Full text of DOI:10.1038/sj.bjp.0703739

British Journal of Pharmacology (2000) 131, 1531 ± 1536
ã
2000 Macmillan Publishers Ltd All rights reserved 0007 ± 1188/00 $15.00
www.nature.com/bjp
Role of selectins on IgE-mediated skin reaction
1
1
1
1
*,1Yukihisa Wada, Akemi Kuzuhara, Mikiko Hanamura, Rie Kida, Tsuyoshi Yoshinaka &
1Tadayuki Saito
1R&D Laboratories, Nippon Organon K.K., 5-90. Tomobuti-cho 1-chome Miyakojima-ku, Osaka 534-0016, Japan
1
Selectins play an important role on leukocytes in®ltration into in¯ammatory tissues. To
understand the role of selectins, we investigated the e€ects of selectin-IgG chimeras and anti selectin
antibodies on the murine IgE-mediated skin in¯ammation model.
2
Biphasic skin reactions were induced by intradermal challenge with ovalbumin (OA) to ears of
actively sensitized mice. This reaction was characterized by immediate and late phase responses
observed as which were induced via a rapid increase in capillary permeability and leukocyte
in®ltration, respectively. The expression of E-selectin mRNA was signi®cantly increased to reach its
highest level at 2 h after OA challenge.
3
E-, P-, and L-selectin-IgG chimeras inhibited the late phase responses, i.e. ear swelling,
neutrophil in®ltration and eosinophil in®ltration at 24 h after OA challenge in a dose-dependent
manner at dose range of 0.1 ± 10 mg kg71, i.v. Antiselectin antibodies did not inhibit the increase of
ear swelling. But anti E- and P-selectin antibodies signi®cantly inhibited neutrophil in®ltration and
eosinophil in®ltration.
4
These results indicate that selectins play an important role on the late phase response of the
murine IgE-mediated skin in¯ammation model by mediating in¯ammatory cell adhesion to
endothelium.
British Journal of Pharmacology (2000) 131, 1531 ± 1536
Keywords: Selectin; cell adhesion; in¯ammation; atopic dermatitis
Abbreviations: EPO, eosinophil peroxidase; IPR, immediate-phase response; LPR, late-phase response; MPO, myeloperoxidase;
OA, ovalbumin; PBS, phosphate-bu€ered saline; sLex, sialyl Lewis x
Introduction
Selectins are a family of three Ca2+-dependent membrane-
bound lectins that interact with not only small sialylated
oligosaccharides such as sialyl Lewis x (sLex) (Foxall et al.,
1992; Varki, 1994), but also glycans displayed on a limited
number of glycoproteins with higher anity. E-selectin,
expressed by cytokine-activated endothelial cells, and P-
selectin, expressed by thrombin activated platelets and
endothelial cells, bind to ligands on myeloid cells and subsets
of lymphocytes. L-selectin, expressed on leukocytes, binds to
constitutive or inducible ligands on endothelial cells. These
selectins participate in the initial phase of adhesion between
in¯ammatory cells and endothelial cells, and play an
important role on leukocytes in®ltration into in¯ammatory
tissues (Berg et al., 1991; Picker et al., 1991; Lorant et al.,
1993; Symon et al., 1994; Akbar et al., 1991).
phils and eosinophils (Solley et al., 1976; Dolovich et al.,
1973). Cell in®ltration into in¯ammatory sites is one of the
causes of chronic allergic in¯ammation (Charlesworth et al.,
1989).
In the present study, we have measured cell in®ltration into
skin tissue in the LPR in a murine IgE-mediated skin
in¯ammation model, and investigated the role of selectin
molecules in this model.
Methods
Animals
Balb/c mice (female, 7 weeks-old) were purchased from Japan
SLC (Hamamatsu, Japan). They were housed in an air-
conditioned room (temperature 23+28C, humidity 55+10%)
with a controlled light-dark cycle (light on 07 : 30 ± 19 : 30 h)
and freely available food and water. All the experiments were
performed according to the Principles for the Care and Use
of Laboratory Animals approved by The Japanese Pharma-
cological Society.
