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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3430-3438
Galectin-3 Activates the NADPH-Oxidase in Exudated but not
Peripheral Blood Neutrophils
By
Anna Karlsson,
Per Follin,
Hakon Leffler, and
Claes Dahlgren
From The Phagocyte Research Laboratory, the Department of Medical
Microbiology and Immunology, University of Göteborg,
Guldhedsgatan 10, S-413 46 Göteborg, Sweden; the Department of
Medical Microbiology and the Department of Infectious Diseases,
University of Linköping, Sweden; and Center for Neurobiology and
Psychiatry, University of California, San Francisco.
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ABSTRACT |
Galectin-3, a lactose-binding mammalian lectin that is secreted from
activated macrophages, basophils, and mast cells, was investigated with
respect to its ability to activate the human neutrophil NADPH-oxidase.
The galectin-3-induced activity was determined with in vivo exudated
cells (obtained from a skin chamber) and compared with that of
peripheral blood neutrophils. Galectin-3 was found to be a potent
activator of the NADPH-oxidase only in exudated neutrophils and the
binding of galectin-3 to the surface of these cells was increased
compared with peripheral blood cells. Different in vitro priming
protocols resulting in degranulation were used to mimic the exudation
process in terms of increasing the receptor exposure on the cell
surface. Galectin-3 could induce an oxidative response similar to that
in exudated cells only after a significant amount of the intracellular
organelles had been mobilized. This increase in oxidative response was
paralleled by an increased binding of galectin-3 to the surface of the
cells. The major conclusion of the study is that galectin-3 is a potent stimulus of the neutrophil respiratory burst, provided that the cells
have first experienced an extravasation process. The results also imply
that the neutrophil response to galectin-3 could be mediated through
receptors mobilized from intracellular granules, and we report the
presence of galectin-3-binding proteins in such organelles.
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INTRODUCTION |
THE HUMAN neutrophils play a key role in
the innate immune response to infection. They act at inflammatory
sites, which they reach after targeting and extravasation from the
peripheral blood stream where they are normally present. Upon
interaction with invading microorganisms or inflammatory mediators they
produce large amounts of toxic oxygen radicals (superoxide anion and
hydrogen peroxide) by activation of the NADPH-oxidase. Although the
mechanism by which such activation is induced and regulated in vivo
remains unclear, multiple extracellular mediators have been found to
activate the neutrophil NADPH-oxidase in vitro, and multiple
intracellular signaling pathways have been implicated.1,2
The degree of activation as well as the subcellular localization of the
toxic oxygen radicals generated is determined by the identity of the agonist and the cell-surface receptor involved in the activation process.3,4
Most in vitro studies of neutrophil function have been performed using
neutrophils isolated from peripheral blood, whereas activation in vivo
occurs after extravasation into the tissue. Earlier published data show
that exudated neutrophils are primed with respect to the function of
the NADPH-oxidase, and as a consequence react with an enhanced response
compared with peripheral blood neutrophils to a variety of
mediators.5-7 Hence, to understand neutrophil activation in
vivo, it is of paramount importance to examine exudated neutrophils.
Among known neutrophil-activating mechanisms is the binding of
bacterial lectins to neutrophil cell-surface glycoproteins; these
glycoproteins presumably act as cell-surface receptors whose activation
often results in a signaling cascade that in turn may cause activation
of the NADPH-oxidase.8 This raises the possibility that
also endogenous mammalian lectins may play a role in neutrophil activation. There are many types of mammalian lectins thought to play
various roles in inflammation,9,10 and one, galectin-3, has
been found to bind to the surface of peripheral blood
neutrophils11 and a variety of other immune
cells.12,13 Galectin-3 has been reported to activate the
NADPH-oxidase in peripheral blood neutrophils but only if the cells
were also treated with cytochalasin B,14 a substance known
to disrupt the microfilament system in the cell.15
Galectin-3, a member of a family of  -galactoside-binding
proteins,12 is produced by activated macrophages. Thus,
neutrophils that have extravasated into inflamed tissue might encounter
galectin-3. To model this situation, we examined the ability of
galectin-3 to activate the NADPH-oxidase in neutrophils that had been
primed by in vivo exudation into a skin chamber. We found that
galectin-3 is a potent activator of exudated neutrophils but not of
peripheral blood neutrophils. Because the exudation-related priming
apparently results in a dramatic increase in neutrophil responsiveness
to galectin-3, we also examined the possibility that this is caused by
mobilization of galectin-3 receptors from intracellular granules to the
cell surface.
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MATERIALS AND METHODS |
Isolation of human neutrophils.
Exudated neutrophils were obtained from chambers placed on unroofed
skin blister lesions on the volar surface of the forearms of healthy
human volunteers as previously described.6 In each experiment, two chambers with three 0.6-mL wells covering the lesions
were used. The chambers were filled with autologous serum, and the
neutrophils were allowed to accumulate in the chambers for 24 hours.
