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TGF-ß1 Reciprocally Controls Chemotaxis of Human Peripheral Blood Monocyte-Derived Dendritic Cells Via Chemokine Receptors

Katsuaki Sato^1,*, Hiroshi Kawasaki^1,, Hitomi Nagayama^*, Makoto Enomoto^*, Chikao Morimoto, Kenji Tadokoro, Takeo Juji and Tsuneo A. Takahashi^2,*

Departments of ^* Cell Processing and Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and Japanese Red Cross Central Blood Center, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined the effect of TGF-ß1 on the chemotactic migratory ability of human monocyte-derived dendritic cells (DCs). Treatment of immature DCs with TGF-ß1 resulted in increased expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXC chemokine receptor-4 (CXCR-4), which were concomitant with enhanced chemotactic migratory responses to their ligands, RANTES (for CCR-1, CCR-3, and CCR-5), macrophage-inflammatory protein-3 (MIP-3) (for CCR-6), or stromal cell-derived growth factor-1 (for CXCR-4). Ligation by TNF- resulted in down-modulation of cell surface expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and the chemotaxis for RANTES, MIP-3, and stromal cell-derived growth factor-1, whereas this stimulation up-regulated the expression of CCR-7 and the chemotactic ability for MIP-3ß. Stimulation of mature DCs with TGF-ß1 also enhanced TNF--induced down-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and chemotaxis to their respective ligands, while this stimulation suppressed TNF--induced expression of CCR-7 and chemotactic migratory ability to MIP-3ß. Our findings suggest that TGF-ß1 reversibly regulates chemotaxis of DCs via regulation of chemokine receptor expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)³ are unique professional major APCs capable of stimulating resting T cells (TCs) in primary immune responses, and are more potent APCs than peripheral blood monocytes/macrophages or B cells (1). DCs also play major roles in autoimmune diseases, graft rejection, HIV infection, and the generation of TC-dependent Abs (1, 2, 3, 4). DCs capture and process Ag in nonlymphoid tissues and then migrate to TC-dependent areas of secondary lymphoid organs via afferent lymph or the bloodstream to prime native TCs and initiate immune responses (5, 6).

Characterization of DCs is difficult because they represent only a small subpopulation that includes interdigitating reticulum cells in lymphoid organs, blood DCs, Langerhans cells in the epidermis of the skin, and dermal DCs (1). Previously, an in vitro culture system revealed that DCs originate from CD34⁺ pluripotent hemopoietic progenitor cells (HPCs) in the bone marrow (BM) and cord blood via myeloid lineage cells in human and murine models (7, 8, 9, 10, 11, 12, 13, 14, 15, 16), and some DCs develop from thymic precursors via lymphoid lineage cells in murine system (17).

Chemokines are extensively involved in inflammatory/immunological responses due to their unique ability to recruit selective leukocyte subsets (18). Chemokines have been implicated in regulation of normal leukocyte recirculation and homing, and also in certain physiological and pathogenic processes, including hemopoiesis, angiogenesis, allergy, autoimmune diseases, and viral infectious diseases (18). Chemokines are a group of 70–90 amino acids, structurally related polypeptides, most of which contain four conserved cysteine residues in their primary amino acid sequence (18). There are two major groups: the CXC chemokines in which the two NH₂-terminal cysteines are separated by a single amino acid, and the CC chemokines, in which the two NH₂-terminal cysteines are adjacent. A third type of chemokine, represented by lymphotactin, contains only two of the four conserved cysteines (18).

The specific effects of chemokines on the target cell types are mediated by a family of single-chain, seven-helix membrane-spanning receptors coupled to heterotrimeric guanine nucleotide-binding protein (G protein) (GPCR), which consists of a Gi, Gß, and G subunit complex (18). Ligand specificities of 14 chemokine receptors have been identified; five of the receptors are specific for CXC chemokines (CXCR1–5) (18), eight of them are specific for CC chemokines (CCR1–8) (18, 19, 20), and the Duffy Ag receptor binds both CXC and CC chemokines (21). In addition, distinct chemokines appear to act on more than one receptor type in vitro (18).

There is increasing interest in the potential role of chemokines and their respective receptors in the biological properties of DCs to clarify the mechanism underlying DC-mediated regulation of immune/inflammatory responses. Previous studies have shown that several chemokine receptors are expressed on some DCs and their progenitor cells at the transcriptional level (22, 23, 24, 25, 26, 27). Recent studies have shown that the chemotactic migratory properties in response to certain chemokines are strictly regulated in the development of DCs from their progenitor cells, and these regulatory mechanisms have been potentially implicated in mediating the trafficking of DCs and their progenitor cells from blood to tissues and then to lymph nodes, where they form a close association with TCs in the process of Ag presentation (26, 27).

