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Infection and Immunity, July 1999, p. 3461-3468, Vol. 67, No. 7
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human
Neutrophils by Interaction with Mobilizable Receptors
Anna
Karlsson*
The Phagocyte Research Laboratory, Department
of Medical Microbiology and Immunology, University of
Göteborg, Göteborg, Sweden
Received 9 November 1998/Returned for modification 8 January
1999/Accepted 16 April 1999
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ABSTRACT |
Wheat germ agglutinin (WGA), a lectin with specificity for
N-acetylglucosamine and sialic acid, was investigated with
respect to its ability to activate the NADPH-oxidase of in
vivo-exudated neutrophils (obtained from a skin chamber), and the
activity was compared to that of peripheral blood neutrophils. The
exudate cells responded to WGA, by both releasing reactive oxygen
species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic
the in vivo-exuded neutrophils with regards to receptor exposure,
peripheral blood neutrophils were induced to mobilize their granules
and vesicles to varying degrees (in vitro priming), prior to challenge
with WGA. The oxidative response to WGA increased with increasing
levels of granule mobilization, and the receptor(s) could be shown to
reside in the secretory vesicles and/or the gelatinase granules in
resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions
containing these organelles. The extra- and intracellular
NADPH-oxidase responses showed differences in sialic acid
dependency, indicating that these two responses are mediated by
different receptor structures.
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INTRODUCTION |
Many neutrophil functions involve
lectin-carbohydrate interactions, e.g., neutrophil adhesion to the
endothelium initially relies on the action of selectins
(carbohydrate-binding proteins that mediate the attachment of
leukocytes to endothelial carbohydrates [46]) and
recognition and phagocytosis of bacteria in the absence of opsonins
most often involve microbial lectins binding to neutrophil glycoconjugate receptors, a process called lectinophagocytosis (41). Also plant lectins possess biological activities in
relation to mammalian cells and have been used to study a wide variety of biological processes. Many plant lectins are toxic, some may aggregate erythrocytes of different blood groups (hemagglutination), and yet others function as mitogens of T and B lymphocytes (42, 45, 52). In relation to neutrophils, lectins with different carbohydrate specificities, e.g., concanavalin A binding to mannose and
wheat germ agglutinin (WGA) binding to N-acetylglucosamine (GlcNAc) and sialic acid, have been shown to induce cellular responses (10, 22, 34, 39).
Human neutrophil granulocytes depend on many adhesion-related events in
order to be able to fulfill their major task of eliminating foreign
intruders by ingestion and killing. A prerequisite for the proper
function of the neutrophil is thus their capability to mobilize new
adhesion molecules and receptors to the cell surface in addition to
releasing proteolytic enzymes and inflammatory mediators in the
vicinity of the cell. The receptor delivery system consists of at least
three different subcellular organelles: the secretory vesicles, the
gelatinase granules, and the specific granules, which can be mobilized
to the cell surface, merging with the plasma membrane, in a
hierarchical manner. Among the membrane components of these
intracellular organelles are adhesion molecules, receptors for opsonins
and chemoattractants, and receptors for bacteria (4, 27,
29). Most studies on neutrophil activation have been conducted on
cells isolated from peripheral blood. However, the neutrophils exert
their function in vivo mainly after extravasation into the tissue. This
process is accompanied by an increased surface expression of various
receptors concomitant with a loss of other surface markers as well as
granule matrix components (48). The fact that neutrophil
extravasation is associated with quantitative as well as qualitative
changes in the cellular responsiveness to external stimuli (6, 18,
28) further stresses the importance in neutrophil research of
performing functional studies not only on resting peripheral blood
neutrophils but also on cells that have mobilized their intracellular
receptor stores. In the context of lectin-phagocyte interaction and the
requirement for receptor mobilization, we have recently shown that
galectin-3, a mammalian, lactose-specific lectin, activates the
neutrophil respiratory burst in exudated cells, whereas no response is
induced in peripheral blood cells (28). We could also
demonstrate that the galectin-3-induced activation is dependent on
granule mobilization. The present study was conducted to analyze the
effect of granule mobilization on the ability of WGA to induce
respiratory burst activity in neutrophils. This lectin has earlier been
shown to be a potent activator of neutrophil oxidative burst, provided
that cytochalasin B was present during the interaction (39),
indicating that this lectin activates neutrophils, provided that the
cells can degranulate. This fact encouraged the use of WGA as a model
activator for studies of mobilizable receptor structures.
