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Heparin Induces Differentiation of CD1a⁺ Dendritic Cells from Monocytes: Phenotypic and Functional Characterization¹

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs) play important roles in initiation and regulation of immune responses. DCs derived from human monocytes can be classified according to presence of CD1a molecules. Although CD1a⁺ DCs can be prepared from monocytes in media containing GM-CSF, IL-4, and FCS, it has been reported that CD1a⁺ DCs could not be easily obtained from monocytes using media containing human serum or plasma. In this study, we demonstrate for the first time that heparin can reliably induce differentiation of CD1a⁺ DCs from monocytes with or without autologous serum or plasma. The development of CD1a⁺ DCs is heparin concentration dependent (0–50 U/ml). Comparing with CD1a^- DCs developed without heparin, CD1a⁺ DCs express higher CD40 and CD80 and lower CD86. Both CD1a⁺ and CD1a^- DCs express similar levels of HLA-DR. CD80, CD86, HLA-DR, and CD40 are proportionally up-regulated when both types of DCs are stimulated with LPS or LPS plus IFN-. The effect of heparin is neutralized by heparin-binding proteins, such as protamine sulfate, platelet factor-4, and -thromboglobulin. Functionally, heparin-treated DCs respond to LPS or LPS plus IFN- with higher IL-10 and less IL-12 production than heparin-untreated DCs. Heparin-treated DCs are more potent in priming allogeneic and autologous CD4⁺ T cells to proliferate and to produce both type 1 and type 2 cytokines. The results of our study show that CD1a⁺ DCs can be prepared from monocytes ex vivo without using xenogeneic serum and may be used for immunotherapy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)³ are professional APCs, and play important roles in the initiation and the regulation of immune responses (1, 2, 3). Previous studies have demonstrated that there are different subsets of DCs in humans (4, 5, 6, 7, 8, 9). These subsets of DCs appear to function differently, and can be distinguished by various cell surface markers (4, 5, 6, 8). Ito et al. (8) reported that three subsets of DCs could be identified in human peripheral blood according to the expression of CD1a and CD11c molecules as CD1a⁺CD11c⁺, CD1a^-CD11c⁺, and CD1a^-CD11c^- DCs. Among CD11c⁺ myeloid DCs in blood, the majority of them were positive for CD1a. It has been suggested that the CD1a⁺CD11c⁺ DCs are precursors of Langerhan’s cells in skin, which play crucial roles in capturing microbial pathogens and eliciting immune response to the invading pathogens (8, 9). Recently, Chang et al. (10) showed that CD1a^- DCs could be prepared from monocytes using the Yssel medium containing 10% FCS, and CD1a⁺ DCs could be obtained by culture of monocytes in the RPMI medium containing FCS. However, it has been difficult to obtain CD1a⁺ DCs from monocytes in culture media without FCS (11, 12).

Although functional differences of various subsets of DCs have been suggested (4, 5, 6), few studies have been performed to compare functional differences between CD1a⁺ and CD1a^- DCs. CD1a molecules are known to play important roles in the presentation of microbial lipid Ags to T cells and the initiation of host adaptive immune defense against microbial pathogens (13, 14). It has been reported that CD1a⁺ and CD1a^- DCs have similar activities to stimulate the proliferation of autologous and allogeneic T cells (10, 12). Nevertheless, this finding has not been confirmed. Neither is it known whether CD1a⁺ and CD1a^- DCs function similarly or differently for their ability to prime T cells.

In recent years, there has been growing interest in using human DCs prepared ex vivo for immunotherapy (15, 16, 17, 18). To avoid the exposure of patients to xenogeneic or allogeneic serum, it is preferable to prepare DCs using autologous plasma or serum. Therefore, it would be useful to have a method that can be reliably applied to generate CD1a⁺ DCs from monocytes using serum-free media or media containing autologous serum or plasma. Recently, we discovered that heparin together with GM-CSF and IL-4 was able to consistently induce the differentiation of majority of monocytes to CD1a⁺ DCs in the presence or absence of autologous serum or plasma. We also found that heparin-treated DCs were more potent than heparin-untreated DCs in priming naive CD4⁺ T cells. The results of our study are reported in this work.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Media and reagents

