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• W2038707943 abstract "The interactions of intercellular adhesion molecules-1 and -3 (ICAM-1 and ICAM-3) with lymphocyte function-associated antigen-1 (LFA-1) have been characterized and compared on the molecular and cellular level. Enzyme-linked immunosorbent-based molecular assays have been utilized to calculate the binding affinities of soluble ICAM-1 (sICAM-1) and soluble ICAM-3 (sICAM-3) for LFA-1. Consistent with previously published data, we found that sICAM-1 binds to LFA-1 with an affinity of ∼60 nm. In contrast, sICAM-3 binds to LFA-1 with an affinity ∼9 times weaker (∼550 nm). Both sICAM-1 and sICAM-3 require divalent cations for binding. Specifically, both Mg2+ and Mn2+ support high affinity adhesion, although interestingly, high concentrations of Ca2+decrease the affinity of each molecule for LFA-1 substantially. Furthermore, a panel of anti-LFA-1 monoclonal antibodies were characterized for their ability to block sICAM-1 and sICAM-3/LFA-1 interactions in molecular and cellular assays to help distinguish binding sites on LFA-1 for both molecules. Finally, molecular and cellular competition experiments demonstrate that sICAM-1 and sICAM-3 compete with each other for binding to LFA-1. The above data demonstrate that sICAM-1 and sICAM-3 share a common binding site or an overlapping binding site on LFA-1 and that the apparent differences in binding sites can be attributed to different affinities of sICAM-1 and sICAM-3 for LFA-1. The interactions of intercellular adhesion molecules-1 and -3 (ICAM-1 and ICAM-3) with lymphocyte function-associated antigen-1 (LFA-1) have been characterized and compared on the molecular and cellular level. Enzyme-linked immunosorbent-based molecular assays have been utilized to calculate the binding affinities of soluble ICAM-1 (sICAM-1) and soluble ICAM-3 (sICAM-3) for LFA-1. Consistent with previously published data, we found that sICAM-1 binds to LFA-1 with an affinity of ∼60 nm. In contrast, sICAM-3 binds to LFA-1 with an affinity ∼9 times weaker (∼550 nm). Both sICAM-1 and sICAM-3 require divalent cations for binding. Specifically, both Mg2+ and Mn2+ support high affinity adhesion, although interestingly, high concentrations of Ca2+decrease the affinity of each molecule for LFA-1 substantially. Furthermore, a panel of anti-LFA-1 monoclonal antibodies were characterized for their ability to block sICAM-1 and sICAM-3/LFA-1 interactions in molecular and cellular assays to help distinguish binding sites on LFA-1 for both molecules. Finally, molecular and cellular competition experiments demonstrate that sICAM-1 and sICAM-3 compete with each other for binding to LFA-1. The above data demonstrate that sICAM-1 and sICAM-3 share a common binding site or an overlapping binding site on LFA-1 and that the apparent differences in binding sites can be attributed to different affinities of sICAM-1 and sICAM-3 for LFA-1. The interactions of intercellular adhesion molecule-1 (ICAM-1) 1The abbreviations used are: ICAM, intercellular adhesion molecules; sICAM, soluble ICAM; LFA-1, lymphocyte function-associated antigen-1; ELISA, enzyme-linked immunosorbent assay; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody. and intercellular adhesion molecule-3 (ICAM-3) with lymphocyte function-associated antigen-1 (LFA-1) are integral to the normal functioning of the immune system (1Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5860) Google Scholar, 2Kishimoto T.K. Rothlein R. Adv. Pharmacol. 1994; 25: 117-169Crossref PubMed Scopus (125) Google Scholar, 3de Fougerolles A.R. Qin X. Springer T.A. J. Exp. Med. 1994; 179: 619-629Crossref PubMed Scopus (156) Google Scholar, 4de Fougerolles A.R. Stacker S.A. Schwarting R. Springer T.A. J. Exp. Med. 1991; 174: 253-267Crossref PubMed Scopus (420) Google Scholar, 5de Fougerolles A.R. Springer T.A. J. Exp. Med. 1992; 175: 185-190Crossref PubMed Scopus (398) Google Scholar, 6Hernandez-Caselles T. Rubio G. Campanero M.R. del Pozo M.A. Muro M. Sanchez-Madrid F. Aparicio P. Eur. J. Immunol. 