Effect of CXC chemokine platelet factor 4 on differentiation and function of monocyte-derived dendritic cells

Chang-Qing Xia1 and Kuo-Jang Kao1

1 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA

Correspondence to: K. J. Kao, Departments of Pathology and Research, Koo Foundation SYS Cancer Center, 125 Lih-Der Road, Pei-Tou District, Taipei, Taiwan. E-mail: kjkao{at}synpacnc.com
Transmitter editor: S. Koyasu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Platelet factor 4 (PF4) is a CXC chemokine secreted by activated platelets. PF4 has been shown to promote monocyte survival and induce the differentiation of monocytes into macrophages. However, the effect of PF4 on differentiation of monocytes into dendritic cells (DC) has yet to be determined. As reported previously, monocytes cultured in RPMI medium containing FCS, granulocyte macrophage colony stimulating factor and IL-4 differentiated into CD1a+ DC. When PF4 was added, the expression of CD1a on DC was inhibited. This inhibitory effect was not observed with the other platelet-derived CXC chemokine, ß-thromboglobulin. The relative number of CD1a DC increased from17 to 92% when the PF4 concentration was increased from 0 to 10 µg/ml. The inhibitory effect of PF4 on CD1a expression was reversed by 50 U/ml heparin. DC developed in the PF4-containing media appeared more adhesive to plastic culture wells and had higher light side scatter by flow cytometry. Immunophenotypically, monocyte-derived DC in the presence of increasing concentrations of PF4 proportionally expressed higher CD86 and lower HLA-DR. The levels of CD11c, CD40 and CD80 remained unchanged with or without PF4. Both CD1a+ DC and CD1a DC were negative for CD14, CD68 and CD83. Functionally, DC developed in the presence of PF4 had their secretion of tumor necrosis factor-{alpha} and IL-12 reduced by 75 ± 10 and 79 ± 13% respectively when they were stimulated by 100 ng/ml lipopolysaccharide and 50 ng/ml IFN-{gamma}. CD1a DC developed in the presence of PF4 were not as active as the control CD1a+ DC in stimulating allogeneic T cells to proliferate. In addition, CD1a DC were less potent in priming naive CD4+ T cells to secrete both type 1 and 2 cytokines. These results indicate that PF4 can influence differentiation and function of monocyte-derived DC.

Keywords: CD1a, dendritic cell, monocyte, platelet factor 4


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Platelet factor 4 (PF4) is a major secretory protein of activated platelets (1). As a member of the CXC chemokines, PF4 participates in inflammatory responses by attracting monocytes and neutrophils (2,3). PF4 inhibits angiogenesis and suppresses tumor growth (46). In addition, it has been shown that PF4 plays an important role in the regulation of hematopoiesis (7) and reduces the chemosensitivity of hematopoietic cells to cytotoxic agents (8). PF4 promotes monocyte survival and induces the differentiation of monocytes into macrophages (9). PF4 has also been demonstrated to modulate B cell (10) and T cell (11) function. However, it is not known whether PF4 can affect the differentiation of monocytes into dendritic cells (DC).

Peripheral blood monocytes can differentiate into DC ex vivo in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 (12). Nevertheless, differentiation of human monocytes into DC in vivo remains uncertain. It has been shown that Langerhans cells can be differentiated from tissue CD14+ cells in the presence of transforming growth factor-ß1 (13). A study in mice also demonstrated that monocytes could differentiate into DC in vivo (14). DC are professional antigen-presenting cells, and play important roles in the initiation and the regulation of immune responses (15). In addition to priming of CD4+ T cells (16), certain subsets of DC can down-regulate T cell activation and may induce immunological tolerance (17).