RPMI 1640 and AIM-V media were purchased from Life Technologies (Grand Island, NY). Human rGM-CSF was obtained from Immunex (Seattle, WA). Recombinant human IL-4 and IFN- were purchased from PeproTech (Rocky Hill, NJ). Fluorescence-conjugated Abs, matched isotype Abs, and cytometric bead array kit for cytokine assay were obtained from BD PharMingen (San Diego, CA). Platelet factor-4 (PF-4) and -thromboglobulin (-TG) were from Hematologic Technologies (Essex Junction, VT). Protamine sulfate, LPS, PMA, ionomycin, and FITC-dextran were from Sigma (St. Louis, MO). CD4⁺ T cell isolation mixture was purchased from StemCell Technologies (Vancouver, Canada). Anti-CD14 microbeads and anti-CD45RA microbeads were purchased from Miltenyi Biotec (Auburn, CA). Paired Abs and cytokine standards for ELISA of IL-10, IL-12, and IFN- were obtained from Pierce-Endogen (Woburn, MA). Porcine heparin was from Elkins Sinn (Cherry Hill, NJ).

Isolation of monocytes from peripheral blood

Venous blood was drawn from healthy volunteer donors and anticoagulated with 1/10 vol of 3.8% sodium citrate. PBMC were prepared by Ficoll-Hypaque density gradient centrifugation of freshly drawn blood. CD14⁺ monocytes were isolated from PBMC by anti-CD14 microbeads according to the instruction of manufacturer. Briefly, 1 x 10⁷ PBMC were incubated with 20 µl anti-CD14 microbeads in 0.1 ml 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 a MS-MACS separation column (Miltenyi Biotec), and CD14⁺ monocytes were isolated after removal of the column from a magnet. The purity of the CD14⁺ cells determined by flow cytometry was always in the range of 90%–95%. The isolated monocytes were used freshly or frozen at -130°C for use on later dates.

Preparation of dendritic cells from monocytes

Monocytes (5 x 10⁵ cells) were seeded into one well of a 12-well plate, and cultured in 1.5 ml AIM-V containing 1000 U/ml GM-CSF and 20 ng/ml IL-4 in the presence or absence of autologous serum with or without heparin for 6 days. Additional 1.5-ml medium containing 1000 U/ml GM-CSF and 20 ng/ml IL-4 was added into each culture on day 3. Protamine sulfate, PF-4, or -TG at respective final concentrations of 0.125 mg/ml, 10 µg/ml, and 10 µg/ml was used to neutralize the heparin effect in some experiments. To induce the maturation of DCs, LPS (100 ng/ml) or LPS (100 ng/ml) plus IFN- (50 ng/ml) was added to cultures on day 6 and incubated for additional 24 h.

For studying the effect of heparin on the development of CD1a⁺ DCs, 50 U/ml heparin was added to separate cultures of monocytes on days 0, 2, and 4. After addition of heparin, all cultures continued for 4 additional days. The cells were harvested and studied for the development of CD1a⁺ DCs by immunofluorescent flow cytometry.

Cytokine production by DC

To study cytokine secretion by heparin-untreated or heparin-treated DCs (1 x 10⁴ cells/well), DCs were stimulated with LPS (100 ng/ml), IFN- (50 ng/ml), or LPS plus IFN- for 24 h in 200 µl AIM-V containing 1% autologous serum in a 96-well plate. The supernatants were harvested and stored at -70°C until assayed for cytokines.

Preparation of CD45RA⁺CD4⁺ T cells

CD4⁺ T cells were prepared from PBMC by negative selection using a CD4⁺ T cell isolation mixture (StemCell Technologies) according to the instruction of the manufacturer. Briefly, 5 x 10⁷ PBMC in 1 ml PBS-1% FCS were incubated with 100 µl CD4⁺ T cell enrichment mixture in the presence of 1% rat serum at 4°C for 15 min. Thereafter, 60 µl magnetic colloid was added to each ml of PBMC, incubated for another 15 min. The cells were loaded onto 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 the isolated CD4⁺ T cells was consistently 95%.