Atopic dermatitis is a common in¯ammatory skin condi-
tion characterized by severe pruritis, chronic relapses with
frequent periods of exacerbation, and distinctive clinical
morphology and distribution of skin lesions. After the
challenge with relevant antigen, sensitized animals or atopic
individuals exhibit immediate responses such as the appear-
ance of weals and ¯ares on the skin. When
a high
concentration of allergen is used, oedema and erythema
usually persist at the challenged site for a 6 ± 24 h period.
These dual responses are of interest because of the similarity
to the clinical manifestations of chronic allergic diseases
(Patterson, 1969; 1983; Ray et al., 1983; Butler et al., 1983;
Katayama et al., 1990; Nagai et al., 1995). Pathogenic
examination of the cutaneous late-phase response (LPR) has
shown in®ltration of mononuclear cells, neutrophils, baso-
Mouse selectin-immunoglobulin fusion proteins
Mouse E-selectin-IgG chimera, mouse P-selectin-IgG chimera
and mouse L-selectin-IgG chimera, were produced as
described previously (Watson et al., 1991; Erbe et al., 1993;
Ohmoto et al., 1996; Wada et al., 1996). Brie¯y the proteins
were dissolved in Ca2+- and Mg2+-free phosphate-bu€ered
saline (PBS (7)). Mouse IgG2a (Zymed, San Francisco, CA,
U.S.A.), dissolved in PBS (7), was used as control. Mouse
E-selectin-IgG chimera was given intravenously 2 h after the
*Author for correspondence.

1532
Y. Wada et al
Role of selectins on IgE-mediated skin reaction
OA challenge. Other test agents were administered intrave-
nously 1 min prior to OA challenge.
their corresponding locations in the nucleotide sequence of
mouse E-selectin were as follows; E-selectin, forward: 5'-
TTCTCCTCGCTGGAGAGAGCACAGC-3' (193 ± 217), re-
verse: 5'-TAGCTCCCAGGATTTGAGGAACATT-3' (919 ±
944). These sequence data are available under EMBL/
GeneBank/DDBJ accession number MUSELAMIB. E-selec-
tin cDNAs were ampli®ed singly. The PCR mixtures of E-
selectin cDNA and respective primers were ampli®ed using a
thermal cycler system (GeneAmp System 2400; Applied
Biosystems, Foster City, CA, U.S.A.). One cycle of PCR
consisted of 45 s 948C, 45 s 588C, and 60 s at 728C, and a
total of 35 cycles was performed. Each PCR reaction mixture
was subjected to 2% agarose gel electrophoresis, and the
ampli®ed products were visualized by staining with ethidium
bromide for 20 min at room temperature. To con®rm the
speci®city of PCR, the bands were also identi®ed by Southern
blot analysis.
Monoclonal anti-E-, P- or L-selectin antibodies
The monoclonal antibodies were purchased from PharMin-
gen (San Diego, CA, U.S.A.). Anti-E-selectin antibody
(10E9.6), anti-P-selectin antibody (RB40, 34), anti-L-selectin
antibody (MEL-14) were dissolved in PBS (7). Mouse
IgG2a, dissolved in PBS (7), was used as control. All anti-
selectin antibodies used, have been reported to have
inhibitory activity on cells to sLex binding in vitro (Bosse &
Vestweber, 1994; Fink et al., 1985; Weller et al., 1992). Anti-
E-selectin antibody was given intravenously 2 h after the OA
challenge. The others were given intravenously 1 min prior to
OA challenge.
