Eur J Biochem 270, 1710–1723 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03528.x Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins Eveliina Ihanus1, Liisa Uotila1, Anne Toivanen1, Michael Stefanidakis1, Pascal Bailly2, Jean-Pierre Cartron2 and Carl G Gahmberg1 Department of Biosciences, Division of Biochemistry, University of Helsinki, Finland; 2INSERM U76, Institut National de Transfusion Sanguine, Paris, France Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding ICAM-4 lacks such a residue, which is replaced by an arginine However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations Inter- estingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2 Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins The five intercellular adhesion molecules ICAM-1–5 currently known in humans, form a family of related cell surface glycoproteins, which mediate cell adhesion by binding to the leukocyte CD11/CD18 integrins All the ICAM proteins have extracellular C-type immunoglobulinlike domains ranging in number from two to nine making them members of the immunoglobulin superfamily [1–4] Despite their structural similarity and integrin binding capability, these proteins have a differential pattern of expression and cellular distribution ICAM-1 which consists of five Ig-like domains, is found on the surface of leukocytes, endothelial cells and various other cells, and can be up-regulated by several proinflammatory cytokines [5,6] ICAM-2 has two Ig-like domains It is constitutively expressed by leukocytes, endothelial cells [6], and platelets [7] ICAM-3 is composed of five Ig-like domains, and it is present at high levels on resting lymphocytes, monocytes, and granulocytes It is the only ICAM significantly expressed on neutrophils [8] The expression of ICAM-4 is restricted to erythrocytes and erythroid precursor cells [9] ICAM-5 is expressed by subsets of neurons, exclusively within the telencephalon of the mammalian brain [10] The predominant cellular ligands for the ICAMs are the leukocyte CD11/CD18 integrins, which consist of four heterodimeric glycoproteins with specific a chains (CD11a, -b, -c, -d) and a common b2 chain (CD18) They play an essential role in mediating adhesion of cells in the immune system [1–4] All five ICAM molecules are able to bind to CD11a/CD18 (LFA-1, aLb2) which is expressed on all leukocytes The first NH2-terminal Ig domain of each ICAM seems to be most important for binding [11–15] ICAM-1, -2 and -4 have been shown to interact also with CD11b/CD18 (Mac-1, aMb2, CR3), which is expressed primarily on the cells of the myelo-monocytic lineage The third Ig-like domain in ICAM-1 [16] and the first NH2terminal Ig domain in ICAM-2 seem to mediate CD11b/ CD18 binding [12] Extensive work has been carried out to localize the ligand binding sites in the leukocyte b2 integrins The a chains of the CD11/CD18 integrins contain an inserted approximately 200-amino-acid intervening domain (I domain), which is homologous to the A domains of von Willebrand factor, repeats in cartilage matrix protein and collagen [17] The I domains of CD11/CD18 integrins have been shown to contain recognition sites for most ligands of leukocyte integrins ICAM-1, -2 and -3 as well as several soluble proteins such as fibrinogen, and the complement component iC3b bind to the I domains of their receptor integrins Correspondence to C G Gahmberg, Department of Biosciences, Division of Biochemistry, P.O Box 56, Viikinkaari 5, FIN-00014 University of Helsinki, Finland Fax: + 358 191 59068, Tel.: + 358 191 59028, E-mail: Carl.Gahmberg@helsinki.fi Abbreviations: ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; CD11a/CD18, LFA-1, leukocyte function associated antigen; CD11b/CD18, Mac-1; LW, Landsteiner– Wiener blood group antigen; GST, glutathione S-transferase (Received 14 January 2003, accepted 20 February 2003) Keywords: adhesion; ICAM, integrin; I domain; red cell Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur J Biochem 270) 1711 By using monoclonal antibodies reacting with integrin I domains and by mutational analysis of the I domains, evidence has been obtained that the binding sites on the I domains for different ligands are overlapping but not identical [18–22] Integrins need divalent cations for their activity, and they have been shown to bind Ca2+ and Mg2+ [23,24] Importantly, the I domains have been shown to bind divalent cations [20,24] ICAM-4 was originally identified as a 42-kDa red cell membrane glycoprotein called the LW (Landsteiner– Wiener) blood group antigen [25] The LW protein has been reported to require intramolecular disulfide bonds and the presence of divalent cations, notably Mg2+, for antigenic activity [26] The LW and Rh blood groups show an interesting phenotypic relationship, as the level of LW expression is greater in RhD-positive than in RhDnegative cells, and extremely rare Rhnull cells, which lack all Rh antigens, are also deficient in the LW protein However, individuals lacking LW antigens have been found among RhD-positive individuals [27] The LW glycoprotein has been renamed ICAM-4 based on strong sequence similarities with the ICAM family and subsequent work, which showed that it binds CD11a/CD18 and CD11b/CD18 integrins [9,28] The ICAM-4 protein contains two immunoglobulin domains of which the first domain is 30% identical to the first domains of ICAM-1, -2 and -3 CD11a/CD18 has been found to bind to the first Ig domain, whereas CD11b/CD18 binding sites encompass both domains of ICAM-4 [14] Unlike the other ICAMs, ICAM-4 does not contain the conserved glutamate residue in the first domain, which is replaced by an arginine 52 residue (Table 1) Mutation of arginine 52 back to glutamate did not affect CD11a/CD18 binding and even reduced the interaction with CD11b/CD18 Instead, site-directed mutagenesis studies identified other residues on the CFG face of the first domain, which are involved in CD11a/CD18 recognition These data suggest that the b2 integrin binding motifs of ICAM-4 differ from those of other ICAMs [14] However, the adjacent residues at arginine 52 in ICAM-4 are identical or similar in the five ICAMs, suggesting a potential role for these residues The noncharged LLG sequence present on ICAM-1 has been reported to function as a ligandbinding motif for CD18 integrins [29] A recent study has suggested that ICAM-4 