From the Department of Medical Biochemistry and
Microbiology, Uppsala University, Biomedical Center, Box 575, S-75123 Uppsala, Sweden and the ¶ Department of Immunology and
Cell Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany
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ABSTRACT |
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Platelet factor 4 (PF-4) is a platelet-derived
The activation and control of polymorphonuclear granulocytes
(PMN)1 are known to play an
essential role in host defense against microbial invaders as well as in
chronic diseases. Several members of the CSs are galactosaminoglycans composed of alternating
glucuronic acid and galactosamine units
( Materials--
BioGel P-10 (superfine) was from Bio-Rad
(Sundbyberg, Sweden). Ficoll-Hypaque, Sephadex G-15, DEAE-Sephacel,
Superose 6, and Superose 12 (prepacked 1.5 × 30 cm) for FPLC were
obtained from Pharmacia (Uppsala, Sweden). Carrier-free
Na235SO4 (1200-1400 Ci/mmol) was
purchased from NEN Life Science Products (Sollentuna, Sweden).
[3H]Acetic anhydride (500 mCi/mmol) and
D-[6-3H]glucosamine (22 Ci/mmol) were
obtained from Amersham International (Solna, Sweden). Sulfate-free
Dulbecco's modified Eagle's medium was from Statens
Veterinärmedicinska Anstalt (Uppsala, Sweden), and fetal calf
serum was from Roche Molecular Biochemicals (Bromma, Sweden). Whatman
3MM filter paper was purchased from Whatman Ltd (Maidstone, Kent,
United Kingdom) and nitrocellulose filter (0.45-µm pore size) from
Sartorius AG (Göttingen, Germany).
Human PF-4 was purified from release supernatants of
thrombin-stimulated platelets in a three-step procedure as described previously (4). The final PF-4 preparation exceeded 99% purity and
contained no protein contaminants detectable on silver-stained SDS-polyacrylamide gels, in an enzyme-linked immunosorbent assay for
CSA from bovine nasal cartilage, dermatan sulfate (CSB) from porcine
skin, and CSC from bovine nucleus pulposus cartilage were generous
gifts from Dr. Anders Malmström (University of Lund, Lund,
Sweden). CSD from shark cartilage and CSE from squid cartilage were
obtained from Seikagaku (Tokyo, Japan), while bovine lung heparin was
received from The Upjohn Co. Human aortic HS (15) was a gift from Dr.
Emadoldin Feyzi (University of Uppsala, Uppsala, Sweden). Heparin
oligosaccharides of defined length used as size-defined standards were
prepared as described previously (16). Hyaluronan fragments for the
same purpose were provided by Dr. Kerstin Lidholt (University of
Uppsala, Uppsala, Sweden). Chondroitinase ABC (from Proteus
vulgaris, EC 4.2.2.4) and chondroitinase AC (from
Arthrobacter aurescens, EC 4.2.2.5) were purchased from
Seikagaku, while protease type XIV (from Streptomyces
griseus, EC 3.4.24.31) and DNase I (EC 3.1.21.1) were obtained
from Sigma (Stockholm, Sweden).
Iodination of PF-4 and PF-4 Receptor Binding Assay--
PF-4 was
iodinated using the chloramine-T method as described for IL-8 (17). To
control the integrity of the labeled chemokine, iodinated PF-4 was
tested for its capacity to induce an exocytosis response in PMN (4),
its ability to form tetramers (5), and its capacity to bind to
different GAGs (filter binding assay, see below). No differences to
unlabeled PF-4 were seen in these assays. Specific receptor binding of
1 µM [125I]PF-4 to PMN was determined as
reported previously (5).
Chemical Radiolabeling of GAGs--
Heparin was
N-[3H]acetylated at free amino groups as
described before (18) to a specific activity of 20,000 dpm
3H/µg of hexuronic acid. Chondroitin sulfates (0.5 mg)
were partially N-deacetylated by hydrazinolysis for 60 min
at 96 °C in 1 ml of hydrazine hydrate (~30% water), 1% hydrazine
sulfate (19) and were then N-[3H]acetylated
with 2.5 mCi of [3H]acetic anhydride, essentially as
described (20). Specific activities of 8 × 105
dpm/µg for CSA, 2 × 105 dpm/µg for CSB, 6 × 105 dpm/µg for CSC, 9 × 105 dpm/µg
for CSD, and 6 × 105 dpm/µg for CSE were achieved.
CSA fragments resulting from a hydrazinolysis for 4 h were
separated on a BioGel P-10 column (1 cm × 140 cm) equilibrated
with 0.5 M NH4HCO3. Pools of
size-defined fragments were collected by reference to standard heparin fragments.
Preparation and Metabolic Labeling of Human Neutrophils--
PMN
cells were routinely isolated from citrated whole blood or fresh buffy
coats of healthy single donors by gradient centrifugation on
Ficoll-Hypaque to a purity consistently greater than 95% as described
previously (21). Viability was examined by trypan blue exclusion and
exceeded 98% in all experiments. Metabolic labeling was performed as
described by Gardiner et al. (10). Briefly, PMN cells were
washed twice with PBS and subsequently incubated at a concentration of
107 cells/ml in sulfate-free Dulbecco's modified Eagle's
medium, supplemented with 5% fetal calf serum, 1% glutamine, and 100 µCi/ml Na235SO4 or 10 µCi/ml
[3H]glucosamine for 5 h at 37 °C in humidified
air. Cells were washed three times with an excess of PBS, and cell
clumps were removed by digestion of cells with 100 µg/ml DNase I in
10 mM Tris/HCl, 10 mM MnCl2, pH
7.5, for 45 min at 37 °C under gentle agitation.