IgE-mediated murine skin inflammatory model
Statistical analysis
IgE-mediated murine skin in¯ammatory model were carried
out as described previously (Nagai et al., 1995). Eight-week-
old female Balb/c mice were sensitized by intraperitoneal
injection with 3 mg of OA and 4 mg of aluminium hydroxide
gel (alum). Reactions to OA were elicited by intracutaneous
injection of 10 mg of OA to each ear of mice 2 weeks after the
sensitization. Various times after the antigen challenge, the
mice were anesthetized by ether and killed. Then, 6 mm
diameter biopsy of the ears were taken and weighed as an
index of tissue swelling. The specimens were homogenized in
50 mM potassium phosphate with 0.5% hexadecyltrimethyl-
ammonium bromide, pH 6.0, and myeloperoxidase (MPO)
activities in the supernatants were measured by the method
described by Bradley et al. (1982) as an index of neutrophil
in®ltration into tissue. In brief, the supernatants were mixed
with MPO substrates bu€er (0.167 mg ml71 o-dianisidine
(Sigma, St. Louis, MO, U.S.A.), 0.0005% H2O2, 50 mM
potassium phosphate (pH 6)), and incubated for 20 min at
258C. MPO activities were measured by determining the
optical density (OD) at 450 nm. In addition, eosinophil
peroxidase (EPO) activities in the supernatants were
measured by the method described by Strath et al. (1985)
as an index of eosinophil in®ltration into tissue. In brief, the
supernatants were mixed with EPO substrates bu€er (1 mM
o-phenylenediamine (Sigma, St. Louis, MO, U.S.A.), 0.5 mM
H2O2, 0.1% Triton X-100, 50 mM potassium phosphate
(pH 5)), and incubated for 30 min at 258C. EPO activities
were measured by determining the OD at 490 nm. For
histological study, sensitized and non-sensitized mice were
challenged with antigen, and the ears were removed 24 h
later. The tissue was ®xed in 10% neutral formalin solution
and embedded in paran using a standard technique. The
paran sections were stained with haematoxylin and eosin.
Results are expressed as mean+s.e.mean. A one-way analysis
of variance with Dunnett's test was used to determine
statistical signi®cance. Statistical signi®cance was de®ned as
P50.05.
Results
Time course of the ear oedema and the expression of
E-selectin
Sensitized Balb/c mice were challenged with 10 mg of OA,
and the time course of the reaction was assessed by
measuring ear weight and MPO activity, as indices of
oedema and cell in®ltration, respectively. The increase of
ear weight was signi®cant at 1 h after the antigen challenge,
and then it decreased gradually (Figure 1). This reaction was
regarded as immediate-phase response (IPR). On the other
hand, MPO activity increased from 4 h after the antigen
challenge and came up to plateau level (Figure 1), and this
was regarded as LPR. These results indicate that the IPR and
LPR were characterized by a rapid increase in capillary
mRNA extraction and reverse transcriptase-PCR
At various times after the antigen challenge, ears were excised
and immediately frozen in a liquid nitrogen, and stored at
7808C until mRNA extraction by oligo- (dT) magnetic
particles (MPG Direct mRNA Puri®cation Kit, CPG Inc.
Lincoln Park, NJ, U.S.A.). The mRNA isolated from the
ears were reverse transcribed into cDNA by SuperScript II
Preampli®cation system (GIBCO ± BRL, Gaithersburg, MD,
U.S.A.). The E-selectin oligonucleotides primers were
synthesized by Greiner Japan (Tokyo, Japan), and b-actin
primers, were used as control, were purchased from
Stratagene (La Jolla, CA, U.S.A.). Primer sequences and
Figure 1 Time course of skin reaction mediated by IgE in ears of
actively sensitized Balb/c mice. Ovalbumin (OA: 3 mg per animal) was
given intraperitoneally with 4 mg per animal of alum 2 weeks before
the challenge of 10 mg per ear of OA. Each point represents the mean
of ®ve animals. Vertical bars indicate s.e.mean. * P50.05, **P50.01,
signi®cantly di€erent from non-sensitized mice.