CD45RA⁺ cells were isolated from CD4⁺ T cells by anti-CD45RA microbeads (Miltenyi Biotec). The procedure was similar to the one used for the isolation of CD14⁺ monocytes, as described above. The purity of isolated CD45RA⁺ cells was always between 90% and 95%.

Mixed lymphocyte culture

For primary allogeneic mixed lymphocyte culture (MLC) assay, immature DCs irradiated with 30 Gy gamma ray were cultured with 1 x 10⁵ allogeneic CD4⁺ T cells in 200 µl AIM-V media containing 1% human AB serum for 5 days in a U-bottom 96-well plate. [³H]Thymidine (1 µCi) was added into each well and incubated for additional 16–18 h. The cells were harvested using a PHD cell harvester (Brandel, Gaithersburg, MD). The incorporation of [³H]thymidine was determined by scintillation counting. All experiments were performed in triplicate incubations.

For autologous MLC, immature DCs were incubated with 100 ng/ml tetanus toxoid (TT) in a tissue culture incubator for 3 h. The DCs were washed three times with PBS and resuspended in AIM-V containing 1% autologous serum. DCs pulsed with or without TT were irradiated by 30 Gy gamma ray and used to stimulate autologous CD4⁺ T cells (1 x 10⁵/well) in triplicates for 5 days. Thereafter, 1 µCi [³H]thymidine was added to each well and incubated for 16–18 h.

To study the production of type 1 and type 2 cytokines by naive CD4⁺ T cells, 1 x 10⁵ CD45RA⁺CD4⁺ T cells were primed with 5 x 10³ allogeneic heparin-treated or heparin-untreated mature DCs for 3 days in 200 µl AIM-V containing 1% AB serum. The T cells were harvested, washed, and restimulated by 25 ng/ml PMA and 1 µg/ml ionomycin for 48 h. The supernatants were harvested and assayed for cytokines.

Assays for cytokines

For quantitation of human IL-10 and IL-12 concentrations by ELISA, each well of immunoassay plates was coated with 50 µl of 5 µg/ml anti-human IL-10 or IL-12 Ab in PBS-azide overnight at 4°C. The plates were washed three times with PBS-0.2% Tween 20 (PBS-Tween) and blocked with 150 µl PBS containing 2% BSA for 30 min at room temperature. Thereafter, 50 µl cytokine standards and unknown samples were added and incubated for 90 min. After incubation, plates were washed and incubated sequentially with 50 µl of 0.5 µg/ml biotinylated anti-cytokine Ab diluted in PBS-Tween, 1% BSA, streptavidin-peroxidase (Sigma), and o-phenylenediamine dihydrochloride peroxidase substrate (Sigma). Paired Abs and recombinant human IL-10 and IL-12 standards were obtained from Pierce-Endogen. The sensitivities of IL-10 and IL-12 assays were 2.5 and 5 pg/ml, respectively.

For measurement of IL-4, IL-5, and IL-10 secreted by CD45RA⁺CD4⁺ T cells, a Cytometric Beads Array Kit from BD PharMingen was used. Concentrations of IFN- secreted by CD45RA⁺CD4⁺ T cells were often too high to be measured by Cytometric Beads Array Kits and were determined by ELISA.

Immunophenotyping by flow cytometry

For immunophenotypic characterization, washed DCs were incubated with Abs labeled with fluorescent dyes diluted in PBS containing 1% BSA and 0.02% NaN₃ at 4°C for 25 min. Matched isotype Abs were used as negative controls. The cells were analyzed by FACScan (BD Biosciences, Mountain View, CA), and the data were analyzed by FCS Express software (De Novo Software, Thornhill, Ontario, Canada).

Phagocytosis of FITC-dextran by DCs

To measure the phagocytic activity of DCs, a previously reported method (11) was used with slight modifications. DCs (5 x 10⁴) were resuspended in 100 µl PBS containing 1% human AB serum and incubated with FITC-dextran (0.1 mg/ml) at 37°C and 0°C for 30 min. The incubations were stopped by adding 2 ml ice-cold PBS containing 1% human serum and 0.02% sodium azide. The cells were washed three times with cold PBS-azide and analyzed on a FACScan flow cytometer.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of autologous serum and heparin-plasma on the development of CD1a⁺ DCs

