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Departments of
* Medical Microbiology and Immunology and
Rheumatology, Phagocyte Research Laboratory, Göteborg University, Göteborg, Sweden; and
Department of Microbiology, Immunology, and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactoside binding capacity
mediated by conserved sequence elements located in their carbohydrate
recognition domain
(CRD)3
(1). Members of the galectin family have been identified
in various tissues of a wide range of organisms, ranging from sponges
and nematodes to humans. At present, 12 mammalian galectins have been
identified and described (2, 3, 4). They are present in cell
cytosol and nuclei as well as in extracellular compartments, suggesting
that they exert many different functions. In fact, galectins have been
found to mediate cell-cell interactions and adhesion, to induce various
intracellular signals that may regulate cell growth and apoptosis, and
to mediate host-pathogen interactions (5, 6, 7). Accordingly,
the expression patterns of different galectins change during
development and may be altered, e.g., in carcinomas and at sites of
inflammation (8, 9). Galectin-1 is a noncovalently linked homodimeric protein with two 14-kDa subunits containing one CRD of 134 amino acids each. Like several other galectins, galectin-1 is produced by many cell types, under both normal and pathological conditions (for a review see Ref. 5) and has been suggested to possess a variety of functions, including inhibition of myoblast adhesion (10), involvement in cell cycle progression (11), and inhibition of fibroblast proliferation (12). So far, the role of galectin-1 in inflammatory processes is not very well defined, but some supportive data exist. Galectin-1 induces apoptosis in immature thymocytes as well as in activated mature T cells (13, 14), processes that grant proper development and resolution of an induced immune response. Furthermore, galectin-1 is up-regulated in inflammatory and activated rat peritoneal macrophages (15) as well as in virally infected CD8+ T cells (16). Colocalization of galectin-1 with complement receptor (CR)3 on the surface of murine macrophages implicates a role in CR3-mediated attachment to extracellular matrix and in phagocytic events (17). The presence of galectin-1 in inflamed tissue and the secretion of the protein by activated inflammatory cells inspired us to investigate the influence of this lectin on neutrophil function.
Neutrophils, together with monocytes/macrophages, play a key role in the innate immune response to infection. These phagocytic cells are activated by inflammatory mediators produced by microorganisms and host inflammatory cells at the inflammatory sites. The neutrophil granulocytes are equipped with various killing systems to eradicate infectious agents, and an appropriate division of these systems into different granules and vesicles grants a versatile and efficient killing of the prey. At least four types of granules/vesicles are present in resting neutrophils: azurophil granules, specific granules, gelatinase granules, and secretory vesicles. These organelles differ from each other not only with regard to content of soluble molecules but also with regard to membrane receptor structures (18).
Following mobilization of these granules to the cell surface, new receptor structures appear on the plasma membrane, allowing for the cell to interact with chemoattractants, extracellular matrix proteins, and other molecules of importance for cell function. Receptor-ligand interactions may lead to triggering of the NADPH-oxidase, which produces oxygen free radicals that are of major importance for the neutrophil bactericidal activity. However, the oxygen radicals may also cause destruction of host tissue, promoting and enhancing an inflammatory process. Several different types of molecules are able to activate the NADPH-oxidase, e.g., vasoactive amines, complement-derived peptides, cytokines, bacterial peptides, and phospholipid-derived substances. We have recently added galectin-3 to this list of inflammatory mediators (19). This endogenous lectin has the ability to activate the neutrophil NADPH-oxidase, provided that the cells have first extravasated into an inflammatory environment. In this study we show that galectin-1 activates neutrophils with a similar pattern to that of galectin-3 but possibly via a different set of receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human galectin-1 was produced in Escherichia
coli BL21 with the galectin-1 coding sequence in the pET-3d
(Novagen, Madison, WI) (10). The bacteria were
cultivated overnight at 37°C in an ampicillin (50 µg/ml)-containing
Luria-Bertani medium and transferred to a new growth medium. The
production of galectin-1 was induced by addition of isopropyl
-D-thiogalacto-pyranoside (0.5 mM). After a
3-h incubation at 37°C the bacteria were harvested by centrifugation,
resuspended in PBS (pH 7.2) containing EDTA (1.8 mM), 2-ME (4 mM), and
PefaBloc (1 mM), and were lysed by sonication. Galectin-1 was purified
from the homogenate by affinity chromatography on lactosyl-Sepharose
and eluted with lactose (150 mM). The lactose was removed from the
lectin preparation by gel filtration on a PD10 column (Pharmacia,
Uppsala, Sweden) and the pure galectin was stored at -70°C. The
purity of the protein was analyzed by SDS-PAGE (20) under
reducing conditions and showed a single band with an apparent molecular
mass of 14 kDa after Coomassie staining.
