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Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine subfamily
of cytokines, activates human neutrophils independently of
intracellular free calcium mobilization or binding to IL-8R. In the
present study, we have identified and partially characterized a
receptor for PF-4 on human neutrophils, which displays weak
cross-reactivity with the IFN-
-inducible protein 10, but not with
other -chemokines such as IL-8, neutrophil-activating peptide 2, or
melanoma growth-stimulatory activity (GRO). Binding studies revealed
that human neutrophils express a high number of receptors
(Bmax 
7.6 x 106
sites/cell) of moderate affinity (Kd 
650
nM). The kinetics of PF-4-binding correlates with the proportion of
PF-4 tetramers in solution and with the activation of neutrophils for
exocytosis. Reduction of PF-4 binding and PF-4-induced exocytosis in
the presence of various glycosaminoglycans or following treatment of
cells with chondroitinase ABC (but not other
glycosaminoglycan-degrading enzymes) altogether demonstrates that the
PF-4 receptor is a proteoglycan of the chondroitin sulfate class.
Cross-linking experiments with radiolabeled PF-4 revealed a
receptor-ligand complex of 250 kDa. Taken together, our data show
that a distinct chondroitin sulfate proteoglycan represents specific
receptors for tetrameric PF-4 on human
neutrophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine subfamily of chemotactic and proinflammatory cytokines
have been recognized to play important roles. The prototype of these
polypeptides, the IL-8 as well as the structurally related polypeptides
melanoma growth-stimulatory activity (GRO), neutrophil-activating
peptide 2 (NAP-2), and others were shown to activate neutrophils, but
not monocytes, to undergo directed chemotactic migration, adherence to
endothelial cells, and degranulation of lysosomal enzymes (reviewed in
Refs. 1 and 2). Moreover, it was found that these mediators elicited
PMN functions through interaction with two common receptors on the cell
surface (3, 4), the two IL-8R, A and B (5, 6), which are now termed
CXCR-1 and CXCR-2 and belong to the family of
seven-transmembrane-domain (7-TMD) receptors.
Since it was shown that the presence of the so-called ELR
(Glu-Leu-Arg-) sequence motif within the N terminus of -chemokines
is critical for binding to and signaling through these receptors
(7, 8, 9), the question arose as to by which structures and mechanisms
those -chemokines that do not contain the ELR motif might elicit
cellular functions. Two representatives of the latter group that are
presently under intense investigation are the IFN--inducible protein
(IP-10) (10) and platelet factor-4 (PF-4) (11, 12). While the former
polypeptide is an inducible product of many different cells (13, 14),
PF-4 is preformed and stored within granules of blood platelets and
mast cells, and becomes released in high amounts upon appropriate
activation (12, 15). To date, no direct evidence has been reported that
neutrophils express specific receptors for either of these mediators.
While this is in principle conceivable with IP-10, because to date no
neutrophil-directed biologic functions have been demonstrated for this
polypeptide, the proven activation of certain PMN functions such as
secondary granule exocytosis and adhesion (16) by PF-4 demands for
structures that can function as cellular binding sites. These
structures cannot be identical to the IL-8R mentioned above, as has
become clear from our previous findings that PF-4 even at extremely
high molar excess (up to 200,000-fold) is unable to compete for
receptor binding with IL-8 (16), as well as from another report by
Clark-Lewis et al. (7), demonstrating that PF-4 can only bind to these
receptors after artificial introduction of an ELR motif into its N
terminus.
Moreover, several further observations indicate that the prospective
PF-4 receptor may not even belong to the family of 7-TMD proteins. Most
strikingly, we did not find PF-4-dependent elevation of intracellular
free calcium concentration in PMN (16), a typical signal that is
induced by all chemokines and other mediators interacting with 7-TMD
receptors (17, 18, 19, 20, 21). In addition, the PF-4 functional spectrum in
neutrophils differs considerably from that elicited by ELR-containing
chemokines. While in earlier studies chemotactic and degranulation
activity as well as enhancement of PMN adhesion to various surfaces has
been claimed for PF-4 (22, 23, 24, 25), more recent studies failed to
demonstrate any PMN-stimulating capacity for this chemokine (7, 26, 27, 28). In an approach to clarify this situation, we could recently
demonstrate that highly purified PF-4 in vitro is in fact not
chemotactic for PMN and does not induce relevant degranulation or
adhesion by itself (16). However, a specialized role for PF-4 different
from that of IL-8 was indicated by its cooperation with physiologically
relevant amounts of TNF-. In the presence of the latter cytokine,
PMN responded to PF-4 by the selective exocytosis of secondary granule
contents (but not of primary granule contents), and underwent enhanced
adhesion to gelatin or plasma proteins. The latter function is probably
a direct consequence of secondary granule exocytosis and might be
important to localize PMN at inflammatory sites. The fact that neither
chemotaxis nor an intracellular Ca2+ signal was generated
even in cooperation with TNF- provided a further hint that the
potential PF-4 receptor(s) was different from 7-TMD proteins.