can also bind through novel motifs to a4b1 and av family integrins [30] In the present study, we wanted to define the role of the CD11a and CD11b I domains in ICAM-4 binding Our results show that ICAM-4 binds specifically to the CD11a and CD11b I domains Table Sequence alignments of the first immunoglobulin domains of ICAM molecules illustrating the glutamate to arginine (shown in bold) difference in ICAM-4 and the surrounding conserved residues (shown as italic) ICAM-4 ICAM-1 ICAM-2 ICAM-3 ICAM-5 NSSLRTPLRQ LLGIETPLPK VGGLETSLNK KIALETSLSK RGGLETSLRR Materials and methods Antibodies The b2 integrin specific mAbs used in these studies include TS1/22, MEM83, MEM30, MEM25, MEM177, 7E3, 60.1, LM2/1, MEM170, 44, 107 and 904 TS1/22 (American Type culture Collection, Rockville, MD), MEM83, MEM30, MEM25 and MEM177 recognize the a chain of CD11a/CD18 TS1/22, MEM83, MEM30 and MEM25 has been mapped to the I domain of the CD11a/CD18 [31,32] The anti-CD11b mAbs 7E3, 60.1, LM2/1 (American Type culture Collection, Rockville, MD), MEM170, 44, 107 and 904 have been described previously [33,34] and are specific for the I domain The ICAM-1 antibodies have been described: GP8911, GP8914 and GP8923 (the Leukocyte Typing Workshop V), LB-2 [35] and RR1/1 [36] The ICAM-1 mAb B-H17, was a generous gift from C VermotDesroches, Diaclone, France The three ICAM-2 mAbs (B-T1, B-R7 and B-S9) have been described previously [37] The mAbs BS46 and BS56 react with the first domain of the ICAM-4 [14,38] A mouse IgG1 negative control was purchased from Silenius (Hawthorn, Australia) and polyclonal goat anti-GST antibodies from Pharmacia Biotech Inc The goat antihuman IgG specific antibody was obtained from Sigma and the peroxidase-conjugated antiGST mAb from Santa Cruz Biotechnology (Santa Cruz, CA) Purification of the CD11a/CD18 and CD11b/CD18 integrins CD11a/CD18 and CD11b/CD18 integrins were purified from human blood buffy coat cell lysates as described previously [12] Expression and purification of the CD11a and CD11b I domains The Escherichia coli strain JM109 transformed with the pGEX-2T (Pharmacia Biotech Inc.) plasmid containing the cDNA fragment of the CD11b I domain was a generous gift from A Arnaout (Massachusetts General Hospital, Boston, MA) [20] The cDNA fragment of CD11a I domain inserted into the expression vector pGEX-5X-3 (Pharmacia Biotech Inc.) was kindly provided by D Altieri and was constructed as reported by Muchowski et al [39] The pGEX-5X-3 plasmid construct containing the cDNA of CD11a I domain was transformed into the E coli strain BL21 The glutathione S-transferase fusion proteins of the I domains were expressed in Escherichia coli cells (strain BL21 for CD11a I domain; strain JM109 for CD11b I domain) as described previously [20,39] Cell pellets derived from to L culture were thawed and lyzed by resuspending in 30 mL of lyzing buffer (10% sucrose, 0.5% Triton X-100, 50 mM Tris, pH 8.0) containing mM EDTA, mM dithiothreitol, mM phenylmethylsulfonyl fluoride, lgỈmL)1 aprotinin and leupeptin After lysozyme (350 lgỈmL)1) treatment the cells were sonicated on ice Triton X-100 was then added to a final concentration of 1%, and the sonication was repeated After centrifugation at 12 000 g for 10 min, the supernatant was incubated with Ó FEBS 2003 1712 E Ihanus et al (Eur J Biochem 270) prewashed glutathione-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) for h at °C The resin was then washed with 50 mL of 50 mM Tris/HCl, pH 8.0, 50 mL of 500 mM NaCl, 1% Triton X-100, 50 mM Tris/HCl, pH 8.0 and again with 50 mL of 50 mM Tris/HCl, pH 8.0 The fusion proteins were eluted with 20 mM reduced glutathione, 50 mM Tris/HCl, pH 8.0 and the samples were then passed through a Bio-Gel P-6DG column to remove glutathione For the experiments, which utilized the I domains separated from the GST moiety, the purified GST fusion proteins of the I domains of CD11a/CD18 and CD11b/ CD18 were cleaved with Factor Xa and thrombin, respectively The GST fusion protein of CD11a I domain was cleaved with biotin-labeled restriction protease Factor Xa (Boehringer Mannheim) The biotin-labeled Factor Xa was removed using a streptavidin gel and the free GST using the glutathione resin To release the recombinant CD11b I domain the fusion protein was treated with thrombin (Sigma) and the cleaved sample was then further purified by ion exchange chromatography on a Mono S HR5/5 column (Pharmacia) using the FPLC system (Pharmacia) Analysis of the purified recombinant I domains on 12% SDS/PAGE revealed a single band of the expected size after staining with Coomassie blue Expression and purification of ICAM-Fc recombinant proteins The ICAM-2Fc and ICAM-4Fc fusion proteins were produced by transient transfection of COS-1 cells by the DEAE-dextran method (Pharmacia) and isolated from the culture supernatants by protein A-Sepharose CL-4B (Pharmacia) chromatography essentially as previously described [14,40] ICAM-1Fc and VCAM-1Fc fusion proteins were obtained from R & D Systems All the recombinant proteins were checked by SDS/PAGE and Western blotting The ICAM-2Fc cDNA vector was kindly provided by D Simmons (John Radcliffe Hospital, Oxford, UK) and the ICAM-4Fc cDNA has been described previously [14] Cells and cell lines Blood samples from common LW and Rh phenotypes (ICAM-4 positive red cells) were obtained from normal volunteers using heparin as an anticoagulant and the LW(a–, b–) blood sample (ICAM-4 negative red cells) was kindly provided by Kathy Burnie (Hematology University Hospital, Ontario, Canada) The LW(a–, b–) cells and the control cells were stored at )70 °C until further used The L929 mouse fibroblast cell line was maintained in IMDM medium supplemented with 10% fetal bovine serum, 100 mL)1 penicillin, and 100 lgỈmL)1 streptomycin The full-length ICAM-1 or ICAM-2 cDNAs in pEF-BOS vector and the full-length ICAM-4 cDNA in pcDNA I vector were separately cotransfected with pCDM8-neo stuffer into L929 mouse fibroblast cells according to standard procedures using either the Lipofectamine reagent kit (Life Technologies, Gaithersburg, MD) or the calcium phosphate precipitation method Stable transfectants were selected in medium containing 0.5 mgỈmL)1 G418 The G418-resistant cell populations were analyzed for ICAM expression with a Becton Dickinson (Immunocytometry Systems, San Jose, CA) FACScan flow cytometer The