From the Department of Medicine, Division of
Infectious Diseases, §§ Greenebaum Cancer
Center, ¶¶ Department of Epidemiology, ¶ Research
Service, Veterans Affairs Medical Center and the
Institute for Human Virology, University of Maryland
School of Medicine, Baltimore, Maryland 21201 and the
Division of Communicable Diseases and
Immunology, Walter Reed Army Institute of Research,
Silver Spring, Maryland 20910-7500
Received for publication, July 29, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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Upon activation with various noncytokine
stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular
sialidase to the plasma membrane, where the sialidase releases
sialic acid from the cell surface. This desialylation enhances PMN
adherence, spreading, deformability, and motility, functions critical
to diapedesis. We now have examined the role of sialidase activity in
PMN adhesion to and migration across the endothelium in
vivo. A polyclonal antibody prepared against Clostridium
perfringens neuraminidase 1) detected surface expression of
sialidase on human PMNs stimulated with IL-8 in vitro and
on murine PMNs stimulated in vivo, but not on that of
unstimulated cells, 2) recognized proteins in human PMN lysates and
granule preparations that were not detected by preimmune antibody, 3)
inhibited bacterial neuraminidase and human PMN sialidase activities
in vitro, and 4) inhibited both pulmonary leukostasis in
mice systemically infused with cobra venom factor and intrapulmonary
transendothelial migration of PMNs into the bronchoalveolar compartment
of mice intranasally challenged with interleukin-8. We conclude that
the chemokine interleukin-8, like other PMN agonists, induces the
translocation of sialidase to the PMN surface and that surface
expression of this sialidase is a prerequisite to PMN recruitment
in vivo. The ability of antibodies raised against a
prokaryotic neuraminidase to recognize eukaryotic sialidase extends the
concept of the neuraminidase superfamily to mammalian enzymes.
Inhibition of mobilized endogenous sialidase may provide a novel
strategy for limiting the inflammatory response.
Sialic acids are found in microbes, invertebrates and in the
tissues of all mammals. Their presence on the cell surface
imparts a negative surface charge and is an important determinant of
that cell's interaction with other cells (e.g. T and B
lymphocyte interaction, aggregation with similar (homotypic) or
distinct (heterotypic) cell types), organic and plastic matrices,
informational molecules (e.g. hormones and cytokines), and
invading microbes (1-5). Sialic acid protects cells and molecules from
immune recognition, clearance, and/or degradation (6-8), and the level
of sialylation determines the rheologic properties, motility, and
metastatic potential of cells (9-12).
Myeloid precursor cells in the bone marrow express abundant sialic
acid. During maturation, a progressive loss of cell-associated sialic
acid is associated with increases in cellular deformability and
motility that permit exit from the bone marrow compartment (10). Once
in the periphery, stimulation of circulating mature myeloid cells is
associated with further loss of cell surface sialic acid, diminished
negative surface charge, and enhanced functional activity (7). Thus,
the sialic acid content of myeloid cells dynamically changes during
both cellular maturation and activation.
Neuraminidases (NANases)1 are
a family of enzymes present in microbes, parasites, and mammalian
tissue (where they are referred to as "sialidases"), which rapidly
remove sialic acid residues, usually present at the nonreducing
terminal position, from sialylated glycoconjugates (13). We observed
that upon activation of polymorphonuclear leukocytes (PMNs), sialidase
present within an intracellular granule compartment translocated to the
plasma membrane, where 95% of the cell-associated sialic acid resides
(9, 14). This sialidase translocation was accompanied by a release of
cell-associated sialic acid, an increase in cellular adhesiveness, and
homotypic aggregation (9). As an integral membrane protein
(i.e. it was not released from the cell), the translocated
sialidase could also desialylate adjacent cells in the local
environment. Thus, during activation, PMNs can rapidly modulate surface
sialic acid in an autocrine and paracrine manner. We propose that the
loss of cell-associated sialic acid through the activity of mobilized sialidase may be a prerequisite event for PMN adherence to and migration across the endothelial barrier into inflamed extravascular tissues.
During acute pulmonary vascular endothelial injury and dysfunction,
including the acute respiratory distress syndrome (ARDS) and immune
complex-mediated lung injury, there is an early entry of PMNs into the
lung microvasculature (15, 16). This PMN recruitment and activation may
be in response to locally generated cytokines, such as IL-8, or to
other chemoattractants, such as C5 cleavage products and leukotriene B4
(16). IL-8, a cytokine produced by many cell types, including
endothelial cells, fibroblasts, respiratory epithelial cells,
macrophages, and PMNs, is a potent chemoattractant for PMNs and binds
to a subfamily of related G-protein-coupled receptors (17). It is found
in high concentrations in the bronchoalveolar lavage fluid of ARDS
patients and in the circulation during clinical and experimental human
sepsis (18, 19). In PMNs, IL-8 also increases surface expression of
adhesion molecules, transendothelial migration, degranulation, and
superoxide anion formation (17).
In order to exit from the circulation into an inflammatory site, PMNs
must adhere to vascular endothelium, markedly alter their shape,
squeeze through interendothelial cell junctions, and disengage from the
undersurface of endothelial cells to enter the underlying tissue (for a
review, see Ref. 20). These functions require dynamic changes in the
amount of sialic acid on the PMN surface.
We previously demonstrated that a variety of PMN agonists (fMLP,
phorbol 12-myristate 13-acetate, and calcium ionophore (A23187)) induce
translocation of sialidase from intracellular storage sites to the
plasma membrane with a release of sialic acid into the culture medium
(9). Since IL-8 also activates PMNs, in this study we determined
whether this endogenous chemokine also provoked surface sialic acid
release. The membrane-associated sialidase from human PMNs has been
neither cloned nor purified. We therefore tested the hypothesis that
the previously described superfamily of prokaryotic NANases (13) might
extend to human PMN sialidase and that antibodies raised against one
bacterial NANase (Clostridium perfringens) may cross-react
with conserved regions of NANase or sialidase obtained from other
species (human or murine PMN sialidase). In this study, we demonstrate
that sialidase inhibition in vivo can profoundly reduce PMN
influx into inflammatory sites in three animal models of PMN recruitment.
