Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0305155 on October 21, 2005

Published online before print October 21, 2005
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(Journal of Leukocyte Biology. 2006;79:59-70.)
© 2006 by Society for Leukocyte Biology

Cytokine-regulated expression and inhibitory function of Fc{gamma}RIIB1 and -B2 receptors in human dendritic cells

Nathalie Guriec1, Catherine Daniel2, Karine Le Ster, Elisabeth Hardy and Christian Berthou

Brest Medical School, Cellular Therapy Laboratory, France

1 Correspondence: Brest Medical School, Cellular Therapy Laboratory, 22, avenue Camille Desmoulins, 29200 Brest, France. E-mail: nguriec{at}voila.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) capture immune complexes (IC) via Fc receptors for immunoglobulin G Fc{gamma}RII and elicit antigen presentation and protective antitumoral immune response in mice. Two protocols are commonly used to differentiate human monocyte-derived DC in vitro. They associate granulocyte macrophage-colony stimulating factor (CM-CSF) with interleukin (IL)-4 or IL-13. In this study, we first assessed the ability of the two types of DC to initiate an immune response against an IC-linked antigen. We evidenced that IL-4 and IL-13 DC display comparable lymphocyte stimulatory capacity and similar lifetimes. We next characterized Fc{gamma}RIIs expressed by pure populations of circulating myeloid DC (BDCA1+DC), IL-4, and IL-13 DC. We highlighted the expression of Fc{gamma}RIIA, -B1, and -B2 by pure populations of BDCA1 myeloid DCs and IL-4 and IL-13 DC. Moreover, IL-4 and IL-13 DC displayed greater Fc{gamma}RIIB expression than monocytes but a comparable Fc{gamma}RIIA. We next investigated the Fc{gamma}RIIB mechanism of action. We evidenced that deleting Fc{gamma}RIIB increased the ability of IC-pulsed DC to stimulate autologous lymphocytes. Fc{gamma}RIIB acted by lowering IC uptake, surface expression of costimulation molecules, and cytokine release. Finally, the balance between activating Fc{gamma}RIIA/inhibitory Fc{gamma}RIIB (B1+B2) could be modulated in vitro by inflammation mediators. By lowering Fc{gamma}RIIB expression without significantly affecting Fc{gamma}RIIA, prostaglandin E2 (PGE-2) appeared to be a major regulator of this balance. IL-1ß and tumor necrosis factor {alpha} were also found to potentiate PGE-2 action. Altogether, our results evidence an inhibitory role for Fc{gamma}RIIB in human DC and provide an easy way to possibly improve in vitro the induction of immune response against IC-linked antigen.

Key Words: phagocytes • immunotherapy • inflammation • immunoglobulin • antigen presentation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are highly differentiated antigen-presenting cells. They play a key role in the initiation and development of immune response to pathogens and tumors [1 , 2 ]. Heat shock proteins, pathogen recognition by Toll-like receptors, and immune complexes (IC) binding to Fc receptors (FcR) are some of the stimuli that can lead to immune response to antigens, including tumor antigens [1 2 3 4 ].

A single class of FcR to immunoglobulin G (IgG); Fc{gamma}RII (CD32) is expressed by monocyte-derived DCs [4 , 5 ]. IC capture by Fc{gamma}RII allows DC maturation and efficient presentation to naive CD8+ T cells of an exogenously derived antigen (e.g., IC-linked antigen [3 4 5 6 7 ]). Furthermore, apoptotic tumor cells opsonized with antibodies have been shown to elicit DC maturation, generation of tumor-specific CD8+ T cells, and protective tumor immunity in vitro and in vivo [8 9 10 ]. DC-based immunotherapies using IC-pulsed DC have, therefore, emerged as an attractive mean to stimulate protective antitumor immunity.