To date, there exist only few reports dealing with cellular binding structures for PF-4. The information available is almost exclusively limited to endothelial and connective tissue cells. As a common feature, there is evidence that heparan sulfate proteoglycans (HSPGs) are involved, as inferred from the sensitivity of PF-4 binding to treatment of cells with heparinase (29, 30). In a more defined approach, Watson and coworkers described interference of PF-4 with basic fibroblast growth factor (bFGF) for binding to the low affinity bFGF receptor (31), which was also identified as a HSPG (31). While it is not clear from this work whether there also exist HSPGs with strict specificity for certain ligands, others could recently provide evidence for the occurrence of HSPGs exhibiting at least a certain degree of selectivity. This was demonstrated by Luster et al. by describing a receptor on endothelial cell lines with cross-reactivity for PF-4 and IP-10, but not with chemokines of the ß-subfamily (32). However, until now, there are no reports dealing with the problem of whether these receptors are simply heterogeneous populations of proteoglycans or whether there may exist structurally defined molecules with individual functions.
In the present study, we have addressed these questions by investigating the binding of PF-4 to neutrophils and by attempting to biochemically characterize specific receptors. Our results indicate that PF-4 interacts with a chondroitin sulfate proteoglycan (CSPG) of defined size. These receptors were found to exhibit specificity for PF-4, which appears to be determined by the oligomeric state of the chemokine. Furthermore, our data suggest that the integrity of this receptor is required for the induction of PF-4-mediated biologic responses in neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rTNF- was a gift from Dr. H. Gallati (Hoffmann-LaRoche,
Basel, Switzerland). Human monocytic rIL-8 (i.e., the 72-residue
isoform), rIP-10, and rGRO were obtained from Pepro Tech (Rocky
Hill, NJ). Human natural PF-4 was purified in our laboratory from
release supernatants of thrombin-stimulated platelets in a three-step
procedure, as previously described (16). Briefly, the major contaminant
ß-thromboglobulin Ag was removed by immunoaffinity chromatography.
PF-4 in the flow-through was further enriched using a heparin-Sepharose
affinity column (Pharmacia/LKB, Freiburg, Germany) and was finally
purified to homogeneity by HPLC on an analytical cyanopropyl column
(4.6 x 250 mm, 5 µm, wide pore; Baker Research Products,
Phillipsburg, NJ). Eluates and fractions were screened for the presence
of PF-4 and potential contamination by ß-thromboglobulin Ag by ELISA,
as decribed elsewhere (16). The final PF-4 preparation exceeded 99%
purity, containing no detectable protein contaminants according to
analyses in silver-stained SDS-polyacrylamide gels and by automated
N-terminal amino acid sequencing (kindly performed by Dr. A. Petersen,
Department of Clinical Medicine, Forschungszentrum Borstel,
Borstel, Germany). Human natural NAP-2 was prepared as described
elsewhere (33).
Preparation of human neutrophils
PMN were routinely isolated from citrated blood of healthy single donors by gradient centrifugation on Ficoll-Hypaque to a purity greater than 95% in all events, as previously described (34). Viability was examined by trypan blue exclusion and exceeded 98% in all experiments.
Measurement of exocytosis
A total of 1 x 107/ml PMN was suspended in
Dulbecco’s PBS (D-PBS)/0.1% BSA (low endotoxin BSA (Serva,
Heidelberg, Germany)). A total of 100 µl volumes of cells was then
distributed to 96-well microassay plates, containing 100 µl of
rTNF- (9 ng/ml) and various concentrations of secondary stimulus
PF-4, in buffer supplemented with CaCl2 and
MgCl2 to yield final concentrations of 0.9 and 0.5 mM,
respectively. In some experiments, PF-4/rTNF- stimuli were
supplemented with various concentrations of glycosaminoglycans (GAG)
(CSA, CSB, or CSC; all from Sigma, Deisenhofen, Germany) appropriately
diluted in D-PBS/0.1% BSA. Controls were performed with rTNF- and
either of the CS alone. After an incubation period of 30 min at 37°C,
the cells were sedimented, and harvested supernatants were assayed for
contents of lactoferrin using a quantitative sandwich ELISA, as
described elsewhere (3). Release rates for lactoferrin were expressed
as the percentages of total contents determined in detergent-treated
PMN lysates prepared in 0.1% hexadecyl-trimethyl-ammonium bromide.
Iodination of chemokines and binding experiments
PF-4 and IL-8 were iodinated using the chloramine T method, as reported previously (3). Routinely, PF-4 was labeled to a sp. act. of 1.5 to 2 Ci/mmol. This tracer served to perform binding experiments designed to establish binding isotherms for PF-4 and was used in competition experiments performed with a fixed dosage of 1 µM 125I-PF-4. In experiments in which very low concentrations of tracer were used, 125I-PF-4 of high sp. act. (450–550 Ci/mmol) was employed. This material served to perform competition experiments with a fixed dosage of 5 nM 125I-PF-4, to trace the incorporation of 0.5 nM 125I-PF-4 into oligomers.
