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(Journal of Leukocyte Biology. 2001;70:329-334.)
© 2001 by Society for Leukocyte Biology

A small-molecule antagonist of LFA-1 blocks a conformational change important for LFA-1 function

Joseph R. Woska, Jr.*, Daw-tsun Shih*, Viviany R. Taqueti*, Nancy Hogg{dagger}, Terence A. Kelly{ddagger} and Takashi K. Kishimoto*

* Department of Biology and
{ddagger} Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut, and
{dagger} Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, London, United Kingdom

Correspondence: Joseph R. Woska, Jr., Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877. E-mail: jwoska{at}rdg.boehringer-ingelheim.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 interactions mediate several important steps in the evolution of an immune response. LFA-1 is normally expressed in a quiescent state on the surface of leukocytes and interacts weakly with its ligands ICAM-1, -2, and -3. LFA-1 activity may be regulated by receptor clustering and by increasing the affinity of LFA-1 for its ligands. Affinity modulation of LFA-1 has been shown to occur via a conformational change in the LFA-1 heterodimer that can be detected by using monoclonal antibody 24 (mAb24). We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that demonstrates selective in vitro and in vivo inhibition of LFA-1/ICAM-1-mediated binding events. We now demonstrate that BIRT 377 blocks the induction of the mAb24 reporter epitope on LFA-1 on the surface of SKW-3 cells treated with various agonists known to induce high-affinity LFA-1. These data imply that BIRT 377 exerts its inhibitory effects by preventing up-regulation of LFA-1 to its high-affinity conformation.

Key Words: adhesion molecules • inflammation • cell-cell interactions • FACS • integrin • mAb epitope


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interaction of lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) with its ligands [intercellular adhesion molecules (ICAMs)-1, -2, and -3] mediates several important steps in the cascade of events leading to an inflammatory response. Leukocyte extravasation, antigen presentation, and T-cell effector functions are all mediated in part by LFA-1 [reviewed in references 1 and 2]. In vitro, monoclonal antibodies (mAbs) directed against LFA-1 block T- and B-cell aggregation, adhesion of leukocytes to the endothelium, mixed lymphocyte responses, antigen- and mitogen-induced proliferation, and T-cell-mediated killing of target cells [reviewed in reference 3]. In vivo, anti-LFA-1 mAbs have proven efficacious in animal models of immune dysfunction and in human disease [reviewed in reference 4]. The most dramatic evidence for an important role of the ß2-integrins in immune function is displayed in patients with type I leukocyte adhesion deficiency (LAD I). LAD I is a heritable disease caused by a variety of mutations in the common CD18 subunit of the ß2-integrins; these mutations prevent cell surface expression of LFA-1, Mac-1 (CD11b, CD18), p150,95 (CD11c, CD18), and {alpha}Dß2 (CD11d, CD18) heterodimers on leukocytes [5 , 6 ]. The lack of ß2-integrin expression leads to the inability of neutrophils to extravasate to sites of inflammation; therefore, these patients are prone to recurrent bacterial infections [6 ]. More recently, mice that are specifically deficient in LFA-1 have been generated [7 8 9 10 ]. CD11a-deficient mice demonstrate impaired leukocyte homotypic aggregation, decreased proliferation induced by mitogen or in the mixed lymphocyte reaction, decreased ability to reject tumors, impaired cytotoxic-T-lymphocyte activity, decreased host-versus-graft reactions, and decreased trafficking to peripheral and mesenteric lymph nodes.

Antagonists of LFA-1 are predicted, on the basis of data obtained both in vitro and in vivo, to be of therapeutic benefit for treatment of various immunological and inflammatory diseases. We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that inhibits the binding of micellar LFA-1 to immobilized soluble ICAM-1 [sICAM-1] with an apparent KD of 26 nM [11 ]. BIRT 377 also inhibits LFA-1-dependent adhesion assays and antigen-induced proliferation of T cells with 50% inhibitory concentrations in the low micromolar range. In vivo, BIRT 377 inhibits superantigen-induced production of interleukin-2 in mice in a dose-dependent fashion. BIRT 377 is specific for LFA-1, as it does not inhibit the binding of micellar Mac-1 to immobilized sICAM-1 [11 ]. The mechanism by which BIRT 377 inhibits LFA-1/ICAM binding has not been reported.

