Red cell binding assays Indicated amounts of the purified CD11b/CD18, recom-binant CD11a and CD11b I domains or control proteins were coated on plastic 96-well plates Nunc, Roskilde, Denm
Trang 1Characterization of ICAM-4 binding to the I domains
of the CD11a/CD18 and CD11b/CD18 leukocyte integrins
Eveliina Ihanus1, Liisa Uotila1, Anne Toivanen1, Michael Stefanidakis1, Pascal Bailly2,
Jean-Pierre Cartron2and Carl G Gahmberg1
1
Department of Biosciences, Division of Biochemistry, University of Helsinki, Finland;2INSERM U76, Institut National de
Transfusion Sanguine, Paris, France
Intercellular adhesion molecule-4 (ICAM-4, LW blood
group antigen), a member of the immunoglobulin
super-familyexpressed on red cells, has been reported to bind to
CD11a/CD18 and CD11b/CD18 leukocyte integrins The
location of the ICAM-4 binding sites on CD11a/CD18 and
CD11b/CD18 are not known CD11/CD18 integrin I
domains have been found to act as major binding sites for
physiological ligands and a negatively charged glutamic acid
in ICAMs is considered important for binding ICAM-4
lacks such a residue, which is replaced byan arginine
However, we demonstrate here that ICAM-4 in red cells and
transfected fibroblasts interacts specificallywith the I
domains of CD11a/CD18 and CD11b/CD18 integrins The
binding was inhibited byanti-I domain and anti-ICAM-4
antibodies and it was dependent on divalent cations
Inter-estingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearlyreduced Using a solid phase assay, we were able to show that isolated I domains directlyand specificallybind to purified recom-binant ICAM-4 in a cation dependent manner Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized bythe ICAM-1 and ICAM-2 Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins
Keywords: adhesion; ICAM, integrin; I domain; red cell
The five intercellular adhesion molecules ICAM-1–5
cur-rentlyknown in humans, form a familyof related cell
surface glycoproteins, which mediate cell adhesion by
binding to the leukocyte CD11/CD18 integrins All the
ICAM proteins have extracellular C-type
immunoglobulin-like domains ranging in number from two to nine making
them members of the immunoglobulin superfamily[1–4]
Despite their structural similarityand integrin binding
capability, these proteins have a differential pattern of
expression and cellular distribution ICAM-1 which consists
of five Ig-like domains, is found on the surface of leukocytes,
endothelial cells and various other cells, and can be
up-regulated byseveral proinflammatorycytokines [5,6]
ICAM-2 has two Ig-like domains It is constitutively
expressed byleukocytes, endothelial cells [6], and platelets
[7] ICAM-3 is composed of five Ig-like domains, and it is
present at high levels on resting lymphocytes, monocytes,
and granulocytes It is the only ICAM significantly
expressed on neutrophils [8] The expression of ICAM-4 is restricted to erythrocytes and erythroid precursor cells [9] ICAM-5 is expressed bysubsets of neurons, exclusively within the telencephalon of the mammalian brain [10] The predominant cellular ligands for the ICAMs are the leukocyte CD11/CD18 integrins, which consist of four heterodimeric glycoproteins with specific a chains (CD11a, -b, -c, -d) and a common b2chain (CD18) Theyplayan essential role in mediating adhesion of cells in the immune system [1–4] All five ICAM molecules are able to bind to CD11a/CD18 (LFA-1, aLb2) which is expressed on all leukocytes The first NH2-terminal Ig domain of each ICAM seems to be most important for binding [11–15] ICAM-1, -2 and -4 have been shown to interact also with CD11b/CD18 (Mac-1, aMb2, CR3), which is expressed primarilyon the cells of the myelo-monocytic lineage The third Ig-like domain in ICAM-1 [16] and the first NH2 -terminal Ig domain in ICAM-2 seem to mediate CD11b/ CD18 binding [12]
Extensive work has been carried out to localize the ligand binding sites in the leukocyte b2integrins The a chains of the CD11/CD18 integrins contain an inserted approxi-mately200-amino-acid intervening domain (I domain), which is homologous to the A domains of von Willebrand factor, repeats in cartilage matrix protein and collagen [17] The I domains of CD11/CD18 integrins have been shown
to contain recognition sites for most ligands of leukocyte integrins ICAM-1, -2 and -3 as well as several soluble proteins such as fibrinogen, and the complement compo-nent iC3b bind to the I domains of their receptor integrins
Correspondence to C G Gahmberg, Department of Biosciences,
Division of Biochemistry, P.O Box 56, Viikinkaari 5,
FIN-00014 Universityof Helsinki, Finland.
Fax: + 358 9 191 59068, Tel.: + 358 9 191 59028,
E-mail: Carl.Gahmberg@helsinki.fi
Abbreviations: ICAM, intercellular adhesion molecule; VCAM,
vascular cell adhesion molecule; CD11a/CD18, LFA-1, leukocyte
function associated antigen; CD11b/CD18, Mac-1; LW, Landsteiner–
Wiener blood group antigen; GST, glutathione S-transferase.