However, the amplitude of immune response is believed to depend on the ratio between activating and inhibitory FcR. In humans, Fc{gamma}RIIs exist as two major isoforms, Fc{gamma}RIIA and -B, which carry out divergent functions. Fc{gamma}RIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic tail, which mediates positive signaling. Activation of Fc{gamma}RIIA results in IC internalization as well as in initiation of immune response. By contrast, an immunoreceptor tyrosine-based inhibitory motif (ITIM) has been found in the Fc{gamma}RIIB cytoplasmic region. Engagement of Fc{gamma}RIIs by IC then leads to cellular activation or inhibition signals, depending on the engaged receptor. Two human Fc{gamma}RIIB inhibitory receptors are encoded by the Fc{gamma}RIIB gene in humans through an alternative splicing mechanism. Fc{gamma}RIIB1 and -B2 only differ by the lack of 19 amino acids in the intracellular domain of Fc{gamma}RIIB2 [11 ]. Both are single-chain receptors with an ITIM domain and negatively regulate B cell receptor (BCR)-, TCR-, and Fc{gamma}R-dependent activation [12 13 14 ]. Recent experiments have demonstrated a better efficacy of tumor eradication with humanized antibodies against tumors in Fc{gamma}RIIB knockout mice than in their wild-type counterparts [15 ]. Fc{gamma}RIIB engagement on human monocytes is associated with the down-regulation of phagocytosis and decreases antigen-presenting capacities [16 , 17 ]. Fc{gamma}RIIB also prevents spontaneous human DC maturation [10 ]. However, no data are available about the expression of Fc{gamma}RIIB isoforms on in vitro-differentiated DC and their regulation. Elucidation of Fc{gamma}RII function and regulation in human DC thus appeared to us as a prerequisite to the development of tumor-specific vaccines. We studied the ability of monocyte-derived DC to induce lymphocyte proliferation depending on the DC differentiation protocol and examined Fc{gamma}RII expression, function, and regulation.

We found that the replacement of interleukin (IL)-13 by IL-4 in DC differentiation protocol allowed the generation of DC with similar lifetimes and comparable abilities to trigger an immune response when pulsed with IC. We observed distinct expression patterns for Fc{gamma}RIIs in blood myeloid DC, monocyte-derived DC. Yet, Fc{gamma}RIIA, -B1, and -B2 receptors were always expressed. Our study also provides clues of the mechanism of action of Fc{gamma}RIIB as well as regulations of this receptor by pro- and anti-inflammatory mediators.

Altogether, our data constitute the grounding for the development of new strategies based on the use of IC in immunotherapy protocols and for testing novel strategies aimed at evaluating the effective link between the humoral immunity and the cellular one.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines, DC preparation and maturation, immunophenotyping, and sorting
The B cell line Raji, monocytic cell line U937, erythroid cell line K562, and fibroblastic cell line MRC5 were grown in RPMI 1640 with L-glutamine and 10% fetal calf serum (FCS). Peripheral blood was obtained from the local blood bank after informed consent of donors. Mononuclear cells were purified by Ficoll separation and then washed twice in phosphate-buffered saline (PBS) plus 1 mM EDTA and 0.5% bovine serum albumin (BSA). Blood DC antigen (BDCA)-1+ myeloid DC were purified from mononuclear cells by using a BDCA-1 isolation kit and following the manufacturer’s instructions (Miltenyi Biotec, Germany). Purity after sorting was checked by flow cytometry analysis. After resuspension in RPMI plus 10% FCS, the mononuclear cells were allowed to adhere on plastic for 2 h at 37°C for monocyte purification. Plastic nonadherent cells were further used as a source of lymphocytes for mixed leukocyte reactions (MLR). Plastic adherent cells (>95% CD14-positive cells, n=8, data not shown) were washed twice with RPMI and differentiated in medium further denoted as complete medium and containing RPMI with L-glutamine, 1% IgG-depleted autologous plasma, 800 U/ml human recombinant granulocyte macrophage-colony stimulating factor (hrGM-CSF), 1000 U/ml hrIL-4, or 10 ng/ml hrIL-13 (all from R&D Systems, UK) to yield immature DC (imDC) at day 6 as described previously [5 , 18 ]. IgG depletion was performed on a protein A sepharose column (Interchim, France) and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Maturation was induced by an additional 3-day incubation of day 6 nonadherent cells in complete medium with 100 ng/ml lipopolysaccharide (LPS; Sigma Chemical Co., St. Louis, MO) or soluble tetanus toxin or IC. Immature DC (imDC) and were immunophenotyped by flow cytometry at days 6 and 9, respectively. Expression of mannose receptor was also checked at day 2. CD1a+ imDC and CD83+ mDC were purified at days 6 and 9, respectively. CD1a+ imDC were purified by positive selection using CD1a microbeads (Miltenyi Biotec) starting from day 6 nonadherent cells. CD83 microbeads (Miltenyi Biotec) were used to perform a positive selection of CD83+ mDC at day 9 starting from nonadherent and adherent cells. In both cases, the selection was carried out in compliance with the manufacturer’s instructions. Then, the sorted DC were immunophenotyped by flow cytometry. Fluorescence-conjugated anti-CD11c antibody was from Dako (France); anti-CD83 and -CD86, from Beckman Coulter (France); anti-CD80, -human leukocyte antigen (HLA) class II, and -CD40, from Becton Dickinson (France); and anti-CD54 and -HLA class I, from Immunotech (France). DC culture supernatants were kept frozen at –80°C.

