Characterization of ICAM-4 binding to the I domains
of the CD11a/CD18 and CD11b/CD18 leukocyte integrins
Eveliina Ihanus
1
, Liisa Uotila
1
, Anne Toivanen
1
, Michael Stefanidakis
1
, Pascal Bailly
2
,
Jean-Pierre Cartron
2
and Carl G. Gahmberg
1
1
Department of Biosciences, Division of Biochemistry, University of Helsinki, Finland;
2
INSERM U76, Institut National de
Transfusion Sanguine, Paris, France
Intercellular adhesion molecule-4 (ICAM-4, LW blood
group antigen), a member of the immunoglobulin super-
family expressed 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

of five Ig-like domains, is found on the surface of leukocytes,
endothelial cells and various other cells, and can be
up-regulated by several proinflammatory cytokines [5,6].
ICAM-2 has two Ig-like domains. It is constitutively
expressed by leukocytes, 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 by subsets 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 b
2
chain (CD18). They play an
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, a
L
b
2
) which is expressed on all
leukocytes. The first NH
2
-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

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 January 2003, accepted 20 February 2003)
Eur. J. Biochem. 270, 1710–1723 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03528.x
By using monoclonal antibodies reacting with integrin
I domains and by mutational 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 Ca
2+
and Mg
2+
[23,24]. Importantly, the I domains have been shown to
bind divalent cations [20,24].
ICAM-4 was originally identified 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, notably Mg
2+
,for
antigenic activity [26]. The LW and Rh blood groups
show an interesting phenotypic relationship, as the level of
LW expression is greater in RhD-positive than in RhD-
negative cells, and extremely rare Rh
null
cells, which lack
all Rh antigens, are also deficient in the LW protein.

4
b
1
and a
v
family integrins [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 specifically to the CD11a
and CD11b I domains.
Materials and methods
Antibodies
The b
2
integrin 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].

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 m
M
Tris, pH 8.0) containing 5 m
M
EDTA, 1 m
M
dithiothreitol, 1 m
M
phenylmethylsulfonyl
fluoride, 5 lgÆmL
)1
aprotinin 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).
ICAM-4
NSSLRTPLRQ
ICAM-1 LLGIETPLPK
ICAM-2 VGGLETSLNK
ICAM-3 KIALETSLSK

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 chromatography on 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 by transient transfection of COS-1 cells by the
DEAE-dextran method (Pharmacia) and isolated from the
culture supernatants by protein A-Sepharose CL-4B (Phar-
macia) chromatography essentially as previously described
[14,40]. ICAM-1Fc and VCAM-1Fc fusion proteins were
obtained from R & D Systems. All the recombinant
proteins were checked by SDS/PAGE and Western blotting.
The ICAM-2Fc cDNA vector was kindly provided by D.
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
–

kidney cell line COS-1 (ATCC) was grown in DMEM
supplemented with 10% fetal bovine serum, 100 UÆmL
)1
penicillin, and 100 lgÆmL
)1
streptomycin.
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/P
i
, pH 7.4. Aliquots of
1 · 10
6
L cells or 1–3 lL of packed red cells were
incubated with 25 lgÆmL
)1
of different anti-ICAM mAbs
for 30–60 min on ice. The cells were washed with NaCl/P
i
and incubated with FITC-conjugated rabbit antimouse
F(ab¢)
2
(Dakopatts a/s, Copenhagen, Denmark) for 30 min
on ice. After washing, 1 · 10
4
cells were analyzed with a
Becton Dickinson (Immunocytometry Systems, San Jose,
CA, USA) FACScan flow cytometer.
Red cell binding assays

and incubated for 2 h at 37 °C. The input of red cells
was quantitated by counting cells in four randomly
chosen fields from duplicate wells. To remove non-
adherent cells, the wells were gently filled with binding
buffer, and the microplate was placed floating upside
down for 40 min in NaCl/P
i
solution before microscopic
observation and scoring the number of attached cells in
four randomly chosen fields from duplicate wells. The
data was presented as a percentage of bound cells
(amount of bound red cells divided by input 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 l
M
)for10minatroom
temperature before starting the adhesion. For the binding
study with or without divalent cations, the adhesion assays
were performed with buffers containing 4 m
M
EDTA,
4m
M
EGTA and 2 m
M
MgCl
2

room temperature. For the experiments with coated GST
I domains the anti-GST antibodies (Pharmacia Biotech
Inc.)dilutedin25m
M
Tris, pH 8.0, 150 m
M
NaCl, 2 m
M
MgCl
2
were adsorbed overnight at 4 °C, at 0.2–1 lgper
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 m
M
Hepes, pH 7.4, 0.5% BSA, 2 m
M
MgCl
2
and 2 m
M
CaCl
2
) prior to addition of the cells
(1.5 · 10
5