When human monocytes are cultured in medium containing FCS in the presence of GM-CSF and IL-4, monocytes always differentiate into CD1a+ DC (1820). Under serum-free conditions or in the presence of autologous serum without FCS, monocytes always differentiate into CD1a DC (2122). Functional studies have demonstrated that CD1a+ DC and CD1a DC have different cytokine production profiles and stimulatory activities in mixed leukocyte cultures (MLC) (2123). Recently, we found that monocytes could differentiate into CD1a+ DC in the presence of autologous serum if heparin was added into the culture media (21). This effect of heparin could be neutralized by PF4. Nevertheless, an excess of PF4 further suppressed the CD1a expression to a level slightly lower than the control (unpublished observation). This finding suggests that PF4 may have a direct effect on differentiation of monocytes into DC. Therefore, it is of interest to determine how PF4 influences the differentiation and function of monocyte-derived DC. The results of our study are reported herein.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Media and reagents
RPMI media were purchased from Life Technologies (Grand Island, NY). PF4 and ß-thromboglobulin (ß-TG) were purchased from Hematologic Technologies (Essex Junction, VT) with purity >95% judged by SDS–PAGE. Both products were prepared using heparin–agarose affinity chromatography. Porcine heparin was from Elkins Sinn (Cherry Hill, NJ). Recombinant human GM-CSF was obtained from Immunex (Seattle, WA). Recombinant human IL-4 and IFN-{gamma} were purchased from PeproTech (Rochy Hill, NJ). Fluorescent-conjugated antibodies, matched isotype antibodies and the CBA kit for assays of cytokines were obtained from BD PharMingen (San Diego, CA). Lipopolysaccharide (LPS), phorbol myristate acetate, ionomycin, heparinase I and FITC–dextran were from Sigma (St Louis, MO). CD4+ T cell isolation cocktail was purchased from Stem Cell Technologies (Vancouver, Canada). Anti-CD14 microbeads and anti-CD45RA microbeads were purchased from Miltenyi Biotech (Auburn, CA). Paired antibodies and cytokine standards for ELISA for IL-2, IL-10, IL-12 (p70), IFN-{gamma} and tumor necrosis factor (TNF)-{alpha} were obtained from Pierce-Endogen (Woburn, MA). The heparin assay kit was from DiaPharma Group (West Chester, OH).

Isolation of monocytes from human peripheral blood
Monocytes were isolated by positive selection as previously reported (21). In brief, peripheral blood mononuclear cells (PBMC) were incubated with 20 µl of anti-CD14 microbeads in 80 µl PBS/1% FCS at 4°C for 15 min. Thereafter, the cells were washed once with PBS and resuspended in 1 ml PBS/1% FCS. The cells were loaded on the magnetic column and CD14+ monocytes were isolated from the column after washing. The purity of the CD14+ cells was always in the range of 90–94%. The isolated monocytes were used freshly or frozen at –130°C for use on later dates.

Isolation of CD4+ T cells and CD45RA+CD4+ T cells from human peripheral blood
CD4+ T cells were prepared from PBMC by negative selection using CD4+ T cell isolation cocktail (Stem Cell Technologies) according to the manufacturer’s instructions. Briefly, 5 x 107 PBMC in 1 ml PBS/1% FCS were incubated with 100 µl CD4+ T cell-enrichment cocktail in the presence of 1% rat serum at 4°C for 15 min. Thereafter, 60 µl magnetic colloid was added to each 1 ml PBMC and incubated for another 15 min. The cells were loaded on to a Stem-Sep column placed in a magnet. CD4+ T cells were eluted by washing the column with 15 ml PBS/1% FCS. The purity of CD4+ T cells was consistently ~95%. CD45RA+CD4+ T cells were purified from the isolated CD4+ T cells using the method described previously (21). The purity of isolated CD45RA+ CD4+ cells was always between 90 and 95%.

Preparation of monocyte-derived DC
Monocytes (5 x 106) were cultured in a 12-well plate in 1.5 ml RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF and 20 ng/ml IL-4 in the absence or presence of different concentrations of PF4 for 6 days. On day 3, 1.5 ml medium containing indicated cytokines and PF4 was added to the culture. For neutralization of PF4, different concentrations of heparin were added simultaneously with PF4. In some experiments, 5 µg/ml ß-TG was used in the culture to study its effect on monocyte differentiation into DC.

To determine whether FCS contains heparin-like activity that promotes the development of CD1a+ DC from monocytes (21), FCS was pretreated with 25 U/ml of heparinase I at room temperature for 1 h, assayed for heparin anti-factor Xa activity and used for the study as described above.

Cytokine production by monocyte-derived DC
To study cytokine secretion by DC, 2.5 x 104 DC were cultured in a well of a 96-well U-bottom plate in 200 µl RPMI 1640 containing 5% FCS in the presence of 100 ng/ml LPS plus 50 ng/ml IFN-{gamma} for 24 h. The supernatants were harvested and stored at –80°C until assayed for cytokines.

