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J. Biol. Chem., Vol. 281, Issue 49, 37904-37912, December 8, 2006

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A Small Molecule Agonist of an Integrin, _L2^*

Wei Yang¹, Christopher V. Carman¹, Minsoo Kim^¶2, Azucena Salas^¶3, Motomu Shimaoka^¶, and Timothy A. Springer⁴

From the CBR Institute for Biomedical Research, Departments of Pathology and ^¶Anesthesia, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, July 19, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The binding of integrin _L2 to its ligand intercellular adhesion molecule-1 is required for immune responses and leukocyte trafficking. Small molecule antagonists of _L2 are under intense investigation as potential anti-inflammatory drugs. We describe for the first time a small molecule integrin agonist. A previously described / I allosteric inhibitor, compound 4, functions as an agonist of _L2 in Ca²⁺ and Mg²⁺and as an antagonist in Mn²⁺. We have characterized the mechanism of activation and its competitive and noncompetitive inhibition by different compounds. Although it stimulates ligand binding, compound 4 nonetheless inhibits lymphocyte transendothelial migration. Agonism by compound 4 results in accumulation of _L2 in the uropod, extreme uropod elongation, and defective de-adhesion. Small molecule integrin agonists open up novel therapeutic possibilities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Integrins are a large family of / heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix adhesion and transduce signals bidirectionally across the plasma membrane. Integrin _L2 (lymphocyte function associated antigen-1 (LFA-1))⁵ belongs to the ₂ integrin subfamily and is constitutively expressed on all leukocytes. _L2 remains in a low affinity state in resting lymphocytes and undergoes dramatic conformational change during lymphocyte activation, which greatly increases its binding affinity for its ligands intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, and -3). Regulation of _L2 activation is pivotal for controlling leukocyte trafficking and immune responses in health and diseases (1-3).

_L2 is an important pharmaceutical target for treating auto-immune and inflammatory diseases (4-8). A humanized antibody to _L2 that blocks its binding to the ligand ICAM-1 has been approved by the FDA for treatment of psoriasis, a T cell-mediated autoimmune disease of the skin (9, 10). Furthermore, small molecule antagonists of _L2 have been discovered and are in development (11-17).

_L2 contains two von Willebrand factor-type A domains, the inserted (I) domains in the _L and the ₂ subunits (18-20). Both _L I and ₂ I domains have a Rossman fold (i.e. a central -sheet surrounded by -helices) with a metal ion-dependent adhesion site (MIDAS) formed by - loops at the "top" face of the domain (20-23). In ligand binding the Mg²⁺ ion in the MIDAS of the _L I domain coordinates directly to a Glu residue that is in the center of the ligand binding sites in domain 1 of ICAM-1 and ICAM-3 (20, 24). The affinity of the _L I domain for ICAMs is regulated by downward axial displacement of its C-terminal 7 helix, which is conformationally linked to reshaping of MIDAS loops and increases affinity for ligand by up to 10,000-fold (25, 26). During activation, the I domain undergoes similar 7 helix downward axial movement, which is induced by the swing out of the hybrid domain (27-30).⁶ Previous data suggested that when activated, the ₂ I domain binds (through the Mg²⁺ in its MIDAS) to the Glu residue (Glu-310) in the C-terminal linker of the _L I domain, exerts a downward pull on its 7 helix, and thereby activates the _L I domain (Fig. 1A) (32, 33).

