FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2036710326> ?p ?o ?g. }

• W2036710326 endingPage "28298" @default.
• W2036710326 startingPage "28290" @default.
• W2036710326 abstract "Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the β2 integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity. Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the β2 integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity. Neutrophils circulate in the bloodstream to sites of inflammation where they adhere and transmigrate through the endothelium as the initial step in combating infection and to facilitate wound healing. Recruitment from the circulation involves a multistep process of cell rolling, activation, and arrest. The heterodimeric integrin receptor LFA-1 1The abbreviations used are: LFA-1, leukocyte function associated antigen 1; ICAM-1, intercellular adhesion molecule-1; PBS, phosphate-buffered saline; MIDAS, metal ion-dependent adhesion site; IDAS, I domain allosteric site; MES, 4-morpholineethanesulfonic acid; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; MFI, mean fluorescent intensities; IL, interleukin. 1The abbreviations used are: LFA-1, leukocyte function associated antigen 1; ICAM-1, intercellular adhesion molecule-1; PBS, phosphate-buffered saline; MIDAS, metal ion-dependent adhesion site; IDAS, I domain allosteric site; MES, 4-morpholineethanesulfonic acid; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; MFI, mean fluorescent intensities; IL, interleukin. is composed of the αL (CD11a) and β2 (CD18) subunits and is constitutively expressed in a low affinity conformation on the plasma membrane of leukocytes (1.Lum A.F. Green C.E. Lee G.R. Staunton D.E. Simon S.I. J. Biol. Chem. 2002; 277: 20660-20670Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 2.Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1279) Google Scholar, 3.Lollo B.A. Chan K.W. Hanson E.M. Moy V.T. Brian A.A. J. Biol. Chem. 1993; 268: 21693-21700Abstract Full Text PDF PubMed Google Scholar). Neutrophils encountering chemokines on inflamed endothelium are activated to shift LFA-1 from the low to high affinity conformation, which supports tight binding to endothelial ICAM-1. Increases in integrin affinity correlate in time with adhesion function as recently demonstrated in aggregation of cells expressing α4β1 and vascular cell adhesion molecule (4.Chigaev A. Zwartz G. Graves S.W. Dwyer D.C. Tsuji H. Foutz T.D. Edwards B.S. Prossnitz E.R. Larson R.S. Sklar L.A. J. Biol. Chem. 2003; 278: 38174-38182Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).ICAM-1 recognizes LFA-1 through an inserted (I) domain in the α subunit. There is strong evidence correlating shifts in I domain conformation to affinity changes in binding ICAM-1. Mutations in I domain residues stabilized distinct structural conformations correlating to LFA-1 affinity. ICAM-1 equilibrium binding constants increase over 4 orders of magnitude ranging between low (i.e. 1600 μm), intermediate (i.e. 9 μm), and high affinity (i.e. 0.15 μm) (5.Shimaoka M. Xiao T. Liu J.H. Yang Y. Dong Y. Jun C.D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J.H. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). Further evidence linking allosteric shifts in I domain conformation to ICAM-1 binding is the activity of a class of allosteric small molecule antagonists engineered to inhibit LFA-1 function (6.Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5231-5236Crossref PubMed Scopus (142) Google Scholar, 7.Lu C. Shimaoka M. Ferzly M. Oxvig C. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2387-2392Crossref PubMed Scopus (119) Google Scholar, 8.Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol. 1999; 292: 1-9Crossref PubMed Scopus (233) Google Scholar, 9.Weitz-Schmidt G. Welzenbach K. Brinkmann V. Kamata T. Kallen J. Bruns C. Cottens S. Takada Y. Hommel U. Nat. Med. 2001; 7: 687-692Crossref PubMed Scopus (957) Google Scholar). Statin-derived small molecules such as lovastatin and LFA703 target the I domain allosteric site (IDAS) and abrogate LFA-1 recognition of ICAM-1 (8.Kallen J. Welzenbach K. Ramage P. Geyl D. Kriwacki R. Legge G. Cottens S. Weitz-Schmidt G. Hommel U. J. Mol. Biol. 1999; 292: 1-9Crossref PubMed Scopus (233) Google Scholar, 10.Welzenbach K. Hommel U. Weitz-Schmidt G. J. Biol. Chem. 2002; 277: 10590-10598Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Another small molecule to the IDAS, BIRT377, was shown to inhibit rolling and adhesion of LFA-1 transfectants to ICAM-1 monolayers (11.Salas A. Shimaoka M. Kogan A.N. Harwood C. Von Andrian U.H. Springer T.A. Immunity. 2004; 20: 393-406Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). BIRT377 and LFA703 appear to exert their actions through shifting LFA-1 into a bent conformation, rendering the I domain inaccessible and LFA-1 into a low affinity state (12.Shimaoka M. Salas A. Yang W. Weitz-Schmidt G. Springer T.A. Immunity. 2003; 19: 391-402Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). A second class of small molecules binds to the I-like domain in the β subunit of LFA-1 and indirectly regulates I domain affinity and ligand binding (10.Welzenbach K. Hommel U. Weitz-Schmidt G. J. Biol. Chem. 2002; 277: 10590-10598Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 12.Shimaoka M. Salas A. Yang W. Weitz-Schmidt G. Springer T.A. Immunity. 2003; 19: 391-402Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The small molecule XVA143 binds to the I-like domain and promotes cellular rolling by inducing an extended conformation that stabilizes an intermediate affinity associated with rolling of LFA-1 expressing transfectants in shear flow (11.Salas A. Shimaoka M. Kogan A.N. Harwood C. Von Andrian U.H. Springer T.A. Immunity. 2004; 20: 393-406Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). We present here a new small molecule allosteric inhibitor that targets the IDAS and downshifts LFA-1 from a high to intermediate affinity. This small molecule is similar to the diaryl sulfide cinnamide antagonists (13.Liu G. Link J.T. Pei Z. Reilly E.B. Leitza S. Nguyen B. Marsh K.C. Okasinski G.F. von Geldern T.W. Ormes M. Fowler K. Gallatin M. J. Med. Chem. 2000; 43: 4025-4040Crossref PubMed Scopus (199) Google Scholar). Allosteric small molecules provide a powerful tool for directing leukocyte adhesion; however, the interrelationships between bond kinetics, LFA-1 conformation, valence in binding ICAM-1, and adhesion stability remain ill-defined.Concomitant with a shift in affinity is a rapid redistribution of LFA-1 into high density clusters on the plasma membrane. We have reported recently that within seconds of activation, LFA-1 on neutrophils reorganizes from a uniform surface distribution to form both small punctate clusters (<1 μm2) and large caps (∼3 μm2) (1.Lum A.F. Green C.E. Lee G.R. Staunton D.E. Simon S.I. J. Biol. Chem. 2002; 277: 20660-20670Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 14.Sigal A. Bleijs D.A. Grabovsky V. van Vliet S.J. Dwir O. Figdor C.G. van Kooyk Y. Alon R. J. Immunol. 2000; 165: 442-452Crossref PubMed Scopus (100) Google Scholar). Clustering of LFA-1 on leukocytes tethered to inflamed endothelium in shear flow is a key step in adhesion strengthening and the transition from cell rolling to arrest (1.Lum A.F. Green C.E. Lee G.R. Staunton D.E. Simon S.I. J. Biol. Chem. 2002; 277: 20660-20670Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 14.Sigal A. Bleijs D.A. Grabovsky V. van Vliet S.J. Dwir O. Figdor C.G. van Kooyk Y. Alon R. J. Immunol. 