Preparation of Surface-expressed PMN-GAGs--
Neutrophil
surface-GAGs were proteolytically released from the cell surface by
digestion with protease type XIV. Cells were suspended to 5 × 107 cells in 1 ml of 0.1 M Tris/HCl, pH
8.0, 1 mM CaCl2 and incubated with 500 µg of
enzyme at 37 °C under agitation. After 18 h of incubation, the
same amount of enzyme was added a second time and the incubation was
continued for another 18 h. The suspension was clarified by
centrifugation (16.000 × g, for 20 min at 4 °C), and proteolytic activity in the supernatant was heat-inactivated at
98 °C for 20 min. GAGs were isolated from the supernatant by ion-exchange chromatography on a DEAE-Sephacel column (1 × 10 cm), equilibrated with 0.2 M
NH4HCO3. After removal of contaminants by
washing with 0.3 M NaCl and re-equilibration of the column with 0.2 M NH4HCO3, GAGs were
eluted with 2 M NH4HCO3 and
subsequently lyophilized. GAGs were quantified by colorimetric
determination of hexuronic acid using the
meta-hydroxydiphenyl method (22) with purified CSA as a
standard. Depending on the respective donor, about 14-16 µg of
GAGs/108 cells could be isolated with a specific
35S-radioactivity of ~3 × 104 dpm/µg
of GAG.
To examine a possible contamination of surface GAGs with
granule-derived material, the integrity of primary and secondary granules was monitored. Therefore, freshly isolated PMN as well as PMN
incubated for 36 h in the presence or absence of protease type XIV
were lysed with detergent and contents of the azurophilic granule
marker elastase as well as the specific granule marker lactoferrin were
determined as described elsewhere (17). In addition, these markers were
assayed also in the supernatant of PMN after 36 h of incubation in
the absence of protease.
Composition of GAG Preparations--
Size determination of
intact 35S-labeled GAG chains was performed by analytical
gel filtration on a Superose 6 column, equilibrated with 50 mM Tris/HCl, pH 7.4, 1 M NaCl, 0.1% Triton
X-100, and calibrated with heparin fragments of defined size
(4-mer ~ 1.3 kDa, 10-mer ~ 3.3 kDa, 20-mer ~ 6.6 kDa, and 26-mer ~ 8.6 kDa) as well as with hyaluronan-fragments
(11.9, 18.9, 30, and 43 kDa). The nature of neutrophil GAGs was deduced
from the susceptibility to deaminative cleavage and to digestion with
chondroitinases as examined by gel chromatography on Superose 12. Deamination with nitrous acid was carried out at pH 1.5 according to
the method of Shively and Conrad (23), which results in degradation of HS to oligosaccharides. Digestions with chondroitinase ABC or AC were
performed with 0.1 unit/ml of the respective enzyme in 50 mM Tris/HCl, pH 8.0, 50 mM sodium acetate,
0.1% BSA for 16 h at 37 °C. About 1-2 × 104
dpm (~0.5 µg) 35S-labeled PMN-GAGs were used in all
incubations in a 200-µl final volume.
Preparation and Analysis of CS
Disaccharides--
35S-Labeled neutrophil GAGs (2 × 105 dpm; ~6.6 µg), were digested with chondroitinase
ABC (0.1 unit/ml) in 200 µl of chondroitinase buffer for 16 h at
37 °C, and the resulting disaccharides were purified on a Sephadex
G-15 column (1 cm × 170 cm) equilibrated with 0.2 M
NH4HCO3. The samples were freeze-dried and
dissolved in water before analysis. PMN-derived GAGs were
quantitatively converted into disaccharides, <5% of the degradation
products being tetrasaccharides. Disaccharides were separated further
with regard to charge by preparative high voltage electrophoresis on Whatman 3MM paper in 1.6 M formic acid (pH 1.7; 40 V/cm)
for 80 min (24, 25), and labeled fractions were recovered by elution with water. Monosulfated disaccharides ( Chondroitinase Protection Assay--
35S-Labeled
neutrophil GAGs (~ 0.5 µg) were preincubated with
increasing concentrations of PF-4 for 20 min at room temperature in 200 µl of chondroitinase buffer and were then digested with 0.1 unit/ml
chondroitinase ABC for 16 h at 37 °C as described above.
Proteins and GAG chains were dissociated by heating samples at 96 °C
for 10 min in buffer containing 50 mM Tris/HCl, pH 7.4, 1 M NaCl, 0.1% Triton X-100, and the products were separated
on a Superose 12 column.