L929 cells expressing either ICAM-1, ICAM-2 or ICAM-4 were cloned by limiting dilution The SV40-transformed African green monkey kidney cell line COS-1 (ATCC) was grown in DMEM supplemented with 10% fetal bovine serum, 100 mL)1 penicillin, and 100 lgỈmL)1 streptomycin Flow cytometry studies Wild type, ICAM-1-, ICAM-2-, or ICAM-4-transfected L cells and ICAM-4-positive or -negative red cells were washed and resuspended in NaCl/Pi, pH 7.4 Aliquots of · 106 L cells or 1–3 lL of packed red cells were incubated with 25 lgỈmL)1 of different anti-ICAM mAbs for 30–60 on ice The cells were washed with NaCl/Pi and incubated with FITC-conjugated rabbit antimouse F(ab¢)2 (Dakopatts a/s, Copenhagen, Denmark) for 30 on ice After washing, · 104 cells were analyzed with a Becton Dickinson (Immunocytometry Systems, San Jose, CA, USA) FACScan flow cytometer Red cell binding assays Indicated amounts of the purified CD11b/CD18, recombinant CD11a and CD11b I domains or control proteins were coated on plastic 96-well plates (Nunc, Roskilde, Denmark) in 25 mM Tris, pH 8.0, 150 mM NaCl, mM MgCl2 by incubation overnight at °C The wells were blocked with 1% BSA for h at room temperature and washed ICAM-4 positive or negative red cells (0.7 · 106 per well) in binding buffer (RPMI 1640 supplemented with 50 mM Hepes, pH 7.4, mM MgCl2, mM CaCl2, and 5% fetal bovine serum) were added to the wells and the plates were then briefly centrifuged (900 g, · min) and incubated for h at 37 °C The input of red cells was quantitated by counting cells in four randomly chosen fields from duplicate wells To remove nonadherent cells, the wells were gently filled with binding buffer, and the microplate was placed floating upside down for 40 in NaCl/Pi solution before microscopic observation and scoring the number of attached cells in four randomly chosen fields from duplicate wells The data was presented as a percentage of bound cells (amount of bound red cells divided by input of cells) For blocking experiments, the cells or protein-coated wells were pretreated with different mAbs (25 lgỈmL)1) or soluble GST/I domain GST (0.2–3 lM) for 10 at room temperature before starting the adhesion For the binding study with or without divalent cations, the adhesion assays were performed with buffers containing mM EDTA, mM EGTA and mM MgCl2, or mM MgCl2 and mM CaCl2 The results of antibody inhibition assays were expressed as a relative percentage of attached cells, where 100% is given as the number of cells bound in the absence of inhibitors The significance was determined by unpaired Student’s t-test Adhesion assays of ICAM transfectants The control protein glycophorin A (1 lg per well) and the purified CD11a/CD18 (1 lg per well) and CD11b/CD18 Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur J Biochem 270) 1713 (0.2 lg per well) were diluted in 25 mM Tris, pH 8.0, 150 mM NaCl, mM MgCl2 and attached to flat-bottom, 96-well microtiter plates (Nunc, Roskilde, Denmark) in 0.01–0.1% n-octyl glucoside by overnight incubation at °C The wells were blocked with 1% BSA for h at room temperature For the experiments with coated GST I domains the anti-GST antibodies (Pharmacia Biotech Inc.) diluted in 25 mM Tris, pH 8.0, 150 mM NaCl, mM MgCl2 were adsorbed overnight at °C, at 0.2–1 lg per well (50 lL per well in triplicate) on flat-bottom, 96-well microtiter plates After blocking nonspecific sites, indicated amounts (0.4–2 lg per well) of GST or recombinant purified GST I domains were added to the wells and incubated for h at room temperature Wells were then washed three times with the binding medium (Iscove’s MDM with 50 mM Hepes, pH 7.4, 0.5% BSA, mM MgCl2 and mM CaCl2) prior to addition of the cells (1.5 · 105 per well, in the binding medium unless otherwise indicated) For blocking experiments, the cells or protein-coated wells were pretreated with different mAbs (25 lgỈmL)1) or soluble GST/I domain GST (0.2–4 lM) for 10 at room temperature After 20 incubation at room temperature the wells were filled with binding buffer, and the microplate was put to float upside down for h in NaCl/Pi solution to remove unbound cells The bound cells (or the total amount of added cells without washing) were lyzed in 100 lL per well phosphatase substrate-containing lyzis buffer (1% Triton X-100 and 50 mM sodium acetate, pH 5.0) and incubated at 37 °C for 30 The reaction was terminated by adding 50 lL per well of M NaOH and the absorbance at 405 nm was measured [41] Solid phase ELISA assay The 96-well plates (Greiner) were coated overnight at °C with 400 ng per well of goat antihuman IgG Fc specific antibody (Sigma) in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl (TBS) After blocking nonspecific sites with 3% BSA in TBS for h at 37 °C the wells were washed three times with TBS, mM CaCl2, mM MgCl2, 0.05% Tween 20, 1% BSA The recombinant proteins (200 ng per well) diluted in TBS, mM CaCl2, mM MgCl2, 1% BSA was then added to the wells and incubated for h at room temperature The wells were washed as before, and 50 lL per well I domain GST or control GST (0–20 lgỈmL)1) diluted in TBS, mM CaCl2, mM MgCl2, 1% BSA was added and incubated at room temperature for h Plates were washed gently three times with TBS, mM CaCl2, mM MgCl2, 0.05% Tween 20, 1% BSA prior to the addition of a peroxidase-conjugated anti-GST mAb (1 : 500 dilution, Santa Cruz Biotechnology) to the wells After h of incubation at 37 °C, the plates were washed as before, and the bound proteins were detected with 100 lL per well 0.5 mgỈmL)1 o-phenylenediamine dihydrochloride added for 10 min, stopped by addition of 50 lL of 12.5% H2SO4 and plates were read in an ELISA reader For inhibition experiments, the soluble GST fusion proteins or protein-coated wells were pretreated with different mAbs (20–40 lgỈmL)1) or inhibitor proteins (ICAMFc proteins, 200 nM; I domains, lM) diluted in TBS, mM CaCl2, mM MgCl2, 1% BSA for 10 at room temperature before the addition of the I-GST protein to the wells Results Purification of CD11/CD18 integrins, recombinant I domains and ICAMFc proteins The purified CD11a/CD18 and CD11b/CD18 preparations were analyzed by SDS/PAGE and no major impurities were observed (not shown) In contrast to the CD11b I domain GST, the majority of the CD11a I domain GST was found in the insoluble fraction of the E coli lyzate Modification of the solubilization conditions and