More than 95% of the cells obtained from the chambers were
neutrophils.
Blood neutrophils were isolated from heparinized whole blood (obtained
from the same person as was carrying the skin chambers) or from buffy
coats from healthy blood donors, using dextran sedimentation and
Ficoll-Paque (Pharmacia, Uppsala, Sweden) gradient
centrifugation.16 All cells were washed and resuspended
(1 × 107/mL) in Krebs-Ringer phosphate buffer
containing glucose (10 mmol/L), Ca2+ (1 mmol/L), and
Mg2+ (1.5 mmol/L) (KRG, pH 7.3). This isolation procedure
allows for cells to be isolated with minimal mobilization
effects.17
Preparation of galectin-3.
Recombinant human galectin-3 (apparent molecular weight of 31 kD; Fig 1) was produced in
Escherichia coli and purified as previously
described.18 The lectin was stored at 4°C in
phosphate-buffered saline (PBS; pH 7.2) containing lactose (150 mmol/L). When used, the lectin preparation was applied to a
gel-filtration column (PD10; Pharmacia) to remove lactose, and diluted
to 400 µg/mL in PBS. The carboxyl-terminal domain fragment of
galectin-3 was prepared by digesting recombinant lectin with
collagenase as described.18 Labeling of galectin-3 with
fluorescein isothiocyanate (FITC) was performed essentially according
to Feizi et al.19
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| Fig 1.
Recombinant human galectin-3. Galectin-3 (3 µg)
produced in E coli and purified as described18 is
shown on a Coomassie-stained SDS-polyacrylamide gel (5% to 20%).
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Neutrophil NADPH-oxidase activity.
The NADPH-oxidase activity was determined using a luminol/isoluminol
enhanced chemiluminescence (CL) system.20 The CL activity was measured in a six-channel Biolumat LB 9505 (Berthold Co, Wildbad, Germany), using disposable 4-mL polypropylene tubes with a 0.95-mL reaction mixture containing 106 neutrophils. The tubes were
equilibrated in the Biolumat for 5 minutes at 37°C, after which the
stimulus (0.05 mL) was added. The light emission was recorded
continuously. To quantify intracellularly and extracellularly generated
reactive oxygen species, respectively, two different reaction mixtures
were used. Tubes used for measurement of extracellular release of
superoxide anion contained neutrophils, horseradish peroxidase (HRP; a
cell impermeable peroxidase; 4 U), and isoluminol (a cell impermeable
CL substrate; 2 × 10 5 mol/L).21 By a
direct comparison of the superoxide dismutase (SOD) inhibitable
reduction of cytochrome C and SOD inhibitable CL, 7.2 × 107 cpm were found to correspond to a production of 1 nmol
of superoxide (a millimolar extinction coefficient for cytochrome C of
21.1 was used). Tubes used for measurement of intracellular generation of reactive oxygen species contained neutrophils, SOD (a cell impermeable scavenger for O2; 50 U),
catalase (a cell impermeable scavenger for
H2O2; 2,000 U), and luminol (a cell permeable
CL substrate; 2 × 105 mol/L).
Mobilization of subcellular organelles.
Three different protocols were used for mobilization of neutrophil
subcellular organelles to the cell surface. The first cell population
was merely incubated at 22°C for 60 minutes without additive.17 The second cell population was treated with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). These
cells were incubated at 15°C for 5 minutes after which fMLP (107 mol/L final concentration) was added and the
incubation was continued for another 10 minutes. The cells were
transferred to a heated water bath (37°C) and were allowed to
incubate for 5 minutes. This treatment results in degranulation without
any activation of the NADPH-oxidase.22 The third cell
population was subjected to stimulation by ionomycin, a calcium
ionophore.23 After preincubation of the neutrophils at
37°C for 5 minutes, ionomycin (5 × 107 mol/L final
concentration) was added and the incubation was continued for 5 minutes. All cell populations were sedimented by centrifugation and the
supernatants were collected for marker analysis. The pellets were
suspended in KRG, washed once to remove any prestimulating agent,
resuspended to 1 × 107 cells/mL in KRG and put on ice
until use, either for cell-surface marker analysis or for NADPH-oxidase
activation studies.
Marker analysis.
