Full text of DOI:10.1038/labinvest.3780407

0023-6837/02/8202-147$03.00/0
LABORATORY INVESTIGATION
Vol. 82, No. 2, p. 147, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Galectin-1 Is Overexpressed in Nasal Polyps under
Budesonide and Inhibits Eosinophil Migration
Carine Delbrouck, Isabelle Doyen, Nathalie Belot, Christine Decaestecker,
Rose Ghanooni, Aurore de Lavareille, Herbert Kaltner, Georges Choufani,
André Danguy, Guy Vandenhoven, Hans-Joachim Gabius, Sergio Hassid, and
Robert Kiss
E.N.T. (CD, RG, GC, SH) and Immunology Departments (A de L), Erasmus University Hospital, and Laboratory of
Histopathology (ID, NB, CD, AD, RK), Faculty of Medicine, Université Libre de Bruxelles, and Medical Department
(GV), N.V. AstraZeneca A.S., Brussels, Belgium; and Institute of Physiological Chemistry (HK, H-JG), Faculty of
Veterinary Medicine, Ludwig-Maximilians-University, Munich, Germany
SUMMARY: Because of the importance of galectins for various cellular activities, the influence of the glucocorticoid budesonide
on the level of expression of galectins-1 and -3 was investigated in human nasal polyposis. Ten nasal polyps obtained from
surgical resection were maintained for 24 hours in the presence of various concentrations of budesonide. As quantitatively
demonstrated by means of computer-assisted microscopy, 250 ng/ml (the highest dose tested) induced a pronounced increase
of galectin-1 expression. This feature was observed in nasal polyps from allergic patients but not in those from nonallergic
patients. Since eosinophils represent the main inflammatory cell population in nasal polyps, we investigated the effect of
galectin-1 on their migration levels by means of quantitative phase-contrast computer-assisted videomicroscopy. Our results
show that galectin-1 (coated on plastic supports) markedly reduced the migration levels of eosinophils in comparison to
P-selectin. On the cellular level, marked modifications in the polymerization/depolymerization dynamics of the actin cytoskeleton
(as revealed by means of computer-assisted fluorescence microscopy) and, to a much lesser extent, an increase in the
adhesiveness of eosinophils to tested substrata were detectable. The present study therefore reveals a new galectin-1–mediated
mechanism of action for glucocorticoid-mediated anti-inflammatory effects. (Lab Invest 2002, 82:147–158).
he cause and pathogenesis of nasal polyposis
have still not been clarified, and nasal polyps
1998). Since glycans of cellular glycoconjugates (gly-
coproteins, glycolipids) are increasingly considered to
present code units to endogenous receptors (Gabius,
2000; Reuter and Gabius, 1999), it is reasonable to
propose that the anti-inflammatory potency of glu-
cocorticoids can involve an as yet undescribed mod-
ulation of eosinophil activity via this information path-
way. This reasoning prompted us to study the
expression and functionality of galectins in this
system.
Based on binding and cross-linking distinct ligands
such as glycans on laminin, IgE glycoforms and IgE
receptor (Fc␧RI), integrins or CD2, CD3, CD7 and
CD45 galectins are implicated in different mecha-
nisms including cell adhesion, growth, and apoptosis
regulation and allergic reactions (Gabius, 1997; Kalt-
ner and Stierstovfer, 1998; Liu, 1993; Perillo et al,
1998). In fact, galectin-1 has been shown to down-
regulate cell matrix adhesion and production of inflam-
matory effectors, such as prostaglandin E2 or IFN-␥
from stimulated T cells or macrophages, and to pro-
vide a perspective to combat activated T cell–depen-
dent diseases via induction of T cell–apoptosis (André
et al, 1999; Levi et al, 1983; Offner et al, 1990;
Rabinovich et al, 1999a, 1999b, 2000; Santucci et al,
2000). Ten-fold overexpression of this gene concur-
rent with onset of glucocorticoid-induced apoptosis in
the susceptible human T cell leukemia line CEM C7
T
could in fact represent a de novo inflammatory growth
on the lateral wall of the nose or arise from the anterior
ethmoidal cells (Bernstein et al, 1995). The predomi-
nant inflammatory cells in nasal polyps are eosino-
phils, which may therefore play prominent roles in the
immunologic and inflammatory processes in nasal
polyps (Bernstein et al, 1995; Stoop et al, 1992, 1993).
Glucocorticoids are already used in hospitals to treat
nasal polyps (Bachert and Gevaert, 1999; Barnes,
1998; Stoop et al, 1992) because they suppress in-
flammation processes by acting at different target
sites. It is widely known that glucocorticoids regulate
gene expression of pro- and anti-inflammatory effec-
tors extending to adhesion molecules such as
E-selectin, intercellular adhesion molecules (ICAMs),
and certain mucins (Cronstein et al, 1992; Holgate and
Mavroleon, 1998; Rothenberg, 1998; Tingsgaard et al,
Received September 4, 2001.
This study was supported by research grants awarded by the Fonds de la
Recherche Scientifique Médicale (FRSM, Belgium), AstraZeneca (Brussels,
Belgium), and the W.-Sander-Shiftung (Munich, Germany).
Address reprint requests to: Dr. Robert Kiss, Laboratoire d’Histopathologie,
Faculté de Médecine, Université Libre de Bruxelles, 808 route de Lennik,
1070 Brussels, Belgium. E-mail: rkiss@ulb.ac.be
Laboratory Investigation • February 2002 • Volume 82 • Number 2 147

Delbrouck et al
implicates this lectin in glucocorticoid-mediated lym-
phocytolysis (Goldstone and Lavin, 1991). Regulation
of galectin-1 by mechanisms at the level of transcrip-
tion, translation, or degradation has also been noted
during postnatal rat lung development, in which stage
and duration of treatment with dexamethasone were
found to be critical (Clerch et al, 1987; Sandford et al,
1993). Transcriptional regulation can be attributed to
the presence of a putative steroid-binding site located
at Ϫ210 in the galectin-1 gene (Gitt and Barondes,
1991). This background information explains why we
addressed the question of whether a glucocorticoid
might modulate galectin-1 expression in human nasal
polyps. For this purpose ex vivo organotypical cul-
tures of human nasal polyps, treated with budesonide
or left untreated, were used. Besides monitoring
galectin-1 we also measured galectin-3, a growth
factor–inducible family member without known
steroid-responsive regulation (Kadrofske et al, 1998).