Two distinct classes of small molecule antagonists of _L2 have been developed as anti-inflammatory agents. One group of antagonists binds the hydrophobic pocket underneath the 7 helix of the _L I domain (e.g. LFA703 or BIRT377), blocks the downward axial movement of the 7 helix, and inhibits ligand binding of _L2 allosterically by stabilizing the _L I domain in the low affinity conformation (11-14, 34). These antagonists are called I allosteric inhibitors. The other group of antagonists appears to bind to the ₂ I domain MIDAS near a key regulatory interface with the _L I domain, blocking communication of conformational change to the _L I domain while at the same time activating conformational rearrangements elsewhere in integrins (35-37). These antagonists, such as compounds 3 and 4 from Genentech and XVA143 from Hoffmann-La Roche, are called / I allosteric inhibitors (Fig. 1B). In this report, however, we describe that compound 4, previously regarded as an / I allosteric inhibitor based on studies in Mn²⁺, actually activates _L2 under physiological conditions in Ca²⁺, and Mg²⁺ and inhibits integrin-dependent functions by perturbing de-adhesion.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Mechanisms of inhibition and chemical structures of / I allosteric antagonists. A, mechanisms of inhibition and impact on integrin conformation of / I allosteric antagonists. / I allosteric inhibitors bind to the 2 I domain MIDAS near a key regulatory interface with the L I domain and block communication of conformational change to the I domain while at the same time activating conformational rearrangements elsewhere in integrins, including swing-out of the hybrid domain. B, chemical structures of / I allosteric antagonists.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Isolation and Culture—K562 transfectants expressing wild-type and mutant _L2 were described (40). Preparation of human peripheral blood mononuclear cells (PBMCs) and interleukin-2-cultured primary lymphocytes was previously described (41). Primary human umbilical vein endothelial cells (HUVECs) were from Cambrex (Walkersville, MD) and cultured as confluent monolayers on fibronectin (10 µg/ml) coated on glass coverslips or T live-cell imaging chambers (Bioptechs, Butler, PA) in EGM-2 complete media (Cambrex, Walkersville, MD).

Binding of Soluble ICAM-1—Binding of soluble ICAM-1-IgA Fc fusion protein complexed with affinity-purified, fluorescein isothiocyanate-conjugated anti-human IgA was measured by flow cytometry (37).

Cell Adhesion to Immobilized ICAM-1—Binding of fluorescently labeled transfectants to immobilized ICAM-1 was as described (40). Briefly, ICAM-1-IgG Fc fusion protein at 10 µg/ml was immobilized on microtiter plates previously coated with 20 µg/ml protein A and blocked with 2% human serum albumin. Binding of transfectants to immobilized ICAM-1 was determined in Hepes, NaCl, glucose, bovine serum albumin (BSA; 20 mM Hepes, pH 7.5, 140 mM NaCl, 2 mg/ml glucose, 1% BSA) supplemented with divalent cations and compounds as indicated. After incubation at 37 °C for 30 min, unbound cells were washed off, and bound cells were quantitated (40).

Flow Chamber Assay—Binding and detachment in shear flow of _L2 transfectants on immobilized ICAM-1 substrates was done in a parallel plate flow chamber as described (42).

Fluorescence Resonance Energy Transfer (FRET) Assay—FRET assay using _L-monomeric cyan fluorescent protein (mCFP)/₂-monomeric yellow fluorescent protein (mYFP) K562 stable transfectants was as described (43).