1993; 23: 2799-2806Crossref PubMed Scopus (89) Google Scholar, 7Campanero M.R. del Pozo M.A. Arroyo A.G. Sanchez-Mateos P. Hernandez-Caselles T. Craig A. Pulido R. Sanchez-Madrid F. J. Cell Biol. 1993; 123: 1007-1016Crossref PubMed Scopus (135) Google Scholar). Although ICAM-1 and ICAM-3 share a similar immunoglobulin-like structure and an overall amino acid identity of ∼48%, with the greatest homology observed in domains 2 and 3 (8de Fougerolles A.R. Klickstein L.B. Springer T.A. J. Exp. Med. 1993; 177: 1187-1192Crossref PubMed Scopus (90) Google Scholar, 9Fawcett J. Holness C.L.L. Needham L.A. Turley H. Gatter K.C. Mason D.Y. Simmons D.L. Nature. 1992; 360: 481-484Crossref PubMed Scopus (302) Google Scholar, 10Vazeux R. Hoffman P.A. Tomita J.K. Dickinson E.S. Jasman L. St. John T. Gallatin W.M. Nature. 1992; 360: 485-488Crossref PubMed Scopus (183) Google Scholar, 11Juan M. Vilella R. Mila J. Yague J. Miralles A. Campbell K.S. Friedrich R.J. Cambier J. Vives J. de Fougerolles A.R. Springer T.A. Eur. J. Immunol. 1993; 28: 1508-1512Crossref Scopus (32) Google Scholar), their differential pattern of expression and cellular distribution suggest different functional roles. ICAM-1 is an inducible molecule that is up-regulated by inflammatory cytokines on endothelium, leukocytes, and multiple other cell types (1Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5860) Google Scholar,2Kishimoto T.K. Rothlein R. Adv. Pharmacol. 1994; 25: 117-169Crossref PubMed Scopus (125) Google Scholar), whereas ICAM-3 is constitutively expressed on leukocytes and absent from endothelium and most other cell types under normal conditions (5de Fougerolles A.R. Springer T.A. J. Exp. Med. 1992; 175: 185-190Crossref PubMed Scopus (398) Google Scholar,9Fawcett J. Holness C.L.L. Needham L.A. Turley H. Gatter K.C. Mason D.Y. Simmons D.L. Nature. 1992; 360: 481-484Crossref PubMed Scopus (302) Google Scholar, 10Vazeux R. Hoffman P.A. Tomita J.K. Dickinson E.S. Jasman L. St. John T. Gallatin W.M. Nature. 1992; 360: 485-488Crossref PubMed Scopus (183) Google Scholar). Since ICAM-3 is constitutively expressed at high levels on unactivated T-cells and antigen presenting cells and has been shown to mediate T-cell activation in vitro (3de Fougerolles A.R. Qin X. Springer T.A. J. Exp. Med. 1994; 179: 619-629Crossref PubMed Scopus (156) Google Scholar, 5de Fougerolles A.R. Springer T.A. J. Exp. Med. 1992; 175: 185-190Crossref PubMed Scopus (398) Google Scholar, 6Hernandez-Caselles T. Rubio G. Campanero M.R. del Pozo M.A. Muro M. Sanchez-Madrid F. Aparicio P. Eur. J. Immunol. 1993; 23: 2799-2806Crossref PubMed Scopus (89) Google Scholar, 7Campanero M.R. del Pozo M.A. Arroyo A.G. Sanchez-Mateos P. Hernandez-Caselles T. Craig A. Pulido R. Sanchez-Madrid F. J. Cell Biol. 1993; 123: 1007-1016Crossref PubMed Scopus (135) Google Scholar, 12del Pozo M.A. Campanero M.R. Sanchez-Mateos P. Arroyo A.G. Pulido R. Munoz C. Hernandez-Caselles T. Aparicio P. Sanchez-Madrid F. Cell Adhes. and Commun. 1994; 2: 211-218Crossref PubMed Scopus (9) Google Scholar), it is believed to primarily play a role in lymphocyte activation during antigen presentation. In fact, ICAM-1 and ICAM-3 appear to function synergistically and/or additively with each other as demonstrated by the ability of monoclonal antibodies (mAb) to ICAM-1 and ICAM-3 to have synergistic or additive effects in vitro in inhibiting the mixed lymphocyte reaction, antigen/mitogen-induced proliferation, and homotypic aggregation assays (3de Fougerolles A.R. Qin X. Springer T.A. J. Exp. Med. 1994; 179: 619-629Crossref PubMed Scopus (156) Google Scholar, 6Hernandez-Caselles T. Rubio G. Campanero M.R. del Pozo M.A. Muro M. Sanchez-Madrid F. Aparicio P. Eur. J. Immunol. 