Filter Binding Assay--
Approximately 0.2 µg of radiolabeled
intact GAGs or size-defined fragments were incubated with PF-4 at the
indicated concentrations in 200 µl of PBS, 0.1% BSA for 2 h at
37 °C. Unbound GAG was removed by filtration through a
nitrocellulose filter, while protein-bound GAG was trapped on the
filter surface (16). The protein-bound GAG was dissociated from the
membrane-trapped proteins in 2 M NaCl and analyzed for
radioactivity in a Characterization of Neutrophil Surface GAGs--
In previous
experiments we had shown that PMN proteoglycans responsible for PF-4
binding were rather resistant toward proteolytic digestion by trypsin
or chymotrypsin (5). For this reason, a more unspecific protease
(Streptomyces protease type XIV) was used for the digestion
of neutrophils. In a first approach, experiments were designed to
explore the time course for the elimination of PF-4 binding sites. A
constant concentration of 500 µg/ml protease was used and the
residual capacity for binding of iodinated PF-4 was monitored at
different time-points as described (5). Binding of 1 µM
[125I]PF-4 to PMN at 4 °C decreased over time with
20% residual binding remaining after 10 h, and 5% residual
binding after 18 h of incubation (data not shown). Therefore, the
incubation time for proteolytic digestion of neutrophil PGs was
extended to 36 h, when PF-4 binding was decreased to background
levels. Under these conditions, about 40-50% of the total
metabolically 35S-labeled material could be mobilized,
while 50-60% remained cell-associated. No difference in the enzymatic
activity of the primary granule marker elastase or content of the
secondary granule marker lactoferrin (both determined in
detergent-treated lysates) was seen between non-treated and
protease-treated cells after 36 h. Furthermore, no marker protein
was found in the cell-free supernatant (data not shown), indicating
that neither primary nor secondary granules had leaked. These results
support the notion that the integrity of granules after the proteolytic
treatment was conserved such that the remaining 35S-labeled
material presumably would be located in neutrophil granules (10,
11).
The released 35S-labeled surface GAGs were further purified
by ion-exchange chromatography, after which the molecular size was determined by gel filtration on a Superose 6 column. PMN-GAGs eluted as
a single broad peak representing molecules of a wide range of molecular
sizes with an average of ~23 kDa (Fig.
1). This value corresponds to a GAG chain
length of about 90 monosaccharide units (calculated with an average
mass of 500 Da for a disaccharide unit), similar in size to a bovine
nasal cartilage CSA preparation used as a standard. In order to
characterize the GAG chains, the metabolically labeled PMN-GAGs were
treated with either nitrous acid at pH 1.5 or with chondroitinase ABC
and AC, respectively, followed by gel chromatography of the products.
Nitrous acid treatment at pH 1.5 did not affect the elution behavior of
the sample, indicating the absence of N-sulfated hexosamine
residues as found in HS (Fig. 2A). In contrast, digestion
with either chondroitinase ABC (Fig. 2B) or chondroitinase
AC (Fig. 2C) resulted in the quantitative conversion of the
chains into small saccharides. From these data, we conclude that
neutrophil surface GAGs consist predominantly of CS and lack detectable
amounts of HS or CSB.
Composition of Neutrophil Surface CS--
For compositional
analysis, the surface PMN-GAGs were extensively digested with
chondroitinase ABC and the resulting fragments separated by gel
filtration on a Sephadex G-15 column. More than 90% of the labeled
material eluted as disaccharides which were further separated by high
voltage paper electrophoresis in order to characterize their sulfation
degree. The migration profile of the disaccharides revealed two peaks
(Fig. 3A). The major peak (P I) with approximately 74% of the total radioactivity
comigrated with a monosulfated disaccharide (
Paper chromatography of the monosulfated disaccharide fraction P I
showed a single peak corresponding to the standard PF-4 Binding to Neutrophil Surface CS Chains--
In order to
characterize the putative PF-4 binding sites on the CS chains, enzyme
protection assays were performed. Purified 35S-labeled
PMN-CS was preincubated with increasing concentrations of PF-4 and
subsequently digested with chondroitinase ABC. At a concentration of 10 µM PF-4, more than 95% of the PMN CS-chains were
protected against digestion with the bacterial eliminase (Fig.
4). However, a stepwise decrease of the
PF-4 concentration led to a corresponding decrease in the amount of
protected polysaccharide: at 1 µM PF-4 59% and at 0.2 µM only 13% of the total radioactivity remained in the
high molecular weight fraction. At 0.04 µM PF-4, protection of the CS chains was completely abrogated and all of the
labeled carbohydrates eluted in a second peak, representing the
breakdown products. Therefore, at sufficiently high concentrations PF-4
can bind to and protect all PMN-derived CS. Corresponding control
experiments performed with the bovine [3H]CSA revealed a
similar PF-4-mediated protection of these chains against digestion with
chondroitinase ABC. However, compared with the PMN-GAGs, the
concentration of PF-4 required for complete protection was
significantly higher (20 µM) indicating a lower affinity
of the chemokine for the bovine CSA. Notably, decreasing the
concentrations of PF-4 led to a reduction in the total amounts of
protected CS chains but did not affect the size of these chains (Fig.
4). Thus, binding of PF-4 to the polysaccharide protected the entire
chains from digestion but did not reveal any limited fragment that
could be identified as a binding site for the chemokine.
Structural Requirements of CS-Binding to PF-4: The Importance of
Chain-length and Disulfated Disaccharide Units--
As PF-4 appeared
to protect the entire neutrophil CS chain from lyase digestion, our
next approach was to identify the minimal fragment size for PF-4
binding. Binding of size-defined, 3H-labeled CSA fragments
to PF-4 was examined using a nitrocellulose filter binding assay (16).