Cell Migration Assays—Lymphocyte transendothelial migration assays were as described (41). Briefly, before each experiment confluent HUVEC monolayers were activated for 12 h with TNF- (100 ng/ml). HUVECs were then washed 3 times in buffer A (Hanks' balanced salt solution supplemented with 20 mM Hepes, pH 7.2, and 1% human serum albumin). Interleukin 2-cultured primary human lymphocytes were pelleted, resuspended at 100,000 cells/ml in 500 µl of buffer A containing compound 4 (1 µM), compound 5 (1 µM), BIRT377 (20 µM), or CBR LFA-1/2 Fab (20 µg/ml) and then added to HUVECs and incubated at 37 °C for 10 or 60 min. Samples were fixed in 3.7% formaldehyde in phosphate-buffered saline for 5 min and stained for leukocyte _L integrin (TS2/4 mAb conjugated to Cy3), endothelial cell ICAM-1 (IC1/11 mAb conjugated to Alexa488), and F-actin (phalliodin-Alexa647; Molecular Probes) as described (41). Imaging was conducted using Bio-Rad Radiance 2000 Laser-scanning confocal microscope system. For each condition complete Z-stacks (0.5 µm thickness) were obtained in each of ten randomly selected fields. Using LaserSharp 2000 software (Bio-Rad) Z-stacks were analyzed (based on previously described criteria (41)) to determine the number of cells in the process of, or having completed diapedesis.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Compound 4 inhibits L2 in Mn2+ but activates L2 in Ca2+ and Mg2+. A-C, soluble multimeric ICAM-1 binding by K562 stable transfectants expressing wild-type L2. Cells were incubated with compounds in Hepes, NaCl, glucose, bovine serum albumin supplemented with 1 mM CaCl2 and 1 mM MgCl2 (A), 2 mM MnCl2 (B), or 1 mM CaCl2, 1 mM MgCl2, and 10 µg/ml CBR LFA-1/2 (C) for 30 min at room temperature. Then fluorescein isothiocyanate-labeled multimeric ICAM-1 was added and incubated with cells for another 30 min at room temperature. The binding was detected by flow cytometry and is expressed as mean fluorescence intensity (MFI). D, soluble multimeric ICAM-1 binding by human PBMCs. Binding was assayed as in A-C with cations, compounds (1 µM), and TS2/14 mAb (10 µg/ml) as indicated. E, static adhesion of L2-expressing K562 cells to immobilized ICAM-1 was as described in "Experimental Procedures" with cations and compounds (1 µM) as indicated. F, adhesion in shear flow of L2-expressing K562 cells to immobilized ICAM-1. K562 cells expressing wild-type L2 were incubated in media containing different divalent cations and compounds (1 µM) as above. Cells were allowed to accumulate on an ICAM-1-Fc-coated substrate at 0.3 dyn/cm2 in the flow chamber for 30 s before increasing the flow rate every 10 s in about 2-fold increments to the indicated wall shear stresses. Bars show the total number of adherent cells, including cells that were rolling (white) or firmly adherent (black).

For live-cell experiments confluent TNF--activated HUVEC monolayers were prepared on Bioptechs T imaging chambers, rinsed three times with buffer A, and maintained at 37 °C. Lymphocytes (100,000) were added to the chambers, and differential interference contract images were acquired (using a Zeiss Axiovert S200 epifluorescence microscope (Germany) equipped with a 63x oil objective coupled to a Hamamatsu Orca CCD (Japan)) at 5-s intervals over a course of 30 min. Cell migration was analyzed by manually tracing the outline of each cell in selected frames (i.e. at 180-s intervals) for each time course. Lines connecting the centroid of each cell outline (auto-matically calculated by OpenLab software) were generated to represent the migration path or "track" followed by each lymphocyte. The total length of the cell tracks was divided by the total time interval during which the track was recorded to calculate average migration velocity. The linear distance between the beginning and endpoint of each track was measured to determine the overall displacement of each cell. Measurement of cell lateral migration parameters was restricted to lymphocytes during their migration over the apical surface of the endothelium and discontinued upon diapedesis across the endothelial monolayer to the subendothelial space. The percentage of diapedesis was obtained by dividing the number of cells that initiated diapedesis by the total number of migrating cells.

To analyze the qualitative details of migration behavior, representative cells were traced at 50-s intervals. The distance separating the centroid of the cell in the initial frame and the centroid of the cell at each subsequent interval was plotted against the cumulative time elapsed.

