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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1158-1166
CHEMOKINES
The CXC-chemokine platelet factor 4 promotes monocyte survival
and induces monocyte differentiation into macrophages
Barbara Scheuerer,
Martin Ernst,
Iris Dürrbaum-Landmann,
Jens Fleischer,
Evelin Grage-Griebenow,
Ernst Brandt,
Hans-Dieter Flad, and
Frank Petersen
From the Department of Immunology and Cell Biology, Research Center
Borstel, D23845 Borstel, Germany.
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Abstract |
Unstimulated monocytes rapidly undergo physiological changes
resulting in programmed cell death (apoptosis) while stimuli promoting
differentiation of these cells into macrophages were shown to inhibit
apoptotic processes. In the present study, we report that the
platelet-derived  -chemokine platelet factor 4 (PF4) induces the
differentiation of monocytes into macrophages, as is evident from
morphological changes as well as from the up-regulation of
differentiation markers (carboxypeptidase M/MAX1 and CD71). Significant
alterations of the phenotype were observed after 72 hours of culture in
the presence of the chemokine and required a minimal concentration of
625 nmol/L PF4. PF4-induced macrophages were characterized
by a lack of HLA-DR antigen on their surface but showed a strong
increase in the expression of the CD28 ligand B7-2. Furthermore, PF4
stimulation prevented monocytes from undergoing spontaneous apoptosis
during 72 hours of culture as determined in an annexin-V-binding
assay. Although PF4 induced the secretion of relevant amounts of
TNF-, neutralizing antibodies directed against TNF- or
granulocyte-macrophage colony-stimulating factor (GM-CSF) did not
revert PF4-induced rescue from programmed cell death, suggesting that
PF4 exerts its antiapoptotic effects in a TNF-- or
GM-CSF-independent fashion. On the basis of these results, we propose
a novel role for PF4 in the control of monocyte differentiation during
an inflammatory process in vivo.
(Blood. 2000;95:1158-1166)
© 2000 by The American Society of Hematology.
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Introduction |
Platelets and many of their products not only
are well characterized with respect to their function in hemostasis,
but are now also recognized to play important roles in the
immunoregulation and differentiation of various cell types. During
acute vascular injury or chronic disease, activated platelets release a
variety of mediators, including 3 members of the chemokine family, the connective tissue-activating peptide III, RANTES, and the platelet factor 4 (PF41).1-3 Although PF4 is a member of
the  - or CXC-chemokine subfamily, sharing a high degree of
similarity in structure and sequence with the other members of this
group, the functional role of PF4 appears to be quite exceptional.
Related CXC chemokines such as interleukin 8 (IL-8),
neutrophil-activating peptide 2, or melanoma growth stimulatory
activity (GRO), were shown to act as potent activators of
polymorphonuclear granulocytes (PMN),4-6 inducing such
biological responses as chemotaxis, degranulation, or adhesion through
binding to common IL-8 receptors. In a recent report, we were able to
show that highly purified PF4 lacks chemotactic activity for PMN, but
in the presence of TNF-, stimulates these cells to undergo such
functions as exocytosis of secondary granule markers or tight adhesion
to different surfaces.7 Investigating PF4-binding sites, we
could demonstrate that PF4-induced functions were elicited not through
binding to IL-8 receptors or another 7-transmembrane-domain molecule,
but through interaction with an integral chondroitin sulfate
proteoglycan expressed on the surface of human PMN.8,9
Unlike other CXC chemokines, which are predominantly active on PMN, PF4
affects a wide range of different cell types. Brindley and
coworkers10 demonstrated the PF4-mediated release
of histamine by basophils, and Hayashi et al11 reported a
role for PF4 in the adherence of eosinophils. Besides eliciting these
proinflammatory functions, which are induced rather rapidly, PF4 was
also shown to be involved in long-term differentiation and regulatory
processes. Han and coworkers reported that PF4 supports the survival of
hematopoietic stem cells as well as of progenitor cells12
and suppresses the development and maturation of cells from the
megakaryopoietic lineage.13 Furthermore, an
antiproliferative activity of the chemokine on endothelial cells and
fibroblasts was reported by several authors.14-16 The
impact of PF4 in the regulation of cell growth was shown in the work of
Tanaka et al,17 who demonstrated the inhibition of tumor
growth and tumor-associated angiogenesis in cells transfected with PF4 cDNA.
In this study, we focus on biological effects elicited
by PF4 in human monocytes. We show that PF4 prevents monocytes
from spontaneous apoptosis and induces their differentiation into
macrophages. These results indicate a potential role for PF4 as a
mediator of long-term effects in the regulation of inflammatory
processes in vivo.