Labeling of galectin-1 with FITC was performed according to Feizi et al. (21).
Isolation of neutrophils
Blood neutrophils were isolated as described by Böyum (22) from heparinized whole blood or buffy coats from healthy volunteers, using dextran sedimentation and Ficoll-Paque (Pharmacia) gradient centrifugation.
Exudated neutrophils were obtained from chambers placed on unroofed skin blister lesions on the volar surface of the forearms, as previously described (23). 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 left to accumulate in the chambers for 24 h.
All cells were resuspended in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM) (Krebs-Ringer glucose, pH 7.3) and stored on ice until use.
Subcellular fractionation
Subcellular fractionation of neutrophils was performed according
to Borregaard et al. (24). In short, neutrophils isolated
from buffy coats were treated with the serine protease inhibitor
diisopropyl fluorophosphate (8 µM) and disintegrated by nitrogen
cavitation (Parr Instrument, Moline, IL), and the postnuclear
supernatant was centrifuged on Percoll gradients. Gelatinase granules
were separated from the classical specific granules as described by
Kjeldsen et al. (25). The gradients were collected in 1-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 (see Marker analysis).
Aliquots were taken for Western blot analysis before the fractions
corresponding to the azurophil granules (
-fraction), specific
granules (1-fraction), gelatinase granules
(2-fraction), and plasma membrane/secretory
vesicles (
-fraction) were pooled and purified from Percoll by
ultracentrifugation (110,000 x g for 2 h at
4°C). The membranes were separated from the matrix by repeated
freeze-thawing and high-speed centrifugation (110,000 x
g for 1.5 h at 4°C). The level of myeloperoxidase
(MPO) contamination in the membrane portion of the -fraction was
determined to ensure that complete separation was achieved.
SDS-PAGE
Samples of the 1-ml fractions as well as of the pooled membrane
and matrix samples from the -, 1-,
2-, and -fractions, respectively, were
diluted in a nonreducing sample buffer, boiled for 5 min, and applied
in volumes corresponding to 1.4 x 106 cells
on SDS-polyacrylamide gels. After electrophoresis the proteins were
either silver-stained (26) or transferred to
polyvinylidene difluoride (PVDF) membranes and probed with
galectin-1 or galectin-3.
Galectin-1 and galectin-3 overlay
The PVDF membranes were incubated overnight at 4°C in a blocking solution containing 1% gelatin and 0.05% Tween 20 in PBS. To detect galectin-binding proteins the blots were incubated with galectin-1 or galectin-3 (40 µg/ml), respectively, for 2 h at room temperature in the blocking solution. After washing twice in PBS-Tween the blots were incubated with Abs against galectin-1 (polyclonal rabbit anti-rat galectin-1; 1/500) or galectin-3 (anti-Mac-2 Abs; culture supernatant from the hybridoma M3/38; 1/10) for 2 h. The blots were washed twice in PBS-Tween before addition of HRP-conjugated Abs (anti-rabbit-HRP and anti-rat-HRP, respectively; DAKO, Glostrup, Denmark) diluted 1/1000 in blocking solution and incubated for 1 h at room temperature. The galectin-binding proteins were subsequently detected by addition of a peroxidase substrate (VIP kit; Vector Laboratories, Burlingame, CA).
