Immunology

and Cell Biology {I99i)

69, 103-110

VLA-2 is a collagen receptor on endotheiial cells p. J. O'CONNELL,* R. FAULL.t G. R. RUSSt AND A. J. F. D'APICE* ^Department of Clinical Immunology. Si Vincent's Hospital. Victoria. 'Department ofNephrology. Royal Melbourne Ho.spifal, Victoria and the ^Transplantation Immunology Laboratory, Queen Elizabeth Hospital. South Australia. Au.stralia (Submitted !4 August 1991. .Accepted for publication 23 March 1991.) Summary In order to identify cell-substrate adhesion receptors on vascular endothelium, murine monoclonal antibodies (MoAb) were raised against human umbilical vein endotheiial cells (HUVE). One anli-HUVE MoAb, RMACl 1. identified the adhesion receptor VLA-2 as it immunoprecipitated a non-covalently linked heterodimer of 160 kD and 130 kD. which was identical to the heterodimer immunoprecipitated by the anti-VLA-2 MoAb. 12F1 and 5E8. Furthermore, proteolytic peptide maps of the VLA-2 a- and [i-chains were highly homologous with those of the RMACl l-recognized molecule. However, unlike other VLA-2 MoAb. RMACl I also identified an 85 kD band which migrated to 90 kD under reducing conditions. This band was most likely a fragment of the 160 kD a-chain as a similar u-chain derived fragment has been demonstrated in the immunoprecipilates of some VLA-4 reactive monoclonals. However the possibility that this may be a novel molecule associated with VLA-2 has not been excluded. M viirn assays of HUVE adhesion to collagen types I and 4, lamininand fibronectin showed that RMACl I blocked adhesion to collagen (types 1 and 4) and laminin, but had no effect on HUVE adhesion to fibronectin. confirming that VLA-2 is a collagen and laminin reeeptor for HUVE.

INTRODUCTION The very late activation antigens (VLA) are a family of human cell surface glycoproteins involved in a wide variety of cell-substrate adhesion interactions (I). At least six VLA receptors all sharing a common P-chain (P') have been described. They appear to be functionally distinct and have differing cell distributions (1). VLA-2, being one of the first VLA receptors to be identified, was so named because of its appearance 'very late' on mitogen-stimulatcd T cells and T cell clones (2,3). It was identified as a non-covalently linked heterodimer with a 165 kD a-chain and a 130 kD P-chain (4,5). With the production of a^ specific MoAb, such as 12F1 (5) and 5E8(6andM. Hemlerpers. comm.), it was realized that VLA-2 was not just a 'very late' activation marker on T cells but was also expressed on a wide variety of cells including platelets, fibroblasts, human neuronal cells and most adherent cell lines tested (5.7.8). VLA2 was initially identified as a collagen receptor

on platelets (9,10) and has been shown to be a laminin receptor on some but not all adherent cell types (II). Endotheiial cells express VLA-2 as well as other members of the VLA family (12,13) and VLA-2 has recently been identified as a receptor for both laminin and collagen on these cells (14). This repott describes the mouse monoclonal antibody RMACl 1 which reacts with a functional epitope on VLA-2 and inhibits HUVE binding to both laminin and collagen. RMACl 1 also precipitates an 85 kD molecule which is most likely a fragment ofthe a--chain. This is similar to the precipitation of an 80 kD a''-chain fragment by anti-VLA-4 antibodtes. MATERIALS AND M E T H O D S Endotheiial cells Fresh umbilical cords were obtained from the delivery ward ofthe Royal Hospital for Women,

Correspondence: Dr A. J. F. d'Apice, Department of Clinical Immunology, St Vincent's Hospital, Victoria Pde, Fitzroy. Vic. 3065, Australia. .4bhrevialions used in lhi.s paper. HUVE. human umbilical vein endotheiial eells; MoAb. monocional antibody; PBS, phosphate buffered saline; RT, room temperature; SAC, Staphyhcoccus aureus Cowan strain I eells; VLA, very late activation antigen.

104

P.J. O'CONNELL£r^L.

