CHEMISTRY. (C) 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 267, No. ... molecule 1; sVCAM-1, soluble form of vascular cell adhesion molecule. 1; FN-40,40-kDa ..... epithelial lymphocytes. Thus 87, like the ...
THEJOURNALOF BIOLOGICAL CHEMISTRY (C)
Vol. 267, No. 12, Issue of April 25, pp. 83664370, 1992 Printed in U.S.A.
1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Adhesion to Vascular Cell Adhesion Molecule1 and Fibronectin COMPARISON OF
a4P1
(VLA-4) AND a4@p, ON THE HUMAN B CELL LINE JY* (Received for publication, December 5, 1991)
Bosco M. C. Chan, MarianoJ. ElicesS, Elizabeth Murphy, and Martin E. HemlerQ From the Dana-Farber Cancer Institute, Harvard Medical School, Boston,Massachusetts 02115
Most mononuclear leukocytes and cell lines express the integrin a4D1 (VLA-4) heterodimer. In this study we have used Northern blotting and immunoprecipitation experiments to demonstrate that a B lymphoblastoid cell line (JY) expressed the integrin f17 subunit in association with a4. These a4B7-positiveJYcells bound poorly or not at all to VLA-4 ligands (soluble form of vascular cell adhesion molecule 1 (sVCAM-1) and the CS1 region of fibronectin). In contrast, a positive variant of JY cells(selected to express a mixture of a4b1and a“&) bound avidly to VLA-4 ligands, and this binding was completely inhibitable by anti-a4 and anti-dl monoclonal antibodies. Thus, expression appears to be acritically important componentof VLA4-mediated binding to its ligands. After either JY or JY-& cells werestimulated for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, the majority of adhesion to VCAM or fibronectin remained a4- and bI-dependent,but a low amount of adhesion to sVCAM- 1 or fibronectin became a4-dependent, B1-independent, thus suggesting a role for a4&. In summary, we have found (i) that a4@7makes little or no contribution to fibronectin or VCAM-1 binding on unstimulated JY cells, (ii) that a“@7perhaps makes a minor contribution to ligand binding on 12-0-tetradecanoylphorbol-13-acetate-stimulated cells, and (iii) that a4B1 is the functionally dominant VCAM-1 andfibronectin receptor even when expressed in relatively low amounts compared to a‘&.
Circulating blood cells including lymphocytes, monocytes, and eosinophils express the integrin VLA-4 and utilize it to mediate binding to VCAM-1‘ expressed on activated endothelium at inflammatory sites (1-7). Thus, in diseases such as asthma, allergy (8,9), arthritis (lo), andatherosclerosis ( l l ) , VLA-4/VCAM-1 interactions could be an important target for therapeutic intervention. In addition, VLA-4 mediates B cell adhesion to VCAM-1
* This work was supported in part by National Institutes of Health Grants CA-42368 and GM-38903 (to M. E. H.) and a Centennial fellowship from the Medical Research Counci of Canada (to B. M. C. (2.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Current address: Cytel Corp., 3525 John Hopkins Court, San Diego, CA 92121. § To whom correspondence should be addressed. ‘The abbreviations used are: VCAM-1, vascular cell adhesion molecule 1; sVCAM-1, soluble form of vascular cell adhesion molecule 1; FN-40,40-kDa chymotryptic fragment of fibronectin; FN-120,120kDa chymotryptic fragment of fibronectin; mAb, monoclonal antibody; MFI, mean fluorescence intensity; TPA, 12-0-tetradecanoylphorbol-13-acetate; kb, kilobase(s).