Binding experiments with PMN were performed as described in detail
elsewhere (3). Briefly, cells were suspended at 2 x
107 cells/ml in D-PBS supplemented with 20 mg/ml BSA
(binding buffer), and duplicate samples of 2 x 106
cells were incubated on ice for 2 h with 125I-PF-4 at
various concentrations in the presence or absence of unlabeled
competitors (PF-4, rGRO, NAP-2, rIL-8, rIP-10). Nonspecific binding
was determined by incubating parallel samples in the presence of a
100-fold molar excess of unlabeled ligand. For practical reasons, the
molar excess in samples containing more than 500 nM labeled PF-4 was
limited to 50-fold (at 1–2 µM 125I-PF-4), to 20-fold (at
2.5–5 µM 125I-PF-4), or to 10-fold (at 10 µM
125I-PF-4). After removal of the unspecifically bound
material by washing, radioactivity was determined in a gamma counter.
The binding data were curve fit with the computer program EBDA/LIGAND
(35) to determine affinity constants (Kd) and
numbers of binding sites (Bmax). Binding of IL-8
was determined as described previously (3).
Cross-linking experiments
For the analysis of PF-4 oligomer formation, 0.5 nM 125I-PF-4 was incubated in PBS with increasing concentrations of the unlabeled peptide for 60 min at room temperature to achieve equilibrium in oligomer formation. Samples were cross-linked by addition of Bis(sulfosuccinimidyl) suberate (BS3) (Pierce, Oud-Beijerland, The Netherlands) from a stock solution of 40 mM in PBS to yield a final concentration of 2 mM. After 30 min, the reaction was quenched by addition of Laemmli buffer, and 50 µl vol were separated electrophoretically on a 15% SDS-polyacrylamide gel under reducing conditions. Rainbow marker proteins (low m.w. marker; Amersham, Braunschweig, Germany) served as m.w. standard. Gels were fixed, dried, and subsequently exposed on x-ray film (Cronex 4; DuPont, Bad Homburg, Germany) for 24 to 48 h. Furthermore, all gels were scanned and quantitatively analyzed for radioactivity using the PhosphorImager System (Molecular Dynamics, Krefeld, Germany) in combination with the program ImageQuaNT Version 4.2 (Molecular Dynamics). The relative amount of monomers and oligomers was then calculated from the amount of radioactivity detected in the individual bands in relation to the total activity present on the respective lane.
For cross-linking of PF-4 to PMN membranes, 4 x 106 cells/tube were incubated with 10 nM 125I-PF-4 in binding buffer alone or in the presence of increasing concentrations of unlabeled PF-4 for 90 min on ice in a total volume of 200 µl. After repeated washing with PBS, cell-bound material was cross-linked with 1.5 mM BS3 in PBS for 1 h on ice. Cells were sedimented by centrifugation and pellets were lysed by adding 250 µl Laemmli buffer containing 1 mM PMSF, 5 µg/ml E-64 (both from Sigma), and 4 mM EDTA, and subsequently homogenized by sonification (10–15 pulses for 1 s at 1.5 W) (Branson, Carouge-Genève, Switzerland). After extraction of proteins at 37°C for 30 min, the supernatants were cleared by centrifugation (230,000 x g, 10 min at 10°C) and directly loaded on a 7.5% SDS-polyacrylamide gel, and electrophoresis was run under nonreducing conditions. Gels were analyzed for radioactivity, as described above.
Enzymatic digestion of PF-4 binding sites
All enzymes were purchased from Sigma. PMN (2 x 107 cells/ml in PBS) were incubated for 30 min at 37°C under agitation with 2000-benzoyl-L-arginine ethyl ester (BAEE) U/ml tosyl-L-lysine-chloromethyl ketone (TLCK)-treated trypsin, 6 U/ml tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated chymotrypsin, or different concentrations of heparinase I (from Flavobacterium heparaninum, EC 4.2.2.7), heparinase III (heparitinase I, from F. heparaninum, EC 4.2.2.8), chondroitinase ABC (protease free, from Proteus vulgaris, EC 4.2.2.4), or hyaluronate lyase (from Streptomyces hyalurolyticus, EC 4.2.2.1), as indicated in the text. After washing, cells were directly used in binding assays or cross-linking experiments.
Statistics
Statistical significance was analyzed by the two-tailed t test. The mean values given in figures and table represent results obtained from three experiments. Each experiment was conducted with neutrophils from a different individual.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments designed to explore the time course of PF-4 binding to
neutrophils were performed by using a constant concentration of 1 µM
of the iodinated chemokine. This dosage was chosen with respect to our
former observation that PF-4 in the 0.3- to 10-µM range was effective
in inducing the exocytosis of secondary granule markers from PMN (16).
Binding of 1 µM 125I-PF-4 to PMN at 4°C increased over
time with 40 to 50% of maximal binding achieved at 10 min, and 95% of
maximal binding achieved after 90 min of incubation (data not shown).
Thus, binding of PF-4 to PMN occurred considerably slower than that of
other -chemokines, e.g., of NAP-2 and IL-8, either of which
displayed 80% of maximal binding at 10 min and achieved equilibrium
after 60 min, as seen in previous work (3). Therefore, in subsequent
equilibrium-binding experiments with PF-4, the incubation time was
extended to 2 h.