PF-4 (5 µM) was incubated with constant quantities of CSA
fragments of various chain lengths, and protein-GAG complexes were
retained by passing the solution through a nitrocellulose filter. As
shown in Fig. 5A, interaction
of PF-4 with CSA-fragments did not exceed background levels (~ 2.5%
bound oligomer) until a fragment length of ~ 8 monosaccharide
units (12.8% bound oligomer). Binding further increased with
increasing oligomer size. In an alternative approach to defining the
minimal PF-4-binding region, the chemokine was mixed with
[3H]CSA that had been partially degraded by extended
hydrazinolysis (27), and the mixture was subjected to digestion with
chondroitinase ABC. Analysis of the undigested [3H]CSA
preparation by gel chromatography showed a broad peak from 10.5 to 18.5 ml (Fig. 5B), representing chain lengths varying between
dimers and ~48-mers, with an average approximate size of a 20-mer
(14.5 ml). The same material that had been treated with chondroitinase
ABC in the presence of PF-4 emerged as two clearly separated peaks. The
first peak, representing the protected fraction, started to elute in
the same range as the untreated control, indicating full protection of
the largest fragments. Fragments were protected down to ~22-26-mer
size. However, fractions corresponding to smaller fragment size showed
a sharp decrease in radioactivity, indicating that chains shorter than
~20 monosaccharide units (eluting after 14.5 ml) were not protected
by PF-4. These smaller fragments were found to be completely degraded
by chondroitinase and eluted in the second peak, representing the
breakdown products. The same labeled GAG fragments in control
digestions lacking PF-4 were completely degraded and eluted exclusively
in the second peak (data not shown). Taken together, the results of the
two experiments indicate that PF-4 binding to CSA requires a minimal saccharide sequence of ~20 monosaccharide units.
The affinity of CS to PF-4 is considered low as compared with that of
heparin or HS (28). As the affinity of GAGs for chemokines appears
related to the charge of the carbohydrate chain (29), we wondered
whether the presence of disulfated disaccharide units in
neutrophil-derived CS would influence the binding to PF-4. Filter
binding assays thus were performed with labeled PMN-CS or CSA at
constant concentration (~1 µg/ml), mixed with purified PF-4 at
increasing concentrations. The binding curves indicated that PMN-CS
bound with higher affinity to PF-4 than did CSA (Fig. 6). Scatchard analysis of the data (Fig.
6, inset) revealed an unusual binding pattern composed of
essentially two phases. Linear plots were obtained only above certain
minimal concentrations of PF-4 (1.25 and 2.5 µM,
respectively, in interactions with PMN-CS and CSA). Based on these
data, PF-4 exposed to both CS types a single class of binding sites
with apparent Kd values of ~0.8 µM
for PMN-CS and ~4.4 µM for CSA, hence more than 5-fold different. However, at concentrations below 1 µM PF-4,
the affinity of the GAG chains for the chemokine ligand decreased
dramatically. Almost identical non-linear binding patterns were
described for the interaction of PF-4 with binding sites on intact
neutrophils, suggesting similar mode of binding of PF-4 to isolated CS
and to intact cells (5). The cellular receptors were shown to
preferentially bind tetrameric PF-4, which was found to occur only at
concentrations exceeding 50 nM.
As the major structural difference between PMN-CS and CSA was the
presence of disulfated disaccharide units in the former species, the
possible influence of these groups on the binding to PF-4 was
considered. For this purpose, binding assays were performed with
various 3H-labeled CSs from different sources and affinity
constants were determined as before. Furthermore, all CS preparations
were analyzed for their content of disulfated disaccharide units.
Bovine intestinal heparin as well as human aortic HS served as
references. As shown in Table I, all of
the GAGs tested bound to PF-4, but with significantly different
affinities. CSA, CSB, and CSC, which did not contain any detectable
disulfated disaccharide units, showed relatively low affinities for
PF-4, Kd values ranging from 2.9 to 4.4 µM. However, CSD, which yielded about 8.7% The composition of neutrophil GAGs has been investigated by
several groups over the last decades. However, the GAGs expressed at
the PMN surface remain poorly defined with regard to structure as well
as functional role(s). The present study was initiated by our
observation that PF-4 binds to a CS proteoglycan on human PMN cells
(5). We therefore aimed at characterizing surface exposed GAGs and
their binding to PF-4. Proteolytic release of cell surface-associated
GAGs under conditions that removed all binding sites for PF-4 but
conserved the macroscopic appearance of the cells resulted in the
isolation of a GAG pool that contained CS, essentially of the CSA type,
but no HS (Fig. 2). These findings are in accordance with earlier
findings that CSA constitutes the major part of neutrophil GAGs (7, 8).
Under the conditions of isolation, only about 40-50% of the total
metabolically 35S-labeled material was released from the
cells, whereas the rest remained cell-associated. Assessment of
cellular integrity by means of marker proteins from either primary or
secondary granules indicated that no leakage had occurred from these
intracellular compartments. It therefore seems reasonable to assume
that the remaining radioactivity would be localized intracellular, in
agreement with earlier publications localizing the majority of
intracellular GAGs to the granules (10, 11). Up to 25% of GAGs from
total PMN extracts was identified as HS, based on susceptibility to nitrous acid treatment (9). Although we cannot exclude the presence of
HS in PMN, we conclude that HS is not expressed at the cell surface and
therefore does not participate in the recognition of PF-4 by these
cells. Such recognition is mediated by CS chains.