To study the production of type 1 and 2 cytokines by naive CD4+ T cells, 1 x 105 CD45RA+CD4+ T cells were primed with 5 x 103 allogeneic DC generated in the presence or absence of 10 µg/ml PF4 for 3 days in 200 µl RPMI 1640 containing 5% FCS. The T cells were harvested, washed, and re-stimulated with 25 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin for 48 h. The supernatants were harvested and assayed for cytokines.

Flow cytometry
For immunophenotypic characterization of DC, washed DC were incubated with antibodies labeled with fluorescent dyes, and diluted in PBS containing 1% BSA and 0.02% NaN3 at 4°C for 25 min. Isotype-matched antibodies were used as negative controls. The cells were analyzed by a FACScan (Becton Dickson) and the data were analyzed by FCS Express software (De Novo Software, Ontario, Canada).

MLC
For the primary allogeneic MLC assay, a specified number of DC irradiated with 30 Gy {gamma}-rays were cultured with 1 x 105 allogeneic CD4+ T cells in each well of a U-bottom 96-well plate in 200 µl RPMI 1640 media containing 5% FCS serum for 6 days. [3H]Thymidine (1 µCi/well) was added into each well and incubated for the last 16 h. The cells were harvested using a PHD cell harvester. The incorporation of [3H]thymidine was determined by scintillation counting.

Assays for cytokines
ELISAs were used to measure concentrations of human IL-10, IL-12 (p70), IFN-{gamma} and TNF-{alpha} produced by DC in the harvested media as previously described (21). Paired antibodies and recombinant human IL-10, IL-12 (p70), IFN-{gamma} and TNF-{alpha} standards were obtained from Endogen (Woburn, MA). The sensitivities of IL-10, IL-12 (p70), IFN-{gamma} and TNF-{alpha} assays were 2.5 pg/ml, 5 pg/ml, 50 ng/ml and 5 pg/ml respectively. IL-4 and IL-5 were measured by CBA beads as previously described (21).

Phagocytosis of FITC–dextran by DC
To study the phagocytic activity of DC, a previously reported method (21) was used with slight modifications. DC (5 x 104) were resuspended in 100 µl PBS/1% FCS, and incubated with FITC–dextran (0.1 mg/ml) at 37 and 0°C for 30 min. The incubations were stopped by adding 2 ml cold PBS containing 1% human serum and 0.02% sodium azide. The cells were washed 3 times with cold PBS/azide and analyzed on a FACScan (Becton Dickson).

Heparin assay
To determine the presence of any heparin-like activities in FCS, FCS used in our experiment was assayed for heparin anti-factor Xa activity using a commercially available chromogenic assay (COATEST LMW Heparin/Heparin) from DiaPharma according to the manufacturer’s instructions.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of PF4 on differentiation of monocytes into DC
First, we studied the effect of different concentrations of PF4 on the differentiation of monocytes into DC in the presence of FCS, GM-CSF and IL-4. After 6 days culture, DC were characterized by flow cytometry for the expression of CD1a and other surface markers. We found that the majority of the monocyte-derived DC expressed a significant level of CD1a in the absence of PF4 (Fig. 1A). In the presence of increasing concentrations of PF4, CD1a expression on DC was significantly suppressed to a background level (Fig. 1E). The degree of suppression was PF4 concentration dependent (Fig. 1). As reported previously by us (21) heparin can induce differentiation of monocytes to CD1a+ DC without FCS. In order to exclude the possibility that the effect of PF4 is mediated through neutralization of any heparin-like factor(s) in FCS, we investigated the possible presence of heparin-like factor(s) in FCS. The effect of pretreatment of FCS with heparinase I on the differentiation of CD1a+ DC from monocytes was studied. We found that there was no detectable heparin-like activity in FCS and that the pretreatment of FCS with 25 U/ml heparinase I that was sufficient to abolish the effect of 50 U/ml heparin (Fig. 2C and D) could not change the effect of FCS on the promotion of development of CD1a+ DC from monocytes (Table 1, and Fig. 2A and B). To determine the specificity of the inhibitory effect of PF4, we compared the effect of PF4 with that of the other platelet CXC chemokine, ß-TG. We found that ß-TG (5 µg/ml), unlike PF4 (5 µg/ml), did not suppress CD1a expression on monocyte-derived DC (Fig. 3).



