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*
ICOS, Bothell, WA 98021; and
Department of Experimental Immunohematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D. CD18 deficiency in humans reduces
inflammatory cell recruitment of leukocytes, while blocking CD18
function with mAbs can limit tissue damage following the reperfusion of
ischemic tissues in experimental animals (2, 4). Lymphocytes bind ICAM by rapidly interconverting LFA-1 from an inactive to active state without changing LFA-1 expression on the cell surface (5). Activation of LFA-1 from the outside of the cell can be accomplished with the divalent cation Mn2+ or certain stimulatory mAbs, and these treatments directly increase LFA-1 affinity for ICAM-1 (6, 7, 8). Physiologic lymphocyte signaling through the TCR induces adhesion, largely by promoting the clustering of LFA-1 on the cell surface to increase avid binding or by promoting integrin-cytoskeletal linkages (7). Soluble ICAM-1, a high-affinity ligand capable of displacing cells bound using high-affinity LFA-1, will not displace TCR-stimulated adhesion, suggesting that low-affinity LFA-1 forms the initial ICAM contacts in these circumstances (6, 9). Chemokines, however, stimulate a rapid increase in LFA-1 affinity that appears to be sufficient to explain lymphocyte arrest on high endothelial venules (10). High-affinity forms of LFA-1 may also represent receptor structures competent to inform the cell of ICAM engagement, so-called outside-in signaling (11).
Structural and mutagenesis experiments have identified regions of LFA-1
important for ligand recognition and the redistribution of LFA-1 on the
cell surface (2). The CD11a subunit of LFA-1 contains an
autonomously folding I domain containing a metal-ion binding site
critical for ligand binding (12), which is inserted in a
predicted 
-propeller structure also important for ligand recognition
(13). The CD18 subunit contains a sequence resembling an
I-like domain important for ICAM-1 adhesion, and several
function-blocking mAbs recognize this region (14, 15). In
both subunits, these ligand recognition regions are suspended on a
cysteine-rich stalk, followed by cytoplasmic tail sequences that
influence LFA-1 affinity and clustering (16, 17, 18). There is
abundant evidence that LFA-1 undergoes allosteric conformational
transitions that can influence ligand binding, and this is summarized
in a model that proposes that inside-out signal transmission influences
a hinge region of the integrin cytoplasmic tails to induce
extracellular conformational changes that reorganize ligand-binding
surfaces (19).
To study LFA-1-dependent adhesion with greater precision, we identified mAbs to LFA-1 induced by cellular stimulation. In this work, we describe a panel of novel activation epitope or neoepitope mAbs that map to the CD18 subunit. Previously described activation epitope mAbs map to the CD11a subunit (7), and so these reagents define additional conformational transitions that occur on a fraction of LFA-1 following physiological activation. These activation epitopes do not require contact with ICAM-1 for induction, but their up-regulation requires cellular signaling and correlates with cellular binding to ICAM-1. Cell lines obtained from individuals with leukocyte adhesion deficiency-1/variant (LAD-12/variant) are defective in the regulation of LFA-1 adhesion (20) and do not induce the neoepitopes, suggesting these mAbs define a conformation that is necessary for ICAM-1 binding. We also find that function-blocking CD18 mAbs prevent the induction of these LFA-1 activation epitopes, and we propose that some CD18 mAbs work, in part, by allosterically preventing a conformational change of LFA-1. The presence and regulation of the neoepitopes on rLFA-1 with high affinity for ICAM-1 suggest these mAbs may mark molecules with higher affinity for ligand than the remainder of cell surface LFA-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LAD-1/variant cell lines. EBV-transformed peripheral blood cells from patients with LAD-1/variant (20) or their unaffected primary relatives were grown in RPMI 1640 with 10% FBS and antibiotics at 37°C in 5% CO2. Col104 and Colo829BL are cell lines derived by EBV transformation of PBLs (American Type Culture Colleciton (ATCC), Manassas, VA). The following chemicals were obtained from Calbiochem (La Jolla, CA): PMA, 2-deoxyglucose, forskolin, 3-isobutyl-1-methylxanthine, dibutyryl cAMP, thapsigargin, ionomycin, cytochalasin D, fMLP, and staurosporine. A308296 is a synthetic chemical that specifically antagonizes the LFA-1-ICAM interaction. Specific inhibitors against phosphodiesterase subtypes 3 (cilostamide) and PDE4 (IC197) were also provided by ICOS (Bothell, WA).