Compositional analysis of the isolated CS chains indicated ~90%
[ PF-4 binds to CS as well as to heparin-related GAGs (12, 13, 28, 30,
31), and is stored as a CS proteoglycan/PF-4 complex in PF-4 is a member of the CXC chemokine family, with a three-dimensional
structure very similar to that of other members of this family. The
monomeric unit, consisting of a C-terminal aliphatic The results of the present study are clearly relevant to the mode of
action of PF-4 at the PMN cell surface. The Kd for
PF-4 interaction with isolated PMN-CS chains (~0.8 µM)
was in a range similar to that determined for the binding to intact PMN
(~0.65 µM). Moreover, the binding curves obtained with
whole cells (5) and with isolated CS showed similar sigmoidal shapes and non-linear Scatchard plots, indicating a decrease in the affinity for PF-4 binding sites at low concentrations of the chemokine. This
phenomenon is most likely caused by the selectivity of neutrophil GAGs
for binding to the tetrameric form of the chemokine. As we have shown
previously, tetramerization of PF-4 takes place only at concentrations
exceeding 50 nM PF-4, and in the absence of PF-4 tetramers
neither binding to cellular receptors, nor functional activation of PMN
is detected (5). Finally, the effects of "oversulfation" of the
PMN-CS on PF-4 binding should be considered. The increase in binding
strength caused by the presence of disulfated disaccharide units would
seem to be of key importance in the control of PF-4 binding and
PF-4-mediated cellular activation. As about half-maximal occupation of
the implicated receptors on PMN is required for the induction of a
measurable cellular response (4), whereas the serum concentrations of
PF-4 do not exceed 1.0-2.5 µM (41), a receptor
substituted simply with CSA (Kd ~ 4.4 µM) would hardly recruit sufficient amounts of the
chemokine to mediate a cellular response. Although it seems likely that the signaling component of the PF-4 receptor is a protein constituent, receptiveness is determined by the composition of the associated CS
chains. We cannot exclude that a PMN-CS proteoglycan serves as a
co-receptor that is coupled to a secondary receptor function. An
important step toward the elucidation of the signaling mechanism will
be the isolation and characterization of the putative receptor core protein.
-chemokine that binds to and activates human neutrophils to undergo
specific functions like exocytosis or adhesion. PF-4 binding has been
shown to be independent of interleukin-8 receptors and could be
inhibited by soluble chondroitin sulfate type glycosaminoglycans or by
pretreatment of cells with chondroitinase ABC. Here we present evidence
that surface-expressed neutrophil glycosaminoglycans are of chondroitin sulfate type and that this species binds to the tetrameric form of
PF-4. The glycosaminoglycans consist of a single type of chain with an
average molecular mass of ~23 kDa and are composed of ~85-90%
chondroitin 4-sulfate disaccharide units type CSA
(
4GlcA
1
3GalNAc(4-O-sulfate)
1
) and of
~10-15% di-O-sulfated disaccharide units. A major
part of these di-O-sulfated disaccharide units are CSE
units (
4GlcA
1
3GalNAc(4,6-O-sulfate)
1
). Binding studies revealed that the interaction of chondroitin sulfate with PF-4 required at least 20 monosaccharide units for significant binding. The di-O-sulfated disaccharide units in neutrophil
glycosaminoglycans clearly promoted the affinity to PF-4, which showed
a Kd ~ 0.8 µM, as the affinities of
bovine cartilage chondroitin sulfate A, porcine skin dermatan sulfate,
or bovine cartilage chondroitin sulfate C, all consisting exclusively
of monosulfated disaccharide units, were found to be 3-5-fold lower.
Taken together, our data indicate that chondroitin sulfate chains
function as physiologically relevant binding sites for PF-4 on
neutrophils and that the affinity of these chains for PF-4 is
controlled by their degree of sulfation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chemokine family like
interleukin-8 (IL-8), neutrophil-activating peptide 2, or melanoma
growth stimulatory activity have been shown to act as potent activators
of PMN by binding to common IL-8 receptors (1). Such binding elicits
diverse biological responses such as chemotaxis, degranulation, or
adhesion. PF-4, another member of the
-subgroup of the chemokine
family, is released in high concentrations from activated platelets (2,
3). The functional role of PF-4 is intriguing. Highly purified PF-4
lacks any apparent biological activity for PMN but will in the presence
of tumor necrosis factor
stimulate these cells to exocytose
secondary granule markers or adhere tightly to different surfaces (4). These PF-4-induced functions are not elicited through binding to IL-8
receptors but by interaction with distinct binding sites different from
all other chemokine receptors known so far (4, 5). The action of PF-4
on PMN was shown to be sensitive to chondroitinase ABC treatment and
could be inhibited by soluble chondroitin sulfate (CS), indicating that
the potential receptor is of CS proteoglycan type (5).
4GlcA
1
3GalNAc
1
)n that are
O-sulfated on one or both
units.2 In contrast to the
glucosaminoglycans heparin and heparan sulfate (HS), they do not
contain N-sulfate groups or L-iduronic acid units (except for CSB), which have been particularly implicated in
protein binding to HS chains (6). The expression of glycosaminoglycans (GAGs) on neutrophils has been described previously by several authors.