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MATERIALS AND METHODS |
Isolation of phagocytic cells.
Exuded neutrophils were
harvested from skin chambers placed on unroofed skin blister lesions on
the volar surfaces of the forearms of healthy human volunteers as
previously described (6, 18). In each experiment, two
chambers with three 0.6-ml wells covering the lesions were used. The
chambers were filled with an autologous serum, and the neutrophils were
allowed to accumulate in the chamber for 24 h. More than 95% of
the cells harvested from the chambers were neutrophils.
Peripheral blood neutrophils were isolated from buffy coats obtained
from the blood bank at Sahlgren's University Hospital, Göteborg,
Sweden. After dextran sedimentation at 1 × g and
hypotonic lysis of the remaining erythrocytes, the neutrophils were
separated from mononuclear leukocytes by centrifugation in a
Ficoll-Paque gradient (5).
The neutrophils were washed twice and resuspended in Krebs-Ringer
phosphate buffer containing glucose (10 mM), Ca
2+ (1 mM),
and Mg
2+ (1.5 mM) (KRG; pH 7.3). The cells were stored on
ice and used
within 4
h.
Depletion of sialic acid.
Neutrophils were incubated with
Clostridium perfringens neuraminidase (0.2 U/ml in KRG) on
ice for 5 min to detach terminal sialic acid residues, after which the
cells were washed once. The function of the neuraminidase was routinely
controlled by measuring the cellular response to a GalNAc
-binding
Actinomyces naeslundii strain, a response that is
dramatically increased after the removal of sialic acid on the
neutrophils, due to the unmasking of a GalNAc-containing receptor
(unpublished observation and reference 47).
Mobilization of subcellular organelles.
Three different
protocols were used for the mobilization of neutrophil subcellular
organelles. The secretory vesicles were partly mobilized by incubating
peripheral blood cells at room temperature (22°) for 1 h without
an additive (1). The secretory vesicles were fully mobilized
by stimulation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Cells were incubated at
15°C for 5 min, after which fMLP (10
7 M final
concentration) was added and the incubation was continued for another
10 min. The cells were transferred to a heated water bath (37°C) and
were allowed to incubate for 5 min. This treatment (referred to as fMLP
5') results in degranulation without activating the NADPH-oxidase
(38). The third protocol used the same fMLP-induced mobilization as described above, the only difference being a prolonged incubation at 37°C (10 min; referred to as fMLP 10') which further increased the degranulation. All cell populations were sedimented by
centrifugation, and the supernatants were collected for marker analysis. The cell pellets were suspended in KRG, washed once, resuspended to 107 cells/ml in KRG and stored on ice until use.
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 and vitamin B
12-binding protein into
the supernatant. CR1 was measured by labeling the cells with mouse
anti-human CD35 (M0710; 10 µl to a cell pellet of 10
6
cells; Dakopatts) and subsequent binding of fluorescein
isothiocyanate-conjugated
goat anti-mouse immunoglobulin (F0479;
1/2000; Dakopatts). To
measure CR3 exposure, the cells were labeled
with phycoerythrin-conjugated
monoclonal antibodies specific for CD11b
(M741; 10 µl to a cell
pellet of 10
6 cells; Dakopatts).
The cells were examined by a FACScan (Becton
Dickinson, Mountain View,
Calif. [
35]). The level of vitamin
B
12-binding protein was determined with the cyanocobalamin
technique
as described by Gottlieb et al. (
20), and
gelatinase was measured
by using an enzyme-linked immunosorbent assay
(ELISA) method (
31).
Neutrophil respiratory burst activity.
The NADPH-oxidase
activity was measured by using luminol- or isoluminol-enhanced
chemiluminescence (CL) systems (14, 36). The CL activity was
measured in a six-channel Biolumat LB 9505 apparatus (Berthold Co.,
Wildbad, Germany), with disposable 4-ml polypropylene tubes. Samples
(0.1 ml; 106 cells) were withdrawn from the neutrophil
suspensions, added to the reaction mixture (0.8 ml; see below), and
placed in the Biolumat apparatus to be equilibrated for 5 min at
37°C. The stimulus (0.1 ml of WGA in KRG; 20 µg/ml) was added, and
the light emission was recorded continuously. In order to quantify
intracellularly and extracellularly generated reactive oxygen species,
two different reaction mixtures were used (for a review see reference
14). Tubes used for the measurement of the
extracellular release of the reactive oxygen species contained
neutrophils, horseradish peroxidase (HRP; a cell-impermeable
peroxidase; 4 U), and isoluminol (a cell-impermeable CL substrate;
2 × 105 M) (36). Tubes used for the
measurement of the intracellular generation of reactive oxygen species
contained neutrophils, superoxide dismutase (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 M).