In addition to protein expression, the levels of
accessible binding sites required for functional ac-
tivity were assessed with the labeled galectins. Thus
expression levels for the galectins and their binding
sites were quantitatively determined by means of
computer-assisted microscopy as described previ-
ously (Camby et al, 2001; Gordower et al, 1999).
RT-PCR analysis and Western blotting served as
positive controls. Our data showed that budesonide
markedly increased the level of galectin-1 expres-
sion in human nasal polyps. Next, we investigated
whether galectin-1 can modulate the levels of mi-
gration, adhesion, and activation of human eosino-
phils. The levels of eosinophil migration on
galectin-1 and P-selectin (as bioactive control)–
coated culture supports were monitored in vitro by
means of quantitative phase-contrast computer-
assisted videomicroscopy (De Hauwer et al, 1997,
1998; Nagy et al, 2001); the levels of eosinophil
adhesion were measured by counting the number of
eosinophils adhering to P-selectin and galectin-1–
coated culture supports. The extent of eosinophil
activation was assessed by means of quantitative
computer-assisted fluorescence microscopy with
the eosinophil cationic protein (ECP) in eosinophils
cultured on P-selectin- and galectin-1–coated
supports.
Results
RT-PCR and Western Blotting Analyses
RT-PCR analysis reported in Figure 1 (bottom) shows
that two polyps (P1 and P2) presented significant
amounts of the amplification products for both
galectin-1 and -3 mRNAs. The two Western blot
panels (top), performed on the two same polyps,
confirm protein expression. They also document the
immunospecificity of the two polyclonal antibodies by
using recombinant galectin-1 and -3 as positive con-
trols (Cϩ), respectively. Galectin-1 Western blot anal-
ysis shows that when present in large amounts,
galectin-1 (subunit mol wt 14 kd) can oligomerize even
under reducing conditions. The lack of crossreactivity
between homologous galectin-1 and galectin-3, re-
Figure 1.
Galectin-1 and -3 expression in polyps analyzed by means of Western blotting and RT-PCR on two different polyp samples (P1 and P2). In Western blotting we used
recombinant galectin-1 and galectin-3 as positive controls (Cϩ). The molecular weight of galectin-1 is 14 kd and galectin-3 is 35 kd. The ␤-actin–specific PCR analysis
served as inherent control to show the integrity of the cDNA and the homogeneity of the sample used for the galectin-specific PCR analyses. The specific primer pairs
used for the two galectins yield amplicon sizes of 322 bp for galectin-1 and 718 bp for galectin-3.
148 Laboratory Investigation • February 2002 • Volume 82 • Number 2

Budesonide Up-Regulation of Galectin-1
ported previously for the polyclonals (Kopitz et al,
1998), was also ascertained (data not shown).
R250). For clarity, the data obtained with the two other
concentrations (ie, 10 and 50 ng/ml) are not reported
because they were associated with weaker levels of
statistical significance than the data for the 250 ng/ml
concentration. The p values reported in Figure 3
concern the statistical significance levels (Ͻ0.05) as-
sociated with the differences observed between the
treated (R250) and the untreated condition (CT). Figure
Morphologic Illustration of the Immunohistochemical
Expression of Galectin-1 in Organotypical Cultures of
Human Nasal Polyps
Although Figure 2A morphologically illustrates the
pattern of immunohistochemical expression of
galectin-1 in a piece of nasal polyp maintained for 24
hours under organotypical culture conditions in the
absence of budesonide, Figure 2B shows this pattern
in the presence of 250 ng of budesonide/ml MEM.
These pieces came from two polyps from allergic
patients showing an actual difference in galectin-1
expression. Evidently, the nasal polyp morphology
and histologic structures were well preserved under
the organotypical culture conditions used. Further-
more, whereas galectin-1 was weakly expressed in
the different tissue layers in the absence of budes-
onide (Fig. 2A), budesonide markedly increased
galectin-1 presence (cf. Fig. 2B). This increase was not
so evident in polyps from nonallergic patients (not
shown) as quantitatively confirmed below.
3
shows that a constant and very significant
budesonide-mediated increase in galectin-1 expres-
sion was observed in the surface epithelium (ϩ38% on
average), the glandular epithelium (ϩ51% on average),
and the connective tissues (ϩ65% on average) of the
nasal polyps from the allergic (ALL) patients. A mar-
ginal budesonide-mediated effect was observed in the
nonallergic (N-ALL) patients. Concerning binding sites
budesonide treatment did not significantly (p Ͼ 0.05)
modify their expression in the nasal polyps regardless
of whether they were obtained from the allergic or the
nonallergic patients. A very significant budesonide-
mediated increase in galectin-3 expression was ob-
served in the connective tissues of the nonallergic
patients. The same feature was observed with respect
to the level of galectin-3 binding site expression in the
connective tissues of the allergic patients. A slight
effect was observed in the surface epithelium of the
nonallergic patients with respect to the galectin-3 level
of expression.
Budesonide-Mediated Effects on Expression of Galectin-1
and -3 and Their Binding Sites
All the budesonide-related data in Figure 3 were
obtained at the 250 ng/ml concentration (labeled
As detailed below, the marked and constant
budesonide-mediated increase in galectin-1 expres-
sion in all the histologic structures of the nasal polyps
from the allergic patients prompted us to investigate
whether galectin-1 was able to significantly modify the
biologic behavior of eosinophils.