1993; 23: 2799-2806Crossref PubMed Scopus (89) Google Scholar, 7Campanero M.R. del Pozo M.A. Arroyo A.G. Sanchez-Mateos P. Hernandez-Caselles T. Craig A. Pulido R. Sanchez-Madrid F. J. Cell Biol. 1993; 123: 1007-1016Crossref PubMed Scopus (135) Google Scholar, 12del Pozo M.A. Campanero M.R. Sanchez-Mateos P. Arroyo A.G. Pulido R. Munoz C. Hernandez-Caselles T. Aparicio P. Sanchez-Madrid F. Cell Adhes. and Commun. 1994; 2: 211-218Crossref PubMed Scopus (9) Google Scholar, 13Bossy D. Buckley C.D. Holness C.L. Littler A.J. Murray N. Collins I. Simmons D.L. Eur. J. Immunol. 1995; 25: 459-465Crossref PubMed Scopus (31) Google Scholar, 14Starling G.C. McLellan A.D. Egner W. Sorg R.V. Fawcett J. Simmons D.L. Hart D.N.J. Eur. J. Immunol. 1995; 25: 2528-2532Crossref PubMed Scopus (64) Google Scholar, 15Teunissen M.B. Koomen C.W. Bos J.D. J. Invest. Dermatol. 1995; 104: 995-998Abstract Full Text PDF PubMed Scopus (16) Google Scholar). In terms of the binding characteristics of ICAM-1 and ICAM-3 for LFA-1, cellular assays have demonstrated that ICAM-1/LFA-1 and ICAM-3/LFA-1 interactions have similar divalent cation and temperature dependences (3de Fougerolles A.R. Qin X. Springer T.A. J. Exp. Med. 1994; 179: 619-629Crossref PubMed Scopus (156) Google Scholar, 16Marlin S.D. Springer T.A. Cell. 1987; 51: 813-819Abstract Full Text PDF PubMed Scopus (1416) Google Scholar). Correlation of surface expression measurements and the binding of T-cells and cell lines to purified LFA-1 suggest that ICAM-1 exhibits a higher affinity for LFA-1 than ICAM-3 (5de Fougerolles A.R. Springer T.A. J. Exp. Med. 1992; 175: 185-190Crossref PubMed Scopus (398) Google Scholar). Mutagenesis studies on LFA-1 have provided evidence that ICAM-1 and ICAM-3 might bind to distinct sites defined within the inserted (I) domain of CD11a (17van Kooyk Y. Binnerts M.E. Edwards C.P. Champe M. Berman P.W. Figdor C.G. Bodary S.C. J. Exp. Med. 1996; 183: 1247-1252Crossref PubMed Scopus (41) Google Scholar, 18Binnerts M.E. van Kooyk Y. Edwards C.P. Champe M. Presta L. Bodary S.C. Figdor C.G. Berman P.W. J. Biol. Chem. 1996; 271: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This concept has been further supported by the observation that certain mAbs to LFA-1 preferentially inhibit the binding of leukocytes to purified ICAM-3 but not to ICAM-1 (18Binnerts M.E. van Kooyk Y. Edwards C.P. Champe M. Presta L. Bodary S.C. Figdor C.G. Berman P.W. J. Biol. Chem. 1996; 271: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 19Binnerts M.E. van Kooyk Y. Simmons D.L. Figdor C.G. Eur. J. Immunol. 1994; 24: 2155-2160Crossref PubMed Scopus (84) Google Scholar, 20Landis R.C. McDowall A. Holness C.L.L. Littler A.J. Simmons D.L. Hogg N. J. Cell Biol. 1994; 126: 529-537Crossref PubMed Scopus (87) Google Scholar). Also, a peptide that maps to the amino terminus of the I domain has been described that inhibits binding of T-cells to ICAM-3 but not ICAM-1 (17van Kooyk Y. Binnerts M.E. Edwards C.P. Champe M. Berman P.W. Figdor C.G. Bodary S.C. J. Exp. Med. 1996; 183: 1247-1252Crossref PubMed Scopus (41) Google Scholar). In this paper, we characterize the interactions of ICAM-1 and ICAM-3 with LFA-1 on the molecular level to understand better the structure and function of the LFA-1/ICAM interaction. Furthermore, we mapped ICAM-1 and ICAM-3 interactions with LFA-1 using mAb and competitive binding assays to determine whether or not ICAM-1 and ICAM-3 share a common binding site on LFA-1. R6.5 (anti-ICAM-1 (21Smith C.W. Rothlein R. Hughes B.J. Mariscalco M.M. Schmalstieg F.C. Anderson D.C. J. Clin. Invest. 1988; 82: 1746-1756Crossref PubMed Scopus (474) Google Scholar)), R7.1/R3.1 (anti-CD11a (22Argenbright L.W. Letts L.G. Rothlein R. J. Leukocyte Biol. 1991; 49: 253-257Crossref PubMed Scopus (130) Google Scholar, 23Ma X.L. Lefer D.J. Lefer A.M. Rothlein R. Circulation. 1992; 86: 937-946Crossref PubMed Scopus (292) Google Scholar)), and R15.7/R3.3 (anti-CD18 (24Entman M.L. Youker K. Shappell S.B. Siegel C. Rothlein R. Dreyer W.J. Schmalstieg F.C. Smith C.W. J. Clin. Invest. 