Subcellular fractionation.
Subcellular fractionation was
performed according to the method described by Borregaard et al.
(3) with some modifications. In short, neutrophils were
disrupted by nitrogen cavitation (Parr Instrument Company, Moline,
Ill.), and the postnuclear supernatant was centrifuged on Percoll
gradients. Plasma membranes were separated from secretory vesicles by
using a flotation gradient as previously described (13).
Gelatinase granules were separated from the classical specific granules
as described by Kjeldsen et al. (32). 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
gradient was determined by marker analysis of the fractions. The level
of vitamin B12-binding protein (marker for the specific
granules) was determined with the cyanocobalamin technique as described
by Gottlieb et al. (20). Gelatinase (marker for the specific
and gelatinase granules) and myeloperoxidase (MPO; marker for the
azurophil granules) levels were measured by using ELISA methods
(31, 49). The alkaline phosphatase (marker for secretory
vesicles and plasma membranes) level was measured by hydrolysis of
p-nitrophenyl phosphate (2 mg/ml) in the presence or absence
of Triton X-100 (0.4%) (15). The HLA class I antigen
(marker for the plasma membrane) level was determined by a mixed ELISA
(2).
SDS-PAGE, Western blotting, and WGA overlay.
Samples of the
plasma membrane (
2), the secretory vesicles (1), the gelatinase
granules (2), and the specific granules (1) were diluted in a
nonreducing sample buffer, boiled for 5 min, and applied to the
gels in volumes corresponding to the fractionated content of 5 × 105 cells. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed according to the method of
Laemmli (33) in 5 to 20% linear gradient polyacrylamide
gels. After electrophoresis the proteins were transferred to
polyvinylidene difluoride (PVDF) membranes by using a Tris-glycine
buffer system (7). The PVDF replicas were blocked in
phosphate-buffered saline (PBS)-Tween (0.05% Tween 20) containing
gelatin (3% [wt/vol]) at 4°C overnight before being incubated with
WGA-HRP (1 µg/ml) in PBS-Tween containing gelatin (1% [wt/vol]) at
4°C overnight. After the replicas were washed (6 times for 5 min
each) in PBS-Tween the bound WGA-HRP was detected by using a peroxidase
substrate (VIP kit; Vector). The reaction was stopped by extensive
washing of the replicas in water.
Inhibitors.
With the exception of ethanol, the inhibitory
substances were diluted in dimethyl sulfoxide and stored in a stock
solution at 
20°C. Fresh working solutions in KRG were prepared new
for every experiment. When the inhibitors were used during measurements of superoxide anion, they were added to the samples during
equilibration and were preincubated with the cells for appropriate
times (see Table 1), after which WGA (2 µg/ml) was added.
Reagents.
The p-nitrophenyl phosphate, Triton
X-100, fMLP, wortmannin, staurosporine, pertussis toxin, isoluminol,
and luminol were obtained from Sigma (Sigma Chemical Co., St. Louis,
Mo.). SDS was from Fluka Chemie AG, Buchs, Switzerland. Catalase, SOD,
and HRP were purchased from Boehringer (Mannheim, Germany). Dextran, Ficoll-Paque, and Percoll were from Pharmacia (Uppsala,
Sweden). WGA and griffonia simplicifolia II (GS-II) were obtained from EY Laboratories, San Mateo, Calif. The molecular weight standard proteins were from Bio-Rad Laboratories, Richmond, Calif. The [57Co]vitamin B12 was supplied by Amersham
Laboratories (Little Chalfont, Buckinghamshire, England). Ionomycin and
calyculin A were purchased from Calbiochem (La Jolla, Calif.).
Carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK) originated
from S. Schlegel, University of Geneva, Geneva, Switzerland, and is
available from Bachem, Basel, Switzerland. Okadaic acid was a generous
gift from Lars Edebo, University of Göteborg, Göteborg,
Sweden. Antibodies for the gelatinase ELISA were a kind gift from Lars
Kjeldsen and Niels Borregaard, Copenhagen, Denmark.
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RESULTS |
WGA-induced production of superoxide anion in exudate
neutrophils.
