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New Product Research Laboratories III,
New Product Research Laboratories IV, and
Basic Technology Research Laboratories, Daiichi Pharmaceutical Co., Tokyo, Japan; and
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Department of Pharmacology and
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3rd Department of Internal Medicine, School of Medicine, Nihon University, Tokyo, Japan
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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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subunit (CD11 a, b, c) noncovalently associated with a common ß
subunit (CD18) (1, 2). The members of this family are LFA-1
(CD11a/CD18), Mac-1 (CD11b/CD18), and p150, 95 (CD11c/CD18) (3). As in
other integrins, association of the CD11 and CD18 subunits is required
for normal surface-membrane expression and function of these receptors
(4, 5). LFA-1 is expressed on all leukocytes and mediates adhesion to a
variety of cell types that express one or more of the LFA-1 ligand’s
ICAM-1 (CD54) (6, 7, 8), ICAM-2 (CD102) (9, 10), and ICAM-3 (CD50)
(11, 12, 13, 14). ICAM-1 is a counter-receptor for the leukocyte integrins LFA-1 and Mac-1 and promotes a wide range of cellular interactions important in inflammation (15, 16, 17, 18). ICAM-1 is a membrane protein with five Ig superfamily extracellular domains, a hydrophobic transmembrane domain, and a short cytoplasmic domain (19, 20). The LFA-1 binding site is located in domain 1 of ICAM-1, although domain 2 appears to play an essential role in maintaining the conformation of domain 1 (21). ICAM-1/LFA-1 interaction includes adhesion of leukocytes to the endothelium, followed by their extravasation at sites of inflammation, costimulatory signaling for T cell activation, and adherence of killer T cells to target cells (3). LFA-1 is maintained in an inactive form on resting leukocytes and becomes activated following signaling through other cell surface receptors such as the TCR/CD3 complex (22). Several groups have reported that the ligand binding site in LFA-1 is located in the I domain. For example, Champe et al. (23) have shown that a number of mAbs that block LFA-1 binding to ICAM-1 map to the I domain of LFA-1. Other reports showed that some point mutations in the I domain significantly reduced LFA-1 binding to ICAM-1 (24, 25). In addition, I domain-IgG chimeras, which are bivalent molecules, specifically bind to ICAM-1 (26). On the other hand, Bajt et al. (27) have reported that the ß subunit is essential for the ligand-binding function of LFA-1. Since the I domain as well as domains V and VI of CD11a (28) have been implicated in the ligand-binding function, it is likely that multiple sites in LFA-1 cooperate in the recognition of ligands. While a purified LFA-1 from leukocytes has been reported to bind to purified ICAM-1 and ICAM-1-expressing cells (22, 29), LFA-1 protein micelles may exhibit a higher avidity due to their multivalency. Therefore, we have produced a recombinant soluble form of LFA-1 (sLFA-12), a truncated form of LFA-1 lacking the transmembrane and cytoplasmic domains, in mammalian cells to facilitate the study of the ICAM-1/LFA-1 interaction (30).
In the present study, we have characterized the ICAM-1/LFA-1 interaction at the molecular level, using a novel technique based on surface plasmon resonance. Our results show that a soluble form of monomeric LFA-1 binds the first two domains of ICAM-1 (D1-D2) expressed as a chimeric IgG fusion protein (D1D2-IgG) with a Kd value of 500 nM and a kdiss value of 0.1 s-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents were obtained from Sigma (St. Louis, MO), unless otherwise indicated. Daigo’s T media and ITES (2 µg/ml insulin, 2 µg/ml transferrin, 122 ng/ml ethanolamine, and 9.14 ng/ml sodium selenite) were purchased from Wako (Osaka, Japan). DMEM/F-12 media, FCS, and G418 were obtained from Life Technologies (Grand Island, NY). The hybridoma lines producing anti-human CD11a mAb (TS1/22, TS2/4) or anti-human CD18 mAb (TS1/18) were obtained from American Type Culture Collection (ATCC, Manassas, VA). Purified MEM-83 (anti-CD11a) and MEM-48 (anti-CD18) were purchased from Sanbio BV (Uden, Netherlands). All anti-ICAM-1 mAbs used in this study were generated and characterized by the authors (30). The mAbs 3D6 and 4E3 directed against epitopes within domain 1 of ICAM-1 have been shown to inhibit the ICAM-1/LFA-1 interaction in a previous study (30).
