VLA4, and may help recruit VLA4-expressing mononuclear leukocytes and eosinophils to inflammatory sites in w (4-6). We isolated a cDNA for VCAMl which encodes a protein with six Ig homology domains (1). We have also characterized a longer form of VCAMl, generated by alternative splicing, and which contains an additional Ig domain (7). Although both forms of VCAMl are functional, the longer form of VCAMl appears to be the major form expressed in cytokine-treated HUVECS (7). The presence of a longer form of VCAMl has also been noted elsewhere (8,9). In this 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. 1498 Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS report we describe the expression and functional characterization of full length seven domain recombinant soluble VCAMl (rsVCAM.1). MATERIALS AND METHODS Cells. HL-60, THPl, Ramos, and JY cells were propagated as described (1,15). PMNs and PBL's were isolated as described (15,16). Antibodies. Phycoerythrein or fluorescein conjugates of mAbs Leu2A(CD8), Leu3A(CD4), Leu4(CD3), Leull(CD16), Leu16(CD20), Leul8(CD45RA), and LeuM3(CD14), were obtained from Becton Dickinson. Fluoresceinated UCHLl(CD45RO) was obtained from Dako. Anti-VCAMl Moab 489(10) was the gift of Dr. John Harlan. Ascites- produced 4B9 was protein A-purified, and Fab2 fragments generated, according to standard methods. The blocking anti-VLA4 mAb HP1/2 (11) was the gift of Dr. Francisco Sanchez-Madrid. Generation of CHO cell lines stablv exoressins rsVCAM1. A recombinant soluble VCAMl expression vector was constructed by standard methods (Fig.lA). The plasmid VCAMl/CDMB, which contains the full length cDNA sequence for VCAMl (1,7), was cleaved and ligated in the presence of two complementary oligonucleotides which supplied a stop codon and a BglII site. The rsVCAM1 cDNA gene was inserted into the mammalian expression v.ector pMDR901 at a Not1 site. pMDR901 is a derivative of pJOD-S (12) which allows insertion of Not1 fragments downstream from the Adenovirus5 major late promoter. The vector also contains an sv40 early promoter/dihydrofolate reductase expression cassette. The rsVCAM1 expression vector, pBN1006, was linearized and electroporated into CHO-DHFR- cells as described (12). Cells were cultured in selective medium, and individual colonies picked into 96 well cluster plates as described (12,13). Cells were placed in serum- free medium overnight, and those secreting high levels of rsVCAM1 selected by standard ELISA assay using anti-VCAMl mAb 489. Purification of VCAMl: rsVCAM1 was purified from CHO cell conditioned medium essentially as described (13) for recombinant soluble endothelial-leukocyte adhesion molecule-l (ELAMl). Briefly, 10 liters of medium were concentrated 25-fold (Amicon PM30) and incubated with lOm1 immunoaffinity resin (Mab 4B9 crosslinked to Affigel 10 at a concentration of 20 mg/ml gel). rsVCAM1 was eluted with 0.1 M glycine pH'3.0, neutralized, dialyzed against Hank's balanced salt solution containing calcium and magnesium, concentrated to about 1 mg/ml, sterile filtered, and stored at - 7o"c. N-terminal SeoUenCing. rsVCAM1 was subjected to automated Edman degradation in an Applied Biosystems 470A gas-phase protein sequencer in the presence of Polybrene (13). Adhesion Assavs. rsVCAM1 was added from a 1 mg/mL stock solution to 100~1 binding buffer (15mM sodium bicarbonate/35mM sodium carbonate pH 9.2) in individual wells of a 96 well bacteriologic plastic plate (Linbro/Titertek), and incubated overnight at 4OC. Plates were blocked with 1% bovine serum albumin (BSA) in PBS for 1 hr at room temperature, washed once with RPM11640 containing 10% fetal bovine serum (RPMI/lO), and adhesion assays performed