increasing the volumes of the bacterial cultures yielded large enough amounts of soluble CD11a I domain GST The purified I domain GST fusion proteins migrated as major bands of  50 kDa After Factor X or thrombin cleavage and removal of GST the I domains appeared as single major bands of approximately 24 kDa (CD11a) and 26 kDa (CD11b) The purities of ICAM-1Fc, ICAM-2Fc and ICAM-4Fc fusion proteins were checked by SDS/PAGE The preparations contained the expected recombinant proteins and the purity of the proteins was >90% (not shown) Binding of red cells to purified I domains The surface expression of ICAMs on ICAM-4 positive, LW(a+, b–), and ICAM-4 negative, LW(a–, b–), red cells was studied by flow cytometry The only ICAM expressed on LW(a+, b–) cells was ICAM-4, while none of the ICAMs were found on LW(a–, b–) cells (not shown) By performing cell adhesion assays with the recombinant I domains, we showed that red cells readily bound to recombinant I domains of both CD11a and CD11b in a concentration-dependent manner (Fig 1A,C) The binding of ICAM-4 positive erythrocytes to CD11a I domain was effectively blocked by monoclonal antibodies to ICAM-4 and the CD11a I domain (Fig 1B) The mAb 7E3 and the mAbs BS46 and BS56 to ICAM-4 partially but significantly inhibited the interaction between ICAM-4 positive red cells and recombinant CD11b I domain (Fig 1D) The ICAM-4 negative red cells retained most or all binding activity to the recombinant CD11b I domain (Fig 1D) The adhesion of these cells was affected neither by mAbs to ICAM-4 nor the activation-dependent mAb 7E3 [42] However, the adhesion of both ICAM-4 positive and negative erythrocytes to coated CD11b I domain was almost completely abrogated in the presence of mAb 60.1, which binds to the recombinant CD11b I domain These data indicate that there might be an unidentified ligand in red cells that could mediate binding to CD11b and probably also to CD11a CD11b I domain inhibits red cell binding to purified integrin Red cells bind poorly to CD11a/CD18 but more efficiently to CD11b/CD18 [28] Therefore, we tested inhibition of erythrocyte binding to CD11b/CD18 by purified CD11b I domain GST Figure shows that half maximal reduction of cell adhesion was achieved at 0.2 lM I domain GST 1714 E Ihanus et al (Eur J Biochem 270) Ó FEBS 2003 Fig Binding of red cells to purified CD11a/CD18 and CD11b/CD18 I domains (A) Indicated amounts of CD11a/CD18 I domain (j) or glycophorin A (GPA) (d) were coated per well (B) Shows the effect of anti-ICAM-4 (BS46 and BS56) and anti-CD11a I domain (TS1/22 and MEM83) mAbs on the binding of ICAM-4 positive and negative red cells to lg of coated CD11a/CD18 I domain (C) Wells were coated with indicated amounts of CD11b/CD18 I domain (j) or ICAM-3 (d) (D) The effect of antibodies on binding of red cells to 0.4 lg of coated CD11b/ CD18 I domain was studied (anti-CD11b I domain mAbs: 7E3 and 60.1) The data in A and C is presented as a percentage of attached cells (amount of bound cells divided by input of cells) The amounts of bound and added red cells were quantitated by counting cells in four randomly chosen fields from duplicate wells The results in B and D are expressed as a relative percentage of bound cells, where 100% is calculated from the total number of ICAM-4 positive red cells bound to the I domain in the absence of pretreatment with mAbs Controls included unrelated mouse IgG antibody (not shown) and wells with coated control protein (GPA) or without coated protein (BSA only) The experiments were repeated 3–5 times with similar results Data are expressed as mean ± SD and statistical significances are shown.wwwP < 0.001, wP < 0.1 Interaction between ICAM transfectants and purified CD11a/CD18 and CD11b/CD18 integrins Fig Inhibition of red cell adhesion to purified CD11b/CD18 by the CD11b I domain GST The binding of erythrocytes to coated purified CD11b/CD18 in the presence of indicated concentrations of soluble CD11b I domain GST (s) or GST (m) are shown Background binding of cells to BSA was substracted The data is presented as a percentage of attached cells The amounts of bound and added red cells were quantitated by counting cells in four randomly chosen fields from duplicate wells The experiment was repeated three times with similar results To obtain further evidence that ICAM-4 binds to CD11a/ CD18 and CD11b/CD18 through the I domains we generated stable mouse L cell transfectants expressing recombinant human ICAM-4 Several ICAM-4 transfectant clones were obtained and the ones expressing high levels of ICAM-4 and strong binding to purified CD18 integrins were chosen for further adhesion assays The stable L cell transfectants expressing human ICAM-1 and ICAM-2 have been established as previously described [41] As expected, the ICAM transfectants reacted only with the corresponding ICAM mAb (Fig 3A) None of the transfectants reacted with mAbs to ICAM-3 or ICAM-5 (not shown) Using purified CD11a/CD18 and CD11b/CD18, we studied the binding of ICAM transfectants to the integrins coated on plastic (Fig 3B) All the ICAM transfectants, but not the wild-type L cells adhered to the coated integrins, but not much to the control protein We found that the ICAM-4 tranfectants could bind much more efficiently to CD11b/CD18 than to CD11a/CD18 coated on plastic wells (approximately 60% and 15% of the total added cells, respectively) Furthermore, the ICAM-4 transfectants adhered more strongly to CD11b/CD18 than ICAM-1 and ICAM-2 transfectants Approximately 30% of these other two ICAM transfectants bound to CD11b/CD18 As the expression levels of ICAM-4 and ICAM-2 L cell Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur J Biochem 270) 1715 transfectant adhesion to coated CD11b/CD18 integrin However the binding of all ICAM transfectants was reduced to 50–60% in the presence of lM CD11b I domain GST Adhesion of ICAM transfectants to purified I domain fusion proteins Fig Cell surface expression of ICAM-1, ICAM-2 and ICAM-4 on L cell transfectants and adhesion assay using purified CD11a/CD18 and CD11b/CD18 integrins (A) Parental L cells (dotted line) and L cells transfected (dark line) with ICAM-1, ICAM-2 and ICAM-4 cDNAs were stained with mAbs anti-ICAM-1 (LB-2), anti-ICAM-2 (B-T1), and anti-ICAM-4 (BS46) followed by FITC-rabbit antimouse IgG F(ab¢)2 fragments and were analyzed by flow cytometry (B) Cell