British Journal of Pharmacology vol 131 (8)

Y. Wada et al
Role of selectins on IgE-mediated skin reaction
1533
permeability and skin thickening with signi®cant in®ltration
of in¯ammatory cells, respectively, in this model.
matory model were studied. Anti selectin antibodies did not
inhibit the increase of ear weight. But anti E- and P-selectin
Histological analysis of the ears, excised at 24 h after the
antigen challenge, is shown in Figure 2. No signi®cant change
was observed in non-sensitized mice. On the other hand, in
sensitized mice, marked in®ltration of neutrophils and
eosinophils was shown as well as a signi®cant increase in
the MPO activity.
The expression of E-selectin mRNA was signi®cantly
increased to reach its highest level at 2 h after OA challenge,
and decreased gradually (Figure 3). No ampli®ed DNA
fragments were observed when PCR performed with mRNA
extracted from the ears of non-sensitized mice. Moreover, the
sequences of these fragments were con®rmed by Southern
blot analysis (data not shown).
Figure 3 RT ± PCR analysis of E-selectin expression in ear tissues of
actively sensitized mice. 7: non-sensitized mice.
The effects of selectin-IgG chimeras and anti-selectin
antibodies on IgE-mediated murine skin inflammatory
model
E€ects of E-, P- and L-selectin-IgG chimera, which bind sLex
in vitro, on the IgE-mediated murine skin in¯ammatory
model were studied. As shown in Figure 4, E-, P- and L-
selectin-IgG chimera inhibited the increase in ear weight,
MPO activity, and EPO activity in a dose-dependent manner
at dose range of 0.1 ± 10 mg kg71, i.v. Further, e€ects of anti
selectin antibodies on the IgE-mediated murine skin in¯am-
Figure 4 E€ects of mouse E-, P- and L-selectin-IgG on IgE-
mediated skin reaction in mice. (A) E€ects on the increase of ear
swelling. (B) E€ects on neutrophil in®ltration. (C) E€ects on
eosinophil in®ltration. Each column represents the mean of ®ve or
six animals. Vertical bars indicate s.e.mean. * P50.05, ** P50.01,
signi®cantly di€erent from IgG treated group. 7: non-sensitized
mice.
Figure 2 Histopathology of IgE-mediated skin lesion in the ears of
actively sensitized mice. Skin sections were stained with haematoxylin
and eosin. (A) Non-sensitized mice. (B) Sensitized mice. (Arrows)
In®ltration of in¯ammatory cells (neutrophils and eosinophils).
Magni®cation:6200.
British Journal of Pharmacology vol 131 (8)

1534
Y. Wada et al
Role of selectins on IgE-mediated skin reaction
antibodies signi®cantly inhibited the MPO and EPO activity
(Figure 5) at dose range of 0.01 ± 10 mg kg71, i.v. Anti L-
selectin antibody signi®cantly inhibited the MPO activity but
it did not inhibit the EPO activity.
molecules on the leukocytes, such as L-selectin, with as yet
unidenti®ed counter structures on the endothelial cells
(Spertini et al., 1991; Smith et al., 1991). A second process
is mediated by the interaction between P-selectin on the
endothelial cells and counter structures on the leukocytes,
such as PSGL-1 (Moore et al., 1995). These two processes are
thought to occur simultaneously. The third process is
mediated by the interaction of E-selectin on the endothelial
cells, synthesized de novo, with as yet unidenti®ed counter
structures on the leukocytes (Bevilacqua et al., 1987; 1989).
In the present study, we have investigated the role of selectin
molecules on the cell in®ltration into skin tissue in IgE-
mediated murine skin in¯ammatory model.