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Materials and methods |
Cytokines
Human natural PF4 was purified to homogeneity from release
supernatants of thrombin-stimulated platelets in a 3-step procedure as
previously described.7 The preparations contained less than 0.125 ng lipopolysaccharide (LPS) per mg PF4 (ie, below 4 pg/mL at 4 µmol/L PF4) as determined by the limulus
amoebocyte lysate assay, ruling out possible side effects caused by
contaminating LPS. PF4 was lyophilized, stored at  80°C, and
reconstituted to stock solutions of 1 mg/mL in 0.1%
trifluoroacetic acid (TFA) prior to use. Recombinant human IL-8
(72-residue isoform), recombinant human RANTES, recombinant human
IP-10, as well as recombinant human fractalkine
(76-residue chemokine domain) were purchased from Pepro Tech Inc (Rocky
Hill, NJ). All recombinant chemokines contained 100 ng LPS/mg protein
or less. Recombinant human macrophage colony-stimulating factor
(M-CSF) was obtained from R&D Systems (Wiesbaden, Germany), and
recombinant human granulocyte-macrophage colony-stimulating factor
(GM-CSF) was purchased from Sandoz (Basel, Switzerland). Recombinant
human TNF- was from Schering (Berlin, Germany).
Antibodies to PF4 and immunoaffinity chromatography
A murine monoclonal antibody against PF4 (clone PF63.1) was
generated in our laboratory following immunization of BALB/c mice with
horse myoglobin-conjugated human PF4, according to standard protocols.
The antibody (immunoglobulin [Ig] G1 isotype) specifically recognized
PF4, as evidenced by total competition of its binding to solid
phase-coated PF4 by excess soluble antigen. The antibody did not show
crossreactivity with either bovine or rabbit PF4, or with the related
human CXC chemokines  -TG Ag, IL-8, IP-10, or GRO as
assayed by the same method. For the preparation of an immunocolumn, 2 mg of the antibody were coupled to 0.3 g cyanogen bromide
(CNBr)-activated Sepharose (Pharmacia, Freiburg,
Germany) according to the recommendations of the
manufacturer to obtain a final gel volume of 1 mL. For
depletion of PF4 from media spiked with 4 µmol/L PF4,
the column was equilibrated with phosphate-buffered saline (PBS)
followed by application of 5 mL culture medium. The respective flow-throughs contained less than 0.06 µmol/L
PF4 and were used for stimulation following sterile filtration.
Cell preparation
Mononuclear cells were isolated from peripheral blood of healthy
volunteer donors by Ficoll-Paque gradient centrifugation. Monocytes and
lymphocytes were separated by counterflow centrifugation as described
earlier.18 The resulting monocyte fraction consisted of
more than 95% CD14+ cells as determined by
immunofluorescence staining with anti-CD14-specific monoclonal
antibody (Leu-M3, clone M -P9, Becton Dickinson, Heidelberg, Germany).
Cell culture and stimulation
Cell cultures were routinely performed in RPMI-1640 (Biochrom,
Berlin, Germany) supplemented with 100 U/mL penicillin G,
100 µg/mL streptomycin, 2 mmol/L
L-glutamine, and 5% heat-inactivated fetal calf serum
(Biochrom). Monocytes (0.5 × 106/500
µL) were cultured in Nunclon polystyrene 24-microwell
plates (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere
with 5% CO2 in the absence or presence of 4 µmol/L PF4. For control purposes, cells receiving no PF4
were always cultured either with medium alone or in the presence of an
amount of TFA corresponding to that contained in PF4-treated samples.
No differences between these samples were observed under any assay
conditions or parameters measured. Further controls were performed by
adding 20 µg/mL heparin (Sigma, Deisenhofen,
Germany) to PF4-stimulated as well as to unstimulated
cells and by culturing monocytes in immunodepleted medium that had been
previously spiked with PF4. To determine whether potential
contamination by endotoxin was responsible for the effects seen with
PF4 and the other chemokine preparations used, blocking experiments
with the LPS antagonist compound 406 were performed. For this,
monocytes were preincubated for 10 minutes at 37°C in the presence
or absence of 100 ng/mL compound 406 (kindly provided by
Prof S. Kusomoto, Department of Chemistry, Faculty of Science, Osaka
University, Osaka, Japan) and subsequently stimulated with the
different chemokines at the concentrations indicated.
In some experiments, cells were cultured in the presence of recombinant
human M-CSF (10 ng/mL), recombinant human GM-CSF (1 ng/mL), or recombinant human TNF- (10 ng/mL). Stimulations in the presence of inhibitory
antibodies were performed with 1 µg/mL neutralizing rat
anti-human GM-CSF antibody (clone BVD2-23B6, IgG2a, PharMingen,
Hamburg, Germany) or an IgG2a isotype control (clone R35-95,
PharMingen), 3 µg/mL neutralizing mouse anti-human TNF- antibody (clone 195, IgG3, Boehringer Mannheim, Mannheim, Germany) or an IgG3 isotype control (clone MIB13, a gift
from Dr J. Gerdes, Research Center Borstel, Borstel, Germany). After a given period of time, the cells were kept on ice for about
1 hour, and culture plates were subsequently washed with ice-cold PBS
to detach the adherent cells.