Production of chimeric forms of ICAM-1 (D1D2-IgG, D1D5-IgG)
Two chimeric soluble forms of ICAM-1, termed D1D2-IgG and D1D5-IgG, were prepared as previously described (30). Briefly, chimeric ICAM-1 was prepared by fusing either the first two Ig domains of ICAM-1 (D1D2: 1–185) or the five Ig domains of ICAM-1 (D1D5: 1–453) to the Fc portion (hinge, CH2, and CH3 domains) of human IgG1 (31) using conventional rDNA techniques. CHO-K1 cells were transfected with the vector pRc/CMV (Invitrogen, San Diego, CA) containing chimeric ICAM-1 cDNA using calcium-phosphate methods. Chimeric fusion proteins were purified from culture supernatants using protein A-Sepharose 4 Fast Flow (Pharmacia, Uppsala, Sweden).
Production and analysis of sLFA-1
sLFA-1 was purified from the culture supernatants of a stable line of sLFA-1-transfected CHO-K1 cells, as previously described (30). Fractions containing sLFA-1 were concentrated using Centriplus 100 microconcentrators (Amicon, Beverly, MA) and dialyzed against HBS(-) buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM MgCl2). Purified sLFA-1 was analyzed by SDS-PAGE and Coomassie blue staining. Protein concentrations were estimated by the AccQ-Tag amino acid composition analysis of acid-hydrolyzed protein samples according to the manufacturer’s instructions (Waters, Milford, MA) and by ELISA, as previously described (30).
Before BIAcore analysis, sLFA-1 (1.2 µg/ml) was further fractionated by gel filtration on a Superose 6 PC3.2/30 SMART column (Pharmacia) in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM MgCl2, and 0.05% Tween-20). The detection of eluted components was monitored by absorbance at 280 nm. The column elution positions of the fractionated sLFA-1 were compared with calibration standards (thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa) (Pharmacia) to determine the m.w.
Expression of membrane-bound LFA-1
Human CD11a (32) and CD18 (33) cDNA were cloned into the EcoRI site of pBluescript II SK (Stratagene, La Jolla, CA), as previously described (30). For the expression of the LFA-1 heterodimer, CD11a and CD18 cDNA fragments were subcloned into the expression vector containing the SV40 early promoter (30). These expression vectors and pSV2neo (ATCC) were then cotransfected into CHO-K1 cells using the calcium-phosphate method, and G418-resistant clones were selected. The expression of LFA-1 on the cell surface was determined by the flow-cytometric analysis on a FACScan (Becton Dickinson, Mountain View, CA) using TS1/22 and TS1/18. LFA-1 stably expressed on the surface of CHO-K1 cells has the same properties as naturally occurring LFA-1 on human leukocytes, e.g., SKW-3 and JY, in terms of both the binding activity to ICAM-1-expressing cells and the reactivity with mAbs directed against LFA-1. A stable line of LFA-1-expressing CHO-K1 cell was cultured in DMEM/F-12 media supplemented with 10% heat-inactivated FCS.
Purification of membrane-bound LFA-1
Membrane-bound LFA-1 (mLFA-1) was purified from LFA-1-transfected CHO-K1 cell lysates using immunoaffinity chromatography, as described by Dustin et al. (29) with modifications. The TS2/4 column (5 ml at 5 mg/ml) was prepared by covalently attaching TS2/4 to N-hydroxysuccinimide (NHS)-activated HiTrap (Pharmacia), according to the manufacturer’s instructions. After neutralization of the fractions from the TS2/4 column, samples were precleared with HiTrap-protein G (Pharmacia) and dialyzed against HBS buffer, following the addition of 0.05% Tween-20. The protein concentration was estimated by BCA protein assay (Pierce, Rockford, IL) and confirmed by AccQ-Tag amino acid composition analysis. The reactivity of mLFA-1 with mAbs was demonstrated by ELISA (30).