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Fig. 1. Effect of different concentrations of PF4 on the differentiation of CD1a+ DC from monocytes. DC were generated by cultures of monocytes in RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF and 20 ng/ml IL-4 for 6 days with different concentrations of PF4: (A) 0, (B) 1, (C) 2.5, (D) 5 and (E) 10 µg/ml. The results are summarized in (F). CD1a+ DC were measured by flow cytometry. Similar results were obtained in two additional experiments.

 


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Fig. 2. Effect of heparinase-treated FCS on the development of CD1a+ DC. FCS was treated with or without heparinase I at a final concentration of 25 U/ml at room temperature for 1 h. Monocytes were cultured in RPMI 1640 medium containing 1000 U/ml GM-CSF, 20 ng/ml IL-4 and 5% FCS (A) or 5% FCS pre-treated with 25 U/ml heparinase I (B) for 6 days. Additional controls were the cultures containing 5% autologous human serum plus 50 U/ml heparin (C), and 5% autologous human serum plus 50 U/ml heparin and 25 U/ml heparinase I (D). The results of (C) and (D) showed that 25 U/ml heparinase I was sufficient to destroy 50 U/ml heparin activity.

 

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Table 1. Effect of heparinase I treatment on heparin activities in FCS
 


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Fig. 3. Specificity of the effect of PF4 on the development of monocyte-derived CD1a DC. Monocytes were cultured for 6 days in RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF and 20 ng/ml IL-4 without PF4 and ß-TG (A), with 5 µg/ml PF4 (B) or with 5 µg/ml ß-TG (C). CD1a expression was examined by immunofluorescent flow cytometry.

 
Morphology and immunophenotypes of DC developed in the absence or presence of PF4
To study the morphology and the immunophenotypic characteristics of DC developed in the presence or absence of PF4, monocytes were cultured for 6 days in medium containing FCS, GM-CSF and IL-4 in the presence or absence of 10 µg/ml PF4. The morphology of DC was evaluated under an inverted microscope. We found that DC that developed in the presence of PF4 were more adhesive to the plastic bottom of the culture plate. The cells were macrophage-like, morphologically (Fig. 4A). In contrast, DC developed in the absence of PF4 were loosely attached to the culture plate. Many cells were growing in aggregates (Fig. 4B). When immunophenotypes of DC were examined by flow cytometry, we found that DC developed in the presence of PF4 showed a higher light side scatter (data not shown), and expressed higher levels of CD86 and lower levels of HLA-DR (Fig. 5). Up-regulation of CD86 and down-regulation of HLA-DR by PF4 was a dose-dependent effect (Fig. 6). Both types of DC expressed similar levels of CD11c, CD40, CD80 and CD83, and did not express any monocyte and macrophage markers, such as CD14 and CD68 (24,25) (Fig. 5). Therefore, the immunophenotypes of the cells developed with and without PF4 were consistent with CD1a+ and CD1a DC respectively.



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Fig. 4. Morphology of DC developed in the presence or absence of PF4. Monocytes were cultured for 6 days in RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF and 20 ng/ml IL-4 in the presence or absence of 10 µg/ml PF4. (A) Morphology of DC developed in the presence of PF4 (x100). (B) DC cultured in the absence of PF4 (x100). The pictures shown in the insets were magnified x320.

 


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Fig. 5. Immunophenotypes of DC developed in the absence or presence of PF4. Monocytes were cultured for 6 days in RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF and 20 ng/ml IL-4 in the absence or presence of 10 µg/ml PF4. Immunophenotypes were characterized by fluorescent-labeled antibodies and flow cytometry. Similar results were obtained from four separate experiments. MFI: mean fluorescent intensity.

 


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Fig. 6. The effect of different concentrations of PF4 on the expression of HLA-DR and CD86 on DC. Monocytes were cultured for 6 days in RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF and 20 ng/ml IL-4 in the presence of different concentrations of PF4 (0–10 µg/ml). Mean fluorescent intensities (MFI) of HLA-DR and CD86 on DC were measured by immunofluorescent flow cytometry and plotted as a function of PF4 concentration.

