Selective, Tight-Binding Inhibitors of Integrin α4β1 That Inhibit Allergic Airway Responses

Lin, Ko-Chung; Ateeq, Humayun S.; Hsiung, Shu‐chi; Chong, Liu; Zimmerman, Craig; Castro, Alfredo; Lee, W C; Hammond, Charles E.; Kalkunte, Satyan; Chen, L L; Pepinsky, R. Blake; Leone, Diane R.; Sprague, Andrew; Abraham, William M.; Gill, Alan; Lobb, Roy R.; Adams, Steven P.

doi:10.1021/jm980673g

Cited by 209 publications

(143 citation statements)

References 34 publications

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“…In addition, the ability of VLA-4 to strengthen lymphocyte adhesion to high-and medium-density VCAM-1 in lymphocytes settled for 1 minute on the integrin ligand was reduced by 2-to 4-fold in LAD EBV cells (data not shown). Notably, VLA-4 tethers capable of immediately arresting tethered lymphocytes on high-density VCAM-1 were mediated by preexistent high-affinity VLA-4 subsets susceptible to blockage by a low dose (1 ng/mL) of the VLA-4 binding peptide, Bio1211 17,19 (data not shown). Although LAD cells normally express such high-affinity VLA-4 subsets, 1 these subsets failed to arrest LAD lymphocytes on VCAM-1 compared with the ability of these subsets to instantaneously arrest as many as 25% of tethered control cells ( Figure 1B and data not shown).…”

Section: Resultsmentioning

confidence: 96%

LAD-III, a leukocyte adhesion deficiency syndrome associated with defective Rap1 activation and impaired stabilization of integrin bonds

Kinashi

Aker

Sokolovsky-Eisenberg

et al. 2004

Blood

106

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Recently, we reported a rare leukocyte adhesion deficiency (LAD) associated with severe defects in integrin activation by chemokine signals, despite normal ligand binding of leukocyte integrins. 1 We now report that the small GTPase, Rap1, a key regulator of inside-out integrin activation is abnormally regulated in LAD Epstein-Barr virus (EBV) lymphocyte cells. Both constitutive and chemokine-triggered activation of Rap1 were abolished in LAD lymphocytes despite normal chemokine signaling. Nevertheless, Rap1 expression and activation by phorbol esters were intact, ruling out an LAD defect in Rap1 guanosine triphosphate (GTP) loading. The very late antigen 4 (VLA-4) integrin abnormally tethered LAD EBV lymphocytes to its ligand vascular cell adhesion molecule 1 (VCAM-1) under shear flow due to impaired generation of high-avidity contacts despite normal ligand binding and intact avidity to surfacebound anti-VLA-4 monoclonal antibody (mAb). Thus, a defect in constitutive Rap1 activation results in an inability of ligandoccupied integrins to generate highavidity binding to ligand under shear flow. This is a first report of an inherited Rap1 activation defect associated with a pathologic disorder in leukocyte integrin function, we herein term it "LAD-III." ( IntroductionLeukocyte arrest at target endothelial sites is nearly exclusively mediated by integrin receptors. 2 Circulating leukocytes maintain their integrins in largely nonadhesive states, rapidly modulated by a variety of inside-out signals. 3 We recently described a human genetic deficiency of leukocyte adhesion to endothelium (LAD), which results from impaired ability of leukocyte integrins to generate high avidity to their endothelial ligands at vascular endothelial contacts in response to endothelial chemoattractant signals. 1 Patient leukocytes, however, express intact integrins and G-protein-coupled chemokine receptors (GPCRs). A growing body of evidence implicates the Ras-related GTPase, Rap1, as a key regulator of integrin activation by virtually all major inside-out stimuli transduced to leukocytes and platelets. [4][5][6][7][8][9] Like other GTPases, it cycles from an inactive guanosine diphosphate (GDP)-bound form to an active guanosine triphosphate (GTP)-bound form, through guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). 10 Suppression of endogenous lymphocyte Rap1 activity by GAP overexpression impairs both very late antigen 4 (VLA-4) and leukocyte function-associated antigen 1 (LFA-1) adhesiveness even in the absence of de novo Rap1 activation by chemoattractants. 11 In this study, we characterized an Epstein-Barr virus (EBV)-transformed B-cell line derived from the LAD patient peripheral blood leukocytes (PBLs) and from age-matched control lymphocytes. We show here that Rap1 is normally expressed in LAD cells, but exhibits an aberrant activation pattern. Suppressed Rap1 activation correlated with a defect in the ability of LAD VLA-4 to generate high-avidity tethers to its endothelial ligand, vascular cell adhe...