In a series of experiments, we consistently found that more CD1a⁺ DCs were obtained when monocytes were cultured in RPMI 1640 containing 10% autologous heparin-plasma than monocytes cultured in the presence of 10% autologous serum (Fig. 1). The results from seven separate experiments showed that CD1a⁺ DCs were 47 ± 9% vs 21 ± 10% (p < 0.05) when monocytes were cultured in the presence of autologous heparin-plasma vs autologous serum.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Development of CD1a+ DCs from monocytes in the culture medium containing autologous serum or heparin-plasma. Purified monocytes were cultured in RPMI 1640 containing 10% autologous serum (A) or heparin-plasma (B) with presence of 1000 U/ml GM-CSF and 20 ng/ml IL-4 for 6 days. CD1a expression was studied by flow cytometry.

To determine whether heparin is responsible for the development of CD1a⁺ DCs, monocytes were cultured in serum-free AIM-V medium containing increasing concentrations of heparin in the presence of GM-CSF and IL-4 for 6 days. It was found that the relative numbers of CD1a⁺ DCs were increased proportionally to the heparin concentration (Fig. 2). The relative number of CD1a⁺ DCs was further increased to 90%, when 2% autologous serum was included in the media containing 50 U/ml heparin (data not shown). However, further increase of serum concentration up to 10% did not enhance the yield of CD1a⁺ DCs.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Effect of different concentrations of heparin on the development of CD1a+ DCs from monocytes. DCs were generated by cultures of monocytes in serum-free AIM-V medium containing 1000 U/ml GM-CSF and 20 ng/ml IL-4 for 6 days at different concentrations of heparin. A, 0 U/ml; B, 10 U/ml; C, 25 U/ml; D, 50 U/ml. CD1a+ DCs were measured by flow cytometry. Similar results were obtained when the experiment was repeated.

To determine the minimal duration of ex vivo culture needed to obtain CD1a⁺ DCs from monocytes, the development of CD1a⁺ DCs was studied as a function of incubation duration in the presence of heparin (50 U/ml) in AIM-V containing 2% autologous serum, GM-CSF, and IL-4. We found that almost all monocytes became CD14 negative after culture for 2 days. At the same time, approximately 50% of the cells began to express variable levels of CD1a (Fig. 3). After treatment for 4 days, 86% of the cells were positive for CD1a (Fig. 3). There was no further significant increase of CD1a⁺ DCs and CD1a level after culture for additional 2 days (Fig. 3). Without presence of heparin, CD1a⁺ DCs were less than 30% after culture for 6 days. The average level of CD1a on DCs prepared in the absence of heparin was also lower than that of CD1a⁺ DCs prepared in the presence of heparin.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Changes of CD1a and CD14 expression after treatment of monocytes with GM-CSF (1000 U/ml) and IL-4 (20 ng/ml) in the absence or presence of heparin (50 U/ml). Cells were harvested at different time points and studied for CD1a and CD14 expression by flow cytometry. The numbers in parentheses are percentages of cells that were positive for CD1a or CD14. The total number of cells in each well did not show any significant change during 6-day culture.

To determine whether heparin is necessary from the beginning of monocyte culture for the production of CD1a⁺ DCs, 50 U/ml heparin was added into separate cultures on days 0, 2, and 4. Thereafter, monocytes were cultured for 4 additional days. We found that to maximally induce differentiation of monocytes to CD1a⁺ DCs, heparin had to be added at the beginning of the culture. Delayed addition of heparin on day 2 reduced the production of CD1a⁺ DCs to 46% (Fig. 4B). Heparin exerted very little effect on the development of CD1a⁺ DCs, when it was added to the culture on day 4 (Fig. 4C).

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Effect of heparin on differentiation of CD1a+ DCs from monocytes after initiation of cultures with 1000 U/ml GM-CSF and 20 ng/ml IL-4 for different durations. Heparin (50 U/ml) was added on day 0 (A), day 2 (B), and day 4 (C) after the initiation of monocyte cultures. After addition of heparin, the cultures continued for additional 4 days. Cells were harvested, and the development of CD1a+ DCs was studied by flow cytometry. Similar results were obtained when the experiment was repeated. The results show the importance of including heparin from the beginning of monocyte culture to obtain a maximal number of CD1a+ DCs.