TGF-ß1, which is a cytokine produced by various types of cells, is a pleiotropic cytokine that has growth-modulatory, immunosuppressive, and inflammatory activities (28, 29, 30). Previous studies have shown that TGF-ß1 is involved in the generation of DCs and Langerhans cells from their progenitor cells (31, 32, 33, 34). Furthermore, Ogata et al. (35) have recently reported that TGF-ß1 not only inhibited the expression of CCR-7 in DCs and DC precursors derived from HPCs, but it also inhibited the migration of these cells in response to macrophage-inflammatory protein (MIP)-3ß in murine models. However, the role of TGF-ß1 in the regulation of chemotaxis of human DCs remains unclear.

We have recently reported that CCR-1, CCR-3, CCR-5, and CXCR-4 are constitutively expressed on the cell surface of human peripheral blood monocyte-derived immature DCs (iDCs) (36). Furthermore, mAb to either CCR-1 or CCR-3, but not CCR-5 and CXCR-4, abolished the chemotactic migratory ability of monocyte-derived iDCs as well as the ability of these cells to activate allogeneic TCs to proliferate and secrete IFN-, indicating that CCR-1 and CCR-3 specifically regulate interactions between TCs and iDCs via chemotactic migratory events during Ag presentation (36).

In this study, we examined the potential roles of TGF-ß1 in the regulation of chemotactic migratory properties of human monocyte-derived DCs via chemokine receptors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Media and reagents

The medium used throughout was RPMI 1640 supplemented with 2 mM L-glutamine, 50 µg/ml streptomycin, 50 U/ml penicillin, and 10% heat-inactivated FCS. GM-CSF was kindly provided by Kirin Brewery (Tokyo, Japan). IL-4, IL-10, TNF-, TGF-ß1, RANTES, MIP-1, eotaxin, stromal cell-derived factor (SDF)-1, MIP-3, and MIP-3ß were purchased from PeproTech (London, U.K.). FITC-labeled dextran (FITC-DX) and lucifer yellow (LY) were purchased from Molecular Probes (Eugene, OR). A mAb to IL-10 was purchased from PharMingen (San Diego, CA). The preparations of mAbs to CCR-1 and CCR-3 (IgG1 isotype), and the specific recognition of these mAbs for the respective CCRs were described previously (36).

In vitro generation and culture of human DCs

DCs were generated from PBMCs, as described previously (9, 10), with some modifications (36, 37, 38). Briefly, PBMCs were obtained from 30 ml of leukocyte-enriched buffy coat from healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden), and the light density fraction from the 42.5–50% interface was recovered. The cells were resuspended in culture medium and allowed to adhere to six-well plates (Costar, Cambridge, MA). After 2 h at 37°C, nonadherent cells were removed and adherent cells were collected, then subsequently negatively selected with anti-CD2 mAb-conjugated immunomagnetic beads (Dynal, Oslo, Norway) and anti-CD19 mAb-conjugated immunomagnetic beads (Dynal) to deplete CD2⁺ cells and CD19⁺ cells, according to the manufacturer’s instructions. The resultant cells (>95% CD14⁺ cells) were used as monocytes and cultured in 3 ml of medium supplemented with GM-CSF (50 ng/ml) and IL-4 (250 ng/ml). After 7 days of culture, iDCs were harvested (36, 37, 38). For preparation of mature DCs (mDCs) from iDCs, the cells were subsequently cultured with TNF- (50 ng/ml) for another 4 days (37, 38). These cell populations exhibited the typical phenotype of DCs, as described previously (36, 37, 38). Cell differentiation was monitored by light microscopy, and the resultant cells were used for subsequent experiments.

Preparation and culture of TCs

TCs were isolated from monocyte-depleted cell population by E-rosetting (39), and TC preparations were typically >95% pure, as indicated by anti-CD3 mAb (Becton Dickinson, Mountain View, CA) staining.

Stimulation of DCs

iDCs or mDCs (10⁷) were unstimulated or stimulated with various concentrations of TGF-ß1 or IL-10 (1–100 ng/ml) in the presence or absence of 1 µg/ml control mouse IgG (Sigma, St. Louis, MO) or anti-IL-10 mAb for the indicated number of days (1–5 days). The cells were washed twice with cold PBS, and used for subsequent experiments.