adhesion of parental L cells and ICAM transfectants to CD11a/CD18, CD11b/CD18 and GPA proteins coated on plastic wells The experiments were repeated three times with similar results The I domains of CD11a and CD11b contain binding sites for ICAM-1 and ICAM-2 Further proof for the interaction of ICAM-4 with these I domains was obtained by comparing the ability of recombinant I domain fusion proteins to support the adherence of L cell transfectants expressing ICAM-1, ICAM-2 or ICAM-4 For our assays we immobilized I domain GST fusion proteins via goat anti-GST antibodies which presumably allowed the I domain GSTs to be presented in favourable orientations and caused more effective binding of the cells As shown in Fig 5, the I domain fusion proteins supported the binding of all three different ICAM transfectants in a concentration-dependent manner For this particular lot of I domains, 0.3–0.8 lg in solution used to coat the wells resulted in good adherence of ICAM L cell transfectants and low background binding of wild-type L cells After substraction of background binding, approximately 30% of the total added ICAM-1 L cells adhered to the I domain of CD11a, while approximately 20% of ICAM-2 and ICAM-4 L cells did so However, at high levels of CD11b I domain fusion proteins, wild-type L cells were adherent as well, a phenomenon noticed also by others for wild-type CHO cells [43] The results indicate that the I domain of CD11b interacts not only with ICAMs but also with an unknown receptor on L cells This interaction with an L cell receptor is not unique for the recombinant CD11b I domain, because wild-type L cells also adhered to high levels of purified CD11b/CD18 integrin (data not shown) Effects of antibodies on binding of ICAM transfectants to purified I domain fusion proteins transfectants were clearly lower than ICAM-1 transfectants, these results suggest that ICAM-4 might be an even more potent ligand for CD11b/CD18 than the other two ICAMs To a certain extent (10–20%) the binding efficiences to ICAM-4 varied between different preparations of CD11b/ CD18 integrins The mAbs to CD11a/CD18, CD11b/CD18 and ICAMs clearly inhibited the binding of ICAM L cells to coated CD18 integrins (data not presented) To examine the role of I domains in ICAM-4 binding in more detail we tested the ability of I domain GST fusion proteins to block the interaction of ICAM transfectants with purified CD11a/CD18 and CD11b/CD18 integrins (Fig 4) They efficiently inhibited the adhesion of ICAM transfectants to CD11a/CD18 and CD11b/CD18 integrins The adhesion of ICAM-4 transfectants to CD11a/CD18 was inhibited by the CD11a I domain GST more efficiently as compared to ICAM-1 and ICAM-2 transfectants The inhibition of ICAM-4 transfectant binding to CD11a/CD18 by soluble CD11a I domain GST was concentrationdependent and 50% inhibition was obtained with an inhibitor concentration of 0.4 lM The soluble CD11b I domain GST was a less active inhibitor of ICAM-4 For further study, we investigated the effects of different mAbs on the interaction of ICAM L cell transfectants with I domain GST fusion proteins of CD11a and CD11b (Figs and 7) The CD11a I domain specific TS1/22 mAb efficiently inhibited the binding of ICAM-1 and -2 transfected L cells to the I domain of CD11a, and partially but significantly inhibited the interaction between ICAM-4 L cells and CD11a I domain The adhesion of all ICAM transfectants to the I domain of CD11a was almost completely blocked by the antiCD11a mAb MEM83 down to GST background level (Fig 6) In an ELISA assay both anti-CD11a mAbs (TS1/22 and MEM83) and all the I domain specific antiCD11b mAbs (LM2/1, MEM170, 60.1, 44a, 107 and 904) reacted with the corresponding I domain GST fusion proteins immobilized via goat anti-GST antibodies (not shown) Pretreatment of the coated CD11b I domain fusion protein with the I domain specific mAbs resulted in efficient inhibition of the adherence of all three different types of ICAM L cells, except for mAb 904 which had no effect on binding of ICAM-2 transfectants (Fig 7) 1716 E Ihanus et al (Eur J Biochem 270) Ó FEBS 2003 Fig Inhibition of adhesion of ICAM transfectants to purified CD18 integrins by corresponding recombinant I domain GST The binding of ICAM transfectants to plastic coated purified CD11a/CD18 or CD11b/CD18 in the presence of indicated concentrations of soluble CD11a I domain GST or CD11b I domain GST (s) or GST (m) are shown Background binding of cells to BSA was substracted The results are expressed as a relative percentage of bound cells, where 100% is given as the total number of cells bound to the CD18 integrins in the absence of soluble competitors (Materials and methods) The experiments were repeated three times with similar results Fig Adhesion of ICAM tranfectants to purified CD11a and CD11b I domain GST fusion proteins Indicated amounts of anti-GST antibodies were coated per well After blocking with BSA, GST fusion proteins of I domains or purified GST were added in amount twice of the anti-GST Both binding of parental L cells to GST I domains (s) or GST (e) and binding of ICAM transfectants to GST I domains (j) or GST (d) are shown The data are presented as a mean percentage of attached cells (amount of bound cells divided by input of cells) ± SD Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur J Biochem 270) 1717 Fig Inhibition of adhesion of ICAM transfectants to purified CD11a I domain GST fusion protein The effects of anti-ICAM and anti-I domain mAbs on adhesion of ICAM transfectants to 0.4 lg of I domain GST captured with 0.2 lg of coated anti-GST Ab Controls include wells with captured GST and the binding of wild-type L cells Background binding of cells to BSA was substracted The results are expressed as a mean relative percentage of bound cells, where 100% is given as the total number of cells bound to the I domain in the absence of pretreatment with mAbs The experiments were repeated 3–5 times with similar results Standard deviations and statistical significances are shown wwwP < 0.002, wwP < 0.02, wP < 0.2 Fig Inhibition of adhesion of ICAM transfectants to purified CD11b I domain GST fusion protein The effects of anti-ICAM and anti-I domain mAbs on adhesion of ICAM transfectants to 0.4 lg of I domain GST captured with 0.2 lg of coated anti-GST Ab Controls include wells with captured GST and the