1990; 85: 1497-1506Crossref PubMed Scopus (190) Google Scholar, 25Lorenz H.M. Harrer T. Lagoo A.S. Baur A. Eger A. Eger G. Kalden J.R. Cell. Immunol. 1993; 147: 110-128Crossref PubMed Scopus (37) Google Scholar)) were previously described. CBR-IC3/1 and CBR-IC3/2 (anti-ICAM-3), TS 1/18 (anti-CD18), TS 1/22, and TS 2/4 (anti-CD11a) were kind gifts from Dr. T. A. Springer. MEM-83 (anti-CD11a) was purchased from Monosan (The Netherlands). YTH81.5 (anti-CD11a) was purchased from Serotec (Washington, D.C.). 25.3.1 (anti-CD11a) was purchased from Immunotech (France). mAb 38 (anti-CD11a) and CLB-LFA-1/2 (anti-CD11a) were purchased from RDI, Inc. (Flanders, NJ). G-25.2 (anti-CD11a) was purchased from Becton Dickinson (San Jose, CA). IB4 (anti-CD18) was purchased from Ancell (Bayport, MN). MHM 23 (anti-CD18) and MHM 24 (anti-CD11a) were purchased from Biomeda (Foster City, CA). Full-length ICAM-3 cDNA in the CDM8 vector was a kind gift of Dr. T. A. Springer. Using the polymerase chain reaction, a soluble form of ICAM-3 was constructed by engineering a stop codon at the putative boundary of domain 5 and the transmembrane domain. Polymerase chain reaction generated a cDNA encoding a mature, soluble form of ICAM-3, consisting of 456 amino acid residues. The cDNA was subsequently cloned into the EcoRI site of the pEE12 vector and expressed in NSO-1 mouse myeloma cells using the Celltech glutamine synthetase gene amplification system (Celltech, Ltd., Berkshire, UK). Briefly, 40 μg of linearized plasmid DNA was introduced into 1 × 107 NSO-1 cells by electroporation. After dilution in nonselective media and incubation at 37 °C for 24 h, selection media without glutamine was added to each well. After 18–25 days post-transfection, wells were screened for the production of sICAM-3 using a sICAM-3 detection ELISA (BioSource International, Camarillo, CA). The highest expressing recombinant was chosen for scale-up and produced sICAM-3 at ∼150 μg/ml. After scale-up, sICAM-3 was purified using conventional immunoaffinity chromatography utilizing the mAb, CBR-IC3/1, the same mAb that was used to characterize and clone ICAM-3 (5de Fougerolles A.R. Springer T.A. J. Exp. Med. 1992; 175: 185-190Crossref PubMed Scopus (398) Google Scholar, 8de Fougerolles A.R. Klickstein L.B. Springer T.A. J. Exp. Med. 1993; 177: 1187-1192Crossref PubMed Scopus (90) Google Scholar). Soluble ICAM-3 was deglycosylated under nondenaturing conditions using recombinant N- glycosidase F (Boehringer Mannheim, GmbH, Germany) according to the instructions recommended by the manufacturer. Detergent and denaturing reagent were omitted from the reaction to retain the binding activity of the molecule. Deglycosylation of the nondenatured sample was monitored by SDS-polyacrylamide gel electrophoresis and compared with a sample which contained SDS and detergent. An unlabeled, soluble ICAM-1 (sICAM-1) and a biotinylated, soluble form of ICAM-1 (CB-sICAM-1) were constructed and purified as described previously (26Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Bormann B.J. J. Immunol. 1996; 156: 4680-4685PubMed Google Scholar,27Marlin S.D. Staunton D.E. Springer T.A. Stratowa C. Sommergruber W. Merluzzi V.J. Nature. 1990; 344: 70-72Crossref PubMed Scopus (238) Google Scholar). Micellar LFA-1 was purified from SKW-3 cells utilizing the protocol published by Dustin et al. (28Dustin M.L. Carpen O. Springer T.A. J. Immunol. 1992; 148: 2654-2663PubMed Google Scholar) and, as described previously, yielding an active form of LFA-1 capable of binding ICAMs with high affinity (26Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Bormann B.J. J. Immunol. 