Tris/HCl, pH 7.4, 150 m
M
NaCl (TBS). After blocking nonspecific sites with 3%
BSA in TBS for 1 h at 37 °C the wells were washed three
times with TBS, 1 m
M
CaCl
2
,1m
M
MgCl
2,
0.05% Tween
20, 1% BSA. The recombinant proteins (200 ng per well)
diluted in TBS, 1 m
M
CaCl
2
,1m
M
MgCl
2
,1%BSAwas
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 m
M

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 n
M
; I domains, 1 l
M
) diluted in
TBS, 1 m
M
CaCl
2
,1m
M
MgCl
2
,1%BSAfor10minat
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 majority of the CD11a I domain GST was found
in the insoluble fraction of the E. coli lyzate. Modification

these cells was affected neither by mAbs 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 completely abrogated
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 probably also to CD11a.
CD11b I domain inhibits red cell binding to purified
integrin
Red cells bind poorly to 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 l
M
I domain GST.
Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur. J. Biochem. 270) 1713
Interaction 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 previously described [41]. As
expected, the ICAM transfectants reacted only with the

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 by counting cells in four randomly chosen fields
from duplicate wells. The experiment was repeated three times with
similar results.
1714 E. Ihanus et al.(Eur. J. Biochem. 270) Ó FEBS 2003
transfectants were clearly lower 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 clearly inhibited 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 ability of I domain GST fusion
proteins to block the interaction of ICAM transfectants
with purified CD11a/CD18 and CD11b/CD18 integrins
(Fig. 4). They efficiently inhibited the adhesion of ICAM
transfectants to CD11a/CD18 and CD11b/CD18 integrins.
The adhesion of ICAM-4 transfectants to CD11a/CD18
was inhibited by the 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
by soluble CD11a I domain GST was concentration-
dependent and 50% inhibition was obtained with an
inhibitor concentration of 0.4 l
M
. The soluble CD11b
I domain GST was a less active inhibitor of ICAM-4

the I domain of CD11b interacts not only with 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 efficiently inhibited the binding of ICAM-1
and -2 transfected L cells to the I domain of CD11a, and
partially but significantly inhibited 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 completely blocked by the anti-
CD11a mAb MEM83 down to GST background level
(Fig. 6). In an ELISA assay both 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).

the ICAM-1 molecule [44]. None of these anti-ICAM-1
mAbs showed inhibitory effects 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 they reacted only with the
mAbs to transfected ICAM (data not shown).
Divalent cation requirements for ICAM/b
2
integrin
interaction
Divalent cations may have multiple effects on integrin-
mediated cell adhesion including enhancement, suppres-
sion, and modification of ligand binding activity. We
have previously shown that Ca
2+
and Mg
2+
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

ations and statistical significances are shown.
wwwP <0.002,wwP <0.02,wP <0.2.
Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur. J. Biochem. 270) 1717
The most efficient binding of all the ICAM L cell
transfectants was observed in the presence of Mn
2+
(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 completely abrogated. 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 inhibitory effects of EDTA
were clearly significant. As can be seen in Fig. 8, MgCl
2
alone or in combination with CaCl
2
supported 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 MgCl
2
and CaCl
2
seems to be required for
high affinity binding 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. CaCl
2

that the adhesion to ICAM-4Fc was mediated by the
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
mostly reflected 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
,2m
M
MgCl
2
,2m
M
CaCl
2
,2m
M
MnCl
2
,or

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.
1718 E. Ihanus et al.(Eur. J. Biochem. 270) Ó FEBS 2003
effectively inhibited 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 substantially blocked 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
ability to inhibit in solid phase assay. MEM170, 44a and 107
were highly active inhibitors of the CD11b I domain GST
interaction with ICAM 4, whereas LM2/1 inhibited weakly
but significantly and 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