Pioneering work by Olsson and co-workers showed that PMN predominantly
express chondroitin 4-sulfate (CSA) (7, 8), and Levitt et
al. demonstrated a minor proportion of HS in these cells (9).
However, as all of these analyses were done with total cell extracts,
little is known about the composition and function of cell
surface-expressed GAGs in PMN. Gardiner and colleagues showed that the
majority of metabolically 35S-labeled compounds occurs as
proteoglycans in neutrophil granules where they may enable proper
storage of granule contents or exert protective functions against
cellular damage (10, 11). Here, we provide evidence that surface
exposed CS chains serve as physiologically relevant receptors for PF-4
on PMN, and propose that this function is critically dependent on the
content of sulfate groups.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thromboglobulin antigen, or by automated N-terminal amino acid
sequencing (kindly performed by Dr. A. Petersen, Department of Clinical
Medicine, Forschungszentrum Borstel, Borstel, Germany).
Di-S) were subsequently analyzed by paper chromatography as described (25). For analysis of the
disulfated disaccharides (
Di-diS), the glycosidic linkage between
the unsaturated hexuronic acid residue and the galactosamine residue
was cleaved by treatment with 10 mM mercuric acetate as described (26) and followed by separation of the products by high
voltage electrophoresis. Papers from high voltage electrophoresis and
chromatography were dried, cut into 1-cm segments, and extracted with
water, and the radioactivity was determined by
-scintillation counting.
-scintillation counter. In some assays, binding
affinities of PF-4 for different GAGs were assessed by transformation
of the data according to Scatchard. As the molar amounts of PF-4 even
at the lowest dosages used in the assay exceeded those of the GAGs by
at least 5-fold, the amount of free PF-4 was set to total PF-4.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
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Fig. 1.
Size determination of neutrophil GAGs.
PMN-derived 35S-labeled GAGs (- -) or
N-[3H]acetyl-labeled CSA (·
·)
(10,000 dpm each) were separated on a Superose 6 gel
filtration column. Elution positions of standard, size-defined
[3H]heparin fragments (1.3, 3.3, 6.6, and 8.6 kDa) and
hyaluronan fragments (11.9, 18.9, 30, and 43 kDa) are indicated by
arrowheads. The void volume (Vo) was
determined with hyaluronan and the total volume
(Vt) with 3H2O.
View larger version (18K):
[in a new window]
Fig. 2.
Susceptibility of neutrophil GAGs to
deamination by nitrous acid and to chondroitinase digestion.
PMN-GAGs (15,000 dpm ~ 0.5 µg) were treated with nitrous acid
at pH 1.5 (A) or digested with 0.1 unit/ml chondroitinase
ABC (B) or chondroitinase AC (C) as described
under "Experimental Procedures". Digestion products (· ·) or
untreated controls (-
-) were separated on a Superose 12 column.
Di-S) standard, while
about 26% of the radioactivity appeared with the disulfated
disaccharide (
Di-diS) fraction. As the radioactivity of this second
peak (P II) represents two [35S]sulfate groups
per disaccharide molecule, the molar proportion of
Di-diS
corresponds to half of the amount of 35S-label in this
fraction. The molar ratio of mono- to disulfated disaccharide units
thus was calculated to 85:15. The occurrence of disulfated disaccharide
units in neutrophil surface CS appeared to be a general phenomenon as
the analysis of disaccharides from two other donors revealed similar
disulfated disaccharide contents of 12 and 13% with minor variations
between individuals (data not shown). Also,
[3H]glucosamine labeling was performed to assess the
amount of non-sulfated disaccharide units in the whole population. No
non-sulfated disaccharides could be detected by chondroitinase ABC
digestion of these preparations followed by high voltage
electrophoresis, indicating that the chains are composed essentially of
sulfated disaccharide units (data not
shown).3
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Fig. 3.
Disaccharide composition of
neutrophil-derived [35S]CS. A,
disaccharides were obtained by complete digestion of PMN-GAGs with
chondroitinase ABC, desalted by gel filtration, and analyzed by high
voltage paper electrophoresis at pH 1.7 as described under
"Experimental Procedures". Migration positions of
3H-labeled standard disaccharides run in parallel lanes are
indicated by arrowheads: Di-S, monosulfated
disaccharide,
4,5 HexA1
3GalNAc(OSO3);
mono-S, monosulfated monosaccharide
GalNAc(OSO3);
Di-diS, disulfated
disaccharide,
4,5
HexA1
3GalNAc(4,6-di-OSO3). Peaks I (P I) and
II (P II) were eluted from the paper stripes as indicated.
B, unsaturated, monosulfated disaccharides in peak I from
panel A were subjected to paper chromatography.
Arrowheads indicate the migration positions of
3H-labeled standards:
Di-4S,
4,5 HexA1
3GalNAc(4-OSO3),
Di-6S,
4,5
HexA1
3GalNAc(6-OSO3). C, mercuric
acetate-treated (·
·) or non-treated (-
-) disaccharides from
peak II in panel A were separated by high voltage
electrophoresis at pH 1.7. Arrowheads indicate the migration
positions of 3H-labeled standards run in parallel lanes:
mono-S, GalNAc(4-OSO3); mono-diS,
GalNAc(4,6-di-OSO3); and
Di-diS,
4,5 HexA1
3GalNAc(4,6-di-OSO3).