1996; 156: 4680-4685PubMed Google Scholar). The purity and structure of sICAM-3 was confirmed by Western blotting. Approximately 1 μg per lane of sICAM-3 or sICAM-1 were electrophoresed on a 4–12% Tris/glycine gel (NOVEX, San Diego, CA) under reducing and denaturing conditions. Proteins were transferred to nitrocellulose and blotted with rabbit polyclonal serum raised against a domain 4 peptide of ICAM-3 or from polyclonal serum raised against sICAM-1. The proteins were then visualized with a goat anti-rabbit Ig alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium pre-mixed substrate solution (Zymed,South San Francisco). sICAM-3 appears as a broad band with an approximate molecular mass of 90–100 kDa, characteristic of a highly glycosylated soluble form of ICAM-3 (see Fig. 6 b, 3rd lane). A soluble form of ICAM-1 was electrophoresed giving a slightly lower apparent molecular mass (see Fig. 6 b, 2nd lane). Aliquots (50 μl) of purified LFA-1, at a concentration of ∼5–8 μg/ml in assay buffer (AB, Dulbecco's phosphate-buffered saline (Life Technologies, Inc.) + 2 mm MgCl2 and 0.1 mmphenylmethylsulfonyl fluoride), diluted below the critical micelle concentration for n-octylglucoside, were allowed to adsorb to wells of a maxisorp microtiter plate (Nunc, Naperville, IL) for 1 h at room temperature. Plates were then washed and blocked for 30 min at 37 °C in 2% bovine serum albumin/AB. For sICAM-3 direct binding assays, purified sICAM-3 in AB was serially diluted 1:2 across the plate in duplicate or triplicate and allowed to incubate at 37 °C for 1 h. After incubation, the plates were washed with AB, and 50 μl of rabbit polyclonal serum raised against a domain 4 peptide diluted 1:100 in 1% bovine serum albumin/AB was added to each well and incubated for 20 min at 37 °C. After washing again with AB, a 1:4000 dilution of goat anti-rabbit Ig horseradish peroxidase conjugate (Zymed) was added to each well and incubated at 37 °C for 15–20 min. After another wash step, 200 μl of 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt substrate (Zymed) was added to each well and incubated at 37 °C for 20 min. The absorbance was then measured with a Molecular Devices Thermomax plate reader (Menlo Park, CA) at 405 nm. For all divalent cation experiments, assays were performed in the presence of either 1 mm Ca2+ or 0.4 mm Mn2+in the AB replacing the 2 mm Mg2+ as needed throughout the assay, including washes. Direct assays involving biotinylated sICAM-1 and a horseradish peroxidase/streptavidin reporter were performed as described (26Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Bormann B.J. J. Immunol. 1996; 156: 4680-4685PubMed Google Scholar). For competitive binding assays, serially diluted competitors (sICAM-1, sICAM-3, mAbs) were added to an equal volume of sICAM-3 or biotinylated sICAM-1 at a constant concentration, and the assays were continued as described above and as described previously (26Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Bormann B.J. J. Immunol. 1996; 156: 4680-4685PubMed Google Scholar). Purified sICAM-3 or sICAM-1 were plated at 10–40 μg/ml (50 μl/well in duplicate) on Linbro EIA II 96-well plates (Flow Laboratories, McLean, VA) and allowed to adsorb for 1 h at room temperature. Purified LFA-1 was plated as above, but at concentrations ranging from 1 to 2 μg/ml. The plates were then washed and blocked with 2% bovine serum albumin/AB for 1 h at 37 °C. Fifty μl of washed SKW-3 cells ± 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma) at 2 × 106cells/ml in complete media (RPMI, 15% fetal calf serum, 10 mm HEPES, Life Technologies, Inc.) were then added to the blocked wells (purified sICAM-1 and sICAM-3 were allowed to preincubate with blocked LFA-1 for 15 min at room temperature before addition of cells). The test samples (mAb at various concentrations) in AB were added to the wells in an equal volume and allowed to incubate at 37 °C for 1 h. Unbound cells were removed by 4 washes with warm RPMI using a multichannel pipettor. Bound cells were then quantified using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, thiazolyl blue reagent. Briefly, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, thiazolyl blue (Sigma) reagent (5 mg/ml in RPMI) was added to each well and incubated at 37 °C for 3–4 h. After incubation, crystals were solubilized in 0.04 n HCl in isopropyl alcohol, and absorbance was measured at 570 nm. All data were analyzed using the SAS statistical software system, version 6.11 (SAS Institute, Cary, NC). ASCII data files containing absorbance measurements from ELISA experiments were converted into SAS data sets. Using the NLIN procedure, data analyses were performed by applying ordinary nonlinear least squares regression techniques to the selected model (see Ref.26Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Bormann B.J. J. Immunol. 