binding of wild-type L cells Background binding of cells to BSA was substracted The results are expressed as a mean relative percentage of bound cells, where 100% is given as the total number of cells bound to the I domain in the absence of pretreatment with mAbs The experiments were repeated three to five times with similar results Standard deviations and statistical significances are shown wwwP < 0.002, wwP < 0.02, wP < 0.2 Several different ICAM-1 mAbs were tested in cellular adhesion assays and variable degrees of inhibition were detected The most efficient inhibition of the binding of ICAM-1 L cell transfectants to the I domains was obtained with the ICAM-1 mAb LB-2 reacting with the first domain of ICAM-1 and the mAbs GP8914 and GP8923 which have been mapped to domains and of the ICAM-1 molecule [44] None of these anti-ICAM-1 mAbs showed inhibitory effects on adhesion of ICAM-2 or ICAM-4 L cells MAbs to ICAM-2 and ICAM-4 inhibited the binding of ICAM-2 and ICAM-4 L cells, respectively The three ICAM L cell transfectants used in the cell adhesion assays were stained with all the above mentioned ICAM mAbs and they reacted only with the mAbs to transfected ICAM (data not shown) Divalent cation requirements for ICAM/b2 integrin interaction Divalent cations may have multiple effects on integrinmediated cell adhesion including enhancement, suppression, and modification of ligand binding activity We have previously shown that Ca2+ and Mg2+ are needed for the maximal binding of CD11a/CD18 and CD11b/ CD18 to ICAM-4 [14,28] Here we have investigated the effect of divalent cations on the binding of ICAM-4 transfectants to the I domains of CD11a and CD11b and compared the results to the divalent cation requirements of ICAM-1 and ICAM-2 transfectants We also analyzed the cation dependence of red cell adhesion to the I domains 1718 E Ihanus et al (Eur J Biochem 270) The most efficient binding of all the ICAM L cell transfectants was observed in the presence of Mn2+ (Fig 8) In the absence of cations, the binding of ICAM-1 transfectants to the CD11a I domain fusion protein and the binding of ICAM-2 transfectants to both I domain fusion proteins were completely abrogated In the presence of EDTA the adhesion of ICAM-4 transfectants to both I domains was efficiently, but not totally abolished, as was also the binding of ICAM-1 transfectants to the CD11b I domain fusion protein The inhibitory effects of EDTA were clearly significant As can be seen in Fig 8, MgCl2 alone or in combination with CaCl2 supported the interaction of ICAM-1 L cells with both I domains and ICAM-2 L cell adherence to the I domain of CD11a However, the presence of both MgCl2 and CaCl2 seems to be required for high affinity binding of ICAM-2 L cell transfectants to the I domain of CD11b as well as for adhesion of ICAM-4 transfectants to both I domains CaCl2 alone was not sufficient to support the maximal binding of any of the ICAM transfectants to the I domain fusion proteins Ó FEBS 2003 Fig Binding of red cells to CD11a/CD18 and CD11b/CD18 I domains in the absence or presence of divalent cations Red cells were washed with cation-free Tris buffer, and then resuspended in serumfree buffer containing either mM MgCl2 and mM CaCl2, mM EDTA, or mM EGTA and mM MgCl2 Plastic wells coated with lg of CD11a I domain or 0.4 lg of CD11b I domain were washed three times with the appropriate buffer before adding the cells The results are expressed as a relative percentage of bound cells, where 100% is calculated from the total number of cells bound to the I domains in the presence of divalent cations Background binding of red cells to BSA and the control protein (GPA) was substracted The experiments were repeated 3–5 times with similar results Standard deviations and statistical significances are shown wwwP < 0.001, wwP< 0.01 Figure shows that the presence of Mg2+ and Ca2+ is needed for efficient adhesion of red cells to the I domains of CD11b and CD11a, while in the absence of Ca2+, chelated using EGTA, the binding of red cells was partially but significantly inhibited A clear reduction of binding was observed if divalent cations were omitted (not shown) or when EDTA was included in the reaction mixture MnCl2 supported the binding of red cells to the I domains efficiently (not shown) Characterization of isolated I domain GST binding to ICAM-4Fc using a solid phase ELISA assay Fig Adhesion of ICAM transfectants to CD11a and CD11b I domain GST fusion proteins in the absence or the presence of divalent cations Stable transfectants were harvested and washed with cation-free Tris buffer, and then resuspended in the Tris buffer containing either mM MgCl2 and mM CaCl2, mM MgCl2, mM CaCl2, mM MnCl2, or mM EDTA Plastic wells precoated with 0.2 lg of anti-GST Ab, were coated with 0.4 lg of CD11a (A) or CD11b (B) I domain GST and washed three times with appropriate buffer before adding the cells Background binding of the cells was substracted The experiments were repeated three to five times with similar results The binding of ICAM-4 directly to CD11a and CD11b I domains was further investigated in a cell-free assay (Figs 10 and 11) The specificity of the adhesion in the solid phase assay was demonstrated by the ability of I domains to bind immobilized ICAM-4Fc in a dose-dependent fashion compared with a lack of binding to either BSA or another closely related Ig-family protein, VCAM-1Fc (Fig 10B,D) The control GST fusion protein, LLG-C4-GST [29], did not interact with ICAM-4Fc or BSA (Fig 10A,C), showing that the adhesion to ICAM-4Fc was mediated by the I domain in the fusion protein We tested the effects of several mAbs on the binding of isolated I domain GST to ICAM-4Fc (Fig 11) and found that the blocking pattern mostly reflected that observed in cellular assays for red cells and ICAM-4 transfectants The MEM83 antibody Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur J Biochem 270) 1719 Fig 10 Specific binding of purified recombinant I domain GST fusion proteins to ICAM-4Fc in a solid phase assay Dose-dependent binding of CD11a I domain GST (A and B) or CD11b I domain GST (C and D) to ICAM-4Fc, VCAM-1Fc and BSA Recombinant ICAM-4Fc or VCAM-1Fc was immobilized via antihuman IgG Fc specific antibody to 96-well plates, which were blocked with BSA Control wells were only blocked with BSA The I domain GST or the control GST were