Measurement of NADPH-oxidase activity
The NADPH-oxidase activity was measured using a
luminol/isoluminol-amplified chemiluminescence (CL) system
(27). The CL was measured in a Biolumat LB 9505 (Berthold,
Wildbad, Germany) using polypropylene tubes with a 900-µl reaction
mixture containing 106 neutrophils. The tubes
were equilibrated for 5 min in the Biolumat at 37°C before the
addition of 100 µl of stimulus. The light emission was recorded
continuously starting 15 s after cell stimulation. To quantify the
intracellularly and extracellularly generated reactive oxygen species,
respectively, two different reaction mixtures were used. The
extracellular release of superoxide anion was measured in tubes
containing neutrophils, HRP (a cell-impermeable peroxidase; 4 U), and
isoluminol (a cell-impermeable CL substrate; 6 x
10-5 M). The intracellular production of
reactive oxygen species was measured in tubes containing neutrophils,
superoxide dismutase (SOD; a cell-impermeable scavenger for
O
2; 50 U), catalase (a cell-impermeable scavenger for
H2O2; 2000 U), and luminol
(a cell-permeable CL substrate; 2 x
10-5 M).
Mobilization of subcellular organelles
To mobilize the neutrophil intracellular granules and vesicles to the plasma membrane three different in vitro mobilization protocols were used. One cell population (1 x 107/ml) was kept in room temperature (22°C) for 1 h. The second and third cell populations (1 x 107/ml) were incubated with the chemotactic tripeptide fMLF (10-7 M) for 10 min at 15°C before being transferred to 37°C and further incubated for either 5 or 15 min, respectively. This treatment resulted in granule/vesicle mobilization without triggering the NADPH-oxidase (28). A population of control cells was kept on ice.
The different cell populations were pelleted and the supernatants were used for analysis of granule markers. The cells were washed once and kept on ice until use, for cell surface analysis of markers or for NADPH-oxidase activation studies. The mobilization of intracellular organelles was monitored by measuring the exposure of CR1 and CR3 on the neutrophil surface, as well as by determining the release of gelatinase, vitamin B12-binding protein, and MPO into the supernatant (see Marker analysis).
Marker analysis
The content of alkaline phosphatase in the obtained fractions was measured by hydrolysis of p-nitrophenyl phosphate in the presence of Triton X-100 (0.4%) (29).
The content of vitamin B12-binding protein was determined with the cyanocobalamin technique as described by Gottlieb et al. (30).
Release of MPO was measured by enzymatic activity. The peroxidase substrate 1,2-phenylenediamine dihydrochloride (DAKO) was dissolved according to the manufacturer’s directions and mixed with H2O2 just before use. Twenty-five microliters from each fraction of the gradient or 100 µl of the supernatants of the in vitro mobilized cells were mixed with 100 µl peroxidase substrate in a 96-well plate and incubated for 30 min at room temperature. The reaction was stopped by adding 100 µl 0.1 M H2SO4 to each well and the absorbance was measured at 492 nm.
Release of gelatinase was determined either by an ELISA according to Kjeldsen et al. (31) (for monitoring the in vitro degranulation protocol) or by SDS-PAGE and immunoblotting (for determination of gelatinase in the subcellular fractions) using polyclonal rabbit anti-gelatinase Abs (1/1000; Chemicon International, Temecula, CA) followed by HRP-conjugated anti-rabbit Ig Abs (1/1000; DAKO). The blots were developed by adding a peroxidase substrate (VIP kit).