Preparation of Polyclonal Anti-NANase Antibody--
New Zealand
White rabbits were immunized with 50 µg of C. perfringens
NANase, type V (Sigma) The initial intravenous injection was
administered in Freund's complete adjuvant, and a booster dose of 50 µg was given intramuscularly on day 14 in Freund's incomplete
adjuvant. Boosts of 50 µg of NANase were given subcutaneously without
any adjuvant at 4, 10, 16, and 22 weeks. At 1 week after the last
boost, serum was obtained, and the IgG was purified by affinity
chromatography on a protein G column (Amersham Biosciences), eluted with glycine-HCl buffer (0.15 M, pH 2.5), and
immediately neutralized with 0.15 M Tris to a pH of
6.8-7.2. The combined fractions were filter-sterilized (0.2 µM), and aliquots of the antibody preparation were stored
at Preparation of Human PMN Lysates and Granules--
Human PMNs
were stimulated for 10 min at 37 °C with phorbol 12-myristate
13-acetate (100 ng/ml) and centrifuged, and the pellets were treated
with 1% Nonidet P-40 and DNase I (final concentration of 10 µg/ml).
The PMN lysates were then subjected to three cycles of freeze/thawing
in methanol and dry ice. For preparation of sialidase in human
neutrophil granules, a human volunteer was treated with recombinant
granulocyte colony-stimulating factor (Amgen, Thousand Oaks, CA) (5 µg/kg at 48 and 24 h prior to pheresis) under a protocol
approved by the University of Maryland at Baltimore Institutional
Review Board, as previously described (21). Following leukapheresis,
cells were separated with Histopaque, and erythrocytes were lysed by
hypotonic lysis. After treatment with diisopropyl fluorophosphate to
inhibit proteolysis, cells were suspended in relaxation buffer (100 mM KCl, 10 mM PIPES, pH 7.4, 3 mM
MgCl2, 3.5 mM NaCl), to which 10 µM phenylmethylsulfonyl fluoride, 1 mM ATP,
and 1 mM EGTA were added. PMNs were subjected to nitrogen cavitation (40 p.s.i. for 15 min), and the cavitate was centrifuged at
500 × g for 5 min to remove unbroken cells and nuclei.
The supernatant was then centrifuged at 10,000 × g for
20 min, and the granule-enriched pellet was resuspended in relaxation
buffer and solubilized at 4 °C with 0.4% deoxycholate, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 20 mM Tris-HCl, pH 8.0, for 1 h. The insoluble material
was centrifuged at 37,000 × g for 1 h at 4 °C,
and the supernatant was dialyzed extensively against 25 M
NaCl, 2 mM EDTA, 10 mM Tris-HCl, pH 8.0, at
4 °C. The protein concentration was determined by a modified Lowry
assay (22).
Western Blot Analysis--
Solubilized protein samples (50 µg)
were boiled in sample buffer containing 62.5 mM Tris-HCl,
pH 6.8, 0.6 mM Immunoprecipitation of PMN Sialidase--
Fresh human PMNs were
lysed with ice-cold lysis buffer containing 50 mM Tris-HCl
(pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 1 mg/ml aprotinin, 1 mM vanadate, 1 mM sodium fluoride, 10 mM disodium pyrophosphate, 500 µM
paranitrophenol, and 1 mM phenyl arsine oxide (all
purchased from Sigma). The PMN lysates were precleared by incubation
with goat anti-rabbit IgG cross-linked to agarose (Sigma) for 1 h
at 4 °C and then incubated overnight with rabbit antibody raised
against C. perfringens neuraminidase or preimmune rabbit
serum. The resultant immune complexes were immobilized by
incubation with goat anti-rabbit IgG cross-linked to agarose for 2 h at 4 °C, centrifuged, washed, boiled for 5 min in sample buffer,
and again centrifuged. The supernatants were then processed for
immunoblotting with the same anti-neuraminidase antibody as described
above. The blots were subsequently incubated with horseradish
peroxidase-conjugated anti-rabbit IgG (Transduction Laboratories) and
developed with ECL.
Detection of Sialidase on the PMN Surface--
PMNs were
prepared from heparinized blood drawn from healthy volunteers as
described (9) or by cardiac puncture of mice. The ability of the
polyclonal anti-NANase antibody to bind to surface sialidase on intact
PMNs harvested from mice 2 h after intraperitoneal injection of
recombinant human IL-8 (Calbiochem) and on human PMNs stimulated
in vitro with either IL-8 or fMLP in the presence of
cytochalasin B was determined by flow cytometry (Facscan II,
Becton-Dickinson, Mountain View, CA) and analyzed with Lysis II
software as described (6). Normal rabbit IgG (Sigma) or rabbit
preimmune IgG was used as the irrelevant controls.
Detection of Changes in Cell Surface Sialic Acid with Two
Distinct Lectins--
Heparinized peripheral blood from healthy
volunteers was treated with 0.83% ammonium chloride (Sigma) for 10 min
at 21 °C to remove erythrocytes. Leukocytes were pelleted, washed at
4 °C in Dulbecco's phosphate-buffered saline without calcium and magnesium (PBS High Performance Anion Exchange Chromatography Coupled with
Pulsed Electrochemical Detector Analysis of Sialyl Residues--
A
Dionex DX600 chromatography system (Dionex Corp., Sunnyvale, CA)
equipped with an electrochemical detector (ED50; Dionex Corp.) was
used. Cell surface sialic acids were released by the treatment of human
PMN suspensions (25 × 10 6 PMNs in 250 µl) with
either medium alone for 15 min, NANase (30 milliunits/ml) for 60 min,
IL-8 (150 ng/ml) for 15 min, or fMLP (10 Measurement of NANase Activity--
The sialidase activity was
detected in PMN extracts (107 PMN/ml) using an established
thiobarbituric acid assay to measure sialic acid release from an
artificial substrate, neuraminyllactose (Sigma) (25). Human PMNs
(107 PMN/ml) were stimulated with IL-8 (0.1-10 ng/ml) for
1 h at 37 °C and lysed, and the lysates were assayed for
sialidase activity. A standard curve of NANase activity was obtained by
testing increasing concentrations of C. perfringens NANase,
type V (1-25 milliunits/ml) in the presence of a fixed concentration
(1 mg/ml) of neuraminyllactose in 0.8 M sodium acetate, pH
5.5, and incubated for 2 h at 37 °C. Alternatively, C. perfringens NANase (2 milliunits) or PMN lysate (100 µg of
protein) was incubated for 1 h at 37 °C with 0.7 mM PNP-NANA (Sigma) as a substrate in 0.8 M sodium acetate, pH
4.6, in a final volume of 200 µl. The reaction was stopped by adding 1 ml of 0.085 M glycine-carbonate, pH 10, and the
nitrophenol released was measured at A405 with
p-nitrophenol used as a standard, as previously described
(26).