Flow cytometry

For surface marker analysis, iDCs or mDCs were cultured with the following mAbs conjugated to FITC or PE for direct fluorescein: CD83 (Coulter Immunology, Hialeah, FL); HLA-DR (Becton Dickinson, Mountain View, CA); CD40, CD86, and CCR-5 (all from PharMingen); CCR-6 (R&D Systems, Minneapolis, MN). The cells were also stained with the corresponding FITC- or PE-conjugated isotype-matched control mAb (all from Becton Dickinson). For indirect staining, the cells were incubated with biotin-conjugated anti-CCR-1 mAb, biotin-conjugated anti-CCR-3 mAb, or biotin-conjugated anti-CXCR-4 mAb (PharMingen) for 30 min at 4°C, washed twice with cold PBS, and subsequently stained with FITC-conjugated avidin (Becton Dickinson) for 30 min at 4°C. Thereafter, the cells were washed twice, and suspended in PBS containing 0.2 µg/ml propidium iodide (Sigma) to exclude dead cells. Analysis of fluorescence staining was performed with a FACScalibur flow cytometer (Becton Dickinson) and CELLQuest Software. The cell surface expression levels in the flow cytometry profiles are expressed as the mean fluorescence intensity (MFI) indices.

Assay for chemotaxis

The in vitro migration of iDCs or mDCs was assessed in a 24-well Transwell cell culture chamber (Costar), as described previously (36, 38). In brief, 8-µm-pore-size polycarbonate filters were precoated with 5 µg of gelatin (WAKO, Osaka, Japan) in a volume of 50 µl on the lower surface, and dried overnight at room temperature. The coated filters were washed in PBS and then dried immediately before use. DCs were pretreated with or without 1 µg/ml of the mAbs to CCR-1, CCR-3, CCR-5, CXCR-4, or control mouse IgG (Sigma) for 30 min at 37°C, and 100 µl of the cell suspension (10⁶) was added to the upper compartment of the chamber. RANTES, MIP-1, eotaxin, SDF-1, MIP-3, or MIP-3ß (1–100 ng/ml) diluted in serum-free culture medium was loaded in the upper compartment and/or the lower compartment. After a 2-h incubation, the filters were fixed with methanol and stained with hematoxylin and eosin (all from WAKO, Osaka, Japan). The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope at a magnification of x200, and each assay was performed in triplicate. The data are expressed as number of migrated cells/high power fields.

Endocytosis assay with FITC-DX and LY

The methods used to determine the endocytotic activity of in vitro generated DCs have previously been described (37, 38). Briefly, FITC-DX or LY was added to a final concentration of 1 mg/ml to the cells, and the cells were cultured for 60 min at 37°C. After incubation, cells were washed four times with ice-cold PBS and analyzed by flow cytometry, as described above.

Mixed leukocyte reaction

A total of 10⁵ responding T cells from an unrelated individual (allogeneic MLR) were cultured in 96-well flat-bottom microplates (Costar) with different numbers (10²–5 x 10⁴) of stimulator cells. Thymidine incorporation was measured on day 5 by an 18-h pulse with [³H]thymidine (1 µCi/well; sp. act., 5 Ci/mmol; Amersham Life Science, Buckinghamshire, U.K.).

Reverse-transcriptase PCR

RNA from each sample (5 x 10⁶) was isolated using Trizol LS reagent (Life Technologies, Gaithersburg, MD). The first strand cDNA kit (SuperScript Preamplification System; Life Technologies) was used to make cDNA (20 µl) from 5 µg of each RNA. Amplification of each cDNA (1 µl) was performed with a SuperTaq Premix kit (Sawady Technology, Tokyo, Japan) using specific primers, as follows: CCR-1, 5'-TCC TCA CGA AAG CCT ACG AGA GTG GAA GC-3' and 5'-CCA CGG AGA GGA AGG GGA GCC ATT TAA C-3'; CCR-3 (23), 5'-TTT GGT GTC ATC ACC AGC AT-3' and 5'-TCA TGC AGC AGT GGG AGT AGG-3'; CCR-5 (23), 5'-GGT GGA ACA AGA TGG ATT AT-3' and 5'-CAT GTG CAC AAC TCT GAC TG-3'; CCR-6 (27), 5'-ATT TCA GCG ATG TTT TCG ACT C-3' and 5'-GGA GAA GCC TGA GGA CTT GTA-3'; CCR-7 (27), 5'-GAT TAC ATC GGA GAC AAC ACC-3' and 5'-TAG TCC AGG CAG AAG AGT CG-3'; CXCR-4, 5'-CTG AGA AGC ATG ACG GAC AAG TAC AGG CT-3' and 5'-CAG ATG AAT GTC CAC CTC GCT TTC CTT TGG-3'. Specific primers for ß-actin (CLP, San Diego, CA) were also used for amplification. The reaction mixture was subjected to 35 cycles of PCR with the following conditions: CCR-1 and CXCR-4, 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. CCR-3 and CCR-5, 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. CCR-6 and CCR-7, 94°C for 1 min, 61.5°C for 2 min, and 72°C for 3 min. PCR products were analyzed by electrophoresis through 2% agarose gels and visualized under UV light after ethidium bromide staining.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TGF-ß1 enhances cell surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in iDCs

