Laboratory of Cancer Immunology, Department of Cryobiology and Cell Therapy, Cancer Research Institute, Barcelona, Spain
Correspondence: Fèlix Rueda, Laboratory of Cancer Immunology, Department of Cryobiology and Cell Therapy, Cancer Research Institute (IRO), Autovia de Castelldefels, Km 2.7, 08907 L’Hospitalet de Llobregat, Barcelona, Spain. E-mail: frueda{at}iro.es
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: inflammation • chemokines • Fc
RIIIA
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Granules contain regulatory cytokines that are
rapidly released upon platelet activation in acute or chronic processes
of vascular damage. Among these regulatory factors, there are members
of the chemokine supergene family of proinflammatory cytokines such as
platelet factor 4 (PF4) [3
]; RANTES [4
];
and connective tissue-activating peptide III (CTAP-III)
[5
, 6
]. PF4 and CTAP-III belong to the
-chemokine subfamily, structurally characterized by the presence of
an extra amino acid between two of the four conserved cysteine residues
(CXC chemokines) [7
]. CTAP-III is secreted as an
inactive precursor form, which is rapidly converted by successive
partial digestions to an N-terminal-truncated active form, the
ß-thromboglobulin (ß-TG)/neutrophil-activating polypeptide-2
(NAP-2) [8
]. ß-TG/NAP-2 is a chemokine with the
characteristic functions of the CXC chemokine family, such as
stimulation of chemotactism and degranulation of neutrophils. These
effects are induced through the interaction with CXCR1 and CXCR2, two
receptors of the interleukin (IL)-8 family [7
,
9
]. Conversely, PF4 does not interact with IL-8
receptors, and its activity has not been associated with intracellular
Ca2+ mobilization, a common characteristic of
the CXC chemokines [10
]. PF4 has been extensively studied in the context of thrombosis and circulatory disorders [3 ], being described as an inhibitor of cellular functions such as hematopoiesis [11 ], angiogenesis [12 ], neoplastic cell growth [13 14 15 ], and T cell suppressor activity [16 ]. Chemotactic activity of PF4 on different cell types in vitro and in vivo during the inflammatory response has been described [17 18 19 20 21 ], and as many of these responder cells are recruited and activated from systemic circulation and from local tissues, PF4 has been proposed to participate in the complex regulation of this process.
Natural killer (NK) cells are CD3- (CD16+CD56+) cytotoxic lymphoid cells, which act as a first-line resistance against a wide range of targets. NK cells are able to secrete cytokines and play a central role in the regulation of several systems [22 , 23 ]. One such cytokine secreted by NK cells is IL-8, a member of the CXC subfamily. IL-8 was initially characterized as a neutrophil chemotactic and activating agent and has been proposed to play a major role in the inflammatory response [24 ].
In this study, we show that PF4, but not ß-TG/NAP-2, induces highly
purified, human NK cells to synthesize bioactive IL-8. This effect is
regulated at the transcriptional level, and it is not suppressed upon
PF4 binding to heparin. PF4 induces a synergistic increase in the IL-8
production in NK cells stimulated through the Fc receptor for
immunoglobulin G FcR-IIIA (1)
, but not when PF4 was
used in combination with IL-2. Pretreatment of NK cells with pertussis
toxin did not block this PF4 effect, suggesting that there is no
implication of the heterotrimeric guanine nucleotide-binding
proteins (G) Go and Gi in the
signal pathway involved in IL-8 production. Finally, we show evidence
that phosphatidylinositol-3 kinase (PI-3K) is a necessary component of
the signaling events triggered by PF4.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NK cell culture and activation
To activate NK cells, we used 50 or 200 IU/ml recombinant human
(rh)IL-2 (Boehringer Mannheim, Mannheim, Germany) or anti-CD16 mAb
(coated at 3 µg/well; Serotec, Oxford, England). Highly purified NK
cells were resuspended at 4 x 106/ml or 2 x
106/ml in Biotarget serum-free medium (Biological
Industries, Kibbutz Beth Haemek, Israel), supplemented with 2 mM
L-glutamine and antibiotics; 30 min before the stimulus, we added 10
µg/ml cycloheximide (CHX), 10 µg/ml actinomycin D, 1 µg/ml
pertussis toxin, or 50 nM wortmannin (Sigma Chemical Co., St. Louis,
MO) to the cell culture. Preliminary data had indicated cell amount and
stimulus concentration required.
