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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4020-4028
By
From the Department of Immunology and Cell Biology, Research Center
Borstel, Borstel, Germany.
Platelet factor 4 (PF-4), a member of the CXC-subfamily of
chemokines, is secreted in high amounts by activated platelets. In
previous studies, we found that PF-4 specifically binds to human
polymorphonuclear granulocytes (PMN), but requires tumor necrosis
factor-
IN THE PAST, EVIDENCE has been
accumulated that inflammation and thrombosis can no longer be regarded
as processes occuring independent of each other, but they have been
recognized as highly overlapping and interactive events. Thus,
activated platelets not only secrete mediators involved in the
regulation of hemostasis, but also various other components modulating
wound repair as well as the functional activities of inflammatory
cells. After platelet activation, blood leukocytes and the vascular
endothelium are the first cellular elements to become exposed to the
platelet release products.1,2 A considerable proportion of
these consists of 2 By contrast, the role of PF-4 is less clear. As we could recently show,
this chemokine neither interacts with receptors CXCR-1 or CXCR-2 on
neutrophils nor does it induce chemotaxis or degranulation in these
cells.12 Likewise, none of the additional classic functions induced by other CXC-chemokines, including intracellular
Ca2+-flux and adhesion to protein-coated surfaces, could be
elicited by PF-4 alone in suspended neutrophils.12 Instead,
a more specialized role for the chemokine was indicated by its
requirement for a costimulus, ie, tumor necrosis factor- However, this model imposes questions as to the conditions that will
allow neutrophil stimulation by PF-4 at the very onset of inflammation,
eg, in a situation in which platelet activation occurs as a consequence
of exogenous mechanical lesion of the tissue. Because proinflammatory
cytokines are virtually absent during this initial stage, alternative
costimulatory mechanisms would be required. In our present report, we
have dealt with this question by investigating neutrophil activation by
PF-4 in a setting more close to in vivo conditions in that we examined
the chemokine's stimulatory potential in the presence of unstimulated
endothelial cells (EC). We observed that, under these conditions, PF-4
did not require exogenous TNF- Cytokines and enzyme-linked immunosorbent assay (ELISA) for PF-4.
Human monocytic recombinant IL-8 (rIL-8; ie, the
72-residue isoform) was obtained from Pepro Tech Inc (Rocky Hill, NJ).
Human natural PF-4 was purified in our laboratory from release
supernatants of thrombin-stimulated platelets in a 3-step procedure as
previously described.12 Briefly, the major contaminant
Antibodies.
A murine MoAb against PF-4 (clone PF1) was generated in our laboratory
after immunization of Balb/c mice with horse myoglobin-conjugated human
PF-4, according to standard protocols. The antibody (IgG1 isotype)
specifically recognized PF-4 as evidenced by total competition of its
binding to solid-phase-coated PF-4 by excess of soluble antigen. The
antibody showed cross-reactivity neither with bovine or rabbit PF-4 nor
with the related human CXC-chemokines Preparation and culture of human neutrophils and EC.
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.7 Viability was examined by trypan-blue exclusion and exceeded 98% in
all experiments. Human EC were isolated from umbilical cord veins by
collagenase treatment and cultured in dishes coated with fibronectin,
as described previously.16,17 The cells were maintained in
M199 (Biochrom, Berlin, Germany) supplemented with 1%
penicillin/streptomycin, 1% L-glutamine (both from Biochrom), 5%
fetal calf serum (FCS), 30 µg/mL EC growth factor (both from
Boehringer Mannheim, Mannheim, Germany), and 20 µg/mL heparin
(Sigma). Cells were subcultured after trypsinization (0.5% trypsin
solution, supplemented with 0.2% EDTA; Biochrom) and used throughout
passages 2 to 4. In experiments performed with fixed cells, PMN or EC
were treated with 3% paraformaldehyde in PBS for 5 minutes and
subsequently washed 4 times with D-PBS/0.1% bovine serum albumin (BSA)
(low endotoxin BSA; Serva, Heidelberg, Germany) before use.
Adhesion assay.
EC were grown in microtiter plates for 2 to 4 days to allow formation
of confluent layers. Cell were then washed 2 times with warm (37°C)
assay buffer (D-PBS/0.1% BSA supplemented with 0.9 mmol/L
CaCl2 and 0.5 mmol/L MgCl2) directly before
use. Then, 150-µL aliquots of PMN (2 × 105 cells)
suspended in assay buffer were added to the washed EC together with
PF-4 or IL-8 at the concentrations indicated. In some experiments,
stimulation with the chemokines described above was performed in the
presence of various antibodies (10 µg/mL each) as indicated in the
text. Because some of the antibody samples contained sodium-azide as a
preservative, the corresponding isotype controls were also supplemented
with azide to identical concentrations. No differences in cellular
reactivity were seen in comparison with azide-free control samples. In
experiments in which PMN or EC were preincubated with PF-4, the
respective cells were washed after 20 minutes at 37°C with an
excess of assay buffer, and PMN were then added to EC as described
above. Neutrophils were allowed to adhere to EC for 20 minutes at
37°C. Nonadherent cells were pelleted to one edge of the wells by
centrifuging the plates at an angle of 45° at 150g for 1 minute at room temperature, unless otherwise indicated. Subsequently,
100-µL aliquots of supernatant were recovered for determination of
lactoferrin (see below), and the pellet of nonadherent cells was
removed by careful aspiration. Cells remaining adherent to EC were
lysed in assay buffer containing 0.1% Triton X-100, and the number of
adherent PMN was determined by measurement of neutrophil-specific
endogenous Measurement of exocytosis.
As mentioned above, supernatants for the determination of PMN granule
marker release were taken immediately after sedimention of nonadherent
cells and subsequently monitored for contents of lactoferrin using a
quantitative sandwich ELISA as described elsewhere.10 Release rates for lactoferrin were expressed as the percentage of total
content determined in lysates of detergent-treated PMN prepared in
0.1% hexadecyl-trimethyl-ammoniumbromide. Because some experiments
were performed with PMN in the presence of fixed EC, side-effects
caused by the potential presence of residual paraformaldehyde had to be
excluded. Therefore, supernatants from fixed EC incubated in the
absence of PMN were collected and used as assay buffer in an exocytosis
assay with PF-4-activated PMN. No difference in exocytosis was seen
between samples receiving this medium as compared with samples
receiving freshly prepared assay buffer.
Flow cytometry.
Neutrophils were incubated with stimuli and EC for 20 minutes at
37°C as described for the adhesion assay described above, and
microtiterplates were subsequently transferred 10 to 15 minutes on ice
to allow detachment. After careful resuspension, PMN were washed with
cold D-PBS followed by incubation with different antibodies (2 µg/mL
each) as indicated in the text for 30 minutes on ice. After labeling
with secondary DTAF-conjugated goat-antimouse IgG (15 µg/mL), samples
were analyzed in a flow cytometer (model FACStar PLUS; Becton
Dickinson, Heidelberg, Germany). To allow comparison of fluorescence
intensities obtained in different experiments, instrument settings were
calibrated with fluorescein-labeled latex beads (Becton Dickinson).
PF-4 stimulates neutrophils to undergo tight adhesion as well as
granule exocytosis in the presence of an EC layer.
In a first set of experiments, the ability of PF-4 to induce PMN
adhesion to a monolayer of unstimulated EC was examined over a wide
range of stimulus concentrations in comparison with that of IL-8.
Moreover, the capacity of PMN to respond by exocytosis under these
conditions was investigated by determining the amount of secondary
granule marker lactoferrin released into the culture supernatants. As
shown in Fig 1A, PF-4 at concentrations of
0.4 µmol/L and greater induced PMN adhesion to EC in a dose-dependent manner, with maximum adhesion (~64% of PMN added) being obtained within a range of 2 to 10 µmol/L of the chemokine. As expected, IL-8
induced PMN adhesion at substantially lower concentrations (in the
nanomolar range), but with an efficacy remarkably inferior to that of
PF-4 (~37% of PMN added; Fig 1B). As a further difference, practically no lactoferrin could be detected in cell culture
supernatants derived from IL-8-stimulated cells, whereas increasing
levels of the granule marker (up to 18% of total content of PMN added) were found with PF-4-stimulated cells (Fig 1A and B). These data provide first evidence that PF-4 has the capacity to induce PMN adhesion to EC without the requirement for an exogenous costimulus and
that the presence of EC enables the chemokine to stimulate PMN for
secondary granule marker exocytosis. Moreover, the diverging efficacies
of PF-4 and IL-8 for induction of cell adhesion, and especially the
failure of IL-8 to stimulate granule exocytosis, suggested that
neutrophil-EC interaction in response to either chemokine involved
different mechanisms.
PF-4-induced adhesion is dependent on L-selectin and leukocyte
function-associated molecule-1 (LFA-1), but not MAC-1.
To address the question of whether PF-4 and IL-8-mediated PMN adhesion
to EC might involve different adhesion molecules, corresponding assays
were performed in the presence of blocking MoAbs directed against
various adhesion molecules known to be either selectively expressed on
PMN (CD11a, CD11b, CD11c, CD18, and CD62L) and on the endothelium
(CD54, CD103, and CD106) or to be coexpressed on both cell types
(CD49d, CD31, and CD15). As shown in Fig 3 (upper panel), PF-4-dependent neutrophil adhesion was substantially inhibited (by more than 76%) after the addition of either anti-CD11a, anti-CD18, or anti-CD62L, which indicated that at least 2 different PMN-expressed adhesion receptors, the
PF-4-mediated neutrophil adhesion and exocytosis require
constitutive and inducible costimulatory signals provided by EC.
With respect to our previous observation that PF-4 in the absence of an
exogenous costimulus is neither able to induce exocytosis in suspended
PMN nor to increase their adherence to plasma- or gelatin-coated
surfaces,12 we wondered by which means such a costimulatory
signal(s) could be provided by EC. First, we sought to get an idea on
whether the contribution by EC just consisted in providing a passive
matrix (eg, by the constitutive expression of surface molecules
interactive with PMN) or whether metabolically active EC were required,
retaining the capacity to respond to PF-4-stimulated PMN or PF-4
itself (eg, by the upregulation of membrane-expressed costimulatory
molecules or by the release of soluble costimulatory factors). In a
first set of experiments, we examined whether PF-4 acted exclusively on
PMN to promote adhesion and exocytosis or whether stimulation of EC was
also required. Whereas PMN pre-exposed to PF-4 (0.4 to 10 µmol/L) and
subsequently washed to remove soluble chemokine still underwent
adhesion as well as exocytosis, correspondingly treated EC did not
promote any of these functions (data not shown), indicating that PF-4 by itself did not elicit a costimulatory signal in EC. To estimate whether functional activation of EC was required at all, these cells
were fixed with paraformaldehyde to render them metabolically inactive.
Subsequently, their capacity to promote PMN adhesion as well as granule
exocytosis in the presence of PF-4 was assayed as initially described.
For comparison, assays using fixed PMN in combination with viable EC
and controls using exclusively viable cells were run in parallel. As
shown in Fig 4A, the ability of viable PMN
to undergo adhesion in response to increasing concentrations of PF-4
was not impaired in the presence of fixed EC, whereas fixation of PMN
led to complete abrogation of their ability to adhere to viable EC.
Thus, PF-4-dependent PMN adhesion does apparently not require
activation of EC, but appears to be controlled by the regulation of
PMN-associated mechanisms. However, as evident from the data presented
in Fig 4B, this may be somehow different with PF-4-dependent granule
marker exocytosis. Here, lactoferrin release by viable PMN cocultured
with fixed EC is markedly reduced as compared with PMN stimulated in
the presence of viable EC. Not only was an approximately 4-fold higher
threshold of PF-4 (0.4 v 0.1 µmol/L) required for the
induction of a measurable response, but also in the presence of fixed
EC an apparent saturation of the response was already reached at a
level amounting to less than 50% of that induced by the same dosages
of PF-4 (2 to 10 µmol/L) in the presence of viable EC. As expected,
fixed PMN were not able to respond to PF-4. These results demonstrate
that, for the induction of a full exocytsosis response by PF-4, at
least 1 of the costimulatory signals contributed by EC can only be
provided by viable cells, whereas PF-4-mediated adhesion appears to be functional in response to constitutive signal(s) provided by EC serving
as a passive matrix.
LFA-1 but not L-selectin is involved in PF-4-mediated granule marker
exocytosis.
As demonstrated by the results given above, the signals contributed by
EC for the induction of adhesion and exocytosis in PMN may be
different. However, these signals appear to be secondary to those that
become elicited by PF-4 in PMN, because prestimulation of PMN with PF-4
is sufficient to promote either of these functions, whereas
prestimulation of EC is uneffective (see above). Thus, we examined
whether the same PMN surface molecules (CD62L and CD11a/CD18) that we
found to be involved in PF-4-mediated adhesion to EC would also
contribute signal(s) for the induction of exocytosis. For this purpose,
adhesion assays were performed in the presence of antibodies directed
against CD11a, CD18, and L-selectin (as described above), and the
proportion of adherent PMN as well as lactoferrin contents in the
cell-free supernatants were determined. As shown in
Fig 5 and consistent with the results
depicted in Fig 3, PF-4-induced neutrophil adhesion was inhibited by
all 3 antibodies. Similarly, in the presence of anti-CD11a and
anti-CD18, the levels of lactoferrin in the supernatants was
substantially reduced (by more than 65% and 83% at 10 µmol/L PF-4
with anti-CD11a and anti-CD18, respectively). However, no inhibition of
exocytosis was obtained in the presence of anti-CD62L. From these data
we conclude that PF-4-induced activation of surface-expressed integrin LFA-1 and its subsequent interaction with adhesion molecules on the
endothelium may elicit costimulatory signals required for PF-4-induced
exocytosis. By contrast, L-selectin appears to have a selective
contribution in the PF-4-mediated adhesion processes.
Although neutrophil activation in response to CXC-chemokines has been
intensively investigated during the last years, a role for platelet
factor 4 as a neutrophil-directed mediator is only beginning to emerge.
Early reports suggesting an activity profile for PF-4 essentially
identical to that of IL-823-26 could not be confirmed in
more recent studies that failed to detect any PMN-stimulating properties of the chemokine.6,27-29 This was most likely
due to the availability of more sophisticated purification methods, leading to the exclusion of contaminating mediators such as NAP-2 from
natural PF-4 preparations.12 Although it now appears clear that PF-4 by itself is not active on suspended neutrophils, we recently
discovered that an appropriate costimulus, such as TNF- The authors thank Drs B. Katzmann and H. Klüter (Institute of
Immunology and Transfusion Medicine, Medical University of Lübeck, Lübeck, Germany) for the generous supply of
platelet concentrates. We are indebted to Dr A. Petersen for performing sequence analyses of the PF-4 preparations and to Dr H. Moll (District Hospital Bad Segeberg, Bad Segeberg, Germany) for supplying us with
umbilical cords. We thank G. Kornrumpf for her expert work of cell
culture and especially acknowledge C. Pongratz and I. von Cube for
perfect technical assistance.
Submitted March 17, 1999; accepted August 18, 1999.
Supported in part by Deutsche Forschungsgemeinschaft,
Sonderforschungsbereich 367, Projekt C4.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Frank Petersen, PhD, Department of
Immunology and Cell Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany; e-mail: fpeters{at}fz-borstel.de.
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TOP
ABSTRACT
INTRODUCTION
(TNF-
-granule proteins that belong to the
CXC-subfamily of chemokines, the platelet factor 4 (PF-4) and the
connective tissue-activating peptide III (CTAP-III), which
both are found in serum at micromolar concentrations.
-thromboglobulin antigen (
inducible protein-10 (IP-10), or MGSA, as assayed by the
same method. MoAbs directed against CD18 (clone MHM23), CD11a
(clone MHM24), CD11b (clone 2LPM19C), CD54 (clone 6.5B5), and CD31
(clone HEC7) as well as murine isotype control antibodies IgG2a and
IgG2b were all purchased from Dako (Hamburg, Germany). Anti-CD49d and
anti-CD11c MoAbs were obtained from T Cell Diagnostics Inc (Woburn,
MA), whereas MoAbs directed against CD62L (clone Dreg56) and CD102
(clone BT-1) were from Coulter-Immunotech (Hamburg, Germany). MoAbs
against CD106 (clone 1.G11B1) were purchased from Endogen (Woburn, MA).
An antibody directed against human IL-2 (clone B0-7)
) was determined in
the cell-free supernatant from the same PMN tested for adhesion (
).
Assay backgrounds were determined in samples of unstimulated cells run
in parallel (adhesion, 8.8% ± 1.8%; exocytosis, 3.5% ± 1.9%)
and were subtracted. Data are given as the mean ± standard deviation (SD) of 3 independent experiments, each performed in
duplicate.
; back), 100 nmol/L IL-8 (
), or left unstimulated (
) were fixed with 3% paraformaldehyde before stimulation with
increasing concentrations of PF-4 or both cell types were left unfixed
(
Discrepancies in binding affinities, receptor densities, and biologic effects.
J Immunol
152:2467, 1994