The chemotactic migratory property of DCs is thought to be crucial for the regulation of immune responses, as well as inflammation and autoimmune diseases. However, little is known about the regulation of chemotaxis of DCs. Previous studies have shown that TGF-ß1 is involved in the development of DCs as well as Langerhans cells from their progenitor cells (31, 32, 33, 34). We have recently reported that human monocyte-derived iDCs constitutively express CCR-1, CCR-3, CCR-5, and CXCR-4 on their cell surface (36). To examine the effect of TGF-ß1 on the expression of these CC and CXCR in iDCs, the cells were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days, and the expressions of these chemokine receptors were analyzed by flow cytometry with respective mAbs. Stimulation of iDCs with TGF-ß1 resulted in the enhancement of the expression levels of CCR-1, CCR-3, CCR-5, and CXCR-4 on their cell surfaces as compared with those of unstimulated iDCs (Fig. 1A).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Effect of TGF-ß1 on the cell surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in iDCs. iDCs (5 x 106) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. The cells were stained with stated mAbs (thick lines) or isotype-matched mAb (thin lines). Cell surface expression was analyzed by FACS. The values shown in the flow cytometry profiles are MFI, and the value of the background FITC staining was less than 8. The results are representative of 10 experiments with similar results.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Dose-response relationship to TGF-ß1-induced surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in iDCs. iDCs (5 x 106) were unstimulated or stimulated with various concentrations of TGF-ß1 (1–100 ng/ml) for 3 days. The cells were stained with stated mAbs (thick lines) or isotype-matched mAb (thin lines). Cell surface expression was analyzed by FACS. The values shown in the flow cytometry profiles are MFI, and the value of the background FITC staining was less than 8. The results are representative of five experiments with similar results.

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Time course-response relationship to TGF-ß1-induced surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in iDCs. iDCs (5 x 106) were unstimulated or stimulated with TGF-ß1 (1–100 ng/ml) for the indicated days (1 to 5 days). The cells were stained with stated mAbs (thick lines) or isotype-matched mAb (thin lines). Cell surface expression was analyzed by FACS. The values shown in the flow cytometry profiles are MFI, and the value of the background FITC staining was less than 8. The results are representative of five experiments with similar results.

To examine the effect of TGF-ß1 on the chemotaxis of iDCs in response to CC and CXC chemokines, the migratory responses of iDCs, which were unstimulated or stimulated TGF-ß1, to RANTES, MIP-1, eotaxin, or SDF-1 using a Transwell cell culture chamber were studied. As shown in Fig. 4A, iDCs migrated in response to these chemokines, and TGF-ß enhanced these chemotactic migrations. The migratory response to these chemokines by these iDCs was chemotactic and not due to chemokinesis because migration was not observed in the absence of a chemokine gradient (Fig. 4A).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Effect of TGF-ß1 on chemotactic migratory ability of iDCs in response to CC and CXC chemokines. A, iDCs (2 x 107) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. The cells (106) were seeded on the filters precoated on the lower surface with 5 µg of gelatin. RANTES, MIP-1, eotaxin, and SDF-1 (1 ng/ml) used as chemoattractant were added to the upper chamber and/or lower chamber. B, iDCs (2 x 107) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. The cells (106) were seeded on the filters precoated on the lower surface with 5 µg of gelatin. RANTES or SDF-1 (0.1–10 ng/ml) used as chemoattractant were added to the lower chamber. C, iDCs (2 x 107) were unstimulated or stimulated with various concentrations of TGF-ß1 (1–100 ng/ml) for 3 days. RANTES or SDF-1 (1 ng/ml) used as a chemoattractant was added to the lower chamber. After a 2-h incubation, the cells that migrated to the lower surface were visually counted. The results are representative of three experiments with similar results.

To address the role of chemokine receptors in the TGF-ß1-induced enhancement of the chemotaxis of iDCs, effect of mAbs to chemokine receptors on the chemotactic migration of unstimulated and TGF-ß1-stimulated iDCs to RANTES or SDF-1 was examined.