The mobilization of subcellular organelles was followed by measuring
the exposure of complement receptors 1 and 3 (CR1 and CR3,
respectively) on the neutrophil surface as well as determining the
release of gelatinase, vitamin B12-binding protein and
myeloperoxidase (MPO) into the supernatant. Exposure of CR1 was
measured by labeling the cells with mouse anti-human CD35 (DAKO,
Glostrup, Denmark; M0710; 10 µL to a cell pellet of 106
cells) and subsequent binding of FITC-conjugated goat anti-mouse Ig
(DAKO F0479; 1/2,000). To measure CR3 exposure, the cells were labeled
with phycoerythrin-conjugated monoclonal antibodies (MoAbs) specific
for CD11b (DAKO M741; 10 µL to a cell pellet of 106
cells). The cells were examined by FACScan (Becton Dickinson, Mountain
View, CA24). Vitamin B12-binding protein was
determined with the cyanocobalamin technique as described by Gottlieb
et al.25 Gelatinase and MPO were measured using
enzyme-linked immunosorbent assay (ELISA) methods.26,27
Surface exposure of galectin-3-binding proteins was assessed in
exudated cells as well as in the four different neutrophil populations
described above (control, 22°C, fMLP, ionomycin). Paraformaldehyde
fixed neutrophils were incubated with FITC-labeled galectin-3 (4 µg
to a pellet of 106 cells) in the presence or absence of
lactose (10 mmol/L) for 30 minutes on ice. After washing twice the
cells were examined by FACScan.
Identification of galectin-3 receptor candidates.
Subcellular fractionation was performed according to the method
described by Borregaard et al28 with some modifications. In
short, peripheral blood neutrophils isolated from buffy coats were
treated with the serine protease inhibitor diisopropylfluorophosphate (DFP; 8 µmol/L), disintegrated by nitrogen cavitation (Parr
Instrument Co, Moline, IL), and the postnuclear supernatant was
centrifuged on Percoll gradients. Plasma membranes were separated from
secretory vesicles in a flotation gradient as previously
described.29 Gelatinase granules were separated from the
classical specific granules as described by Kjeldsen et
al.30 The gradients were collected in 1.5-mL fractions by
aspiration from the bottom of the centrifuge tube and the localization
of subcellular organelles in the gradients was determined by marker
analysis of the fractions.
Cytosol was prepared by centrifugation of cavitated neutrophils (as
described above) at 100,000g for 90 minutes (4°C) and collection of the supernatant containing the cytosolic proteins.
Samples of the plasma membrane ( 2), the secretory vesicles (1),
the gelatinase granules (2), the specific granules (1), and the
azurophil granules ( ), respectively, were diluted in nonreducing
sample buffer, boiled for 5 minutes, and applied to the gels in volumes
corresponding to the fractionated content of 5 × 106
cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli31 in 5% to
20% gradient gels. After electrophoresis the proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a Tris-Glycine buffer system.32 The membranes were blocked by incubation
in PBS-Tween (0.05% vol/vol) containing gelatin (3% wt/vol) for 6 hours at room temperature before blotting with galectin-3 (8 µg/mL) in PBS-Tween containing gelatin (1% wt/vol) at 4°C overnight. The
membranes were washed 5 × 5 minutes in PBS-Tween and incubated with
anti-galectin-3 antibodies (anti-Mac-2 antibodies; culture supernatant from the hybridoma M3/38; 1/25) in PBS-Tween containing gelatin (1% wt/vol) for 6 hours at room temperature. After washing twice, the membranes were incubated in HRP-labeled rabbit anti-rat Ig
antibodies (DAKO P0450; 1/1,000) for 1 hour at room
temperature and developed by adding peroxidase substrate (VIP Kit;
Vector Laboratories, Burlingame, CA).
Reagents.
The fMLP, FITC, ATP, EGTA, piperazine-N,N -bis(2-ethane sulfonic acid),
isoluminol, and luminol were obtained from Sigma (Sigma Chemical Co, St
Louis, MO). The SDS was from Fluka Chemie AG (Buchs, Switzerland).
Catalase, SOD, and HRP were purchased from Boehringer Mannheim
(Mannheim, Germany). Dextran, Ficoll-Paque, and Percoll were from
Pharmacia (Uppsala, Sweden). The molecular weight standard proteins
were from Bio-Rad Laboratories (Richmond, CA). The
[57Co]vitamin B12 was supplied by Amersham
Laboratories (Amersham, Buckinghamshire, UK). Ionomycin was purchased
from Calbiochem (La Jolla, CA). Antibodies for the gelatinase-ELISA
were a kind gift from Drs Lars Kjeldsen and Niels Borregaard
(Copenhagen, Denmark).
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RESULTS |
Galectin-3-induced NADPH-oxidase activity.
The ability of galectin-3 to induce superoxide anion production in
human neutrophils was investigated. Galectin-3 was added to human
neutrophils obtained from a skin chamber (exudated cells) or to
neutrophils isolated from peripheral blood. The lectin induced a
pronounced extracellular release (Fig 2a) as well as an intracellular production (Fig 2d) of superoxide anion in exudated
neutrophils, whereas there was no response induced in peripheral blood
cells (Fig 2a and d). The extracellular response induced by galectin-3 was around 25% of that induced by 107 mol/L fMLP (data
not shown). To assess the influence of contaminating lipopolysaccharide
in the galectin-3 preparation on the response, the lectin was
preincubated for 10 minutes with polymyxin B.33,34 No
alteration of the response was seen in the presence of polymyxin B,
indicating that the neutrophil activity was induced by galectin-3 alone
(data not shown).