For the BIAcore analysis, mLFA-1 was fractionated by gel filtration on a Superose 6 HR10/30 FPLC column (Pharmacia) in HBS buffer. Fractions (0.5 ml) were collected at a flow rate of 0.1 ml/min. The column elution positions of the fractionated LFA-1 were compared with calibration standards, as already described. LFA-1 was quantitated by ELISA using MEM48 (anti-CD18) and TS2/4 (anti-CD11a).
BIAcore analysis
The interaction of LFA-1 with immobilized ICAM-1 was studied on
a BIAcore 2000 biosensor (Pharmacia Biosensor AB, Uppsala, Sweden). All
experiments were performed at 25°C. All of the proteins for injection
were dialyzed against HBS buffer and diluted with HBS buffer. To
immobilize D1D2-IgG to a CM5 sensor chip (Pharmacia Biosensor AB),
polyclonal goat anti-human IgG (
-chain) Ab (Zymed, San
Francisco, CA) was coupled to the sensor chip (about 11,000 RU) using
the amine-coupling kit (Pharmacia Biosensor AB), as described (34),
except that the Ab was injected at 50 µg/ml in 10 mM Na acetate (pH
4.5). After injection of D1D2-IgG at 50 µg/ml for immobilization via
the goat anti-human IgG Ab, LFA-1 was injected at a flow rate of 20
µl/min. The sensor surface was regenerated at the end of each
experiment with 10 mM HCl.
Analysis of the binding data in BIAcore
The analysis of kinetic data for LFA-1 binding to captured D1D2-IgG was performed using standard kinetic equations described by Karlsson et al. (34). The portion of the sensorgram that corresponds to the dissociation of sLFA-1 from immobilized D1D2-IgG was analyzed to obtain the dissociation rate constant (kdiss). Nonlinear curve fitting was conducted with the BIA evaluation 2.0 program (Pharmacia Biosensor AB). The association rate constant (kass) was determined by nonlinear curve fitting to the association phase data using the model of one site. Kd was calculated from the ratio kdiss/kass.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The interaction between LFA-1 and ICAM-1 was studied using a
soluble form of human LFA-1 (sLFA-1) and a chimeric molecule consisting
of the amino-terminal two Ig domains of human ICAM-1 (D1D2) fused to
the Fc portion (hinge, CH2, and CH3 domains) of
human IgG1 (D1D2-IgG) on a BIAcore biosensor. Specific binding of
sLFA-1 to D1D2-IgG was demonstrated by a solid-phase binding assay
(30). D1D2-IgG was indirectly immobilized on the sensor surface through
the covalently coupled goat anti-human IgG Ab, which binds the IgG
portion of D1D2-IgG. This has the advantage that all of the immobilized
chimeric ICAM-1 is present in the same orientation on the sensor
surface. When sLFA-1 was injected over the sensor surface with
D1D2-IgG, a large response was observed for sLFA-1 (Fig. 1
A). In contrast, sLFA-1
induced little or no response when it was injected over a control
sensor surface on which D1D2-IgG was not captured. Saturation of
immobilized D1D2-IgG with anti-ICAM-1 mAb, 3D6, which blocks sLFA-1
binding (30), resulted in a decrease in the response to the baseline
level, suggesting that the observed interaction between sLFA-1 and
D1D2-IgG on a BIAcore biosensor is specific. Sensorgrams
obtained in a typical experiment are overlaid in Figure 1B.
When sLFA-1 (250–500 nM) was injected over the sensor surface with
D1D2-IgG (300 RU), it increased the response in a dose-dependent
manner. Interaction of sLFA-1 with immobilized D1D2-IgG might be
multiphasic in that the plots of both the ln(RUo/RU) versus time and
the ln(abs(dRU/dt)) versus time do not give linear plots.