1996; 156: 4680-4685PubMed Google Scholar), using the Marquardt-Levenberg minimization method (35SAS Institute SAS-STAT Users Guide Version 6. 4th Ed. SAS Institute Inc., Cary, NC1989Google Scholar). An ELISA-based assay was utilized to measure the binding interaction between sICAM-1 or sICAM-3 and immunopurified, immobilized LFA-1. Fig. 1 shows two representative experiments, demonstrating the binding of sICAM-3 and biotinylated sICAM-1 to LFA-1. Previous results have demonstrated that biotinylation of sICAM-1 does not affect its affinity for LFA-1 (26Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Bormann B.J. J. Immunol. 1996; 156: 4680-4685PubMed Google Scholar). Fig. 1 a shows a representative sICAM-3 direct binding assay. As the concentration of sICAM-3 is increased in the assay, the absorbance measured at 405 nm increases producing a typical sigmoidal shaped curve. These data were analyzed with the equilibrium model for direct binding (see “Materials and Methods” given in the Appendix of Ref.26Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Bormann B.J. J. Immunol. 1996; 156: 4680-4685PubMed Google Scholar) giving a dissociation constant (Kd) of 553 (±47) nm (where the number in parentheses is the S.E. (n = 4). Fig. 1 b shows a representative direct binding experiment for biotinylated sICAM-1 binding to immunopurified, immobilized LFA-1 in a similar ELISA-based format. The Kd for this interaction was determined to be 62 (±3) nm (n = 4). The affinity of sICAM-3 for LFA-1 is approximately a log weaker than that of sICAM-1 for LFA-1 as the midpoint of the curve for sICAM-3 is shifted to higher concentrations. Purified sICAM-3 was then tested for its ability to effectively support the binding of an LFA-1 bearing cell line. Fig. 2 a shows the binding of the T-cell lymphoma SKW-3 to immobilized sICAM-3. Treatment of the cells with PMA (50 ng/ml) in the presence of divalent cations resulted in a 2-fold increase in binding presumably due to an increase in avidity resulting from integrin clustering and a change in the activation state of LFA-1 from a low to high affinity state (29Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1286) Google Scholar, 30van Kooyk Y. van de Wiel-van Kemenade P. Weder P. Kuijpers T.W. Figdor C.G. Nature. 1989; 342: 811-813Crossref PubMed Scopus (396) Google Scholar, 31Hogg N. Cabanas C. Dransfield I. Lipsky P.E. Rothlein R. Kishimoto T.K. Faanes R.B. Smith C.W. Structure, Function and Regulation of Molecules Involved in Leukocyte Adhesion. Springer-Verlag Inc., New York1993: 3-13Crossref Google Scholar, 32Stewart M.P. Cabanas C. Hogg N. J. Immunol. 1996; 156: 1810-1817PubMed Google Scholar, 33Lollo B.A. Chan K.W.H. Hanson E.M. Moy V.T. Brian A.A. J. Biol. Chem. 1993; 268: 21693-21700Abstract Full Text PDF PubMed Google Scholar). The binding of SKW-3 cells to immobilized sICAM-3 was inhibited by the blocking anti-CD11a mAb, (R3.1). Both the anti-ICAM-3 mAbs (CBR-IC3/1 and CBR-IC3/2) when used together effectively decreased the number of cells bound to purified sICAM-3 by ∼70%. The combination of these antibodies is needed to block the adhesion of LFA-1 bearing cells to purified sICAM-3, similar to what has been demonstrated by de Fougerolles et al. (3de Fougerolles A.R. Qin X. Springer T.A. J. Exp. Med. 1994; 179: 619-629Crossref PubMed Scopus (156) Google Scholar). The reason for this phenomenon is unknown, but it has been speculated that neither