Specificity of PF4 effect
Human PF4 (Diagnostica Stago, Boehringer Mannheim) used
throughout this study had a purity higher than 95% (single band on
sodium dodecyl sulfate-polyacrylamide gel electrophoresis). To
corroborate the functional specificity of PF4, each dose-response
experiment included a control condition in which the highest dose of
200 ng/ml PF4 was preincubated with 16 µg/ml neutralizing polyclonal
anti-human PF4 rabbit serum (Diagnostica Stago) for 30 min at 4°C.
The excess of Ab and PF4-Ab conjugates was removed by a 2-h incubation
with protein A-sepharose CL-4B (Pharmacia LKB Biotechnology,
Piscataway, NJ) at 4°C, thus avoiding the effect of Ig on NK cells
[25
]. After confirming the lack of any detectable PF4 by
enzyme-linked immunosorbent assay (ELISA; ELISA Asserachrom PF4,
Diagnostica Stago), supernatants were added to the cell culture. At the
indicated time, cells were harvested and tested for IL-8 production.
PF4 binding to heparin
Heparin-sepharose CL-6B (20 µL; Pharmacia LKB Biotechnology)
was prewashed in serum-free medium and incubated with 200 ng/ml or 40
ng/ml PF4. After stirring for at least 90 min at 4°C, tubes were
centrifuged, and pellets were extensively washed with culture medium
before incubating with purified NK cells at 4 x
106/ml. After 48 h incubation, cells were centrifuged,
and supernatants were tested for antigenic IL-8 content by ELISA.
Assay of IL-8 production by activated NK cells
Antigenic IL-8 levels were quantified by ELISA (Amersham,
Arlington Heights, IL) according to the instructions recommended by the
manufacturer. Supernatants of PF4-activated NK cells were serially
diluted and assayed in triplicate. Isolation of polymorphonuclear
leukocytes (PMN; responding cells) and chemotactic assay (modified
Boyden chamber chemotaxis) for functional IL-8 was performed as
described elsewhere [26
]. To block IL-8 activity, PF4-NK
supernatants were incubated with 2 µg/ml neutralizing anti-IL-8 Ab
(R&D Systems Inc., Minneapolis, MN) and were procesed as described
above for determining PF4-specific activity. Each assay was referred to
a standard curve of chemotactic activity induced by rIL-8 (from 0 to 25
ng/ml; R&D Systems Inc). The results are presented as percentages of
the maximal activity in the standard curve.
Reverse transcriptase-polymerase chain reaction (RT-PCR) of
IL-8-mRNA
Total RNA was extracted from NK cells by the
guanidinium-isothiocyanate phenol-chloroform method
[27
]. A total cDNA was obtained by incubating 0.5 µg
total RNA with 200 units RT at 37°C for 60 min. For the specific
amplification of ß-actin, we used the following primers:
5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' and
5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3' (Clontech Laboratories Inc.,
Palo Alto, CA), which yield a 838-bp product. IL-8-specific sequence
was amplified with the following primers: 5'-ATGACTTCCAAGCTGGCCGTGCT-3'
and 5'-TCTCAGCCCTCTTCAAAAACTTCTC-3' [28
], yielding a
289-bp product. We performed 25 cycles of amplification for IL-8 and 20
cycles for ß-actin under identical conditions and in separate
reaction tubes from the same transcription reaction. Each cycle
included 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C.
PCR products (20 µL) were electrophoresed on a 2% agarose gel and
were stained with ethidium bromide. Bands were quantified by
densitometry of negatives (Polaroid black/white print film type 667)
with a Bio-Profile 1D-2D image analyzer.
3H-Thymidine uptake and assay of cytotoxic activity
NK cells with or without PF4 (range from 0 to 200 ng/ml) were
cultured with 50 IU/ml rhIL-2 during 48 h in a 96-well microtiter
plate. Cell proliferation was measured by the uptake of
methyl-3H-thymidine (0.5 µCi per well; Amersham) for an
additional 18 h. For cytotoxic activity, NK cells were cultured
with 200 IU/ml rhIL-2 for 48 h. IL-2-dependent cytotoxic activity
[lymphokine-activated killer (LAK)] was measured in a 4-h standard
51Cr release assay using the Raji cell line as a target
cells [29
].
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 View larger version (14K): [in a new window] |
Figure 1. ELISA determination of the antigenic levels of IL-8 on highly purified
2 x 106 NK cells/ml. Experimental points are the
average of triplicates from a single experiment out of the four
performed. (A) Dose-response experiment of IL-8 secretion by NK cells
cultured with the indicated doses of PF4 (•) or ß-TG;3>
( ). (B) Time-course representation of IL-8 secretion by NK cells
incubated with 50 ng/ml PF4 (•) or culture medium (CM;3>; ).