Melbourne. HUVE cells were isolated from the umbilical vein by the method of Gimbrone (15) and were serially passaged as described by Thornton et al. (16). In brief, the cells were plated on to 75 mm^ tissue culture flasks (Costar, MA) and cultured in RPMl (Flow Laboratories, Australia) supplemented with 20% fetal calf serum (Flow Laboratories), 20 tig/mL endothelial cell growth supplement (Sigma. USA), 100 U/mL bovine heparin. 4 mmol/L glutamine, 100 U/mL penicillin and 1 ng/mL streptomycin. When the cells reached confluence they were dislodged with 0.1% EDTA (Sigma, USA) in Hanks balanced salt solution without calcium and magnesium (Commonwealth Serum Laboratories. Australia) and passaged after 1:4 dilution. The flasks were incubated at 37°C in 5% COi and the cells were used on their third to ninth subculture. Production ofRMACII The MoAb RMACl I. an lgG2a antibody, was produced by immunizing Balb/c mice intraperitoneally with HUVE cells on days 31 and 3 prior to fusion. Sensitized spleen cells were then fused with the murine myeloma cell line Sp2/0-Agl4 (17) and grown in selective medium. Hybridomas recognizing anti-HUVE cell determinants were detected by enzyme linked immunoabsorbent assay, where the primary antigen was paraformaldehyde (0.1%) fixed HUVE cells. Selected positive hybrids were cloned by limiting dilution and RMACl 1 was obtained from mouse ascites by ammonium sulfate precipitation. Monoclonal antibodies W6/32 (igGi) recognizes a non-polymorphic determinant on MHC class I molecules and RM2.I84 (IgG2a) which recognizes a polymorphism of CR3 (18) were used as negative controls. The MoAb 12F1 (5)and 5E8(6)recognize the a-chain of VLA-2 and were provided by Dr Pischel. San Francisco and Dr Zyistra, Buflalo NY respectively. Isolation of platelets Trisodium citrate (2.3%) (Ajax Chemicals. Aust.) was added to venous blood at 1:10 dilution and the mixture was centrifuged at iOO,? for 10 min. The platelet-rich plasma was decanted, diluted with RPMI-1640 and centrifuged at 2000 g for 20 min. The platelet pellet

was washed twice and contaminating leucocytes pelleted by centrifugation at 100 g for 10 min. The purity of the platelet preparation was assessed using a Neubauer haemocytometer and phase contrast microscopy. The platelet rich supernatant was then centrifuged at 2000 ^ for 10 min and resuspended to the desired concentration. Cell surface iodination HUVE cells were dislodged by incubating in 0.1% EDTA in Hank's balanced salt solution without calcium and magnesium and washed with phosphate buffered saline (PBS). Platelets were isolated as described. An aliquot 1-2 X 10^ HUVE cells or I X 10' platelets per sample were resuspended in 150 |JL of PBS and iodinated with NA ['-5|] (Amershani. UK) by the lactoperoxidase method (19). The cells were washed three times with PBS containing 1 mmol/L potassium iodide and 0.02% NaN3. Cell membrane molecules were solubilized by mixing the cells in lysis bufl'er (0.5% Triton X-IOO and 2 mmol/L PMSF in PBS pH 7.4) for 90 min at 4T. The suspension was then centrifuged at 2500 f> for 15 min at 4°C to remove nuclear debris. Immunoprecipitation and SOS-PAGE analysis Soluble cell membrane molecules were precieared by the addition of saturating amounts of an irrelevant monoclonal antibody for 1 h at 4 T followed by 200 (,iL of 10% suspension of Staphvlococcus aureu.s. Cowan strain 1 cells (SAC) (Sigma, USA) in PBS (pH 7.4) with 0.5% Triton X-100. 4.8 mmoi/L potassium iodide and 0.02% NaN3. After incubating overnight at 4°C the precleared cell membrane extract was incubated with 5 pL of RMACl 1 ascites or an irrelevant MoAb for 1 h at 4 T and then for 30 min with 200 nL of 10% SAC. The SAC was washed twice with 0.1 mmol/L Tris, 0.4 mmol/L NaCI bufler pH 8.3 plus 0.02% NaN.i and resuspended and boiled for 3 min in 0.1 mnioi/L Tris pH6.8 containing 10% glycerol and 2% SDS. The samples were electrophoresed in the presence or absence of 50 mmol/L dithiothrietol (Biorad). After boiling, the samples were centrifuged for 3-4 min in a Beckman microcentrifugc and the supernatants applied to 7.5% SDS-polyacrylamide gels. After electrophoresis the gels were fixed, dried and autoradiographcd using Kodak XRP-5 X-ray film and Dupont Cronex Hi-plus intensifying screens.