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The results in Figure 1
B suggest that there are at
least two types of binding activities in the sLFA-1 preparation: one
dissociates fast and the other dissociates slowly. The kinetic analysis
of the binding data indicated that association and dissociation of
sLFA-1 to immobilized D1D2-IgG were biphasic. It seemed likely that the
slow dissociation was due to the binding of aggregated sLFA-1. We,
therefore, performed the gel filtration of the sLFA-1 preparation to
separate the monomeric sLFA-1 and aggregated sLFA-1, and then
distinguished each response on a BIAcore biosensor. sLFA-1 was
fractionated into two peaks on Superose 6 (Fig. 2A). The main peak (Fig. 2A, peak 2) corresponded to monomeric sLFA-1, since it
eluted from the column in the size range expected for the monomeric
form of sLFA-1 (258 kDa). The expected m.w. of the shoulder peak (Fig. 2A, peak 1) was consistent with the molecular size of the
dimerized sLFA-1 (516 kDa).
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C) and simultaneously injected
over a control sensor surface with only goat anti-human IgG Ab to
distinguish specific reactions from nonspecific ones. When sLFA-1 from
the major peak (fr.10, peak 2) was injected over the
D1D2-IgG-immobilized surface (Fig. 2C), the component of
both the slow association and slow dissociation was reduced
significantly in comparison with the unfractionated sLFA-1 (Fig. 2B). The association rate constant
(kass) and the dissociation rate constant
(kdiss) for the reaction of the monomeric sLFA-1
were 2 x 105 M-1 s-1 and
1 x 10-1 s-1, respectively. The
equilibrium dissociation constant (Kd) for the
monomeric sLFA-1 was calculated to be 500 nM. Furthermore, the sLFA-1
in the minor peak (fr.8, peak 1) was bound with a high avidity, as
expected for a multimeric interaction (Fig. 2C). No binding
activity was detected from the other peak, which eluted from the column
later than the main peak, indicating that these were lower m.w.
contaminants. These results clearly indicated that the binding of
sLFA-1 to ICAM-1 is monophasic and that the multiphasic interaction of
sLFA-1 with D1D2-IgG was due to the presence of approximately 10%
multimeric sLFA-1 in the sLFA-1 preparation. Interaction of mLFA-1 with chimeric ICAM-1
To exclude the possibility that the truncation of the cytoplasmic
and transmembrane domain of LFA-1 affects the receptor-ligand
interaction, we repeated the BIAcore analysis using mLFA-1, a
full-length heterodimeric receptor. We purified mLFA-1 from CHO-K1
cells transfected with CD11a/CD18 by TS2/4 affinity chromatography. The
interaction of the immunoaffinity-purified mLFA-1 with the ICAM-1
chimera was analyzed using a BIAcore biosensor. D1D2-IgG was
immobilized on the sensor chip via the goat anti-human IgG Ab. When
immobilized D1D2-IgG was saturated with 3D6, the response was reduced
to almost the level seen in the control flowcell, whereas the
irrelevant Ab had no effect (Fig. 3A). Various concentrations of
mLFA-1 (100–200 nM) were injected over the surface, while regenerating
the surface at the end of each experiment. The overlay plot for the
mLFA-1 interaction with chimeric ICAM-1 at different concentrations of
mLFA-1 is shown in Figure 3B. The plot of the dissociation
phase from 300 to 600 s was calculated using the BIAcore software.
Dissociation is expressed as the natural log (ln) of the drop in
resonance units (RUo/RU). Association (0 to 300 s) is expressed as
the natural log (ln) of the absolute value of the rate of change of
resonance units (abs(dRU/dt)) (RUo, resonance units at beginning of
dissociation; RU, resonance units at the indicated time; abs, absolute
value). When the ln(RUo/RU) versus time and the ln(abs(dRU/dt)) versus
time were plotted, these graphs do not give linear plots. This result
indicates that the binding of mLFA-1 to the immobilized chimeric ICAM-1
might be multiphasic during both the association and dissociation
phases. We, therefore, analyzed binding of monomeric mLFA-1
fractionated by gel-filtration chromatography.