Galectin-1–Mediated Effects on Eosinophil Migration
Figure 4A illustrates (at a ϫ200 magnification) the
morphologic aspects of human eosinophils plated on
a P-selectin substratum: they appear as refringent
white dots. Figure 4B presents the migration pattern of
these eosinophils over a 12-hour period of quantitative
videomicroscopy on this P-selectin substratum. Fig-
ure 5A schematically illustrates the two quantitative
variables that we used to characterize the level of
eosinophil migration. The AS variable is the average
speed (the full trajectory length divided by the time
used to cover it), whereas the maximum relative dis-
tance from the origin (MRDO) variable measures the
greatest linear distance covered by each eosinophil
(since the beginning of the experiment) normalized by
the duration of the cell observation. These two vari-
ables therefore enable very distinct migratory behavior
patterns to be distinguished. While the cell in the
upper left part of Figure 5A (“d1”) covered a trajectory
with a very short MRDO, even if the cell migration
speed could be increased, the trajectory covered over
the 12 hours of observation by the second cell (“d2”)
had a MRDO value about five times higher. As detailed
below, Figure 5 (B to D) compares the MRDO and AS
value distributions (in terms of median and quartiles)
obtained on eosinophil trajectories with regard to the
Figure 2.
Morphologic illustrations (ϫ200) of the immunohistochemical expression of
galectin-1 in two nasal polyp pieces maintained for 24 hours under organo-
typical culture conditions in the absence of budesonide (A) and the presence
of 250 ng of budesonide/ml MEM (B).
Laboratory Investigation • February 2002 • Volume 82 • Number 2 149

Delbrouck et al
Figure 3.
Quantitative evaluation (by means of computer-assisted microscopy) of the staining intensity (determined by means of the mean optical density variable) reflecting
the expression of galectin-1 and -3 and their binding sites (BS) in nasal polyp samples treated with a concentration of 250 ng/ml (R250) of budesonide or left untreated
(CT). This evaluation was performed on the surface and glandular epithelium and the connective tissue of the nasal polyp samples, and reported separately for the
polyps from the nonallergic (N-ALL, n ϭ 5) and the allergic patients (ALL, n ϭ 5). The data are reported as the mean and its standard error evaluated on 50
measurements (ie, 10 fields evaluated for each sample). MOD ϭ mean optical density; a.u. ϭ arbitrary units.
different supports and reports statistical comparisons
(Mann-Whitney tests) with respect to the P-selectin–
coated support. Figure 5B shows that the largest
relative distance covered by eosinophils (ie, the
MRDO variable) on the plastic support was very similar
(p Ͼ 0.05) to that on the P-selectin–coated support. In
sharp contrast, the MRDO values characterizing eo-
sinophils migrating on galectin-1–coated supports
were markedly reduced when compared with the
values obtained on P-selectin–coated supports (Fig.
5B). The average speed of eosinophil migration was
very similar (p Ͼ 0.05) on plastic versus P-selectin–
coated supports over the first 6 hours of observation
(Fig. 5C), whereas it significantly decreased on plastic
alone between the 7th and the 12th hour of observa-
tion (Fig. 5D). The migration speed of eosinophils on
galectin-1–coated supports was noticeably greater
over the first 6 hours of observation (Fig. 5C) and then
it decreased markedly (Fig. 5D).
Galectin-1–Mediated Effects on Eosinophil Adhesion and
Actin Polymerization/Depolymerization Dynamics
The marked galectin-1–induced decrease both in the
greatest relative distance covered by the eosinophils
and in their speeds of migration during 12 hours of
observation (Fig. 5), prompted us to investigate
whether this inhibitory effect on eosinophil migration
corresponded to a similarly dramatic galectin-1–in-
duced increase in eosinophil adhesion. Naturally, high
150 Laboratory Investigation • February 2002 • Volume 82 • Number 2

Budesonide Up-Regulation of Galectin-1
Figure 4.
A, shows the morphologic appearances (ϫ200) and B illustrates the trajectories of human eosinophils when migrating on a P-selectin–coated substratum. C to E
document the immunofluorescence caused by the presence of eosinophil cationic protein (ECP) (red fluorescence) in eosinophils plated on plastic alone (C), and on
P-selectin–coated (D) and galectin-1–coated (E) culture supports. DAPI staining (blue fluorescence) identifies the cell nuclei.
levels of adhesion will prevent high levels of migration
(Palecek et al, 1997). Figure 6A shows that this was
definitely not the case. In fact, only a slight increase in
eosinophil adhesion level was observed when com-
paring cell behavior on the galectin-1–coated support
and the plastic after 2 hours of observation (hatched
bars). After 12 hours of observation (black bars), a
significant increase in eosinophil adhesion level was
seen for the lowest (G-0.1 ϭ 0.1 ␮g/cm2) and the
intermediate (G-1 ϭ 1 ␮g/cm2) galectin-1 concentra-
tions but not for the highest concentration (G-10 ϭ 10
␮g/cm2), when these galectin-coated supports were
compared with the plastic or the P-selectin–coated
ones. Thus, the marked galectin-1–induced decrease
in eosinophil migration cannot be explained through a
galectin-1–induced increase in their levels of adhesion
to their substrata. We therefore investigated whether
galectin-1 is able to modulate the organization of the
actin cytoskeleton in eosinophils. Indeed, modifica-
tions to the actin polymerization/depolymerization dy-
namics result in marked modifications of cell migration
(Lauffenburger and Horwitz, 1996). Figure 7A illus-
trates the morphologic aspect of the filamentous actin
cytoskeleton pattern in human eosinophils plated on a
P-selectin–coated support, whereas Figure 7B illus-
trates this pattern with respect to the glomerular actin
cytoskeleton. The influence of P-selectin and
galectin-1 with respect to glomerular versus filamen-
tous actin amounts in human eosinophils is shown in
Figure 7C. These data show that after 2 hours
(hatched bars) of eosinophil plating on plastic alone,
the glomerular (red fluorescence) to filamentous
(green fluorescence) actin ratio was largely shifted to
the filamentous actin (ratio G/F Ͻ 0.5). With respect to
plastic supports, plating these eosinophils for 2 hours
on either P-selectin–coated or galectin-1–coated sur-
faces markedly decreased the polymerization of
G-actin (ratio G/F Ͼ 1.5), with the maximum effects
seen for the two highest galectin-1 concentrations
tested (Fig. 7C). After 12 hours of plating, the ratio
between glomerular and filamentous actin reached an
equilibrium for the lowest galectin-1 concentration
(ratio Х 1), whereas glomerular actin was predomi-
nantly detected for the two highest galectin-1 concen-
trations tested here. Glomerular actin prevailed in
eosinophils plated for 12 hours on P-selectin (Fig. 7C).