It has been previously reported that the IgE-mediated
biphasic skin reaction in passively sensitized mice with
monoclonal anti-DNP IgE antibody could serve as an animal
model for atopic dermatitis (Katayama et al., 1990; Nagai et
al., 1995). In this study, we demonstrate that the skin
reaction model with actively sensitized mice could also serve
as an animal model for atopic dermatitis. The IPR, which is
characterized oedema, and LPR, which is characterized cell
in®ltration, were signi®cantly observed in this actively
Discussion
Extravasation of leukocytes to sites of in¯ammation is
thought to consist of at least three sequential processes
(Butcher, 1991). Firstly, circulating leukocytes undergo
margination, whereby they move from the centre to the
periphery of the blood vessel, and begin to bind reversibly to
the endothelium. This process is referred to as rolling, as they
roll along the endothelial wall. This initial binding may be
followed, during a second step, by the induction of ®rm
adhesion (Luscinskas et al., 1991; Ebisawa et al., 1992), when
the leucocytes stick to the endothelial wall. Subsequently, the
leukocytes transmigrate through the endothelial cell mono-
layer into the tissue. In vitro studies indicate that the ®rst
stage consists of at least three sequential processes. One part
of this process is mediated by the interaction of adhesion
Figure 5 E€ects of anti-mouse E-, P- and L-selectin antibodies on IgE-mediated skin reaction in mice. (A) E€ects on the increase
of ear swelling. (B) E€ects on neutrophil in®ltration. (C) E€ects on eosinophil in®ltration. Each column represents the mean of ®ve
or six animals. Vertical bars indicate s.e.mean. * P50.05, ** P50.01, signi®cantly di€erent from control immunoglobulin treated
group. 7: non-sensitized mice.
British Journal of Pharmacology vol 131 (8)

Y. Wada et al
Role of selectins on IgE-mediated skin reaction
1535
sensitized model used. In this study, we demonstrate that the
skin reaction model with actively sensitized mice could also
serve as an animal model for atopic dermatitis.
inhibited neutrophil in®ltration, but it did not inhibit the
eosinophil in®ltration. The reason for the lack of e€ect of the
anti L-selectin antibody in inhibiting the eosinophil in®ltra-
tion is not clear. It is known that leucocytes express L-
selectin constitutively, and the amount of neutrophils is
extremely large compared with the eosinophils in peripheral
blood. One possible explanation is that most anti L-selectin
antibodies bind L-selectin molecules on neutrophils. If this is
so, it could not bind eosinophils, and had no e€ect on
in®ltration of eosinophils. On the other hand, E-selectin and
P-selectin were expressed by cytokine-activated endothelial
cells, so anti E- and P-selectin antibodies bound endothelium
and inhibited the in¯ammatory cells adhesion to endothe-
lium. These results indicate that both selectins and their
counter structures, which might be small sialylated oligosac-
charides such as sLex which are displayed on a limited
number of glycoproteins or glycolipids, play important roles
on cell in®ltration into in¯ammatory sites.
It was reported that marked vascular endothelial expres-
sion of E-selectin in association with in¯ammatory in®ltra-
tion was observed in antigen-stimulated human skin (Cotran
et al., 1986; Messadi et al., 1987) and atopic dematitis
(Groves et al., 1991). E-selectin mRNA expression was also
found in the skin tissue excised 2 h after the antigen challenge
in our model. In vitro, E-selectin has been found to be
induced on human endothelial cells after incubation with
TNF-a, LPS, or IL-1 (Bevilacqua et al., 1987). It has been
demonstrated that human dermal mast cells contained stores
of TNF-a within granules, which can be released rapidly into
the extracellular space upon degranulation (Walsh et al.,
1991). Taken together, it might be suggested that in our
model, E-selectin could be expressed by TNF-a released from
skin mast cells induced by the interaction between antigen
and IgE on mast cells.
In conclusion, it is suggested that this murine IgE-mediated
skin model of atopic dermatitis is a useful model for the
study of mechanisms in dermatitis. The results indicate that
selectins play important roles in the LPR of this model.