As shown in Figure 1
, specific PF-4
binding to PMN using increasing concentrations of 125I-PF-4
was measurable from 0.15 µM on and was near saturation with 10 µM
of the ligand. Nonspecific binding (data not shown) continued to
increase in a linear manner even at higher concentrations of labeled
PF-4. Scatchard analysis of the data (Fig. 1, inset)
revealed an unusual binding pattern composed of essentially two phases.
A typical linear relationship between the transformed data was only
obtained when data derived from PF-4 binding at 300 nM (corresponding
to 8.1 pmol of bound 125I-PF-4/2 x 106
cells) and at higher concentrations were considered. Based on this set
of data, PMN expressed a single class of binding sites with an apparent
Kd of 648 ± 14 nM and a
Bmax of 7.6 x 106 ± 1 x
106 sites/cell. By contrast, transformed data derived from
PF-4 binding below 300 nM of the ligand did not fit into this
relationship, but revealed a decrease of binding affinity with
decreasing concentration of the chemokine. Examination of the data by
Hill transformation yielded a Hill coefficient (nH) of 3.701,
indicating significant positive cooperation in the binding of PF-4 to
its receptor(s).
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In additional experiments, we sought to obtain more direct
evidence for positive cooperation in PF-4 binding to its receptor(s),
as well as to examine whether specific PF-4 binding sites were shared
by other -chemokines. To approach the former issue, a very low
(nonbinding) concentration of 125I-PF-4 at 5 nM was
incubated with PMN in the presence of increasing concentrations of
unlabeled PF-4, and its binding kinetics was analyzed. As shown in
Figure 2A, at dosages of cold
PF-4 higher than 50 nM, 125I-PF-4 binding dramatically
increased over background levels, reaching a maximum with 1.25 µM of
cold PF-4. Further enhancement of cold ligand concentration led to a
dose-dependent decrease in the amount of bound 125I-PF-4
down to background levels. The initial increase in
125I-PF-4 binding in the presence of cold PF-4 confirmed
that positive cooperation existed between PF-4 molecules, leading to
enhanced interaction with binding sites, while competition of this
binding by more elevated dosages of cold PF-4 indicated that the
binding was specific. The selectivity of cooperation between PF-4
molecules was confirmed in experiments in which 5 nM
125I-PF-4 was coincubated with increasing concentrations of
other unlabeled -chemokines (IL-8, NAP-2, GRO, IP-10). Under
these conditions, none of the chemokines enhanced or competed the
binding of labeled PF-4 (Fig. 2A).
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B). By contrast, neither unlabeled rIL-8, nor rGRO or
NAP-2 used as competitors at dosages up to 30 µM changed the binding
of 125I-PF-4 to PMN. Unexpectedly, the
non-ELR--chemokine IP-10 at concentrations from 1.1 to 30 µM
competed for binding with labeled PF-4, although with a potency about
threefold lower than PF-4 competed for itself. Moreover, competition by
IP-10 was only partial, achieving a maximum of 71% (at 10 µM) as
compared with total competition by PF-4 itself (at 30 µM). A further
increase in cold IP-10 concentration did not result in further
reduction of bound 125I-PF-4, suggesting that IP-10
interacted with only part of the PF-4 receptors. Thus, within the range
of different -chemokines tested, binding sites were selective for
non-ELR- chemokines PF-4 and IP-10, while ELR chemokines exhibited
no detectable binding to these receptors. PF-4 binding and biologic activity for neutrophils correlate with the formation of PF-4 tetramers in solution
With respect to former reports that PF-4 tends to undergo
noncovalent oligomerization with increasing concentration (36, 37), we
wondered whether the phenomenon of positive cooperation in PF-4 binding
to PMN could be caused by the formation of oligomers, exhibiting
enhanced affinity for specific receptors. To examine whether
oligomerization took place within the concentration range of PF-4 used
for receptor-binding experiments, a constant concentration of
radiolabeled PF-4 (0.5 nM) was incubated alone or in the presence of
increasing concentrations of the unlabeled chemokine. Potentially
formed oligomers were then stabilized by adding cross-linker
BS3, and identical volumes of samples were subsequently
separated by SDS-PAGE. In Figure 3C, the appearance of a
representative polyacrylamide gel following visualization of
radioactive protein bands by autoradiography is given. At
concentrations of up to 50 nM, PF-4 existed in a monomer-dimer
equilibrium, as indicated by the presence of two bands with a molecular
mass of 7.9 and 16.2 kDa, respectively (lanes 2 and
3). From a concentration of 250 nM on, two further bands
with apparent molecular masses of 26.3 and 36.2 kDa became detectable
(lane 4), indicating the formation of PF-4 trimers
and tetramers, respectively. With further increasing concentrations of
cold PF-4 added, tetramer formation was clearly at the expense of
monomers, while the relative proportions of dimers and trimers remained
largely unchanged (lanes 5–7). The proportion of a
minor amount of high m.w. material appearing at the top of the gel in
the presence of cross-linker (compare to untreated control) remained
constant over the entire concentration range of PF-4. Scanning analysis
of radioactive bands and determination of their individual contribution
to total radioactivity detected on the respective lanes revealed
negative correlation of the amounts of tetramers and monomers present.