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| Fig 2.
Galectin-3-induced activation of the NADPH-oxidase in
exudated neutrophils. The NADPH-oxidase activity in peripheral blood neutrophils (106 cells; dashed lines) or exudated
neutrophils (106 cells; solid lines) was assessed. The
figure shows the neutrophil responses to galectin-3 (20 µg/mL; a and
d), galectin-3 (20 µg/mL) in the presence of lactose (10 mmol/L; b
and e), and collagenase digested galectin-3 (CRD; 20 µg/mL; c and
f). The extracellular release of superoxide anion (a through c) was
measured in the presence of HRP (4 U) and isoluminol
(5 × 105 mol/L) while the intracellular production
of superoxide (d through f) was measured in the presence of SOD (50 U), catalase (2,000 U), and luminol (5 × 105 mol/L).
Responses are given as chemiluminescence (CL) units in megacounts per
minute (Mcpm).
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The NADPH-oxidase was not activated by galectin-1, another member of
the galectin family which is also implicated in
inflammation35 (data not shown).
Features of galectin-3 required for activation of exudated
neutrophils.
Galectin-3 consists of a C-terminal carbohydrate recognition domain
(CRD, about 130 amino acids) with affinity for lactose and related
saccharides, and an N-terminal region without known carbohydrate-binding activity (about 120 amino acids).12
The lactose-binding activity of the CRD was required for galectin-3 activation of exudated neutrophils, because the galectin-3-induced NADPH-oxidase activity was inhibited by the addition of soluble lactose
(Fig 2b and e). The N-terminal domain, previously shown to be required
for aggregation and cooperative binding of galectin-3,18 was also required for activation of the neutrophil NADPH-oxidase, illustrated by the fact that galectin-3 CRD, lacking the N-terminal domain, could not induce oxidase activation in exudated cells (Fig 2c
and f).
Surface exposure of galectin-3-binding sites.
Many neutrophil receptors that are stored in the granules and secretory
vesicles in peripheral blood cells are mobilized to the plasma membrane
during the extravasation process.36 To determine if this
applies also for galectin-3 attachment sites, we determined the binding
of FITC-labeled galectin-3 to fixed intact cells by FACS analysis. The
histogram in Fig 3 depicts a representative experiment (of three), showing a marked increase in galectin-3 binding
to exudated cells as compared to peripheral blood cells (control),
supporting that galectin-3-binding receptor structures may be stored
intracellularly in resting cells with the potential to be upregulated
to the cell surface upon cell activation. The sugar specificity of the
binding was confirmed by the fact that it was inhibited by lactose (Fig
3).
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| Fig 3.
Presence of galectin-3-binding sites on the surface of
resting and exudated neutrophils. Exudated cells were compared with resting peripheral blood cells regarding ability to bind galectin-3. The cells (106) were paraformaldehyde fixed, incubated with
FITC-labeled galectin-3 in the presence or absence of lactose (50 mmol/L), and analyzed by flow cytometry. The figure shows a histogram
from one representative experiment out of three.
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In vitro mobilization of subcellular organelles.
To examine the extent to which mobilization of intracellular granules
to the cell surface might explain the increased responsiveness of the
exudated neutrophils to galectin-3, a sequential mobilization of
vesicles and granules was induced in vitro in peripheral blood neutrophils by three different priming protocols: 22°C treatment, fMLP treatment, and ionomycin treatment (see Materials and Methods for
details).
The upregulation of membrane proteins to the cell surface, the release
of granule matrix proteins, as well as the ability of
galectin-3 to activate the NADPH-oxidase in the different cell populations were determined (Fig
4). The most easily mobilized of the neutrophil organelles, the secretory vesicles, are partly mobilized by in vitro "aging" of neutrophils17,37;
ie, the secretory vesicles supply the plasma membrane with new receptors (shown for CR1 and CR3 in Fig 4b) during storage of the cells
at 22°C without addition of any exogenous stimulus. In addition, a
minor release of gelatinase was seen in these cells (Fig 4a). When this
cell population was stimulated with galectin-3, a slight activation of
the NADPH-oxidase was obtained (Fig 4c). In comparison, the
fMLP-treated cells had further upregulated their CR1 and CR3 (Fig 4b)
concomitant with a 20% release of the total cell content of
gelatinase. Because the specific granule marker vitamin
B12-binding protein was not released in these cells (Fig
4a), the released gelatinase must be derived from gelatinase granules
only, which thus have been mobilized to around 40% (as 50% of the
neutrophil gelatinase is localized in these organelles38). The galectin-3-induced extracellular release as well as intracellular production of oxygen radicals in these cells was considerably increased
compared with control cells (Fig 4c). Further release of gelatinase
together with part of the specific granule marker vitamin
B12-binding protein was achieved by stimulating the
neutrophils with the calcium ionophore ionomycin (Fig 4a). When
ionomycin-treated neutrophils were stimulated with galectin-3, the
extracellular response was even higher than corresponding response in
fMLP-treated cells, while the intracellular response was slightly
decreased (possibly as a result of translocation of part of the
specific granule localized NADPH-oxidase to the plasma membrane leading to a depletion of the intracellular oxidase pool) (Fig 4c).