Online Supplemental Material—Supplemental Videos 1 and 2 are representative videos of lymphocyte migration in the absence (Video 1) and presence (Video 2) of compound 4 as described in Figs. 7, C and D, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Compound 4 Activates _L2 in Physiologic Cations (Ca²⁺/Mg²⁺) but Inhibits in Mn²⁺
K562 cells expressing _L2 showed little binding to soluble multimeric ICAM-1 in Ca²⁺/Mg²⁺ (Fig. 2A), whereas binding was greatly increased by Mn²⁺ (Fig. 2B) or the activating mAb CBR LFA-1/2 (Fig. 2C). In Mn²⁺, compounds 3-5 potently inhibited soluble, multimeric ICAM-1 binding by _L2 (Fig. 2B), consistent with previous observations (17, 37). However, in physiologic cations (i.e. 1mM Ca²⁺ and 1 mM Mg²) we found, unexpectedly, that compound 4 greatly increased ligand binding, whereas compounds 3 and 5 had no effect (Fig. 2A). Furthermore, activation of _L2 binding to ICAM-1 in Ca²⁺/Mg²⁺ by CBR LFA-1/2 mAb was further increased by compound 4 but inhibited by compounds 3 and 5 (Fig. 2C).

Next we assessed the effects of these compounds on physiologic leukocytes (i.e. primary human PBMCs). The PBMCs showed weak binding to soluble multimeric ICAM-1 in Ca²⁺/Mg²⁺ alone and significant binding in Mn²⁺ alone (Fig. 2D). Consistent with our observations with K562 transfectants (Fig. 2, A-C), compound 4 strongly increased binding of soluble ICAM-1 to PBMCs in Ca²⁺/Mg²⁺ but inhibited Mn²⁺-induced binding (Fig. 2D). Both compound 4- and Mn²⁺-induced ICAM-1 binding was _L2-dependent, as such binding was completely inhibited by the _L I domain-specific blocking antibody TS2/14 (Fig. 2D).

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Inhibition of agonism by compound 4 with compound 5 and LFA703. Soluble, multimeric ICAM-1 binding by L2-expressing K562 cells was determined as described in Fig. 2A in 1 mM CaCl2, 1 mM MgCl2 after co-incubation with the indicated concentrations of compound 4 and compound 5 (A) or LFA703 (B).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Compound 4 and Mn2+ activateL2 by different mechanisms. Soluble ICAM-1 binding by K562 transfectants expressing wild-type or mutant L2 was as described in Fig. 2A in the presence of cations and compounds (1 µM) as indicated. MFI, mean fluorescence intensity.

The Activating Effect of Compound 4 Is Inhibited by Compound 5 Competitively—Compound 4 and compound 5 have homologous structures, and our previous findings suggested that both compounds bind to the MIDAS of the ₂ I domain (37). However, in Ca²⁺/Mg²⁺, compound 4 was activating, whereas compound 5 was inhibitory to wild type _L2 (Fig. 2). Therefore, we studied whether ICAM-1 binding to _L2 in Ca²⁺/Mg²⁺ stimulated by compound 4 could be competitively inhibited by compound 5. We found that _L2 activation by 50 nM compound 4 was reversed by compound 5 in a dose-dependent manner (Fig. 3A). Importantly, the inhibitory dose-response curve of compound 5 was shifted significantly to the right in the presence of a higher concentration (1 µM) of compound 4 (Fig. 3A). Such concentration dependence demonstrates a competitive mode of inhibition. Binding to ICAM-1 stimulated by compound 4 was also inhibited by an I allosteric inhibitor, LFA703, that binds the hydrophobic pocket underneath the 7 helix of the _L I domain (Fig. 3B). However, the inhibitory dose-response curve of LFA703 was identical with 50 and 1000 nM compound 4, demonstrating non-competitive inhibition.