The following mAbs were obtained from ATCC, unless otherwise specified: OKT3, CD28.1 (BD PharMingen, San Diego, CA), TS1/22, 1B7 (ICOS; an IgG1 isotype control), TS1/18, 22F12C (ICOS), H52, IB4, TS2/4, 240Q (ICOS), 164B (anti-ICAM-1; ICOS), 92C12F (anti-ICAM-2; ICOS), and ICR8.1 (anti-ICAM-3; ICOS). Fab fragment of 240Q were also conventionally prepared. ICAM-1/Ig fusion protein contains domains 1–5 of ICAM-1 fused to IgG1 and is a dimeric protein (21).
Isolation of mAbs to LFA-1 activation epitopes
BALB/c mice were immunized with human rLFA-1 (rhLFA-1) (21). Conventionally cloned hybridoma supernatants that recognized rhLFA-1 in an ELISA were tested for differential staining of Hut-78 cells by FACS in the presence and absence of 1 mM MnCl2. Seven noncompeting activation epitope mAbs were purified by protein A chromatography. We characterized three mAbs, 327C, 327A, and 330E, in detail. Fab fragment of 327A and 327C were prepared by conventional methods and purified by HPLC to >98% purity. For some experiments, mAb or Fab fragment were labeled with biotin using N-hydroxy-succinamide biotin or labeled with the fluorochrome conjugate Alexa488 (Molecular Probes, Eugene, OR).
Cell staining and FACS analysis
Resting peripheral blood T cells were purified from volunteers by density gradient centrifugation, followed by negative immunodepletion using magnetic beads (Dynal Biotech, Great Neck, NY). These cells were 98% positive for TCR staining and do not express detectable Mac-1 (CD11b/CD18). In a typical experiment, 1 x 105 cells in RPMI 1640 with 5% heat-inactivated FBS were mixed with a staining mAb at 10 µg/ml. Medium with or without various stimuli was added and the cells were warmed to 37°C for 10 min, then the cells were washed with ice-cold Dulbecco’s PBS (D-PBS) with 1% BSA, fixed in 1% formaldehyde in D-PBS, and analyzed on the FACS. For indirect staining, the cells were counterstained on ice with either F(ab')2 goat anti-mouse IgG-Alexa488 at 2 µg/ml (Molecular Probes), or streptavidin-FITC at 10 µg/ml. To stimulate lymphocytes through the TCR, cells were treated with biotinylated CD3 or CD28 mAbs on ice for 10 min at concentrations indicated in figure legends, then with 10 µg/ml streptavidin in the presence of Alexa488-conjugated staining mAb at 37°C for 10 min. PMA was used to stimulate lymphocytes at 10 ng/ml. mAb pretreatment of lymphocytes was typically done on ice for 10 min with 20 µg/ml mAb, followed by the addition of staining mAb and stimulus at 37°C for 10 min. Neutrophils were purified using density gradient centrifugation. fMLP was used at 10 ng/ml to stimulate polymorphonuclear cells (PMNs). Cell-staining experiments were performed with at least three donors.
Epitope mapping of activation epitope Abs
cDNA encoding aa 23–457 or 411–700 from human CD18 were PCR amplified from a full-length cDNA clone and inserted in pDisplay (Promega, Madison, WI). The resulting plasmids were transfected into COS7 cells by calcium phosphate transfection. Cells were stained with 10 µg/ml mAb, followed by counterstaining with F(ab')2 goat anti-mouse IgG-Alexa488 at 2 µg/ml (Molecular Probes) and analyzed on FACS.