To demonstrate heparin was indeed responsible for the differentiation of CD1a⁺ DCs from monocytes, heparin-binding proteins such as protamine sulfate (0.125 mg/ml), PF-4 (10 µg/ml), and -TG (10 µg/ml) were used to neutralize heparin. The results shown in Fig. 5 indicate that protamine sulfate, PF-4, and -TG can neutralize the effect of heparin on the development of CD1a⁺ DCs.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Inhibition of heparin-induced development of CD1a+ DCs by heparin-binding proteins. In this experiment, monocytes were cultured in AIM-V medium containing 2% autologous serum, GM-CSF (1000 U/ml), and IL-4 (20 ng/ml) for 4 days. A, 25 U/ml heparin and no heparin-binding proteins; B, no heparin and no heparin-binding proteins; C, 25 U/ml heparin and 0.125 mg/ml protamine sulfate; D, 25 U/ml heparin and 10 µg/ml PF-4; E, 25 U/ml heparin and 10 µg/ml -TG. The effect of heparin to induce the development of CD1a+ DCs was neutralized by presence of the heparin-binding proteins.

To determine immunophenotypic differences between DCs cultured in the presence and absence of heparin, we studied the expression of CD14, CD80, CD86, CD40, CD83, and HLA-DR on both types of DCs by immunofluorescent flow cytometry. The mean fluorescent intensities of each marker from three separate experiments were analyzed by paired t test and showed that CD1a⁺ DCs developed in the presence of heparin expressed higher levels of CD40 (51 ± 3 vs 20 ± 5, p = 0.01) and CD80 (20 ± 2 vs 11 ± 3, p = 0.015) and a lower level of CD86 (10 ± 2 vs 55 ± 14, p = 0.026) comparing with CD1a-DCs developed in the absence of heparin (Fig. 6). When the small number of heparin-treated and CD1a^- DCs were analyzed, the levels of CD40, CD80, and CD86 were the same as heparin-untreated and CD1a^- DCs (data not shown). Both CD80 and CD86 were up-regulated, when heparin-treated CD1a⁺ DCs and heparin-untreated CD1a^- DCs were stimulated with LPS to induce maturation. Nevertheless, CD80 and CD86 on CD1a⁺ mature DCs remained higher and lower than those on CD1a^- mature DCs, respectively (data not shown). Both CD1a⁺ and CD1a^- immature DCs expressed similar levels of HLA-DR without statistical difference and were negative for both CD14 and CD83 (Fig. 6). When immature DCs were stimulated by LPS or LPS plus IFN-, CD83 was similarly up-regulated on both types of DCs (Fig. 7, B and D). Maturation of both types of DCs did not lead to significant change of CD1a expression (Fig. 7).

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Immunophenotypic characterization of DCs developed in the absence or presence of heparin. All DCs were stained simultaneously by PE-labeled anti-CD1a Ab and FITC-labeled Ab to different cell surface markers, and were analyzed by flow cytometry. A total of 86% of DCs generated without heparin treatment were CD1a-, and 87% of DCs with heparin treatment were CD1a+. These CD1a- and CD1a+ cells were gated and analyzed, respectively. The results are shown in histograms. Similar results were obtained in two additional repeated experiments. MFI, mean fluorescent intensity. The results from three experiments analyzed by paired t test showed higher CD40 (51 ± 3 vs 20 ± 5, p = 0.01) and CD80 (20 ± 2 vs 11 ± 3, p = 0.015), and lower CD86 (10 ± 2 vs 55 ± 14, p = 0.026) on CD1a+ DCs. There were no significant differences for CD14, CD83, and HLA-DR between CD1a- and CD1a+ DCs.

View larger version (52K): [in this window] [in a new window]   FIGURE 7. Expression of CD1a and CD83 on DCs developed in the absence (A and B) or presence (C and D) of heparin (50 u/ml), and with (B and D) or without (A and C) stimulation with LPS (100 ng/ml) for 24 h. In this experiment, monocytes were cultured in AIM-V medium containing 2% autologous serum, GM-CSF (1000 U/ml), and IL-4 (20 ng/ml) with or without heparin for 6 days, and cells were stimulated with or without LPS for 24 h. The results showed the induction of CD1a expression by the treatment of heparin and the induction of CD83 by LPS stimulation. Similar results were obtained when the experiment was repeated.