The exposure of CR1 (CD35) and CR3 (CD11b/CD18) on the neutrophil cell surface was assessed by immunostaining and FACS analysis. Exposure of CR1 was measured by labeling paraformaldehyde-fixed cells (4% at 4°C for 5 min) with mouse anti-human CD35 (10 µl/106 cells; DAKO M710) for 1 h at 4°C and subsequent binding of FITC-labeled goat anti-mouse Ig (1/2000; DAKO F0479) for another hour (32). Measuring of CR3 exposure was performed by incubating the cells at 4°C with PE-conjugated anti-CR3 Abs (CD11b, 10 µl/106 cells; BD Biosciences, Mountain View, CA). To investigate the level of galectin-1 binding to neutrophils the fixed cells were incubated with FITC-labeled galectin-1 (40 µg/ml) for 1 h at 4°C and washed twice with FACSwash (PBS, 0.02% NaN3, EDTA 10-4 M). The analysis of the fluorescent markers was performed by FACScan (BD Biosciences).
Reagents
The isopropyl -D-thiogalacto-pyranoside, fMLF,
FITC, LPS, isoluminol, and luminol were obtained from Sigma-Aldrich
(St. Louis, MO). The SDS was obtained from Fluka (Buchs, Switzerland).
PefaBloc, catalase, SOD, and HRP were purchased from Boehringer
Mannheim (Mannheim, Germany). Dextran, Ficoll-Paque, and Percoll were
obtained from Pharmacia. The molecular mass standard proteins were
purchased from Bio-Rad (Richmond, CA). The
[57Co]vitamin B12 was
supplied by Amersham (Little Chalfont, Buckinghamshire, U.K.). Abs for
the gelatinase ELISA were a kind gift from Drs. L. Kjeldsen and N.
Borregaard (Rigshospitalet, Copenhagen, Denmark).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We investigated the influence of galectin-1 on peripheral blood neutrophils as compared with exudated neutrophils that have been exposed to inflammatory mediators and stress during extravasation in vivo. An experimental model was used where neutrophils were allowed to migrate into an aseptic inflammatory environment, where they were collected. Peripheral blood neutrophils isolated from the same donors were used as control. Production of superoxide anion by the intracellular neutrophil NADPH-oxidase was determined by CL.
Galectin-1 did not induce any superoxide production in peripheral blood
neutrophils (Fig. 1
). However,
stimulation of exudated cells with galectin-1 gave rise to a
substantial oxidative burst. The presence of lactose inhibited the
activation, suggesting a dependency on the CRD of the lectin for
activity.
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The exudation process is accompanied by an increased exposure of
receptor structures on the neutrophil cell surface caused by
mobilization and fusion of receptor-storing granules with the plasma
membrane (33). We investigated the presence of
galectin-1-binding epitopes on the surface of peripheral blood and
exudated neutrophils. Binding of FITC-labeled galectin-1 to exudated
neutrophils was enhanced as compared with peripheral blood cells (Fig. 2). One possible explanation to these
results is the presence of galectin-1-binding epitopes that are not
exposed on the plasma membrane of peripheral blood neutrophils but are
stored elsewhere in the cell. The exudated cells may thus have
mobilized such hidden receptors to the cell surface and bind galectin-1
correspondingly.
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To determine the subcellular localization of potential galectin-1
receptors, peripheral blood neutrophils were fractionated on Percoll
gradients. Using known markers for the different subsets of granules,
the fractions containing each organelle were identified (Fig. 3, upper panel). The azurophil
granules (-fraction) were recovered from the densest fractions
(1, 2, 3, 4, 5), the specific granules
(1-fraction) were found in fractions 10–14,
the gelatinase granules (2-fraction) were
found in fractions 15–17, while the light membranes, composed of
secretory vesicles and plasma membrane (-fraction) were recovered in
fractions 19–22. The lightest fractions (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) consist
of cytosol (25).
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(lower panel), and the
distribution of these proteins differed between the fractions. The
major bands detected were found to be a 28-kDa protein in the dense
fractions (2, 3, 4, 5, 6), a 75-kDa protein in fractions 7–18, and
a 45-kDa protein in the light fractions (19, 20, 21, 22, 23, 24, 25, 26).