Effect of Anti-NANase-IgG on Cobra Venom Factor (CVF)-induced
Pulmonary Leukostasis--
The systemic infusion of CVF activates
complement, generates the C5 cleavage product, C5a, and induces
pulmonary leukostasis (27). To assess the effect of sialidase
neutralization on CVF-mediated leukostasis, we pretreated 6-8-week-old
ICR mice (Charles River, Wilmington, MA) with either nonimmune rabbit
IgG or anti-NANase-IgG. After 1 h, CVF (Quidel, San Diego, CA) or
PBS was injected intravenously in a total volume of 0.1 ml. PMN
recruitment into the lung was determined by both biochemical and
histologic techniques. The right lung was used to quantify
myeloperoxidase (MPO) activity/0.1 g of tissue (28) (see below),
whereas the left lung was processed for histology with
hematoxylin/eosin staining (29).
Tissue MPO Assay--
MPO is a heme-containing enzyme found in
the azurophilic granules of PMNs and has been successfully used as a
biochemical marker of intrapulmonary PMNs (27). Quantification of the
number of PMN per 0.1 g of tissue was done according to the method
of Suzuki et al. (28) on lung homogenates. The MPO activity
was interpolated from a standard curve using purified human MPO (Sigma) and correlated with the MPO activity of murine PMNs prepared by Ficoll-Hypaque density gradient centrifugation (380 g, 20 °C, 40 min) of blood obtained by cardiac puncture.
Preliminary experiments confirmed earlier observations that CVF-induced
pulmonary leukostasis was maximal at 0.5 h after injection and
then abated (27). At 0.5 h, mice were sacrificed and bled, and MPO
activity in the nonperfused lung homogenates was measured. In
preliminary experiments, we observed that perfusing the lungs in this
model removed all PMNs within the blood vessels and thus abolished any
effects of CVF on pulmonary leukostasis (data not shown). Accordingly,
in these experiments lung MPO was determined on unperfused lungs.
Effect of Anti-NANase IgG on IL-8-induced PMN Recruitment into
the Bronchoalveolar Compartment--
Since IL-8 is detected in the
lungs after bacterial infection (30) and during ARDS (16), we developed
an animal model to assess PMN extravasation from the pulmonary
vasculature into the interstitium and alveolar space in response to
this chemokine. Following anesthesia with ketamine hydrochloride (80 mg/kg) and xylazine (8 mg/kg) given intraperitoneally (29), ICR
mice were administered intravenously either anti-NANase IgG or
nonimmune rabbit IgG (Sigma). After 1 h, the mice were given
equivalent volumes (50 µl) of either nonpyrogenic normal saline (see
Fig. 3, NS) or rhu IL-8 (2.5 µg/mouse) into the left nasal
opening. PMN recruitment into the lung was followed over a 4-h period, since preliminary experiments indicated that 4 h was the time of
maximal PMN influx. To exclude intravascular PMN, lungs were perfused
with 5 ml of phosphate-buffered saline containing 1 unit/ml of heparin
via the right ventricle.
Effect of Anti-NANase IgG on Circulating and Tissue PMN
Levels--
To determine whether anti-NANase antibodies that bound to
murine PMNs in vitro resulted in accelerated clearance of
PMNs from the circulation, we measured the level of circulating PMNs in mice 1.5 and 5 h after intravenous administration of either normal rabbit IgG or rabbit anti-NANase antibody. The number of PMNs was
manually counted in a hematocytometer. At 5 h, tissue leukostasis in the liver and spleen was measured by the MPO assay.
Mouse Peritonitis Model--
Since the mechanism(s) of PMN
extravasation from the systemic and pulmonary circulation appear to be
distinct (3, 24), we also determined whether sialidase also might be
required for optimal PMN recruitment into the peritoneal cavity. Mice
were given rhu IL-8 intraperitoneally, and the PMN influx was followed at various time points by direct counts of PMNs harvested by peritoneal lavage. In preliminary experiments, the PMN influx was optimal 4 h
after IL-8 challenge. In this model, we utilized soluble sialic acid
(Sigma) (31) as a competitive inhibitor of sialidase administered either as a single bolus prior to the IL-8 challenge or in multiple doses before and after rhu IL-8 administration. In control animals, equivalent volumes (0.1 ml) of pyrogen-free, normal saline were administered at the same time points in lieu of sialic acid.
Statistical Analysis--
Comparisons of the -fold increase were
done with the two-tailed Mann-Whitney U test. All data are
expressed as median ± quartiles.
Effect of IL-8 on PMN Release of Cell-associated Sialic
Acid--
We have demonstrated that PMN sialidase resides with a
granule subpopulation (9). Since IL-8 not only attracts but also stimulates PMN exocytosis (17), we determined whether IL-8 also provoked surface sialic acid release. Sialic acid release from the
surface of human PMNs could be demonstrated by a decrease in the
amount of the sialic acid-binding lectin, L. flavus, and an increase in PNA lectin binding on the cell
surface of IL-8 treated nonpermeabilized cells (Fig.