2000; 165: 442-452Crossref PubMed Scopus (100) Google Scholar). To emulate affinity and molecular scale clustering of LFA-1 on activated leukocytes, we engineered a cell-free LFA-1 expression system by fusing the α-β subunits at the C terminus with an inserted leucine zipper motif. The C termini of two heterodimers were bound to each Fab arm of an anti-leucine zipper antibody covalently attached to the surface of a latex microsphere. We tested the hypothesis that two adjacent LFA-1 binding to an ICAM-1 homodimer could facilitate rebinding and exponentially prolong bond lifetime. Binding of fluorescent ICAM-1 to LFA-1 on beads was monitored in real time by flow cytometry while shifting LFA-1 conformation and affinity state with soluble agonists and antagonists.Dissociation of monomeric ICAM-1 from high affinity LFA-1 was ∼10-fold faster than dimeric ICAM-1. This difference was attributed to the ability of the dissociated leg of the ICAM-1 dimer to rebind to an adjacent LFA-1 as it is held in proximity by the remaining LFA-1 bond. Adhesion of neutrophils to beads presenting dimeric ICAM-1 in shear flow was sustained beyond 10 min, while monomeric ICAM-1 beads dissociated within 100 s. These data highlight the physiological significance of regulation of both LFA-1 affinity and LFA-1 spatial proximity in tuning bond lifetime and adhesion stability when binding to ICAM-1 homodimer.MATERIALS AND METHODSReagents—The following antibodies were used: anti-leucine zipper (324C), anti-CD18 activating antibody (240Q) (1.Lum A.F. Green C.E. Lee G.R. Staunton D.E. Simon S.I. J. Biol. Chem. 2002; 277: 20660-20670Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 6.Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5231-5236Crossref PubMed Scopus (142) Google Scholar, 15.Lupher Jr., M.L. Harris E.A. Beals C.R. Sui L.M. Liddington R.C. Staunton D.E. J. Immunol. 2001; 167: 1431-1439Crossref PubMed Scopus (57) Google Scholar, 16.Beals C.R. Edwards A.C. Gottschalk R.J. Kuijpers T.W. Staunton D.E. J. Immunol. 2001; 167: 6113-6122Crossref PubMed Scopus (74) Google Scholar), steric blocking anti-LFA-1 (TS1/22) (17.Lu C. Shimaoka M. Salas A. Springer T.A. J. Immunol. 2004; 173: 3972-3978Crossref PubMed Scopus (38) Google Scholar), anti-LFA-1 TS2/4 (18.Huang C. Springer T.A. J. Biol. Chem. 1995; 270: 19008-19016Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), recombinant LFA-1 heterodimer with an inserted leucine zipper, ICAM-1/Ig produced as a chimeric human IgG containing two full-length ICAM-1 (molecular mass is 150 kDa as confirmed by native PAGE), anti-CD18 327C (1.Lum A.F. Green C.E. Lee G.R. Staunton D.E. Simon S.I. J. Biol. Chem. 2002; 277: 20660-20670Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 15.Lupher Jr., M.L. Harris E.A. Beals C.R. Sui L.M. Liddington R.C. Staunton D.E. J. Immunol. 2001; 167: 1431-1439Crossref PubMed Scopus (57) Google Scholar), and LFA-1 small molecule allosteric inhibitor IC487475 of the p-arylthio cinnamides series that targets the I domain allosteric site of LFA-1. Inhibitory anti-CD18 TS1/18 was purchased from Pierce. Blocking anti-Mac-1 2LPM19c was purchased from DakoCytomation, Glostrop, Denmark. Lovastatin sodium was purchased from Calbiochem. Anti-CD18 KIM127 (11.Salas A. Shimaoka M. Kogan A.N. Harwood C. Von Andrian U.H. Springer T.A. Immunity. 2004; 20: 393-406Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 19.Robinson M.K. Andrew D. Rosen H. Brown D. Ortlepp S. Stephens P. Butcher E.C. J. Immunol. 1992; 148: 1080-1085PubMed Google Scholar, 20.Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (257) Google Scholar, 21.Lu C. Shimaoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar), which detects extended conformations of LFA-1, was a gift from Martin Robinson (Exploratory Research Cell Tech Therapeutics Ltd., Bath Road Slough, UK). Recombinant human ICAM-1 (monomeric full-length, molecular mass is 85 kDa as confirmed by native polyacrylamide gel) and chemotactic stimulus interleukin-8 (IL-8) were purchased from R & D Systems, Minneapolis, MN. Anti-CD18 FITC, anti-CD54 FITC, and mouse IgG1 were purchased from Caltag Laboratories, Burlingame, CA. Goat anti-mouse FITC was purchased from Kirkegaard & Perry Laboratories, Inc. Gaithersburg, MD. Dimethyl sulfoxide (Me2SO) was bought from Sigma.Assembly of LFA-1 on the Microsphere—Amino microsphere latex beads (diameter = 6 μm, 2.7% solids, latex) (Biosciences, Piscataway, NJ) were mixed (Eppendorf Thermomixer R, Brinkmann Instruments) with a sulfosuccinimidyl maleimide-N-hydroxysuccinimide ester cross-linker (0.125 × 10–3 m) (Pierce) as described by the manufacturer's protocol. A sulfhydryl group was added to anti-leucine zipper 324C by incubating 324C with a sulfhydryl linker (Pierce). 324C-SH was mixed (450 rpm) with the amino cross-linker beads for 2 h at 8 °C. Beads were washed and stored at a final concentration of 107 beads/ml at 4 °C. Directly prior to experimentation, LFA-1 (20 μg/ml) was mixed with 105 beads/100-μl sample at 37 °C at 450 rpm for 30 min. The bivalent binding structure of the antibody allows for clustering of the LFA-1 into a dimer-like configuration presented on the bead surface. The cell-free system offers the opportunity to prescribe LFA-1 distribution in a manner that shifts the binding spectrum toward bivalent driven adhesion of ICAM-1.LFA-1 Activation, Inhibition, and Detection—Cell-free LFA-1 on beads was washed and resuspended in 100 μl of phosphate-buffered saline (PBS) without Ca2+ or Mg2+ (Invitrogen) and incubated (all incubations were performed at 37 °C at 450 rpm for 30 min unless otherwise noted) with CaCl2 (1.5 mm), MgCl2 (3 mm), and/or 240Q (10 μg/ml). Without washing, fluorescently labeled (AlexaFluor488, Molecular Probes, Eugene, OR) dimeric ICAM-1 (20 μg/ml) was added to the beads and incubated. Beads were washed and resuspended in 150 μl of PBS, and mean fluorescence intensity was detected by flow cytometry. For inhibition of binding, IC487475 (1 μm), TS1/22, TS1/18, or TS2/4 (20 μg/ml) was added prior to activation and incubated at 37 °C, 450 rpm for 15 min. Without wash, MgCl2 (3 mm) and ICAM-1 (20 μg/ml) were introduced and incubated for 20 min.Binding kinetics of ICAM-1-AlexaFluor488 (10 μg/ml) to beads were measured by adding ICAM-1 immediately prior to flow cytometry readings. ICAM-1 association and dissociation were measured for 3 × 105 beads in 300 μl of PBS aliquots for up to 40 min at room temperature. In the indicated samples, there was a 10-min incubation at room temperature with MgCl2 (or MnCl2, 3 mm) and/or 240Q (10 μg/ml) before the addition of ICAM-1 with the activator remaining in the solution throughout the flow cytometry reading. Dissociation of bound ICAM-1 was continuously detected after the introduction of small molecule inhibitor IC487475 (1 μm), small molecule lovastatin (100 μm), addition of TS1/22 (100 μg/ml), or dilution in the presence of unlabeled ICAM-1. The inhibitory reagents were introduced through polyethylene tubing inserted into the cytometer tube. For dissociation of bound ICAM-1 with dilution, 1 ml of PBS was added containing unlabeled dimeric ICAM-1/Ig 20 times the concentration of the labeled ICAM-1.To detect conformation, LFA-1 beads were treated with by MgCl2 (3 mm), 240Q (10 μg/ml), or IC487475 (1 μm) during a 15-min incubation. KIM127 was incubated with the samples for an additional 15 min. Samples were washed and incubated for 15 min with goat anti-mouse FITC, and mean fluorescent intensities (MFI) were read by flow cytometry (FACScan flow cytometer, Pharmingen).Neutrophil Isolation and Activation—Whole blood was drawn from healthy subjects by venipuncture into sterile syringes with heparin (10 units/ml of blood, Elkins-Sinn, Inc., Cherry Hill, NJ) as described previously (1.Lum A.F. Green C.E. Lee