The WGA-induced NADPH-oxidase activity in
exudate neutrophils was followed by luminol- or
isoluminol-amplified CL. The terminal component of the
NADPH-oxidase, the b cytochrome, resides both in the plasma
membrane and in the membranes of subcellular granules and vesicles
(9, 50). Superoxide anion produced by the plasma membrane-localized NADPH-oxidase will be released from the cell into
the extracellular milieu and can be exclusively measured with
isoluminol, a membrane-impermeable CL amplifier (36). The superoxide anion produced by the granule-localized NADPH-oxidase can be
measured by using the membrane-permeable CL amplifier luminol in
combination with SOD and catalase, which consume the extracellularly produced radicals. WGA induced an extensive oxidative response in
exudate neutrophils giving rise to superoxide anion production both
extra- and intracellularly, while no significant superoxide anion
production was detected in the peripheral blood cells (Fig. 1). Thus, the extravasation process
transferred the neutrophils from a nonresponding to a responding state
with regard to WGA stimulation.
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FIG. 1.
WGA-induced NADPH-oxidase activation in exudate and
peripheral blood neutrophils. Shown is the time course of the CL
response induced by WGA (2 µg/ml) in exudate (closed circles) and
peripheral blood (open circles) neutrophils (106 cells).
The extracellular responses were measured in the presence of isoluminol
(5 × 105 M) and HRP (4 U), while the intracellular
responses were measured in the presence of luminol (5 × 105 M), SOD (50 U), and catalase (2,000 U). The CL is
given in increments of 106 cpm (Mcpm). The figure shows a
representative experiment (n = 6).
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Mobilization of neutrophil granules.
Most of the receptors in
peripheral blood neutrophils are localized in mobilizable subcellular
organelles such as the secretory vesicles or the gelatinase and
specific granules (4). Exudate cells have mobilized their
secretory vesicles and parts of their gelatinase granules and specific
granules (48), suggesting that the WGA receptors mediating
the NADPH-oxidase activation need to be mobilized from at least one of
these subcellular organelles in order to be available for lectin
binding. To investigate this hypothesis and make an attempt to disclose
the identity of the storage organelle(s), the sequential mobilization
of vesicles and granules was induced in peripheral blood neutrophils by
the use of three different preactivation protocols. The mobilization of
membrane proteins to the cell surface, the release of granule matrix
proteins, and the ability of WGA to activate the NADPH-oxidase were
determined in these preactivated cell populations.
The extent to which neutrophil intracellular organelles were mobilized
to the cell surface was assessed by determining the
upregulation of CR1
and CR3 on the neutrophil cell surface. In
resting cells, CR1 is
localized in the secretory vesicles, the
most easily mobilized
neutrophil organelles. During incubation
at room temperature for 1 h, the CR1 expression on the cell surface
increased by 50% (Fig.
2). Pretreatment of the cells with fMLP
at 15°C and heating to 37°C for 5 and 10 min, respectively, further
increased the CR1 expression (around 150 and 200%, respectively).
CR3
is localized in both the secretory vesicles, the gelatinase
granules,
and the specific granules. Thus, CR3 mobilization (Fig.
2) could
reflect the degranulation of either of these three organelles.
Therefore, the release of specific markers for the gelatinase
and
specific granules was measured in the extracellular fluid
after
incubation under the different conditions used. The mobilization
of
gelatinase granules was monitored by measuring the release
of
gelatinase (50% of which is localized to these organelles,
the rest
being localized in the specific granules). Incubation
at room
temperature for 1 h induced a release of around 7% of
the
cellular content of gelatinase (Fig.
2). Under these conditions,
very
little release of vitamin B
12-binding protein could be
detected
(Fig.
2), indicating that the secreted gelatinase was almost
entirely
derived from the gelatinase granules and not from the specific
granules. Thus, room temperature incubation of the cells induced
a
release of approximately 15% of the gelatinase granules. Incubation
with fMLP and heating for 5 min further upregulated the gelatinase
granules (around 30%), while the specific granules were almost
entirely retained (Fig.
2). The treatment of the peripheral blood
cells
with fMLP and heating for 10 min mobilized a substantial
part of the
gelatinase granules together with around 10% of the
specific granules,
shown by the release of vitamin B
12-binding
protein (Fig.
2).
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FIG. 2.