Immunofluorescence
Unconjugated murine monoclonal antibodies directed against the
following human antigens were used for immunofluorescence labeling of
monocytes: anticarboxypeptidase M/MAX1 (clone 7A2, IgG1), kindly provided by Dr R. Andreesen (Department of Hematology and
Oncology, University of Regensburg, Germany), anti-CD80 (clone MAB104,
IgG1, Coulter-Immunotech, Hamburg, Germany), anti-CD86 (clone FUN1), anti-CD95 (clone DX2, both IgG1, PharMingen), and IgG1
control antibody (Dako Diagnostika, Hamburg, Germany).
The following fluorescein isothiocyanate (FITC)-conjugated antibodies
were used: anti-HLA-DR (clone L243, IgG2a), anti-CD14 (Leu-M3, clone
M-P9, IgG2b), and IgG2a control antibody (all purchased from Becton
Dickinson), anti-CD14 (clone M5E2, IgG2a, PharMingen), IgG2b control
antibody (Dako Diagnostika), and dichlorotriazinyl-amino-fluorescein (DTAF)-conjugated goat F(ab')2
anti-mouse IgG Fc (Dianova, Hamburg, Germany). Phycoerythrin
(PE)-conjugated antibodies were anti-CD86 (clone IT2.2, IgG2b),
anti-CD95 (clone DX2, IgG1), IgG2b control antibody (all
purchased from PharMingen), and IgG1 control antibody (Dako Diagnostika).
For direct immunofluorescence labeling, cells were incubated with the
respective FITC- or PE-conjugated antibodies at concentrations according to the manufacturers' advice for 15 to 30 minutes on ice in PBS, washed with PBS, and fixed with PBS containing
1.5% paraformaldehyde. For indirect
immunofluorescence labeling, cells were incubated with the unconjugated
antibodies and washed as described, followed by incubation with
DTAF-conjugated goat anti-mouse antibody. After 15 to 30 minutes of
incubation, the cells were washed again and finally resuspended
and fixed. Flow cytometry analysis was performed with a FACStar
Plus (Becton Dickinson).
Evaluation of cell viability and detection of apoptosis
Determination of apoptotic cells was done by double-labeling with
annexin-V-FITC and propidium iodide (PI). Annexin-V binds to
phosphatidylserine residues, which are translocated from the inner to
the outer leaflet of the plasma membrane during the early stages of
apoptosis.19,20 The amount of necrotic cells within the
annexin-V-positive stained cells was determined by
counterstaining with PI (final concentration 1 µg/mL).21,22
Labeling of apoptotic cells was performed by using an annexin-V kit
(Bender MedSystems, Boehringer Ingelheim, Heidelberg, Germany).
Briefly, monocytes were incubated with annexin-V-FITC in binding
buffer (provided by the manufacturer) for 10 minutes on ice, washed,
and resuspended in the same buffer as described by the manufacturer.
Propidiumiodide was added immediately before flow cytometry analysis.
Measurement of monokine release into culture supernatants
After 24, 48, and 72 hours of cell culture, the supernatants were
harvested and stored at 20°C until analysis. The
concentration of TNF- in the supernatants was determined by use of a
quantitative enzyme-linked immunosorbent assay (ELISA), provided by Dr
H. Gallati (Intex, Muttenz, Switzerland) and performed as recommended
by the manufacturer. Quantitative ELISAs for the determination of IL-6,
GM-CSF (both from PharMingen), and IL-1 (R&D Systems) were performed
according to the manufacturers' advice.
Statistics
Wilcoxon matched pairs signed rank test was used for statistical analysis.
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Results |
PF4 induces morphological changes in human monocytes
To investigate the effects of PF4 on morphology and surface
marker expression of monocytes, freshly isolated monocytes were cultured in the presence of PF4 or left unstimulated. Since we have
shown previously that PF4 at concentrations of 4 µmol/L
induces a strong biological response in human neutrophils,7
this concentration was chosen for a first approach to investigate
potential effects of the chemokine on the morphology of human monocytes.
During 72 hours of culture, the PF4-stimulated monocytes
showed a dramatic increase in total cellular size and acquired a macrophagelike morphology, including the formation of pseudopodia (Figure 1A). Furthermore, these cells
became highly adhesive to the plastic surface. In contrast,
unstimulated monocytes were far less adherent and appeared irregularly
shaped, were of considerably lower size, and did not show any
development of pseudopodia (Figure 1B). The increase in cell size in
PF4-stimulated monocytes, as evident from microscopical examination,
was confirmed by flow cytometry analysis, in which these cells gave
rise to a significantly higher forward scatter signal as compared with
unstimulated cells (data not shown).
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| Fig 1.
PF4-induced changes in monocyte morphology.
Purified human monocytes were (A) cultured for 72 hours in the presence
of 4 µmol/L PF4 or (B) left untreated. Photographs were
taken by phase-contrast microscopy at the same magnification
(× 20).