Cell adhesion to ICAM-1
ICAM-1/Ig was used to coat 96-well ELISA plates (Immulon; Nunc, Naperville, IL) at 5 µg/ml. Cell adhesion assays were performed in triplicate, as described previously (22). Lymphocytes or EBV-transformed cell lines were added to the wells at 1 x 105 cells/well and stimulated with PMA (10 ng/ml) or by cross-linking OKT3-stained cells (10 µg/ml) with an anti-IgG2a-specific mAb (10 µg/ml; BD PharMingen) at 37°C. Adherent cells were cross-linked to the plate by the addition of 2.5% glutaraldehyde in D-PBS at room temperature for 2 h, the plate was washed exhaustively in water, and adherent cells were stained with crystal violet. Light absorption was measured at 490 nm, which is proportional to the number of adherent cells.
Interaction of neoepitope mAb or ICAM-1 with rhLFA-1
Plates were coated with 5 µg/ml TS2/4, a nonblocking mAb to LFA-1, by overnight incubation in 50 µl at 4°C. Nonspecific binding was blocked with 5% gelatin in PBS. rhLFA-1 at 2.5 µg/ml was bound to the plates for 1 h and plate washed in PBS with 0.05% Tween 80. Biotinylated ICAM-1/Ig or biotinylated 327C was serially diluted in PBS with 0.05% gelatin and bound to rhLFA-1-coated plates for 1 h at room temperature. The plate was washed three times, and a 1/1000 dilution of streptavidin-europium (Wallac, Gaithersburg, MD) in PBS with 0.5% gelatin was added for 30 min. The plate was washed six times, and bound europium was measured by the addition of Enhance solution (Delphia) and time-resolved fluorescence was measured in a 96-well plate reader (Wallac, Gaithersburg, MD). Cation concentration was manipulated by washing bound rhLFA-1 with PBS with 0.5% gelatin and 1 mM EDTA for 15 min, washing in PBS/Tween 20, then incubating with PBS with 0.5% gelatin with 100 µM EDTA, or 1 mM CaCl2, MgCl2, or the stimulatory mAb 240Q (20 µg/ml) for 30 min. These agents were included in subsequent binding with ICAM-1/Ig or biotinylated mAb. Nonspecific binding of 327C was measured by preincubating rhLFA-1 with 50 µg/ml nonbiotinylated 327C. Nonspecific binding of ICAM-1/Ig was determined by preincubating rhLFA-1 with 50 µg/ml blocking mAb 22F12C. Nonspecific binding was <5% of total binding throughout the concentration range reported. To control for the nonspecific effects of cations, a biotinylated mAb to another epitope of rhLFA-1, 324C, was incubated to bound rhLFA-1, and its binding was detected with streptavidin-europium.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A panel of anti-LFA-1 mAbs was tested for selective
recognition of PMA-activated human peripheral blood T lymphocytes by
flow cytometry, and three noncross-competing mAbs, 327A, 327C, and
330E, were selected for further study. These mAbs bound to purified
rLFA-1, indicating they identify an activation epitope present on this
integrin. Neoepitope mAb staining was blocked by certain CD18 mAbs (see
below), suggesting that the mAbs do not cross-react with other T
lymphocyte cell surface Ags. Initial experiments demonstrated that the
recognition of their corresponding epitopes required cell stimulation,
extracellular divalent cations, physiologic temperatures, and cellular
metabolism (Fig. 1
). These requirements
are identical to the requirements for LFA-1-dependent adhesion
(5), and suggested a parallel between neoepitope
up-regulation and cell adhesion to ICAM-1. In these experiments, we
used directly conjugated mAb with a defined fluorochrome-to-Ab coupling
ratio, and so by comparing the staining intensity with that of a
directly conjugated mAb recognizing total LFA-1, we can estimate the
fraction of LFA-1 bearing these activation epitopes. We estimate that
20% of the cell surface LFA-1 possessed neoepitopes after PMA
stimulation and 10% of LFA-1 possessed neoepitopes after CD3
cross-linking (data not shown). Stimulatory Abs induce a single
high-affinity state between cell-free LFA-1 and ICAM-1 (9, 23), so we treated peripheral blood T cells with Fab fragment of
the activating mAb 240Q, and found that the fraction of LFA-1 stained
with these neoepitope mAbs increased to 80% of cell surface LFA-1.
These results indicate that cell stimulation results in changes
detected by these neoepitope mAbs in a fraction of LFA-1, but that most
LFA-1 is competent to assume this state.