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A), while the purity of the
mLFA-1 was greater than 95%, as determined by Coomassie blue staining
of SDS-PAGE, indicating that mLFA-1 is heterogeneous in terms of
molecular size. To determine whether the multimerization of mLFA-1 was
critical for its binding characteristics to ICAM-1, we analyzed the
binding activity of each fraction containing mLFA-1 on a BIAcore
biosensor. Fractions containing mLFA-1 ranging from 250 to 300 kDa
apparent molecular mass, which correspond to monomeric mLFA-1, were
concentrated fivefold in Centricon 30 microconcentrators (Amicon), and
immediately tested for their ability to bind to chimeric ICAM-1 on a
BIAcore biosensor. As shown in Figure 4B, the
monomer-enriched fractions (fr.26, 27) showed a fast dissociation as
compared with the higher molecular size fractions (fr.20, 22). The
result was quite similar to that obtained by sLFA-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). The
monomeric peak of sLFA-1 was used for affinity and kinetic
measurements, suggesting that this study has estimated the true
affinity. Divalent cations such as Mg2+ regulate ligand interactions through selective binding to several sites on integrins and are thought to directly associate with the ligand binding site and control access to a cryptic binding site through altering the conformation of the integrin (38, 39). Although Mg2+ is directly involved in the affinity of LFA-1 for its ligand, Ca2+ correlates with avidity regulation of LFA-1 by clustering LFA-1 molecules at the cell surface of T cells, thereby facilitating LFA-1-ligand interaction (40). Other reports showed that Ca2+ inhibits Mg2+-induced T cell adhesion by inhibiting the expression of the Mg2+-induced 24 epitope on LFA-1 (41). Addition of 1 mM Ca2+ inhibited the binding of LFA-1 to immobilized chimeric ICAM-1 by 20% in our BIAcore analysis (data not shown). We, therefore, performed affinity and kinetic measurements in the absence of Ca2+, but in the presence of Mg2+ to induce high affinity state of LFA-1.
In the BIAcore analysis, sLFA-1 (250–500 nM) was bound to the
immobilized D1D2-IgG (300 RU), but little or no binding was observed
when ICAM-1 was not present on the sensor surface. Binding specificity
was clearly demonstrated by the fact that binding of sLFA-1 to
immobilized D1D2-IgG was completely inhibited when ICAM-1 on the sensor
surface was pretreated with 3D6 mAb that binds domain 1 of ICAM-1 and
blocks LFA-1 binding (Fig. 1A). In the association phase, a
fast association was observed at the very beginning of the reaction,
followed by a slow association phase, and the reaction did not reach
the equilibrium state during the injection period (Fig. 1B).
Since we showed that the sLFA-1 preparation contained approximately
10% of the dimeric sLFA-1 (Fig. 2A), the slow association
in the reaction might be due to the binding of sLFA-1 to D1D2-IgG as a
dimer with two binding sites to replace the sLFA-1 monomer binding to
the surface. It appears likely that the dimeric forms of sLFA-1 slowly
diffuse due to their higher molecular size. The binding activity of the
monomeric sLFA-1 was not likely to be detected in conventional binding
assays as a result of its kdiss value.
When we used the mAb affinity-purified mLFA-1, the multiphasic
association and dissociation steps were observed during the biosensor
kinetic analysis of the interaction between mLFA-1 and the chimeric
ICAM-1. However, mLFA-1 fractionated in lower molecular size fractions
have kinetics similar to that of the monomeric sLFA-1 (Fig. 4B). The monomer-enriched fractions of mLFA-1 dissociated
from the immobilized D1D5-IgG faster than the multimeric mLFA-1. We
confirmed that similar results were obtained with the
D1D2-IgG-immobilized surface (data not shown) and that D1D5 (D1D5-IgG)
has the same potency as D1D2 (D1D2-IgG) with binding to sLFA-1, as
previously described (30). We also tried to analyze the binding of D1D5
to mLFA-1 immobilized on the sensor surface indirectly using TS2/4 or
MEM48. Binding of monomeric D1D5 fractionated by size-exclusion
chromatography to immobilized mLFA-1 revealed rapid binding kinetics.