Effects of different mobilization protocols on the
surface exposure of complement receptors and secretion of granule
markers. Neutrophils were isolated, resuspended in KRG, and divided
into four portions treated as follows: (i) incubated on ice (control),
(ii) incubated at 22°C for 1 h, (iii) incubated with fMLP
(107 M) at 15°C for 10 min and then at 37°C for 5 min, and (iv) incubated with fMLP (107 M) at 15°C for
10 min and then at 37°C for 10 min. The top panel 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
value of each cell population and expressed as a percentage of the
value obtained with control (resting) cells. The bottom panel shows the
release into the medium of markers for gelatinase granules (gelatinase)
and specific granules (vitamin B12-binding protein). The
values are given as the percent released marker of the total amount in
control cells. Data are means plus standard deviations (error bars)
from three experiments.
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The WGA-induced NADPH-oxidase activity increased in parallel with the
mobilization of subcellular organelles, i.e., after
incubating the
cells at room temperature for 1 h the activity
was considerably
increased compared to resting cells; after mobilization
induced by fMLP
and heating for 5 min the activity was markedly
higher, and the
response was raised even further in cells stimulated
with fMLP and
heated for 10 min (Fig.
3).
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FIG. 3.
Effects of different mobilization protocols on the
WGA-induced activation of peripheral blood neutrophils. Shown is the
time course of the CL response induced by WGA (2 µg/ml) in peripheral
blood neutrophils (105 cells) treated as follows: (i)
incubated on ice (control; open circles), (ii) incubated at 22°C for
1 h (closed circles), (iii) incubated with fMLP (107
M) at 15°C for 10 min and then at 37°C for 5 min (open triangles),
and (iv) incubated with fMLP (107 M) at 15°C for 10 min
and then at 37°C for 10 min (closed triangles). The extracellular
responses were measured in the presence of isoluminol (5 × 105 M) and HRP (4 U), while the intracellular responses
were measured in the presence of luminol (5 × 105
M), SOD (50 U), and catalase (2,000 U). The CL is given in increments
of 106 cpm (Mcpm). The figure shows a representative
experiment (n = 5).
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The increase in superoxide anion production in the different cell
populations after WGA stimulation correlated with the mobilization
of
the secretory vesicles (CR1 expression) and the gelatinase
granules
(release of gelatinase) (Fig.
4),
indicating that the
receptor responsible for activation probably
resides in either
or both of these subcellular organelles in resting
peripheral
blood cells.
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FIG. 4.
Correlation between neutrophil degranulation and
NADPH-oxidase activity. Mean values (n = 5) of
extracellular (open squares) and intracellular (filled diamonds) CL
responses obtained with pretreated (22°C, fMLP 5', and fMLP 10')
neutrophils are plotted against mean values (n = 5) of
CR1 mobilization (top), CR3 mobilization (middle), and gelatinase
release (bottom). The correlation coefficients
(r2) for the calculated linear regressions are
given.
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Binding of WGA to proteins from subcellular fractions of human
neutrophils.
To investigate the presence of WGA-binding proteins
in neutrophil subcellular organelles, cells were fractionated on two
different Percoll gradients and subcellular fractions corresponding to
the specific granules (1), the gelatinase granules (2), the
secretory vesicles (1), and the plasma membrane (2) were
collected (Fig. 5). Proteins from these
fractions were separated by SDS-PAGE and blotted onto a PVDF membrane.
WGA-binding glycoproteins were detected by incubation of
the replicas with WGA-HRP and subsequent development with a peroxidase
substrate.
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FIG. 5.
Subcellular fractionation of human neutrophils. Shown is
the distribution of marker molecules in discontinuous Percoll
gradients. Postnuclear supernatants were fractionated on a three-step
gradient (A) or a flotation gradient (B), and fractions of 1.5 ml were
collected from the bottoms of the respective centrifuge tubes. The
fractions from the three-step gradient were analyzed for
myeloperoxidase (marker for azurophil granules;  ; open circles),
vitamin B12-binding protein (marker for the specific
granules; 1; closed squares), gelatinase (marker for specific and
gelatinase granules; 1 and 2; open squares), and alkaline
phosphatase measured in the presence of detergent (marker for secretory
vesicles and plasma membrane; closed triangles). The fractions from the
flotation gradient were analyzed for myeloperoxidase, vitamin
B12-binding protein, latent alkaline phosphatase (marker
for the secretory vesicles; 1; open diamonds), and HLA class I
(marker for the plasma membrane; 2; closed diamonds). Abscissa,
fraction number; ordinate, amount of marker (arbitrary units [AU]).