 
Effect of neutralization of PF4 by heparin on CD1a expression
PF4 is a heparin-binding protein. The biological effect of PF4 can be neutralized by heparin. To further characterize the inhibitory effect of PF4 on the development of CD1a+ DC, we studied the effect of heparin (0–50 U/ml) on the induction of development of CD1a DC by 5 µg/ml PF4. Our results showed that heparin was able to neutralize the inhibitory effect of PF4 on the expression of CD1a by DC (Fig. 7). The effect of PF4 was completely neutralized by 50 U/ml of heparin (Fig. 7E). The morphologic characteristics of DC reverted to those that developed without PF4 (data not shown).



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Fig. 7. Neutralization of the effect of PF4 on generation of CD1a+ DC by heparin. In this experiment, monocytes were cultured in RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF, 20 ng/ml IL-4 and 5 µg/ml PF4 for 6 days with different concentrations of heparin: (A) 0, (B) 0.5, (C) 2, (D) 10 and (E) 50 U/ml. CD1a expression was progressively suppressed by increasing concentrations of heparin. Similar results were obtained when the experiment was repeated.

 
Phagocytosis of FITC–dextran by DC prepared in the absence or presence of PF4
To determine whether DC developed in the absence or presence of PF4 have the same antigen-capturing capability or not, we compared the phagocytic activities between two types of DC. The results showed that there was no significant difference between both types of DC in the phagocytosis of FITC–dextran (Fig. 8).



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Fig. 8. Phagocytosis of FITC–dextran by DC developed in the absence or presence of PF4. Monocytes were cultured for 6 days in RPMI 1640 medium containing 5% FCS, 1000 U/ml GM-CSF and 20 ng/ml IL-4 in the absence (A) or presence (B) of 10 µg/ml PF4. The dotted lines and the solid lines represent the uptake of FITC–dextran at 0 and 37°C respectively. Similar results were obtained when the experiment was repeated.

 
Production of IL-10, IL-12 (p70) and TNF-{alpha} by DC prepared in the absence or presence of PF4
To investigate whether DC prepared in the absence or the presence of PF4 have the same cytokine production activities, we measured the production of IL-10, IL-12 (p70) and TNF-{alpha} by DC stimulated with LPS and IFN-{gamma}. As shown in Fig. 8, CD1a DC developed in the presence of PF4 had their production of IL-12 and TNF-{alpha} reduced by 79 ± 13% (mean ± SD, n = 3) and 75 ± 10% (mean ± SD, n = 3) respectively, compared to CD1a+ DC prepared without PF4. Both types of DC had similar activity to secrete IL-10.

Stimulatory activity of DC in MLC
When CD1a and CD1a+ DC prepared with and without PF4 were studied for their activity to stimulate proliferation of allogeneic CD4+ T cells, we found that PF4-induced CD1a DC had a lower stimulatory activity than the control CD1a+ DC (Fig. 10).



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Fig. 10. Stimulator activity of DC developed in the absence or presence of PF4 in allogeneic MLC. Different doses of DC generated with or without PF4 (10 µg/ml) were irradiated with 30 Gy {gamma}-rays and cultured with allogeneic CD4+ T cells (1 x 105/well) for 5 days. Each incubation was then treated with 1 µCi [3H]thymidine for 16 h. Cells were harvested and [3H]thymidine incorporation was measured by scintillation counting. Each value is the mean ± SD of triplicate incubations. Similar results were obtained when the experiment was repeated two additional times.

 
Cytokine production profiles of CD45RA+CD4+ T cells primed with DC developed with or without PF4
To determine whether DC developed with or without PF4 differed in their abilities to prime naive CD4+ T cells, allogeneic CD45RA+CD4+ T cells (1 x 105cells) were cultured in the presence of {gamma}-irradiated DC (1 x 103 cells) for 3 days. The primed T cells were then stimulated with phorbol myristate acetate (25 ng/ml) and ionomycin (1 µg/ml) for 2 days. The media from the cultures of secondary stimulation were harvested and assayed for T cell cytokines. The results summarized in Table 2 indicated that CD1a DC developed in the presence of PF4 were less potent than control CD1a+ DC in priming T cells to produce both type 1 and 2 cytokines. This finding is in parallel with the lower activity of CD1a DC to stimulate proliferation of CD4+ T cells (Fig. 10). Furthermore, PF4-treated CD1a DC appeared to prime CD4+ T cells for relatively higher Th2 response. Thus, PF4 could potentially indirectly affect the T cell immune response (Table 2).