In addition, neutrophil and eosinophil in®ltration into skin
tissue was signi®cantly inhibited by selectin-IgG chimera and
anti E- and P-selectin antibodies. Anti L-selectin antibody
References
AKBAR, A.N., SALMON, M. & JANOSSY, G. (1991). The synergy
between naive and memory T cells during activation. Immunol.
Today, 12, 184 ± 188.
BERG, E.L., YOSHINO, T., ROTT, L.S., ROBINSON, M.K., WARNOCK,
R.A., KISHIMOTO, T.K., PICKER, L.J. & BUTCHER, E.C. (1991).
The cutaneous lymphocyte antigen is a skin lymphocyte homing
receptor for the vascular lectin endothelial cell-leukocyte
adhesion molecule 1. J. Exp. Med., 174, 1461 ± 1466.
ERBE, D.V., WATSON, S.R., PRESTA, L.G., WOLITZKY, B.A.,
FOXALL, C., BRANDLEY, B.K. & LASKY, L.A. (1993). P- and E-
selectin use common sites for carbohydrate ligand recognition
and cell adhesion. J. Cell Biol., 120, 1227 ± 1235.
FINK, P.J., GALLATIN, W.M., REICHERT, R.A., BUTCHER, E.C. &
WEISSMAN, I.L. (1985). Homing receptor-bearing thymocytes, an
immunocompetent cortical subpopulation. Nature, 313, 233 ±
235.
BEVILACQUA, M.P., POBER, J.S., MENDRICK, D.L., COTRAN, R.S. &
GIMBRONE JR., M.A. (1987). Identi®cation of an inducible
endothelial-leukocyte adhesion molecule. Proc. Natl. Acad. Sci.
U.S.A., 84, 9238 ± 9242.
FOXALL, C., WATSON, S.R., DOWBENKO, D., FENNIE, C., LASKY,
L.A., KISO, M., HASEGAWA, A., ASA, D. & BRANDLEY, B.K.
(1992). The three members of the selectin receptor family
recognize a common carbohydrate epitope, the sialyl Lewis(x)
oligosaccharide. J. Cell Biol., 117, 895 ± 902.
GROVES, R.W., ALLEN, M.H., BARKER, J.N., HASKARD, D.O. &
MACDONALD, D.M. (1991). Endothelial leucocyte adhesion
molecule-1 (ELAM-1) expression in cutaneous in¯ammation.
Br. J. Dermatol., 124, 117 ± 123.
KATAYAMA, I., TANEI, R., YOKOZEKI, H., NISHIOKA, K. & DOHI,
Y. (1990). Induction of eczematous skin reaction in experimen-
tally induced hyperplastic skin of Balb/C mice by monoclonal
anti-DNP IgE antibody: possible implications for skin lesion
formation in atopic dermatitis. Int. Arch. Allergy Appl. Immunol.,
93, 148 ± 154.
BEVILACQUA, M.P., STENGELIN, S., GIMBRONE JR., M.A. & SEED,
B. (1989). Endothelial leukocyte adhesion molecule 1: an
inducible receptor for neutrophils related to complement
regulatory proteins and lectins. Science, 243, 1160 ± 1165.
BOSSE, R. & VESTWEBER, D. (1994). Only simultaneous blocking of
the L- and P-selectin completely inhibits neutrophil migration
into mouse peritoneum. Eur. J. Immunol., 24, 3019 ± 3024.
BRADLEY, P.P., PRIEBAT, D.A., CHRISTENSEN, R.D. & ROTHSTEIN,
G. (1982). Measurement of cutaneous in¯ammation: estimation
of neutrophil content with an enzyme marker. J. Invest.
Dermatol., 78, 206 ± 209.
BUTCHER, E.C. (1991). Leukocyte-endothelial cell recognition: three
(or more) steps to speci®city and diversity. Cell, 67, 1033 ± 1036.