Because IL-10 and IL-12 secreted by DCs are involved in directing immune response of T cells (2, 19, 20, 21), the secretion of IL-12 and IL-10 by heparin-untreated and heparin-treated DCs was studied. The results from one of the three separate experiments are shown in Fig. 8. We consistently found that heparin-treated DCs secreted more IL-10 than IL-12 in response to LPS stimulation (p = 0.008). Heparin-untreated DCs stimulated with LPS plus IFN- secreted more IL-12 than IL-10 (p = 0.05). Heparin-untreated DCs secreted less IL-10 (p = 0.05) and higher IL-12 (p = 0.05) than heparin-treated DCs in response to the stimulation by LPS plus IFN-. When DCs were stimulated by LPS alone, heparin-treated DCs secreted more IL-10 than heparin-untreated DCs (Fig. 8, p = 0.04), and there was no significant difference between heparin-untreated and heparin-treated DCs in response to the stimulation by LPS alone for IL-12 secretion (p = 0.9). Interestingly, we also noted that the production of IL-12 was conversely related to the production of IL-10.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Effect of LPS and/or IFN- stimulation on the production of IL-12 and IL-10 by heparin-untreated and heparin-treated DCs. DCs (1 x 104) were treated with LPS (100 ng/ml) and/or IFN- (50 ng/ml) for 24 h. Culture media were harvested and assayed for IL-10 and IL-12 by ELISA. The experiments were repeated using DCs from two other individuals. Similar results were obtained. Results from one of the three experiments are shown in Fig. 8. Statistical analysis of results from three separate experiments by paired t test indicates that heparin-treated DCs produced more IL-10 than heparin-untreated DCs after stimulation with LPS (p = 0.04) or LPS plus IFN- (p = 0.05). Heparin-untreated DCs produced more IL-12 than heparin-treated DCs after stimulation with LPS plus IFN- (p = 0.05), but not by LPS alone (p = 0.9). *, p > 0.05; , p 0.05.

One important function of immature DCs is phagocytosis of exogenous Ags. After phagocytosis, the Ags are processed by DCs and antigenic peptides are presented by the MHC class II molecules by which Ag-specific CD4⁺ T cells are activated in conjunction with costimulatory signals. To compare the phagocytic activities of CD1a⁺ and CD1a^- DCs, phagocytosis of FITC-dextran by these two types of DCs was studied. CD1a⁺ and CD1a^- cells in heparin-treated and heparin-untreated DCs were gated respectively for analysis. We found that CD1a⁺ or CD1a^- immature DCs generated in the presence or the absence of heparin had similar degree of phagocytic activity (Fig. 9). After induction of maturation by LPS, the phagocytosis of FITC-dextran by both types of DCs was significantly reduced (Fig. 9).

View larger version (35K): [in this window] [in a new window]   FIGURE 9. Phagocytosis of FITC-dextran by immature or mature DCs generated in the absence or presence of heparin. Mature DCs were prepared by stimulation of immature DCs with LPS (100 ng/ml) for 24 h. The histograms of dotted lines represent the uptake of FITC-dextran at 0°C. The upper panels (A and C) are immature DCs, and the lower panels (B and D) are mature DCs. The left panels (A and B) are CD1a- DCs generated in the absence of heparin (50 U/ml), and the right panels (C and D) are CD1a+ DCs generated in the presence of heparin. DCs were stained with anti-CD1a labeled with PE. Heparin-treated CD1a+ and heparin-untreated CD1a- DCs were gated and analyzed.

As described above, DCs developed in the presence and the absence of heparin have different phenotypes and cytokine production profiles. Therefore, it is of interest to learn whether these two types of DCs can prime allogeneic or autologous CD4⁺ T cells similarly or differently. We first studied the stimulator activity of DCs to allogeneic CD4⁺ T cells in MLC. We found that heparin-treated DCs were significantly more potent than heparin-untreated DCs in stimulation of allogeneic CD4⁺ T cells to proliferate (Fig. 10A). For stimulation of autologous CD4⁺ T cells to proliferate, heparin-treated DCs pulsed with TT were slightly more potent than heparin-untreated DCs (Fig. 10B).