Di-4S disaccharide (Fig. 3B), whereas no material co-migrating
with the
Di-6S standard was seen. In order to assign the sulfation pattern of the disulfated disaccharide in fraction P II, the
disaccharides were treated with mercuric acetate (26) and the resulting
products were separated by high voltage electrophoresis. About 85% of
the labeled material displayed an increased migration after Hg-acetate cleavage as compared with the untreated control (Fig. 3C).
This peak corresponds to the disulfated monosaccharide
GalNAc(4,6-OSO3), originating from the disaccharide
HexA1
3GalNAc(4,6-OSO3) (
Di-diSE). The
remaining 15% of the generated monosaccharides migrated slower than
the original disaccharide and correspond to a monosulfated monosaccharide, originating from either
HexA(2-OSO3)1
3GalNAc(4-OSO3) (
Di-diSB) or
HexA(2-OSO3)1
3GalNAc(6-OSO3)
(
Di-diSD). In summary, the neutrophil surface GAGs
contain predominantly (up to 90%) chondroitin-4-sulfate disaccharide
sequences, while 12-15% of the chains consists of disulfated
disaccharide units, mainly due to the presence of GalNAc-4,6-disulfate residues.
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Fig. 4.
Protection by PF-4 of PMN-derived CS during
digestion with chondroitinase ABC. 35S-Labeled
neutrophil GAGs (15,000 dpm ~ 0.5 µg) were preincubated with
10 µM (- -), 1 µM (
), 0.2 µM (-
-), or 0.04 µM (-
-) PF-4 before
digestion with 0.1 unit/ml chondroitinase ABC for 16 h at
37 °C. Undigested GAGs (-
-) were run in parallel incubations, and
the products were separated on a Superose 12 column.
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Fig. 5.
Determination of the minimal CSA chain length
for binding to PF-4. A, nitrocellulose filter binding.
3H-Labeled even-numbered CSA fragments (~ 0.2 µg) were
incubated with 5 µM PF-4 for 2 h at 37 °C.
Binding was assessed using the nitrocellulose filter assay described
under "Experimental Procedures." The data represent mean ± S.D. of three independent experiments. B, lyase protection.
3H-Labeled partially degraded CSA (~25,000 dpm) was
incubated with 5 µM PF-4 and digested by chondroitinase
ABC (- -) or left undigested (·
·). Analysis of the products
was performed as described in Fig. 4.
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Fig. 6.
Equilibrium binding of PF-4 to
neutrophil-derived CS and to CSA. Approximately 0.2 µg of
35S-labeled PMN-CS (- -) or of 3H-labeled CSA
(-
-) were incubated with increasing concentrations of PF-4 for
2 h at 37 °C, and binding was assessed by the nitrocellulose
filter assay. Inset, data were transformed according to
Scatchard (dotted lines). Only values representing >5000
dpm bound for PMN-GAGs and >3600 dpm bound for CSA were used for the
determination of affinity constants (solid lines). The data
represent mean ± S.D. of three independent experiments, each
performed in duplicate.
Di-diS
upon chondroitinase digestion, bound to PF-4 with an affinity
comparable to that of PMN-CS (Kd values of 0.6 and
0.8 µM, respectively). Moreover, CSE, with the highest
content of
Di-diS (30.7%), scored the lowest Kd:
0.3. Interestingly, binding of PF-4 to HS from aorta revealed an
approximately 2.5-fold lower affinity (Kd ~ 2.3 µM) as compared with that of PMN-CS. By contrast,
heparin, the most negatively charged carbohydrate tested, bound PF-4
with appreciably higher affinity than any of the other GAGs tested (Table I).
Binding affinities of PF-4 for different glycosaminoglycans
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4GlcA
1
3GalNAc(4-OSO3)
1
] (CSA) units, in
agreement with previous findings (7-9). In addition, however, we
identified a significant proportion of disulfated disaccharide units,
most of which contained 4,6-di-O-sulfated GalNAc units (CSE
type), whereas a minor portion carried sulfate groups on both
monosaccharide units.
-granules of
platelets (32, 33). The affinity of PF-4 for different GAGs has been
postulated to decrease in the order heparin
HS
DS > CSC > CSA (28). In previous work, PF-4 displayed the highest
affinity for heparin of all the chemokines tested (13); a
Kd of 30 nM (12) is in fair agreement with the value (60 nM) determined in the present study.
However, the postulated generalized order of affinities for GAGs needs to be modified to account for the effects of minor variations in the
degree of sulfation. Although PMN-derived CS contained only ~13%
di-O-sulfated disaccharides, it bound PF-4 with an affinity (Kd ~ 0.8 µM) more than 5-fold
higher than that of the strictly monosulfated CSA from nasal cartilage
(Kd ~ 4.4 µM), and more than 2-fold
higher than that of HS from human aorta HS (Kd ~ 2.3 µM). The positions of the additional sulfate residues
appear to be less important for the increased affinity, as in CSD, with
an affinity for PF-4 (Kd ~ 0.6 µM)
comparable to that of PMN-derived CS, the disulfated disaccharides have
the GlcA(2-OSO3)
1
3GalNAc(6-OSO3) and not
the GlcA
1
3GalNAc(4,6-OSO3) structure as
predominantly found in PMN-CS.