diluted in Tris buffered saline containing mM CaCl2, mM MgCl2 and 1% BSA and incubated for h at room temperature Data shown are from one representative experiment out of 3–5 I domain GST + ICAM-4Fc (j), control GST + ICAM-4Fc (m), I domain GST + BSA (s), control GST + BSA (e), I domain GST + VCAM-1Fc (d) effectively inhibited the CD11a I domain/ICAM)4Fc interaction while the TS1/22 blocked to a lesser degree as did also the ICAM-4 mAb BS46 The mAbs MEM25 and MEM30 substantially blocked the CD11a I domain GST binding to captured ICAM-4Fc, whereas the MEM177 was nonblocking Five CD11b I domain specific mAbs were tested for their ability to inhibit in solid phase assay MEM170, 44a and 107 were highly active inhibitors of the CD11b I domain GST interaction with ICAM 4, whereas LM2/1 inhibited weakly but significantly and mAb 904 had no effect However, the failure of mAb 904 to inhibit CD11b I domain/ICAM-4 interaction was unexpected, as the mAb was an efficient blocker of adhesion between ICAM-4 L cell transfectants and coated CD11b I domain GST We checked that all the mAbs to the I domains and the anti-ICAM-4 mAb bound to corresponding coated and soluble recombinant proteins (data not shown) As a further approach to characterize the interaction between ICAM-4 and the I domains we examined the effect of soluble recombinant ICAMFc proteins and the isolated I domains on the binding of I domain GST fusion proteins to plastic captured ICAM-4Fc in solid phase ELISA assay (Fig 11) The results indicated that recombinant CD11a I domain lacking GST was a potent competitor of the interaction between coated ICAM-4Fc and the CD11a I domain GST fusion protein, whereas the CD11b I domain only inhibited weakly However, soluble CD11b I domain readily inhibited the binding of captured ICAM-4Fc to CD11b I domain GST, while the soluble CD11a I domain did not have any effect Furthermore, soluble ICAM-4Fc was highly active in competing with the coated ICAM-4Fc for the recombinant I domain GST fusion proteins and the soluble ICAM-1Fc and ICAM-2Fc fusion proteins were even more efficient competitors Discussion In the present study, we have used several techniques to show that the I domains of the CD11a/CD18 and CD11b/ CD18 leukocyte integrins contain binding sites for ICAM-4 Our results show that ICAM-4 expressing red cells bound specifically and dose-dependently to isolated recombinant CD11a and CD11b I domains The effective inhibition of binding of ICAM-4 positive red cells by anti-ICAM-4 antibodies, indicate a major role for ICAM-4 in binding of red cells to the I domains The efficient inhibition of the 1720 E Ihanus et al (Eur J Biochem 270) Fig 11 Effect of monoclonal antibodies, soluble competitor proteins and EDTA on binding of recombinant I domain GST to coated ICAM4Fc in a solid phase assay CD11a I domain GST (A) and CD11b I domain GST (B) binding to ICAM-4Fc was examined in the presence of mAbs (20–40 lgỈmL)1) against the ICAM-4 (BS46), CD11a I domain (TS1/22, MEM83, MEM30, MEM25, MEM177), CD11b I domain (LM2/1, MEM170, 44a, 107, 904) or soluble competitor proteins; CD11a and CD11b I domains of which the GST part was removed (1 lM), ICAM-Fc proteins (200 nM) or mM EDTA The results are shown as a percentage of I domain GST binding in the absence of soluble inhibitors or EDTA The experiments were repeated 3–5 times with similar results Data are from one representative experiment Standard deviations and statistical significances are shown wwwP < 0.005, wwP< 0.05, wP < 0.5 The significance was determined by unpaired Student’s t-test interaction between purified CD11b/CD18 and erythrocytes by soluble CD11b I domain GST is consistent with the idea of this subdomain being a ligand binding area for a red cell receptor However, it is probable that other erythrocyte receptors for the CD11a and CD11b I domains exist because of the residual binding of the ICAM-4 negative cells The mAb 60.1 reacting with the CD11b I domain almost completely inhibited the binding of both ICAM-4 positive and negative red cells To the best of our knowledge the epitope for mAb 60.1 within the I domain has not been mapped in detail but the epitope for activation dependent mAb 7E3 has been localized to the amino-terminal region of the CD11b I domain [42] overlapping partially with the Ó FEBS 2003 metal ion-dependent adhesion site A clear reduction in adhesion was also obtained with the CD11b I domain specific LM2/1 mAb (not shown) which has been shown to inhibit the binding of red cells to the CD11b/CD18 integrin [28] The soluble I domains also clearly reduced the binding of all ICAM transfectants to purified CD11a/CD18 and CD11b/CD18 However, the ICAM-4 binding to CD11b/ CD18 was reduced to 60% in the presence of the corresponding I domain compared to 20% for the interaction between ICAM-4 and CD11a/CD18 This indicates differences in binding of ICAM-4 to these two integrins The coated I domain fusion proteins were able to mediate dose-dependent binding of all three different ICAM transfectants Various anti-I domain mAbs were tested in cellular binding assays to find out possible differences in binding of ICAM-4 and the other two ICAMs for the I domains Many of these mAbs displayed similar inhibitory profiles in assays investigating the effects of the mAbs on the interaction of ICAM L cell transfectants with the I domains However, the CD11a I domain specific mAb TS1/22 which efficiently blocked the binding of ICAM-1 and -2 L cells only partially inhibited the interaction between ICAM-4 L cells and CD11a I domain The adhesion of all ICAM transfectants to the I domain of CD11a was almost completely blocked by the I domain specific MEM83 Previous reports have shown that MEM83 stimulates cellular CD11a/CD18 binding to purified ICAM-1 [31,45] but inhibits the interaction between ICAM-3 and CD11a/CD18 [46,47] Using a flow cell assay MEM83 has been reported to increase the number of rolling cells expressing a membrane-anchored form of the CD11a I domain on ICAM-1 bilayer membranes but to decrease it on ICAM-3 bilayers Furthermore, the MEM83 decreased the rolling velocity of the cells on ICAM-1-containing membranes indicating an enhanced avidity [48] The different results may be due to differences in assay systems We have investigated the static adhesion of ICAM expressing transfected cells to the isolated recombinant I domains