G.R. Staunton D.E. Simon S.I. J. Biol. Chem. 2002; 277: 20660-20670Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Neutrophils were isolated from whole blood using a density gradient media (Robbins Scientific Corp., Sunnyvale, CA). Neutrophils were washed once with HEPES buffer (10 mm KCl, 110 mm NaCl, 10 mm glucose, 1 mm MgCl2, and 30 mm HEPES, pH 7.4) and were maintained at room temperature in a calcium-free HEPES buffer for up to 4 h. Neutrophils were activated by addition of antibody 240Q (10 μg/ml) for 10 min at room temperature in HEPES buffer, human serum albumin (0.1%), and CaCl2 (1.5 mm). Kinetic experiments were performed as described for the LFA-1 microspheres.ICAM-1 Bead Assembly—Carboxylate microspheres (diameter = 10 μm) were purchased from Polysciences, Inc. (Warrington, PA). 500 μlof beads were washed twice in 1.5 ml of MES buffer, pH 5.0 (Sigma), resuspended in 200 μl of MES, and sonicated for 15 min. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, hydrochloride (Molecular Probes, Eugene, OR), was added at 1 mm, and beads were incubated for 5 min at room temperature and at 500 rpm. Monomeric ICAM-1 (70 μg/ml) or dimeric ICAM-1/Ig (32 μg/ml) was mixed with the beads for 1 h at room temperature and at 500 rpm. Glycine (Sigma) was added (10 mm), and beads were mixed for 30 min at room temperature at 500 rpm. Blockaid blocking solution (Molecular Probes) was added (200 μl) and incubation continued for an additional 15 min. ICAM-1 beads were washed in PBS and resuspended in 1.5 ml of PBS. Site densities were obtained by Quantum Simply Cellular Beads (Bangs Laboratories, Fishers, IN) to be ∼6000 sites/μm2 for monomeric and dimeric beads as identified by anti-CD54.Adhesion of Neutrophils to Beads Presenting Monomeric and Dimeric ICAM-1—Neutrophils were mixed with 10-μm fluorescent latex beads (Fluoresbrite Carboxyl YG 10 Micron Microspheres) with ICAM-1 derivatized to their surface. Samples contained 1 × 106 neutrophils/ml, 2 × 106 beads/ml, and a small magnetic stir bar. Mac-1 blocking antibody 2LPM19c (test volume 10 μl) and/or 240Q (10 μg/ml) was preincubated for 10 min with the cell suspension without beads. For samples stimulated with IL-8 (5 nm), beads and stimulus were added immediately prior to reading on the flow cytometer. Samples were maintained at 37 °C within a mixing chamber with a magnetic motor as described previously (22.Tsang Y.T. Neelamegham S. Hu Y. Berg E.L. Burns A.R. Smith C.W. Simon S.I. J. Immunol. 1997; 159: 4566-4577PubMed Google Scholar). The magnetic motor coupled with a magnetic stir bar created a shear field (shear stress ∼1.0 dyne/cm2) within the test tube and initiated collisional interactions. Neutrophil capture of ICAM-1-coated beads was continuously monitored by their characteristic forward and right angle light scatter properties and gated in order to exclude unbound beads. Neutrophil-bead adhesion was quantitated on green fluorescence on fluorescence histograms. Quantal increases in fluorescence appeared as peaks in the fluorescence histogram corresponding to populations of neutrophils binding increasing numbers of beads (22.Tsang Y.T. Neelamegham S. Hu Y. Berg E.L. Burns A.R. Smith C.W. Simon S.I. J. Immunol. 1997; 159: 4566-4577PubMed Google Scholar). To distinguish relative levels of bead capture within the stimulated neutrophil population, neutrophil-bead interactions were quantitated as the average number of beads per neutrophil according to Equation 1, beads/neutrophil=∑i=15i(NBi)N+∑j=15NBj(Eq. 1) where N represents the number of nonadherent neutrophils, and NBi represents the number of neutrophil-bead aggregates bound to between 1 and 5 beads. Aggregates larger than 5 beads were not notably seen in this assay.Dissociation of ICAM-1 beads from neutrophils was induced after 2 min by removing the cytometer sample