Thus, within the range of 0.25 to 31.25 µM of cold PF-4 added, the
relative amount of tetramers increased from about 7 to 63%, while at
the same time that of monomers decreased from about 67 to 10%.
Comparison of these data with those obtained for
concentration-dependent binding of PF-4 to neutrophils (Fig. 3A) as well as with the dose-response kinetics of
PF-4-induced exocytosis of lactoferrin from the same cells (Fig. 3B) revealed that both binding and biologic activity were
not detectable in the absence of PF-4 tetramers. In fact, the threshold
concentration of 0.25 µM PF-4 for tetramer formation was similar
(within the limits of the assay) to that obtained for binding (0.15
µM) and for the induction of exocytosis (0.31 µM), and either of
the latter parameters continued to increase with increasing tetramer
concentration.
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To obtain initial information whether PF-4 binding sites were
biochemically similar to other -chemokine receptors on neutrophils,
the effects of proteolytic enzymes, high ionic strength, and low pH on
the specific binding of PF-4 and IL-8 to PMN were compared. As shown in
Table I, pretreatment of PMN with 6 U/ml
chymotrypsin for 30 min at 37°C had no effect on the subsequent
binding of 125I-PF-4 (at 4°C), while the binding of
125I-labeled IL-8 was reduced by more than 50%. Likewise,
PF-4 binding was not affected following pretreatment of cells with 2000
BAEE U/ml of TLCK-treated trypsin (data not shown). A drastic effect on
PF-4 binding was, however, observed when cells preloaded with
125I-PF-4 were subsequently washed with buffer of high
ionic strength (PBS containing 0.5 M NaCl). Under these conditions,
PF-4 binding was reduced by more than 95%, while IL-8 binding remained
totally unaffected (Table I). Surprisingly, washing a parallel sample
of the same chemokine-loaded cells with an acidic buffer of pH 3 (a
procedure commonly used to detach polypeptide ligands from membrane
receptors) did not reduce the binding of 125I-PF-4, while
no detectable 125I-labeled IL-8 remained associated with
the cells (Table I). Altogether these results demonstrate that
interaction of PF-4 and IL-8 with their respective receptors is
governed by quite different principles. Resistance of PF-4 binding to
the action of proteases and to acidic conditions, as well as its
susceptibility to high ionic strength, all suggest biochemical
characteristics of the PF-4 receptor(s) diverging from those of the
common seven-transmembrane-segment receptors known for chemokines.
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The data presented above provided further evidence that PF-4
binding sites on PMN were quite different from those for other
-chemokines. In particular, our observation that high salt buffer
abrogated the binding of PF-4 pointed to the involvement of
proteoglycans, inasmuch as others have previously demonstrated that
binding of chemokines to, for example, heparan sulfate proteoglycans
(HSPGs) exhibited significant lability under corresponding experimental
conditions (32). To investigate whether cell surface-associated
proteoglycans could be responsible for PF-4 binding, neutrophils were
incubated with different GAG-degrading enzymes for 30 min and
subsequently examined for their capacity to specifically bind
radiolabeled PF-4 as well as to respond to PF-4 by exocytosis of
secondary granule contents. As shown in Figure 4A, pretreatment of cells with
increasing concentrations (up to 5 U/ml) of hyaluronidase, heparitinase
I, or heparinase I did not affect 125I-PF-4 binding as
compared with untreated control cells. However, a drastic decrease in
binding of the chemokine was obtained following incubation with
chondroitinase ABC. Reduction by about 50% was achieved with only 0.08
U/ml of the enzyme, and binding was reduced to background levels after
treatment with 2 U/ml. Furthermore, treatment of PMN with 1 U/ml
chondroitinase ABC, but not heparinase III, reduced significantly
PF-4-induced exocytosis in TNF--costimulated cells (Fig. 4B). Upon stimulation with up to 2.5 µM PF-4, the biologic
response in chondroitinase-digested PMN was reduced to background
levels, while at more elevated dosages of PF-4 residual responses
amounting to 29% (at 5 µM PF-4) and 47% (at 10 µM PF-4) of that
of untreated control cells were obtained. These data clearly
demonstrate that CSPG are responsible for the binding of PF-4 to PMN,
and binding to CSPGs is involved in PF-4-induced neutrophil activation.
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A,
specific binding of 1 µM 125I-PF-4 was inhibited in the
presence of all three types of CS, i.e., by 83% with CSA, by 95% with
CSB, and by 86% with CSC as compared with the untreated control.
Because preincubation of PMN with CS, followed by washing, was without
effect on subsequent PF-4 binding to the cells (data not shown), direct
complex formation of these GAG with PF-4 is likely to be responsible
for their inhibitory effects. Corresponding results were obtained when
the effect of CS on PF-4-induced lactoferrin release was examined (Fig. 5B). In this study, as compared with untreated control
cells, inhibition by 71%, by 90%, and by 82% was observed with CSA,
CSB, and CSC, respectively. Altogether these results demonstrate that
CSPGs are efficient competitors of PF-4 binding and PF-4 biologic
activity, providing further evidence that CSPG-like structures
may function as cellular receptors for the chemokine.