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Activation epitope up-regulation in T cells by PMA treatment or CD3
stimulation was not accompanied by an increase in total cell surface
LFA-1, measured with mAbs specific for CD18 or CD11a (Fig. 1
G). Neutrophils, however, selectively up-regulate Mac-1 by
3-fold after cell activation. PMA or fMLP treatment induces the
neoepitopes 50-fold on neutrophils, indicating most of the increased
neoepitope display is due to a conformational alteration rather than an
increase in total cell surface expression of CD18 integrins (Fig. 1H). Therefore, these LFA-1 neoepitopes are induced in
leukocytes stimulated through structurally distinct receptors.
Activation epitope induction also followed physiological stimulation of
T cells by cross-linking the TCR. Stimulation of the CD28 costimulatory
molecule alone also weakly induced neoepitope production, and this
observation parallels a report that CD28 cross-linking of the Jurkat
cell line increased LFA-1-dependent cell adhesion (24).
CD28 costimulation increased the sensitivity of neoepitope induction to
lower levels of CD3 cross-linking (Fig. 2A). We investigated the
specificity of the CD3 response by cross-linking cells with mAbs
directed to CD4, CD7, CD8, CD9, CD96, and several nonblocking mAbs to
LFA-1, and in all cases the neoepitopes were not up-regulated (data not
shown).
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). Cells treated with
LFA-1-stimulatory mAbs achieve maximal staining within 1 min,
indicating that the time course of neoepitope mAb staining reflects the
induction of the neoepitope and is not limited by the on-rate of the
staining mAb (data not shown). LFA-1 activation epitope induction requires cellular signal transduction and is defective in LAD-1/variant
The cellular signals that control LFA-1 avidity are not entirely
defined, but include protein tyrosine phosphorylation, cAMP levels, and
intracellular calcium levels, all signaling mediators that have
important roles in the lymphocyte response to Ag (5, 25).
We examined whether compounds that perturb these signaling pathways
influenced the neoepitope induction. CD3-induced 327C neoepitope
induction was attenuated in cells treated with the cell-permeable cAMP
analogue, dibutyryl cAMP, or with the nonspecific phosphodiesterase
inhibitor 3-isobutyl-1-methylxanthine (Fig. 3A). The inhibition of
neoepitope appearance mirrors the effects these inhibitors have on cell
adhesion at similar concentration (5). We also used
selective inhibitors of PDE3 and PDE4 enzymes, both expressed in T
lymphocytes (26), and showed that a combination of the two
inhibitors was more effective at suppressing induction of the 327C
neoepitope than either agent alone. We used ionomycin and thapsigargin
to elevate intracellular Ca2+ levels, and both
agents elevated neoepitope formation (Fig. 3B). Both
compounds have been shown to increase lymphocyte cell adhesion to
ICAM-1 with dose-response relationships very similar to those presented
in this study for neoepitope staining (25). Finally, we
showed that the protein kinase inhibitor staurosporine inhibited 327C
neoepitope induction by PMA (Fig. 3C), and similar
concentrations of this inhibitor reduced lymphocyte ICAM-1 cell
adhesion (5). Each of the three neoepitope mAbs showed
similar responses to T cell agonists and antagonists (data not shown).
Taken together, these results indicate that LFA-1 neoepitope induction
involves cellular signal transduction events, and the neoepitope
markers may be used to identify the cell signaling pathways that
regulate LFA-1 activity.
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2 (CD18) or 3
families of integrins (20). The defect is felt to lie on
signaling pathways that affect stimulus-response coupling to these
integrins. We examined EBV-transformed lymphoblastoid cell lines
derived from patients with LAD-1/variant for the induction of these
neoepitopes. Staining of control cell lines with neoepitope mAb was
increased by treatment of the cells with PMA. However, patient cell
lines had lower levels of staining with the neoepitope mAb at rest and
after PMA stimulation (Fig. 4, A and B) despite having similar levels of cell
surface LFA-1. LAD-1/variant cell lines induced the neoepitopes and
adhered to ICAM-1 if they were treated with the CD18-activating mAb
240Q (Fig. 4C, and data not shown), indicating that in this
condition LFA-1 will display the neoepitopes and adhere to ICAM-1 if
cellular signaling is bypassed. Therefore, these neoepitope mAbs are
capable of identifying cells defective in signal transmission to CD18
integrins.