Compound 4 and Mn²⁺ Activate _L2 by Different Mechanisms—The interaction between the ₂ MIDAS and an acidic residue in the C-terminal linker of I domains, e.g. Glu-310 in _L, is indispensable for Mn²⁺-induced activation of ₂ integrins (32, 33, 44). Mutation of either the metal-coordinating MIDAS residue Ser-114 in the ₂ I domain or Glu-310 in the _L I domain C-terminal linker totally abolished Mn²⁺-induced ICAM-1 binding (Fig. 4). Mutation of another nearby acidic residue in the C-terminal linker of the _L I domain, _L-E316, only partially reduced Mn²⁺-induced ligand binding and served as a control (Fig. 4). Consistent with our previous conclusion that compound 4 binds to the MIDAS of the ₂ I domain (37), the ₂ Ser-114 mutation completely abolished both inhibition of ICAM-1 binding in Mn²⁺ by compound 4 and stimulation of ICAM-1 binding in Ca²⁺/Mg²⁺ by compound 4 (Fig. 4). Despite the absolute requirement for _L-Glu-310 in Mn²⁺-induced ICAM-1 binding by _L2, compound 4 was able to activate binding to ICAM-1 by the _L-E310A mutant, demonstrating that compound 4 activates _L2 by a mechanism that is distinct from that of Mn²⁺.

Susceptibility to _L2 Inhibitory Antibodies—mAbs exist that inhibit _L2 function by distinct mechanisms. Whereas some mAbs bind to the _L I domain and competitively block ICAM-1 binding, other _L I domain and ₂ I domain mAbs block ICAM-1 binding indirectly through allosteric mechanisms (34, 45, 46). We compared inhibition by a panel of these mAbs of CBR LFA-1/2-activated _L2 (wild type + mAb); _L-Glu-310C/₂-A210C (CC), an _L2 mutant that is constitutively activated by introducing an intersubunit disulfide bond between residue 210 in a ₂ I domain MIDAS loop and the _L-Glu-310 residue (33); _L2 activated by compound 4 in Ca²⁺/Mg²⁺ (wild type + #4); and _L2 activated by a disulfide bond mutationally introduced into the _L I domain (HA) (Table 1). The _L-E310C/₂-A210C mutant and wild-type _L2 activated by compound 4 showed almost identical susceptibility, i.e. they were inhibited by both the competitive _L I domain mAbs and the allosteric TS2/14 _L I domain mAb, were partially inhibited by mAb to Glu-175 in the specificity-determining loop of the ₂ I domain, and were resistant to mAbs to residues in the 1 helix (133) and 7 helix (332 and 339) of the ₂ I domain.

View this table: [in this window] [in a new window]   TABLE 1 Inhibition by L I and 2 I domain antibodies of multimeric ICAM-1 binding to L2 mutants Wild-type (WT) L2 in K562 transfectants was activated by preincubation with 10 µg/ml mAb CBR LFA-1/2 (WT + mAb) or 1 µM compound 4 (WT + #4). CC, L-E310C/2-A210C. HA, L2 with the high affinity K287C/K294C I domain mutation. Binding to soluble, multimeric ICAM-1 in medium containing 1 mM CaCl2 and 1 mM MgCl2 was in the presence of the indicated mAb. All mAbs bound to L-E310C/2-A210C, K287C/K294C, and wild-type L2 with or without compound 4 equally well (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Effect of compounds on the conformation of L2. A and B, effect of compounds on expression of activation epitopes on L2. L2-Expressing K562 cells were stained with m24 (A) or KIM127 (B) in Hepes buffer containing 1 mM CaCl2, 1 mM MgCl2 and compounds at 37 °C for 30 min followed by immunofluorescence flow cytometry. C, binding of compounds induces spatial separation of L2 cytoplasmic domains. FRET was measured in L-mCFP/2-mYFP K562 transfectants after treatment with compounds (1 µM) or 1 mM Mn2+ and soluble monomeric ICAM-1 (slCMA-1) 100 µg/ml) as indicated. Data are the mean ± S.E. for 8 to 10 cells. *, p < 0.05 versus control. MFI, mean fluorescence intensity.