tube for not more than 10 s during reading and adding inhibitor (1 μm IC487475, 100 μm lovastatin, 100 μg/ml TS1/22, or 4 μl of 1:100 Me2SO:PBS). Cytometer reading resumed and dissociation was modeled as rate of bead/polymorphonuclear leukocyte disaggregation.Data Analysis—Data were analyzed using Graphpad Prism version 4.0 for Windows (Graphpad Software Inc., San Diego). Constants koff (dissociation rate constant) and kobs (observed rate constant) were obtained by performing one-phase exponential decay (Y = specific binding × e–kT + nonspecific binding) and one-phase exponential association (Y = Ymax (1 – e–kT)) curve fits of the real time data, respectively. The kon (association rate constant) was calculated as kon = (kobs – koff)/(ICAM-1-Alexa). Data points were calculated by taking the average fluorescence over 5–25 s, depending on the rate of change. KD, the equilibrium affinity constant, was calculated as koff/kon for kinetic experiments with ICAM-1 binding to the LFA-1 beads. KD was calculated as the EC50 value in a dose-response curve for ICAM-1 binding to the neutrophils. Statistical significance was determined (p ≤ 0.05) by a one-way analysis of variance with a Newman-Keuls multiple comparison post-test or by two-way analysis of variance.RESULTSLFA-1 expressed on the leukocyte membrane can shift its conformation in response to inside-out signaling via chemokine stimulation or extracellularly by addition of divalent cations (23.Hogg N. Henderson R. Leitinger B. McDowall A. Porter J. Stanley P. Immunol. Rev. 2002; 186: 164-171Crossref PubMed Scopus (144) Google Scholar), antibodies (16.Beals C.R. Edwards A.C. Gottschalk R.J. Kuijpers T.W. Staunton D.E. J. Immunol. 2001; 167: 6113-6122Crossref PubMed Scopus (74) Google Scholar), and small molecules (12.Shimaoka M. Salas A. Yang W. Weitz-Schmidt G. Springer T.A. Immunity. 2003; 19: 391-402Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 24.Kelly T.A. Jeanfavre D.D. McNeil D.W. Woska Jr., J.R. Reilly P.L. Mainolfi E.A. Kishimoto K.M. Nabozny G.H. Zinter R. Bormann B.J. Rothlein R. J. Immunol. 1999; 163: 5173-5177PubMed Google Scholar). LFA-1 heterodimer captured at the C terminus by an antibody covalently attached to microspheres was predominantly expressed in a low affinity state but retained the flexibility to shift conformation and boost affinity for ICAM-1. Cell-free LFA-1 increased binding to dimeric ICAM-1/Ig by 150% over base line in response to the addition of divalent cations Mg2+ or Mn2+, but by only 20% in response to Ca2+ (Fig. 1). Addition of mAb 240Q, which is associated with allosterically stabilizing CD18 into a ligand binding conformation (1.Lum A.F. Green C.E. Lee G.R. Staunton D.E. Simon S.I. J. Biol. Chem. 2002; 277: 20660-20670Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 6.Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5231-5236Crossref PubMed Scopus (142) Google Scholar, 15.Lupher Jr., M.L. Harris E.A. Beals C.R. Sui L.M. Liddington R.C. Staunton D.E. J. Immunol. 2001; 167: 1431-1439Crossref PubMed Scopus (57) Google Scholar, 16.Beals C.R. Edwards A.C. Gottschalk R.J. Kuijpers T.W. Staunton D.E. J. Immunol. 2001; 167: 6113-6122Crossref PubMed Scopus (74) Google Scholar), did not itself induce activation of LFA-1 on beads but in conjunction with Mg2+ augmented ICAM-1 binding by 50% above stimulation with Mg2+ alone.ICAM-1/Ig binding was inhibited by pretreating Mg2+-activated LFA-1 with mAb TS1/22, which when bound to its epitope on the I domain sterically blocks ICAM-1 recognition (17.Lu C. Shimaoka M. Salas A. Springer T.A. J. Immunol. 2004; 173: 3972-3978Crossref PubMed Scopus (38) Google Scholar, 20.Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (257) Google Scholar, 25.Huang C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3156-3161Crossref PubMed Scopus (60) Google Scholar). Addition of the allosteric small molecule IC487475, which binds with high affinity to the I domain (i.e. ∼10 nm), also abrogated ICAM-1 binding stimulated by Mg2+ (Fig. 1b). Pretreatment with mAb TS1/18, which binds to an allosterically sensitive domain on the β subunit (25.Huang C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3156-3161Crossref PubMed Scopus (60) Google Scholar), blocked 65% of ICAM-1 binding in the presence of Mg2+. A nonblocking control, anti-CD11a TS2/4 (18.Huang C. Springer T.A. J. Biol. Chem. 1995; 270: 19008-19016Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), increased ICAM-1 binding in the presence of Mg2+ by 25% (data not shown). These data indicate that cell-free LFA-1 retains the capacity to increase affinity for the ICAM-1/Ig dimer through activation by divalent cation or by allosteric mAb 240Q. Moreover, ICAM-1 binding can be sterically blocked by TS1/22 or allosterically inhibited by binding of a small molecule to the I domain.Antibodies that recognize specific epitopes on the β subunit can report on integrin conformation and activation. KIM127 binding has been correlated recently with the extended conformation of LFA-1 and is associated with intermediate or high affinity ligand binding (11.Salas A. Shimaoka M. Kogan A.N. Harwood C. Von Andrian U.H. Springer T.A. Immunity. 2004; 20: 393-406Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 19.Robinson M.K. Andrew D. Rosen H. Brown D. Ortlepp S. Stephens P. Butcher E.C. J. Immunol. 1992; 148: 1080-1085PubMed Google Scholar, 20.Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (257) Google Scholar, 21.Lu C. Shimaoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar). This activation reporter was used to determine the mechanism by which IC487475 and 240Q allosterically alter the affinity for ICAM-1. In the absence of divalent cations, binding of KIM127 was 2-fold above an IgG isotype-matched control, indicating that a fraction of the LFA-1 adopted a conformation other than the bent low affinity state in the absence of stimulation (data not shown). Activation of LFA-1 with Mg2+ increased KIM127 binding by 40%, which was augmented to 60% upon addition of 240Q (Fig. 1c). IC487475 did not significantly decrease LFA-1 recognition by KIM127 despite its ability to abrogate Mg2+-induced ICAM-1 binding. This suggests that IC487475 can downshift LFA-1 affinity at the IDAS in the absence of inducing a bent conformation of the heterodimer.Kinetic Analysis of ICAM-1 Binding by Fluorescence Flow Cytometry—Leukocytes can shift LFA-1 conformation and increase avidity within seconds of contact at vascular sites of inflammation (26.Ley K. Immunol. Rev. 2002; 186: 8-18Crossref PubMed Scopus" @default.
• W2036710326 created "2016-06-24" @default.
• W2036710326 creator A5019384731 @default.
• W2036710326 creator A5026441929 @default.
• W2036710326 creator A5040402621 @default.
• W2036710326 creator A5051233762 @default.
• W2036710326 creator A5079839111 @default.
• W2036710326 date "2005-08-01" @default.
• W2036710326 modified "2023-09-30" @default.
• W2036710326 title "Leukocyte Function-associated Antigen 1-mediated Adhesion Stability Is Dynamically Regulated through Affinity and Valency during Bond Formation with Intercellular Adhesion Molecule-1" @default.
• W2036710326 cites W125310781 @default.
• W2036710326 cites W1479880028 @default.
• W2036710326 cites W1489928516 @default.
• W2036710326 cites W1499176694 @default.
• W2036710326 cites W1551890509 @default.
• W2036710326 cites W1613698407 @default.
• W2036710326 cites W1646222158 @default.
• W2036710326 cites W1988373084 @default.
• W2036710326 cites W1996257032 @default.
• W2036710326 cites W1996629582 @default.
• W2036710326 cites W2006657990 @default.
• W2036710326 cites W2015313829 @default.
• W2036710326 cites W2015826818 @default.
