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(Journal of Leukocyte Biology. 2002;72:590-597.)
© 2002 by Society for Leukocyte Biology

Platelet factor 4 induces human natural killer cells to synthesize and release interleukin-8

Francesc Martí, Esther Bertran, Montserrat Llucià, Esther Villén, Matilde Peiró, Joan Garcia and Fèlix Rueda

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We provide evidence that platelet factor 4 (PF4), but not the related chemokine neutrophil-activating polypeptide-2, induced highly purified human natural killer (NK) cells to produce interleukin (IL)-8 in a time- and dosage-dependent manner. This ability was retained even while PF4 was bound to heparin. PF4 increased the steady state level of IL-8 mRNA, likely implying a transcriptional effect of PF4. Stimulation of NK cells through the Fc receptor for immunoglobulin G-IIIA was found to synergistically increase the effect of PF4 on IL-8 production but did not affect IL-2-related activities such as cytotoxic activity and proliferation. Pertussis toxin did not block the PF4-derived IL-8 production in NK cells, but this response was sensitive to wortmannin, implicating a role of phosphatidylinositol 3-kinase in the intracellular signaling pathway triggered by PF4. Our results characterize a new capacity for PF4 and provide further evidence for the pivotal role of NK cells in the environment of inflammation.

Key Words: inflammation • chemokines • Fc{gamma}RIIIA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to the well-characterized function in the hemostatic process, platelets play an important role in the regulation of the inflammatory process and of tissue repair [1 , 2 ]. {alpha}-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 {alpha}-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 Fc{gamma}R-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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
NK cell purification
Peripheral blood mononuclear cells were separated on a Ficoll-Metriozate gradient of diluted buffy coats obtained from healthy donors. NK cells were obtained by negative selection. Briefly, nonadherent, nylon wool-passed cells were incubated with a pool of the following monoclonal antibodies (mAb): anti-CD5, anti-CD3, anti-CD14 (Reactivos Cromatests, Laboratorios Knickerbocker, Barcelona, Spain), and anti-CD20 (kindly provided by Dr. P. Martin, Fred Hutchinson Cancer Research Center, Seattle, WA) for 30 min at 4°C. After two Hanks’ balanced saline solution washes, cells were bound to prewashed sheep, anti-mouse, Ig-coupled magnetic beads (Dynal, Oslo, Norway) for 30 min at 4°C. Cells collected with a magnet were discarded. After extensive washing, cells in suspension resulted in a highly enriched population of CD3-/CD56+ and/or CD16+ NK cells (>95%) as determined by three-color flow cytometry analysis (Epics XL-MCL cytometer, Coulter, Miami, FL).

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 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PF4 induced NK cells to release IL-8
To determine whether PF4 had any effect on the NK cell production of IL-8, highly purified, human NK cells were incubated with increasing doses of PF4 (from 0 to 200 ng/ml). Supernatants were harvested at 48 h and then tested for antigenic IL-8 production. Our results (Fig. 1 A ) show that PF4 induced NK cells to release IL-8 in a dose-dependent manner without requiring any additional stimulation. Detectable response was found at doses as low as 10 ng/ml PF4, and the maximum response was in the range of 100–200 ng/ml. Specificity of PF4 effect was demonstrated, as NK-derived IL-8 production was abrogated when PF4 was removed from test samples by incubation with neutralizing Ab. Moreover, parallel experiments carried out with ß-TG/NAP-2 instead of PF4 did not result in any increase on IL-8 production.



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  		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> ({blacksquare}). (B) Time-course representation of IL-8 secretion by NK cells incubated with 50 ng/ml PF4 (•) or culture medium (CM;3>; {blacksquare}). Supernatants were collected and analyzed after 48 h of incubation with cytokines. As a control, we include an experimental condition ({blacktriangleup}) 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.

 
Time-course of NK-derived IL-8 in response to PF4
IL-8 released by NK cells in response to 50 ng/ml PF4 was detectable after 10 h of culture. IL-8 spontaneous release in the absence of any stimulus was uniformly detected in all experiments but never exceeded 10% of the quantities released by NK cells in response to the optimal dose of PF4. PF4-induced IL-8 secretion peaked at 96 h with 50 ng/ml per 2 x 105 cells in six different experiments. Incubation of NK cells with PF4 in the presence of 10 µg/ml cycloheximide completely abrogated the production of antigenic IL-8 (see Fig. 1B ). This long-lasting secretion has previously been unreported in any leukocyte subset [30 31 32 33 ]. To determine whether NK cell-derived IL-8 was functional, the same supernatants from time-course assays were tested for their ability to induce a chemotactic response on PMN. Our results showed that supernatants from NK cells stimulated with PF4 possessed PMN-chemotactic activity in a way that correlated with antigenic IL-8 production (Fig. 2 ). We attributed the chemotactic activity to IL-8, as it was almost eliminated when supernatants were incubated with neutralizing anti-IL-8 Ab. However, despite this, partial activity (20–30%) remained at later time points, suggesting the existence of other PMN chemotactic agents induced by PF4 in NK cells. No chemotactic effect on PMN was directly promoted by PF4 at the dose used in these experiments.



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  		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.