1). Increasing concentrations of
bacterial neuraminidase induced a dose-dependent loss of
lectin binding to the PMN surface (Fig. 1A). This loss of
surface fluorescence from PMNs treated with one concentration of
neuraminidase was reversed by the competitive sialidase inhibitor,
2-deoxy-NANA (Fig. 1B). IL-8 decreased lectin binding to
PMNs compared with untreated PMN controls (Fig. 1C). In
contrast to the L. flavus lectin, the PNA lectin binds to
desialylated glycoproteins that have a terminal
Sialic acid release from the surface of PMNs could also be demonstrated
by HPLC. Following exposure of 25 × 106 human PMNs to
IL-8, fMLP, or medium alone, the amounts of sialyl residues released
from PMNs into the supernatant were below the level of detection of the
Dionex DX600 chromatography system, a highly sensitive detection system
for sialic acid. Only after NANase treatment was the release of sialyl
residues consistently detectable. As an alternative strategy to
quantify PMN desialylation, we therefore measured how much
NANase-releasable sialyl residues remained on the PMN surface after
treatment with agonists. Following treatment with IL-8 or fMLP or
exposure to medium, the pellets of each of the three PMN preparations
were then subjected to NANase treatment. The amount of sialic acid
released into the supernatant by NANase treatment of cells initially
incubated in medium alone was defined as the total NANase-releasable
sialic acid. After treatment with IL-8 and fMLP, there were decreases
in cell surface-associated sialyl residues of 14 ± 5 and 10 ± 1%, respectively (p = 0.06 and <0.01,
respectively, compared with PMNs exposed to medium alone, by
one-tailed, unpaired t test). Thus, as described for exogenous PMN agonists (9), IL-8 induced PMN desialylation. Therefore,
IL-8-induced PMN desialylation could be demonstrated by both HPLC and
flow cytometry with two distinct lectins. This loss of PMN surface
sialic acid residues following activation with an endogenous mediator,
IL-8, was compatible with translocation of sialidase activity to the
plasma membrane with attendant surface desialylation.
Ability of Anti-NANase IgG to Recognize Human PMN
Sialidase--
To detect the mobilization of human PMN sialidase, we
prepared antibodies to clostridial NANase for use in both Western blot analysis of PMN lysates and granule preparations and in analysis of
intact cells by flow cytometry. Coomassie Blue staining of proteins
from clostridial NANase revealed a major band with an apparent
Mr of 91,000 (Fig.
2, lane 2).
Immunoblotting with the polyclonal anti-NANase IgG antibody revealed
multiple PMN lysate (lane 4) and granule
(lane 8) proteins as well as a strong reaction with bacterial NANase protein (lane 3). In
contrast, preimmune IgG did not react with either preparation
(lanes 5 and 9). Since we failed to
detect a band in the PMN lysate that co-migrated with the
immunoreactive bands in the granule preparation (lane 8), we used immunoprecipitation with anti-NANase antibody to
enrich for and detect a cross-reactive protein in the PMN lysate
(lane 6). Nonimmune IgG did not detect these
bands (lane 7) by immunoprecipitation. The
anti-NANase antibody neither recognized human purified MPO (Sigma) on
Western blot nor interfered with its enzymatic activity in
vitro (data not shown). Conversely, human purified MPO did not
block the ability of anti-NANase antibody to recognize NANase on
Western blot analysis, whereas the addition of clostridial NANase to
the same antibody preparation decreased NANase immunoreactive signal
(data not shown). Thus, our antibody raised against a bacterial NANase
recognized epitopes on human proteins that reside within PMN granules,
whereas it did not react with a second protein that is known to reside
within the same PMN granule population.
Binding of Anti-NANase to Intact Murine and Human PMNs--
The
rabbit anti-NANase antibody also was used with flow cytometry to
document the presence or absence of translocated enzyme on the surface
of resting and IL-8-activated PMNs isolated both from humans and mice.
Human PMNs were stimulated in vitro, and murine PMNs were
stimulated in vivo with IL-8. Incubation of unstimulated human PMNs with normal rabbit IgG and anti-NANase IgG each yielded similar binding intensities (Fig.
3A). In contrast, IL-8
increased anti-NANase IgG binding to the PMN surface (Fig.
3B), whereas normal rabbit IgG binding was unchanged. Normal
rabbit IgG binding to stimulated and nonstimulated PMNs were no
different than that observed with nonspecific binding (i.e.
binding of secondary antibody in the absence of primary antibody).
To determine whether anti-NANase antibody could bind to PMNs stimulated
in vivo, mice were treated with either IL-8 or PBS 2 h
prior to cardiac puncture, when PMNs were harvested and studied for
anti-NANase IgG and control IgG binding. There was no difference between the binding of nonimmune rabbit IgG and anti-NANase IgG to PMN
from saline-injected mice (Fig. 3C). After IL-8 challenge, PMN surface expression of an epitope was recognized with anti-NANase antibody increased (Fig. 3D). These data indicate that a
clostridial NANase cross-reactive antigen is expressed on the surface
of PMNs only upon activation both in vitro and in
vivo. Thus, areas of homology in the neuraminidase/sialidase
superfamily exist such that antibodies against clostridial NANase also
recognized epitopes in an endogenous sialidase of PMNs isolated from
both mice and humans.
Inhibition of Clostridial NANase and PMN Sialidase Activities by
Anti-NANase Antibody--
The anti-NANase antibody recognized both
clostridial NANase and human PMN proteins (Figs. 2 and 3). To establish
whether this same antibody could inhibit NANase/sialidase catalytic
activity in vitro, clostridial NANase (25 milliunits/ml) and
sialidase-containing PMN lysates were incubated for 1.5 h at room
temperature with different concentrations of anti-NANase IgG or
preimmune IgG, followed by the addition of these enzyme/antibody
mixtures to the artificial NANase substrate, neuraminyllactose, for
1 h at 37 °C, and liberated sialic acid was measured (9) (Table
I). At an IgG antibody concentration of
10 µg/ml, there was 56% inhibition of bacterial neuraminidase
activity compared with no inhibition with preimmune IgG. Whereas the
anti-NANase IgG inhibited sialidase activity in PMN lysates in a
dose-related manner unlike preimmune IgG, it was unable to achieve 50%
inhibition, even with the addition of higher anti-NANase IgG
levels.
Anti-NANase Antibodies Inhibited CVF-induced Pulmonary
Leukostasis--
Since the polyclonal anti-NANase antibodies inhibited
both clostridial NANase and PMN sialidase activities in
vitro, we examined whether the same antibody preparation exerted
any inhibitory activity in vivo. To determine whether
anti-NANase antibodies were effective in blocking an early step in
non-IL-8-induced intrapulmonary recruitment of PMNs (i.e.
PMN adhesion to the lung microvascular endothelium), mice were infused
with CVF intravenously, and pulmonary leukostasis was quantified (27).
In control mice, a mean ± S.D. of 6.6 ± 0.72 × 106 PMN/0.1 g of nonperfused lung tissue (n = 4) was found. With CVF treatment, there was a 4-5-fold increase in
PMNs to 32 ± 19.9 × 10 6 PMN/0.1 g of lung
tissue at 0.5 h, the time of peak PMN recruitment established in
an earlier study (27) and confirmed in preliminary studies (data not
shown). Anti-NANase antibody reduced CVF-induced pulmonary leukostasis
compared with treatment with nonimmune IgG (Fig.