(Received 14 January2003, accepted 20 February2003)
Trang 2Byusing monoclonal antibodies reacting with integrin
I domains and bymutational analysis of the I domains,
evidence has been obtained that the binding sites on the
I domains for different ligands are overlapping but not
identical [18–22] Integrins need divalent cations for their
activity, and they have been shown to bind Ca2+and Mg2+
[23,24] Importantly, the I domains have been shown to
bind divalent cations [20,24]
ICAM-4 was originallyidentified as a 42-kDa red cell
membrane glycoprotein called the LW (Landsteiner–
Wiener) blood group antigen [25] The LW protein has
been reported to require intramolecular disulfide bonds
and the presence of divalent cations, notablyMg2+, for
antigenic activity[26] The LW and Rh blood groups
show an interesting phenotypic relationship, as the level of
LW expression is greater in positive than in
RhD-negative cells, and extremelyrare Rhnull cells, which lack
all Rh antigens, are also deficient in the LW protein
However, individuals lacking LW antigens have been
found among RhD-positive individuals [27] The LW
glycoprotein has been renamed ICAM-4 based on strong
sequence similarities with the ICAM familyand
subse-quent work, which showed that it binds CD11a/CD18
and CD11b/CD18 integrins [9,28] The ICAM-4 protein
contains two immunoglobulin domains of which the first
domain is 30% identical to the first domains of ICAM-1,
-2 and -3 CD11a/CD18 has been found to bind to the
first Ig domain, whereas CD11b/CD18 binding sites
encompass both domains of ICAM-4 [14] Unlike the
other ICAMs, ICAM-4 does not contain the conserved
glutamate residue in the first domain, which is replaced by
an arginine 52 residue (Table 1) Mutation of arginine 52
back to glutamate did not affect CD11a/CD18 binding
and even reduced the interaction with CD11b/CD18
Instead, site-directed mutagenesis studies identified other
residues on the CFG face of the first domain, which are
involved in CD11a/CD18 recognition These data suggest
that the b2integrin binding motifs of ICAM-4 differ from
those of other ICAMs [14] However, the adjacent
residues at arginine 52 in ICAM-4 are identical or similar
in the five ICAMs, suggesting a potential role for these
residues The noncharged LLG sequence present on
ICAM-1 has been reported to function as a
ligand-binding motif for CD18 integrins [29] A recent studyhas
suggested that ICAM-4 can also bind through novel
motifs to a4b1 and avfamilyintegrins [30]
In the present study, we wanted to define the role of the
CD11a and CD11b I domains in ICAM-4 binding Our
results show that ICAM-4 binds specificallyto the CD11a
and CD11b I domains
Materials and methods
Antibodies The b2integrin specific mAbs used in these studies include TS1/22, MEM83, MEM30, MEM25, MEM177, 7E3, 60.1, LM2/1, MEM170, 44, 107 and 904 TS1/22 (American Type culture Collection, Rockville, MD), MEM83, MEM30, MEM25 and MEM177 recognize the a chain of CD11a/CD18 TS1/22, MEM83, MEM30 and MEM25 has been mapped to the I domain of the CD11a/CD18 [31,32] The anti-CD11b mAbs 7E3, 60.1, LM2/1 (American Type culture Collection, Rockville, MD), MEM170, 44, 107 and
904 have been described previously[33,34] and are specific for the I domain The ICAM-1 antibodies have been described: GP8911, GP8914 and GP8923 (the Leukocyte Typing Workshop V), LB-2 [35] and RR1/1 [36] The ICAM-1 mAb B-H17, was a generous gift from C Vermot-Desroches, Diaclone, France The three ICAM-2 mAbs (B-T1, B-R7 and B-S9) have been described previously[37] The mAbs BS46 and BS56 react with the first domain of the ICAM-4 [14,38] A mouse IgG1 negative control was purchased from Silenius (Hawthorn, Australia) and poly-clonal goat anti-GST antibodies from Pharmacia Biotech Inc The goat antihuman IgG specific antibodywas obtained from Sigma and the peroxidase-conjugated anti-GST mAb from Santa Cruz Biotechnology(Santa Cruz, CA)
Purification of the CD11a/CD18 and CD11b/CD18 integrins
CD11a/CD18 and CD11b/CD18 integrins were purified from human blood buffycoat cell lysates as described previously[12]
Expression and purification of the CD11a and CD11b I domains
The Escherichia coli strain JM109 transformed with the pGEX-2T (Pharmacia Biotech Inc.) plasmid containing the cDNA fragment of the CD11b I domain was a generous gift from A Arnaout (Massachusetts General Hospital, Boston, MA) [20] The cDNA fragment of CD11a I domain inserted into the expression vector pGEX-5X-3 (Pharmacia Biotech Inc.) was kindlyprovided byD Altieri and was constructed as reported byMuchowski et al [39] The pGEX-5X-3 plasmid construct containing the cDNA of CD11a I domain was transformed into the E coli strain BL21 The glutathione S-transferase fusion proteins of the
I domains were expressed in Escherichia coli cells (strain BL21 for CD11a I domain; strain JM109 for CD11b
I domain) as described previously[20,39] Cell pellets derived from 1 to 4 L culture were thawed and lyzed by resuspending in 30 mL of lyzing buffer (10% sucrose, 0.5% Triton X-100, 50 mM Tris, pH 8.0) containing 5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 lgÆmL)1aprotinin and leupeptin After lysozyme (350 lgÆmL)1) treatment the cells were sonicated on ice Triton X-100 was then added to a final concentration of 1%, and the sonication was repeated After centrifugation
at 12 000 g for 10 min, the supernatant was incubated with
Table 1 Sequence alignments of the first immunoglobulin domains of
ICAM molecules illustrating the glutamate to arginine (shown in bold)
difference in ICAM-4 and the surrounding conserved residues (shown as
italic).