105

VLA-2 ON ENDOTHELIAL CELLS

Froteolytic peptide mapping

Proteins purified by one dimensional SDSPAGE were cut from the SDS polyacrylamide gels and placed in the sample well ofa second 15% SDS gel. The gels were overlaid with varying concentrations of S. aureus V8 protease (Sigma, USA) which was dissolved in nonreducing sample buffer and the gel slices were incubated for 30 min at room temperature prior to electrophoresis on the slab gel. When the protease solution had migrated approximately half way through the stacking gel. the current was turned off for a further 30-min incubation. The electrophoresis, staining, drying and autoradiographing ofthe gel then proceeded in the usual manner. Densitometry scanning ofthe a-chain molecules was performed on an LKB Ultrascan XL enhanced laser densitometer. HWE-sttbstrate adhesion assay Ninety-six well microtitre plates were incubated overnight at room temperature (RT) with 10 Hg/mL fibronectin (Boehringer Mannheim. Germany). 100 jig/mL collagen type I {Sigma. St Louis, MO), 100 jig/mL collagen type 4 (Sigma). 20 fig/mLlaminin (Sigma) or 0.5% gelatin, which were all diluted in Hank's balanced salt solution. After washing with PBS, the plates were blocked with 1% bovine serum albumin in PBS for I h at RT. HUVE were eluted from culture flasks (as above), resuspended at a concentration of 2 X 10^ cells/mL and incubated for 10 min at RT with the relevant MoAb, which were

added to the cells as 1:200 dilution of ascites. After washing the plates with PBS. 2X10^ cells were added to each well and incubated at 37°C for 2 h. Non-adherent cells were washed off by inversion of the plate and 'flicking". PBS was then added to the wells and the procedure repeated twice. Adherent cells were quantified by counting the number of HUVE remaining in five random high-power fields in each well and expressed as a percentage ofthe number of adherent cells in the control wells. Experiments were performed in triplicate. RESULTS RMACn recognizes VLA-2 Lysates of [i^nj-labelled HUVE or platelets were immunoprecipitated with RMACl 1 and electrophoresed by SDS-PAGE. Non-reduced immunoprecipitates of HUVE or platelet lysates always identified two non-covalently linked bands of 150 and 11 5 kD which migrated as two bands of 160 and 130 kD under reducing conditions (Fig. I). In some but not all precipitates, a third band of 85 kD (Fig. 1. lane 5) was also identified. This 85 kD band migrated to 90-95 kD under reducing conditions (Fig. I, lane 4) and could be identified in both HUVE and platelet precipitates (Fig. I. lanes 4 and 5). To identify the RMACl 1 recognized molecule as VLA-2. HUVE lysates were immunoprecipitated with RMACl I or the anti-VLA-2 MoAb 12F1 and electrophoresed by one-dimensional

Table I. Substrate

Experiment

Adherent HUVE/IOhpf W6/32 RMACl I

Inhibition (%)

Fibronectin

1 2 3 Mean (s.d.) 1 2 3 Mean (s.d.)

388 388 279 351 456 488 380 441 488 416 388 430 336 284 308 309

-26.8 -16.5 -19.7 -21.0 34.2 45.1 25.0 34.7 59.0 34.6 29.6 41.1 50.0 36.6 4.5 30.4

Collagen type 1

Collagen type 4

1

2 3 Mean (s.d.) Laminin

1

2 3 Mean (s.d.)