M
CaCl
2
,1m
M
MgCl
2
and1%BSAandincubatedfor2hatroomtemperature.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).
Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur. J. Biochem. 270) 1719
interaction between purified CD11b/CD18 and erythrocytes
by soluble CD11b I domain GST is consistent with the idea
of this subdomain being a ligand binding area for a red cell
receptor. However, it is probable that other erythrocyte
receptors for the CD11a and CD11b I domains exist
because of the residual binding of the ICAM-4 negative
cells. The mAb 60.1 reacting with the CD11b I domain
almost completely inhibited the binding of both ICAM-4
positive and negative red cells. To the best of our knowledge
the epitope for mAb 60.1 within the I domain has not been
mapped in detail but the epitope for activation dependent
mAb 7E3 has been localized to the amino-terminal region
of the CD11b I domain [42] overlapping partially with the
metal ion-dependent adhesion site. A clear reduction in
adhesion was also obtained with the CD11b I domain
specific LM2/1 mAb (not shown) which has been shown to
inhibit the binding of red cells to the CD11b/CD18 integrin

on ICAM-3 bilayers. Furthermore, the MEM83 decreased
the rolling velocity of the cells on ICAM-1-containing
membranes indicating an enhanced avidity [48]. The differ-
ent results may be due to differences in assay systems. We
have investigated the static adhesion of ICAM expressing
transfected cells to the isolated recombinant I domains. The
previous results were performed with coated CD11a/CD18
or using cells expressing either a whole CD11a/CD18
integrin or a membrane-anchored form of the CD11a
I domain. Altogether, our data with both red cells and
ICAM-4 transfected L cells suggest that the epitope within
the CD11a I domain recognized by the MEM83 antibody is
involved in ICAM-4 binding.
Treatment of the coated CD11b I domain fusion protein
with I domain specific anti-CD11b antibodies resulted in an
efficient inhibition of the adherence of all three types of
ICAM L cells, except that mAb 904 had no effect on
binding of ICAM-2 transfectants. The LM2/1 mAb did not
inhibit the binding of ICAM-4 L cells as efficiently as did
the other anti-I domain mAbs. The degrees of inhibition
between mAbs may vary due to different mechanisms of
action. Several studies have pointed out that blocking mAbs
can inhibit ICAM/integrin interactions either by direct
competition for the ligand binding site or by an indirect
mechanism through binding to a regulatory site located
outside the actual ligand binding site [46,49,50]. Most
function-blocking mAbs directed against integrins may act
allosterically by stabilizing the low-affinity state of the
Fig. 11. Effect of monoclonal antibodies, soluble competitor proteins
and EDTA on binding of recombinant I domain GST to coated ICAM-

binding site on the opposite side of the metal ion-dependent
adhesion site (MIDAS) motif, whereas the binding interface
for mAb 107 seems to overlap that of physiologic ligands
and clearly engages the metal ion-dependent adhesion site
area [51–53].
The presence of Mg
2+
and Ca
2+
or Mn
2+
is required for
maximal binding efficiency of ICAM-4 transfectants and
red cells to the I domains of CD11a and CD11b. Our results
indicate that Mg
2+
or Mn
2+
, but not Ca
2+
are necessary
for the interaction of CD11a I domain with ICAM-1 and
ICAM-2 transfectants and for the CD11b I domain binding
to the ICAM-1 transfectants. The presence of both MgCl
2
and CaCl
2
gives better binding of ICAM-2 L cell transfect-
ants to the I domain of CD11b as well as for adhesion of
ICAM-4 transfectants and red cells to both I domains.

haemostasis where in the developing thrombus, erythro-
cytes interact with activated neutrophils and monocytes or
during wound healing to mediate removal of red cells
from the thrombus by phagocytic macrophages. The very
recent finding that ICAM-4 also binds to the platelet
integrin IIb/IIIa indicates an important role for this
molecule in haemostasis [55]. Another recent report
described the capture and adhesion of normal erythrocytes
to activated neutrophils and platelets, as well as to fibrin,
at depressed venous shear rates. In agreement with our
present and previous data the results suggested that the
binding of red cells to neutrophils might be mediated
through ICAM-4 and CD11b/CD18 [56].
Acknowledgements
We thank Leena Kuoppasalmi, Aili Grundstro
¨
m, Outi Nikkila
¨
,Tuula
Nurminen and Saija Ma
¨
kinen for excellent technical assistance, and
Yvonne Heinila
¨
for secretarial work. We also want to thank Erkki
Koivunen and Tanja-Maria Ranta for providing the LLG-C4-GST
protein. These studies were supported by the University of Helsinki, the
Academy of Finland, the Sigrid Juse
´
lius Foundation, the Magnus