4A). Microscopic examination
of lung sections from CVF-infused mice revealed PMNs within alveolar
septal wall capillaries but not within alveoli (Fig. 4B).
Pretreatment with anti-NANase IgG markedly reduced the intravascular
PMNs (Fig. 4C), whereas normal rabbit IgG did not (Fig.
4B). These data are compatible with our hypothesis that
endogenous PMN sialidase activity is required for increased PMN
adhesion to the endothelial surface in CVF-induced pulmonary
leukostasis. Interestingly, when examined at 4 h, a time at which
the CVF-induced leukostasis was no longer evident, there was no
difference in the PMN level in the lungs of mice treated with immune or
nonimmune IgG. This suggests that in the absence of PMN activation and
surface expression of sialidase, the enzyme was no longer accessible to
the anti-NANase antibody.
Effect of Anti-NANase IgG on IL-8-induced Recruitment of PMNs into
the Brochoalveolar Compartment--
Since immunoblockade of endogenous
sialidase activity diminished PMN-to-endothelial adhesion in response
to systemic complement activation, we asked whether this same
intervention might restrain paracellular movement of PMNs across the
endothelial barrier in response to an extravascular chemotactic
gradient. We have previously demonstrated that both sialic acid and
2-deoxy-NANA, competitive inhibitors of NANase, inhibit sialidase
activity of human PMN in vitro (9). To extend these findings
to our in vivo system, we studied whether the anti-NANase
IgG, which recognized sialidase on the surface of activated murine and
human PMNs and inhibited catalytic activity in vitro, also
was capable of modifying in vivo PMN behavior mediated
through desialylation. In preliminary experiments, intranasal
administration of IL-8 induced a time-dependent increase in
MPO activity in homogenates of perfused lungs for up to 4 h (data
not shown). This increase in lung MPO coincided with PMN migration
across the pulmonary vascular endothelial barrier into alveoli detected
by light microscopy.
The mean ± S.D. base-line PMN content in perfused lungs of mice
was 9.3 ± 0.4 × 105 cells/0.1 g of tissue
(n = 4). At 4 h after intranasal saline administration, lung PMN content increased 2-fold over base line; IL-8
administration induced >4-fold increase (Fig.
5A). Pretreatment with
anti-NANase antibody intravenously decreased the IL-8-induced recruitment of PMNs to the lungs >2-fold, compared with pretreatment with nonimmune IgG. Histologic examination of the lungs following IL-8
administration revealed intra-alveolar PMNs (Fig. 5, B and C). This mouse model involving an intranasal challenge with
IL-8 is therefore an in vivo model of transendothelial PMN
migration across the pulmonary alveolar-capillary barrier. The decrease in MPO activity in the anti-NANase IgG-treated lungs reflected the
lower number of intra-alveolar PMNs on histologic examination of lung
sections (Fig. 5, compare B and D).
Therefore, not only is endogenous sialidase activity required for
sequestration of PMNs within the pulmonary microvasculature; it is also
a prerequisite for their mobilization into the alveolar space.
Effect of Anti-NANase Antibody on Levels of Circulating and
Intrapulmonary PMNs--
Intravenous infusion of anti-NANase antibody
to mice did not decrease circulating PMNs. At 1.5 h after
intravenous administration of anti-NANase antibody, there was a
mean ± S.D. of 1.9 ± 1.1 × 106 PMN/ml of
blood (n = 5) compared with a preinfusion level of 1.8 ± 0.8 × 106 PMN/ml, and at 5 h there
were 2.7 ± 1.1 × 106 PMN/ml. At 5 h, no
differences in either liver or spleen MPO could be demonstrated between
mice receiving anti-NANase antibody and those receiving normal rabbit
IgG (0.53 ± 0.1 × 106 PMN/0.1 g of tissue
versus 0.8 ± 0.54 × 106 PMN/0.1 g of
tissue in the liver (n = 5) and 17.5 ± 5.3 × 106 PMN/0.1 g of tissue versus 19.8 ± 6.3 × 106 PMN/0.1 g of tissue in the spleen
(n = 5)).
Systemic Infusion of a Competitive Sialidase Inhibitor, Sialic
Acid, Decreases Intraperitoneal Recruitment of
PMNs--
Intraperitoneal administration of IL-8 increased peritoneal
total leukocytes (2.3-fold) and, more specifically, PMNs (6-fold) at
4 h compared with the saline-treated controls (Table
II). A single dose of sialic acid, a
competitive inhibitor of sialidase activity (31), 5 min prior to IL-8
reduced the intraperitoneal recruitment of total leukocytes and PMNs,
but these reductions did not achieve statistical significance compared
with those seen with saline administration before IL-8 treatment. In
contrast, following multiple doses of sialic acid, intraperitoneal
total leukocytes and PMNs were both significantly decreased. These
combined data suggest that the sustained presence of sialic acid within the circulation effectively reduced recruitment of leukocytes, especially PMNs, into an inflammatory site. Thus, immunologic and
pharmacologic interventions that target PMN surface sialidase diminished PMN recruitment to more than one relevant body
compartment.
These studies have demonstrated that targeting endogenous PMN
sialidase activity through either immunoblockade or competitive inhibition altered PMN trafficking to an inflamed site. These interventions decreased PMN recruitment in response to two distinct inflammatory stimuli: a chemokine (IL-8) or CVF-generated
complement cleavage products. The ability of IL-8 to mobilize sialidase
to the PMN surface (Fig. 3) and to increase the release of
cell-associated sialic acid (Fig. 2) was comparable with what we have
reported for exogenous, nonphysiological agonists (9). Whereas some desialylating stimuli are not chemoattractants (e.g. phorbol
12-myristate 13-acetate and calcium ionophore), the ability of
the chemokine, IL-8, to augment transendothelial migration of PMNs
in vivo may be related in part to its sialidase-mobilizing
activity. Thus, these studies extend the concept of PMN
activation-induced translocation of sialidase and surface desialylation
in response to endogenous stimuli (IL-8) and suggest that this response
may be central to PMN recruitment in vivo.