The galectin-1-binding structures were further characterized and
compared with earlier identified galectin-3-binding proteins
(34). The membrane and matrix portions of the pooled
fractions were prepared by freeze-thawing and ultracentrifugation, and
the total protein content of the pooled fractions was examined by
silver-staining as shown in Fig. 4A. The protein patterns are
markedly different between the -, -, and -fractions, while the
1 and 2 patterns are
similar to a great extent, in agreement with previously published data
(25).
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B. In the
-fraction the major part of the galectin-1-binding 28-kDa protein
was found in the matrix portion, suggesting that it is a soluble
protein. The 75-kDa galectin-1-binding protein was present in both the
membrane and matrix portions of the 1-fraction
and in the membrane portion of the 2-fraction,
suggesting that it is either a peripheral membrane protein loosely
attached to the granule membrane or a "sticky" matrix protein. The
45-kDa galectin-1-binding protein located in the light fractions of the
gradient (Fig. 3, lower panel) was detected as a faint band
in the membrane portion of the -fraction. An 
45-kDa band was also
present in the cytosol (the lightest fractions; Fig. 3). A comparison
with the silver-stained gel (Fig. 4A) indicated that the
major galectin-1 binding bands were not major components of the
neutrophil granules as the galectin-1 binding pattern and the total
protein content of the silver-stained gel were largely diverging.
Besides the above-mentioned proteins, several major and minor bands
were shown to bind galectin-1 when assessing the concentrated membranes
and matrices. In the -fraction a faint band with an apparent
molecular mass of 66 kDa was present in the membrane portion. This band
was also detected in the silver-stained gel (Fig. 4A). In
the matrix portion of the -fraction several bands were found, among
which three were distinct, namely 75, 55, and 45 kDa proteins,
besides the above-mentioned 28-kDa protein. None of these three bands
was found in the silver-stained gel, indicating that they are minor
components of the -fraction. In the
1-fraction, few bands besides the 75-kDa
protein were found. The most distinct membrane protein detected by
galectin-1 was a protein of >116 kDa. In the matrix portion of the
1-fraction a band of 50 kDa was present in
addition to the 75-kDa protein, while the matrix portion of the
2-fraction contained no major
galectin-1-binding proteins. In the 2 membrane
fraction, the major galectin-1-binding protein beside the 75-kDa
protein was the >116-kDa protein also found in the
1-fraction. This protein was also detected in
the membrane portion of the -fraction.
We have earlier shown that galectin-3 binds to two members of the
CD66/carcinoembryonic Ag-related cell adhesion molecule (CEACAM) family
in the 1- and
2-fractions. To investigate whether the
galectin-1-binding proteins in the same fractions could correspond to
any of the CD66/CEACAM molecules, these fractions were overlaid with
either galectin-1 or anti-carcinoembryonic Ag Ab and the patterns
of the binding were compared. The results (data not shown) suggest that
the 75-kDa galectin-1-binding protein in the -fractions could be a
CEACAM member. This has to be further investigated.
The membrane portion of the -fraction also contained the 45- and
75-kDa proteins mentioned earlier. No strong bands were present in the
matrix portion. A broad band with a molecular size of 25 kDa was
present in all membrane portions and in the matrix portions of the -
and 1-fractions. These may comprise a
degradation product of larger proteins or several small proteins not
possible to separate on the 10% SDS-PAGE.
In the presence of lactose (50 mM), the staining of all galectin-1-binding bands was weaker but not entirely abolished (data not shown). This partial inhibition is probably due to low affinity of galecin-1 for the soluble disaccharide as compared with the complex sugars present on the glycoproteins. When the blots wereincubated only with anti-galectin-1 Ab and secondary Ab (and no galectin-1), none of the bands described above was detected (data not shown).