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Table 2. Cytokine production by CD45RA+CD4+ T cells primed with PF4-treated CD1a DC and control CD1a+ DC
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
PF4 is a major chemokine secreted by activated platelets (13). Although the monocyte chemotactic property of PF4 has been reported (3) and PF4 can promote differentiation of monocytes into macrophages (9), the effect of PF4 on differentiation of monocytes into DC has not been studied. Because DC can be derived from monocytes and play critical roles in immune responses, it is of interest to understand how PF4 affects the differentiation of monocytes into DC. The results of our study demonstrate that PF4 can inhibit the expression of CD1a on monocyte-derived DC. In the culture containing 10 µg/ml PF4, the majority of monocytes differentiated into CD1a DC (Fig. 1) and became more adherent to the plastic plate. Morphologically, CD1a DC appeared polygonal and macrophage-like (Fig. 4A). In contrast, monocytes in the absence of PF4 predominantly differentiated into CD1a+ DC which were less adherent and grew in aggregates (Fig. 4B). An earlier study showed that PF4 could induce monocytes to differentiate into macrophages (9).

To confirm that CD1a cells derived from monocytes in the presence of GM-CSF, IL-4 and PF4 were DC, we characterized the developed cells for the expression of CD14 and CD68 (Fig. 5), and found that the cells were negative for both monocyte/macrophage markers (24,25). Moreover, CD1a cells were positive for immunophenotypic markers of DC, such as HLA-DR, CD86 and CD11c (Fig. 5). The cells were also capable of phagocytosis (Fig. 8) and were potent antigen-presenting cells (Fig. 10). These findings supported the observation that the CD1a cells developed in the presence of GM-CSF, IL-4 and PF4 were DC.

PF4 is a heparin-binding protein. It is well known that most of the biological activities of PF4 can be neutralized by heparin (79,21). We therefore studied the effect of heparin on PF4-induced differentiation of monocytes into DC. As expected, the effect of PF4 was abolished by heparin (Fig. 7). CD1a DC can also be derived from human monocytes when monocytes are cultured in the presence of GM-CSF, IL-4 and autologous serum without the use of FCS (21,26,27). Our recent study further demonstrated that the addition of 50 U/ml heparin induced the differentiation of monocytes into CD1a+ DC in the presence of autologous serum without FCS (21). These findings suggest the possible presence of heparin-like factor(s) in FCS. Thus, neutralization of the heparin-like factor(s) by PF4 might have contributed to the development of CD1a DC from monocytes.

To investigate this possibility, we studied the effect of PF4 on the development of CD1a+ DC from monocytes in the presence of 5% FCS that had been pretreated with heparinase I. We measured the heparin anti-factor Xa activity in FCS and did not detect any (Table 1). We then found that 25 U/ml heparinase I was able to nullify up to 50 U/ml of heparin (Table 1). We then cultured monocytes in 5% FCS that had been pretreated with heparinase I. Our results showed that the treatment of FCS with 25 U/ml heparinase I was unable to prevent differentiation of monocytes into CD1a+ DC in the presence of GM-CSF and IL-4 (Fig. 2). These results indicate that the factor(s) responsible for differentiation of monocytes to CD1a+ DC cannot be destroyed by heparinase I. It is likely that the factor(s) in FCS is not heparin-like substances.

Thus, the effect of PF4 to induce differentiation of monocytes into CD1a DC in the presence of FCS was not mediated by neutralization of heparin-like factor(s) in FCS. It has been reported that monocytes possess chondroitin sulfate proteoglycan on the cell surface (28,29) and those chondroitin sulfate proteoglycans may serve as receptors for tetameric PF4 (30). Recently, the presence of PF4 receptors on endothelial cells has been suggested (31). Sulpice et al. (31) reported the direct effect of PF4 on signal transduction in endothelial cells. For these reasons, PF4 likely exerts its effect directly on monocytes through binding to its receptors. Nevertheless, the exact biochemical entity of PF4 receptors on monocytes is not known and remains to be further characterized.