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The appearance of activation epitopes on LFA-1 could reflect
conformational alterations independent of ligand binding or could
require ligand binding—so-called ligand-induced binding site
mAbs. Lymphocyte stimulation could induce LFA-1 to adhere to ICAMs on
neighboring cells and thereby induce LIBS neoepitopes. We stimulated T
cells with PMA in the presence of the CD11a I domain-directed blocking
mAb TS1/22 (27), which abrogates binding to all ligands.
The 327A, 327C, and 330E neoepitopes were still up-regulated,
indicating that these activation epitopes are not dependent on ICAM
interaction for their expression (Fig. 5A, and data not shown).
Similarly, a cocktail of blocking mAbs to ICAM-1, ICAM-2, or ICAM-3 did
not interfere with stimulus-induced neoepitope formation.
LFA-1-adhesive events depend on the CD11a subunit I domain, so we used
a potent I domain-specific antagonist of LFA-1 adhesion
(21) to block LFA-1-dependent adhesion. In the presence of
this inhibitor, T cell activation still elicited the activation
epitopes (Fig. 5B). Taken together, these results indicate
that these neoepitopes are induced by cell stimulation before ligand
contact, and are not necessarily induced as a consequence of ligand
binding. These properties are distinct from the widely used LFA-1
activation epitope Ab mAb24, which maps to the CD11a subunit, and which
requires ICAM contact for induction on physiologically activated T
cells (28).
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C, and data not shown). Soluble ICAM-1/Ig may induce the
activation epitopes on unstimulated cells, perhaps by an induced-fit
mechanism, stabilizing a form of LFA-1 energetically favored to bind
ligand. CD18-blocking Abs prevent the induction of the activation epitopes, in part, by an allosteric mechanism
The CD18-blocking mAbs TS1/18 and IB4 each recognize distinct
epitopes of CD18, yet either will block all of the 327A, 327C, and 330E
epitopes on T cells (Fig. 6A,
and data not shown). This response is specific because these
neoepitopes were not affected by blocking mAb to the LFA-1 CD11a
subunit or nonblocking mAb to CD18 (Fig. 5A, and data not
shown). The possibility that each of the function-blocking mAbs
competitively displaces three distinct neoepitope mAbs is unlikely,
since none of the activation epitope mAbs will block T cell
adhesion.
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B). Since
the major epitope of the function-blocking TS1/18 mAb is physically
distinct from the 330E neoepitope, we conclude that TS1/18 inhibits
330E epitope formation through an allosteric mechanism. Similar data
showed the 330E neoepitope is distinct from a second blocking Ab IB4
(data not shown). Moreover, T cells can be costained with both TS1/18
and 330E if cells are first treated with an activating mAb, indicating
directly that TS1/18 and 330E bind to distinct sites (data not shown).
Blocking CD18 mAbs may also inhibit binding of the 327A and 327C
epitopes by allosteric change acting within the I-like domain of the
CD18 subunit, although these experiments do not address this
conjecture. These data suggest that CD18-blocking mAbs function, in
part, by suppressing a conformational change involving the CD18
chain. The activation epitopes can be modulated on rLFA-1, and this correlates with apparent affinity of LFA-1 for ICAM-1
The affinity of cellular LFA-1 for ICAMs is low relative to other
receptor-ligand interactions, and this has made it difficult to
distinguish how great a role affinity modulation plays in
LFA-1-dependent cell adhesion (11). We therefore examined
the binding of the activation epitope mAb to immobilized rhLFA-1
(21). rhLFA-1 was captured with a nonblocking mAb to
microtiter dishes and then specifically bound with biotinylated 327A,
327C, and 330E neoepitope mAbs or biotinylated rICAM-1/Ig. The apparent
affinity of the 327C neoepitope mAb for rhLFA-1 increased in the
presence of Mg2+ or Mg2+
plus the stimulatory mAb 240Q relative to its affinity in
Ca2+ or EDTA-containing buffer (Fig. 7A). rhLFA-1 had no measurable
binding to ICAM-1/Ig in the presence of EDTA or
Ca2+ (data not shown), but LFA-1 interaction with
ICAM-1/Ig was easily measurable in Mg2+. The
apparent binding affinity increased significantly by the addition of
the stimulatory mAb 240Q. The presence of EDTA,
Mg2+, or Ca2+ did not
affect the apparent binding affinity for a mAb directed to another
epitope present on rhLFA-1 (Fig. 7C). Thus, rhLFA-1 assumes
a conformation capable of binding neoepitope mAbs as well as ligand,
and 327C and ICAM-1 binding for LFA-1 increased in parallel under
several conditions. Similar results were obtained using the neoepitope
Ab 327A (not shown). These binding experiments do not strictly measure
affinity, as the ligands ICAM-1/Ig and 327C are both dimeric, but it is
unlikely that LFA-1 is undergoing changes in clustering after
immobilization on a surface. These experiments support the hypothesis
that neoepitope-positive LFA-1 on the lymphocyte cell surface has
higher affinity for ICAM-1.