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| Fig 4.
Effect of different priming protocols on
secretion or surface exposure of different granule markers, and on the
oxidative response to galectin-3. Peripheral blood neutrophils were
pretreated as stated in the Materials and Methods section. The top
panel (a) shows release into the medium of markers for gelatinase
granules (gelatinase), specific granules (gelatinase and vitamin
B12-binding protein), and azurophil granules (MPO). The
values are given as percent released marker of the total amount in
control cells. The middle panel (b) shows the surface exposure of the
membrane components CR1 (mobilized from the secretory vesicles) and CR3 (mobilized from secretory vesicles, gelatinase granules, and specific granules), calculated from the mean fluorescence intensity of each cell
population and expressed in percent of the value obtained with control
cells ( ). The lower panel (c) shows the extracellular and
intracellular production of superoxide anion in response to galectin-3
(20 µg/mL). The responses are measured as in Fig 2 and are given as
chemiluminescence (CL) units in megacounts per minute (Mcpm). Data are
given as mean + SD, n = 4. ( ), 22°C; ( ), fMLP; ( ),
ionomycin.
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The galectin-3-induced NADPH-oxidase responses in in vitro-primed
neutrophils were inhibited by lactose, and required the presence of the
N-terminal domain of galectin-3 (Fig 5).Hence, these responses are dependent on the same features of galectin-3 as are the responses of in vivo-exudated neutrophils.
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| Fig 5.
Galectin-3-induced activation of the NADPH-oxidase in in
vitro primed neutrophils. The figure shows the time courses of the CL
responses in primed neutrophils (106 cells) induced by
galectin-3 (20 µg/mL; a and d), galectin-3 in the presence of lactose
(10 mmol/L; b and e), or collagenase digested galectin-3 (CRD; 20 µg/mL; c and f). The experiment was done with the primed cell
population giving the largest response to galectin-3 (Fig 4), ie,
ionomycin-treated cells for extracellular responses (a through c), and
fMLP treated cells for intracellular responses (d through f). The
responses were measured as in Fig 2 and are given as chemiluminescence
(CL) units in megacounts per minute (Mcpm).
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The granule marker expression/release induced by the sequential granule
mobilization was paralleled by an increased binding of galectin-3 to
the cell surface, shown by FACS analysis of cells labeled with
FITC-galectin-3 (Fig 6). This binding of
galectin-3 was inhibited by addition of lactose (data not shown).
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| Fig 6.
Binding of galectin-3 to resting and in vitro primed
neutrophils. In vitro-primed cells (see Materials and Methods) and
resting peripheral blood cells (106) were paraformaldehyde
fixed, incubated with FITC-labeled galectin-3, and analyzed by flow
cytometry. Panel (a) shows a representative histogram while panel (b)
depicts the relative amounts of bound galectin-3, calculated from the
mean fluorescence intensity of each cell population and expressed in
percent of the value obtained with control cells (). The results are
given as mean + SD, n = 8.
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Identification of galectin-3-binding proteins in neutrophil
organelles.
Provided that primed neutrophils are able to respond to galectin-3 due
to the exposure of receptors mobilized from intracellular stores,
galectin-3-binding residues could be expected to be found in such
organelles. To investigate the presence of galectin-3-binding proteins
in subcellular organelles, neutrophils were disintegrated and
fractionated on discontinuous Percoll gradients. Two different gradients were used; the three-step gradient separates the gelatinase granules from the specific granules (Fig
7a) whereas the flotation gradient allows
separation of the plasma membrane from the secretory vesicles (Fig 7b).
The azurophil granules were collected from the bottom of the three-step
gradient (Fig 7a). Proteins from the different organelles and the
cytosol were separated by SDS-PAGE (Fig 8a)and transferred to a blotting membrane. The blot was overlaid with
galectin-3 and bound galectin-3 was detected using anti-galectin-3 and
secondary antibody. Eight galectin-3-binding proteins were revealed
(Fig 8b), and the binding was inhibited in the presence of lactose (Fig
8c).
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| Fig 7.