View larger version (21K): [in this window] [in a new window]   FIGURE 10. Stimulator activity of heparin-treated and heparin-untreated DCs in allogeneic and autologous MLC. A, Shows the results of allogeneic MLC. Different doses (625, 1250, 2500, 5000 per well) of immature heparin-treated (•) and heparin-untreated () DCs were irradiated with 30 Gy gamma ray and cultured with allogeneic CD4+ T cells (1 x 105/well) for 5 days. Each incubation then was treated with 1 µCi [3H]thymidine for 16 h. Similar results were obtained when DCs from two other individuals were studied. B, Shows the results of autologous MLC. Immature heparin-untreated DCs ( and ) and heparin-treated DCs (• and ) were pulsed with (filled symbols) or without (open symbols) TT (100 ng/ml) for 3 h at 37°C and washed three times before use. MLC was performed as described above. Each value is mean ± SD of triplicate incubations. Similar results were obtained when the experiment was repeated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Prompted by this intriguing observation, we investigated the effect of heparin on the differentiation of DCs from monocytes, and discovered for the first time that heparin had dramatic effect on the promotion of monocytes to differentiate into CD1a⁺ DCs in the presence of GM-CSF and IL-4 without any serum or plasma (Fig. 2). When 2% autologous serum was included in the cultures, the relative number of CD1a⁺ DCs was increased from 80% to 90%. We also found that heparin had to be included in the culture from the very beginning to induce maximal development of CD1a⁺ DCs (Fig. 4). Delayed addition of heparin reduced the development of CD1a⁺ DCs (Fig. 4). It is likely that exposure of monocytes to GM-CSF and IL-4 initiates the differentiation of monocytes to CD1a^- DCs. The initiation of such differentiation may have rendered the differentiating monocytes refractory to heparin. The results of our study indicate that the addition of heparin from the beginning of cultures is essential for majority of monocytes to differentiate into CD1a⁺ DCs.

Although many heparin-binding proteins are known to be present in plasma or serum (31, 32), our study shows that heparin can induce the differentiation of CD1a⁺ DCs without plasma or serum (Fig. 2). In addition, specific heparin binding sites are known to be present on monocytes (30). Thus, the effect of heparin on the differentiation of CD1a⁺ DCs is most likely mediated by the direct binding of heparin to monocytes. This conclusion is further supported by the finding that heparin-binding proteins, such as protamine sulfate, PF-4, and -TG, could neutralize the effect of heparin and prevent the differentiation of monocytes into CD1a⁺ DCs (Fig. 5). All these three heparin-binding proteins are known to inhibit the anticoagulation activity of heparin. In addition, it has been reported that GM-CSF and IL-4 could interact with heparin by which their activities and signaling are modulated (33, 34).Therefore, the interaction of heparin with GM-CSF and IL-4 may play some role in the development of CD1a⁺ DCs and should be considered.

In our study, we found that heparin-untreated DCs were able to produce more IL-12 than IL-10 and higher level of IL-12 than heparin-treated DCs, when they were stimulated with LPS plus IFN- (Fig. 8). This finding suggested that heparin-untreated DCs might be more potent than heparin-treated DCs in priming naive T cells for type 1 cytokine reponse and cell-mediated immunity. The higher level of IL-10 produced by heparin-treated DCs (Fig. 8) may favor type 2 T cell response. Nevertheless, we found that heparin-treated DCs were significantly more potent than heparin-untreated DCs in priming allogeneic naive CD4⁺ T cells for the production of both type 1 and type 2 T cell cytokines (Table I). The ratios of IFN- and IL-5 (Table I) indicate that heparin-treated DCs have relatively higher capacity to prime naive CD4⁺ T cells for type 2 response. This finding is consistent with the results shown in Fig. 8 in which heparin-treated DCs produced higher amount of IL-10 than heparin-untreated DCs when they were stimulated with LPS or LPS plus IFN-.