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All subcellular fractions showed positive staining for WGA-binding
proteins, but the pattern of binding varied significantly
(Fig.
6). However, one protein, a 30-kDa
WGA-binding protein,
was common among the subcellular fractions,
although the amount
in the plasma membrane was very small compared to
the other fractions.
In addition, the specific (
1) and gelatinase
(
2) granules contained
a vast amount of WGA-binding proteins, the
major ones being of
195, 140, 120, 65, 60, and 40 kDa. The specific
granule (
1) fraction
in addition contained a 160-kDa WGA-binding
protein, while two
proteins of 100 and 50 kDa were specific for the
gelatinase granules
(
2). The plasma membrane (
2) and secretory
vesicle (
1) fractions
contained a 20-kDa protein as the major
WGA-binding protein in
addition to the 30-kDa protein.
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FIG. 6.
Analysis of neutrophil proteins that are recognized by
WGA. Proteins from the neutrophil-specific granules (1), gelatinase
granules (2), secretory vesicles (1), and plasma membrane (2),
corresponding to the fractionated content of 5 × 105
cells, were separated on SDS-PAGE gels which were stained with
Coomassie blue (left) or blotted onto a PVDF membrane (right). The blot
was incubated with WGA-HRP (1 µg/ml) and developed with peroxidase
substrate (VIP kit; Vector). Molecular sizes are given to the left in
kilodaltons.
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As stated above, the fact that the NADPH-oxidase responses to WGA
gradually increase in parallel with the mobilization of
the secretory
vesicles and gelatinase or specific granules suggests
that the receptor
mediating the responses may be present in all
mobilizable organelles.
This makes the 30-kDa protein an attractive
receptor
candidate.
Inhibition of the WGA-induced NADPH activity.
The WGA-induced
response was determined in the presence of free
N-acetylglucosamine (GlcNAc; 10 mM). The GlcNAc totally
inhibited both extra- and intracellular CL (data not shown), indicating that WGA indeed interacts with the neutrophils in a
carbohydrate-specific manner.
WGA binds not only GlcNAc but also sialic acid. To determine if sialic
acid-containing glycoconjugates were involved in the
WGA-induced
activation of neutrophils, the cells were treated
with neuraminidase.
The cleavage of sialic acid residues from
the cell surfaces of
responding neutrophils (shown with fMLP 10'
cells; Fig.
7) significantly inhibited the release of
reactive
oxygen species (reduced by around 75%), while the
intracellular
response was not at all affected (Fig.
7). These results
indicate
that the WGA-induced activation of the oxidase leading to an
extracellular
release of oxygen metabolites involves a surface
glycoconjugate
receptor containing sialic acid. In addition, the
receptors (or
receptor epitopes) mediating the two NADPH-oxidase
responses (extra-
and intracellular, respectively) can be concluded to
be different
from each other.
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FIG. 7.
Effect of neuraminidase on WGA-induced NADPH-oxidase
activation. Responding (fMLP 10') neutrophils were treated with 0.2 U
of neuraminidase per ml for 5 min on ice and washed twice. The results
are expressed as the peak values of the CL responses induced by WGA (2 µg/ml) in control (open bars) or neuraminidase-treated (closed bars)
neutrophils (105 cells). The extracellular responses were
measured in the presence of isoluminol (5 × 105 M)
and HRP (4 U), while the intracellular responses were measured in the
presence of luminol (5 × 105 M), SOD (50 U), and
catalase (2,000 U). Shown are the means plus standard deviations (error
bars) of four experiments. The CL is given in increments of
106 cpm (Mcpm).
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Since the intracellular response to WGA was not inhibited by
neuraminidase, the interaction causing this response may involve
a
neutrophil GlcNAc residue functioning as the binding epitope
of the
receptor. The WGA binding to this residue was mimicked
by another
lectin, GlcNAc-binding GS-II. However, GS-II (10 µg/ml)
did not
induce NADPH-oxidase activity in in vitro-primed cells,
and neither
could this lectin inhibit the activation by WGA (data
not
shown).
WGA-induced signal transduction events.
The signal
transduction pathways engaged by the WGA-binding receptors were
analyzed by using inhibitors of different signal-transducing molecules
(Table 1).
To investigate whether either of the signal transduction
pathways involved a heterotrimeric G-protein, responding
cells (fMLP
10') were preincubated in the presence or absence of
pertussis
toxin (0.5 µg/ml) for 120 min at 37°C. When the cells
were challenged
with WGA, the extracellular response was ablated
independent of
whether pertussis toxin was present or not (Fig.