LFA-1 is normally expressed in a quiescent or inactive state on the surface of leukocytes [14–16; reviewed in references 12 and 13]. In vitro, LFA-1 can be induced to an active form by two mechanisms: changes in avidity (i.e., receptor clustering) and changes in the affinity of LFA-1 for its ligand. Engagement of the T-cell receptor (TCR) by antigen or antibody results in a transient conversion of LFA-1 to a high-avidity state [14 , 15 ]. This high-avidity state may also be induced by treatment of leukocytes with phorbol esters [14 , 17 , 18 ] or cross-linking of other cell surface receptors [reviewed in references 12 and 13]. LFA-1 may also be converted to an active form by the addition of the divalent cations Mg2+ and Mn2+ [19 , 20 ]. Stewart et al. [21 ] have demonstrated that high concentrations of Mg2+ plus EGTA can induce LFA-1-mediated adhesion resulting from the conversion of LFA-1 from a low-affinity conformation to a high-affinity conformation. This high-affinity state can be monitored by assessing the ability of sICAM-1 to bind LFA-1 and by measuring the binding of the activation reporter mAb24 [19 ]. Some anti-ß2-integrin mAbs, such as KIM 185 and KIM 127, activate LFA-1-dependent adhesion [22 , 23 ] and induce expression of the mAb24 epitope [24 ]. Conversely, phorbol myristate acetate (PMA) and TCR cross-linking do not induce a high-affinity conformation of LFA-1 (as evidenced by the lack of mAb24 expression) but stimulate adhesion by clustering LFA-1 in the cell membrane [21 , 25 ]. Kucik et al. [26 ] have demonstrated that treatment of MP cells (a B-cell line) with PMA or low doses of cytochalasin D results in the transient release of LFA-1 from cytoskeletal restraints. This allows the free diffusion of LFA-1 through the cell membrane, which could then lead to increased ligand/receptor interactions and high-avidity binding. Membrane ICAM-1 and sICAM-1 have been shown to induce high-affinity LFA-1 in T cells and SKW-3 cells (a T-cell lymphoma) [24 , 27 ]. It is plausible, therefore, that cross-linking of the TCR induces high-avidity binding between LFA-1 and ICAM-1 in vivo through receptor clustering, and ICAM-1 may subsequently induce or stabilize a transiently expressed high-affinity conformation in a percentage of the LFA-1 molecules, strengthening adhesion and possibly providing differential signals important to the T cell.

In this report, we present evidence that BIRT 377 inhibits the induction of the high-affinity conformation of membrane-bound LFA-1. This is demonstrated by the absence of mAb24 epitope expression on BIRT 377-treated SKW-3 cells which have been stimulated with various agents that are known to increase mAb24 epitope expression. This inhibition was specific for LFA-1 in that K562 cells transfected with Mac-1 express the mAb24 epitope in the presence or absence of BIRT 377. The results presented herein suggest that BIRT 377 may act as an allosteric antagonist of LFA-1 and should help in characterizing the structural requirements underlying affinity modulation of LFA-1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and mAbs.
SKW-3 cells were provided by the biotechnology group at Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT) and were grown in RPMI 1640 supplemented with 10% fetal calf serum and antibiotics (Gibco, Grand Island, NY). K562 Mac-1 transfectants, a kind gift of Dr. Michael Dustin (Washington University, St. Louis, MO), were grown in RPMI 1640 supplemented with 10% fetal calf serum, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 800 µg/mL of G418 (Gibco). mAb24 has been described previously (19). Activating-mAbs KIM 185 and KIM 127 were a kind gift of M. Robinson (CellTech Chiroscience plc, Berkshire, Great Britain).