Table I shows that both the anti-CCR-1 mAb and anti-CCR-3 mAb inhibited chemotactic migration of unstimulated and TGF-ß1-stimulated iDCs to RANTES, whereas anti-CCR-5 exhibited a slight suppression. The combinations of mAbs to CCR-1, CCR-3, and CCR-5 exhibited greater inhibition on the migratory capacity of these cells to RANTES than those of each mAb to CCRs. On the other hand, anti-CXCR-4, but not mAbs to CCRs, suppressed SDF-1-induced chemotactic migratory responses of these cells, whereas this mAb did not suppress RANTES-induced chemotaxis of these cells.

View this table: [in this window] [in a new window]   Table I. Effect of mAbs to chemokine receptors on TGF-ß1-induced enhancement of chemotaxis of iDCs to RANTES and SDF-11

Sozzani et al. (40) have previously reported that IL-10 enhanced the expression of CCR-1, CCR-2, and CCR-5 in human monocytes at the transcriptional level, and these phenonena were involved in the enhanced chemotactic migratory capacity for monocyte chemotactic protein-1 and MIP-1ß. We have also reported that stimulation of iDCs with IL-10 resulted in up-regulation of their chemotactic migratory ability to RANTES (38). On the other hand, TGF-ß1 reportedly enhanced the ability of macrophages to produce IL-10 in murine systems (28, 29). To examine whether IL-10 mediates TGF-ß1-induced expression of CC and CXCR, iDCs were unstimulated or stimulated with IL-10 or TGF-ß1 in the presence or absence of anti-IL-10 mAb (Table II). Stimulation of iDCs with IL-10 resulted in increased expressions of CCR-1, CCR-3, CCR-5, and CXCR-4, whereas anti-IL-10 mAb suppressed them. On the other hand, anti-IL-10 mAb did not affect TGF-ß1-induced expression of CC and CXCR.

TGF-ß1 up-regulates TNF--induced down-modulation of the expressions of CC and CXCR and the chemotaxis to their ligands

This study shows that TGF-ß1 exhibited the ability to up-regulate the cell surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in iDCs. To examine whether TGF-ß1 affects the transcription of these chemokine receptors, iDCs were unstimulated or stimulated with TGF-ß1, and the expressions of their mRNA were analyzed by RT-PCR. As shown in Fig. 5B, the transcripts of these chemokine receptors were enhanced by TGF-ß1 as compared with those of unstimulated iDCs, and these results were paralleled by their cell surface expressions (Fig. 5A).

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Effect of TGF-ß1 on the expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in mDCs. iDCs or mDCs (5 x 106) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. A, The cells were stained with stated mAbs (thick lines) or isotype-matched mAb (thin lines). Cell surface expression was analyzed by FACS. The values shown in the flow cytometry profiles are MFI, and the value of the background FITC staining was less than 8. B, RNA was extracted, and the expression of CCR-1, CCR-3, CCR-5, and CXCR-4 mRNA was determined by RT-PCR. PCR products for CCR-1 (440 bp), CCR-3 (444 bp), CCR-5 (1117 bp), CXCR-4 (810 bp), and ß-actin (645 bp) are shown. The results of RT-PCR for ß-actin demonstrate the loading of equal amounts of DNA on the gel. The results are representative of five experiments with similar results.

We further examined the effect of TGF-ß1 on chemotaxis of mDCs to RANTES and SDF-1 (Fig. 6). The chemotactic migratory abilities of mDCs to RANTES and SDF-1 were lower than those of iDCs. On the other hand, TGF-ß1 increased the ability of mDCs to migrate for RANTES and SDF-1, and these abilities were comparable with those of unstimulated iDCs.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Effect of TGF-ß1 on chemotactic migratory ability of mDCs in response to RANTES and SDF-1. iDCs (A) or mDCs (B) (2 x 107) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. The cells (106) were seeded on the filters precoated on the lower surface with 5 µg of gelatin. RANTES or SDF-1 (0.1–10 ng/ml) used as chemoattractant was added to the lower chamber. After a 2-h incubation, the cells that migrated to the lower surface were visually counted. The results are representative of three experiments with similar results.

We examined the influence of TGF-ß1 on phenotypic changes in iDCs and mDCs. As shown in Fig. 7A, TGF-ß1 had little or no effect on the cell surface expressions of CD40, CD86, and HLA-DR in iDCs. Furthermore, TNF--induced activation/maturation-associated enhancement of the cell surface expressions of CD40, CD83, CD86, and HLA-DR was not affected by TGF-ß1 in mDCs.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Effect of TGF-ß1 on phenotypic and functional changes of iDCs and mDCs. iDCs or mDCs (2 x 107) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. A, Cell surface phenotypic analysis of iDCs or mDCs. The cells were stained with the stated mAbs or or isotype-matched mAbs (thin lines), and cell surface expression was analyzed by FACS. The values shown in the flow cytometry profiles are MFI, and the value of the background FITC or PE staining was less than 8. B, Pinocytic activities of iDCs or mDCs. The cells were unstained (thin lines) or stained with FITC-DX or LY (thick lines). Pinocytosis was analyzed by FACS. The values shown in the flow cytometry profiles are MFI. C, Allogeneic TC stimulatory capacities of iDCs or mDCs. TCs were cultured with different numbers of iDCs or mDCs. The proliferative response was measured on day 5. Values are the mean ± SD obtained for triplicate cultures. The results are representative of three experiments with similar results.