8). The receptor
inducing the activation
of plasma membrane-bound NADPH-oxidase
thus seems to be sensitive to
the prolonged incubation, and consequently
no conclusion can
be drawn as to the involvement of G-proteins
in this
response. The intracellular response to WGA was also heat
sensitive,
although not to the same degree. Pertussis toxin significantly
inhibited the remaining intracellular response (Fig.
8), indicating
that a heterotrimeric G-protein may be involved in the signaling
pathway launched by the WGA receptor to activate intracellular
NADPH-oxidase.
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FIG. 8.
Effect of pertussis toxin on WGA-induced NADPH-oxidase
activation. Responding (fMLP 10') neutrophils were incubated at 4°C
or 37°C in the absence (open bars) or presence (closed bars) of
pertussis toxin (0.5 µg/ml) for 120 min prior to activation. The
results induced by WGA (2 µg/ml) or fMLP (107 M;
extracellular response) (inset) in 105 neutrophils are
expressed as a percentage of the CL response in control (4°C) cells
stimulated correspondingly. The extracellular responses were measured
in the presence of isoluminol (5 × 105 M) and HRP
(4 U), while the intracellular responses were measured in the presence
of luminol (5 × 105 M), SOD (50 U), and catalase
(2,000 U). Shown are the means plus standard deviations (error bars) of
six experiments. Student's t test for paired samples was
used for the statistical analysis of the data. ns, not significant.
|
|
Protein kinase C activity was required for the WGA-induced
NADPH-oxidase activity both intra- and extracellularly, as shown
by the
sensitivity for staurosporine (Table
1). The inhibition
of signaling
through phospholipase D, either by the addition of
ethanol or by
the use of zLYCK (
30,
51), inhibited both the
extra- and
intracellular oxidase activities. Okadaic acid as well
as calyculin A
was used to inhibit protein phosphatases 1 and
2A. In the presence
of these inhibitors, the extracellular response
was enhanced,
while the intracellular response either was slightly
higher than the
control (okadaic acid) or decreased (calyculin
A). Phosphatidylinositol
3-kinase was involved in both responses
as indicated by the
sensitivity to
wortmannin.
| |
DISCUSSION |
This study shows that the binding of WGA to glycoconjugates on
human neutrophils isolated after in vivo exudation results in an
activation of the superoxide anion and hydrogen peroxide-generating NADPH-oxidase. During the process of extravasation, new receptors are
exposed due to the mobilization of intracellular organelles to the cell
surface. These organelles are mobilized to different degrees, the
secretory vesicles being the most easily mobilized followed by the
gelatinase granules and the specific granules, in that order, while the
azurophil granules are essentially retained during the extravasation
process (48). It has earlier been shown that exudate cells
exhibit an enhanced responsiveness to the chemotactic factor fMLP as
well as to complement-derived opsonins, and that these functional
changes are due to the upregulation of fMLP receptors as well as
complement receptors from the vesicle and granule pools (6).
In addition, we have recently shown that the human lectin galectin-3
activates exudate neutrophils but not peripheral blood neutrophils due
to an increased exposure of galectin-3 receptors on the cell surfaces
of exudate cells (28). In light of this, the data presented
here can be interpreted to mean that the receptors for WGA are also
stored in mobilizable organelles. This is supported by earlier studies
of the WGA-induced activation of neutrophil NADPH-oxidase, in which
the presence of cytochalasin B was a prerequisite for activation
(39). Cytochalasin B is a fungal metabolite known to block
motile functions such as locomotion and phagocytosis by inhibiting the
polymerization of contractile microfilaments. However, it also
facilitates the secretion of granule constituents by removing actin
filaments which normally block the access of the granules to the plasma
membrane (24). The response to WGA in cytochalasin B-treated
cells could thus be explained by a facilitated mobilization of granules
associated with an upregulation of intracellular receptors.
To more specifically localize the WGA receptor(s) involved in oxidase
activation, three different in vitro protocols were used to
sequentially mobilize the vesicles or granules in peripheral blood
cells. Both the intra- and extracellular NADPH-oxidase responses induced by WGA increased in parallel with the degree of granule and
vesicle mobilization. Based on these results, the receptors mediating
the oxidative responses are suggested to be localized in the secretory
vesicles and/or the gelatinase granules, possibly with an additional
pool in the specific granules.