Cell adhesion assays
Ninety-six-well plates were coated with 10 µg/mL of sICAM-1 [constructed and purified as described in reference 28] in Dulbecco’s phosphate-buffered saline (DPBS; Gibco) at room temperature for 1 h. The plates were then blocked with DPBS containing 2% bovine serum albumin for 1 h at 37°C or overnight at 4°C. SKW-3 cells were counted and resuspended at a density of ~106/mL in 9 mL of DPBS supplemented with 4.5 g/L of glucose and incubated with 1.5 µL of a 10-µg/µL stock solution of Calcein-AM (Molecular Probes, Eugene, OR) for 10 min at 37°C. After incubation, the cells were washed once with Hanks’ balanced salt solution [HBSS (Mg2+ and Ca2+ free); Gibco] containing 10 mM EDTA and then twice with HBSS alone. Cells were then resuspended in activation medium {HBSS + 5 mM Mg2+/1 mM EGTA; HBSS + 1 mM Mg2+/1 mM Ca2+ [either with or without 100 ng/mL of PMA (Sigma, St. Louis, MO) or 1–10 µg/mL of KIM 185/127]; or RPMI medium (with or without 100 ng/mL of PMA or 1–10 µg/mL of KIM 185/127)}. A 50-µL volume of cell suspension was then incubated for 1 h at 37°C with an equal volume of medium or serial dilutions of BIRT 377 dissolved in the appropriate activation medium. After incubation, the plates were washed three times with warmed RPMI using a multichannel pipette. Fluorescence was measured before and after washing with a PerSeptive Biosystems (Framingham, MA) Cytofluor series 4000 plate reader with a 485-nm excitation/530-nm emission filter.

The percentage of cell binding was calculated as follows: (mean fluorescence after washing/mean fluorescence before washing) x 100. The percentage of binding inhibition was calculated as follows: [(mean fluorescence of control cells - mean fluorescence of treated cells)/mean fluorescence of control cells] x 100. Background fluorescence was subtracted from each sample reading.

Flow cytometry
For direct–immunofluorescence analysis, cells were washed once in HBSS plus 10 mM EDTA and then twice in HBSS and resuspended at a density of 107/mL in activation medium (see above). A 100-µL volume of cell suspension was incubated with fluorescein isothiocyanate (FITC)-conjugated mAb24 or an isotype-matched control at 3 µg/mL at room temperature or 37°C for 30 min in the presence of dimethyl sulfoxide, compound, or activating mAbs at indicated concentrations. Cells were then washed twice in PBS-azide, fixed with 1% paraformaldehyde, and analyzed using a Becton Dickinson (San Jose, CA) FACScan flow cytometer.

For indirect–immunofluorescence analysis, cells were treated as described above and then incubated with the primary antibody at 2 µg/mL for 30 min at room temperature or 37°C in activation medium (see above). Cells were washed twice in PBS-azide and then incubated with 50 µL of a 1:50 dilution of a goat anti-mouse immunoglobulin G-phycoerythrin conjugate (BioSource International, Camarillo, CA) for 20 min at 4°C in the dark. Cells were then washed, fixed in 1% paraformaldehyde, and analyzed as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LFA-1 present on T lymphocytes can be induced to a high-affinity state by treatment with high levels of the divalent cation Mg2+ and EGTA [20 , 21 ], by treatment with Mn2+ [20 ] or by treatment with activating mAbs such as KIM 185 and KIM 127 [22 , 23 ]. Conversely, treatment of cells with phorbol esters does not lead to conversion of LFA-1 to a high-affinity state [21 ] but can activate integrin-mediated binding through receptor clustering [21 , 25 ]. Treatment of SKW-3 T-cell lymphoma cells under any of the above-described conditions induced binding of the cells to immobilized sICAM-1, as shown in Figure 1 . The presence of phorbol esters such as PMA in RPMI medium containing ~0.4 mM Mg2+ and 2.4 mM Ca2+ resulted in the adhesion of ~65% of the input cells to sICAM-1. Treatment with Mg2+ and EGTA resulted in adhesion of ~75% of the input cells to sICAM-1. Treatment of cells with 1–10 µg/mL of KIM 185 in the presence of divalent cations at physiologic concentrations resulted in ~45% adhesion. Similar results were obtained with KIM 127 (data not shown).



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  		Figure 1. Binding of SKW-3 cells to immobilized sICAM-1 under various activating conditions. Data are expressed as percentages of input cells binding after three washes and are the means of values from four experiments ± the SE.