CBR-IC3/1 nor CBR-IC3/2 map to the binding site on ICAM-3 for LFA-1 and therefore a combination of both mAbs is needed to sterically hinder the binding of ICAM-3 to LFA-1. These observations demonstrate the specificity of the sICAM-3/LFA-1 interaction. The binding of SKW-3 cells to immobilized sICAM-1 was also characterized (see Fig. 2 b). A 2–3-fold increase in binding was observed when cells were treated with PMA. Both a blocking anti-ICAM-1 (R6.5) and the blocking anti-CD11a mAb R3.1 inhibited binding of the SKW-3 cells to immobilized sICAM-1, demonstrating the specificity of the sICAM-1/LFA-1 interaction under the same assay conditions. The divalent cation requirements for binding of both sICAM-3 and sICAM-1 to LFA-1 were then characterized on the molecular level. On the cellular level, these requirements have been well characterized for LFA-1-mediated binding to both ICAM-1 (16Marlin S.D. Springer T.A. Cell. 1987; 51: 813-819Abstract Full Text PDF PubMed Scopus (1416) Google Scholar, 34Rothlein R. Springer T.A. J. Exp. Med. 1986; 163: 1132-1149Crossref PubMed Scopus (401) Google Scholar) and ICAM-3 (3de Fougerolles A.R. Qin X. Springer T.A. J. Exp. Med. 1994; 179: 619-629Crossref PubMed Scopus (156) Google Scholar) and have shown that cellular LFA-1 requires Mg2+ or Mn2+to be functional in binding ICAM-1 or ICAM-3. Previous direct binding experiments (Fig. 1, a and b) were performed in the presence of Mg2+ (2 mm) to ensure high affinity binding. The Mg2+ ions in all buffers throughout the assay were then replaced with an excess of Mn2+ (400 μm) or Ca2+ (1 mm) ions, and Kd values were determined for sICAM-1 and sICAM-3 binding to LFA-1 in a direct format. In the presence of Mn2+, high affinity binding is observed comparable to that of binding in the presence of Mg2+ for both sICAM-3 and sICAM-1 binding to LFA-1. The calculated Kd for sICAM-3/LFA-1 binding in the presence of Mn2+ was 579 (±123) nm (n = 3) (see representative experiment, Fig. 3 a). The calculated Kd for sICAM-1/LFA-1 binding in the presence of Mn2+ was 118 (±16) nm(n = 3) (see representative experiment, Fig. 3 b). Both values are similar to the binding observed for sICAM-3 and sICAM-1 in the presence of Mg2+. When each ELISA was performed in the presence of Ca2+, replacing the Mg2+, binding was observed but only at very high concentrations of sICAM-3 and sICAM-1 (>100 μm). Due to the limits of protein solubility, a complete curve could not be generated and a Kd could not be calculated. It can, however, be estimated that the affinities of sICAM-1 and sICAM-3 for LFA-1 in the presence of high concentrations of Ca2+ are at least 100-fold weaker than their respective affinities calculated in the presence of Mg2+ or Mn2+. These data are consistent with the observations that have been characterized for both interactions on the cellular level. To distinguish whether ICAM-3 or ICAM-1 has unique binding sites on LFA-1, a panel of anti-CD11a and anti-CD18 mAbs were evaluated in molecular binding assays. Fig. 4,a and b, shows the inhibitory profiles of these mAbs in the sICAM-3/LFA-1 and sICAM-1/LFA-1 molecular binding assays, respectively (each ICAM was used at 3 × its respectiveKd, normalizing their unequal affinities). A similar inhibition profile can be seen with both ICAMs. The one exception is MEM-83, which gives slight inhibition in the sICAM-3 assay and none in the sICAM-1 assay. Two previous reports have shown that MEM-83 and YTH81.5, which map to the I domain of CD11a, selectively inhibit ICAM-3/LFA-1-mediated adhesion and not ICAM-1/LFA-1-mediated adhesion (18Binnerts M.E. van Kooyk Y. Edwards C.P. Champe M. Presta L. Bodary S.C. Figdor C.G. Berman P.W. J. Biol. Chem. 1996; 271: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 19Binnerts M.E. van Kooyk Y. Simmons D.L. Figdor C.G. Eur. J. Immunol. 