Apoptosis quantitation by flow cytometry
Apoptosis was evidenced by flow cytometry analysis of annexin V and propidium iodide staining. The cells (5x105) were stained according to the manufacturer’s instructions (Beckman Coulter).

Immunoprecipitation, blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Pure DC populations were suspended in lysis buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1 mM Na3VO4) for 10 min on ice and washed, and postnuclear extracts were solubilized further in immunoprecipitation buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2 mM Na3VO4, 100 mM NaF, 4 mM NaH2PO4, 1 µM phenylmethylsulfonyl fluoride, 1% Triton, 0.5% Nonidet P-40, 5 mg/ml leupeptin, 5 mg/ml aprotinin). The purity of membrane protein purification was controlled by caspase-3 Western blotting, as caspase-3 is known to be mostly cytosolic (Upstate Biotechnology, Lake Placid, NY, n=6, data not shown). Equal amounts of membrane proteins were immunoprecipitated by using anti-Fc{gamma}RII KB61 and AT10 antibodies (generous gift from Dr. Karen Pulford, John Radcliffe Hospital, Oxford, UK, and Abcyss SA, France, respectively), separated on 10% polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Hybond P, Amersham Biosciences, France), probed with anti-Fc{gamma}RII 2E1 antibody (Beckman Coulter), and developed by enhanced chemiluminescence [17 ]; its signal was quantified with densitometry software (Bioprofil, Vilbert Lourmat, France). DC conditioned media were also tenfold concentrated using centricon (molecular weight cut-off=3000, Amicon, France) at day 6 for imDC and day 9 for mDC. Positive controls for Western blotting and immunoprecipitation experiments were the Raji cell line for Fc{gamma}RIIB expression, U937 cell line for Fc{gamma}RIIA, and K562 for soluble Fc{gamma}RIIA (sFc{gamma}RIIA) [19 , 20 ]. MRC5 cell line was used as a negative control.

Total RNA extraction from sorted DC or cell lines and RT were performed as described previously. Primers for Fc{gamma}RIIA, Fc{gamma}RIIB, and ß-actin and PCR conditions have been described previously [17 , 21 ]. Positive and negative controls were the same as for immunoprecipitation experiments. PCR products were run on a 10% polyacrylamide gel. Equal efficiency of RT was checked by analysis of ß-actin expression, and results of Fc{gamma}RIIA and -B expression were normalized to ß-actin.

Transfection
A Fc{gamma}RIIB antisense and a sense construct were generated by RT-PCR using cDNA prepared from the Raji cell line as template. A 200-base pair fragment encompassing the second extracytoplasmic domain up to the first intracytoplasmic one and common to Fc{gamma}RIIB1 and -B2 was obtained by enzymatic digestion of the PCR product (BamHI+PstI). Then, the fragment was inserted into the PMG expression vector (Tebu, France). Constructs were under control of a cytomegalovirus promoter (encephalomyocarditis virus-internal ribosomal entry site). Plasmid DNA was prepared by alkaline lysis followed with Triton-X114 purification to remove LPS, and then endotoxins levels were checked as described previously [22 ]. Plasmid DNA was dissolved in HEPES saline buffer (150 mM NaCl, 20 mM HEPES, pH 7.4). As mannose receptor is expressed by human monocytes after a 48-h incubation with GM-CSF + IL-4/IL-13 (data not shown [23 ]), sense and antisense constructs were transfected at day 2 using mannose polyethylenimine conjugates (manPEI). Mannose was linked to commercially available PEI (Exgen 500, Euromedex, France) via a phenylisothiocyanate bridge made by using mannopyranosyphenyl isothiocyanate as a coupling reagent as described [23 ]. Coupling efficiency and purification were controlled as reported [23 ]. manPEI was suspended in HEPES saline buffer, and a 1/1 ratio between plasmid DNA and manPEI was used for transfection [23 ]. Six-well plates were gently spinned before addition of DNA/manPEI complexes in complete medium. Transfection medium was replaced with complete medium after 4 h of incubation. Hygromycin selection of transfectants was started 24 h after transfection, and a Ficoll separation was performed at day 4 to remove dead cells, healthy were then incubated in complete medium with hygromycin for 2 additional days. At day 6, nonhealthy cells were removed by Ficoll separation. Viability was then checked by trypan blue exclusion and cytometry analysis of Annexin V and propidium iodide. As expected [23 ], antibiotic selection gave rise to 15–20% resistant cells with more than 95% healthy cells. mRNA levels of Fc{gamma}RIIA, -B1, and -B2 were determined by specific RT-PCR. Fc{gamma}RIIA, -B1, and -B2 protein expression levels by nonadherent transfectants at day 6 were checked by immunoprecipitation, and maturation status was controlled by flow cytometry analysis of CD40, -80, -83, and -86 staining.