 
The high-affinity state of LFA-1 induced by these conditions can be detected using the metabolic energy-dependent, divalent cation-sensitive antibody mAb24, which binds to LFA-1 and Mac-1 at what has been described as an "activation-induced" epitope [19 ]. Figure 2A shows that treatment of SKW-3 cells with Mg2+ led to the induction of high-affinity LFA-1 as assessed by measurement of mAb24 binding by flow cytometry. Previous reports indicated that mAb24 epitope expression follows a conformational change in LFA-1 [19 , 20 ]. Addition of EGTA (to chelate Ca2+) to the Mg2+-treated cells increased mAb24 expression by more than twofold. This suggests that Ca2+ inhibits this conformational shift to a high-affinity state, as has been reported by Dransfield et al. [20 ]. Treatment of cells with EDTA, 1 mM Ca2+, physiologic concentrations (1 mM each) of Mg2+ and Ca2+, or RPMI medium (data not shown) did not induce mAb24 expression. It is interesting that treatment of the cells with PMA in the presence of physiologic concentrations of the divalent cations resulted in a small but detectable increase in mAb24 expression. Also, addition of PMA to Mg2+/EGTA-treated cells resulted in a twofold increase in the binding of mAb24 (data not shown). It has been hypothesized that PMA treatment does not directly lead to mAb24 induction but, rather, PMA exerts its effect indirectly by promoting homotypic, ICAM- and LFA-1-mediated adhesion of adjacent SKW-3 cells [27 ]. Because binding of ICAM-1 to LFA-1 induces expression of the mAb24 epitope [24 , 27 ], this epitope has been classified as a ligand-induced binding site [LIBS].



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  		Figure 2. (A) Representative indirect-immunofluorescence experiment demonstrating the induction of mAb24 on SKW-3 cells under various activating conditions. Data are expressed as relative median fluorescence (n = 7). (B) Representative direct immunofluorescence experiment demonstrating mAb24 induction by KIM 185 monoclonal antibody treatment of SKW-3 cells in the presence of 1 mM Mg2+ and 1 mM Ca2+ (n = 2).

 
The ability of the activating mAb KIM 185 to induce the conversion of LFA-1 to its high-affinity state was assessed by flow cytometry utilizing FITC-conjugated mAb24. Figure 2B demonstrates that treatment of SKW-3 cells with KIM 185 [or KIM 127 (data not shown)] resulted in a level of mAb24 expression >10-fold that observed with physiologic divalent-cation concentrations alone. The median fluorescence was similar to that obtained with Mg2+/EGTA treatment (data not shown). The percentage of LFA-1 molecules induced to convert to their high-affinity conformation was calculated by comparing surface expression of mAb24 with total LFA-1 expression (quantitated using an anti-CD18 mAb that is not affected by LFA-1 activation). These observations demonstrated (data not shown)] that only about 15–30% of the LFA-1 molecules could be induced to convert to the high-affinity conformation by using divalent cations and activating mAbs.

We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that inhibits the binding of LFA-1 to sICAM-1 in molecular and cellular assays [11 ]. One characteristic of this compound is the ability to block the adhesion of SKW-3 cells to immobilized sICAM-1 in a dose-dependent manner, regardless of the activation conditions used (Fig. 3 ). To investigate the mechanism by which BIRT 377 inhibits LFA-1 activity, the compound was incubated with SKW-3 cells in the presence of various inducers of LFA-1 activation, and mAb24 expression was analyzed by flow cytometry. Figure 4 shows that 10 µM BIRT 377 blocked mAb24 epitope expression induced by Mg2+/EGTA (median relative fluorescence, 340 with Mg2+/EGTA and 32 with BIRT 377). BIRT 377 also blocked the modest increase in expression observed after PMA treatment (data not shown). Furthermore, using FITC-labeled mAb24, it was determined that BIRT 377 inhibits mAb24 expression induced by the KIM 185-activating mAb (Fig. 5 ). These data strongly suggest that BIRT 377 is preventing the conformational change necessary for conversion of LFA-1 to its high-affinity state.



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  		Figure 3. SKW-3 cell binding experiment with immobilized sICAM-1, showing inhibition profiles for compound BIRT 377 under various activating conditions. Closed circles represent PMA-treated cells, open circles represent Mg2+/EGTA-treated cells, and closed triangles represent KIM mAb-treated cells. Data points represent the means of values from three experiments ± the SE.

 


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  		Figure 4. Histogram from a representative indirect-immunofluorescence experiment (one of four), demonstrating inhibition of mAb24 induction in SKW-3 cells under activating conditions with and without BIRT 377 at 10 µM. Thin solid line, secondary-antibody control (median fluorescence = 5); thick solid line, 5 mM Mg2+/1 mM EGTA (median fluorescence = 340); dotted line, 10 mM EDTA (median fluorescence = 6); dashed line, Mg-EGTA plus BIRT 377 (median fluorescence = 32).