Thus, the relatively similar (p Ͼ 0.05) migration rate for
eosinophils plated on plastic alone as opposed to
P-selectin–coated surfaces (Fig. 5) was associated
with two completely opposite forms of actin cytoskel-
eton organization, namely a highly organized filamen-
tous actin cytoskeleton for eosinophils plated on plas-
tic alone (ratio G/F Ͻ 0.5) and a weakly organized
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Delbrouck et al
Figure 5.
A, schematically illustrates the trajectories of two cells with different types of behavior pattern. Although the first moved randomly and remained in a small area, the
second established a more extended trajectory (about five times the cell size). Each cell trajectory was characterized by two parameters. The first was the average
speed (the AS variable) defined by the length of the cell trajectory divided by the time taken by the cell to cover it, whereas the second was the maximum relative
distance from the origin (the MRDO variable). This parameter is defined by the greatest linear distance between the reference point (t ϭ 0) and any subsequent
position of the cell (distances d1 and d2 in the figure) divided by the duration of the time of observation of the cell. B, documents the variation (black squares ϭ
median, lower/upper bars ϭ lower/upper quartiles; ie, 25/75%) in the MRDO values observed during the entire experimental period (12 hours) for eosinophils plated
on different supports (PLAS ϭ plastic, P-Sel ϭ 0.2 ␮g/cm2 P-selectin, G-xx ϭ xx ␮g/cm2 galectin-1 with densities of 0.1, 1, and 10 ␮g/cm2). The levels of significance
(p values) were computed in relation to the P-selectin–coated support. Similarly, C and D report the variation in AS values observed during the first and last 6 hours
of the experiment, respectively.
filamentous actin for eosinophils plated on the P-
␮g/cm2) this effect was no longer reliably observable
selectin–exposing surface (ratio G/F Ͼ 1.5).
at this time point.
Galectin-1–Mediated Effects on Eosinophil Activation
Discussion
Figure 4 (C to E) morphologically illustrate the levels of
activation of eosinophils plated on plastic alone and
on P-selectin–coated and galectin-1–coated surfaces
as monitored by immunofluorescence targeted
against ECP. The ECP staining intensity was deter-
mined quantitatively by means of computer-assisted
fluorescence microscopy, as detailed in Figure 6B.
Plating eosinophils on the surface with P-selectin as
compared with plastic alone activated them signifi-
cantly after 2 hours of plating (hatched bars), whereas
no activation was seen after 12 hours of plating (black
bars). Galectin-1 at all three of the concentrations
tested activated the eosinophils markedly stronger
than P-selectin. This effect remained observable after
12 hours of plating at the lowest dose tested for
galectin-1 (0.1 ␮g/cm2). At higher doses (1 and 10
As detailed in the “Introduction,” nasal polyposis is a
relevant model for the study of eosinophil-mediated
inflammatory processes. As stated by Bernstein et al
(1995), various hypotheses have been proposed to
explain the cause of nasal polyps, with the two most
plausible and widely discussed theories being allergy
and inflammation. Eosinophil infiltration is a striking
feature of nasal polyps: eosinophils are markedly more
numerous in most nasal polyps than in the mucosa of
the middle and inferior turbinates (Bernstein et al,
1995; Stoop et al, 1992, 1993). Increased numbers of
macrophages and other lymphocytes are also ob-
served in nasal polyps as compared with normal nasal
mucosa (Bernstein et al, 1995; Stoop et al, 1992,
1993). These types of inflammatory cells, particularly
eosinophils because of their large majority, increase
152 Laboratory Investigation • February 2002 • Volume 82 • Number 2

Budesonide Up-Regulation of Galectin-1
Figure 7.
A and B, illustrate immunofluorescence of the fibrillary actin cytoskeleton
(green) and the glomerular actin cytoskeleton (red) in human eosinophils
plated on a P-selectin–coated support. C, shows (similarly to Fig. 6) the
influence of the type of support on the relative amounts of glomerular versus
filamentous actin in the human eosinophils reported here based on the ratio of
the two immunofluorescence intensities (red/green). The levels of significance
(p values) were computed in relation to data obtained with the P-selectin
support.
Figure 6.
A, shows the numbers (means Ϯ standard errors) of adherent eosinophils
after 2 (hatched columns) and 12 (black columns) hours when plated at a
concentration of 75,000 cells/ml RPMI on five distinct substrata (legend
similar to the one for Fig. 5). Similarly, B reports the levels of ECP-dependent
immunofluorescence intensity for each coating condition. The levels of
significance (p -values) were computed in relation to data obtained with
untreated plastic support.
with the cytokines and chemokines, less is known
about glucocorticoid-mediated anti-inflammatory ef-
fects with respect to adhesion molecule expression.