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To better define the biochemical characteristics, i.e., the
molecular size and number of species of PF-4 binding sites,
radiolabeled PF-4 was covalently cross-linked to intact PMN and the
solubilized membrane fraction subsequently separated by SDS-PAGE under
nonreducing conditions. To examine the specificity of
125I-PF-4 incorporation into radioactive bands detected on
the gel, parallel samples of cells were cross-linked in the presence of
1) 10 nM 125I-PF-4 (a dosage exhibiting no specific binding
to PMN; see Fig. 1), 2) a mixture of 10 nM 125I-PF-4 and 1
µM cold PF-4 (in which, due to oligomerization, high specific binding
was to be expected; see Fig. 2A), and 3) a mixture of 10 nM
125I-PF-4 and 10 µM cold PF-4 (in which, due to the
excess of unlabeled PF-4 oligomers, competition of specific binding
should occur; see Fig. 2A). As expected, cross-linking with
10 nM labeled PF-4 alone did not lead to detectable radioactivity on
the gel, except for that caused by free ligand itself at the bottom of
the gel (Fig. 6, lane 1). By
contrast, a broad band typical for proteoglycans and corresponding to
an approximate molecular mass of 250 kDa became visible with 10 nM
125I-PF-4/1 µM PF-4 (lane 2). This band
disappeared upon increasing the dosage of cold PF-4 to 10 µM
(lane 3). In a parallel set of experiments, PMN were
pretreated with GAG-degrading enzymes (each at 2 U/ml for 30 min at
37°) before cross-linking with 10 nM 125I-PF-4/1 µM
PF-4. Neither hyaluronidase nor heparinase treatment abolished the
250-kDa band, while treatment with chondroitinase ABC led to its
disappearance (data not shown). However, a lot of poorly separated
material remaining at the top of lane 2 (Fig. 6)
and forming a broad smear indicated that at least part of the
receptor-ligand complexes had become precipitated during the
cross-linking procedure. Thus, it cannot be excluded that further bands
representing specific binding sites may be hidden within this
unseparated material. Exactly the same results were obtained when
cross-linked cellular proteins were separated under reducing conditions
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine
receptors/cell for IL-8 (3), which comprise two related molecular
species, termed CXCR-1 and CXCR-2. Both molecules are G protein-coupled
7-TMD receptors displaying similarly high affinities for IL-8 and
diverging affinities for other -chemokines such as NAP-2 and GRO
(3, 4). However, as we could recently show by cross-competition
analyses, these receptors are not able to bind PF-4 (16). In our
present approach using labeled PF-4 for direct binding analyses, we
nevertheless found a surprisingly high number of PF-4 binding sites
(7.6 x 106/cell) of relatively moderate affinity
(Kd = 650 nM). Even with respect to our findings
that PF-4 apparently binds as a tetramer, resulting in a calculated
density of 1.9 x 106 sites/cell, this amount
outnumbers that of IL-8R by more than 30-fold. An explanation for this
enormous number of binding sites could be derived from the biochemical
composition of the receptor. First, indirect evidence for its
proteoglycan nature arose from experiments in which the sensitivity of
PF-4 binding to different dissociating agents was examined. At variance
with IL-8, cell-associated PF-4 could be dissociated by brief exposure
to high salt buffer (0.5 M NaCl), indicating the predominant
involvement of ionic bonds in receptor binding. Moreover, the stability
of PF-4 binding to treatment with moderately acidic buffer (pH 3)
suggested a crucial role for moieties of considerably low pI. More
direct evidence that the PF-4 binding sites are indeed located within
the (usually negatively charged) carbohydrate portion of a CSPG was
provided by our observation that pretreatment of intact neutrophils
with chondroitinase ABC (but not with heparinase I, heparitinase I, nor
hyaluronidase) strongly reduced PF-4 binding. Because several
carbohydrate side chains may be expressed on a single PG core protein,
and one side chain will possibly bind more than one PF-4 tetramer, the
actual number of receptor molecules is probably much lower than that
inferable from the calculated number of binding sites. Due to the heterogeneity in length of their GAG chains, proteoglycans do not migrate as sharp bands in SDS-PAGE, and therefore, determination of the molecular size of the PF-4 receptor was difficult. Our approach to covalently cross-link iodinated PF-4 to intact neutrophils revealed a specifically labeled receptor-ligand complex migrating at approximately 250 kDa in SDS-PAGE, a size that has also been found for a variety of different PGs such as syndecans or glypican (38, 39). Current studies are underway for the identification and characterization of the receptor core protein. Nevertheless, the strict requirement for detergent to achieve solubilization indicates that PF-4 receptors are integral membrane molecules.