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LFA-1 clustering may be required for efficient cell adhesion, so
we examined the relationship between LFA-1 clustering on T lymphocytes
and the appearance of these LFA-1 neoepitopes. First, when we
cross-linked LFA-1 with nonblocking mAb directed to either CD11a or
CD18, the activation epitopes were not induced (data not shown). Next,
we attempted to limit the lateral mobility of LFA-1 by lightly fixing
cells with 1% formaldehyde and found that the activation epitopes
could still be induced. Unfixed cells treated with Abs displayed LFA-1
with a punctate appearance consistent with receptor capping. In
contrast, cells fixed in 1% formaldehyde had a relatively smooth
uniform distribution of LFA-1 when detected with a mAb to CD11a or to
CD18 (Fig. 8A). Fixation with
0.25% or 1% formaldehyde abrogated PMA-dependent 327C neoepitope
induction, but did not significantly affect 327C neoepitope induction
with Fab fragments derived from the stimulatory mAb 240Q, indicating
that extracellular activation of LFA-1 was not impaired by the fixation
process (Fig. 8C). Thus, neoepitope induction occurred on
cells in the absence of gross LFA-1 clustering. Fixed cells, positive
for the 327C neoepitope with a uniform cellular distribution (Fig. 8A), were also able to specifically bind to ICAM-1-coated
microtiter dishes, further strengthening the association between 327C
neoepitope and cell adhesion, even when the lateral mobility of LFA-1
was limited by prior fixation (Fig. 8B). Finally, we limited
actin microfilament-dependent integrin clustering by treating cells
with cytochalasin D and examined the effects of this treatment on
neoepitope induction. Cytochalasin D limits the reorganization of LFA-1
that is induced in lymphocytes by CD3 and PMA treatment, and impairs
LFA-1-dependent cell adhesion (29). We found that 327C
neoepitope induction was not altered over a wide range of cytochalasin
D concentrations in response to CD3 cross-linking and only partially
inhibited neoepitope induction in response to PMA, while adhesion to
ICAM-1 was eliminated (Fig. 8, D and E). We infer
that the LFA-1 clustering that follows lymphocyte stimulation is not
critically involved in the induction of these CD18 neoepitopes, and
that these epitopes are not likely to report integrin clustering per
se. Since cytochalasin D inhibits cell adhesion without ablating
neoepitope up-regulation, neoepitope formation is not sufficient for
cell adhesion, and is consistent with reports that postreceptor events
are also necessary for cell adhesion (29).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IIb3 integrin
(19). Additionally, conformation-sensitive mAbs recognize
subpopulations of integrins. For instance, the CBRM1/5 mAb identifies a
conformational change in the ligand-binding I domain of CD11b
(30), and other mAbs recognize integrins following ligand
engagement. Finally, the crystal structure of the collagen-binding
2 integrin I domain undergoes an important
structural alteration when bound to collagen triple helical peptide
(31). In the particular case of LFA-1, the mAb24 Ab
detects a site on the CD11a subunit that when physiologically induced
requires ICAM contact (28). The NKL1-16 mAb recognizes an
epitope on the CD11a subunit associated with LFA-1 clustering
(32). Zang et al. (33) recently identified
another activation mAb, YTA-1, that recognizes an epitope requiring
both subunits and blocks interaction with ICAM-1. Alterations within
the LFA-1 I domain occur upon ICAM binding, and mutation in these
regions alters LFA-1 affinity and signal-initiated inside-out signaling
(21). The neoepitopes recognized by 327C, 327A, and 330E