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PF4-mediated up-regulation of differentiation markers on human
monocytes
The PF4-induced changes in morphology of the cultured
monocytes toward a macrophagelike cell type raised the question of
whether this phenomenon represented a true cellular differentiation or simply a shape change of the cells during culture. Since
carboxypeptidase M/MAX1 expression is known to increase during
maturation of monocytes to macrophages,23,24 the expression
of this differentiation antigen was used as a marker. In a first set of
experiments, dose-response experiments with increasing amounts of PF4
were performed, and carboxypeptidase M/MAX1 expression was monitored
after 72 hours of culture. As depicted in Figure
2A, at concentrations of up to 312 nmol/L PF4, no difference was observed in the surface
density of the marker (expressed as median fluorescence intensity
[MFI]) in comparison with unstimulated cells (MFI of approximately
30). However, PF4 concentrations from 625 nmol/L or higher
provoked about a 6-fold increase in the expression of carboxypeptidase M/MAX1 on monocytes. Surprisingly, the surface expression of the differentiation marker could not be further enhanced by increasing the
dosage of PF4 up to 10 µmol/L, indicating that this
phenomenon did not follow regular dose-response kinetics. Since a
concentration of 4 µmol/L PF4 was found to be optimal,
this dosage was chosen for all further experiments.
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| Fig 2.
Concentration and time-dependent effects of PF4 on the
surface expression of carboxypeptidase M/MAX1 on human monocytes.
(A) Concentration-kinetics. Monocytes were cultured for 72 hours in the
presence of increasing concentrations PF4 ( ) or left untreated
(-----). Cells were subsequently analyzed by flow cytometry for
carboxypeptidase M/MAX1 expression, given as median fluorescence
intensity as described under "Materials and Methods." (B)
Time-kinetics. Monocytes were incubated for different time periods in
the absence ( ) or presence () of a constant concentration of PF4
(4 µmol/L) and analyzed as described above. Data from 1 representative experiment out of 3 are shown.
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Next, monocytes were stimulated for different time periods with a
constant concentration of PF4 (4 µmol/L) or left
unstimulated, and expression of carboxypeptidase M/MAX1 was determined.
As depicted in Figure 2B, the density of the marker remained low
(greater than MFI 30) for up to 2 days of culture, showing only
marginal variations between unstimulated and stimulated cells. When the cell culture period was extended to 3 days, significant differences became apparent: although only a rather low increase of the marker expression (up to MFI 25) could be observed in unstimulated cells, this
increase was markedly higher in PF4-stimulated cells (up to MFI 170).
Interestingly, at this time point, also the macrophagelike morphology
became evident as controlled by microscopical examination. In the
presence of the chemokine carboxypeptidase M/MAX1, levels increased
further, up to 240 in MFI after 8 days, whereas marker expression
remained low on unstimulated cells. It should be mentioned that in
unstimulated cells the proportion of viable cells (as determined by
trypan blue exclusion) decreased after 3 days of culture to
approximately 70% and after 8 days was down to approximately 30%,
while in PF4-treated samples, cell viability exceeded 95% at all time points.
Principally the same results for the time-and-dose response kinetics of
monocytes stimulated with PF4 (increase after 72 hours at concentration
greater than 312 nmol/L PF4) were obtained when we
analyzed the expression of the transferrin receptor (CD71) as an
alternative differentiation marker (data not shown). To test whether
the observed effects were specific for PF4, control experiments were
performed in which the PF4-containing culture medium (1) was
supplemented with heparin and (2) was passed over an immunoaffinity
column to deplete the added PF4. Neither morphological changes nor
up-regulation of MAX1 expression were seen in cells stimulated with PF4
in the presence of 20 µg/mL heparin or in cells
receiving medium immunodepleted from the added PF4 (data not shown).
Taken together, our data provide evidence that PF4 induces a
change in the monocyte phenotype toward a macrophagelike
cell type.
PF4 regulates changes in the expression of surface markers on
human monocytes
To further characterize the phenotype of PF4-treated monocytes, the
cells were labeled with various monoclonal antibodies directed against
cell-surface markers known to become modulated in response to
inflammatory stimuli or during monocyte differentiation. Cells were
stained immediately after isolation and then following 72 hours of
culture in the presence or absence of PF4 and thereafter analyzed by
flow cytometry. The expression of carboxypeptidase M/MAX1 was used as a
functional positive control (Figure 3A). As
already shown above, PF4 induced a strong up-regulation of the latter
antigen after 72 hours of culture (up to MFI 445), as compared with
unstimulated control cells cultured for the same time (MFI 30) or with
freshly isolated cells (MFI 15). Just the opposite effect was observed
when analyzing the expression of HLA-DR: although the density of this
marker was slightly elevated after culture in the absence of PF4 (from
30 to 74 in MFI, Figure 3B) without relevant changes in the number of
positive cells, PF4 stimulation resulted in a significant
down-regulation of this marker to the levels of the isotype control.
Analysis of the LPS/LPS-binding protein receptor CD14 (Leu M3), which
was found to be present on all freshly isolated cells (MFI 102),
revealed a decrease in its expression upon culture irrespective of
whether PF4 was present or not (Figure 3C). However, in untreated
cultures, CD14 expression was reduced to background levels whereas
about 36% of the monocytes stimulated with the chemokine showed a
significant residual expression (MFI 91).
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| Fig 3.
Effects of PF4 on the surface expression of
different monocyte markers.