Comparison of the pattern of galectin-1-binding proteins with that of
galectin-3-binding proteins in the subcellular fractions (Fig. 4, B and C) showed great differences. Galectin-3 did
not bind any matrix proteins in the subcellular organelles, while
galectin-1 bound several such soluble proteins. Furthermore, galectin-3
bound to no proteins in the -fraction but preferably to proteins in
the -fractions. The two major galectin-3-binding bands have the same
molecular mass as the two major components that were isolated earlier
by affinity purification on galectin-3 Sepharose, and which we have
identified as CD66b (CEACAM8; 100 kDa) and CD66a (CEACAM1; 160 kDa)
(34).
Galectin-1 binding correlates with granule mobilization in in vitro primed neutrophils
Although galectin-1 binds to a number of proteins present in the neutrophil, it is reasonable to believe that only one or a few may be responsible for inducing a signal leading to cell activation. To investigate in which subcellular organelle the potential galectin-1 receptor is localized, we attempted to correlate the ability of galectin-1 to activate the neutrophil NADPH-oxidase with the degree of degranulation, and thus the status of receptor exposure.
We used an in vitro priming model in which different stimulation
protocols were used to generate cells that had mobilized their granules
to different (increasing) extents. To monitor the degranulation, we
measured the release of granule markers into the extracellular space
(i.e., vitamin B12-binding protein and
gelatinase; Fig. 5A) as well
as the increased surface exposure of membrane proteins (i.e., CR1 and
CR3; Fig. 5B). The data suggest that incubating neutrophils
at room temperature for 1 h resulted in the release of part of the
secretory vesicles, shown as a significantly increased exposure of CR1
(marker for the secretory vesicle; Ref. 35). The release
of gelatinase was slightly raised above background in these cells.
During incubation with fMLF for 5 min at 37°C (after the 15°C
incubation for 10 min) more secretory vesicles and part of the
gelatinase granules were incorporated into the plasma membrane,
indicated by the significantly increased release of gelatinase and
increased exposure of CR3 on the plasma membrane. Prolongation of the
incubation with fMLF to 15 min at 37°C resulted in fusion of a more
substantial part of the gelatinase granules with the plasma membrane,
seen as an increase in the amount of gelatinase released (from 5
to 60%). Under none of the conditions used could any
degranulation of the specific granules be detected (vitamin
B12-binding protein; Fig. 5A), nor
were the azurophil granules mobilized as measured by release of MPO
(data not shown). Mobilization of the specific and azurophil granules
is difficult to achieve without activating the NADPH-oxidase;
therefore, such experiments were not performed.
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B). Galectin-1-induced NADPH-oxidase activation correlates with granule mobilization
The in vitro primed cell populations were investigated with regard
to NADPH-oxidase activation induced by galectin-1. First we assessed
the intracellularly produced superoxide anion by luminol-amplified CL.
Interestingly, an increase in granule mobilization paralleled an
increase in the galectin-1-induced activation of granule-localized
NADPH-oxidase (Fig. 6). As with the
exudated cells (Fig. 1), the activity in in vitro primed neutrophils
was lactose sensitive (Fig. 6).
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, A–C). In all
three cases a positive correlation between marker up-regulation/release
and superoxide production was achieved, suggesting that degranulation
and galectin-1-induced NADPH-oxidase activation are interdependent
phenomena. Galectin-1-induced oxidase activation also correlated with
galectin-1 binding (Fig. 8D), further supporting the
hypothesis that the activating galectin-1 receptor needs to be
mobilized to the cell surface to be functional. The fact that the
oxidase activity correlated to CR1 up-regulation as well as to
gelatinase release indicates that the activating receptor is localized
in the secretory vesicles (holding CR1) and probably also in the
gelatinase granules.
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The NADPH-oxidase response was measured in primed (a 10
min-incubation at 15°C with fMLF followed by a 15-min incubation at
37°C) neutrophils using galectin-1 of concentrations between 5 and
640 µg/ml. The intra- and extracellular galectin-1-induced
NADPH-oxidase activation showed similar dose-response patterns (Fig. 9). No saturating concentration could be
achieved within the range of concentration used.