do not require ICAM for their induction, and therefore identify
neoepitopes of CD18 that mature before ligand engagement. The time
course of the appearance of these neoepitopes, their independence from
ligand engagement, and their mapping to CD18 indicate that these mAbs
are distinct from previously reported neoepitopes. Activating mAbs
induce conformational changes that are propagated to both the CD11a
subunits (mAb24) and to the amino-terminal region and cysteine-rich
tail of CD18 (these neoepitopes), indicating that LFA-1 activation
leads to widely distributed conformational changes in both integrin
subunits. Signal-induced alterations also occur in a subset of LFA-1
involving several regions of CD18. A widespread conformational change
in LFA-1 was refined in a model that positions the I-like domain of the
CD18 subunit adjacent to the proposed -propeller domain of the CD11a
subunit, further suggesting this interface is a functional linkage for
the propagation of inside-out signals (33). The neoepitopes correlate closely with the ability of lymphocytes to adhere to ICAM-1 when stimulated several different ways, and neoepitope induction appears to share several cell-signaling intermediates with LFA-1-dependent adhesion. Since these mAbs recognize stimulated T cells or PMNs, they should prove useful tools to address physiological leukocyte signal transduction from a variety of receptors and may prove to be a useful surrogate of cell adhesion. The correlation between activation epitope induction and cell adhesion is, however, not perfect. Small molecule antagonists or blocking mAbs directed to the CD11a I domain inhibit ICAM-1 binding without influencing signal-dependent CD18 neoepitopes. This observation is consistent with evidence that the CD11a I domain plays a necessary role in LFA-1 function, and suggests some signal-induced CD18 conformational alterations are independent of CD11a I domain interaction with ligand. Cytochalasin D efficiently disrupts cell adhesion with minimal effects on these neoepitopes. This may merely reflect the multicomponent nature of cell adhesion, with cytochalasin D inhibiting steps subsequent to neoepitope induction (29). Taken together, these results indicate that these neoepitopes are necessary, but are not sufficient for ICAM binding.
Function-blocking CD18 mAbs have been used to show CD18 plays an
important role in cell recirculation and inflammatory cell infiltration
(34), and mAbs of this sort are being studied as therapy
for the treatment of inflammatory disease. CD18 function-blocking mAbs
may competitively block integrin-ligand contact, as contact sites
recognized by these blocking mAbs lie in the I-like domain of the CD18
subunit that is necessary for ligand binding (14, 15). We
found that several function-blocking CD18 mAbs prevented the
stimulus-induced binding of 327A, 327C, and 330E activation epitope
mAbs. In the case of 330E, we are able to show that the CD18
function-blocking mAbs bind to a site distinct from the 330E mAb, and
therefore the inhibition of 330E binding occurs by an allosteric
mechanism. 330E detects denatured CD18 upon immunoblotting (data not
shown) and maps to the cysteine-rich stalk region, suggesting that
integrin activation is associated with stalk unfolding. Since the CD18
neoepitope mAbs are highly correlated with cell adhesion, we propose
that CD18 function-blocking mAbs work, in part, by allosterically
preventing a conformational change of the CD18 subunit of LFA-1 that
may be necessary for ligand binding. The
1-blocking mAb 13 allosterically blocks ligand
binding, so there is precedent for allosteric inhibition by
anti-integrin mAbs (35).