with inverted centrifugation as described (1,4). Blood leukocyte adhesion was quantified by fluorescence as described (13). For 1499 Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS antibody inhibition assays, rsVCAMl-coated plates were incubated with blocking Moab 4B9 at 10 ,ug/ml for 30 min at room temperature, or VLA4-expressing cells incubated with supernatants from HP1/2- secreting hybridoma cells diluted l/10, and adhesion assays performed as above. For "panning" experiments 20 ,ug rsVCAM1 in 1 ml binding buffer (see above) was incubated on 35mm bacteriologic plastic Petri dishes (Falcon #1008) overnight at 4OC and blocked as above. Cells (1 ml, 2x106/ml) were incubated at room temperature in RPMI/lO for 10 min, plates washed 3X with RPMI/lO, and bound cells eluted with PBS/l%BSA/5mM EDTA, counted and processed for FACS analysis, which was performed as described (13). The percentage of T, B, NK, CD4 and CD8 cells in either the starting or VCAMl-bound populations was determined from one color FACS analyses. The percentage of memory (UCHLl+) and naive (CD45R+) T cells was determined from two color FACS analyses of CD3+ cells. RESULTS AND DISCUSSION Generation, Purification, and Characterization of rsVCAM1. Figure 1A illustrates the construction of rsVCAM1. Insertion of a stop codon at the leucine which is the first amino acid of the putative transmembrane domain (1,7), generated a construct lacking both transmembrane and cytoplasmic domains. rsVCAM1 is secreted into the medium conditioned by stably transfected CHO cells, and is readily purified by immunoaffinity chromatography to >95% purity (Fig. 1B). About 0.4 nmol rsVCAM1 was subjected to 20 cycles of N-terminal sequencing. Its sequence (FKIETTPESRYLAQIGDSVS) was exactly as Wild Type SPELLV rsVCAH1 TCTCCTCAGTGAG&XT S P E * Bg12 Fisure 1. A) Construction of rSVCAM1. The domain structure of VCAMl consists of seven Ig homology domains (Dl-7), a transmembrane domain (TM), and a short cytoplasmic tail (CY). A stoo codon was inserted at the putative d&main 7/transmembrane boundary. B) SDS- PAGE of immunoaffinity purified rsVCAM1. Molecular weights (x10') are illustrated at the left. 1500 Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1400 0 CONTROL n rsVCAM q 489 q HP112 0 HL-60 RAMOS THP.l JY PBL PMN Fiaure 2. Cell adhesion to immobilized rsVCAM1. The adhesion of HL60, Ramos, THPl, JY, PMN, and PBLs is shown, either to BSA- coated control plates, or to rsVCAMl-coated plates, either alone or in the presence of blocking mAbs 4B9 (anti-VCAMl) or HPl/2 (anti-VLA4). For inhibition of THPl binding, 489 Fab2 (10 pug/ml) was used. predicted from the cDNA (1). The secretion of rsVCAM1 as a soluble protein, and its N-terminal sequence, confirm putative assignments of the signal sequence, transmembrane and cytoplasmic regions (1,7). Immobilized rsVCAM1 is a Functional Adhesion Molecule. Using an optimal coating concentration of 0.5 Mug/well, the adhesion to immoblized rsVCAM1 of various human cell types was examined (Fig. 2). VLA4-expressing cell lines which are known to bind to VCAMl, such as HL60, Ramos, and THPl (1,4) bind well to rsVCAM1. Moreover, adhesion is completely blocked by either the anti-VCAMl Mab 4B9 (10) or the anti-VLA4 Moab HPl/2 (11). In contrast, the lymphoblastoid cell line JY, which does not express VLA4 and which does not bind cell surface VCAMl (1,4), does not adhere to immobilized rsVCAM1. Human Leukocvte bindino to immobilized rsVCAM1. PMN do not bind to VCAMl expressed either on COS cells or HUVECs (1,10,14), do not express cell surface VLA4 (l), and do not bind to rsVCAM1 (Fig. 2). In contrast, lymphocytes bind to cell surface VCAMl (3,10,14), and 1501 Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE I. T CELL SUBSET ANALYSISa CD4 % CD8 % RATIO MBvtOHY% NAIVE % RATIO PBLs (Initial) 61.2 f 5.2 38.8 f 3.3 1.58 44.8 f 0.9 55.2 + 2.3 0.81 PBLs (VCAMI-bound) 52.0 f 3.3 48.0 f 3.6 1.08 67.8 zk 2.9 32.2 f 1.8 2.10 aThe percentage of T cell subsets (mean f S.E.M.) in the initial or VCAMl-bound PBLs was determined by one or two color FACS analysis as described in the methods section. monocyte-depleted PBL populations bind specifically to immobilized rsVCAM1 (Fig. 2). We examined the PBL subpopulations binding to rsVCAM1 in more detail by "panning" on rsVCAMl-coated Petri dishes, followed by FACS analysis of bound cell populations, In four experiments, the starting PBL population contained an average of 76.4+2.6% (mean+S.E.M.) T cells, 7.8+0.4% B cells, and 9.0+1.8% NK cells, while the VCAMl-bound population contained 61.0+3.0% T cells, 13.7+0.7% B cells, and 14.5+2.1% NK cells. In control experiments, negligible binding of the PBL starting populations to either BSA-coated dishes or 4B9-blocked rsVCAMl-coated dishes was observed (not shown). The results indicate that T(CD3+), B(CD20+), and NK(CDl6+) cells, all of which express VLA4, bind to immobilized rsVCAM1, which shows a modest preference for B and NK cells. We next examined subpopulations of T cells binding to rsVCAM1 (Table I). In four experiments, the starting PBL population contained 61.2% CD4+ and 38.8% CD8+ T cells (CD4/CD8 ratio of 1.58), and 44.8% memory (UCHLl+) and 55.2% naive (CD45RO+) T cells (memory/naive ratio of 0.81). However, in the VCAMl-bound population we found that both the CD4/CD8 and memory/naive ratios had changed significantly (Table I). The bound population contained 52.0% CD4+ T cells and 48.0% CD8+ T cells (CD4/CD8 ratio of 1.08), with 67.7% memory T cells and 32.3% naive T cells (memory/naive ratio of 2.10). The results show that rsVCAM1 preferentially binds memory versus naive T cells, and CD8+ versus CD4+ memory T cells. 1502 Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Since CD8+ cells express predominantly the naive phenotype (15), it is apparent that rsVCAM1 shows a significant preference for CD~+ memory T cells. In parallel studies we found that, using the same PBL starting populations, immobilized rsELAM1 preferentially binds CD4+ memory T cells (13), indicating that the results observed are not due to an artifact of the panning procedures, and that the two adhesion molecules show functional differences. The preferential accumulation of the memory (UCHLl+) T cell subset to inflammatory lesions in vivo (16) has been attributed to their enhanced adhesive capacity for endothelium, noted in vitro (16,17). The preferential binding of cells of the memory phenotype to both VCAMl and ELAMl further emphasizes this possibility, while the propensity of VCAMl and ELAMl to bind preferentially CD8+ and CD4+ memory T cells, respectively, provides an added level of selectivity that may contribute to pathophysiology of lymphocyte recruitment. In summary, we have generated milligram quantities of a soluble monomeric form of human VCAMl. rsVCAM1 can serve as a functional adhesion protein demonstrating the same specificity as vCAM1 expressed at the surface of HUVECs, and should prove useful for the evaluation of the effects of VCAMl/VLA4 cognate recognition on leukocyte function. ACKNOWLEDGMENTS We thank Jaqueline Ashook for help with FACS analyses, John Harlan for Mab 4B9, and Francisco Sanchez-Madrid for mAb HPl/2. REFERENCES 1. Osborn, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S Chi-Rosso, G., and Lobb, R. (1989) Cell, 59, 1203-1211. 2:'Osborn, L. (1990) Cell, 62, 3-6. 3. 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