blood group glycoprotein is homologous to intercellular adhesion
molecules. Proc. Natl Acad. Sci. USA 91, 5306–5310.
10. Yoshihara, Y., Oka, S., Nemoto, Y., Watanabe, Y., Nagata, S.,
Kagamiyama, H. & Mori, K. (1994) An ICAM-related neuron
glycoprotein, telencephalin, with brain segment-specific expres-
sion. Neuron 12, 541–553.
11. Staunton, D.E., Dustin, M.L., Erickson, H.P. & Springer, T.A.
(1990) The arrangement of the immunoglobulin-like domains of
ICAM-1 and the binding sites for LFA-1 and rhinovirus. Cell 61,
243–254.
12. Li, R., Nortamo, P., Valmu, L., Tolvanen, M., Kantor, C. &
Gahmberg, C.G. (1993) A peptide from ICAM-2 binds to the
leukocyte integrin CD11a/CD18 and inhibits endothelial cell
adhesion. J. Biol. Chem. 268, 17513–17518.
13. Holness, C.L., Bates, P.A., Littler, A.J., Buckley, C.D.,
McDowall,A.,Bossy,D.,Hogg,N.&Simmons,D.L.(1995)
Analysis of the binding site on intercellular adhesion molecule 3
for the leukocyte integrin lymphocyte function-associated 1.
J. Biol. Chem. 270, 877–884.
14. Hermand, P., Huet, M., Callebaut, I., Gane, P., Ihanus, E.,
Gahmberg, C.G., Cartron, J P. & Bailly, P. (2000) Binding sites of
leukocyte b2 integrins (LFA-1, Mac-1) on the human ICAM-4/
LW blood group protein. J. Biol. Chem. 275, 26002–26010.
Ó FEBS 2003 Interaction of ICAM-4 with leukocyte integrins (Eur. J. Biochem. 270) 1721
15. Tian, L., Kilgannon, P., Yoshihara, Y., Mori, K., Gallatin, W.M.,
Carpe
´
n, O. & Gahmberg, C.G. (2000) Binding of T lymphocytes
to hippocampal neurons through ICAM-5 (telencephalin) and
characterization of its interaction with the leukocyte integrin

binding of leukocyte integrins to their ligands. In Leukocyte
Adhesion. Basic and Clinical Aspects. Excerpta Medica, Interna-
tional Congress Series 1010 (Gahmberg, C.G., Mandrup-Poulsen,
T., Wogensen Bach, L. & Ho
¨
kfelt, B., eds), pp. 205–219. Elsevier
Science Publishers, BV, Amsterdam.
24. Griggs, D.W., Schmidt, C.M. & Carron, C.P. (1998) Character-
istics of cation binding to the I domains of LFA-1 and MAC-1.
J. Biol. Chem. 273, 22113–22119.
25. Mallinson, G., Martin, P.G., Anstee, D.J., Tanner, M.J.A.,
Merry, A.H., Tills, D. & Sonneborn, H.H. (1986) Identification
and partial characterization of the human erythrocyte membrane
component(s) that express the antigens of the LW blood-group
system. Biochem. J. 234, 649–652.
26. Bloy, C., Hermand, P., Blanchard, D., Cherif-Zahar, B., Goos-
sens, D. & Cartron, J P. (1990) Surface orientation and antigen
properties of Rh and LW polypeptides of the human erythrocyte
membrane. J. Biol. Chem. 265, 21482–21487.
27. Cartron, J P. (1994) Defining the Rh blood group antigens.
Biochemistry and molecular genetics. Blood Rev. 8, 199–212.
28. Bailly, P., Tontti, E., Hermand, P., Cartron, J P. & Gahmberg,
C.G. (1995) The red cell LW blood group protein is an inter-
cellular adhesion molecule which binds to CD11/CD18 leukocyte
integrins. Eur. J. Immunol. 25, 3316–3320.
29. Koivunen, E., Ranta, T M., Annila, A., Taube, S., Uppala, A.,
Jokinen, M., van Willigen, G., Ihanus, E. & Gahmberg, C.G.
(2001) Inhibition of b
2
integrin-mediated leukocyte cell adhesion