 


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  		Figure 5. Representative direct-immunofluorescence experiment demonstrating inhibition of mAb24 induction in KIM 185-treated SKW-3 cells by dimethyl sulfoxide (DMSO) (solid bars) or 10 µM BIRT 377 (hatched bars) (n = 2).

 
It is interesting that BIRT 377 does not inhibit the binding of micellar ß2-integrin Mac-1 to sICAM-1 [11 ]. mAb24 also binds to an induced epitope on Mac-1 and therefore can also be utilized as a marker of a high-affinity receptor for this integrin, which is closely related to LFA-1. Mac-1-transfected K562 cells were analyzed for their ability to express mAb24 under various activation conditions in the presence or absence of BIRT 377. Figure 6 shows that, like LFA-1, Mg2+/EGTA induces high-affinity Mac-1, as assessed by measuring mAb24 binding by flow cytometry. Unlike the case for LFA-1, this high-affinity conformation could not be blocked by incubation with BIRT 377. This demonstrates that BIRT 377 is not directly blocking the binding of mAb24 to its epitope on the ß2-integrins. These data lend support to the theory that the mode of action of BIRT 377 on LFA-1 is likely allosteric in nature and that this compound exerts its inhibitory effects on mAb24 and ICAM-1 binding by preventing the shift of LFA-1 to its high-affinity conformation. Additional evidence suggesting an allosteric mechanism of inhibition by BIRT 377 can be surmised from photo-affinity labeling experiments with recombinant CD11a I domain, in which a benzophenone photoprobe was demonstrated to bind to proline 281 of the I domain, at a site distal from the amino acids that make up the ICAM-1 binding site [28a]. Subsequent studies have established that the compounds bind near the C-terminal {alpha}-helix at a face opposite the MIDAS domain, the region known to interact directly with ICAM-1.



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  		Figure 6. Representative indirect-immunofluorescence experiment demonstrating mAb24 induction in Mac-1 K562 transfectants under various activating conditions with dimethyl sulfoxide (DMSO) (solid bar), 10 µM BIRT 377 (cross-hatched bar), or 50 µM BIRT 377 (horizontally striped bar) (n = 2).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have characterized the mechanism of action of BIRT 377, a low-molecular-weight antagonist of LFA-1-mediated adhesion. This compound is a potent inhibitor of LFA-1/ICAM-1 interactions in molecular and cellular in vitro assays and superantigen-induced interleukin-2 production in vivo [11 ]. We have demonstrated that this compound exerts its inhibitory effects by interfering with a conformational shift of a population of LFA-1 molecules present on the surface of SKW-3 cells to a high-affinity state. This shift is required for high-affinity binding to ICAM-1 and can be detected using mAb24. Soluble ICAM-1 can directly induce or potentially stabilize the mAb24 epitope, suggesting that it is an LIBS [24 , 27 ], and, therefore, ICAM-1 binding might represent the only physiologically relevant mechanism of affinity modulation. This conformational change can be mimicked by treatment of cells with Mg2+/EGTA, Mn2+, or the activating mAbs KIM 127 and KIM 185 [20 21 22 23 ]. These agents, which induce binding of SKW-3 cells to immobilized sICAM-1, also stimulate binding of the activation-specific mAb24. LFA-1-dependent adhesion mediated by divalent cations, activating mAbs, or PMA is blocked in a dose-dependent manner by BIRT 377. It is interesting that the binding of mAb24 induced by all of the above-mentioned agonists is reduced on incubation with BIRT 377, suggesting that BIRT 377 inhibits the conformational shift necessary for LFA-1-dependent adhesion.

The ability of BIRT 377 to inhibit PMA-induced adhesion suggests that LFA-1 on the surface of PMA-treated SKW-3 cells may not all be in a low-affinity state. As demonstrated in Figure 2A , PMA treatment of SKW-3 cells resulted in a reproducible induction of mAb24 epitope expression. It is possible that the low-affinity interactions induced by PMA lead to a ligand-induced shift of LFA-1 to its high-affinity conformation (and expression of the mAb24 epitope), as first suggested and demonstrated by Cabanas and Hogg [27 ]. These high-affinity LFA-1 molecules could conceivably be mediating adhesion of PMA-induced SKW-3 cells to immobilized sICAM-1, as shown in Figure 1 . This would explain the inhibition by BIRT 377. It is possible that true low-affinity-mediated adhesion cannot be isolated in a static assay because of the presence of ligand. Alternatively, BIRT 377 treatment of LFA-1 might change the conformation of LFA-1 enough to perturb all ligand interactions. We have reported previously that BIRT 377 inhibits the binding of two blocking mAbs that map to the I domain of LFA-1, regardless of the conformation state of LFA-1 [11 ]. It is also possible that BIRT 377 interferes with LFA-1 receptor clustering, which would also explain the inhibition of PMA-induced adhesion. Additional research is required to dissect the exact mechanism involved.