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Section: Resultsmentioning

confidence: 96%

LAD-III, a leukocyte adhesion deficiency syndrome associated with defective Rap1 activation and impaired stabilization of integrin bonds

Kinashi

Aker

Sokolovsky-Eisenberg

et al. 2004

Blood

106

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“…This interaction was also detected in cells plated on VCAM-1 (data not shown). FRET was significantly decreased when two small molecule, LDV ligand mimetic inhibitors of ␣4␤1 integrin [S976162 and S9916197 (Gläsner et al, 2005;Lin et al, 1999) see supplementary material Table S1 and Fig. S2 for characterisation] were added to pre-spread cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were then washed three times in medium and either fixed for FLIM analysis or imaged live in phenol-red free growth media at 37°C. For integrin antagonist studies, cells were pre-treated with concentrations and times specified in figure legend with V0519 (Peyman et al, 2000), S9916197 (Gläsner et al, 2005) or S976162 (Lin et al, 1999) and subjected to FLIM analysis.…”

Section: Bead Coating and Incubationmentioning

confidence: 99%

Quantification of integrin receptor agonism by fluorescence lifetime imaging

Parsons

Messent

Humphries

et al. 2008

Journal of Cell Science

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Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin adhesion receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to agonists. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using FLIM to measure FRET. Talin-β1 and paxillin-α4 association was both ligand- and receptor activation state-dependent, and sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules and provides a new, quantitative assay for the screening of activity of small molecule integrin inhibitors.

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“…Recombinant 7-domain human vascular cell adhesion molecule (VCAM)-1, soluble VCAM-1, and BIO-1211, a VLA-4-specific blocker, 17 were kindly provided by B. Pepinsky (Biogen Idec). Intercellular adhesion molecule (ICAM)-Fc, VCAM-1-Fc, and CXC chemokine ligand (CXCL)12 were purchased from R&D Systems.…”

Section: Reagents and Antibodiesmentioning

confidence: 99%

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions

Manevich-Mendelson

Feigelson

Pasvolsky

et al. 2009

Blood

105

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Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out IntroductionIntegrins constitute the major and largest family of cell adhesion receptors. 1 In hematopoietic cells, these heterodimers rapidly undergo dramatic allosteric conformational changes in response to various activation signals. 2 Talins are key integrin activators implicated in these processes in essentially all integrin-containing cell types. 3 Activated integrins are essential for platelet aggregation, firm leukocyte adhesiveness to vascular endothelium, and lymphocyte arrest on antigen-presenting cells. 4 Recent evidence suggests that Kindlins, a group of 3 structurally related adaptors, cooperate with talin in activating integrins in different cell types through binding to distinct motifs on the short tails of the integrin ␤ subunits. [5][6][7][8] In contrast to Kindlins-1 and -2, Kindlin-3 expression is restricted to the hematopoietic system. 7 Whereas loss of talin1 is embryonically lethal, deletion of Kindlin-3 is not; 7 yet, deletion of Kindlin-3 in mice results in severe defects in platelet and leukocyte integrin activation and in reduced lymphocyte counts, 7,9 osteoporosis, 7 and abnormal erythrocyte function, which is ultimately fatal. 10 Although a recent study has directly implicated murine Kindlin-3 in neutrophil and monocyte integrin adhesiveness to inflamed endothelium in vitro and in vivo, 9 the role of Kindlin-3 in inside-out (chemokine-mediated) and outside-in (ligand-induced) activation of lymphocyte integrins has remained unexplored.Leukocyte adhesion deficiency (LAD)-III is a rare autosomal recessive syndrome, which is manifested as a combined defect in ␤ 3 , ␤ 2 , and ␤ 1 integrin activation in platelets, neutrophils, and lymphocytes. 11 We previously reported 3 LAD-III cases from families of Turkish origin, associated with a homozygous splice junction mutation that results in defective expression of a key Rap-1-specific guanine nucleotide exchange factor (Rap-1/2 GEF), CalDAG-GEFI (CDGI), in platelets, neutrophils, and resting lymphocytes. 12 We recently showed that these patients suffer from an additional mutation, a homozygous nonsense stop ...

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Selective, Tight-Binding Inhibitors of Integrin α4β1 That Inhibit Allergic Airway Responses

Cited by 209 publications

References 34 publications

LAD-III, a leukocyte adhesion deficiency syndrome associated with defective Rap1 activation and impaired stabilization of integrin bonds

LAD-III, a leukocyte adhesion deficiency syndrome associated with defective Rap1 activation and impaired stabilization of integrin bonds

Quantification of integrin receptor agonism by fluorescence lifetime imaging

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions

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