Biol. 143, 1523–1534.
35. Patarroyo, M., Clark, E.A., Prieto, J., Kantor, C. & Gahmberg,
C.G. (1987) Identification of a novel adhesion molecule in human
leukocytes by monoclonal antibody LB-2. FEBS Lett. 210, 127–131.
36. Rothlein, R., Dustin, M.L., Marlin, S.D. & Springer, T.A. (1986)
A human intercellular adhesion molecule (ICAM-1) distinct from
LFA-1. J. Immunol. 137, 1270–1274.
37. Xie, J., Li, R., Kotovuori, P., Vermot-Desroches, C., Wijdenes, J.,
Arnaout, M.A., Nortamo, P. & Gahmberg, C.G. (1995) Inter-
cellular adhesion molecule-2 (CD102) binds to the leukocyte
integrin CD11b/CD18 through the A domain. J. Immunol. 155,
3619–3628.
38. Sonneborn, H.H., Uthemann, H., Tills, D., Lomas, C.G., Shaw,
M.A. & Tippett, P. (1984) Monoclonal anti-LWab. Biotest. Bull.
2, 145–148.
39. Muchowski, P.J., Zhang, L., Chang, E.R., Soule, H.R., Plow, E.F.
& Moyle, M. (1994) Functional interaction between the integrin
antagonist neutrophil inhibitory factor and the I domain of
CD11b/CD18. J. Biol. Chem. 269, 26419–26423.
40. Simmons, D.L. (1993) Cloning cell surface molecules by transient
expression in mammalian cells. In Cellular Interactions in
Development. A Practical Approach (Stevenson, B. ed.) pp. 93–128,
Oxford University Press, Oxford.
41. Tian, L., Yoshihara, Y., Mizuno, T., Mori, K. & Gahmberg, C.G.
(1997) The neuronal glycoprotein telenchephalin is a cellular
ligand for the CD11a/CD18 leukocyte integrin. J. Immunol. 158,
928–936.
42. Plescia, J., Conte, M.S., Van Meter, G., Ambrosini, G. & Altieri,
D.C. (1998) Molecular identification of the cross-reacting epitope
on a

1722 E. Ihanus et al.(Eur. J. Biochem. 270) Ó FEBS 2003
47. Woska, J.R., Jr, Morelock, M.M., Durham Jeanfavre, D., Cavi-
ness, G.O., Bormann, B J. & Rothlein, R. (1998) Molecular
comparison of soluble intercellular adhesion molecule (sICAM)-1
and sICAM-3 binding to lymphocyte function-associated antigen-
1. J. Biol. Chem. 273, 4725–4733.
48. Knorr, R. & Dustin, M.L. (1997) The lymphocyte function-
associated antigen 1, I domain is a transient binding module
for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in
hydrodynamic flow. J. Exp. Med. 186, 719–730.
49. Huth, J.R., Olejniczak, E.T., Mendoza, R., Liang, H., Harris,
E.A., Lupher, M.L., Jr, Wilson, A.E., Fesik, S.W. & Staunton,
D.E. (2000) NMR and mutagenesis evidence for an I domain
allosteric site that regulates lymphocyte function-associated anti-
gen 1 ligand binding. Proc. Natl Acad. Sci. USA 97, 5231–5236.
50. Lupher, M.L., Jr, Harris, E.A.S., Beals, C.R., Sui, L., Liddington,
R.C. & Staunton, D.E. (2001) Cellular activation of leukocyte
function-associated antigen-1 and its affinity are regulated at the I
domain allosteric site. J. Immunol. 167, 1431–1439.
51. Zhang, L. & Plow, E.F. (1999) Amino acid sequences within the
alpha subunit of integrin alpha M beta 2 (Mac-1) critical for
specific recognition of C3bi. Biochemistry 38, 8064–8071.
52. Xiong, J P., Li, R., Essafi, M., Stehle, T. & Arnaout, M.A. (2000)
An isoleucine-based allosteric switch controls affinity and shape
shifting in integrin CD11b A-domain. J. Biol. Chem. 275, 38762–
38767.
53. Li, R., Haruta, I., Rieu, P., Sugimori, T., Xiong, J P. &
Arnaout, M.A. (2002) Characterization of a conformationally
sensitive murine monoclonal antibody directed to the metal ion-
dependent adhesion site face of integrin CD11b. J. Immunol. 168,