The previous results were performed with coated CD11a/CD18 or using cells expressing either a whole CD11a/CD18 integrin or a membrane-anchored form of the CD11a I domain Altogether, our data with both red cells and ICAM-4 transfected L cells suggest that the epitope within the CD11a I domain recognized by the MEM83 antibody is involved in ICAM-4 binding Treatment of the coated CD11b I domain fusion protein with I domain specific anti-CD11b antibodies resulted in an efficient inhibition of the adherence of all three types of ICAM L cells, except that mAb 904 had no effect on binding of ICAM-2 transfectants The LM2/1 mAb did not inhibit the binding of ICAM-4 L cells as efficiently as did the other anti-I domain mAbs The degrees of inhibition between mAbs may vary due to different mechanisms of action Several studies have pointed out that blocking mAbs can inhibit ICAM/integrin interactions either by direct competition for the ligand binding site or by an indirect mechanism through binding to a regulatory site located outside the actual ligand binding site [46,49,50] Most function-blocking mAbs directed against integrins may act allosterically by stabilizing the low-affinity state of the Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur J Biochem 270) 1721 receptor and thus preventing the conformational change to the active high affinity form The activation-independent mAbs LM2/1 and 904 have indeed been suggested to recognize a common or overlapping epitope distant from the ligand binding region [51] MAbs 44a and 107 have been reported to share the property of stabilizing the integrin in the low affinity state [52,53] However, the epitopes for mAb 44a reside at considerable distance from the ligand binding site on the opposite side of the metal ion-dependent adhesion site (MIDAS) motif, whereas the binding interface for mAb 107 seems to overlap that of physiologic ligands and clearly engages the metal ion-dependent adhesion site area [51–53] The presence of Mg2+ and Ca2+ or Mn2+ is required for maximal binding efficiency of ICAM-4 transfectants and red cells to the I domains of CD11a and CD11b Our results indicate that Mg2+ or Mn2+, but not Ca2+ are necessary for the interaction of CD11a I domain with ICAM-1 and ICAM-2 transfectants and for the CD11b I domain binding to the ICAM-1 transfectants The presence of both MgCl2 and CaCl2 gives better binding of ICAM-2 L cell transfectants to the I domain of CD11b as well as for adhesion of ICAM-4 transfectants and red cells to both I domains The finding that the soluble I domains were able to compete only with the respective I domain GST fusion protein for binding to the captured ICAM-4Fc strengthens the earlier results that the binding sites for the CD11a and CD11b I domains in ICAM-4 are different although they could be partially overlapping [14] The ICAM-4 binding region in both I domains clearly overlaps with the region recognized by ICAM-1 and ICAM-2 Soluble ICAM-1Fc and ICAM-2Fc proteins prevented the binding of immobilized ICAM-4Fc to the I domains almost completely whereas the soluble ICAM-4Fc was a less active competitor, perhaps due to lower affinity The observations described in this report have fundamental importance for the detailed understanding of the recognition between ICAM-4 and CD11a/CD18 and CD11b/CD18 integrins The physiological role of ICAM-4 is not known, but it may function as an erythroid recognition protein for macrophage integrins in erythroblastic islands in bone marrow during erythropoiesis The expression level of ICAM-4 has been reported to be higher on erythroid precursor cells and is gradually decreased during the later stages of differentiation finally down to the level observed in mature red cells [54] In addition, the expression of the ICAM-4 integrin receptors on macrophages suggests a possible function for this interaction in red cell turnover to eliminate aged red cells from the circulation by spleen macrophages The ICAM4/b2-integrin interaction may also be important during haemostasis where in the developing thrombus, erythrocytes interact with activated neutrophils and monocytes or during wound healing to mediate removal of red cells from the thrombus by phagocytic macrophages The very recent finding that ICAM-4 also binds to the platelet integrin IIb/IIIa indicates an important role for this molecule in haemostasis [55] Another recent report described the capture and adhesion of normal erythrocytes to activated neutrophils and platelets, as well as to fibrin, at depressed venous shear rates In agreement with our present and previous data the results suggested that the binding of red cells to neutrophils might be mediated through ICAM-4 and CD11b/CD18 [56] Acknowledgements We thank Leena Kuoppasalmi, Aili Grundstrom, Outi Nikkila, Tuula ă ă Nurminen and Saija Makinen for excellent technical assistance, and ă Yvonne Heinila for secretarial work We also want to thank Erkki ă Koivunen and Tanja-Maria Ranta for providing the LLG-C4-GST protein These studies were supported by the University of Helsinki, the ´ Academy of Finland, the Sigrid Juselius Foundation, the Magnus Ehrnrooth Foundation and the Finnish Cancer Society References Arnaout, M.A (1990) Structure and function of the leukocyte adhesion molecules CD11/CD18 Blood 75, 1037–1050 Gahmberg, C.G., Tolvanen, M & Kotovuori, P (1997) Leukocyte adhesion Structure and function of human leukocyte b2-integrins and their cellular ligands Eur J Biochem 245, 215–232 Gahmberg, C.G (1997) Leukocyte adhesion CD11/CD18 integrins and intercellular adhesion molecules Curr Opin Cell Biol 9, 643–650 Hayflick, J.S., Kilgannon, P & Gallatin, W.M (1998) The intercellular adhesion molecule (ICAM) family of proteins New members and novel functions Immunol Res 17, 313–327 Rothlein, R., Czajkowski, M., O’Neill, M.M., Marlin, S.D., Mainolfi, E & Merluzzi, 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antibodies, indicate a major role for ICAM-4 in binding of. .. possible differences in binding of ICAM-4 and the other two ICAMs for the I domains Many of these mAbs displayed similar inhibitory profiles in assays investigating the effects of the mAbs on the. .. binding sites for ICAM-1 and ICAM-2 Further proof for the interaction of ICAM-4 with these I domains was obtained by comparing the ability of recombinant I domain fusion proteins to support the