Immunofluorescence-staining of human monocytes with antibodies directed
against carboxypeptidase (A) M/MAX1, (B) HLA-DR, (C) CD14, (D) CD80, or
(E) CD86 was performed either with freshly isolated cells (left column)
or with cells cultured for 72 hours in the absence (middle column) or
presence (right column) of 4 µmol/L PF4. Cells were
analyzed for the respective surface marker expression (gray histograms)
or isotype controls (open histograms) by flow cytometry. Percentage
values refer to the relative number of positive cells and values within
brackets to mean fluorescence intensity of these cells. Data are
derived from 1 representative experiment out of at least 6. Significant
differences between numbers of positive cells from PF4-treated and
untreated cells after culture were observed in panel A (n = 10,
P < .006), panel B (n = 9, P < .008), panel C
(n = 9, P < .005), and panel E (n = 11,
P < .006).
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B7-1 (CD80) and B7-2 (CD86) are costimulatory molecules that are
required for the activation of T cells by monocytes. According to our
data, these surface markers, which were only weakly expressed on
freshly isolated monocytes, are differentially affected by PF4-treatment: while CD80 expression remained largely unchanged after
culture in the presence and in the absence of PF4 (Figure 3D),
stimulation with the chemokine mediated a strong up-regulation of the
CD86 antigen from 18% to 76% positive cells with an MFI of 4 and 28, respectively (Figure 3E).
In summary, our data show that PF4-induced differentiation of human
monocytes is accompanied by various changes of the phenotype of these
cells. As will be discussed later, the PF4-mediated loss of HLA-DR, the
preservation of CD14, as well as the up-regulation of CD86 may indicate
a specific functional role of these cells.
PF4 prevents monocytes from undergoing spontaneous
apoptosis
From our observation that considerably higher numbers of dead and
damaged cells were present in unstimulated long-term cultures than in
cultures run in the presence of PF4 (Figure 2B), the question arose as
to how the chemokine would influence the survival of these cells. To
assess the proportion of apoptotic cells, monocytes cultured for 72 hours in the presence or absence of PF4 were subsequently labeled with
annexin-V. Necrotic cells were identified by counterstaining with PI as
described in "Materials and Methods." Recombinant human GM-CSF (1 ng/mL), which is known to inhibit spontaneous apoptosis in
monocytes, was used as a positive control. As shown in Figure 4A, in the absence of PF4, about 59% of
the monocytes developed an apoptotic staining pattern
(PIlow and annexin-Vhigh) whereas 26% of the
population appeared to be necrotic (PIhigh and
annexin-Vhigh). By contrast, PF4-stimulated monocytes were
efficiently prevented from undergoing apoptosis. The number of
apoptotic cells in PF4-stimulated cultures was reduced to 4%, whereas
the number of necrotic cells was decreased to 9% (Figure 4B). Results
almost identical to those obtained with PF4 were seen with GM-CSF (4%
apoptotic cells and 8% necrotic cells) (Figure 4C). Comparable results
were achieved following 7-amino actinomycin-D (7-AAD) labeling of
monocytes or DNA-laddering in electrophoresis (data not shown). The
effects induced by PF4 (but not those induced by GM-CSF) could be
completely blocked by the addition of heparin (20 µg/mL)
to cell cultures as well as by immunodepletion of PF4 from the medium
(data not shown). Thus, our data clearly show that PF4 not only changes surface marker expression in monocytes but also prevents these cells
from undergoing spontaneous apoptosis.
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| Fig 4.
PF4 prevents monocytes from undergoing spontaneous
apoptosis.
Monocytes were cultured for 72 hours (A) in medium alone, (B) in the
presence of 4 µmol/L PF4, or (C) in 1 ng/mL GM-CSF. After simultaneous staining with
annexin-V-FITC and PI, cells were analyzed by flow cytometry, as
described under "Materials and Methods." The upper right
quadrant represents necrotic cells; the lower right quadrant represents
apoptotic cells; and the lower left quadrant represents viable,
nonapoptotic cells. Data are derived from 1 representative experiment
out of 13. Statistical differences between numbers of apoptotic cells
of untreated and PF4-stimulated or GM-CSF-stimulated cells were
observed with P < .002 (n = 13).
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Since signaling via CD95 (Fas)/CD95 ligand represents one of the
mechanisms known to induce apoptosis, we examined the expression of
CD95 on human monocytes during culture in the presence or
absence of PF4. Surprisingly, after 72 hours of culture, we found an
efficient down-regulation of CD95 to levels of the isotype control in
unstimulated cells, whereas the expression of the antigen remained
unchanged on PF4-activated monocytes (about 80% positive cells with an
MFI of 30; data not shown). The fact that CD95 expression was
significantly higher in cells that were protected from apoptosis by PF4
as compared with the unstimulated controls (n = 9,
P < .008) may indicate that signaling through CD95
does not play a central role in this regulatory process.