in germinal centers (12, 13) and adhesion of hematopoietic progenitor cells to VCAM-1 on bone marrow stroma (14, 15). Also, binding of melanoma cell VLA-4 to VCAM-1 on activated endothelium is hypothesized to contribute to metastasis (16). Besides binding to VCAM-1, VLA-4also mediates adhesion to a domain (called CSl) within the Hep-2 region of fibronectin (17-19). Thisinteraction may have important functional relevance during maturation of bone marrow progenitor cells (20), during localization of sensitized T lymphocytes to antigenic sites (21), and during embryogenesis (22). In other experiments, VLA-4 has been found to trigger homotypic aggregation (23, 24), by a mechanism that probably does not involve binding to VCAM-1 or fibronectin (25). Like other integrins, VLA-4 is composed of an a subunit (a4) and a 3/ subunit (8’). However, preliminary evidence has indicated that human a4 might sometimes associate with a B subunitother than B1. For example, ahumanB cell line no (called JY) expresses a highlevel of a4, butlittleor detectable p1subunit (5), and freshly isolated human B lymphocytes contain approximately 2-fold more a4 than p1 (26). It is assumed that this previously observed “excess” a4 is associated with another /3 subunit, other than pl, because integrin heterodimer formation hasbeen established as being an obligatory prerequisite for cell surface expression (27-30). On some mouselymphoid cells a4has been shown to associate with at least one other 8 subunit (called pp) which may be involved in lymphocyte homing (31, 32). A possible human homologue of the murine @p is p7 (33, 34), but it has not yet been determined whether human a4 might associate with the p7 subunit. During studies of the functions of VLA-4 (a4@’heterodimer), antibodies directed toward the a4 subunit have often been utilized, and thus difficulties in interpretation could arise if a4 associates with multiple /3 subunits. In this regard, it has not yet been determined whether another a4 heterodimer (ie. not containing&) might mediate adhesion functions similar to those mediated byVLA-4 (a4&).Furthermore, while the a4 subunit of VLA-4 has clearly been shown to be required for cell binding to VCAM-1 and fibronectin, direct evidence for involvement has been lacking. In this paper we have made use of &-negative J Y cells and J Y cells selected for p1 expression (JY-8, cells) to study the contribution of to VLA-4 functions. Also, we have identified B7 as the subunit associated with a4 on &-negative JY cells, and compared its functional contribution with that of 8’. MATERIALSANDMETHODS
Antibodies and Zntegrin Ligands-Monoclonal antibodies used were B-5G10 (35) and HP2/1 (36) (anti-VLA-4);A-1A5 (37), TS2/16 (381, mAb 13 (39), 4B4 (40), and B l E l l (41) (anti-VLA-&); P1D6 (42) (anti-VLA-5); J-2A2 (43) and P3 (44) (negative control antibodies). Rabbit anti-/3, antibody was provided by Dr. Chris Parker (DanaFarber Cancer Institute), andwas prepared against a synthetic pep-
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Functional Comparison of tide (RREYSRFEKEQQQLNWKQDS) derived from the cytoplasmic domain of @7 (33,34). The FN-120 chymotryptic fragment of fibronectin was purchased from Telios Pharmaceuticals (La Jolla,CA), and theFN-40 fragment was prepared as previously described (45). Briefly, fibronectin was isolated from human plasma using gelatin-Sepharose andthen cleaved with chymotrypsin. The resulting 40-kDa fragment was purified using heparin-Sepharose, followed by DEAE chromatography (45). The 25-amino acid CS1 peptide (DELPQLVTLPHPNLHGPEILDVPST) derived from fibronectin and recombinant soluble VCAM-1 (sVCAM-1) were gifts from Dr. R.Lobb (Biogen Inc., Cambridge, MA). The latterwas prepared by genetic alteration of the native molecule (the form with seven Ig domains) such that the transmembrane and cytoplasmic regions are deleted (46). Establishment and Analysis of JY-@,Cells-The B lymphoblastoid cell line JY was previously described as lacking @but ,, expressing a4 (5). Attempts were made to transfect JY cells with p1 cDNA in the pFneo expression vector, and select for @,-positiveJY cells. However, in multiple experiments, the transfection procedure itself resulted in selection for P1 expression, regardless of the presence or absence of cDNA. This was possibly due to a selective advantage conferred by endogenous P1on cells grown for extended periods at low viability. Thus, a few subcloned JY cell populations were found to express variable levels of cell surface pl, as assessed by flow cytometry. The @,-positivecells were further enriched by magnetic bead selection, using the anti-@, mAbA-1A5. When grown in normal conditions (RPMI 1640 supplemented with 10% fetalbovine serum) the elevated expression of O1 (in JY-8, cells), or lack of p1expression (in JYcells), was each remarkably stable despite continuous culture for nearly 6 months. Cell AdhesionAssays-Cell attachment to FN-40, FN-120, CS1 peptide, and sVCAM-1,was carriedout essentially as previously described (3), except that bound cells were detected by fluorescence. Briefly, protein ligands were coated onto 96-well microtiter plates (Flow Labs), and then0.1% bovine serum albumin was added to block nonspecific adhesion. Cells were labeled by incubation with the fluorescent dye BCECF (Molecular Probes, Eugene, OR) for 30 min and then washed once in PBS containing 0.5 mM EDTA, once in 0.1% bovine serum albumin in RPMI, and finally resuspended in the same solution. The labeled cells ( 5 X 10') were then added to each well of a ligand-coated microtiter plate and incubated for 25 min a t 37 "C followed by three washes with 0.1% bovine serum albumin in RPMI. For inhibition studies, monoclonal or polyclonal antibodies were present during the 25-min adhesion period. After washing, cells remaining attached to the plate were analyzed using a Fluorescence Concentration Analyzer machine (IDEXX Co., Portland, ME). After subtraction of background cell binding (assessed using bovine serum albumin-coated wells), values for cells bound/mm2 were calculated from the equation,Cells bound/mm2 = BF/TF X 50,000 cells added per we11/38-mm2 area per well, where B F = bound fluorescence after washing and TF = total initial fluorescence. For example, if 100% binding were observed, then 50,000/38 = 1316 cells bound/mm2. We ascertained that fluorescence was linearly proportional to cell number [from 50,000 cells (1316 cells/mm2) down to 150 cells (4 cells/mm2)]. Background binding was typically less than 10% of the total, and assays arereported as themean of triplicate determinations.Standard deviations typically ranged from 20 to 50 cells/mm2. Also, for every assay, a microscope was used to frequently monitor cell attachment by visual inspection. Radiolabeling,Immunoprecipitation, and Northern Blot AnalysisCells were surface-labeled with lZ5Iusing lactoperoxidase and lysed in the presence of 0.5% Nonidet P-40. Then immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses (on 7% polyacrylamide gels) were carried out as previously described (47). For Northern blot analysis, total RNA was prepared as previously described (48), separated by electrophoresis on formaldehyde-agarose gels, transferred to Zetabind nylon membrane (Cuno Inc., Meriden, CT), and then RNA wasprobed with a 2.3-kb fragment of p7 cDNA (33) that had been labeled with 32Pby random priming.