Most of these studies relate to ICAMs or vascular
cellular adhesion molecules. For example, Tingsgaard
et al (1998) used 100 ␮g of budesonide administered
twice daily in each nostril of 11 patients suffering from
nasal polyposis and observed a budesonide-mediated
down-regulation of ICAM-1 expression in the vascular
endothelium in the nasal polyps. These authors argue
that this effect might interfere with leukocyte extrava-
sation and partially account for the anti-inflammatory
effect of local glucocorticoid treatment in human nasal
polyposis. In the present study, we used organotypical
cultures of human nasal polyps and, as a validation of
this methodology, we also observed a statistically
significant budesonide-mediated decrease in the im-
munohistochemical expression of ICAM-1 in the cul-
tured polyp pieces (not shown). In contrast, budes-
onide did not significantly modify the level of
expression of P-selectin in these organotypical nasal
polyp cultures (not shown). P-selectin acts as a potent
adhesion receptor (located on platelets and endothe-
lial cells) for distinct glycoproteins on the eosinophil
surface (Varki, 1997; Vestweber and Blanks, 1999). As
outlined in the “Introduction,” galectin-1 could be
considered as a further target of glucocorticoids. Our
data clearly indicate that the budesonide markedly
the potential for immunologic reactivity in polyps as
compared with turbinates (Bernstein et al, 1995). Cy-
tokines and growth factors released from the resident
cells of the nasal epithelium might also recruit inflam-
matory cells into nasal polyps, although the exact
order of inflammatory cell interactions in the develop-
ment of nasal polyposis is not yet precisely under-
stood (Bernstein et al, 1995). The large majority of
studies published on the anti-inflammatory effects of
glucocorticoids focus on their action on the levels of
expression of cytokines, chemokines, and growth
factors (Bachert and Gevaert, 1999; Barnes, 1998). In
aggregate, glucocorticoids mediate their anti-
inflammatory effects by increasing the transcription of
anti-inflammatory genes (including, for example,
lipocortin-1 and the leukocyte inhibitory protein) and
by decreasing the transcription of inflammatory ones
(including, for example, granulocyte macrophage-
CSF, exotoxin, IL-2, IL-3, IL-4, IL-11, IL-13, TNF-␣,
RANTES and, in the first place, IL-5, all of which play
major roles in eosinophil pathobiology) (Barnes, 1998;
Holgate and Mavroleon, 1998; Rothenberg, 1998).
Orchestrated by the hormones and mediators, adhe-
sion molecules are actors in the migration of inflam-
matory cells towards an inflammatory site. Compared
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Delbrouck et al
increased the level of expression of galectin-1 in the
different histologic nasal polyp structures analyzed
from the five allergic patients. The fact that this was
not observed in the nasal polyps from the five nonal-
lergic patients has to be investigated. Presentation of
accessible binding sites is not affected, and
galectin-3, too, is apparently not subject to glucocor-
ticoid regulation. The pronounced budesonide-
mediated increase in galectin-1 expression seems to
translate into a notable decrease in eosinophil migra-
tion, a feature that was not observed with P-selectin.
Thus, galectin-1 acted as a nonpermissive substrate
with respect to processes relevant for eosinophil mi-
gration. Biophysical measurements with atomic force
microscopy make it unlikely that a firm and nearly
irreversible adhesion is the reason for this effect
(Dettmann et al, 2000), pointing to intracellular reac-
tions instead. Interestingly, galectin-1 triggers the
same reaction on tumor astrocytes (Camby et al,
manuscript submitted for publication). Based on the
biophysical data, we turned our attention to monitor-
ing the organization of the actin cytoskeleton as a
potential target site of galectin-1–initiated signaling.
Indeed, obvious effects were detected in this respect
with F- and G-actin–specific probes. Preliminary data
that we recently obtained (not shown) suggest that the
galectin-1–induced alterations of the organization of
the actin cytoskeleton in eosinophils might be medi-
ated through the activation of certain Rho GTPases.
They represent one of the major classes of molecules
controlling the organization of the actin cytoskeleton
(Aspenström, 1999). In addition to the impact of
galectin-1 on eosinophil migration, we also describe
for the first time an effect on activation. As revealed by
an obvious increase in the level of ECP expression, the
highest two galectin-1 concentrations markedly acti-
vated the eosinophils plated on the galectin-coated
surface. This feature was observed after 2 hours of
plating and disappeared after 12 hours (except for the
lowest coating concentration). Indeed, after 12 hours
of high galectin-1 concentrations, the eosinophil ECP
levels were similar to those observed in eosinophils
plated onto plastic, ie, in nonactivated eosinophils.
These data suggest that eosinophil activation was
apparently favored in the lower section of the tested
concentration range of galectin-1 relative to the de-
granulation process, which seems to be sensitive to
the highest-dose regimens. By increasing the level of
galectin-1 expression, budesonide decreased the mi-
gration level of the eosinophils and, as shown by the
ECP expression level, caused (at high doses) an
acceleration in their degranulation process. This ac-
celeration may adversely affect the maintenance of a
chronic inflammatory state, thereby adding to the
anti-inflammatory effects of budesonide resulting from
the slowing down eosinophil motility. In conclusion,
the present data describe a new functionality of
galectin-1 on eosinophils, in the course of glucocorti-
coid treatment, different from the proapoptotic activity
on susceptible T cells.
Materials and Methods
Clinical Data and Histopathologic Characteristics
Medical records and samples of nasal polyps were
obtained for 10 patients, with the polyposis diagnoses
made according to the classification that we reported
previously (Hassid et al, 1997). The 10 nasal polyps in
the present study involved massive polyposis and
came from 5 allergic and 5 nonallergic patients. The
allergic versus nonallergic status of the patients was
determined on the basis of the skin prick test and total
and specific IgE expression (Dreborg, 1993).