A novel finding in this study was the ability of antibody elicited
against a bacterial NANase to recognize mammalian sialidase(s) in both
human and murine PMNs by both Western analysis and flow cytometry
(Figs. 2 and 3). With intact nonpermeable cells, the sialidase was
accessible only on the surface of activated PMNs in vitro
and in vivo. The same anti-clostridial NANase antibody that
recognized and bound sialidase on the surface of intact PMNs as well as
in PMN lysates and granule preparations and that neutralized the
catalytic activity of both the bacterial NANase and PMN sialidase in vitro also altered the trafficking of murine PMNs
in vivo. The polyclonal antibody selectively targeted
sialidase, since it failed to recognize or inhibit the activity of
another PMN granular resident enzyme, MPO. That polyclonal antibody
preparations from multiple rabbits exhibited similar binding and
functional properties with regard to human and murine PMN sialidase
activity suggests that the anti-clostridial NANase antibodies recognize mammalian sialidase. These studies therefore extend the concept of a
prokaryotic NANase (sialidase) superfamily (13) to the eukaryotic
sialidases that cleave sialyl residues from PMN surface glycoconjugates.
The anti-clostridial NANase antibody was active in animal models of PMN
recruitment to both intrapulmonary and extrapulmonary sites.
CVF-generated complement cleavage products and IL-8 each evoked
different types of PMN behavior within the lungs (pulmonary intravascular leukostasis by systemic complement activation
versus transendothelial migration of PMN into alveoli in
response to an IL-8 gradient), with distinct kinetic profiles (peak PMN
recruitment at 0.5 and 4.0 h, respectively). Pretreatment of mice
with anti-NANase antibodies prevented pulmonary leukostasis
(i.e. PMN adhesion in the absence of migration). This is an
early and rapid event that may occur following systemic complement
activation such as demonstrated here experimentally with cobra venom
factor (Fig. 4) or clinically following hemodialysis (32). This
antibody also reduced extravasation of PMNs into the alveoli of
IL-8-treated lungs. Whereas this model may also require PMN adhesion,
it also demands that PMNs become highly deformed and motile as they
squeeze through the interendothelial junction in response to a local
chemotactic gradient.
The recruitment of PMNs to an extrapulmonary inflammatory site was
inhibited by competitive pseudosubstrate inhibition of neuraminidase/sialidase activity (Table II). Thus, PMN recruitment to
both pulmonary and extrapulmonary inflammatory sites by two distinct
stimuli could be inhibited by either immunologic or pharmacologic blockade.
Sialidases differ in the type of sialyl glycosidic linkages they cleave
as well as in their tissue and subcellular distributions (26, 33-35).
Many microbial sialidases have been cloned and sequenced (13).
Mammalian sialidase has been cloned from the cytosol of rat muscle (35)
and Chinese hamster ovary cells (33); however, a role for these gene
products in the immune response has not been proposed. Recently, a
nonlysosome-associated human sialidase was cloned from the major
histocompatibility complex region of human Epstein-Barr
virus-infected lymphoblastoid cells (26). The molecular mass of
this enzyme (16 kDa), believed to be similar to human lymphocyte
sialidase, is different from the apparent molecular masses of the
multiple protein bands in PMNs detected by the polyclonal anti-NANase
antibody (26). Sialidase is present in lymphocyte lysosomes as a
multienzyme complex that translocates to the surface upon cell
activation. Phosphorylation of an internalization signal on the
translocated sialidase prevents its endocytosis (36). Anti-NANase IgG
appears to recognize the larger of two sialidase isoforms produced by
C. perfringens (37).
IL-8 treatment of PMNs resulted in decreased cell-associated sialic
acid detected by HPLC and the loss of binding of a sialic acid-binding
lectin to the cell surface with a concomitant increase in binding of a
lectin (PNA) known to react strongly with desialylated glycoconjugates
(Fig. 1). IL-8 also induced the mobilization of a molecule to the PMN
surface, which was recognized by an antibody raised against clostridial
neuraminidase but not by preimmune serum (Fig. 3). Functional
changes in both PMN and peripheral blood mononuclear cells have been
demonstrated with up to 30% desialylation (9, 38, 39). On the basis of
the present studies, we cannot determine whether inhibition of
endogenous sialidase activity altered PMN trafficking through
modulation of global desialylation with changes in net surface charge,
and/or by interfering with the removal of sialyl residues from specific glycoconjugates essential to PMN adhesion and migration, such as a
Since the removal of cell-associated sialic acid makes PMNs more
adherent, deformable, and motile (11), a hypothetical schema may be
proposed by which sialidase might regulate multiple steps in
diapedesis. This enzyme shares the same intracellular compartment as
cellular stores of CD11b/CD18. Sialidase may cleave the selectin (CD62L)-mediated low affinity adhesion to sialylated counterstructures that precedes the integrin-mediated tight adherence between PMNs and
endothelium. Sialidase may also play a direct or indirect role in the
functional activation of the integrins and/or the immunoglobulin-like
adhesion molecules on endothelial cells (e.g. intercellular
adhesion molecule 1) necessary for firm adhesion to occur. The
mobilization of sialidase may facilitate the spreading of PMNs onto the
adjacent endothelium, an essential step that precedes transendothelial
migration. This may occur by removal of sialyl residues from specific,
heavily sialylated molecules (e.g. leukosialin, also
known as CD43) known to play a role in PMN adhesion to surfaces (40,
41). Alternatively, the desialylation of multiple glycoconjugate
species on both the PMN and/or adjacent endothelial cell surface may
sufficiently decrease the net negative surface charge of either or both
cell types with a reduction of repulsive forces.
PMN movement to and its migration through the interendothelial cell
junction requires reversible and dynamic changes in adhesion. This
could occur through the alternating activities of sialidase and
sialyltransferase enzymes. The translocated sialidase could desialylate
cells adjacent to the activated PMN, perhaps along its leading edge,
promoting PMN contact with the endothelial surface. Conversely,
restoration of the preactivated state and release from the endothelium
could rapidly occur if sialyltransferases (such as those previously
described in human PMNs (42)) added sialyl residues back to
glycoconjugates on the cell surface, perhaps at polarized sites on the
PMN surface. Since sialidase is not released from the plasma membrane
into the environment, this mechanism may localize the inflammatory
response to the site of the responding cell. Thus, removal and
restoration of sialyl residues from and to glycoconjugates on the
surface may be a rapidly adaptable, highly orchestrated, yet spatially
restricted process through which PMNs respond to chemotactic gradients
across the endothelium without disrupting endothelial barrier integrity.