Despite that heparin-untreated DCs produced more IL-12 than heparin-treated DCs after stimulation with LPS plus IFN- (Fig. 8), heparin-treated DCs were more effective in priming allogeneic naive CD4⁺ T cells for both type 1 and type 2 responses (Table I). It is not known why heparin-treated DCs were more potent than heparin-untreated DCs for priming naive allogeneic T cells to produce higher level of type 1 cytokine. Neither is it known whether the observed functional difference between heparin-treated and heparin-untreated DCs has anything to do with the expression of CD1a. Nevertheless, it was noted that higher levels of CD40 and CD80 and lower level of CD86 were expressed on CD1a⁺ DCs (Fig. 6). According to earlier animal study (35), differential expression of higher level of CD80 could direct the differentiation of CD4⁺ Th precursor cells to Th1 pathway. Thus, higher level of CD80 expression on heparin-treated DCs might have played some roles in priming naive CD4⁺ T cells for more vigorous production of IFN-.

In addition, the results of our study showed that heparin-untreated DCs were much less potent than heparin-treated DCs to stimulate proliferation of CD4⁺ T cell in allogeneic MLC (Fig. 10). When we measured IL-2 level in the supernatant 3 days after initiation of the allogeneic MLC, it was found that the level of IL-2 in the supernatant of CD4⁺ T cells stimulated by heparin-treated DCs was higher than in the supernatant of CD4⁺ T cells stimulated by heparin-untreated DCs (740 pg/ml vs 342 pg/ml). This finding supported greater CD4⁺ T cell proliferation in the MLC using heparin-treated DCs as stimulatory cells. For autologous MLC, heparin-treated DCs were only slightly more active than heparin-untreated DCs to stimulate proliferation of naive and memory CD4⁺ T cells specific for TT Ags (Fig. 10). All these findings indicate that heparin-treated DCs could be more potent APCs than heparin-untreated DCs.

Because DCs play a pivotal role in immune responses, a great deal of research effort has been focused on the potential clinical application of these cells to immunotherapy of malignant neoplasms, viral infection, and other clinical conditions (15, 16, 17, 18, 36, 37). To avoid exposing patients to xenogenic serum, DCs are often prepared ex vivo by cultures of monocytes in the presence of autologous serum or in the absence of any serum. However, monocytes cultured in the media without serum or with autologous or allogeneic human serum/plasma mainly differentiated into CD1a^- DCs (11, 12). Despite that activation of heparin-treated DCs ex vivo with LPS plus IFN- produced lower IL-12 and higher IL-10 than heparin-untreated DCs (Fig. 8), the results reported in this work indicate that heparin-treated DCs have higher priming activity for T cells to produce both type 1 and type 2 cytokines (Table I). Although immune polarization to Th1 response has been associated with the development of cytotoxic T cells and protective antitumor immunity (38), the induction of Th2 response could confer humoral immunity for Ab-mediated cytotoxicity (39), which may provide additional immune protection against tumor growth. Because heparin-treated DCs could prime T cells for type 1 and type 2 cytokine responses (Table I) and are more potent than heparin-untreated DCs to stimulate proliferation of T cells (Fig. 10), heparin-treated DCs may be more efficacious than heparin-untreated DCs for the induction of cellular and humoral immunity against tumor growth. In addition, heparin-treated DCs are able to produce a higher level of IL-10, which has recently been shown to promote the development of Th1 type of effector cells (40) and the maintenance of antitumor CD8⁺ T cells (41). All these findings indicate that treatment of monocytes with GM-CSF and IL-4 in the presence of heparin could be a practical and reliable approach for preparation of potent Ag-presenting DCs that can be clinically applied for immunotherapy.

	Acknowledgments

 
We thank Sandra Donahue and Lori Boggs for their excellent technical assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant HL-58809 and Florida Biomedical Research Program Grant BM-012.

² Address correspondence and reprint requests to Dr. Kuo-Jang Kao, Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Box 100275, Gainesville, FL 32610. E-mail address: kjkao{at}ufl.edu

³ Abbreviations used in this paper: DC, dendritic cell; MLC, mixed lymphocyte culture; PF-4, platelet factor 4; -TG, -thromboglobulin; TT, tetanus toxoid.

Received for publication August 17, 2001. Accepted for publication November 20, 2001.

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