The biophysical mechanism of LFA-1/ICAM interaction on cells has
received considerable scrutiny. Competition of the binding of soluble
ICAM-1 to cell surface LFA-1 with mAbs suggested that PMA induces
10% of LFA-1 to a higher affinity state (36). However,
others have argued that LFA-1-dependent adhesion on lymphocytes is not
governed by affinity changes, but is largely dependent upon receptor
clustering, resulting in high avidity binding. This argument is
supported by the observation that high-affinity soluble ICAM-1 fails to
block the adhesion of cells stimulated with PMA or TCR cross-linking
(6, 9). Chemokines, however, clearly induce a transient
increase in LFA-1 affinity that contributes to adhesion
(10). We find that only 10% of cell surface LFA-1
binds our neoepitope mAb following stimulation and may define a subset
of LFA-1 with a special role in ligand recognition. For instance, it
could be LFA-1 in a higher affinity state than the remainder of cell
surface LFA-1, the subset of LFA-1 forming a high-affinity interaction
with ICAM-1, or a conformation associated with outside-in signaling
from ligand engagement.
LFA-1 undergoes significant reorganization on lymphocyte cell surface after stimulation and multivalent interactions can increase cell adhesion. We showed that Fab fragments of two of these neoepitope mAbs showed increased expression of stimulated T cells, ruling out that increased staining is due to clustering of LFA-1 increasing the avidity of low-affinity Ab. However, lateral association of LFA-1 could induce the appearance of novel epitopes, and so we looked for evidence that these neoepitopes were associated with LFA-1 clustering. Previous studies have shown that low concentrations of cytochalasin D relieve the interaction of LFA-1 with the cytoskeleton, and promote the lateral mobility of LFA-1 increasing cell adhesion (37). Mutations in the CD18 cytoplasmic tail reduce cell surface LFA-1 clustering in parallel with cell adhesion (38), and the tail of CD18, inserted in chimeric integrin molecules, controls integrin clustering, spreading, and adhesion independent of the affinity of the receptor for ligand (18). First, we were unable to induce the neoepitopes by deliberately cross-linking LFA-1 with nonblocking mAb. Second, treatment of resting lymphocytes with cytochalasin D induces clustering of LFA-1 (29), but these neoepitopes were not induced. Third, the 327C neoepitope can be modulated on rhLFA-1 on plastic, in which clustering is unlikely to occur. Finally, cell fixation that inhibits LFA-1 lateral mobility did not prevent the induction of neoepitopes by a stimulatory mAb. So, while this work does not address the overall importance of LFA-1 clustering in cell adhesion, it does indicate that these CD18 neoepitopes correlate poorly with integrin clustering. Instead, our results suggest these neoepitopes identify LFA-1 with a higher affinity for ICAM-1. rLFA-1 has a much higher affinity for ICAM-1 (23) than that estimated for LFA-1 on resting cells (36) and is recognized by all of the neoepitope mAbs that are used in this study. We found that treatment of rhLFA-1 with Mg2+ or Mg2+ plus an activating mAb increases the affinity for ICAM-1 and for the 327C neoepitope Ab. Next, a high concentration of soluble ICAM-1/Ig or Mn2+ treatment will induce the neoepitopes on unstimulated cells, suggesting neoepitope-positive LFA-1 is stabilized in a conformation favorable for binding ligand. Finally, the introduction of a mutation in the CD11a cytoplasmic domain of LFA-1 that increases LFA-1-dependent adhesion, probably due to an increase in affinity, also induces these neoepitopes (16, 18) (data not shown).
We demonstrate the stimulus-induced appearance of several novel CD18 activation epitopes on leukocytes. These mAbs identify a subpopulation of this cell surface integrin that correlates with cell adhesion, and most likely identify LFA-1 with increased affinity for ICAM-1. Although anti-CD18 inhibition of cellular adhesion may act by blocking direct interaction with ICAMs, these neoepitope mAbs suggest that anti-CD18 Abs also act by blocking a conformational change of LFA-1. In addition to providing important tools for the dissection of signaling pathways involved in LFA-1 activation, our work also indicates a role for additional conformational changes in CD18 as well as CD11a in LFA-1-dependent adhesion.
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2 Abbreviations used in this paper: LAD-1, leukocyte adhesion deficiency-1; D-PBS, Dulbecco’s PBS; MFI, median fluorescence intensity; PMN, polymorphonuclear cell; rh, recombinant human.
Received for publication May 2, 2001. Accepted for publication September 20, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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