A number of WGA-binding proteins could be detected in neutrophil
subcellular organelle fractions. The literature reports a vast amount
of neutrophil proteins isolated by WGA affinity chromatography or
binding WGA on protein blots. Several receptors expose WGA-binding carbohydrates, e.g., the fMLP, C5a, interleukin 8, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors, as
well as Mac-1 (CR3) (8, 16, 21, 26, 40, 44), and some of
these may correspond to proteins that are detected by WGA in this
study. Whether any of these receptors can be activated not only by
their natural ligand but also by a lectin binding to the carbohydrate residues on the receptor remains to be elucidated.
The activation of the NADPH-oxidase can be induced along several
different signal transduction pathways. The stimulation of neutrophils
with fMLP through the fMLP receptor is a G-protein-dependent event
that results in phospholipase C activation leading to the release
of intracellular free calcium and protein kinase C activation. The NADPH-oxidase activity launched by fMLP is exclusively
extracellular (12). In contrast, the elevation of
intracellular calcium by the Ca2+ ionophore ionomycin
induces intracellular NADPH-oxidase activation only. Some stimuli such
as opsonins from the complement system (25), the
mannose-specific lectin concanavalin A (34), and influenza A
viruses (23) give rise to an intracellular production of
reactive oxygen species upon interaction with neutrophils. The signals
generated by WGA mediate both the extracellular release and the
intracellular production of superoxide anion, placing this lectin in
the same category of stimulants as galectin-3 (28) and
protein kinase C activators such as phorbol myristate acetate (37) and dioctanoylglycerol (17).
Based on neuraminidase sensitivity, the activation of the two pools of
NADPH-oxidase (the plasma membrane- and specific granule-localized pools mediating the extracellular release and intracellular production of reactive oxygen species, respectively) can be concluded to be
induced by different receptor structures, and the intracellular signals
induced by these receptors differ, shown by the differences in their
sensitivities to inhibitors of signal-transducing molecules. Different
protein phosphatases may be involved in the intra- and extracellular
responses. Okadaic acid greatly increased the extracellular response,
while the intracellular response was much less affected. Calyculin A,
on the other hand, induced changes in both the extra- and intracellular
responses, although in different directions, enhancing the
extracellular response and inhibiting the intracellular. Taken
together, these data may indicate that the extracellular response
involves the activation of protein phosphatase 2A, which is inhibited
by both okadaic acid and calyculin A, while the intracellular response
may involve protein phosphatase 1, which is inhibited by calyculin A
but not as potently by okadaic acid (11).
The lack of sensitivity of the intracellular response to neuraminidase
treatment does not necessarily mean that sialic acid is not involved in
this activation. The sensitivity to neuraminidase has been shown to
differ depending on the structure of the sialic acid-containing
glycoconjugate as well as the surrounding membrane structures, and
large fractions of sialic acid may remain on the cell surface after
neuraminidase treatment (19, 43). In addition, agonists such
as the sialic acid-specific lectin maackia amurensis agglutinin (MAA)
as well as sialic acid-binding Actinomyces spp., can also
activate cells after neuraminidase treatment (unpublished observation).
In conclusion, the data presented here point to the importance of
taking into account the fact that neutrophils exhibit markedly different levels of responsiveness to various stimulating agents, physiological as well as nonphysiological, depending on their states of
priming. By responding properly to priming agents, the neutrophils may
limit their receptor exposure to include only those receptors needed at
one precise moment, and the neutrophil activation and degranulation
processes can thus be modulated to limit the inflammatory process.
| |
ACKNOWLEDGMENTS |
I thank Per Follin for kind help with the exudate chambers. The
skillful technical assistance of Lisbeth Björck and Marie Samuelsson is gratefully acknowledged.
The work was supported by the Fredrik and Ingrid Thuring Foundation,
the Swedish Society for Medicine, the Lars Hierta Foundation, the
Magnus Bergvall Foundation, and the Anna-Greta Crafoord Foundation for
Rheumatological Research.
| |
FOOTNOTES |
*
Mailing address: The Phagocyte Research Laboratory,
Department of Medical Microbiology and Immunology, University of
Göteborg, Guldhedsgatan 10, S-413 46 Göteborg, Sweden.
Phone: 46-31-342 46 35. Fax: 46-31-82 88 98. E-mail:
anna.karlsson{at}microbio.gu.se.
Editor:
J. R. McGhee
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Infection and Immunity, July 1999, p. 3461-3468, Vol. 67, No. 7
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