Therapeutic interventions targeting this enzyme may provide a novel
strategy for limiting the inflammatory response. There is an induction
of sialidase and concomitant loss of sialyl residues from
the PMN surface following stimulation with a wide range of agonists,
including the chemokine, IL-8. Modulation of the activity of an
endogenous mobilized sialidase by either antibody or a competitive inhibitor alters the trafficking of PMNs to the lungs in response to
diverse stimuli. This strategy may have several advantages: by
targeting multiple stages of diapedesis, inhibition of sialidase may
provide a greater effect than may occur by inhibiting one step
(e.g. the anti-selectin strategy (43)). Further, by
inhibiting the effects of multiple, diverse chemotactic stimuli that
are known to be generated in the lung, this approach may be preferable to targeting only one of these mediators (e.g. IL-8,
leukotriene B4, or C5a). Since sialidase is not expressed on resting
cells (Fig. 3) and since PMN sialidase remains bound to the activated cell, the anti-inflammatory effect of sialidase inhibitors should be
restricted to activated PMNs and localized to sites of inflammation.
The presence of neuraminidase in diverse bacterial, viral, and
parasitic pathogens may represent a form of molecular mimicry whereby
these microbes take advantage of the pivotal role of surface sialic
acid modulation by endogenous sialidase in immune responses and in
cellular interactions described in this report. A similar dynamic
modulation of surface sialic acid also may be critical to other
processes requiring cell-to-cell contact, such as cell differentiation,
antigen presentation, embryogenesis, and tissue remodeling as well as
the metastatic potential of malignant cells. The potential therapeutic
application of modulation of sialic acid content in these conditions
merits further investigation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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20 °C.
-mercaptoethanol, 2% (w/v) SDS, 10%
(v/v) glycerol and separated by 10% SDS-PAGE and then transferred to
polyvinylidene difluoride and nitrocellulose membranes (23). Proteins
immobilized to polyvinylidene difluoride were stained with Coomassie
Blue. After electrotransfer, Western blot analysis using anti-NANase
antibodies was performed on the nitrocellulose membrane, which had been
blocked overnight at 4 °C in 0.5% bovine serum albumin, 0.05%
Tween 20 in PBS, pH 7.4 (PBT buffer). Sialidase activity was detected
by incubating the membrane with 5 ml of PBT containing polyclonal IgG
antibodies raised against clostridial NANase (dilution 1:50) for 1 h at room temperature and then washed twice (10 min each) with 5 ml of
PBT. Bound anti-sialidase antibodies were detected by incubating the membrane with horseradish peroxidase-conjugated goat anti-rabbit IgG
diluted at 1:5000 in PBT for 45 min at room temperature. The membrane
was washed twice as described above, and bound peroxidase activity was
detected with 3,3'-diaminobenzidine in the presence of
CaCl2 as described (24).
) (Invitrogen) supplemented with
0.2% heat-inactivated fetal calf serum (Hyclone, Logan,
UT), and repelleted. The leukocytes were resuspended to a concentration
of 5 × 106 cells/ml in PBS
with 0.2%
heat-inactivated fetal calf serum and were exposed to IL-8, NANase, or
medium alone. After treatment, cells were washed and labeled with
fluorescein isothiocyanate (FITC)-labeled Limax flavus
lectin (EY Laboratories, San Mateo, CA), which binds to sialyl residues
in a non-glycosidic linkage-specific manner or with the lectin from
Arachis hypogaea (peanut agglutinin lectin (PNA), EY
Laboratories), which binds to terminal
-galactose residues on
glycoconjugates (usually after removal of terminal sialyl residues). Samples of surface lectin-labeled cells were then acquired and analyzed
on a FACScalibur flow cytometer equipped with Cell Quest software
(Becton Dickinson, San Jose, CA). Based on forward and right angle
light scatter properties (linear scale), an electronic gate was placed
around the PMNs exclusively. The measurement of green fluorescence
(MPFL) (log scale) was obtained from unstained, nonstimulated, and
NANase- or IL-8-treated PMNs.
6 M)
for 5 min, all at 37 °C. Supernatants were obtained for analysis of
sialyl residues, and cell pellets were resuspended to their original
volume in culture medium, and each of the four PMN preparations was
then treated with NANase (30 milliunits/ml for 60 min) to further
release sialyl residues not released by the initial treatments. The
sialic acid content of the two supernatants was analyzed with a
CarboPac-PA1 column (4 × 250 mm), with 0.1 M sodium
hydroxide and 1 M sodium acetate in 0.1 M NaOH
as eluents. A gradient of 5-20% over 0-15 min was run at a flow rate
of 1 ml/min. Under this condition, N-acetylneuraminic acid
was eluted at 9.3 min. Sialic acid samples were quantified by
integration of the peak area using a standard
N-acetylneuraminic acid solution as the reference, with an
area of 6.8 corresponding to 1 nM sialic acid.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-galactose residue.
Consequently, instead of the decreased fluorescence observed with the
sialic acid-binding lectin following NANase treatment or IL-8
mobilization of an endogenous sialidase (Fig. 1C), we
observed an increased binding of FITC-labeled PNA following these same
desialylating treatments (Fig. 1D).
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Fig. 1.
IL-8 decreases binding of the sialic
acid-binding lectin, L. flavus, to the surface of
human PMNs. PMNs were isolated from the peripheral blood of
healthy human donors. Following treatment with NANase, IL-8, or medium
alone (unstimulated), PMNs were stained with FITC-labeled L. flavus lectin (A-C) or FITC-labeled A. hypogaea (PNA) lectin and analyzed by flow cytometry.
A, a progressive loss of surface fluorescence was observed
following treatment with increasing doses (10-100 milliunits/ml) of
NANase; B, the addition of the competitive NANase inhibitor,
2-deoxy-NANA, reversed the loss of fluorescence that occurred with the
NANase treatment. No similar reversal was observed when NANase was
co-incubated with 2,3-keto-octonic acid (KDO) a molecule
with similar size and charge as 2-deoxy-NANA, but without NANase
inhibitory activity (data not shown); C, treatment of PMNs
with either IL-8 (150 ng/ml) or NANase caused a loss of fluorescence
from the surface of PMNs compared with unstimulated cells.