Supernatants were collected and analyzed after 48 h of incubation
with cytokines. As a control, we include an experimental condition
( ) in which 200 ng/ml PF4 (the highest dose tested) was preincubated
with a neutralizing polyclonal anti-human PF4 serum, and the complex
was removed with protein A-sepharose.
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 View larger version (31K): [in a new window] |
Figure 2. Measure of the chemotactic activity on PMNs of functional IL-8 secreted
by PF4-activated NK cells. NK cells/ml (2x106) were
incubated with 50 ng/ml PF4 at different time points, cells were
collected by centrifugation, and the supernatant was split into three
aliquots. One aliquot was preincubated with an anti-IL-8 antiserum
before chemotactic testing (IL-8 depleted supernatant), another one was
preincubated with an isotypic antibody as control for the IL-8
depletion (Mock treated supernatant), and the third one was used for
the determination of the chemotactic response induced on PMN
(Supernatant). IL-8-induced chemotactic activity was measured by
counting the number of PMNs that migrated to the lower chamber in a
modified Boyden chamber assay (see Materials and Methods). Each
experimental point is the average count of five random fields. A
representative experiment of the three performed is shown.
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 View larger version (35K): [in a new window] |
Figure 3. PF4 conjugation to heparin does not block production of NK-derived
IL-8. PF4 (200 or 40 ng/ml) was incubated with 20 µL
heparin-sepharose at 4°C. After 90 min, the conjugate was
centrifuged, extensively washed, and coincubated with 2 x
106 NK cells. After 48 h incubation, cells were
centrifuged, and supernatants were tested for antigenic IL-8 content by
ELISA. Open bars show levels of IL-8 secreted by PF4-activated NK cells
(control). Striped bars show levels of IL-8 secreted by NK cells
activated with the PF4-heparin sepharose conjugate (PF4 +
HEP.SEPH). A representative experiment of the four performed is
shown.
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 View larger version (38K): [in a new window] |
Figure 4. Effect of PF4 on IL-8 synthesis by NK cells. (Upper panels) NK cells
(1.5x107) per condition were cultured with PF4, processed
for RNA extraction, and RT-PCR-analyzed as described in Material and
Methods. (Lower panels) Bands were quantified by densitometry, and the
index IL-8 mRNA/ß-actin mRNA, calculated as an estimate for specific
IL-8 mRNA-relative levels to normalize variability originated during
sample processing. To determine relative increases in IL-8 mRNA levels
produced by PF4 activation, the different experimental points were
referred to IL-8 mRNA levels measured in the control NK cultures not
activated with PF4 (point 0, arbitrarily stated as 1 relative unit of
IL-8 mRNA). Left: Effect of a 30-min preincubation with 10 µg/ml CHX
on the IL-8-mRNA production by NK cells activated with 50 ng/ml PF4. NK
cells were incubated for 6 h with 10 µg/ml CHX plus 50 ng/ml PF4
(CHX+PF4) and culture medium (CM) or 50 ng/ml PF4 (PF4) as controls.
Middle: Dose-response effect of increasing concentrations of PF4 on the
IL-8-mRNA production by NK cells cultured for 6 h with the
indicated doses of PF4. Right: Time-response effect on the IL-8-mRNA
production by PF4-activated NK cells. NK cells were cultured with 100
ng/ml PF4 and processed at indicated times. In all cases, one
representative experiment out of three is shown.
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-RIIIA ligand or IL-2 on the
PF4-induced release of IL-8-RIIIA ligand and IL-2 [30
]. When NK cells were
cultured with precoated anti-CD16 mAb (specific for Fc-RIIIA) in the
presence of PF4, the highest amount of IL-8 released was four- to
fivefold above the level induced by anti-CD16 mAb alone and 10- to
15-fold higher than the level with PF4 alone (Fig. 5 A
). Unlike the effect observed through Fc-RIIIA, no differences
in the amount of NK-derived IL-8 were detected when NK cells were
cultured with PF4 in the presence of rIL-2 (Fig. 5B)
. To determine if
PF4 induced similar effects upon other IL-2-dependent responses, NK
cells were preincubated for 30 min with PF4, and then tested for
IL-2-dependent cytotoxic activity (LAK) and 3H-thymidine
uptake. Our results show that PF4 did not modulate either activity
(Fig. 6
), therefore excluding the possibility that PF4 could block the
binding of IL-2 to its receptor, as has been described for other
cytokines [34
, 35
].