(63)

(55)

(52)

(26)

492 452 334 426 (82) 300 268 285 284 (16) 200 272 273 248 168 180 294 214

(42)

(70)

NS (5.3) O.OI (10.1)

0.01 (15.7)

NS (23.4)

106

P. J.O•CONNELL£7^4Z..

200

I

116 97

200 116

97 116

97

66

66

66

42 42

1

2

3

4

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Fig.l. SDS-PAGE ofRMACll immunoprecipitates from [""i].|abelled HUVEand platelet lysates. Lanes land 3 show HUVE lysates immunoprccipitated with a control Mo.Ab (RM2.184) and lanes 2 and 4 show HUVE lysates immunoprecipitated wiih RMACl t under non-reducing (lane 2) and reducing conditions (lane 4). Lane 5 shows platelet lysates immunoprecipitated with RMACl 1 under non-reducing conditions.

SDS-PAGE (Fig. 2). In immunoprecipitations of both HUVE (Fig. 2) and platelet lysates. the aand P-chains of the RMACl 1 molecule were identical to the VLA-2 molecule identified by 12F1 (Fig. 2). The 85 kD band seen in RMACl I precipitates however, was never seen in repeated 12F1 or 5E8 precipitates. To further demonstrate that RMACl 1 recognized VLA-2, one-dimensional peptide maps using S. aureu.s V8 protease were performed on the RMACl 1 and I2F1 immunoprecipitates. Proteolytic digestions of radiolabel!ed a- and pchains from HUVE lysates showed that both pchains were equally resistant to proteolysis with V8 protease (Fig. 3a). The peptide maps of the RMACl I and 12F1 a-chains revealed that the peptide maps of the RMACU a-chain were highly homologous with the peptide map of the

I2F1 (VLA-2) a-chain at two different protease concentrations (Fig. 3b). Furthermore, profiles produced by densitometric scanning of the RMACl 1 and I2F1 a-chain products were almost identical which confirms the homologous appearance seen on the autoradiographs (Fig. 3c). Supporting evidence that the RMACl 1-and 12Fl-identified molecules were identical was obtained from sequential immunoprecipitations of radiolabeiled HUVE lysate which showed that RMACl I completely precleared the molecule recognized by 12FI and in separate experiments precleared the molecule recognized by the anti-VLA-2 MoAb 5E8. Furthermore the migration patterns of the RMACl 1 molecule on 2D-IEF/SDS-PAGE were identical to those of VLA-2 (data not shown).

107

VLA-2 ON ENtX)THELIAL CELLS

200-

116-

Fewer cells (approximately 30% less) adhered to iaminin and gelatin and had a more spindly appearance. The anti-HLA class I MoAb ^dlbl had no effect on adhesion. RMACl I inhibited adhesionof HUVE to collagen types 1 and 4 and laminin by approximately 50% compared to W6/32 (Table I) but did not inhibit adhesion to fibronectin. Inhibition was concentration dependent with maximum inhibition occurring at a dilution of ascitic fluid of approximately 1:200. In other experiments, an anti-VLA-5 (fibronectin receptor) MoAb inhibited adhesion to fibronectin but not to collagen or laminin. The appearance of non-adherent cells was clearly different from those in the control wells when they were examined before washing. They remained round and failed to spread on the substrate. A number of the cells that remained after washing the wells containing RMACl I still had this appearance, while the adherent cells in the control wells were spread and flattened.

DISCUSSION

42

Fig. 2. SDS-PAGE of [""l]-labelled HUVE lysates immunoprecipitated with RMACl 1 (lane l)and I2FI (lane 2) and electrophoresed on a 7.5% polyaerytamide gel under non-reducing conditions.

RMACl I inhibits the adherence ofHVVE to hoth collagen and laminin Cultured HUVE adhered and spread well on purified fibronectin and collagen types I and 4.