-helix lying on
top of a three-stranded antiparallel
-sheet (34), forms dimers and
tetramers (35). Basic amino acid residues implicated with GAG binding
are predominantly located in the
-helix, but also in loops of the
-sheets (12, 36), in such a way that a PF-4 tetramer will display a
belt of positively charged residues around the entire molecule (37).
This arrangement may help to explain some intriguing observations
pertaining to the molecular dimensions of GAG-PF-4 interactions.
Although a short heparin fragment of ~6/8-mer size is shown to bind
PF-4 (16), maximal affinity is attained only for >20-mers (16, 38).
These extended sequences are believed to interact with both dimer
subunits of a tetramer (38), as has been found for HS (14). The
interaction between CS and PF-4 is too weak to show up at the 6/8-mer
level, but is clearly evident for ~20-mer sequences (Fig.
5A), thus suggesting similar modes of binding for CS, HS and
heparin. Moreover, even more extended, ~9-kDa (~40 monosaccharide
units), sequences of HS were protected against heparitinase digestion
in the presence of PF-4 (14). These large fragments were shown to
contain sulfated domains positioned at both ends, separated by a
central, mainly N-acetylated region, and would be expected
to wrap around the entire circumference of a PF-4 tetramer (14). CS
fragments of similar initial size were also protected by PF-4 against
digestion with chondroitinase ABC (Fig. 5B). Unexpectedly,
however, more extended CS chains (average ~19 kDa) were either
completely degraded by the enzyme or remained seemingly intact,
depending on the relative proportions of CS and PF-4 (Fig. 4). A likely
explanation to this finding is that the extended CS chain interacts
with more than one PF-4 tetramer, in such a manner that essentially the
entire length of the chain is engaged in protein binding. Provided that sufficient amounts of PF-4 are present to saturate all CS chains in the
mixture, these will be completely protected against enzymatic cleavage;
unbound CS chains will be completely degraded. A HS chain will behave
differently, due to its less homogeneous structure, highly sulfated
domains being interspersed by essentially unsulfated sequences (39).
The latter structures will be less prone to protein binding, hence
protection, and will therefore be preferentially cleaved during
incubation with the appropriate endoglycosidase. Composite domain
structure for HS fragments interacting with cytokines have been
postulated not only for PF-4 (14), but also for interferon-
(40) and
IL-8 (29).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Anders Malmström (University of Lund, Lund, Sweden) for providing CSA, CSB (dermatan sulfate), and CSC. We are also grateful to Dr. Kerstin Lidholt and Dr. Emadoldin Feyzi (University of Uppsala, Uppsala, Sweden) for supplying us with size-defined hyaluronan-fragments and human aortic HS, respectively. We especially thank Prof. Ingemar Björk (Swedish University of Agriculture Science, Uppsala, Sweden) for helping us with the interpretation of the binding data.
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FOOTNOTES |
---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Sonderforschungbereich 367, Projekt C4; by Swedish Medical Research Council Grant 2309; and by Polysackaridforskning AB (Uppsala, Sweden).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.
§ Recipient of Fellowship PE 724/1-1 from the Deutsche Forsch ungsgemeinschaft.
To whom all correspondence and reprint requests should be
addressed. Tel.: 46-18-471-4367; Fax: 46-18-471-4209; E-mail:
dorothe.spillmann{at}medkem.uu.se.
2
The nomenclature to define the various CS
species is based on the notion that all variants contain a significant
proportion of 4GlcA
1
3GalNAc(4-OSO3)
1
disaccharide units. Although CSA contains no additional sulfated
disaccharide unit, the other CS species do; the characteristic units
are:
4IdoA
1
3GalNAc(4-OSO3)
1
(additional
sulfated substituents may occur) for CSB,
4GlcA
1
3GalNAc(6-OSO3)
1
for CSC,
4GlcA(2-OSO3)
1
3GalNAc(6-OSO3)
1
for CSD, and
4GlcA
1
3GalNAc(4,6-OSO3)
1
for CSE.
3 Metabolic labeling of PMN-GAGs with [3H]glucosamine is hampered by the short metabolical labeling period possible and by the reduced metabolic turnover of these terminally differentiated cells. A general use of this labeling procedure for analytical purposes is therefore practically unfeasible.
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ABBREVIATIONS |
---|
The abbreviations used are:
PMN, polymorphnuclear cells;
BSA, bovine serum albumin;
CS, chondroitin
sulfate;
DS, dermatan sulfate;
Di-4S,
4,5
HexA1
3GalNAc(4-OSO3);
Di-6S,
4,5
HexA1
3GalNAc(6-OSO3);
Di-diSE,
4,5 HexA1
3GalNAc(4,6-OSO3);
Di-diS, disulfated disaccharide;
Di-S, monosulfated disaccharide;
GAG, glycosaminoglycan;
GalNAc, N-acetylgalactosamine;
HexA, hexuronic acid;
HS, heparan sulfate;
IL-8, interleukin-8;
mono-diS, disulfated monosaccharide;
mono-S, monosulfated monosaccharide;
PBS, phosphate-buffered saline;
PF-4, platelet factor 4.
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REFERENCES |
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