Ex Vivo Organotypical Cultures of Human Nasal Polyps
The 10 nasal polyps were maintained under organo-
typical culture conditions in a manner identical to the
methodology that we have described for human brain
(Camby et al, 1996), prostate (Janssen et al, 1997),
colon (Philippart et al, 2000), and other cancer tissues
(Gabius et al, 2001). Briefly, after their surgical re-
moval, the nasal polyp specimens were rinsed twice in
MEM (Gibco-Life Technologies, Merelbeke, Belgium)
and immediately cut in half. One half was used for
histopathologic diagnosis, while the other half was
immediately divided into pieces measuring approxi-
mately 2 mm3. To overcome the problem of biologic
heterogeneity, each experimental condition included
between 10 and 15 randomly selected tissue pieces
for each nasal polyp. These pieces were cultured for
24 hours in 30 mm (diameter) ϫ10 mm (height) Petri
dishes (Nunc-Life Technologies, Merelbeke, Belgium)
containing 3 ml of culture medium in the form of MEM
supplemented with a mixture of 0.6 mg/ml glutamine,
200 IU/ml penicillin, 200 IU/ml streptomycin, and 0.1
mg/ml gentamicin (all from Gibco). The effects of three
concentrations (10, 50, and 250 ng/ml) of budesonide
(Rhinocord®; AstraZeneca, Brussels, Belgium) were
studied. For this purpose, budesonide was first dis-
solved in a minimal amount of dimethyl sulfoxide, with
the final dimethyl sulfoxide concentration of less than
0.1%. An equal volume of this solution was added to
the control cultures. After 24 hours of culture, the
samples were fixed in buffered formalin for 5 days.
After paraffin embedding, 5-␮m sections were pre-
pared for quantitative histochemistry as detailed
below.
Determination of Expression of Galectin-1 and -3 and
Their Accessible Binding Sites in Human Nasal Polyps by
Means of Histochemistry
Specific polyclonal antibodies and the label-free or
biotinylated galectins were prepared as described in
detail previously (Camby et al, 2001; Gabius et al,
1991; Gordower et al, 1999). For each treatment
condition, 10 to 15 tissue pieces were embedded in
the same paraffin block from which 5-␮m–thick sec-
tions were cut. These sections were processed with
the anti-galectin-1 and -3 antibodies or the biotinyl-
ated galectins. Incubation with the marker-containing
solutions was performed at 25 Ϯ 1° C for 30 minutes
154 Laboratory Investigation • February 2002 • Volume 82 • Number 2

Budesonide Up-Regulation of Galectin-1
at a concentration of 10 gm/ml. The extent of the
specifically bound probes was visualized by means of
avidin-biotin-peroxidase complex kit reagents (Vector
Laboratories, Burlingame, California), with diamino-
benzidine/H2O2 as chromogenic substrates. The con-
trol reactions included the omission of the incubation
step with the probe (antibody or galectin) and also with
each of the secondary reagents to exclude any stain-
ing by binding of kit reagents, such as the glycopro-
teins avidin or peroxidase. Counterstaining with hema-
toxylin concluded the processing (see Fig. 1).
To evaluate the staining profiles, two quantitative
variables were determined by means of a SAMBA
2005 computer-assisted microscope system (Samba
Technologies, Grenoble, France) with a ϫ40 (aperture
0.50) magnification lens. The labeling index reports the
percentage of tissue area specifically stained by a
given histochemical marker. The mean optical density
(MOD) denotes staining intensity computed on the
immunopositive areas only. Both the way in which we
used the computer-assisted system to quantify the
histochemical staining and the standardization proce-
dures dealing with the manner in which we used the
computer-assisted microscopy have been detailed
elsewhere (Camby et al, 2001; Goldschmidt et al,
1996; Hassid et al, 1997). A negative histologic control
slide (routine processing without the incubation step
involving the primary antibody or the biotinylated
galectin) was analyzed for each specimen under
study. The software used on the computer-assisted
microscope automatically subtracted the labeling in-
dex and MOD values of the negative control sample
from each corresponding positive one.
All the PCR analyses were performed in 50-␮l
reaction media containing 2 ␮l of cDNA, 0.5 U of Taq
polymerase (Qiagen, Hilden, Germany), 50 mM KCl, 20
mM Tris/HCl (pH 9.0), 3 mM MgCl2, dNTPs (0.5 mM
each), and 0.4 ␮M of both sense and antisense prim-
ers. The amplification was performed as follows: 94° C
for 4 minutes, followed by 40 cycles at 94° C for 1
minute, 60° C for 1 minute, and 72° C for 2 minutes.
After a final extension period of 10 minutes at 72° C,
the amplified products were separated by gel electro-
phoresis in 2% Tris-acetate-EDTA (TAE) gels and
visualized by ethidium bromide staining under ultravi-
olet light.
Western Blotting Analyses
Nasal polyp extracts were prepared by sonication in
PBS (20 mM) containing 1 mM PMSF and 10 ␮g of
aprotinin/ml. Nasal polyp lysates containing 10 ␮g/ml
of extracted proteins (determined by the bicinchoninic
acid protein assay; Pierce, Polylabo, Antwerp, Bel-
gium) were loaded into each lane of a 12% polyacryl-
amide gel under denaturing and reductive conditions.
After electrophoresis, the proteins were transferred
onto a Polyscreen polyvinylidene difluoride membrane
(NEN Life Science Products, Boston, Massachusetts)
by tank blotting. Galectins were immunodetected by
affinity-purified rabbit polyclonal anti-galectin-1 and -3
specific antibodies (1 ␮g/ml PBS containing 5% of
milk powder) in conjunction with the conjugate of goat
anti-rabbit immunoglobulin G and horseradish perox-
idase (0.2 ␮g/ml; NEN). Control experiments included
the omission of the incubation step with the anti-
galectin-1 or -3 antibodies (negative control; not
shown) and the electrophoresis of 0.1 ␮g/ml of recom-
binant galectin-1 or -3 as positive controls (Fig. 1).