To examine whether TGF-ß1 affects the allogeneic TC-stimulatory abilities of iDCs and mDCs, allogeneic TCs were cultured with various numbers of these cells, and the proliferative responses were measured on day 5. Fig. 7C shows that iDCs stimulated allogeneic TCs to proliferate in a dose-dependent fashion, and the allostimulatory capacity of mDCs was more potent than that of iDCs. On the other hand, TGF-ß1 had little or no effect on the abilities of iDC and mDCs to stimulate allogeneic TC proliferation as compared with unstimulated cells.

TGF-ß1 induces the expression of CCR-6 and chemotaxis for MIP-3 in iDCs and mDCs

Chan et al. (41) have recently reported that the transcript of CCR-6 was expressed in iDCs, whereas its product on the cell surface was not observed in these cells. On the other hand, Yang et al. (42) have recently shown that iDCs generated from monocytes cultured with GM-CSF, IL-4, and TGF-ß1 expressed both CCR-6 mRNA and protein on the cell surface, and these cells responded to MIP-3. We therefore tested the effect of TGF-ß1 on expression of CCR-6 in iDCs and mDCs (Fig. 8, A and B). The transcriptional expression of CCR-6 was observed in iDCs, whereas the cell surface expression of CCR-6 was not detected. Stimulation of iDCs with TGF-ß1 resulted in the increased expressions of the transcript and the product of CCR-6. On the other hand, the transcriptional and cell surface expression od CCR-6 were decreased in mDCs, while TGF-ß1 stimulated mDCs to express CCR-6.

View larger version (24K): [in this window] [in a new window]   FIGURE 8. Effect of TGF-ß1 on chemotactic migratory capacity of iDCs in response to MIP-3 via CCR-6. iDCs or mDCs (2 x 107) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. A, RNA was extracted, and the expression of CCR-6 mRNA was determined by RT-PCR. PCR products for CCR-6 (1021 bp) and ß-actin (645 bp) are shown. The results of RT-PCR for ß-actin demonstrate the loading of equal amounts of DNA on the gel. B, The cells were stained with stated mAbs (thick lines) or isotype-matched mAb (thin lines). Cell surface expression was analyzed by FACS. The values shown in the flow cytometry profiles are MFI, and the value of the background PE staining was less than 8. C, The cells (106) were seeded on the filters precoated on the lower surface with 5 µg of gelatin. MIP-3 (0.1–10 ng/ml) used as chemoattractant was added to the lower chamber. After a 2-h incubation, the cells that migrated to the lower surface were visually counted. The results are representative of three experiments with similar results.

TGF-ß1 suppresses TNF--induced expression of CCR-7 and chemotaxis for MIP-3ß in mDCs

Previous studies have shown that CCR-7 is exclusively expressed in mDCs at the transcriptional level, and these cells migrate to MIP-3ß via CCR-7 (26, 27). We therefore tested the effect of TGF-ß1 on expression of the transcript of CCR-7 in mDCs (Fig. 9A). The transcriptional expression of CCR-7 was observed in mDCs, but not in iDCs. On the other hand, TGF-ß1 suppressed this expression in mDCs. We also examined the effect of TGF-ß1 on the chemotactic migratory ability of mDCs in response to MIP-3ß. Fig. 9B shows that mDCs migrated in response to MIP-3ß, while treatment of mDCs with TGF-ß1 suppressed this migration.