1994; 24: 2155-2160Crossref PubMed Scopus (84) Google Scholar, 20Landis R.C. McDowall A. Holness C.L.L. Littler A.J. Simmons D.L. Hogg N. J. Cell Biol. 1994; 126: 529-537Crossref PubMed Scopus (87) Google Scholar). In this report, YTH81.5 inhibits both sICAM-1 and sICAM-3 binding to immobilized LFA-1 in a dose-dependent manner (see Fig. 5 a), whereas MEM-83 only shows activity in the sICAM-3 assay (see Fig. 5 b). In cellular assays involving either sICAM-3 or sICAM-1, neither mAb showed any activity up to 300 and 50 μg/ml of YTH81.5 and MEM-83, respectively (data not shown).Figure 5a, sICAM-3 and biotinylated sICAM-1 binding to purified LFA-1 in the presence of YTH81.5 mAb. Each point represents the mean percent binding compared with an assay buffer control of 2–4 experiments ± S.E. b, sICAM-3 and biotinylated sICAM-1 binding to purified LFA-1 in the presence of MEM-83 mAb. Each point represents the mean percent binding compared with an assay buffer control of 2–5 experiments ± S.E.View Large Image Figure ViewerDownload (PPT) Since ICAM-3 is the most highly glycosylated member of the ICAMs with 15 putative N-linked glycosylation sites within the protein (8de Fougerolles A.R. Klickstein L.B. Springer T.A. J. Exp. Med. 1993; 177: 1187-1192Crossref PubMed Scopus (90) Google Scholar, 9Fawcett J. Holness C.L.L. Needham L.A. Turley H. Gatter K.C. Mason D.Y. Simmons D.L. Nature. 1992; 360: 481-484Crossref PubMed Scopus (302) Google Scholar, 10Vazeux R. Hoffman P.A. Tomita J.K. Dickinson E.S. Jasman L. St. John T. Gallatin W.M. Nature. 1992; 360: 485-488Crossref PubMed Scopus (183) Google Scholar), the role of glycosylation in the binding of ICAM-3 to LFA-1 was evaluated using molecular assays. De-glycosylation of sICAM-3 was performed with recombinant N-glycosidase F in the presence or absence of denaturing agents and detergent. In Fig. 6 a, samples of sICAM-3 (de-glycosylated in the presence (lane 3) or absence (lane 2) of SDS/detergent) were electrophoresed and blotted with polyclonal serum directed against domain 4 of sICAM-3. As can be seen, both samples were digested" @default.
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• W2038707943 title "Molecular Comparison of Soluble Intercellular Adhesion Molecule (sICAM)-1 and sICAM-3 Binding to Lymphocyte Function-associated Antigen-1" @default.
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• W2038707943 cites W1489928516 @default.
• W2038707943 cites W1499176694 @default.
• W2038707943 cites W1510771960 @default.
• W2038707943 cites W169876379 @default.
• W2038707943 cites W1966286149 @default.
• W2038707943 cites W1971832587 @default.
• W2038707943 cites W1975081780 @default.
• W2038707943 cites W1988019232 @default.
• W2038707943 cites W1988136983 @default.
• W2038707943 cites W2006821793 @default.
• W2038707943 cites W2008106258 @default.
• W2038707943 cites W2020455973 @default.
• W2038707943 cites W2023155608 @default.
• W2038707943 cites W2034234555 @default.
• W2038707943 cites W2037301837 @default.
• W2038707943 cites W2039805883 @default.
• W2038707943 cites W2042245332 @default.
• W2038707943 cites W2052043921 @default.
• W2038707943 cites W2057784785 @default.
• W2038707943 cites W2060217665 @default.
• W2038707943 cites W2061357570 @default.
• W2038707943 cites W2073383011 @default.
• W2038707943 cites W2075380738 @default.
• W2038707943 cites W2076625163 @default.
• W2038707943 cites W2081029926 @default.
• W2038707943 cites W2084762695 @default.
• W2038707943 cites W2093830420 @default.
• W2038707943 cites W2099369265 @default.
• W2038707943 cites W2103493503 @default.
• W2038707943 cites W2114104400 @default.
• W2038707943 cites W2121107920 @default.
• W2038707943 cites W2135252042 @default.
• W2038707943 cites W2136437500 @default.
• W2038707943 cites W2140305004 @default.
• W2038707943 cites W2143125251 @default.
• W2038707943 cites W2144518949 @default.
• W2038707943 cites W2157279574 @default.
• W2038707943 cites W2163521672 @default.
• W2038707943 cites W2169528096 @default.
• W2038707943 cites W2220655641 @default.
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