We previously developed a FRET method to monitor the spatial proximity of _L and ₂ cytoplasmic domains in living cells by fusing mCFP and mYFP to the C termini of _L and ₂, respectively (43). Efficient FRET can only be observed when the cytoplasmic tails of _L and ₂ (and, therefore, the fused mCFP and mYFP) are in close proximity. Consistent with our previous observations (43), we found here that stable K562 cell transfectants expressing _L-mCFP/₂-mYFP exhibited a significant FRET signal under basal conditions and that FRET was significantly decreased by treatment with Mn²⁺ plus soluble monomeric ICAM-1 (Fig. 5C). Exposure to either compound 4 or 5 in Ca²⁺/Mg²⁺ also statistically significantly reduced FRET, although to a somewhat lesser extent. These data suggest that compounds 4 and 5, consistent with induction of exposure of the m24 and KIM127 epitopes (Fig. 5, A and B), induce spatial separation of the _L and ₂ cytoplasmic domains (Fig. 5C).

Compounds 4 and 5 Inhibit Lymphocyte Transendothelial Migration by Distinct Mechanisms—To assess the effects of compounds on transendothelial migration, i.e. diapedesis, we monitored migration of interleukin-2-cultured primary human lymphocytes through TNF--activated HUVEC monolayers in medium with Ca²⁺/Mg²⁺ by confocal microscopy. Under control conditions, efficient lymphocyte transendothelial migration was observed (45% by 10 min and 70% by 60 min). Compared with control, compound 4, compound 5, and BIRT377, an I allosteric antagonist (Fig. 6A), all inhibited transendothelial migration by greater than 2-fold. Interestingly, Fab fragments of the _L2-activating antibody, CBR LFA-1/2, also produced a comparable inhibition of diapedesis (Fig. 6A).

Despite similarity in overall extent of inhibition of diapedesis, morphological analysis (as described under "Experimental Procedures") revealed dramatic differences among these antagonists (Fig. 6, B-D). Under control conditions (Me₂SO), the majority of the cells were polarized, whereas the remaining cells were equally divided into round and spread populations. In the presence of either compound 5 or BIRT377, the polarized cell population was reduced by greater than 2-fold, and the round cell population was dominant (Fig. 6, C and D). In stark contrast, for both compound 4 and CBR LFA-1/2 Fab treatments, the major cell population was in an unphysiologic "extremely polarized" (X-polarized) state in which the uropod was extended in length and dramatically enriched in _L2, concomitant with depletion of _L2 from other regions of the cell (Fig. 6, B-D).

View larger version (64K): [in this window] [in a new window]   FIGURE 6. Effect of compounds on lymphocyte diapedesis. Interleukin-2-cultured human lymphocytes were incubated with TNF- activated HUVEC monolayers for 10 or 60 min (A) or 10 min (B-D) in the absence or presence of compounds or CBR LFA-1/2 Fab, fixed, and stained as described under "Experimental Procedures." For each experiment a minimum of 100 lymphocytes from randomly selected fields were carefully analyzed to determine stage of diapedesis and morphology as described under "Experimental Procedures." A, quantitation of transendothelial migration (TEM). The number of cells having either initiated or completed diapedesis is expressed as a percentage of total cells. Values represent mean ± S.E. of 3-6 independent experiments. DMSO, dimethyl sulfoxide. B-D, morphologic characterization of lymphocytes. Lymphocytes and HUVECs were fixed and stained for L integrin (green) and F-actin (red). Representative micrographs demonstrate each of four principal morphologic categories (round, spread, polarized, and X-polarized) observed among the apically adherent cells. C, the number of cells displaying each of the morphologies is expressed as a percentage of the total. Values represent mean ± S.E. of 3-8 experiments. D, representative fields used for the quantitation shown in C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The interaction between _L2 and ICAM-1 plays a critical role in the formation of the immunological synapse in immune responses and in leukocyte adhesion and extravasation through endothelium. _L2 is a clinically validated target for the treatment of autoimmune disease, and small molecule antagonists of _L2 are under intense investigation. Here, we show that a class of compounds previously classified as / I allosteric antagonists includes among its members a compound that is an agonist of _L2 in the presence of physiologic divalent cations, i.e. Ca²⁺ and Mg²⁺. In contrast, compound 4 is an antagonist in Mn²⁺, as previously reported (17, 37). Agonism in Ca²⁺/Mg²⁺ and antagonism in Mn²⁺ was consistently observed in soluble multimeric ICAM-1 binding assays, static cell adhesion, and flow chamber assays and with both K562 transfectants expressing _L2 and physiologic leukocytes, i.e. PBMCs. In parallel assays the structurally homologous compounds 3 and 5 (XVA143) exhibit only antagonistic properties. The finding that compound 4 can act as both an agonist and antagonist support our previous conclusion that it is an allosteric effector (37) and does not mimic and directly compete binding of ICAM-1 (17, 47).