As detailed previously (Hassid et al, 1997), three
types of histologic structure were analyzed for each
experimental condition, ie, the surface epithelium, the
glandular epithelium, and the connective tissue (Fig.
2). Ten fields of between 60,000 and 120,000 ␮m2
each were scanned for each histologic structure.
Eosinophil Purification
Eosinophils were purified from blood of healthy volun-
teers as previously described (Woerly et al, 1999).
Briefly, granulocyte pellet, containing mainly neutro-
phils, eosinophils, and erythrocytes, was isolated by
density gradient centrifugation on Lymphoprep (Ny-
comed, Oslo, Norway). Erythrocytes were eliminated
by hypotonic saline lysis. Eosinophils were further
purified by incubation with anti-CD16– and anti-CD3–
bound immunomagnetic beads (Miltenyi Biotec
Gmbh, Bergisch Glabdach, Germany) to eliminate
contaminating neutrophils and lymphocytes, respec-
tively. Eosinophils were separated from magnetically
labeled neutrophils and lymphocytes by passage of
cells on a magnetic cell sorter column (Miltenyi Biotec
Gmbh). The purity of eosinophils was always Ͼ95%.
The eosinophils so purified were then placed in culture
media (RPMI; Gibco).
RT-PCR Technique for Galectin-1 and Galectin-3 mRNA
Detection in Human Nasal Polyps
Total RNA was extracted from two distinct nasal
polyps by means of Tripure isolation reagent (Roche
Diagnostics, Mannheim, Germany). Five micrograms
of RNA were used as a template for cDNA synthesis.
Reverse transcription was performed for 50 minutes at
42° C in RT buffer (50 mM Tris/HCl, pH 8.3, 75 mM KCl,
3 mM MgCl2), 10 mM dithiothreitol, dNTPs (0.5 mM
each), random hexamer primers (5 ng/␮l), and 200
Superscript reverse transcriptase (Gibco, Karlsruhe,
Germany). The reaction was terminated by incubation
for 10 minutes at 70° C. cDNA integrity was confirmed
by ␤-actin–specific PCR analysis. The following prim-
ers were used for RT-PCR analysis: galectin-1 sense
ϭ 5' AAC CTG GAG AGT GCC TTC GA 3' and
galectin-1 antisense ϭ 5' GTA GTT GAT GGC CTC
CAG GT 3'; galectin-3 sense ϭ 5' ATG GCA GAC AAT
TTT TCG CTC C 3' and galectin-3 antisense ϭ 5' ATG
TCA CCA GAA ATT CCC AGT T 3'.
Description of the Video Cell-Tracking System Used to
Characterize the Influence of P-Selectin and Galectin-1
on In Vitro Eosinophil Motility Features
The eosinophil motility levels were characterized by
means of a device that is described in detail elsewhere
Laboratory Investigation • February 2002 • Volume 82 • Number 2 155

Delbrouck et al
(De Hauwer et al, 1997, 1998; Nagy et al, 2001).
Briefly, this instrument consists of an inverted-phase
contrast microscope equipped with a black and white
CCD camera, an incubator maintaining the tempera-
ture at 37° C, and a computer containing a frame
grabber and image-processing software that analyses
digitized frames. The software that we had previously
developed enabled each living cell in the culture under
study to be monitored separately and automatically on
the basis of specific morphologic characteristics that,
in turn, enabled the cells to be identified against their
background (the plastic Falcon dishes). Once the
segmentation procedure was complete, each cell was
transformed into its center of gravity. Because an
image was digitized every minute by the computer, the
video system was able to track the trajectory of each
cell by analyzing the movement of its center of gravity.
From these trajectories the average speed (the AS
quantitative variable) and the maximum relative dis-
tance from the origin (the MRDO quantitative variable)
were calculated for each cell analyzed. The AS vari-
able represents the speed of migration, whereas the
MRDO variable describes the greatest linear distance
found between the original and subsequent positions
of a cell, divided by the observation time (De Hauwer
et al, 1997, 1998). All the experiments were performed
over 12 hours with a cell concentration of 75,000
eosinophil/ml RPMI, with one image being recorded
every minute.
Eosinophils were plated on different supports, ie,
regular plastic (control 1) or plastic supports pre-
coated with (a) 0.2 ␮g P-selectin/cm2 (control 2), (b)
galectin-1 at 0.1 ␮g/cm2, (c) galectin-1 at 1 ␮g/cm2,
and (d) galectin-1 at 10 ␮g/cm2. A 0.2 ␮g/cm2 coating
density has already been used by Cheresh et al (1989)
for fibronectin and by Stroeken et al (1998) for laminin.
The 1 and 10 ␮g/cm2 galectin-1 coating concentra-
tions were used to mimic the budesonide-mediated
increase in galectin-1 expression (see “Results”).
Experiments at each condition were performed in
triplicate. A minimum of 202 and a maximum of 315
eosinophil trajectories were analyzed in each experi-
mental condition on the basis of the triplicate analy-
ses. The triplicate analyses were performed indepen-
dently three times for three distinct eosinophil donors.
(Pharmacia and Upjohn, Uppsala, Sweden), which
binds to human ECP and eosinophil protein
X/eosinophil-derived neurotoxin. EG2 therefore stains
activated eosinophils (Tai et al, 1984). The eosinophils
were plated at a concentration of 75,000 cells/ml
RPMI on five distinct substrates, ie, regular plastic or
plastic supports precoated with (a) 0.2 ␮g P-selectin/
cm2, and galectin-1 at (b) 0.1 ␮g/cm2, (c) galectin-1 at
1 ␮g/cm2, or (d) galectin-1 at 10 ␮g/cm2. The ECP
intensity was recorded in 100 individual eosinophils
per each experimental condition by means of quanti-
tative computer-assisted fluorescence microscopy
(using a goat anti-mouse secondary antibody conju-
gated with Alexa 594 red fluorochrome; Molecular
Probes Inc., Eugene, Oregon) after either 2 or 12 hours
of plating on the above-mentioned substrata.