View larger version (13K): [in this window] [in a new window]   FIGURE 9. Effect of TGF-ß1 on chemotactic migratory capacity of mDCs in response to MIP-3ß via CCR-7. iDCs or mDCs (2 x 107) were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for 3 days. A, RNA was extracted, and the expression of CCR-7 mRNA was determined by RT-PCR. PCR products for CCR-7 (1067 bp) and ß-actin (645 bp) are shown. The results of RT-PCR for ß-actin demonstrate the loading of equal amounts of DNA on the gel. B, The cells (106) were seeded on the filters precoated on the lower surface with 5 µg of gelatin. MIP-3ß (0.1–10 ng/ml) used as chemoattractant was added to the lower chamber. After a 2-h incubation, the cells that migrated to the lower surface were visually counted. The results are representative of three experiments with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The transcriptional and cell surface expressions of CCR-1, CCR-5, and CXCR-4 were previously shown in various types of human DCs (22, 23, 24, 26, 36, 41). However, there are conflicting reports about the expression of CCR-3 in these cells (22, 23, 24, 41). In accordance with previous reports (23, 24, 41), we showed that iDCs constitutively expressed the transcripts and products on the cell surface of CCR-3 as well as CCR-1, CCR-5, and CXCR-4 ( Figs. 1–3, 5, and 6). Furthermore, we observed that iDCs migrated to their respective ligands such as RANTES (for CCR-1, CCR-3, and CCR-5), MIP-1 (for CCR-1, CCR-2, and CCR-5), eotaxin (for CCR-3), and SDF-1 (for CXCR-4) (Fig. 4). These results suggest that these CC and CXCR may be functionally expressed on the cell surfaces of iDCs.

We showed that stimulation of iDCs with TGF-ß1 enhanced the cell surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 ( Figs. 1–3), and this stimulation up-regulated the chemotactic migratory ability of iDCs for RANTES, MIP-1, eotaxin, and SDF-1 (Fig. 4). Furthermore, mAbs to CCR-1, CCR-3, and CCR-5 significantly inhibited the chemotaxis of TGF-ß1-stimulated iDCs as well as unstimulated iDCs in response to RANTES (Table I). We also demonstrated that anti-CXCR-4 mAb suppressed the SDF-1-induced chemotaxis of these cells (Table I). These results suggest that the TGF-ß1-induced up-regulation of chemotaxis of iDCs to CC and CXC chemokines may be mediated by increased expression of their respective receptors.

It has been shown that TGF-ß1 stimulates macrophages to produce IL-10, and several biological effects of TGF-ß1 are mediated by IL-10 in murine systems (28, 29). We showed that mAb to IL-10 failed to abrogate TGF-ß1-induced enhancement of the cell surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in iDCs (Table I). These results suggest that TGF-ß1-induced enhancement of the expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 is not mediated by IL-10. Thus, TGF-ß1 may directly regulate the expressions of these chemokine receptors in iDCs.

Greaves et al. (25) have previously reported that the transcript of CCR-6 was expressed in CD34⁺ HPC-derived DCs, whereas this expression was undetectable in monocyte-derived DCs. In contrast, recent studies have shown that the transcriptional expression of CCR-6 was observed in monocyte-derived iDCs (41, 42). In accordance with previous reports (41, 42), the transcript of CCR-6 was constitutively expressed in monocyte-derived iDCs (Fig. 8A), although its cell surface expression was not detected in these cells (Fig. 8B). Furthermore, stimulation of iDCs with TGF-ß1 induced the enhancement of the expression of CCR-6 transcript and product (Fig. 8, A and B), and these cells responded to MIP-3 (Fig. 8C). These results suggest that TGF-ß1-induced posttranscriptional regulation of CCR-6 may lead to its functional cell surface expression iDCs.

The expressions of CCR-1, CCR-3, CCR-5, and CCR-6 were detectable on the cell surface (Figs. 5A and 8B), whereas little or no transcriptional expression of these CCRs was observed in mDCs (Figs. 5B and 8A). The reason that the levels of transcriptional expression of these CCRs were not completely consisted with the levels of their cell surface expressions remains unclear, but one possible explanation is that the kinetics of decreased cell surface expressions of these CCRs may not be different from those of their transcriptional expressions. Alternatively, other molecular mechanism(s) as well as the transcriptional regulation may be involved in the reduced cell surface expressions of these CCRs (43). Further study will be needed to examine this possibility.

We showed that stimulation of mDCs with TGF-ß1 resulted in increased expression of the transcripts and products on the cell surfaces of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4 compared with unstimulated mDCs (Fig. 6). Furthermore, the chemotactic migratory abilities of TGF-ß1-stimulated mDCs to RANTES, MIP-3, and SDF-1 were more potent than those of mDCs (Fig. 7). These findings suggest that TGF-ß1 may also enhance the chemotaxis to RANTES, MIP-3, and SDF-1 via up-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4 in mDC. On the other hand, the transcriptional expression of CCR-7 was suppressed by TGF-ß1 in mDCs, and this expression was associated with their ability to migrate to MIP-3ß (Fig. 9). These findings suggest that TGF-ß1 may impair the chemotactic migratory ability of mDCs for MIP-3ß via down-regulated expression of CCR-7. In contrast to TGF-ß1-mediated regulation of CC and CXCR in human monocyte-derived DCs in our experiments, Ogata et al. (35) have recently reported that the expression of CCR-7 transcript was inhibited in TGF-ß1-treated murine BM-derived DCs when GM-CSF plus TNF--cultured HPCs were treated with TGF-ß1 for the first 6–7 days, but not at days 13–14, whereas the transcriptional expression of CCR-1 was not affected by TGF-ß1 in these cells. This discrepancy might be due to species differences.