PF4-induced secretion of TNF- is not involved in the
protection against spontaneous apoptosis
In principle, PF4-mediated inhibition of monocyte apoptosis
could be induced either by direct action of the chemokine on the cells
or indirectly by stimulating the release of secondary mediators acting
in an autocrine manner. We and others have recently shown that low
concentrations of exogenous TNF- prevent monocytes from undergoing
apoptosis.22,25 Similar effects have been described for
hematopoietic growth factors, such as GM-CSF26 and
M-CSF.27 In the following experiments, a potential role for
autocrine acting factors was investigated first by direct measurement
of different cytokines (TNF-, IL-1, GM-CSF) in the culture
supernatants and then by the performance of blocking experiments using
inhibitory monoclonal antibodies to these cytokines.
In a first set of experiments, human monocytes were stimulated
for 24 hours with PF4 and the amount of TNF- secreted into the
culture supernatant was compared with that present in suspensions of unstimulated cells (Figure 5). Although
the levels of TNF- secreted displayed high variability between
individual donors (from 30 to 1,250 pg/mL in unstimulated
cells, and from 200 to 8,200 pg/mL in stimulated cells),
the concentration of TNF- from every single donor was always
elevated in supernatants derived from cells treated with PF4 (2.7-fold
to 93-fold) as compared with the respective unstimulated control. In
the same supernatants, neither IL-1 nor GM-CSF was detectable (data
not shown).
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| Fig 5.
PF4 induces TNF- release from monocytes.
Monocytes from 12 individual donors (indicated by different symbols)
were cultured for 24 hours in the presence of 4 µmol/L
PF4 or left untreated. TNF- release in the supernatants was detected
by ELISA.
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In the next step, we examined whether monocyte-secreted TNF- was
responsible for PF4-induced protection from spontaneous apoptosis. For
this approach, TNF-- and PF4-stimulated monocytes as well as
unstimulated control cells were cultured in the presence and absence of
inhibitory antibodies directed against TNF- and were subsequently
analyzed for apoptotic cells by annexin-V. As expected, stimulation
with PF4 as well as with TNF- significantly reduced the number of
cells undergoing apoptosis (from about 70% in unstimulated cultures to
18% with PF4- and 10% with TNF- stimulation, Figure
6). Although this effect could be
completely reverted by the addition of neutralizing anti-TNF-
antibodies in the TNF--stimulated control sample, these antibodies
were without effect on PF4-activated cells. These results clearly show
that PF4-mediated protection against spontaneous apoptosis was not due
to the induced release of TNF-. To exclude a potential effect of low
concentrations of GM-CSF not detectable by ELISA, parallel experiments
were performed with this growth factor in the presence or absence of
respective neutralizing antibodies. Although we could confirm that
GM-CSF effectively blocks spontaneous apoptosis in monocytes and that this could be reversed in the presence of the anti-GM-CSF
antibody, these antibodies had no effect on PF4-stimulated cells
(data not shown). Furthermore, analysis of the immune phenotype
of cells stimulated with PF4, GM-CSF, or M-CSF revealed
that all 3 stimuli induced an increased expression of
carboxypeptidase M/MAX1 after 72 hours of stimulation (92% to 98%
positive cells with an MFI between 150 and 250; data not shown).
However, cells showed a strikingly different pattern in the expression
of HLA-DR: although this surface marker was down-regulated on
PF4-treated cells (Figure 3B), HLA-DR appeared to be highly expressed
on GM-CSF- and M-CSF-activated monocytes (70% to 80% positive cells
with an MFI between 80 and 180; data not shown).
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| Fig 6.
Effect of neutralizing antibodies against TNF- on PF4-
and TNF--mediated rescue from monocyte apoptosis.
Monocytes were cultured for 72 hours in medium alone (left panel), with
4 µmol/L PF4 (middle panel), or with 10 ng/mL TNF- (right panel) in the presence (dark columns)
or absence (white columns) of neutralizing antibodies directed against
TNF-. Cells were subsequently labeled with annexin-V and PI, and the
percentage of apoptotic cells was determined by flow cytometry. Data
from 1 representative experiment out of 6 are shown (n.d. = not
determined).
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PF4-mediated effects on human monocytes cannot be
mimicked by other chemokines
To determine whether the induction of these cellular responses was
unique to PF4 or instead represented a more general property of all
chemokines, we tested several other chemokines representative of the ELR-CXC type (IL-8), non-ELR-CXC type (IP-10), CC type (RANTES), and CX3C type (fractalkine) at
physiological (10 nmol/L) and pharmacological (100 nm and
1 µmol/L) concentrations for
their ability to prevent spontaneous apoptosis in monocytes and to
induce monocyte differentiation into macrophages. Data represented in Table 1 demonstrate that at
concentrations of 10 and 100 nmol/L, none of the
chemokines had any effect on these functions. However, at a
pharmacological concentration of 1 µmol/L, a weak
although statistically not significant reduction of monocyte apoptosis was seen in cells treated with IP-10 (48% apoptotic cells), RANTES (47% apoptotic cells), and fractalkine (62% apoptotic cells) as compared with the untreated control cells (74% apoptotic cells). To
determine whether this effect was specifically induced by the chemokines or was rather due to potential endotoxin contamination of
the preparations used, we performed control experiments in the presence
of the potent endotoxin antagonist compound 406.28 In these
assays, protection against apoptosis induced by all 3 stimuli was
abolished by the endotoxin antagonist, indicating that the observed
effects were caused by LPS contamination and not by the chemokines
themselves (data not shown). However, our natural PF4 tested under
the same conditions retained its activity in the presence of
compound 406. IL-8 at a concentration of 1 µmol/L
significantly reduced monocyte apoptosis to half of the level that
was induced by PF4, in a way insensitive to the LPS antagonist.