Trang 3prewashed glutathione-Sepharose 4B (Pharmacia Biotech,
Uppsala, Sweden) for 2 h at 4C The resin was then
washed with 50 mL of 50 mMTris/HCl, pH 8.0, 50 mL of
500 mMNaCl, 1% Triton X-100, 50 mMTris/HCl, pH 8.0
and again with 50 mL of 50 mM Tris/HCl, pH 8.0 The
fusion proteins were eluted with 20 mM reduced
gluta-thione, 50 mMTris/HCl, pH 8.0 and the samples were then
passed through a Bio-Gel P-6DG column to remove
glutathione
For the experiments, which utilized the I domains
separated from the GST moiety, the purified GST fusion
proteins of the I domains of CD11a/CD18 and CD11b/
CD18 were cleaved with Factor Xa and thrombin,
respect-ively The GST fusion protein of CD11a I domain was
cleaved with biotin-labeled restriction protease Factor Xa
(Boehringer Mannheim) The biotin-labeled Factor Xa was
removed using a streptavidin gel and the free GST using the
glutathione resin To release the recombinant CD11b
I domain the fusion protein was treated with thrombin
(Sigma) and the cleaved sample was then further purified by
ion exchange chromatographyon a Mono S HR5/5 column
(Pharmacia) using the FPLC system (Pharmacia) Analysis
of the purified recombinant I domains on 12% SDS/PAGE
revealed a single band of the expected size after staining with
Coomassie blue
Expression and purification of ICAM-Fc recombinant
proteins
The ICAM-2Fc and ICAM-4Fc fusion proteins were
produced bytransient transfection of COS-1 cells bythe
DEAE-dextran method (Pharmacia) and isolated from the
culture supernatants byprotein A-Sepharose CL-4B
(Phar-macia) chromatographyessentiallyas previouslydescribed
[14,40] ICAM-1Fc and VCAM-1Fc fusion proteins were
obtained from R & D Systems All the recombinant
proteins were checked bySDS/PAGE and Western blotting
The ICAM-2Fc cDNA vector was kindlyprovided byD
Simmons (John Radcliffe Hospital, Oxford, UK) and the
ICAM-4Fc cDNA has been described previously[14]
Cells and cell lines
Blood samples from common LW and Rh phenotypes
(ICAM-4 positive red cells) were obtained from normal
volunteers using heparin as an anticoagulant and the
LW(a–, b–) blood sample (ICAM-4 negative red cells) was
kindlyprovided byKathyBurnie (HematologyUniversity
Hospital, Ontario, Canada) The LW(a–, b–) cells and the
control cells were stored at)70 C until further used
The L929 mouse fibroblast cell line was maintained in
IMDM medium supplemented with 10% fetal bovine serum,
100 UÆmL)1penicillin, and 100 lgÆmL)1streptomycin The
full-length ICAM-1 or ICAM-2 cDNAs in pEF-BOS vector
and the full-length ICAM-4 cDNA in pcDNA I vector were
separatelycotransfected with pCDM8-neo stuffer into L929
mouse fibroblast cells according to standard procedures
using either the Lipofectamine reagent kit (Life
Techno-logies, Gaithersburg, MD) or the calcium phosphate
preci-pitation method Stable transfectants were selected in
medium containing 0.5 mgÆmL)1G418 The G418-resistant
cell populations were analyzed for ICAM expression with a
Becton Dickinson (Immunocytometry Systems, San Jose, CA) FACScan flow cytometer The L929 cells expressing either ICAM-1, ICAM-2 or ICAM-4 were cloned bylimiting dilution The SV40-transformed African green monkey kidneycell line COS-1 (ATCC) was grown in DMEM supplemented with 10% fetal bovine serum, 100 UÆmL)1 penicillin, and 100 lgÆmL)1streptomycin
Flow cytometry studies Wild type, ICAM-1-, ICAM-2-, or ICAM-4-transfected
L cells and ICAM-4-positive or -negative red cells were washed and resuspended in NaCl/Pi, pH 7.4 Aliquots of
1· 106L cells or 1–3 lL of packed red cells were incubated with 25 lgÆmL)1of different anti-ICAM mAbs for 30–60 min on ice The cells were washed with NaCl/Pi and incubated with FITC-conjugated rabbit antimouse F(ab¢)2(Dakopatts a/s, Copenhagen, Denmark) for 30 min
on ice After washing, 1· 104cells were analyzed with a Becton Dickinson (Immunocytometry Systems, San Jose,
CA, USA) FACScan flow cytometer
Red cell binding assays Indicated amounts of the purified CD11b/CD18, recom-binant CD11a and CD11b I domains or control proteins were coated on plastic 96-well plates (Nunc, Roskilde, Denmark) in 25 mM Tris, pH 8.0, 150 mM NaCl, 2 mM MgCl2 byincubation overnight at 4C The wells were blocked with 1% BSA for 2 h at room temperature and washed ICAM-4 positive or negative red cells (0.7· 106per well) in binding buffer (RPMI 1640 supplemented with 50 mM Hepes, pH 7.4, 2 mM MgCl2, 2 mM CaCl2, and 5% fetal bovine serum) were added to the wells and the plates were then brieflycentrifuged (900 g, 2· 1 min) and incubated for 2 h at 37C The input of red cells was quantitated bycounting cells in four randomly chosen fields from duplicate wells To remove non-adherent cells, the wells were gentlyfilled with binding buffer, and the microplate was placed floating upside down for 40 min in NaCl/Pi solution before microscopic observation and scoring the number of attached cells in four randomlychosen fields from duplicate wells The data was presented as a percentage of bound cells (amount of bound red cells divided byinput of cells) For blocking experiments, the cells or protein-coated wells were pretreated with different mAbs (25 lgÆmL)1) or soluble GST/I domain GST (0.2–3 lM) for 10 min at room temperature before starting the adhesion For the binding studywith or without divalent cations, the adhesion assays were performed with buffers containing 4 mM EDTA,