Distribution of marker molecules in discontinuous Percoll
gradients. A postnuclear supernatant was divided in two and centrifuged on either a three-step Percoll gradient (a) or a flotation gradient (b)
and fractions of 1.5 mL were collected from the bottom of the
centrifuge tube. The fractions from the three-step gradient were
analyzed for myeloperoxidase (marker for azurophil granules; ;  ),
vitamin B12-binding protein (marker for the specific
granules; 1;  ), gelatinase (marker for specific and gelatinase
granules; 1 and 2; ), and total alkaline phosphatase (marker
for secretory vesicles and plasma membrane; 1 and 2; ). The
fractions from the flotation gradient were analyzed for latent alkaline
phosphatase (marker for the secretory vesicles; 1;  ) and
nonlatent alkaline phosphatase (marker for the plasma membrane; 2;
 ). Abscissa, fraction number; ordinate, amount of marker (arbitrary
units). Samples for electrophoresis studies were prepared by pooling
fractions from the three-step gradient (a) as follows: , 1-3; 1,
6-8; 2 10-12; and from the flotation gradient (b) as follows: 1, 10-13; 2 15-18.
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| Fig 8.
Subcellular localization of galectin-3-binding proteins
in neutrophils. The organelles were obtained by fractionation of
disintegrated peripheral blood neutrophils on a three-step Percoll
gradient (, 1, and 2)30 and a flotation gradient
(1 and 2),29 respectively (Fig 7). A
Coomassie-stained SDS-polyacrylamide gel (5% to 20%; a) with
separated proteins from the neutrophil plasma membrane (2),
secretory vesicles (1), gelatinase granules (2), specific
granules (1), and azurophil granules (), respectively, corresponding to the fractionated content of 5 × 106
cells, is shown together with corresponding Western blots (b and c).
The blots were incubated with galectin-3 (8 µg/mL) in the absence (b)
or presence (c) of lactose (100 mmol/L), followed by incubation with
antibodies directed against galectin-3 (anti-Mac-2 antibodies; culture
supernatant from the hybridoma M3/38; 1/25) and finally with
HRP-labeled rabbit anti-rat Ig antibodies (DAKO P0450; 1/1,000). The
blots were developed with peroxidase substrate (VIP Kit; Vector).
Molecular sizes are given in kilodaltons.
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A protein with an apparent molecular weight of 31 kD which was mainly
detected in the plasma membrane fraction (Fig 8b; 2) and stained
also in the presence of lactose (Fig 8c; 2) was identified as
galectin-3 based on the fact that this protein was detected also when
no galectin-3 but only the primary and secondary antibodies were added
to the Western blot (Fig 9; 2). Because
the neutrophil cytosol contains galectin-3 (Fig 9; cytosol), we
hypothesize that the lectin is bound to the plasma membrane after
disintegration of the cells and that it follows the membrane through
the fractionation procedure. This hypothesis gains support from the
fact that only a small amount of the 31-kD protein is found in the
secretory vesicles (Fig 8; 1), although these organelles are plasma
membrane derived.39
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| Fig 9.
A Western blot with separated proteins from neutrophil
plasma membrane (2) and cytosol, corresponding to the fractionated content of 5 × 106 and 1 × 106 cells,
respectively. The blot was incubated with antibodies directed against
galectin-3 (anti-Mac-2 antibodies; culture supernatant from the
hybridoma M3/38; 1/25) followed by HRP-labeled rabbit anti-rat Ig
antibodies (DAKO P0450; 1/1,000), after which the blot was developed
with peroxidase substrate (VIP Kit; Vector). Molecular sizes are given
in kilodaltons.
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The plasma membrane fraction (2) contains one major
galectin-3-binding protein of approximately 150 kD. The secretory
vesicles also contain a 150-kD protein in addition to four proteins of 95, 107, 120, and >200 kD. The gelatinase (2) and specific (1) granules stain equally for galectin-3-binding proteins, exhibiting a
150- and a 120-kD protein in addition to a 110- and a >200-kD protein. The >200-kD protein in the secretory vesicles differs in
molecular weight from that in the gelatinase/specific granules. No
galectin-3-binding proteins were detected in the azurophil granules
().
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DISCUSSION |
We show that galectin-3 is capable of inducing a respiratory burst in
neutrophils, provided that the cells have experienced the extravasation
process. That exudated cells are primed is evident, not only in
response to galectin-3, but to various inflammatory mediators,
chemoattractants, and other ligands.6,7,40-42 The need for
prior extravasation to achieve a responding cell (eg, to galectin-3)
may be a regulatory mechanism, granting a cellular response (such as
release of toxic oxygen radicals) only at sites where it is functional
and necessary, eg, in inflamed or infected areas. Activation of
neutrophils at other sites could result in an undesirable release of
oxygen metabolites and proteases that would risk damage to healthy
tissue.