Quantitative Immunofluorescence Analysis of the In Vitro
Determination of Actin Polymerization/Depolymerization
Dynamics
The conversion of filamentous (F) into monomeric or
glomerular (G) actin is a key element in the dynamic
changes involved in cell motility (Theriot and Mitchi-
son, 1991). The influence on actin polymerization of
culturing eosinophils on plastic alone, or on P-selectin
or galectin-1 precoated plastic surfaces, was there-
fore investigated with fluorescent probes specific to
either F- or G-actin. The fluorescent phallotoxin deriv-
ative phallacidin was chosen to label the F-actin, and
fluorescent DNase I was chosen for the G-actin
(Knowles and McCulloch, 1992). Both compounds
bind to actin subunits in
a stoichiometric ratio
(Knowles and McCulloch, 1992). The experimental
protocol is as follows: eosinophil concentrations of
75,000 cells/ml RPMI were plated for 2 hours on
plastic, 0.2 ␮g/cm2 P-selectin, and 0.1 ␮g/cm2, 1
␮g/cm2, or 10 ␮g/cm2 galectin-1 precoated plastic
supports. After this 2-hour period of plating, the cells
were washed with PBS, pH 7.4, and fixed for 15
minutes with formaldehyde 4% in PBS. After three
washes in PBS, the cells were permeabilized for 1
minute at 4° C with Tris-buffered saline (10 mM Tris
and 0.15 M NaCl, pH 7.4) containing 0.01% Triton
X-100, 2 mM MgCl2, 0.2 mM dithiothreitol, and 10%
glycerol (v/v). After three rinses in PBS, the cells were
incubated for 2 hours at room temperature in a mixture
of DNase I conjugated with Texas Red (DNaseI-TR;
Molecular Probes) at a final concentration of 10 ␮g/ml
PBS and phallacidin conjugated with the fluorescein-
like fluorochrome BODIPY FL (Phall-FL; Molecular
Probes) at a final concentration of 5 U/ml PBS. Meth-
anol from the phallacidin stock solution was evapo-
rated before dilution in PBS. After three to five rinses in
PBS, the slides were mounted with Vectashield
mounting medium (Vector Laboratories). The quantifi-
cation of the intensities of green (F-actin) and red
(G-actin) fluorescence was performed using a soft-
ware developed by SAMBA Technologies. The
computer-assisted microscope included a Zeiss Axio-
plan microscope (Zaventem, Belgium) equipped with
excitation/emission filters of 590/615 nm for the
Eosinophil Adhesion Assay
Eosinophils were plated at a concentration of 75,000
cells/ml RPMI on different substrata, ie, regular plastic
or plastic supports precoated with (a) 0.2 ␮g P-
selectin/cm2, (b) galectin-1 at 0.1 ␮g/cm2, (c)
galectin-1 at 1 ␮g/cm2, or (4) galectin-1 at 10 ␮g/cm2.
The numbers of adherent eosinophils were recorded
after 2 and 12 hours of culture. Ten fields of 1 mm2
were analyzed per experimental condition, all of which
were performed in triplicate.
Eosinophil Activation Assay
The extent of eosinophil activation was monitored with
the EG2 monoclonal anti-human ECP/EPX antibody
156 Laboratory Investigation • February 2002 • Volume 82 • Number 2

Budesonide Up-Regulation of Galectin-1
of corticosteroids: The glucocorticoid receptor regulates
leukocyte adhesion to endothelial cells and expression of
endothelial-leukocyte adhesion molecule 1 and intercellular
adhesion molecule 1. Proc Natl Acad Sci USA 89:9991–9995.
DNaseI-TR and of 505/512 for the Phall-FL, and with a
Hamamatsu C4880 Dual-mode cooled CCD camera
(Tokyo, Japan). For experiments at each experimental
condition (performed in triplicate), photographs of 200
cells were taken (for both fluorochromes) with a ϫ100
lens and at identical exposures for each (red or green)
fluorescence.
De Hauwer C, Camby I, Darro F, Decaestecker C, Gras T,
Salmon I, Kiss R, and Van Ham P (1997). Dynamic charac-
terization of glioblastoma cell motility. Biochem Biophys Res
Commun 232:267–272.
Statistical Analyses
De Hauwer C, Camby I, Darro F, Migeotte I, Decaestecker C,
Verbeek C, Danguy A, Pasteels J-L, Brotchi J, Salmon I, Van
Ham P, and Kiss R (1998). Gastrin inhibits motility, decreases
cell death and increases cell proliferation in human glioblas-
toma cell lines. J Neurobiol 37:373–382.
Statistical comparisons of the data were performed
with the Student t test (one-way variance analysis for
two groups) after a check of the equality of variance
using the Levene test and by the normal distribution
fitting of the data by means of the ␹2 test of goodness
of fit. When these parametric conditions were not
satisfied, the nonparametric Mann-Whitney test was
performed. All the statistical analyses were performed
using Statistica (Statsoft, Tulsa, Oklahoma).
Dettmann W, Grandbois M, André S, Benoit M, Wehle AK,
Kaltner H, Gabius H-J, and Gaub HE (2000). Differences in
zero-force and force-driven kinetics of ligand dissociation for
␤-galactoside-specific proteins (plant and animal lectins,
immunoglobulin G) monitored by plasmon resonance and
dynamic single molecule force microscopy. Arch Biochem
Biophys 383:157–170.
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