To our knowledge, the present report is the first to show specific
binding of a chemokine to proteoglycans on neutrophils. Although others
have shown previously that PF-4 binds to membrane-associated HSPGs on
bovine aortic endothelial cells (30) as well as to human umbilical cord
vein-derived cells (29), none of these reports provided information on
the number of species or the molecular size of the proteoglycans
involved. Interestingly, Rot and coworkers recently could demonstrate
interaction of IL-8 with proteoglycans on endothelium in vivo (40, 41, 42),
and Luster et al. found that PF-4 and IP-10 share the same HSPGs for
binding to human endothelial cell lines (32). Our data demonstrating
that human neutrophils likewise express proteoglycans cross-reactive to
PF-4 and IP-10 are nevertheless surprising, since we found CSPG rather
than HSPG to be responsible for binding of the chemokines. In addition,
we found IP-10 to induce an exocytosis response in TNF--costimulated
PMN within a similar concentration range as characteristic for PF-4,
although with considerably lower efficacy (unpublished data). This
indicates that IP-10 is able to induce functional activation of
neutrophils through a pathway similar or identical to that activated by
PF-4. On the other hand, it is rather unlikely that IP-10 constitutes a
physiologically relevant activator of PMN functions, since its binding
and biologic activity occur at dosages much higher than those that are
observed to become released by its producer cells (43). Considering
this, PF-4 receptors will be physiologically monospecific for PF-4, a
chemokine found at micromolar concentrations. In general, our results
strengthen the view that cell-associated proteoglycans may exhibit
selectivity for certain chemokines, since no competition of PF-4
binding was found with the closely related -chemokines IL-8, NAP-2,
and GRO.
That chemokines in fact may exhibit specificity for individual GAG also
was shown recently by Witt and coworkers (44), who demonstrated
divergent binding affinities of IL-8, NAP-2, GRO, and PF-4 for
subpopulations of fractionated heparin and heparan sulfates. With
respect to these observations, we were not surprised to find that
exogenous CS (A, B, and C) in solution prevented the binding of PF-4 to
neutrophils, and inhibited PF-4/TNF--mediated neutrophil exocytosis.
Obviously, the soluble GAG acted as scavengers (by binding free PF-4)
and did not directly impair the capacity of cell-associated receptors
to interact with the chemokine, since PF-4 binding and
PF-4/TNF--mediated biologic activity remained unchanged in cells
preexposed to and subsequently washed free of soluble GAG.
Interestingly, Webb et al. (45) reported quite different phenomena when
investigating the effect of exogenous GAG in solution on IL-8-mediated
neutrophil activation. These authors described enhancement of
IL-8-mediated chemotactic migration as well as of intracellular calcium
fluxes, suggesting that GAG-bound IL-8 was still able to interact with
its receptor, and that matrix PGs could serve to immobilize and present
IL-8 to migrating PMN. It is thus conceivable that the distinct
physical natures of IL-8 receptors (7-TMD proteins) and PF-4 receptors
(CSPGs) will have a different impact on the ability of the bound ligand
to interact with accessory molecules such as exogenous GAG.
A further parameter determining the selectivity of chemokine receptors
for their ligands may be the oligomeric state of the chemokines
themselves. Noncovalent oligomer formation has been found to represent
a typical feature of all chemokines analyzed to date. While with
increasing concentration IL-8, NAP-2, GRO, and MIP-1ß form dimers
in solution (46, 47, 48, 49), PF-4 forms tetramers (36, 37) and IP-10
associates in oligomers of even higher order (32). The question whether
dimerization of IL-8 is essential for its biologic activity on
neutrophils has been investigated exhaustively, until Rajarathnam et
al. (50) could show that synthetic IL-8 analogues modified in a way to
prevent oligomer formation retained full biologic activity and
receptor-binding capacity. According to our results, conditions appear
to be quite different with PF-4. Scatchard analyses of PF-4 binding to
its receptor revealed significant positive cooperation, indicating that
PF-4 oligomer formation might be essential for binding. This assumption
was further supported by the observation that a marked shift in
monomer-dimer-tetramer equilibrium took place over the concentration
range of PF-4 used for the binding experiments. As visible after
covalent cross-linking and SDS-PAGE analysis, the proportion of
tetramers in solution dramatically increased with increasing PF-4
concentration at the expense of monomers, while that of dimers remained
unchanged. As has been reported by other authors before (36), PF-4
trimers (that became detectable in parallel to tetramers) are most
likely due to incomplete cross-linking of tetramers, and thus are not
normally present in solution. More direct evidence for a role of
oligomers in PF-4 binding to its receptor could be derived from the
analysis of competition kinetics, that resulted in an unusual
bell-shaped binding curve: while 125I-PF-4 alone did not
bind at low concentrations (<150 nM), addition of increasing dosages
of cold PF-4 led to a successive increase in cell-bound radioactivity,
suggesting that mixed oligomers of iodinated and cold PF-4 had formed
and interacted with the receptor. Displacement of the ligand at further
increased dosages of cold PF-4 was most likely due to the formation of
unlabeled oligomers that competed for binding. Similar bell-shaped
competition kinetics was reported recently by Hoogewerf et al. for the
binding of oligomeric IL-8 and other chemokines to isolated heparin as
well to GAG on the surface of endothelial cells (51). Furthermore,
these authors suggested that the oligomeric state of chemokines may
play a crucial role for their sequestration by GAG-coated surfaces. It
should be noted that in the presence of increasing concentrations of
IP-10, the binding of labeled PF-4 was not enhanced. This indicates
that IP-10 does not associate with PF-4 to generate functional
heterooligomers. Finally, since PF-4 did not significantly bind to or
stimulate biologic activity in neutrophils at dosages (up to 50 nM) in
which only monomers and dimers were present in solution, but only from
concentrations on which tetramers became detectable (250 nM), all
evidence obtained to date is in favor of the tetramer representing the
functional PF-4 oligomer.