During the extravasation process, a number of different inflammatory
mediators are generated5,43 and most of these substances have been shown to induce in vitro priming of
neutrophils.44 Although the molecular mechanism(s)
responsible for induction of the primed state is unclear, a review of
the current literature makes it obvious that several
pathways/mechanisms work in parallel.17,37,44-46 Suggested
mechanisms include signaling events such as tyrosine phosphorylation,46 intracellular free calcium
increases,44 and activation of
phospholipases.45 The exposure of new receptors is another
attractive model as molecular base for an augmented response. We know
from an earlier study36 that intracellular organelles are
mobilized to the cell surface during extravasation, resulting in an
increased exposure of various receptors. Human neutrophils contain at
least four such organelles: the azurophil granules, serving primarily
an intracellular role, and the specific granules, the gelatinase
granules and the secretory vesicles, functioning as secretory
organelles.39 These secretory organelles are mobilized
during in vivo exudation; the secretory vesicles are completely
mobilized together with 40% of the gelatinase granules and 20% of the
specific granules while virtually all azurophil granules are
retained.36 The secretory organelles all function as easily
accessible reservoirs of plasma membrane proteins, including various
receptors,38,39 which have been shown to occur in increased amounts on the surface of exudated cells.7,36,42
By use of in vitro priming protocols we also investigated whether the
primed response to galectin-3 in exudated cells could be accounted for
by receptor mobilization. Indeed, the ability to respond to galectin-3
paralleled the mobilization state in the in vitro-primed cells, and,
although correlationally, our data suggest that galectin-3-binding
receptors would reside in the gelatinase and possibly also the specific
granules in unperturbed (resting) cells. This conclusion is based on
the following: strong oxidative responses were induced by galectin-3
only after a significant portion of the gelatinase granules (40%) had
been mobilized to the cell surface, and the extracellular response was
further enhanced after the specific granules (25%) and additional
gelatinase granules (calculated to about 20%) had been mobilized (Fig
4).
There are additional facts supporting receptor upregulation as a
probable cause of the primed response to galectin-3. The study by
Yamaoka et al14 shows that galectin-3-induced
NADPH-oxidase activity in peripheral blood neutrophils was dependent on
the pretreatment of the cells with cytochalasin B, a fungal metabolite that blocks the polymerization of contractile
microfilaments15 thereby facilitating
degranulation.47 Further, when resting, exudated, and in
vitro-primed neutrophils were labeled with fluorescent galectin-3, the
amount of bound lectin increased with increasing granule mobilization.
However, we want to emphasize that we cannot at this time conclude
whether the galectin-3-induced activation is dependent on a certain
density/avidity of attachment sites, allowing for aggregation of the
lectin to occur, or whether a specific receptor structure has to appear
on the cell surface to make the cells responsive.
Our data suggest that the potential galectin-3 receptor(s) should be
found in intracellular organelles in resting cells, and we could also
identify galectin-3-binding glycoproteins in the secretory vesicles,
gelatinase granules, specific granules, and in the plasma membrane. It
remains to be determined whether these galectin-3-binding proteins are
similar to any earlier identified galectin-3-binding
protein14,48,49 and if they have signaling capacity. The
fact that galectin-3 binds to many different proteins (of which the
majority probably will lack signaling capacity) makes it hard to
determine binding characteristics (number of binding sites and
affinity) of the signaling receptor, even after the identity of the
protein has been established. Yamaoka et al14 found two
galectin-3-binding proteins after cell-surface iodination of resting
neutrophils (not treated with cytochalasin B). One of them was
suggested to be the nonspecific cross-reacting antigen, NCA-160.
Interestingly, the NCA-160 is localized mainly in the specific granules
in resting neutrophils50 and is thus a possible activation-mediating receptor.
Galectin-3 was found to be present in human neutrophils, detected in
the plasma membrane and cytosol fractions (Figs 8 and 9). We
hypothesize that galectin-3 is present in the cytosol in intact
neutrophils, but that it attaches to the outside of the plasma
membrane-derived vesicles after disintegration of the cells. Galectin-3 is probably not bound to any higher extent to the plasma membrane of intact cells, since the secretory vesicles, which are
derived from the plasma membrane and largely contain the same membrane
markers, do not contain any substantial amounts of the lectin (Fig 8).
Finally, we conclude that two different signals are induced by
galectin-3 in neutrophils, one that induces assembly of the NADPH-oxidase in the plasma membrane (giving extracellular release of
oxygen radicals) and one that induces assembly of the NADPH-oxidase in
the specific granule membrane (giving rise to intracellularly produced
oxygen radicals).4,51,52 Whether these signals are generated by a single galectin-3 receptor, or if two different receptors are involved, remains to be determined. Recent evidence from
both plant and animal cells suggest a role for intracellular reactive
oxygen species as intracellular messengers, mediating activation of
transcription factors and protein phosphorylation events.53
How the galectin-3-induced intracellular oxygen radicals may take part
in a signal transduction cascade that leads to functional effects in
the cell is an intriguing question for future studies.
| |
FOOTNOTES |
Submitted July 21, 1997;
accepted December 18, 1997.
Supported by the Swedish Medical Research Council, the King Gustaf V
80-Year Foundation, the Fredrik and Ingrid Thurings Foundation, the
Lars Hierta Foundation, the Swedish Society for Medical Research, the
Tore Nilsson Foundation, the Swedish Society for Medicine, the
Anna-Greta Crafoord Foundation for Rheumatological Research, and the
Swedish Association Against Rheumatism.