Compounds 3-5 (XVA143) have very similar structures and appear to have overlapping binding sites. The ability of all three compounds to stabilize non-covalent association of the _L and ₂ subunits in SDS-PAGE is not dependent on the _L I domain and is absolutely dependent on divalent cations and the ₂ I domain MIDAS residue Ser-114. Mn²⁺ and Ca²⁺/Mg²⁺ each support stabilization of _L2 and _M2 noncovalent complexes in SDS-PAGE. All three compounds inhibit ligand binding by _M2 as well as _L2 (37). Antagonism and agonism by compound 4 appear to occur at the same binding site, since the closely related compound 5 competitively antagonizes agonism by compound 4, and agonism requires ₂ I domain residue Ser-114.

View larger version (54K): [in this window] [in a new window]   FIGURE 7. Dynamics of lymphocyte lateral migration and diapedesis across endothelium. Live-cell imaging and analysis of lymphocytes migrating on TNF--activated HUVEC monolayers was as described under "Experimental Procedures." For each condition, greater than 50 cells, taken from four separate imaging experiments (see representative experiments in supplemental Videos 1 and 2) were analyzed. A and B, two-dimensional tracks of lymphocytes migrating over a 30-min period under control conditions (A) and in the presence of 1 µM compound 4 (B). Tracks of cells that initiated diapedesis during the imaging time course are terminated at the point of initiation of diapedesis and are depicted in red. C-E, kinetics of migration of representative lymphocytes. C-D, left panels are selected frames from representative live-cell imaging experiments under control condition (C, see Video 1) and in the presence of compound 4 (D, see Video 2). Representative cells (boxed region in left panels) were tracked at 50-s intervals. The outline (red) of the cell position at relative time 0 is shown in all panels. Note that in control condition (C) the migrating cell steadily increases its distance from its origin over time, whereas in the presence of compound 4 (D) the cell repeatedly moves away from and then contracts back toward the origin. E, the distances from the origin of the centroids of the two migrating cells shown in C and D are plotted against time for control (black) and compound 4 (red) conditions. The control cell is only tracked for 7 min because after this it left the boxed region in Fig. 7C.

Our working model for agonism by compound 4 is as follows. Ca²⁺ and Mn²⁺ compete for binding to the Adjacent to MIDAS (ADMIDAS) metal ion binding site and by binding to this site inhibit and stimulate ligand binding, respectively, and coordinate with alternative ADMIDAS residues (48). In both Ca²⁺/Mg²⁺ and Mn²⁺, compounds 3-5 (XVA143) bind to the ₂ MIDAS and block its interaction with _L-Glu-310. In Ca²⁺/Mg²⁺, the complex between compound 4 and the ₂ I domain is slightly altered compared with its conformation in Mn²⁺ so that it is complementary to and can bind to the _L I domain or its linker and induce the open conformation of the _L I domain through interactions that do not involve, but functionally substitute for, the _L-Glu-310: ₂-MIDAS interaction.