The circumstance that PF-4 receptors appear to selectively bind
tetramers imposes several problems on the correct determination of
receptor affinity. Calculation of dissociation constants
(Kd) based on Scatchard transformation of
binding data is limited to bimolecular interactions defined by the
equilibrium according to the mass action law [Rec.] + [Ligand] 
[RecLigand]. However, PF-4 has to oligomerize before binding,
and the proportion of tetramers formed is a result of various
monomer-dimer-tetramer equilibria. Because the association of PF-4 into
tetramers is far from complete at concentrations in which binding to
receptors occurs (e.g., 16% at 1.25 µM), the calculated affinity of
Kd = 650 nM can only be addressed as an apparent
Kd, but does not give the true affinity of
receptors for tetramers. Nevertheless, it may provide a measure for the
relevance of PF-4 binding under physiologic conditions. Affinities of
PF-4 for other GAG were reported to be significantly higher. Witt et
al. (44) demontrated that PF-4 binds to heparin with a
Kd of 30 nM, and Gallagher and coworkers
reported divergent affinities (Kd of 15.9 and
200 nM, respectively) for its binding to heparan sulfate (52). Although
in comparison with these findings the affinity of PF-4 for CSPG on PMN
appears rather low, local concentrations of PF-4 after platelet
activation (normal serum concentration: 1.3–2.5 µM) (53) are likely
to be more than sufficient to effect more than half-maximal receptor
occupation and biologic responses in neutrophils. Furthermore,
restriction of the receptor to tetramer binding could serve as a
mechanism to protect neutrophils from improper activation, e.g., by low
amounts of PF-4 that constantly arise from decaying platelets. The
sharp decrease in the relative proportion of tetramers with decreasing
PF-4 concentration will probably also help to focus neutrophil
activation to distinct inflammatory sites, i.e., to areas in which
massive platelet aggregation, followed by substantial release of the
chemokine, takes place. Even a slight decrease in PF-4 levels in the
close vicinity of such sites would greatly reduce the neutrophil
inflammatory response and protect intact tissue from becoming
destroyed.
It is still an open question whether the PF-4 receptor described has a signaling function on its own. One could imagine that the CSPGs that we have identified are not signaling by themselves, but that they function as coreceptors, presenting the bound PF-4 to a second receptor with signaling capacity. Such a relationship has been described for bFGF, which binds to a HSPG on fibroblast and becomes then presented to its signaling receptor (54). Interestingly, it was found that PF-4 can also bind to this coreceptor and thereby modulate bFGF biologic activity (31). The coreceptor hypothesis might be supported by our observation that PF-4-induced exocytosis in chondroitinase ABC-treated cells was completely abrogated only at low concentrations of the stimulus, while there was still residual activity with high dosages of PF-4. On the other hand, to avoid cytotoxic side effects, we limited the concentration of the enzyme to 1 U/ml, and this dosage might not have been sufficient to effect total degradation of all GAG chains. Thus, a direct signaling function of the receptor cannot be excluded. Nevertheless, our data demonstrating that removal of cell-associated CS by chondroitinase treatment as well as the presence of excess soluble CS both inhibit PF-4 binding and PF-4-induced exocytosis strongly suggest that interaction of PF-4 with membrane-bound CS proteoglycans has a crucial impact on PF-4-mediated neutrophil activation.
Our present investigations are directed to the identification of the receptor(s) and the mechanism that are involved in PF-4 receptor signaling.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 575, S-75123 Uppsala, Sweden.
3 Address correspondence and reprint requests to Dr. Ernst Brandt, Department of Immunology and Cell Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail address:
4 Abbreviations used in this paper: PMN, polymorphonuclear; BS3, Bis(sulfosuccinimidyl) suberate; bFGF, basic fibroblast growth factor; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycan; D-PBS, Dulbecco’s PBS; GAG, glycosaminoglycan; GRO, melanoma growth-stimulatory activity ; HSPG, heparan sulfate proteoglycan; 125I-PF-4, 125I-labeled platelet factor 4; IP-10, IFN--inducible protein 10; NAP-2, neutrophil-activating peptide 2; PF-4, platelet factor 4; 7-TMD, seven-transmembrane domain.
Received for publication March 19, 1998. Accepted for publication June 17, 1998.
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), rIL-8 (
), NAP-2 (
), or GRO
). Data
represent total bound radioactivity and are given as mean ± SD of
three independent experiments.
),
heparinase III (
), or hyaluronidase (