BUTLER, J.M., PETERS, J.E., HIRSHMAN, C.A., WHITE JR., C.R.,
MARGOLIN, L.B. & HANIFIN, J.M. (1983). Pruritic dermatitis in
asthmatic basenji-greyhound dogs: a model for human atopic
dermatitis. J. Am. Acad. Dermatol., 8, 33 ± 38.
LORANT, D.E., TOPHAM, M.K., WHATLEY, R.E., MCEVER, R.P.,
MCINTYRE, T.M., PRESCOT, S.M. & ZIMMERMAN, G.A. (1993).
In¯ammatory roles of P-selectin. J. Clin. Invest., 92, 559 ± 570.
LUSCINSKAS, F.W., CYBULSY, M.I., KIELY, J.M., PECKINS, C.S.,
DAVIS, V.M. & GIMBRONE JR., M.A. (1991). Cytokine-activated
human endothelial monolayers support enhanced neutrophil
transmigration via a mechanism involving both endothelial-
leukocyte adhesion molecule-1 and intercellular adhesion
molecule-1. J. Immunol., 146, 1617 ± 1625.
MESSADI, D.V., POBER, J.S., FIERS, W., GIMBRONE JR., M.A. &
MURPHY, G.F. (1987). Induction of an activation antigen on
postcapillary venular endothelium in human skin organ culture.
J. Immunol., 139, 1557 ± 1562.
CHARLESWORTH, E.N., HOOD, A.F., SOTER, N.A., KAGEY SOBO-
TOKA, A., NORMAN, P.S.
& LICHTENSTEIN, L.M. (1989).
Cutaneous late-phase response to allergen. Mediator release
and in¯ammatory cell in®ltration. J. Clin. Invest., 83, 1519 ±
1526.
COTRAN, R.S., GIMBRONE JR., M.A., BEVILACQUA, M.P., MEN-
DRICK, D.L. & POBER, J.S. (1986). Induction and detection of a
human endothelial activation antigen in vivo. J. Exp. Med., 164,
661 ± 666.
MOORE, K.L., PATEL, K.D., BRUEHL, R.E., LI, F., JOHNSON, D.A.,
LICHENSTEIN, H.S., CUMMINGS, R.D., BAINTON, D.F.
&
DOLOVICH, J., HARGREAVE, F.E., CHALMERS, R., SHIER, K.J.,
GAULDIE, J. & BIENENSTOCK, J. (1973). Late cutaneous allergic
responses in isolated IgE-dependent reactions. J. Allergy Clin.
Immunol., 52, 38 ± 46.
EBISAWA, M., BOCHNER, B.S., GEORAS, S.N. & SCHLEIMER, R.P.
(1992). Eosinophil transendothelial migration induced by
cytokines. I. Role of endothelial and eosinophil adhesion
molecules in IL-1 beta-induced transendothelial migration. J.
Immunol., 149, 4021 ± 4028.
MCEVER, R.P. (1995). P-selectin glycoprotein ligand-1 mediates
rolling of human neutrophils on P-selectin. J. Cell Biol., 128,
661 ± 671.
NAGAI, H., SAKURAI, T., INAGAKI, N. & MORI, H. (1995). An
immunopharmacological study of the biphasic allergic skin
reaction in mice. Biol. Pharm. Bull., 18, 239 ± 245.
British Journal of Pharmacology vol 131 (8)

1536
Y. Wada et al
Role of selectins on IgE-mediated skin reaction
OHMOTO, H., NAKAMURA, K., INOUE, T., KONDO, N., INOUE, Y.,
YOSHINO, K., KONDO, H., ISHIDA, H., KISO, M. & HASEGAWA,
A. (1996). Studies on selectin blocker. 1. Structure-activity
relationships of sialyl Lewis X analogs. J. Med. Chem., 39,
1339 ± 1343.
STRATH, M., WARREN, D.J. & SANDERSON, C.J. (1985). Detection
of eosinophils using an eosinophil peroxidase assay. Its use as an
assay for eosinophil di€erentiation factors. J. Immunol. Methods,
83, 209 ± 215.