According to CD1a and CD11c surface markers, three different subsets of DC (CD1a+CD11c+, CD1aCD11c+ and CD1aCD11c) in peripheral blood have been identified (32). It has also been reported that CD34+ hematopoietic stem cells or monocytes could differentiate into CD1a+ and CD1a DC (2123,26,27,33). Monocytes differentiate into CD1a+ or CD1a DC depending on the culture condition (21,22,26,27). Among CD11c+ myeloid DC in blood, the majority of them are positive for CD1a (32). It has been shown that CD1a+ DC could be precursors of the Langerhans cells in skin which play critical roles in capturing microbial pathogens and eliciting immune responses to the invading pathogens in host (34). Our previous work showed that CD1a DC wad less potent in antigen-presenting activity than CD1a+ DC (21). The suppression of the development of CD1a+ DC (Fig. 1) and their function (Figs 9 and 10) by PF4 suggests that PF4 may play a role in the attenuation of immune responses.



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Fig. 9. Secretion of IL-10, IL-12 (p70) and TNF-{alpha} by DC developed in the absence or presence of PF4 in response to stimulation by LPS plus IFN-{gamma}. DC (2.5 x 104) were prepared with or without 10 µg/ml PF4. DC were cultured in 200 µl RPMI 1640 medium containing 5% FCS, and stimulated with 100 ng/ml LPS and 50 ng/ml IFN-{gamma} for 24 h. Thereafter, the supernatants were harvested, and assayed for IL-10, IL-12 and TNF-{alpha}.

 
The down-regulatory roles of PF4 are further supported by the recent finding that PF4 could inhibit proliferation and cytokine release of activated human T cells (35). Moreover, we found the CD1a DC generated in the presence of PF4 expressed lower levels of HLA-DR than CD1a+ DC (Fig. 5). The down-regulation of HLA-DR by PF4 has also been observed during the differentiation of monocytes into macrophages (9). The level of HLA-DR expressed on DC appeared to be inversely correlated with the concentration of PF4 (data not shown). HLA-DR is essential for DC to present antigenic peptides to CD4+ T cells. According to the two-signal hypothesis, this engagement provides signal ‘1’ for the activation of CD4+ T cells (36). The reduced HLA-DR level on DC generated in the presence of PF4 could potentially lead to reduced activity to stimulate and prime T cells as observed in our MLC study (Fig. 10 and Table 2). It is not known how the difference in the expression of CD86 might have contributed to the observed functional differences in CD1a+ and CD1a DC for priming T cells (3739).

The results our study show that relatively high concentrations of PF4 in the range of mg/ml are needed to exert its effect on differentiation of monocytes to DC. This finding suggests that the affinity of PF4 receptors on monocytes is low. Although high concentrations of PF4 are required for its effect, concentrations at the level of mg/ml should be achievable in vivo at tissue injury sites with platelet activation. When we measured PF4 concentrations in sera of normal individuals by ELISA, we found concentrations between 50 and 80 µg/ml.

In summary, the results of our study suggest that platelet activation at the sites of tissue injury and/or inflammation could lead to secretion of PF4. PF4 exerts a chemotactic effect on various types of inflammatory cells including monocytes (2), and may influence the development and activity of monocyte-derived DC. Thereby, immune responses are modulated. In order to conclusively define the immunomodulatory roles of PF4 and its effect on differentiation of monocytes into DC in vivo, production and further characterization of PF4 gene knockout mice will be necessary.


    Acknowledgements
 
The authors are grateful for the excellent technical assistance provided by Ms Sandra C. Donohue. This work is supported by the Chiles Endowment Biomedical Research Program (BM012) of the Florida Department of Health. The opinions, findings and conclusions or recommendations expressed in this publication are those of the author(s), and do not necessarily reflect the views of the Biomedical Research Program of the Florida Department of Health.


    Abbreviations
 
ß-TG—ß-thromboglobulin

DC—dendritic cell

GM-CSF—granulocyte-macrophage colony stimulating factor

LPS—lipopolysaccharide

PF4—platelet factor 4

MLC—mixed leukocyte culture

PBMC—peripheral blood mononuclear cell

TNF—tumor necrosis factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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