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7 µM and
an equilibration time of t1/2 10 h
(36). Although this opens the possibility that galectin-1
occurs as a monomer in the low micromolar range, this is not likely to
have occurred in the present study. When diluted from a concentrated
stock solution (120 µM) and immediately analyzed further, rat
galectin-1 behaves as a dimer on size exclusion chromatography at least
down to 1 µM (tests <1 µM have not been conducted; data
not shown). Because incubation times in the present case were <20 min,
galectin-1 most likely would have remained a dimer at all
concentrations tested.
In contrast to galectin-1, galectin-3 showed totally different
dose-response patterns, with its maximal activity reached by 20
(intracellular response) and 80 µg/ml (extracellular response), as
shown in Fig. 9. Taken together, these results support the biochemical
data (Figs. 3 and 4) suggesting that galectin-1 and galectin-3 use
different receptor structures to induce a neutrophil response. Based on
the dose-response curves, the intra- and extracellular responses
induced by galectin-3 may be suggested to involve separate galectin-3
receptors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the extravasation process, neutrophils are exposed to inflammatory mediators such as ILs, complement factors, and lipid mediators. This induces cellular activities that enhance the neutrophil adhesion, diapedesis, and chemotaxis, as well as the oxidative responsiveness to stimuli. Such enhancement of cellular activity is referred to as priming. The molecular background to this phenomenon has been extensively discussed. The involvement of calcium, protein phosphorylation, and several other mechanisms has been suggested (37, 38), as well as the mobilization of granules accompanied by increased receptor exposure (39). As stated above, we show in this work that the latter effect is of major importance in the priming of the galectin-1-induced responses in human neutrophils.
Monitoring the granule mobilization using in vitro stimulation protocols allowed us to correlate receptor up-regulation with the neutrophil responsiveness to galectin-1. We found a positive correlation between superoxide production and release of gelatinase, as well as up-regulation of CR1 and CR3, while no prior mobilization of the specific granules was necessary. This suggests that the receptors turning the cells from a nonresponding to a responding state are stored in easily mobilized organelles such as the secretory vesicles (and possibly the gelatinase granules). The fact that there are large similarities with respect to the content of membrane proteins between the gelatinase and specific granules suggests that, even though no mobilization of the specific granules is required for a galectin-1 response, these organelles may still constitute a storage pool of galectin-1 receptors.
Both the secretory vesicles and the gelatinase granules contained major
galectin-1-binding proteins, as detected by lectin overlay and
immunoblotting. Galectin-1-binding proteins were present in the
membranes of the specific granules, the gelatinase granules, and the
secretory vesicles, as well as in the matrix of the azurophil granules
and the specific granules. Because the activating receptor is probably
localized to the secretory vesicles and possibly the gelatinase
granules, potential receptor candidates would be the 75- or 120-kDa
proteins in the gelatinase granule membrane or the 45-kDa protein in
the membrane of the -fraction. However, we do not rule out that the
receptor is present in amounts that are not detectable with the lectin
overlay assay. It is also possible that the receptor is modified during
or after mobilization to the cell surface. Such a receptor would not be
detected in its nonmodified, non-galectin-1-binding configuration. The
definite identification of the receptor used by galectin-1 has thus to
await a more detailed investigation. In addition to the membrane-bound
galectin-1-binding proteins, we found matrix proteins with
galectin-1-binding capacity. These proteins probably have no receptor
function but may act as modulators of activation by interfering with
galectin-1 binding to the signaling receptor.