SYMON, F.A., WALSH, G.M., WATSON, S.R. & WARDLAW, A.J.
(1994). Eosinophil adhesion to nasal polyp endothelium is P-
selectin-dependent. J. Exp. Med., 180, 371 ± 376.
PATTERSON, R. (1969). Laboratory models of reaginic allergy. Prog.
Allergy, 13, 332 ± 407.
PATTERSON, R., HARRIS, K.E. & PRUZANSKY, J.J. (1983). Induction
of IgE-mediated cutaneous, cellular, and airway reactivity in
rhesus monkeys by Ascaris suum infection. J. Lab. Clin. Med.,
101, 864 ± 872.
PICKER, L.J., KISHIMOTO, T.K., SMITH, C.W., WARNOCK, R.A. &
BUTCHER, E.C. (1991). ELAM-1 is an adhesion molecule for
skin-homing T cells. Nature, 349, 796 ± 799.
VARKI, A. (1994). Selectin ligands. Proc. Natl. Acad. Sci. U.S.A., 91,
7390 ± 7397.
WADA, Y., SAITO, T., MATSUDA, N., OHMOTO, H., YOSHINO, K.,
OHASHI, M., KONDO, H., ISHIDA, H., KISO, M. & HASEGAWA, A.
(1996). Studies on selectin blockers. 2. Novel selectin blocker as
potential therapeutics for in¯ammatory disorders. J. Med.
Chem., 39, 2055 ± 2059.
RAY, M.C., THARP, M.D., SULLIVAN, T.J. & TIGELAAR, R.E. (1983).
Contact hypersensitivity reactions to dinitro¯uorobenzene
mediated by monoclonal IgE anti-DNP antibodies. J. Immunol.,
131, 1096 ± 1102.
SMITH, C.W., KISHIMOTO, T.K., ABBASS, O., HUGHES, B., ROTH-
LEIN, R., MCINTIRE, L.V., BUTCHER, E. & ANDERSON, D.C.
(1991). Chemotactic factors regulate lectin adhesion molecule 1
(LECAM-1)-dependent neutrophil adhesion to cytokine-stimu-
lated endothelial cells in vitro. J. Clin. Invest., 87, 609 ± 618.
SOLLEY, G.O., GLEICH, G.J., JORDON, R.E. & SCHROETER, A.L.
(1976). The late phase of the immediate wheal and ¯are skin
reaction. Its dependence upon IgE antibodies. J. Clin. Invest., 58,
408 ± 420.
WALSH, L.J., TRINCHIERI, G., WALDORF, H.A., WHITAKER, D. &
MURPHY, G.F. (1991). Human dermal mast cells contain and
release tumor necrosis factor alpha, which induces endothelial
leukocyte adhesion molecule 1. Proc. Natl. Acad. Sci. U.S.A., 88,
4220 ± 4224.
WATSON, S.R., FENNIE, C. & LASKY, L.A. (1991). Neutrophil in¯ux
into an in¯ammatory site inhibited by a soluble homing receptor-
IgG chimaera. Nature, 349, 164 ± 167.
WELLER, A., ISENMANN, S. & VESTWEBER, D. (1992). Cloning of
the mouse endothelial selectins. Expression of both E-and P-
selectin is inducible by tumor necrosis factor alpha. J. Biol.
Chem., 267, 15176 ± 15183.
SPERTINI, O., LUSCINSKAS, F.W., KANSAS, G.S., MUNRO, J.M.,
GRIFFIN, J.D., GIMBRONE JR., M.A. & TEDDER, T.F. (1991).
Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts
with an inducible endothelial cell ligand to support leukocyte
adhesion. J. Immunol., 147, 2565 ± 2573.
(Received June 19, 2000
Revised September 25, 2000
Accepted September 26, 2000)
British Journal of Pharmacology vol 131 (8)