It has been shown that TGF-ß1 negatively modulates several functions of various cell types (28, 29). Lee et al. (30) have previously reported that TGF-ß1-transduced murine BM-derived DCs exhibited lower allogeneic TC stimulatory ability than control vector-transduced cells, although these types of cells expressed similar expression levels of MHC products and adhesion/costimulatory molecules. We showed that TGF-ß1 did not affect phenotypes and functions of iDCs and mDCs with respect to the abilities for the endocytosis and the allogeneic TC stimulation (Fig. 8). Furthermore, we observed that TC proliferation was suppressed when allogeneic TCs were cultured with iDCs or mDCs in the presence of TGF-ß1 (data not shown), suggesting that TGF-ß1 may not directly regulate Ag-processing/presenting functions of monocyte-derived DCs, although TGF-ß1 may suppress DC-mediated activation of TCs. These phenomena led us to hypothesize that TGF-ß1 may specifically regulate the chemotactic migratory responses of iDCs and mDCs by regulating the expression of chemokine receptors.

The role of TGF-ß1 in the trafficking of human DCs in vivo remains unclear.

Recently, the responsiveness of iDCs to proinflammatory-related chemokines, including RANTES via respective CCRs, has been shown to possibly involve the trafficking of these cells from peripheral tissue into inflammatory sites (26, 27). Subsequently, iDCs may be converted into mDCs by extracellular stimuli, including IL-1, TNF-, LPS, and CD40 ligation in the inflammation sites, and these cells may lose their responsiveness to these chemokines (26, 27). In turn, the expression of CCR-7 might be induced in mDCs, and these cells may acquire responsiveness to MIP-3ß, which is specifically secreted by TC-rich areas in lymph nodes (26, 27). These properties may account for accumulation of Ag-loaded mDCs in TC-dependent areas of secondary lymphoid tissue (26, 27). We showed that TGF-ß1 enhanced the RANTES-, MIP-3-, and SDF-1-mediated chemotaxis via up-regulation of respective receptors in iDCs and mDCs (Figs. 6 and 7), while the chemotactic migratory property of mDCs for MIP-3ß was inhibited by TGF-ß1 via down-regulation of CCR-7 expression (Fig. 9). It has been shown that TGF-ß1 is produced by various cell types, including TCs, monocytes/macrophages, and granulocytes, and the production of TGF-ß1 was observed in inflammation sites (44, 45, 46). Collectively, our findings imply that TGF-ß1 is potentially involved in the trafficking properties of iDCs as well as mDCs via regulating chemotaxis to CC and CXC chemokines.

In summary, our findings provide a novel mechanism for the trafficking of human DCs. The chemokines and their respective receptors play important roles in inflammatory and allergic diseases, as well as immune responses (18). In addition, DCs are extensively involved in autoimmune diseases, graft rejection, and viral infections (1, 2, 3, 4). On the other hand, previous studies have shown the induction of MHC products-mediated immune responses after stimulation with tumor Ag-pulsed DCs in clinical trials (47, 48). Recently, the effective targeting of tumor Ag-loading DCs into tumor tissues or lymph nodes around these sites has been shown to be useful for tumor immunotherapy. Defining the precise mechanisms of the chemotactic migratory ability of DCs may provide further insights into the role of these cells in immune-related diseases, and facilitate the use of DCs in vaccinations for cancer treatment.

	Acknowledgments

 
We thank Dr. H. Tsuda for helpful suggestions in RT-PCR, and M. Midorikawa and R. Adachi for secretarial assistance.

	Footnotes

 
¹ K.S. and H.K. contributed equally to this study.

² Address correspondence and reprint requests to Dr. Tsuneo A. Takahashi, Department of Cell Processing, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address:

³ Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; CXCR, CXC chemokine receptor; FITC-DX, FITC labeled-dextran; HPC, hemopoietic progenitor cell; iDC, immature DC; LY, lucifer yellow; mDC, mature DC; MFI, mean fluorescence intensity; MIP, macrophage-inflammatory protein; SDF, stromal cell-derived factor; TC, T cell.

Received for publication August 23, 1999. Accepted for publication December 13, 1999.

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