An interesting observation is that only ~15–30% of the LFA-1 molecules expressed on SKW-3 cells could be induced to convert to a high-affinity conformation with the various agonists utilized in this study. These findings agree with those previously reported for SKW-3 cells [24 ], T lymphoblasts (M. Stewart and N. Hogg, unpublished observations), and T-cell hybridoma cell lines [16 ]. This phenomenon has also been described for neutrophils, in which a similar percentage of Mac-1 molecules can be induced to shift to a high-affinity conformation in response to phorbol ester or chemoattractant stimulation [29 ]. The reason(s) for this saturation of high-affinity LFA-1 molecules at ~15–30% is unclear. Dustin [24 ] has proposed that either this number represents an equilibrium point between high- and low-affinity LFA-1 molecules or a cellular cofactor(s) necessary for high-affinity LFA-1 conversion is present in limited amounts. Identification of this cofactor(s) and its function will be helpful in the further characterization of affinity modulation.

The structural basis for conformational changes in LFA-1 is unknown. The CD11a I domain has been predicted to be tethered to one face of the ß-propeller-like structure [30 ]. It is probable that the I domain changes its position relative to the rest of the LFA-1 heterodimer in response to agonists, thereby revealing a high-affinity ligand binding site or potentially enlarging the ligand binding interface and thereby promoting higher-affinity binding. Whatever the mechanism, we argue that the altered site now allows further access to ligand and anti-cation-LIBS (anti-CLIBS) antibodies like mAb24. Evidence supporting this model has been published by McDowall et al. [31 ], who have shown that soluble I domain inhibits high-affinity-mediated adhesion to immobilized ICAM-1 by competing with cell-bound LFA-1 I domain for the ß-propeller domain. This competition prevents a conformational shift (which can be detected with the mAb24 antibody) that is necessary for high-affinity binding. Studies using BIRT 377 suggest that it may bind to a region within the I domain of CD11a [11 ], whereas the mAb24 epitope maps outside the I domain [32 ]. In addition, the mAb24 epitope is a revealed epitope rather than a neoepitope, which further suggests that an alteration in the quaternary structure of LFA-1 occurs on activation [33 ]. Thus, it is likely that BIRT 377 inhibits mAb24 epitope expression through an allosteric mechanism by inhibiting a conformational change within LFA-1.

Other integrins, notably the platelet {alpha}IIbß3 receptor, have been demonstrated to undergo conformational changes on ligand engagement. Similar to mAb24 induction, soluble ligand or ligand-mimetic peptides can induce conformational changes in the ß1- and ß3-integrin heterodimers, resulting in the binding of anti-CLIBS mAbs [34 35 36 37 38 39 ]. RGD-containing peptides have been demonstrated to bind to {alpha}IIbß3 and cause an increase in the affinity of the receptor for fibrinogen [40 ]. Divalent cations and activating mAbs can also induce the expression of the CLIBS epitopes on ß1- and ß3-integrins [36 , 37 , 41 , 42 ]. A number of small-molecule antagonists and peptidomimetics of other integrins have been described. It is interesting that the majority of recently described {alpha}IIbß3 antagonists induce a conformational change in the receptor, leading to the binding of anti-CLIBS antibodies [43 ]. This is also the case for a recently identified {alpha}4ß1 antagonist [44 ]. BIRT 377, therefore, is unique in its mode of inhibition because it does not induce anti-CLIBS binding (mAb24) but instead actually prevents it. The BIRT 377 compound will be a novel and useful reagent in the dissection of the mechanism of integrin activation.


   ACKNOWLEDGEMENTS
 
The authors thank Carol Stearns for performing flow cytometry analyses.


   FOOTNOTES
 
This manuscript is dedicated to Gabrielle.

Received January 9, 2001; revised March 6, 2001; accepted March 8, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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