However, we could not analyze the interaction kinetically, because the
mLFA-1 baseline gradually decreased during the experiment as a result
of dissociation of mLFA-1 from the sensor surface (data not shown). The
interaction between the sICAM-1 and immobilized mLFA-1 has been studied
in a number of laboratories. Dissociation constants ranging from 100 nM
(35) to 130 nM (36) have been reported using conventional
receptor-binding assays. Lollo et al. showed that the affinity of LFA-1
for ICAM-1 on T cells activated by phorbol esters was approximately 400
nM (42). These values are of the same order of magnitude as the 500 nM
measured in this study. Surface plasmon resonance technology has a
great advantage in that we can analyze both the association phase and
dissociation phase of the interaction; thus, this is the first kinetic
analysis of the interaction of ICAM-1 with LFA-1 using a BIAcore
biosensor.
Recent studies have provided affinity and kinetic data on the
interactions of CD2 with its ligands CD48 (43) and CD58 (44, 45). These
studies have concluded that monomeric CD2 binds CD48 and CD58 with an
affinity in the 100 µM range. CD80 has been shown to bind CD28 with a
low affinity (Kd, 4 µM) and very fast kinetics
(kdiss 
1.6 s-1) (46). These
kinetic studies of cell-cell recognition molecules have revealed that
rapid binding kinetics may be a general feature of the molecular
interactions mediating cell-cell recognition. Nicholson et al. (47)
have shown that CD62L (L-selectin) binds immobilized GlyCAM-1
with a very low affinity (Kd, 108 µM) and a
very fast dissociation rate constant (10 s-1). The
extremely fast kdiss of CD62L/GlyCAM-1
interaction may have an influence on the duration of leukocyte tethers
and the velocity of leukocyte rolling. The affinity measured in the
present study for monomeric LFA-1 binding to ICAM-1 is much higher than
that measured for these adhesive interactions (Table I). Monomeric interaction of LFA-1 in
high affinity state with ICAM-1 has an affinity with 500 nM, while the
affinity measured for LFA-1 binding to ICAM-1 on unstimulated T cells
is very low, about 100 µM (42). As compared with CD62L, LFA-1 has
high affinity and forms long-lived bonds with ICAM-1, suggesting a
potential mechanism for firm adhesion. Stimulation of leukocytes with
physiologic stimuli or PMA induces clustering of LFA-1 as well as
conformational changes of LFA-1 itself into high affinity state.
Despite a slow dissociation rate constant of LFA-1, monomeric
interaction of LFA-1 would not be sufficient for firm adhesion.
Clustering of high affinity LFA-1 would induce cooperative interaction
of each molecule, increase in the avidity, and thus induce firm
adhesion.
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Footnotes |
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2 Abbreviations used in this paper: sLFA-1, soluble LFA-1; D1D2, domains 1–2 of ICAM-1; D1D5, domains 1–5 of ICAM-1; mLFA-1, membrane-bound LFA-1; RU, resonance unit; sICAM-1, soluble ICAM-1; GlyCAM-1, glycosylation-dependent cell adhesion molecule-1.
Received for publication January 14, 1998. Accepted for publication June 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits and a common ß-subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med. 158:1785.Lß2, CD11a/CD18) and MAC-1 (Mß2, CD11b/CD18). J. Biol. Chem. 270:94. subunit contains an ICAM-1 binding site in domains V and VI. ENBO J. 13:1790.[Medline]
subunit: an integrin with an embedded domain defining a protein superfamily. J. Cell Biol. 108:703. subunits. ENBO J. 8:3759.[Medline]
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