 View larger version (21K): [in a new window] |
Figure 5. Opposite actions of FcRIIIA and IL-2 on the IL-8 production by
PF4-activated NK cells. (A) PF4 and the FcRIII-A act synergistically
to overinduce IL-8 production by NK cells. Five duplicates containing
2 x 106/ml NK cells each were preincubated for 30 min
with increasing concentrations (0, 8, 16, 40, and 200 ng/ml) of PF4.
Cells from one of the duplicated series were then added to anti-CD16
mAb-precoated wells (3 µg/well) and were incubated for 48 h, and
cells in the other group were kept in culture and did not receive any
additional activation. (B) IL-2 does not overinduce IL-8 production by
PF4-activated NK cells. NK cells (2x106/ml) were
stimulated with 50 ng/ml PF4 (open bars) or 50 ng/ml PF4 plus 50 IU/ml
IL-2 (striped bars) for 48 h. Supernatants were collected, and
antigenic IL-8 was quantified by ELISA. Results shown are the average
of triplicates from a single experiment representative of the four
performed.
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 View larger version (15K): [in a new window] |
Figure 6. The chemokine PF4 does not induce any effect on proliferation or
IL-2-cytotoxic activitiy (LAK) of NK cells. (A) Cell proliferation was
measured by the uptake of 0.5 µCi per well of
methyl-3H-thymidine (3HTdR) for an additional
18 h. Results are expressed as cpm x 103. (B)
Purified NK cells from a representative donor were pretreated for
18 h with PF4 at various doses, washed, and assayed for LAK
activity (see Materials and Methods). After incubation for 4 h at
37°C, supernatants were harvested and counted on a gamma counter.
Results are expressed as lytic units (LU)/109 large
granular lymphocytes.
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 View larger version (24K): [in a new window] |
Figure 7. Effects of wortmannin and pertussis toxin on the IL-8 synthesis by NK
cells in response to PF4 activation. (A) Pertussis toxin did not block
IL-8 synthesis by NK cells in response to PF4. (B) Wortmannin inhibits
IL-8 synthesis by NK cells in response to PF4. NK cells
(2x106/ml) were preincubated with 1 µg/ml pertussis
toxin or 50 nM wortmannin for 30 min and were then incubated with 50
ng/ml PF4 for an additional 48 h. Supernatants were harvested, and
antigenic IL-8 was measured by ELISA. Results are expressed as the
percentage of IL-8 secreted, considering 100% as the amount of IL-8
released in response to PF4 alone. Shown is one experiment
representative of the five performed. PT, Cells preincubated with
pertussis toxin; Wort, Cells preincubated with wortmannin. CM, Cells
maintained in culture without additional stimulation.
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-RIIIA [38
]. To determine the relevance of PI-3K in
PF4-induced production of IL-8, NK cells were preincubated with
wortmannin (50 nM), a fungal metabolite that is a specific PI-3K
inhibitor at nM concentrations. As it is shown in Figure 7B
, wortmannin
reduced PF4-mediated IL-8 release by up to 80%. These results suggest
that activation of PI-3K is a necessary step to induce the
PF4-triggered production of IL-8 in NK cells. |
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Plasma concentration of PF4 and ß-TG/NAP-2 has been estimated to be
over 2 and 6 ng/ml respectively [3
, 40
]. In
this work, we have been unable to detect any effect on NK activity at
those concentrations. Nevertheless, when PF4 and ß-TG/NAP-2 are
released from platelet -granules after activation by tissue injury,
vessel-wall damage, or inflammation, plasmatic concentration is
increased by 1000-fold, and furthermore, released PF4 is rapidly bound
to the heparan sulfate residues of the vascular endothelium, where it
is locally concentrated [41
, 42
]. In this
form, it may exert its function on neutrophil granulocytes and
monocytes [43
44
45
].
In this work, we demonstrated that PF4, free or complexed with heparin,
at the concentration expected to be found in the place of inflammation,
may act on NK cells to induce the production of IL-8, a potent
chemotactic factor for neutrophils that has been implicated in vascular
repair by induction of endothelial cell activation [46
,
47
]. The physiological relevance of the observed PF4
activity on NK cells has been clearly demonstrated, as heparin-bound
PF4 conserved the ability of soluble PF4 to induce IL-8 production by
NK cells. Physiologically, after platelet activation, released PF4
binds with great affinity and avidity to glycosaminoglycans of the cell
surface and extracellular matrix through its C-terminal -helix
[48
]. This feature is responsible for the rapid
clearance of PF4 from circulation [40
] and suggests that
its hypothetical, physiologic role might involve crynopexy, a process
whereby soluble factors are excreted, stored, and stabilized in the
extracellular matrix [49
].