4 mMEGTA and 2 mMMgCl2, or 2 mMMgCl2and 2 mM CaCl2 The results of antibodyinhibition assays were expressed as a relative percentage of attached cells, where 100% is given as the number of cells bound in the absence of inhibitors The significance was determined byunpaired Student’s t-test
Adhesion assays of ICAM transfectants The control protein glycophorin A (1 lg per well) and the purified CD11a/CD18 (1 lg per well) and CD11b/CD18
Trang 4(0.2 lg per well) were diluted in 25 mM Tris, pH 8.0,
150 mMNaCl, 2 mMMgCl2and attached to flat-bottom,
96-well microtiter plates (Nunc, Roskilde, Denmark) in
0.01–0.1% n-octyl glucoside by overnight incubation at
4C The wells were blocked with 1% BSA for 2 h at
room temperature For the experiments with coated GST
I domains the anti-GST antibodies (Pharmacia Biotech
Inc.) diluted in 25 mMTris, pH 8.0, 150 mMNaCl, 2 mM
MgCl2 were adsorbed overnight at 4C, at 0.2–1 lg per
well (50 lL per well in triplicate) on flat-bottom, 96-well
microtiter plates After blocking nonspecific sites,
indica-ted amounts (0.4–2 lg per well) of GST or recombinant
purified GST I domains were added to the wells and
incubated for 2 h at room temperature Wells were then
washed three times with the binding medium (Iscove’s
MDM with 50 mM Hepes, pH 7.4, 0.5% BSA, 2 mM
MgCl2 and 2 mM CaCl2) prior to addition of the cells
(1.5· 105 per well, in the binding medium unless
other-wise indicated) For blocking experiments, the cells or
protein-coated wells were pretreated with different mAbs
(25 lgÆmL)1) or soluble GST/I domain GST (0.2–4 lM)
for 10 min at room temperature After 20 min incubation
at room temperature the wells were filled with binding
buffer, and the microplate was put to float upside down
for 1 h in NaCl/Pisolution to remove unbound cells The
bound cells (or the total amount of added cells without
washing) were lyzed in 100 lL per well phosphatase
substrate-containing lyzis buffer (1% Triton X-100 and
50 mM sodium acetate, pH 5.0) and incubated at 37C
for 30 min The reaction was terminated byadding 50 lL
per well of 1MNaOH and the absorbance at 405 nm was
measured [41]
Solid phase ELISA assay
The 96-well plates (Greiner) were coated overnight at 4C
with 400 ng per well of goat antihuman IgG Fc specific
antibody(Sigma) in 50 mM Tris/HCl, pH 7.4, 150 mM
NaCl (TBS) After blocking nonspecific sites with 3%
BSA in TBS for 1 h at 37C the wells were washed three
times with TBS, 1 mMCaCl2, 1 mMMgCl2,0.05% Tween
20, 1% BSA The recombinant proteins (200 ng per well)
diluted in TBS, 1 mMCaCl2, 1 mMMgCl2, 1% BSA was
then added to the wells and incubated for 2 h at room
temperature The wells were washed as before, and 50 lL
per well I domain GST or control GST (0–20 lgÆmL)1)
diluted in TBS, 1 mMCaCl2, 1 mMMgCl2, 1% BSA was
added and incubated at room temperature for 2 h Plates
were washed gentlythree times with TBS, 1 mM CaCl2,
1 mM MgCl2, 0.05% Tween 20, 1% BSA prior to the
addition of a peroxidase-conjugated anti-GST mAb
(1 : 500 dilution, Santa Cruz Biotechnology) to the wells
After 1 h of incubation at 37C, the plates were washed
as before, and the bound proteins were detected with
100 lL per well 0.5 mgÆmL)1o-phenylenediamine
dihydro-chloride added for 10 min, stopped byaddition of 50 lL
of 12.5% H2SO4 and plates were read in an ELISA
reader For inhibition experiments, the soluble GST
fusion proteins or protein-coated wells were pretreated
with different mAbs (20–40 lgÆmL)1) or inhibitor proteins
(ICAMFc proteins, 200 nM; I domains, 1 lM) diluted in
TBS, 1 mMCaCl, 1 mMMgCl, 1% BSA for 10 min at
room temperature before the addition of the I-GST protein to the wells
Results
Purification of CD11/CD18 integrins, recombinant
I domains and ICAMFc proteins The purified CD11a/CD18 and CD11b/CD18 preparations were analyzed by SDS/PAGE and no major impurities were observed (not shown) In contrast to the CD11b I domain GST, the majorityof the CD11a I domain GST was found
in the insoluble fraction of the E coli lyzate Modification
of the solubilization conditions and increasing the volumes
of the bacterial cultures yielded large enough amounts of soluble CD11a I domain GST The purified I domain GST fusion proteins migrated as major bands of 50 kDa After Factor X or thrombin cleavage and removal of GST the
I domains appeared as single major bands of approximately
24 kDa (CD11a) and 26 kDa (CD11b) The purities of ICAM-1Fc, ICAM-2Fc and ICAM-4Fc fusion proteins were checked bySDS/PAGE The preparations contained the expected recombinant proteins and the purityof the proteins was >90% (not shown)