Despite agonistic stimulation of ligand binding, compound 4 can still block physiologic functions of _L2 that require cycles of adhesion and detachment. It has been proposed that integrins are active at the leading edge, whereas they are inactive at the trailing edge of migrating leukocytes (49, 50). Inactivation of integrins at the trailing edge is thought to be important for detaching the uropod (51). Indeed, sustained activation of ₁ or ₂ via activating antibodies (52, 53) or blockade of Rho signaling (54) suppressed eosinophil and monocyte transmigration by preventing the trailing edge from being detached.

We found that although compounds 4 and 5, BIRT377, and CBR LFA-1/2 all inhibit lymphocyte transmigration across the endothelium cell layer, they do so by different mechanisms. Compound 5 and BIRT377 distinctly promoted a predominant round cell population, with greatly reduced spreading and polarization consistent with a reduction in overall adhesiveness. In contrast, compound 4 and CBR LFA-1/2 Fab induced the migrating lymphocytes to display unusually long uropods that were highly enriched in _L2, consistent with increased adhesion and decreased de-adhesion in the trailing edge. This was confirmed by live-cell imaging analysis that demonstrated frustrated lateral migration induced by compound 4, in which failure of the uropod to detach limited lymphocyte migration. Thus, compound 5 (XVA143) blocks transendothelial migration by reducing adhesion, whereas compound 4 and CBR LFA-1/2 Fab block transendothelial migration by activating _L2 and interfering with uropod detachment. In a related finding, mutant mice expressing constitutively active _L2 were impaired in T cell migration, T cell proliferation stimulated by antigen presenting cells, cytotoxic T cell activity, T-dependent humoral immune responses, and neutrophil recruitment during aseptic peritonitis, although signaling through _L2 was not affected (31). The above observations are consistent with the previous report that compound 4 is a potent inhibitor of the mixed lymphocyte reaction (17). Our study demonstrates for the first time a small molecule integrin allosteric agonist that functions as an anti-inflammatory drug through a novel mechanism of action, perturbation of integrin de-adhesion.

Compound 4 is the first small molecule agonist reported for any integrin. Integrin agonists open up novel opportunities for therapeutics that increase rather than decrease integrin-dependent adhesion. For example, immune recognition of tumor cells is LFA-1-dependent, and agonists might enhance immune responses, including cytotoxic killing of tumor cells. Although we have found that agonism of _L2 decreases cell migration, and mice with permanently up-regulated _L2 are functionally impaired, appropriate dosing could allow cycles of agonism at peak drug levels to be alternated with cell migration during intervening troughs. There is extensive precedent with G-protein-coupled receptors for closely related compounds to act as agonists and antagonists (inverse agonists), and both types of compounds have important therapeutic applications.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants CA31798 (to T. A. S.) and AI63421 (to M. S.) and a grant from the Arthritis Foundation (to C. V. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Videos 1 and 2.

¹ These authors contributed equally to this work.

² Current address: Division of Surgical Research, Rhode Island Hospital, Brown University School of Medicine, 593 Eddy Street, Middlehouse 207, Providence, RI 02903.

³ Current address: Instituto de Investigaciones Biomédicas de Barcelona-CSIC, Roselló 161, 7^a planta, 08036 Barcelona, Spain.

⁴ To whom correspondence should be addressed: The CBR Institute for Bio-medical Research, Dept. of Pathology, Harvard Medical School, 200 Long-wood Ave., Boston, MA 02115. Tel.: 617-278-3200; Fax: 617-278-3232; E-mail: springeroffice{at}cbr.med.harvard.edu.

⁵ The abbreviations used are: LFA-1, lymphocyte function associated anti-gen-1; ICAM, intercellular adhesion molecule; MIDAS, metal ion-dependent adhesion site; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; HUVEC, human umbilical vein endothelial cell; FRET, fluorescence resonance energy transfer; TNF, tumor necrosis factor; mCFP, monomeric cyan fluorescent protein; mYFP, monomeric yellow fluorescent protein.

⁶ Nishida, N., Xie, C., Shimaoka, M., Cheng, Y., Walz, T., and Springer, T. A., (2006) Immunity 25, 583-594.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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