Comparing the data presented in this work with our previously published results on galectin-3 (19), the two lectins appear to share the NADPH-oxidase-activating potential, provided that the neutrophils have been primed before activation. Our studies show that different degrees of granule mobilization are required for the cells to be fully responsive to galectin-1 and galectin-3, respectively, as well as that the dose-response curves are quite different between the two lectins. This suggests that galectin-1 and galectin-3 engage different receptors during neutrophil activation. This is supported by our finding that the galectin-1- and galectin-3-binding patterns differ, with regard to both granule localization and protein identity. In contrast to galectin-1, which seems to bind a receptor localized in the secretory vesicle (and possibly also the gelatinase granules), the receptor(s) for galectin-3 is localized in the gelatinase and specific granules (19). Furthermore, the sizes of the galectin-3-binding proteins in the gelatinase and specific granules are different from the galectin-1-binding proteins in these (and the other) organelles. Although the receptor structures for galectin-1 and galectin-3 differ, they appear to induce signals in the cell, resulting in the activation of the same effector function, i.e., oxidase activation. This is not surprising, because the ability of different receptors to activate the same enzymatic reactions is a well-known fact, discussed extensively by Jordan et al. (40).
The involvement of galectin-1 in inflammatory processes has previously been implicated by Rabinovich et al. (41), who showed that galectin-1 inhibits T cell adhesion to the extracellular matrix, as well as proinflammatory cytokine secretion. Our data support the involvement of galectin-1 in inflammation by implying that this lectin may influence the early innate immune response involving phagocytic cells recruited to an inflammatory site. Galectin-1 has been isolated from various tissues, e.g., skeletal, cardiac and smooth muscle cells (42), human placenta (42, 43), peripheral lymph nodes, ovarian carcinoma (44), and human endothelial cells (45), and has been shown to possess diverse biological effects at these different sites. The production of galectin-1 by the endothelium, and thus the possibility that galectin-1 may be present in peripheral blood, is an interesting notion in aspects of inflammation and in relation to our results. A stimulation of neutrophils and especially activation of the respiratory burst in circulation may be of potential danger to the vascular wall. In such a scenario, the need for neutrophil receptor up-regulation for the induction of galectin-1 responsiveness may play a regulatory role, preventing an exposure of the endothelial cells to toxic oxygen metabolites produced by the neutrophil.
Galectin-1 not only induces extracellular release of toxic oxygen radicals by the plasma membrane-bound NADPH-oxidase but also induces production of intracellular oxygen radicals. The role of such intracellular oxygen metabolites has not yet been proven, but there are some suggested functions. First, intracellularly produced oxygen radicals may have bactericidal effects, and thus galectin-1 may work as a potentiator of the antibacterial functions of the cells. Furthermore, it has recently been shown that the intracellular activation of the NADPH-oxidase in neutrophils induces apoptosis (46). Galectin-1 has previously been shown to be involved in the apoptosis of T cells (13), and whether the galectin-1-induced oxidative response in neutrophils influences apoptotic events will be an intriguing matter to investigate.
The recent findings of galectins in different species, in different tissues, and exerting many varying functions indicate that these molecules are conserved structures that play important parts as mediators in various biological processes. Whether the function is decided by the binding specificity of the different galectins or by the distribution has still to be clarified. However, the fact that some galectins (galectin-1 and galectin-3) have potent inflammatory effects in vitro while others do not (e.g., galectin-4; data not shown) indicates specificity based on fine variations in carbohydrate binding. Whether galectin-1 and galectin-3 have different functions in inflammatory processes, possibly dependent on distribution, is an interesting subject for future studies.
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2 Address correspondence and reprint requests to Dr. Jenny Almkvist, Department of Medical Microbiology and Immunology, Phagocyte Research Laboratory, Box 435, S-405 30 Göteborg, Sweden. E-mail address: jenny.almkvist{at}microbio.gu.se
3 Abbreviations used in this paper: CRD, carbohydrate recognition domain; CR, complement receptor; MPO, myeloperoxidase; PVDF, polyvinylidene difluoride; SOD, superoxide dismutase; CL, chemiluminescence; CEACAM, carcinoembryonic Ag-related cell adhesion molecule.
Received for publication October 1, 2001. Accepted for publication February 6, 2002.
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), vitamin B12-binding protein
(
), alkaline phosphatase (
), and gelatinase (immunoblot),
analyzed as described in Materials and Methods. After
analysis, the azurophil granules (