D, the loss of sialic acid from the surface of PMNs also
could be demonstrated by an increase in PNA-binding activity following
treatment with either NANase or IL-8. In contrast to the L. flavus lectin, the PNA lectin reacts with desialylated
glycoconjugates. Each cytometric analysis is representative of at least
two experiments.
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Fig. 2.
Immunoprecipitation and immunoblotting of
human PMN lysates (PMN) and granules
(gran) with antibodies raised against clostridial
neuraminidase (NA). Solubilized PMN proteins were
resolved by 10% SDS-PAGE and electrotransferred to polyvinylidene
difluoride. Coomassie Blue stain was used to detect NANase
(lane 2). Putative NANase/sialidase was detected
by incubating the transfers with polyclonal IgG antibodies against
clostridial neuraminidase (anti-NA) or preimmune IgG
(IgG). Bound antibodies were detected by incubation with
horseradish peroxidase-conjugated goat anti-rabbit IgG and developed
with enhanced chemiluminescence. Each lane was loaded with
molecular mass markers (lane 1), clostridial
NANase (0.1 µg) (lanes 2 and 3), PMN
lysate (50 µg) (lanes 4 and 5), and
PMN granules (50 µg) (lanes 8 and
9). To enrich for and detect a cross-reactive PMN protein
with a gel mobility comparable with those detected in granules and
NANase, we performed immunoprecipitation with anti-NANase
(lane 6) and nonimmune (lane
7) antibodies. The heavy chains of the immune and nonimmune
antibodies are evident in both lanes. The position of the molecular
mass standards (kDa) are shown on the left. Each blot is
representative of three separate experiments.
View larger version (22K):
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Fig. 3.
Flow cytometric analysis of human
(A and B) and murine (C
and D) PMNs treated in vitro or
in vivo with either normal saline or IL-8. Human
PMNs were treated with medium alone (A) or rhu IL-8 (250 ng/ml) (B) for 45 min at 37 °C. Murine PMNs were purified
from blood 2 h after intraperitoneal administration of either
normal saline (NS) (C) or IL-8 (750 ng)
(D). PMNs were incubated with nonimmune rabbit IgG (Sigma)
or polyclonal rabbit anti-NANase IgG followed by FITC-labeled goat
anti-rabbit IgG as the secondary antibody. Nonspecific binding
(NSB) of fluorescently labeled secondary antibody was
determined in the absence of cell treatment with anti-NANase or normal
rabbit IgG. An increase in the intensity of binding of anti-NANase
antibodies to activated PMNs was examined by a shift in mean channel
fluorescence.
Anti-C. perfringens neuraminidase antibodies inhibit both C. perfringens neuraminidase and human neutrophil sialidase activities
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Fig. 4.
Anti-NANase antibody reduced pulmonary
leukostasis in CVF-treated mice. At 1 h after intravenous
(i.v.) pretreatment with either nonimmune or
anti-NANase IgG (20 µg/g), mice were infused with CVF (25 units/kg)
intravenously (A). MPO activity was measured in nonperfused
lungs as a biochemical marker of intrapulmonary PMNs. Saline-infused
mice had no increase in lung MPO activity compared with untreated mice
(data not shown). Lung sections were obtained from mice pretreated with
either nonimmune IgG (B, hematoxylin/eosin (H&E)
stain, ×560 magnification) or with anti-NANase IgG (C,
hematoxylin/eosin stain, ×560 magnification). The arrows
point to intravascular PMNs.
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Fig. 5.
Anti-NANase antibodies reduce PMN
transendothelial migration into the alveoli of IL-8-treated mice.
Mice pretreated intravenously (i.v.) with either nonimmune
IgG or anti-NANase IgG (each given at 20 µg/g, body weight) were
administered either normal saline or IL-8 (2.5 µg/mouse) in 50 µl
intranasally (i.n.) 1 h later. A, MPO
activity was measured at 4 h as a biochemical marker of PMNs. The
-fold increase over base-line levels was calculated by dividing the
number of PMN/0.1 g of tissue of each animal by the base-line value for
the particular treatment. B and C, lung sections
were obtained from IL-8-challenged mice following pretreatment with
either nonimmune IgG (B, hematoxylin/eosin
(H&E) stain, ×560 magnification; C,
hematoxylin/eosin stain, ×900 magnification) or anti-NANase IgG
(D, ×560 magnification). The arrows indicate
intra-alveolar PMNs.
A competitive inhibitor of sialidase, sialic acid, inhibits
IL-8-induced influx of PMN into the mouse peritoneum
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-integrin or the IL-8 receptor. We are currently
examining the sialyl content of specific cell adhesion molecules
following IL-8 treatment.
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ACKNOWLEDGEMENTS |
---|
We thank Kathleen Glaser, Lynnette Young, and Carrie Redinger for technical assistance and Dr. Steven Wasserman for consultation with statistics.
![]() |
FOOTNOTES |
---|
* This work was supported by the National Institutes of Health Grant AI42818-01A1 (to A. S. C., S. E. G., and B.-E. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Division of Infectious Diseases, Dept. of Medicine, MSTF Suite 900, 10 S. Pine St., Baltimore, MD 21201. Tel.: 410-328-7394; Fax: 410-328-6896; E-mail: across@umm.edu.
Present address: Dept. of Medicine, Division of Infectious
Diseases and Clinical Microbiology, Adnan Menderes University School of
Medicine, Aydin 09100, Turkey.
** Present address: Dept. of Hematology and Oncology, Charite/Campus Virchow-Klinikum, Humboldt University, 13125 Berlin, Germany.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M207591200
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ABBREVIATIONS |
---|
The abbreviations used are:
NANase, neuraminidase;
PMN, polymorphonuclear leukocytes;
ARDS, acute
respiratory distress syndrome;
fMLP, formyl-methionyl-leucyl-phenylalanine;
PNP-NANA, 2-O-(p-nitrophenyl)-D-N-acetylneuramic
acid;
2-deoxy-NANA, 2,3-deoxy-N-acetyl-neuraminic
acid;
CVF, cobra venom factor;
IL, interleukin;
PIPES, 1,4-piperazinediethanesulfonic acid;
FITC, fluorescein isothiocyanate;
PNA, peanut agglutinin lectin;
MPO, myeloperoxidase;
HPLC, high
pressure liquid chromatography;
PBS, phosphate-buffered saline.
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