Different effects of heparin and heparan sulfates on chemokine-mediated activities have been previously reported. The associaton between PF4 and heparin prevented the effects of PF4 in angiostatic activity [12 , 50 ] and megakaryocytopoiesis [51 ]. On the contrary, binding of PF4 to heparan-sulfate receptors on different cells of the immunological system induced the effect of PF4 [44 ] and some immunoreactive chemokines [52 53 54 ].
The important synergy of PF4 in the Fc-RIIIA activation has not been
previously described in other systems and suggests a role of PF4 in
amplification of immunological response mediated by antibodies or
immunocomplexes, which could be important in inflammatory processes and
autoimmunity.
IL-2 is one of the more important activators of NK cells. The effects
of IL-2 on different parameters of NK cells, such as increasing the
cytotoxic activity as well as proliferation, have been widely reported
(reviewed in ref. [23
]). We tested whether PF4 acts in
addition or in synergy with IL-2 on NK cells, but contrarily to the
effect observed with precoated, anti-CD16 mAb (specific for
Fc-RIIIA) in the presence of PF4; there were no differences in the
activities induced by IL-2 on NK cells. Taub et al. [55
]
reported a tendency of several chemokines to induce migration by
resting but not IL-2-activated NK cells. Collectively, these findings
suggest that NK cells could up-regulate IL-8 synthesis through
different pathways but also support the idea that at least some
mechanisms for IL-8 production in NK cells are independent of
proliferative or cytotoxic responses. These results are apparently in
desagreement with previous studies [56
57
58
]. It must be
pointed out, however, that we have used primary and quiescent NK cells,
and cell lines or NK cells previously stimulated could respond
differently in each case.
Most chemokines act via seven-transmembrane domain receptors that transmit signals through heterodimeric guanosine 5'-triphosphate-binding proteins [7 ]. In this study, we reported that the PF4 effect was not carried out by means of a Gi and Go protein-coupled receptor, as pertussis toxin was not able to block the IL-8 synthesis induced by PF4. Unlike other chemokines, PF4 is known to bind the target cell through a nonseven-transmembrane domain receptor. In fact, Petersen et al. [44 ] demonstrated that the PF4 effect on human neutrophils occurred through a receptor with characteristics of a chondroitin sulfate proteoglycan. Further studies are required to demonstrate whether the effect of PF4 in NK is mediated by the interaction with one of such receptors. There is strong evidence for a role of PI-3K in chemokine-mediated signal transduction [7 , 36 ]. In accordance, we present results suggesting that PI-3K is a necessary step to induce PF4-triggered IL-8 production by NK cells.
As to the physiological relevance of our results, one must ask when and
under what conditions NK cells may interact with PF4. Platelet
activation and release of granule content occur at the inflammatory
focus in the initial steps of the process. There is evidence of the
important role of the platelet-released chemokines PF4 and ß-TG/NAP-2
at the early time points of the inflammatory process
[40
]. Different cell subsets migrate from the vascular
compartment to the site of inflammation in a sequential order. This
process starts by the recognition of a foreign agent by phagocytic
leukocytes, such as granulocytes (PMN) [1
,
6
]. NAP-2 could induce the chemotaxis of neutrophil to
the site of inflammation through the interaction with IL-8 receptors
CXCR1 and CXCR2, and PF4 will interact through a specific condroitin
sulfate receptor [44
] favoring the firm attachment of
neutrophils to endothelial cells and subsequently, inducing their
granule exocytosis [46
]. In light of our results, we
suggest that NK cells directly interact with PF4. Different chemokines
play a critical role in the polarization and recruitment of NK cells
into inflammatory sites [59
, 60
], where the
concentration of PF4 can increase to a range of 12–500 ng/ml. At this
condition, NK cells release IL-8, which has been recognized as a strong
T cell chemotactic factor [61
]. Therefore, our results
support the hypothesis that PF4 (alone or in synergy with
Fc-RIIIA-ligand) might amplify the inflammatory response of NK cells
through the regulation of IL-8 production. On the other hand, activated
platelets can also directly interact with leukocytes simultaneous to
the release of their granule content, and the NK cell was found to be
the main lymphocyte subset to which the activated platelet binds
[62
]. Thus, we describe a new capacity for PF4 and
provide further evidence for the role of NK cells in the regulation of
the inflammatory response.
Received September 7, 2000; revised June 12, 2001; accepted May 5, 2002.
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