None of the chemokines tested (except for PF4) was able to
induce monocyte differentiation or morphological changes of
these cells.
| |
Discussion |
In the present study, we report on the discovery of novel biological
activities of the platelet-derived CXC chemokine PF4 for human
monocytes. Although rapidly inducible monocyte functions like
chemotaxis or phagocytosis can be mediated by several
chemokines,5,6 chemokines like MCP-1 and other chemotactic
factors were shown to be incapable of rescuing monocytes from
apoptosis.26 Here we show for the first time that PF4
elicits long-term biological effects in these cells, such as prolonged
survival and differentiation into macrophages. Under physiological
conditions, monocytes circulate for 3 to 4 days in the human blood and
then emigrate into the tissue where they either differentiate into
macrophages or undergo apoptosis.29 During maturation,
monocytes increase in cell size and granularity and develop
pseudopodia, reflecting a variety of functional changes within these
cells.29-31 In general, the differentiation process takes
about 7 to 10 days, but the cytokines M-CSF and GM-CSF are known to
accelerate this development.27,32,33 Although
platelets have been recognized to play an important role in the acute
activation of monocytes, eg, by the release of pro-inflammatory cytokines such as IL-1 or the chemokine RANTES,3,34 their functional relevance for monocyte differentiation has remained ill
defined and is only beginning to emerge. Recently Ammon et al35 described the development of macrophages
upon coculture of monocytes with intact platelets, and further analysis
revealed that the active components were undefined lipid constituents
of the platelet membrane. We, however, show that a highly purified protein constituent, the PF4, is sufficient to induce differentiation of monocytes into macrophages and does so in a time- and
concentration-dependent manner. PF4-treated monocytes exhibited an
increased cell size, enhanced adhesiveness, and developed pseudopodia.
Furthermore, diffentiation markers carboxypeptidase M/MAX1 and CD71
(transferrin receptor) were significantly up-regulated. Differentiation
became visible after 72 hours of culture and required a dosage of at least 625 nmol/L PF4. Surprisingly, already moderately
higher concentrations of the chemokine (about 1.25 µmol/L) did not lead to further enhancement of marker
expression, indicating that the induction of this function did not
follow classic dose-response kinetics but rather represented an on/off
mechanism. The fact that PF4 (like GM-CSF) induces a differentiation
within 3 days instead of 7 to 10 days as described for serum-derived
macrophages, indicates that the chemokine actively accelerates the
differentiation process. No other chemokine tested, including IL-8,
IP-10, RANTES, and fractalkine, was able to induce monocyte
differentiation even at pharmacological concentrations, indicating that
within the chemokine family, this function may be a unique property
of PF4. Although no data exist in the literature concerning
PF4 concentrations at a site of acute platelet activation in
vivo, normal serum concentrations of PF4 (1 to 2.5 µmol/L36; seen also in our own
observations) are thus sufficient to induce a full
monocyte response. Apart from this, concentrations near a
thrombus, where platelet-monocyte interaction may occur, are likely to
be much higher.
The mechanisms involved in the regulation of monocyte apoptosis are
only partially understood. Today, several authors claim the existence
of 2 distinct pathways: one by which inactivated monocytes are
sentenced to programmed cell death and another by which activated cells
undergoing differentiating processes are rescued from apoptosis. First
reports by Mangan et al22,26 described the constitutive
occurence of apoptotic monocytes in cultures with low serum content.
Addition of LPS, GM-CSF, M-CSF,27 or the monokines TNF-
and IL-1, which represent monocyte-activating factors, could inhibit
this process and lead to prolonged survival of the monocytes. Since LPS
and M-CSF are able to induce the release of monokines, it has been
suggested that paracrine and autocrine mechanisms control monocyte
apoptosis. Our results show that PF4, like GM-CSF or
M-CSF, effectively blocks programmed cell death in human monocytes.
Stimulation of these cells with other chemokines, such as IP-10,
RANTES, or fractalkine, were without effect on monocyte apoptosis.
Surprisingly, IL-8 at a concentration of 1 µmol/L
significantly reduced monocyte apoptosis to half of the level induced
by PF4. Since unlike PF4, which is found at micromolar concentrations,
IL-8-induced effects occurred at dosages much higher than those that
were reported to occur physiologically, it appears rather unlikely that
IL-8 constitutes a physiologically relevant modulator of monocyte
apoptosis. Considering this, the latter function will be
physiologically monospecific for PF4 among chemokines. Furthermore,
our observation that IL-8-mediated prevention of apoptosis occurred
independently of monocyte differentiation indicates that PF4 and IL-8
may act through different signaling pathways.