 
Heparin-immobilized PF4 retained the capacity to induce IL-8 synthesis
Our results demonstrated that soluble PF4 was sufficient to induce NK cells to release bioactive IL-8, but physiologically, PF4 binds with great affinity and avidity to glycosaminoglycans of the cell surface and extracellular matrix, a feature responsible for the rapid clearance of PF4 from circulation. To test whether heparine-bound PF4 induced a different response than soluble PF4, we next incubated different doses of PF4 with a heparin-sepharose matrix and then tested the induction of NK-derived IL-8 release in parallel with identical doses of soluble PF4. Heparin-bound PF4 was at least as efficient as soluble PF4 with respect to induction of IL-8 production by NK cells (Fig. 3 ). Heparin-sepharose without PF4 did not induce any effect (data not shown).



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  		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.

 
Transcriptional effect of PF4
Incubation of NK cells with PF4 in the presence of 10 µg/ml cycloheximide completely abrogated the production of antigenic IL-8, but also resulted in a steady-state increase of IL-8 mRNA when compared with the level of IL-8 mRNA induced by PF4 alone (Fig. 4 , left). These results suggest that PF4 increases the IL-8 production through the a regulation of the mRNA synthesis and/or stabilization, rather than through the synthesis of any other peptide. To further elucidate the effect of PF4 on IL-8 production by NK cells, we next analyzed IL-8-mRNA steady-state levels in NK cells in response to PF4 at adose- and time-dependent manner. In accordance with previous reports [30 ], a small but significant level of specific IL-8 mRNA was consistently detected in each sample of untreated NK cells. The level of IL-8-mRNA transcription in response to PF4 peaked at 100 ng/ml (Fig. 4 , middle), in correspondence with the peak of antigenic IL-8 released (Fig. 1) . As shown in Figure 4 , Right, specific IL-8-mRNA expression in 100 ng/ml PF4-stimulated cells resulted in a bell-shaped, time-course curve. Densitometric measures showed a detectable increase of IL-8 mRNA after 1 h stimulation, peaking at 6 h with an average four- to fivefold increase above untreated NK cells.



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  		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.

 
Costimulatory effect of Fc{gamma}-RIIIA ligand or IL-2 on the PF4-induced release of IL-8
It has been described previously that synthesis of IL-8 is induced on NK cells upon activation through different stimuli, two of which are Fc{gamma}-RIIIA ligand and IL-2 [30 ]. When NK cells were cultured with precoated anti-CD16 mAb (specific for Fc{gamma}-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{gamma}-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 ].



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  		Figure 5. Opposite actions of Fc{gamma}RIIIA and IL-2 on the IL-8 production by PF4-activated NK cells. (A) PF4 and the Fc{gamma}RIII-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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  		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.

 
Pertussis toxin does not block the PF4 activity observed in NK cells
Several studies (reviewed in ref [7 ]) showed that chemokine receptors exhibit a seven-membrane spanning region, a characteristic of receptors linked to G proteins. To evaluate if PF4 triggers cellular events through one of such receptors, we studied the effect of the bacterial pertussis toxin (1 µg/ml) in our system. As it is shown in Figure 7 A , pertussis toxin was not able to block the effect of PF4 on NK cells, which demonstrates that response to PF4 is not coupled to a pertussis toxin-sensitive Gi, Go class of G proteins.



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  		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.

 
Evidence for the role of the PI-3K pathway on the release of NK-derived IL-8 in response to PF4
PI-3K and its metabolic products have been described to play a key role in the signaling transduction of different chemokines [36 , 37 ]. Moreover, the same pathway has been implicated in the release of NK granules upon activation through Fc{gamma}-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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report that PF4 induces NK cells to produce IL-8 at the mRNA and the protein level. As NK cell preparations that yielded more than 0.5% of contaminating monocytes or 1% of PMN granulocytes were discarded, the possibility that monocytes or PMNs, the major producers of IL-8 [30 31 32 33 , 39 ], were responsible for any effect observed in this study is unlikely. Functional specificity was established by the abrogation of the PF4 effect in controls, where the highest dose of PF4 (200 ng/ml) was preincubated with neutralizing anti-human PF4 polyclonal antibodies. PF4 induces a sharp increase in IL-8 synthesis in the first 24 h, followed by a continuous increase up to 96 h. Kinetics of IL-8 transcription preceded that observed for protein synthesis, peaking at 6 h after PF4 stimulus. At 12 h, steady-state levels of IL-8 mRNA still remained threefold higher than at time zero.

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 {alpha}-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 {alpha}-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{gamma}-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{gamma}-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{gamma}-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.


   ACKNOWLEDGEMENTS
 
F. M. and E. B. contributed equally to this study and were supported by grants from the Cancer Research Institute (IRO). The authors thank Dr. P. Martin (Fred Hutchinson Cancer Research Center, Seattle, WA) for providing the anti-CD20 mAb (clone IF-5), the Blood Bank (Ciutat Sanitària i Universitària de Bellvitge) for providing buffy coats, Rafel Cardoner for his assistance in the RNA scanning, and Stanislao Navarro for assistance in review of the manuscript.

Received September 7, 2000; revised June 12, 2001; accepted May 5, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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