VLA-2 was one of the first VLA proteins to be described. It was first identified on cytotoxic Tcell clones by the anti-VLA-p MoAb A-1A5, which recognized the 130 kD p'-subunit noncovalently linked to the 210 kDa'-and the 165 kD a'-chain (3). The identity of the VLA-2 molecule was confirmed by the MoAb 12FI. which reacted with the a-chaIn of VLA-2 and immunoprecipitated a non-covalently linked heterodimer of 165 and 130 kD (5). The 130 kD P-chain was identical to the P'-chain recognized by A-IA5, which indicates that all the a--chain recognized by I2F1 was associated with the AIA5 p'-chain (5). Screening of tissues, cells and cell lines showed that 12F1 reacted with platelets, fibroblasts. the T-cell leukaemic line HSB, neuroblastoma cells and most adherent cell lines tested (3,7,8). VLA-2 has been identified as a collagen receptor (9,10) and corresponds to the glycoprotein-complex Ia-Ila on platelets (20). It is one of five VLA molecules, with VLA-1, 3, 5 and 6, expressed on HUVE (13.14). VLA-2 has been identified as the major laminin receptor on HUVE although VLA-3 and VLA-6 (both laminin receptors) are also expressed on these cells (4). The data presented in this report show that RMAC11 recognizes the VLA-2 a-chain and inhibits HUVE binding to collagen types 1 and 4 and laminin. RMACl I immunoprecipitated a non-covalently linked heterodimer of 160 and

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Fig. 3. Proicolylicpcplidcmapsofthc l2Fl-aiid RMACl l-dctiiicd «-and {i-chains. [i--M]-labcllcd bands from HUVE immunoprecipitates were cut from an SDS-PAGE gel. The gel pieces containing the a- and p-chains were treated with 0.5 ^g or 5 \ig ofS. aureus V8 protease. The digests were then electrophoresed on a 1 5% SDS ptilyacrylamide gel. (a) shows the peptide maps ofthe 12Fl (lane I and 3) and RMACI1 (lane 2 and 4) (Vchains and (b) shows the peptide maps ofthe 12FI (lane 5 and 7) and RMACI 1 (lane 6 and 8) a-chains. Autoradiographs ofthc RMACI I and 12FI a-chain peptide maps were subjected to densitometric scanning. Section (c) shows densitometer profiles ofthe RMACI 1 a-chain (a) and the 12FI a-chain (b) after they had been subjected to 0.5 jigof V8 protease (from laties 5 and 6). 130 kD (Fig. I), and one-dimensional SDSPAGE ofRMACI 1 imniunoprecipitations were identical to those ofthe VLA-2 molecule recognized by 12FI (Fig. 2). Furthermore, proteolytic peptide maps ofthe 12Fl-and RMAClI-recog-

nized a- and ^-chains were highly homologous (Fig. 3). In addition to the 160 kD/130 kD a^/p' heterodimer. RMACI 1 immunoprecipitates often contained an 85-kD band, which migrated as a

109

VLA-2 ON ENDOTHELIAL CELLS

90-95 kD band under reducing conditions. This band was not seen in 12FI or 5E8 precipitates. A similar band has been seen with VLA-4 precipitates (8) and was recently identified with another anti-VLA-2 antibody which also inhibited substrate binding. Immunoprecipitations of VLA-1 and -2 from PHA-activatcd T-cells. with the anti-VLA-^' MoAb. A-1A5, have identified a similar band (21). but this was previously considered to be due to the co-precipitation of VLA4 from these cells (8). The 80-kD band in VLA-4 immunoprecipitates was shown to be derived from the 150-kD a^-chain as it was recognized by anti-a'' antisera; it had an identical NHvterminal sequence to a*^ and trypsin cleaved a** into an 80 and a 70 kD band respectively (8). Furthermore, the cDNA clones of the VLA-4 achain have identified a potential protease cleavage site near the middle of the coding region (22). It therefore seems reasonable to suggest thai the 85-kD molecule immunoprecipitated by RMACl I is probably also a product ofthe a-chain and bears the RMACl l-a- reactive epitope while the I2FI and 5E8-rcactive epitopes are not contained within the 85-kD fragment. The possibility that the 85-kD fragment is a novel chain which is non-covalently associated with the a-/p' complex has not been positively excluded by these experiments. If the 85-kD molecule is a fragment ofthe a--chain. it is not clear whether this is due to endogenous proteoiysis with biological significance or an artifact in the preparation ofthe lysate. It should be noted, however, that unlike VLA-4. a potential proteolytic cleavage site has not been identified within the primary structure of VLA-2 (22). HUVE express VLA-2 as well as other members ofthe VLA family (12). VLA-2 has been shown to be a collagen and laminin receptor on a number of cell types, e.g. platelets (10.11.14). These results confirm that VLA-2 is a receptor for collagen types 1 and 4 and laminin on HUVE. Adhesion is not completely inhibited by RMAC 11 in these experiments, presumably due to t he contribution to adhesion by other collagen and laminin receptors such as VLA-1. 3 and 6 beingunaffected by RMACll. RMACl I clearly recognizes a functional epitope on VLA-2 and the immunoprecipitation studies localize the epitope and probably the functional binding sites for iaminin and collagen to the 85-kD fragment ofthe a--chain. Like platelets, endothelial cells express a large number of adhesion-related molecules including the vitronectin receptor. VLA-2. 3. 5 and 6. Three (and possibly four) of these receptors are able to bind laminin and three bind fibronectin.

The reason for such reeeptor redundancy is unclear. Laminin has been shown not to be a single protein but a mixture of proteins and Ruoslahti's group has proposed that different laminin binding integrins may be required to bind different forms of laminin (14). At this point, however, it remains unclear what role each ofthe laminin receptors has in the binding of endothelium to the basement membrane.

ACKNOWLEDGEMENTS This work was supported in part by a grant from the National Health and Medical Research Council of Australia, Dr P. O'Connell was a recipient ofa NH and MRC postgraduate medical scholarship.

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10. Staatz, W. D., Rajpara, S. M.. Wayner, E. A.. Carter. W. G. and Santoro. S. A. 1989. The membrane glycoprotein la-lla (VLA-2) complex mediates the Mg"'^ ^-dependent adhesion of platelets to collagen. / Cell. Biol. 108: 19171924. 11. Elices. M. J. and Hemler. M. E. 1989. The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others. Proc. NatlAcad. ScL US.4 86: 9906-9910. 12. Giltay, J. C , Brinkman. H-J. M.. Modderman. P. W.. von dem Bome A. E. G. and van Mourik, J. A. 1989. Human vascular endothelial cells express a membrane protein complex immunochemically indistinguishable from tbe platelet VLA-2 (glycoprotein la-lla) complex. Blood 73: 1235-1241. 13. Abelda, S. M.. Daise. M.. Levine. E. M. and Buck. C. A. 1989. Identification and characterization of cell-substratum adhesion receptors on cultured human endothelial cells../. Clin. lnvt'.\l. 83: 19922002. 14. Languino. L. R.. Gehlsen, K. R.. Wayner, E., Carter. W. G.. Engvall. E. and Ruoslahti. E. 1989. Endothelial cells use a2(i| integrin as a laminin receptor. / Cell. Biol. 109: 2455-2462.

15. Gimbrone. M. A. 1976. Culture of vascular endothelium. P>V}i. Hemosi. Thromh. 3: I. 16. Thornton. S. C . Mueller. S. N. and Levine, E. M. 1983. Human endothelial cells: use of heparin in cloning and long term serial cultivation. Science 222: 623. 17. Schulman. M.. Wilde. C. D. and Kohler G. 1978. A cell line for making hybridomas secreting specific antibodies. Naliirc 276: 269. 18. Russ, G. R.. Haddad. A. P.. Tail. B. D.. d'Apice. A. J. E. 1985. Polymorphism of the complement receptor for C3bi. J. Clin. Inve.si. 76: 1965-1970. 19. Goding, J. W. 1983. Monoclonal .iniihodies: Prim ipte.s and Practice. Academic Press. London. 20. Pischel. K.. Bluestein. H. and Woods. V. 1988. Platelet glycoproteins la. Ic and II are physicochemically indistinguishable from tbe very late activation antigens adhesion-related proteins of lymphocytes and olher cell types../. (///(. Invest. 81: 505-513. 21. Hemler. M. E., Ware, C. F, and Strominger, J. L. 1983. Characterization of a novel differentiation antigen complex recognized by a monoclonal antibody (A-IA5): unique activation-specific molecular forms on stimulated T cells.,/. Immunol. 131: 334-340.