PF4-mediated abrogation of apoptosis was accompanied by the secretion
of TNF- but not IL-1 or GM-CSF. However, the lack of
the capability of neutralizing antibodies directed against TNF-
to revert PF4-induced protection against apoptosis clearly demonstrated
that this monokine is not involved in the PF4-mediated process. Since
we did not find IL-1 or GM-CSF in culture supernatants of cells
stimulated with PF4, and anti-GM-CSF antibodies did not antagonize the
PF4-mediated effect, a participation of these monokines in regulating
PF4-induced functions appears unlikely. At present, we cannot
decide whether still other factors are involved as autocrine effectors
or whether PF4 is able to directly induce monocyte rescue from programmed cell death. A well-characterized pathway leading to
apoptosis is mediated by interaction of Fas ligand (FasL) with Fas receptor (CD95).37,38 Both Fas and FasL are
expressed on almost all freshly isolated
monocytes.39,40 Therefore, monocyte apoptosis can be
triggered by activating Fas on monocytes after interaction with FasL on
other leukocytes, or, vice versa, monocytes can induce apoptosis in
other Fas-expressing cells.39 However, our
observation that CD95 is completely lost after 3 days of culture on
untreated cells whereas its expression is conserved on PF4-treated cells argues against the involvement of the Fas/FasL pathway in our system. Our results are in line with results reported by
others that various factors (IL-1, TNF-, GM-CSF, and LPS) that
promote prolonged survival of monocytes do not mediate a
down-regulation of CD95.38,39 Most recently, Heidenreich et
al21 suggested that down-regulation of CD14 represents a
trigger for the induction of monocyte apoptosis. Indeed we
could confirm that the loss of this surface marker is
accompanied by spontaneous apoptosis in unstimulated cells, but on the
other hand, we also observed down-regulation (to a lesser extent) of
CD14 in PF4-treated cells that showed prolonged survival.
From our data, we are not able to decide whether the residual low
expression of CD14 on the latter cells may be sufficient for their
survival or whether the regulation of CD14 is not involved in
PF4-mediated inhibition of apoptosis. Since it has long been known that
high concentrations of human serum allow monocytes to survive in
long-term culture, the question may be raised whether this could
be due to PF4 present in the serum. We have investigated this
by using PF4-immunodepleted human serum (at 20%) for 3-day monocyte
cultures and found no difference in the numbers of apoptotic cells as
compared with cultures performed with nondepleted serum (unpublished
results). Therefore, the serum constituents protecting monocytes from
apoptosis are apparently not identical to PF4.
Besides the up-regulation of differentiation markers, monocytes
stimulated with PF4 undergo changes in the expression of several other surface molecules. Unlike unstimulated, M-CSF-stimulated, or
GM-CSF-stimulated monocytes (data not shown), PF4-treated cells displayed a strong down-regulation of HLA-DR antigen, which could suggest a substantial loss of their antigen-presenting capacity for T
cells. Simultaneously, the costimulatory molecule CD86 (B7-2), but
not CD80 (B7-1), was up-regulated during culture with PF4. B7-1 and
B7-2 are ligands for 2 receptors on T cells (CD28 and CTLA-4)
that function as accessory molecules in monocyte-dependent T-cell
activation.41,42 However, whether there is a regulatory role for B7-molecules in the absence of HLA-DR is not clear. Our data
suggest that PF4 induces the differentiation of monocytes into a
subtype of macrophages that is different from that induced in vitro by
GM-CSF, since the latter cells showed a high expression of HLA-DR but
no modulation of the B7-antigens.43 Current investigations are on the way to clarify the regulatory role of PF4-stimulated monocytes in the activation of T-cell functions.
In conclusion, our results provide direct evidence that
platelet-derived PF4 prolongs monocyte survival and induces the
differentiation of monocytes into macrophages. This indicates that
defined platelet products are involved in long-term regulatory
processes of these cells and might support the differentiation of
infiltrating monocytes into macrophages in vivo during intermediate and
late stages of an inflammatory process.
| |
Acknowledgments |
We wish to thank Drs C. Schlenke and H. Klüter (Institute of
Immunology and Transfusion Medicine, Medical University of
Lübeck, Germany) for the generous supply of platelet
concentrates. We acknowledge Dr A. Petersen (Research Center
Borstel, Borstel, Germany) for performing sequence analysis of the PF4
preparations. We thank Dr R. Andreesen (Department of Hematology and
Oncology, University of Regensburg, Germany) for providing antibodies
directed against carboxypeptidase M/MAX1. We especially thank Mrs R. Bergmann and Mrs E. Kaltenhäuser for perfect technical assistance
and Dr L. Bock for the preparation of PF4.
| |
Footnotes |
Submitted May 27, 1999; accepted October 12, 1999.
Supported in part by Deutsche Forschungsgemeinschaft,
Sonderforschungsbereich 415, Projekt A5.
Reprints: Frank Petersen, Department of Immunology and Cell
Biology,
Research Center Borstel, Parkallee 22, D-23845 Borstel,
Germany; e-mail: fpeters{at}fz-borstel.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction