* Department of Biology and
Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut, and
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
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adhesion molecules • inflammation • cell-cell interactions • FACS • integrin • mAb epitope
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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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.
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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).
 View larger version (13K): [in a new window] |
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.
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 View larger version (14K): [in a new window] |
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).
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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.
 View larger version (19K): [in a new window] |
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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 View larger version (19K): [in a new window] |
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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 View larger version (19K): [in a new window] |
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).
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-helix at a face opposite the
MIDAS domain, the region known to interact directly with ICAM-1.
 View larger version (22K): [in a new window] |
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).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
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 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
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
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.
Received January 9, 2001; revised March 6, 2001; accepted March 8, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits EMBO J 8,3759-3765[Medline]-subunit into a ß-propeller domain Proc. Natl. Acad. Sci. USA 94,65-724ß1 Integrin-dependent cell adhesion is regulated be a low affinity receptor pool that is conformationally responsive to ligand J. Biol. Chem. 270,28740-28750IIbß3 (platelet GPIIb-IIIa) Cell 65,409-416[Medline]6ß1 Proc. Natl. Acad. Sci. USA 90,9051-90554ß1 that inhibit allergic airway responses J. Med. Chem. 42,920-934[Medline]This article has been cited by other articles:
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