• W2036710326 cites W2017602598 @default.
• W2036710326 cites W2020455973 @default.
• W2036710326 cites W2022431638 @default.
• W2036710326 cites W2032896166 @default.
• W2036710326 cites W2037401620 @default.
• W2036710326 cites W2039805883 @default.
• W2036710326 cites W2044163981 @default.
• W2036710326 cites W2045619217 @default.
• W2036710326 cites W2047963682 @default.
• W2036710326 cites W2056565413 @default.
• W2036710326 cites W2057979638 @default.
• W2036710326 cites W2063220206 @default.
• W2036710326 cites W2063559096 @default.
• W2036710326 cites W2081029926 @default.
• W2036710326 cites W2085979652 @default.
• W2036710326 cites W2095272173 @default.
• W2036710326 cites W2109483333 @default.
• W2036710326 cites W2112573306 @default.
• W2036710326 cites W2113177113 @default.
• W2036710326 cites W2114224767 @default.
• W2036710326 cites W2122945822 @default.
• W2036710326 cites W2125435732 @default.
• W2036710326 cites W2133169381 @default.
• W2036710326 cites W2135987235 @default.
• W2036710326 cites W2153005017 @default.
• W2036710326 cites W2153032621 @default.
• W2036710326 cites W2160506150 @default.
• W2036710326 cites W2162452021 @default.
• W2036710326 cites W2162906577 @default.
• W2036710326 cites W2164240234 @default.
• W2036710326 cites W2170389449 @default.
• W2036710326 doi "https://doi.org/10.1074/jbc.m501662200" @default.
• W2036710326 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15955822" @default.
• W2036710326 hasPublicationYear "2005" @default.
• W2036710326 type Work @default.
• W2036710326 sameAs 2036710326 @default.
• W2036710326 citedByCount "48" @default.
• W2036710326 countsByYear W20367103262012 @default.
• W2036710326 countsByYear W20367103262013 @default.
• W2036710326 countsByYear W20367103262014 @default.
• W2036710326 countsByYear W20367103262015 @default.
• W2036710326 countsByYear W20367103262016 @default.
• W2036710326 countsByYear W20367103262018 @default.
• W2036710326 crossrefType "journal-article" @default.
• W2036710326 hasAuthorship W2036710326A5019384731 @default.
• W2036710326 hasAuthorship W2036710326A5026441929 @default.
• W2036710326 hasAuthorship W2036710326A5040402621 @default.
• W2036710326 hasAuthorship W2036710326A5051233762 @default.
• W2036710326 hasAuthorship W2036710326A5079839111 @default.
• W2036710326 hasBestOaLocation W20367103261 @default.
• W2036710326 hasConcept C12554922 @default.
• W2036710326 hasConcept C134659918 @default.
• W2036710326 hasConcept C138885662 @default.
• W2036710326 hasConcept C139007053 @default.
• W2036710326 hasConcept C14036430 @default.
• W2036710326 hasConcept C147483822 @default.
• W2036710326 hasConcept C16224149 @default.
• W2036710326 hasConcept C178790620 @default.
• W2036710326 hasConcept C185592680 @default.
• W2036710326 hasConcept C203014093 @default.
• W2036710326 hasConcept C2909375385 @default.
• W2036710326 hasConcept C41895202 @default.
• W2036710326 hasConcept C55493867 @default.
• W2036710326 hasConcept C79879829 @default.
• W2036710326 hasConcept C84416704 @default.
• W2036710326 hasConcept C85789140 @default.
• W2036710326 hasConcept C86803240 @default.
• W2036710326 hasConcept C95444343 @default.
• W2036710326 hasConceptScore W2036710326C12554922 @default.
• W2036710326 hasConceptScore W2036710326C134659918 @default.
• W2036710326 hasConceptScore W2036710326C138885662 @default.
• W2036710326 hasConceptScore W2036710326C139007053 @default.
• W2036710326 hasConceptScore W2036710326C14036430 @default.
• W2036710326 hasConceptScore W2036710326C147483822 @default.
• W2036710326 hasConceptScore W2036710326C16224149 @default.

• next