Binding of red cells to purified I domains The surface expression of ICAMs on ICAM-4 positive, LW(a+, b–), and ICAM-4 negative, LW(a–, b–), red cells was studied byflow cytometry The onlyICAM expressed on LW(a+, b–) cells was ICAM-4, while none
of the ICAMs were found on LW(a–, b–) cells (not shown)
Byperforming cell adhesion assays with the recombinant
I domains, we showed that red cells readilybound to recombinant I domains of both CD11a and CD11b in a concentration-dependent manner (Fig 1A,C) The binding
of ICAM-4 positive erythrocytes to CD11a I domain was effectivelyblocked bymonoclonal antibodies to ICAM-4 and the CD11a I domain (Fig 1B) The mAb 7E3 and the mAbs BS46 and BS56 to ICAM-4 partiallybut significantly inhibited the interaction between ICAM-4 positive red cells and recombinant CD11b I domain (Fig 1D) The ICAM-4 negative red cells retained most or all binding activityto the recombinant CD11b I domain (Fig 1D) The adhesion of these cells was affected neither bymAbs to ICAM-4 nor the activation-dependent mAb 7E3 [42] However, the adhesion
of both ICAM-4 positive and negative erythrocytes to coated CD11b I domain was almost completelyabrogated
in the presence of mAb 60.1, which binds to the recombin-ant CD11b I domain These data indicate that there might
be an unidentified ligand in red cells that could mediate binding to CD11b and probablyalso to CD11a
CD11b I domain inhibits red cell binding to purified integrin
Red cells bind poorlyto CD11a/CD18 but more efficiently
to CD11b/CD18 [28] Therefore, we tested inhibition of erythrocyte binding to CD11b/CD18 by purified CD11b
I domain GST Figure 2 shows that half maximal reduction
of cell adhesion was achieved at 0.2 lMI domain GST
Trang 5Interaction between ICAM transfectants and purified CD11a/CD18 and CD11b/CD18 integrins
To obtain further evidence that ICAM-4 binds to CD11a/ CD18 and CD11b/CD18 through the I domains we generated stable mouse L cell transfectants expressing recombinant human ICAM-4 Several ICAM-4 transfect-ant clones were obtained and the ones expressing high levels
of ICAM-4 and strong binding to purified CD18 integrins were chosen for further adhesion assays The stable L cell transfectants expressing human ICAM-1 and ICAM-2 have been established as previouslydescribed [41] As expected, the ICAM transfectants reacted onlywith the corresponding ICAM mAb (Fig 3A) None of the trans-fectants reacted with mAbs to ICAM-3 or ICAM-5 (not shown)
Using purified CD11a/CD18 and CD11b/CD18, we studied the binding of ICAM transfectants to the integrins coated on plastic (Fig 3B) All the ICAM transfectants, but not the wild-type L cells adhered to the coated integrins, but not much to the control protein We found that the ICAM-4 tranfectants could bind much more efficientlyto CD11b/CD18 than to CD11a/CD18 coated on plastic wells (approximately60% and 15% of the total added cells, respectively) Furthermore, the ICAM-4 transfectants adhered more stronglyto CD11b/CD18 than ICAM-1 and ICAM-2 transfectants Approximately30% of these other two ICAM transfectants bound to CD11b/CD18
As the expression levels of ICAM-4 and ICAM-2 L cell
Fig 1 Binding of red cells to purified CD11a/CD18 and CD11b/CD18 I domains (A) Indicated amounts of CD11a/CD18 I domain (j) or glycophorin A (GPA) (d) were coated per well (B) Shows the effect of anti-ICAM-4 (BS46 and BS56) and anti-CD11a I domain (TS1/22 and MEM83) mAbs on the binding of ICAM-4 positive and negative red cells to 1 lg of coated CD11a/CD18 I domain (C) Wells were coated with indicated amounts of CD11b/CD18 I domain (j) or ICAM-3 (d) (D) The effect of antibodies on binding of red cells to 0.4 lg of coated CD11b/ CD18 I domain was studied (anti-CD11b I domain mAbs: 7E3 and 60.1) The data in A and C is presented as a percentage of attached cells (amount of bound cells divided byinput of cells) The amounts of bound and added red cells were quantitated bycounting cells in four randomly chosen fields from duplicate wells The results in B and D are expressed as a relative percentage of bound cells, where 100% is calculated from the total number of ICAM-4 positive red cells bound to the I domain in the absence of pretreatment with mAbs Controls included unrelated mouse IgG antibody(not shown) and wells with coated control protein (GPA) or without coated protein (BSA only) The experiments were repeated 3–5 times with similar results Data are expressed as mean ± SD and statistical significances are shown.wwwP < 0.001, wP < 0.1.
Fig 2 Inhibition of red cell adhesion to purified CD11b/CD18 by
the CD11b I domain GST The binding of erythrocytes to coated
purified CD11b/CD18 in the presence of indicated concentrations of
soluble CD11b I domain GST (s) or GST (m) are shown
Back-ground binding of cells to BSA was substracted The data is presented
as a percentage of attached cells The amounts of bound and added red
cells were quantitated bycounting cells in four randomlychosen fields
from duplicate wells The experiment was repeated three times with
similar results.
Trang 6transfectants were clearlylower than ICAM-1 transfectants,
these results suggest that ICAM-4 might be an even more
potent ligand for CD11b/CD18 than the other two ICAMs
To a certain extent (10–20%) the binding efficiences to
ICAM-4 varied between different preparations of CD11b/
CD18 integrins The mAbs to CD11a/CD18, CD11b/CD18
and ICAMs clearlyinhibited the binding of ICAM L cells
to coated CD18 integrins (data not presented)
To examine the role of I domains in ICAM-4 binding in
more detail we tested the abilityof I domain GST fusion
proteins to block the interaction of ICAM transfectants
with purified CD11a/CD18 and CD11b/CD18 integrins
(Fig 4) Theyefficientlyinhibited the adhesion of ICAM
transfectants to CD11a/CD18 and CD11b/CD18 integrins
The adhesion of ICAM-4 transfectants to CD11a/CD18
was inhibited bythe CD11a I domain GST more efficiently
as compared to ICAM-1 and ICAM-2 transfectants The
inhibition of ICAM-4 transfectant binding to CD11a/CD18
bysoluble CD11a I domain GST was
concentration-dependent and 50% inhibition was obtained with an
inhibitor concentration of 0.4 lM The soluble CD11b
I domain GST was a less active inhibitor of ICAM-4
transfectant adhesion to coated CD11b/CD18 integrin However the binding of all ICAM transfectants was reduced to 50–60% in the presence of 1 lM CD11b
I domain GST
Adhesion of ICAM transfectants to purified I domain fusion proteins
The I domains of CD11a and CD11b contain binding sites for ICAM-1 and ICAM-2 Further proof for the interaction
of ICAM-4 with these I domains was obtained bycompar-ing the abilityof recombinant I domain fusion proteins to support the adherence of L cell transfectants expressing ICAM-1, ICAM-2 or ICAM-4 For our assays we immo-bilized I domain GST fusion proteins via goat anti-GST antibodies which presumablyallowed the I domain GSTs
to be presented in favourable orientations and caused more effective binding of the cells As shown in Fig 5, the
I domain fusion proteins supported the binding of all three different ICAM transfectants in a concentration-dependent manner For this particular lot of I domains, 0.3–0.8 lg in solution used to coat the wells resulted in good adherence of ICAM L cell transfectants and low background binding of wild-type L cells After substraction of background binding, approximately30% of the total added ICAM-1 L cells adhered to the I domain of CD11a, while approximately 20% of ICAM-2 and ICAM-4 L cells did so However, at high levels of CD11b I domain fusion proteins, wild-type
L cells were adherent as well, a phenomenon noticed also by others for wild-type CHO cells [43] The results indicate that the I domain of CD11b interacts not onlywith ICAMs but also with an unknown receptor on L cells This interaction with an L cell receptor is not unique for the recombinant CD11b I domain, because wild-type L cells also adhered to high levels of purified CD11b/CD18 integrin (data not shown)
Effects of antibodies on binding of ICAM transfectants
to purified I domain fusion proteins For further study, we investigated the effects of different mAbs on the interaction of ICAM L cell transfectants with I domain GST fusion proteins of CD11a and CD11b (Figs 6 and 7) The CD11a I domain specific TS1/22 mAb efficientlyinhibited the binding of ICAM-1 and -2 transfected L cells to the I domain of CD11a, and partiallybut significantlyinhibited the interaction between ICAM-4 L cells and CD11a I domain The adhesion of all ICAM transfectants to the I domain of CD11a was almost completelyblocked bythe anti-CD11a mAb MEM83 down to GST background level (Fig 6) In an ELISA assayboth anti-CD11a mAbs (TS1/22 and MEM83) and all the I domain specific anti-CD11b mAbs (LM2/1, MEM170, 60.1, 44a, 107 and 904) reacted with the corresponding I domain GST fusion proteins immobilized via goat anti-GST antibodies (not shown)
Pretreatment of the coated CD11b I domain fusion protein with the I domain specific mAbs resulted in efficient inhibition of the adherence of all three different types of ICAM L cells, except for mAb 904 which had no effect on binding of ICAM-2 transfectants (Fig 7)
Fig 3 Cell surface expression of ICAM-1, ICAM-2 and ICAM-4 on
L cell transfectants and adhesion assay using purified CD11a/CD18 and
CD11b/CD18 integrins (A) Parental L cells (dotted line) and L cells
transfected (dark line) with ICAM-1, ICAM-2 and ICAM-4 cDNAs
were stained with mAbs anti-ICAM-1 (LB-2), anti-ICAM-2 (B-T1),
and anti-ICAM-4 (BS46) followed byFITC-rabbit antimouse IgG
F(ab¢) 2 fragments and were analyzed by flow cytometry (B) Cell
adhesion of parental L cells and ICAM transfectants to CD11a/CD18,
CD11b/CD18 and GPA proteins coated on plastic wells The
experi-ments were repeated three times with similar results.
Trang 7Fig 4 Inhibition of adhesion of ICAM transfectants to purified CD18 integrins by corresponding recombinant I domain GST The binding of ICAM transfectants to plastic coated purified CD11a/CD18 or CD11b/CD18 in the presence of indicated concentrations of soluble CD11a I domain GST
or CD11b I domain GST (s) or GST (m) are shown Background binding of cells to BSA was substracted The results are expressed as a relative percentage of bound cells, where 100% is given as the total number of cells bound to the CD18 integrins in the absence of soluble competitors (Materials and methods) The experiments were repeated three times with similar results.
Fig 5 Adhesion of ICAM tranfectants to purified CD11a and CD11b I domain GST fusion proteins Indicated amounts of anti-GST antibodies were coated per well After blocking with BSA, GST fusion proteins of I domains or purified GST were added in amount twice of the anti-GST Both binding of parental L cells to GST I domains (s) or GST (e) and binding of ICAM transfectants to GST I domains (j) or GST (d) are shown The data are presented as a mean percentage of attached cells (amount of bound cells divided byinput of cells) ± SD.
Trang 8Several different ICAM-1 mAbs were tested in cellular
adhesion assays and variable degrees of inhibition were
detected The most efficient inhibition of the binding of
ICAM-1 L cell transfectants to the I domains was
obtained with the ICAM-1 mAb LB-2 reacting with the
first domain of ICAM-1 and the mAbs GP8914 and
GP8923 which have been mapped to domains 4 and 5 of
the ICAM-1 molecule [44] None of these anti-ICAM-1
mAbs showed inhibitoryeffects on adhesion of ICAM-2
or ICAM-4 L cells MAbs to ICAM-2 and ICAM-4
inhibited the binding of ICAM-2 and ICAM-4 L cells,
respectively The three ICAM L cell transfectants used in
the cell adhesion assays were stained with all the above
mentioned ICAM mAbs and theyreacted onlywith the
mAbs to transfected ICAM (data not shown)
Divalent cation requirements for ICAM/b2integrin interaction
Divalent cations mayhave multiple effects on integrin-mediated cell adhesion including enhancement, suppres-sion, and modification of ligand binding activity We have previouslyshown that Ca2+and Mg2+are needed for the maximal binding of CD11a/CD18 and CD11b/ CD18 to ICAM-4 [14,28] Here we have investigated the effect of divalent cations on the binding of ICAM-4 transfectants to the I domains of CD11a and CD11b and compared the results to the divalent cation requirements
of ICAM-1 and ICAM-2 transfectants We also analyzed the cation dependence of red cell adhesion to the
I domains
Fig 6 Inhibition of adhesion of ICAM
trans-fectants to purified CD11a I domain GST
fusion protein The effects of anti-ICAM and
anti-I domain mAbs on adhesion of ICAM
transfectants to 0.4 lg of I domain GST
captured with 0.2 lg of coated anti-GST Ab.
Controls include wells with captured GST and
the binding of wild-type L cells Background
binding of cells to BSA was substracted The
results are expressed as a mean relative
per-centage of bound cells, where 100% is given as
the total number of cells bound to the I
domain in the absence of pretreatment with
mAbs The experiments were repeated 3–5
times with similar results Standard deviations
and statistical significances are shown.
wwwP < 0.002, wwP < 0.02, wP < 0.2.
Fig 7 Inhibition of adhesion of ICAM
trans-fectants to purified CD11b I domain GST
fusion protein The effects of anti-ICAM and
anti-I domain mAbs on adhesion of ICAM
transfectants to 0.4 lg of I domain GST
captured with 0.2 lg of coated anti-GST Ab.
Controls include wells with captured GST and
the binding of wild-type L cells Background
binding of cells to BSA was substracted The
results are expressed as a mean relative
per-centage of bound cells, where 100% is given as
the total number of cells bound to the I
domain in the absence of pretreatment with
mAbs The experiments were repeated three to
five times with similar results Standard
devi-ations and statistical significances are shown.
wwwP < 0.002, wwP < 0.02, wP < 0.2.
Trang 9The most efficient binding of all the ICAM L cell
transfectants was observed in the presence of Mn2+
(Fig 8) In the absence of cations, the binding of ICAM-1
transfectants to the CD11a I domain fusion protein and the
binding of ICAM-2 transfectants to both I domain fusion
proteins were completelyabrogated In the presence of
EDTA the adhesion of ICAM-4 transfectants to both
I domains was efficiently, but not totally abolished, as was
also the binding of ICAM-1 transfectants to the CD11b
I domain fusion protein The inhibitoryeffects of EDTA
were clearlysignificant As can be seen in Fig 8, MgCl2
alone or in combination with CaCl2supported the
interac-tion of ICAM-1 L cells with both I domains and ICAM-2
L cell adherence to the I domain of CD11a However, the
presence of both MgCl2and CaCl2seems to be required for
high affinitybinding of ICAM-2 L cell transfectants to the
I domain of CD11b as well as for adhesion of ICAM-4
transfectants to both I domains CaCl2 alone was not
sufficient to support the maximal binding of anyof the
ICAM transfectants to the I domain fusion proteins
Figure 9 shows that the presence of Mg2+and Ca2+is needed for efficient adhesion of red cells to the I domains of CD11b and CD11a, while in the absence of Ca2+, chelated using EGTA, the binding of red cells was partiallybut significantlyinhibited A clear reduction of binding was observed if divalent cations were omitted (not shown) or when EDTA was included in the reaction mixture MnCl2 supported the binding of red cells to the I domains efficiently(not shown)
Characterization of isolated I domain GST binding
to ICAM-4Fc using a solid phase ELISA assay The binding of ICAM-4 directlyto CD11a and CD11b
I domains was further investigated in a cell-free assay (Figs 10 and 11) The specificityof the adhesion in the solid phase assaywas demonstrated bythe abilityof I domains to bind immobilized ICAM-4Fc in a dose-dependent fashion compared with a lack of binding to either BSA or another closelyrelated Ig-familyprotein, VCAM-1Fc (Fig 10B,D) The control GST fusion protein, LLG-C4-GST [29], did not interact with ICAM-4Fc or BSA (Fig 10A,C), showing that the adhesion to ICAM-4Fc was mediated bythe
I domain in the fusion protein We tested the effects of several mAbs on the binding of isolated I domain GST to ICAM-4Fc (Fig 11) and found that the blocking pattern mostlyreflected that observed in cellular assays for red cells and ICAM-4 transfectants The MEM83 antibody
Fig 8 Adhesion of ICAM transfectants to CD11a and CD11b I domain
GST fusion proteins in the absence or the presence of divalent cations.