(3,
RESULTS
Selection of JY Cells Expressing the P1 Subunit-As demonstrated by flow cytometry, the B lymphoblastoid cell line JY expressed substantial levels of cell surface a4 but approximately 17-fold less 8, (Fig. 1).This result suggested that at least 94% of the a4was associated with a different B subunit.
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a4p1and a4P7
LOG FLUORESCENCE INTENSlTY
FIG. 1. Flow cytometric analysisof J Y and JY-B1 cells. Both JY and JY-(3, cells were stained with either negative control monoclonal antibody (Pa, indicated in every panel with a dotted line), anti(A-lA5), anti-a4 (B-5G10), or with anti-a' (PlD6), followedby fluorescein isothiocyanate-labeled rabbit anti-mouse second antibody. Flow cytometry was carried out using a FACScan instrument (Becton Dickinson, Mountainview, CA) as previously described (3).
LIGAND (ug/well)
FIG. 2. Comparison of adhesive properties of J Y and JY-8, cells. As described under "Materials and Methods," identical numbers of JY and JY-8, cells were tested for adhesion to the FN-40 fragment of fibronectin, the FN-120 fragment of fibronectin, the CS1 peptide derived from the FN-40 fragment, and sVCAM-1. Each of the ligands was coated onto the surface of 96-well plastic dishes at the indicated doses.
From these J Y cells, a variantpopulation of cells was selected with PI expression elevated over 10-fold as demonstrated by flow cytometry (Fig. 1).At the same time, a4 expression was not increased in the JY-Pl cells, and in fact was diminished slightly compared to the level in J Y cells. Expression of a5 was also observed in the JY-P, cells, although it was undetectable in J Y cells. Expression of a', a*,a3,and a6 was not observed in either the J Y or JY-P, cells (not shown). From mean fluorescence intensity values, it was estimated that approximately 50% of the newly expressed PI in JY-PI cells was associated with a6 (-18 MFI units), leaving the other 50% to be associated with cy4. Because only part of the total a4 (-42 MFI units) appeared to be associated with P1 (-18 MFI units), it is assumed that the remaining a4 (-24 MFI units) may be a associated with a different /3 subunit. Adhesion Properties of Unstimulated JY and JY-PI CellsTo analyze the functional consequences of PI expression in J Y cells, adhesion assays were carried out as shown in Fig. 2. Unselected J Y cells adhered rather poorly to theFN-40 fragment of fibronectin ( A ) ,to theFN-120 fragment of fibronectin ( B ) , to the 25-amino acid CS1 peptide derived from a sequence within FN-40 ( C ) ,or to sVCAM-1 (D). In contrast, JY-B1 cells showed substantially greater adhesion at nearly all doses of each of the ligands tested. In the relatively short time period of the adhesion assay (25 min), no spreading was observed for either J Y or JY-P, cells. To determine the involvement of specific integrins in adhesion to each ligand, antibody inhibition experiments were
Functional Comparison
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of
performed. As shown in Fig. 3A, attachment of JY-@,cells to FN-40, CS1 peptide, and sVCAM-1 was uninhibited in the presence of either controlmAb, or the a n t i d MAb PlD6, but adhesion was almost completely inhibited by the anti-a4MAb HP2/1. In contrast, adhesion to the FN-120 fragment of fibronectin was substantially inhibited by the a n t i d mAb, but only minimally inhibited by the anti-a4 mAb. Adhesion to all of these substrates was highly inhibited by the anti-@, mAb BlE11. In anotherexperiment (Fig. 3B), attachment to sVCAM-1 by JY-P1 cells was almost completely abolished by two different anti-& antibodies (mAb 13, 4B4), but not by nonblocking anti-& antibodies (A-1A5, TS2/16; not shown) or by negative control antibodies (P3). Together the results in Fig. 3 are consistent with VLA-5-mediated binding to FN120, and VLA-4-mediated binding to FN-40, the CS1 peptide, and to sVCAM-1 for JY-P, cells. In contrast, attachment of JY cells to thevarious ligands was too low (