Address reprint requests to Anna Karlsson, PhD, The Phagocyte Research
Laboratory, Department of Medical Microbiology and Immunology,
Guldhedsgatan 10, S-413 46 Göteborg, Sweden.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The skillful technical assistance of Lisbeth Björck and Marie
Samuelsson is gratefully acknowledged.
| |
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S. G. Correa, C. E. Sotomayor, M. P. Aoki, C. A. Maldonado, and G. A. Rabinovich
Opposite effects of galectin-1 on alternative metabolic pathways of L-arginine in resident, inflammatory, and activated macrophages
Glycobiology,
February 1, 2003;
13(2):
119 - 128.
[Abstract]
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E. Feuk-Lagerstedt, M. Samuelsson, W. Mosgoeller, C. Movitz, A. Rosqvist, J. Bergstrom, T. Larsson, M. Steiner, R. Prohaska, and A. Karlsson
The presence of stomatin in detergent-insoluble domains of neutrophil granule membranes
J. Leukoc. Biol.,
November 1, 2002;
72(5):
970 - 977.
[Abstract]
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V. del Pozo, M. Rojo, M. L. Rubio, I. Cortegano, B. Cardaba, S. Gallardo, M. Ortega, E. Civantos, E. Lopez, C. Martin-Mosquero, et al.
Gene Therapy with Galectin-3 Inhibits Bronchial Obstruction and Inflammation in Antigen-challenged Rats through Interleukin-5 Gene Downregulation
Am. J. Respir. Crit. Care Med.,
September 1, 2002;
166(5):
732 - 737.
[Abstract]
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J. Bylund, A. Karlsson, F. Boulay, and C. Dahlgren
Lipopolysaccharide-Induced Granule Mobilization and Priming of the Neutrophil Response to Helicobacter pylori Peptide Hp(2-20), Which Activates Formyl Peptide Receptor-Like 1
Infect. Immun.,
June 1, 2002;
70(6):
2908 - 2914.
[Abstract]
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G. A. Rabinovich, N. Rubinstein, and L. Fainboim
Unlocking the secrets of galectins: a challenge at the frontier of glyco-immunology
J. Leukoc. Biol.,
May 1, 2002;
71(5):
741 - 752.
[Abstract]
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J. Almkvist, C. Dahlgren, H. Leffler, and A. Karlsson
Activation of the Neutrophil Nicotinamide Adenine Dinucleotide Phosphate Oxidase by Galectin-1
J. Immunol.,
April 15, 2002;
168(8):
4034 - 4041.
[Abstract]
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D. Granfeldt, M. Samuelsson, and A. Karlsson
Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase
J. Leukoc. Biol.,
April 1, 2002;
71(4):
611 - 617.
[Abstract]
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S. Sato, N. Ouellet, I. Pelletier, M. Simard, A. Rancourt, and M. G. Bergeron
Role of Galectin-3 as an Adhesion Molecule for Neutrophil Extravasation During Streptococcal Pneumonia
J. Immunol.,
February 15, 2002;
168(4):
1813 - 1822.
[Abstract]
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J. Almkvist, J. Faldt, C. Dahlgren, H. Leffler, and A. Karlsson
Lipopolysaccharide-Induced Gelatinase Granule Mobilization Primes Neutrophils for Activation by Galectin-3 and Formylmethionyl-Leu-Phe
Infect. Immun.,
February 1, 2001;
69(2):
832 - 837.
[Abstract]
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C. Dahlgren, A. Karlsson, and F. Sendo
Neutrophil secretory vesicles are the intracellular reservoir for GPI-80, a protein with adhesion-regulating potential
J. Leukoc. Biol.,
January 1, 2001;
69(1):
57 - 62.
[Abstract]
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P. Nangia-Makker, Y. Honjo, R. Sarvis, S. Akahani, V. Hogan, K. J. Pienta, and A. Raz
Galectin-3 Induces Endothelial Cell Morphogenesis and Angiogenesis
Am. J. Pathol.,
March 1, 2000;
156(3):
899 - 909.
[Abstract]
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C. Dahlgren, T. Christophe, F. Boulay, P. N. Madianos, M. J. Rabiet, and A. Karlsson
The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A4 receptor
Blood,
March 1, 2000;
95(5):
1810 - 1818.
[Abstract]
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E. Feuk-Lagerstedt, E. T. Jordan, H. Leffler, C. Dahlgren, and A. Karlsson
Identification of CD66a and CD66b as the Major Galectin-3 Receptor Candidates in Human Neutrophils
J. Immunol.,
November 15, 1999;
163(10):
5592 - 5598.
[Abstract]
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A. Karlsson
Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors
Infect. Immun.,
July 1, 1999;
67(7):
3461 - 3468.
[Abstract]
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Abstract
Introduction
Abstract