Stable transfectants were harvested and washed with cation-free Tris
buffer, and then resuspended in the Tris buffer containing either 2 m M
MgCl 2 and 2 m M CaCl 2 , 2 m M MgCl 2 , 2 m M CaCl 2 , 2 m M MnCl 2 , or
4 m M EDTA Plastic wells precoated with 0.2 lg of anti-GST Ab,
were coated with 0.4 lg of CD11a (A) or CD11b (B) I domain GST
and washed three times with appropriate buffer before adding the cells.
Background binding of the cells was substracted The experiments
were repeated three to five times with similar results.
Fig 9 Binding of red cells to CD11a/CD18 and CD11b/CD18 I domains in the absence or presence of divalent cations Red cells were washed with cation-free Tris buffer, and then resuspended in serum-free buffer containing either 2 m M MgCl 2 and 2 m M CaCl 2 , 4 m M
EDTA, or 4 m M EGTA and 2 m M MgCl 2 Plastic wells coated with
1 lg of CD11a I domain or 0.4 lg of CD11b I domain were washed three times with the appropriate buffer before adding the cells The results are expressed as a relative percentage of bound cells, where 100% is calculated from the total number of cells bound to the I domains in the presence of divalent cations Background binding of red cells to BSA and the control protein (GPA) was substracted The experiments were repeated 3–5 times with similar results Standard deviations and statistical significances are shown wwwP < 0.001, wwP< 0.01.
Trang 10effectivelyinhibited the CD11a I domain/ICAM)4Fc
in-teraction while the TS1/22 blocked to a lesser degree as did
also the ICAM-4 mAb BS46 The mAbs MEM25 and
MEM30 substantiallyblocked the CD11a I domain GST
binding to captured ICAM-4Fc, whereas the MEM177 was
nonblocking
Five CD11b I domain specific mAbs were tested for their
abilityto inhibit in solid phase assay MEM170, 44a and 107
were highlyactive inhibitors of the CD11b I domain GST
interaction with ICAM 4, whereas LM2/1 inhibited weakly
but significantlyand mAb 904 had no effect However, the
failure of mAb 904 to inhibit CD11b I domain/ICAM-4
interaction was unexpected, as the mAb was an efficient
blocker of adhesion between ICAM-4 L cell transfectants
and coated CD11b I domain GST We checked that all the
mAbs to the I domains and the anti-ICAM-4 mAb bound
to corresponding coated and soluble recombinant proteins
(data not shown)
As a further approach to characterize the interaction
between ICAM-4 and the I domains we examined the effect
of soluble recombinant ICAMFc proteins and the isolated
I domains on the binding of I domain GST fusion proteins
to plastic captured ICAM-4Fc in solid phase ELISA assay
(Fig 11) The results indicated that recombinant CD11a
I domain lacking GST was a potent competitor of the
interaction between coated ICAM-4Fc and the CD11a
I domain GST fusion protein, whereas the CD11b I domain
onlyinhibited weakly However, soluble CD11b I domain
readilyinhibited the binding of captured ICAM-4Fc to
CD11b I domain GST, while the soluble CD11a I domain
did not have anyeffect Furthermore, soluble ICAM-4Fc
was highlyactive in competing with the coated ICAM-4Fc
for the recombinant I domain GST fusion proteins and the
soluble ICAM-1Fc and ICAM-2Fc fusion proteins were
even more efficient competitors
Discussion
In the present study, we have used several techniques to
show that the I domains of the CD11a/CD18 and CD11b/
CD18 leukocyte integrins contain binding sites for ICAM-4
Our results show that ICAM-4 expressing red cells bound
specificallyand dose-dependentlyto isolated recombinant
CD11a and CD11b I domains The effective inhibition of
binding of ICAM-4 positive red cells byanti-ICAM-4
antibodies, indicate a major role for ICAM-4 in binding of
red cells to the I domains The efficient inhibition of the
Fig 10 Specific binding of purified recombinant I domain GST fusion
proteins to ICAM-4Fc in a solid phase assay Dose-dependent binding
of CD11a I domain GST (A and B) or CD11b I domain GST (C and
D) to ICAM-4Fc, VCAM-1Fc and BSA Recombinant ICAM-4Fc or
VCAM-1Fc was immobilized via antihuman IgG Fc specific antibody
to 96-well plates, which were blocked with BSA Control wells were
onlyblocked with BSA The I domain GST or the control GST were
diluted in Tris buffered saline containing 1 m M CaCl 2 , 1 m M MgCl 2
and 1% BSA and incubated for 2 h at room temperature Data
shown are from one representative experiment out of 3–5.
I domain GST + ICAM-4Fc (j), control GST + ICAM-4Fc (m),
I domain GST + BSA (s), control GST + BSA (e), I domain
GST + VCAM-1Fc (d).