MLR assays
Autologous MLR were performed to evaluate antigen-presenting capacity of human DC for a soluble or an IC-linked antigen [24 ]. U-bottom 96-well plates were coated with tetanus toxin IC (tetanus toxin, Roche Diagnostics, France; human polyclonal IgG against tetanus toxin, EFS, France), obtained as described previously (ratio 1/20, 10–8 M tetanus toxin [4 , 22 ]). MLR assays were performed in triplicate by cocultivation of day 6 DC at various dilutions with lymphocytes (range 1/10–1/100) in X-vivo 15 medium (Bio-Whittakker, France). Proliferation was assessed by using tritiated thymidine and a ß counter (Tri-Carb, Packard, Downers Grove, IL).

Quantitation of IC uptake
Nonadherent, day 6 transfectants were washed twice in RPMI and incubated for 24 h in complete medium in plates coated overnight with tetanus toxin IC. Cells were then fixed with PBS with 3% paraformaldehyde and washed twice in PBS with 1% BSA and 5 mM EDTA to separate surface-bound IgG [5 ]. They were further permeabilized with PBS containing 1% BSA and 0.3% saponin before staining with fluorescein isothiocyanate-conjugated goat anti-human antibody (Beckman Coulter [9 ]). IC uptake from independent experiments was analyzed by flow cytometry on CD11c-positive cells [9 ].

IL-10, p70 IL-12, and interferon-{gamma} (IFN-{gamma}) release by DC after Fc{gamma}R-mediated activation
Cytokine concentrations in frozen DC culture supernatants from various experiments were measured in duplicate by cytokine-specific sandwich enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).

Modulation of Fc{gamma}RII expression assays
Cytokines were purchased by R&D Systems and prostaglandin E2 (PGE-2) and polyIC (synthetic analog of double-strand RNA), by Sigma Chemical Co. Oligodeoxynucleotide (ODN) 2006 and ODN 2216 were synthesized and purified by Proligos (France). As described previously, the concentrations used ranged from 10 to 500 U/ml for IFN-{gamma} and tumor necrosis factor {alpha} (TNF-{alpha}), 0.2 to 20 ng/ml IL-1ß, 1 to 100 ng/ml vascular endothelial growth factor (vEGF) and transforming growth factor-ß1 (TGF-ß1), 20 to 200 ng/ml IL-10, 10 to 500 ng/ml IL-6, 0.1 to 10 ng/ml PGE-2, 5 to 100 µg/ml polyIC, and 0.5 to 10 µg/ml ODN [25 26 27 28 29 30 31 32 33 ]. To avoid any cytokine interference, DC were washed three times with RPMI medium before incubation for 24 h at 1 x 106 cells/ml in a medium containing RPMI, L-glutamine, 1% IgG-depleted autologous plasma, and mediators but neither GM-CSF nor IL-4/13 (such a deprivation affects neither Fc{gamma}RII expression levels nor patterns, n=6, data not shown). Finally, the cells were sorted on CD1a or CD83 according to their differentiation stage, and their purity was checked by flow cytometry.

Statistical analysis
Data are expressed as mean ± SEM. Nonparametric statistical methods (Wilcoxon test) and two-tailed P values were used for comparison.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DC characterization and functionality
In immunotherapy protocols, an efficient differentiation of monocytes toward DC is essential. Previous studies demonstrated that IL-13 can be substituted successfully for IL-4 to yield DC [18 ]. The ability of IL-4 and IL-13 DC to stimulate T cell was similar as well as cytokines released by T cells [18 , 34 , 35 ]. Hereafter, the DC differentiated with GM-CSF + IL-4, and those differentiated with GM-CSF + IL-13 will be referred to as IL-4 DC and IL-13 DC, respectively. At day 6 of differentiation, with IL-4 or IL-13, we reproducibly obtained up to 80% CD1a-positive, nonadherent cells corresponding to imDC; at day 9, we obtained up to 95% of CD83-positive cells corresponding to mDC (Fig. 1 ). imDC and mDC were also analyzed for Fc{gamma}R expression other than Fc{gamma}RII (n=8, Fig. 1 ). Fc{gamma}RI and Fc{gamma}RIII were not expressed by IL-4 imDC and mDC. IL-13 allowed DC to differentiate with an identical Fc{gamma}R expression profile. Moreover, Fc{gamma}RII expression was lower on mDC than on imDC. One should note that this difference was even more pronounced when maturation was induced by IC instead of LPS (Fig. 1) .



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  		Figure 1. DC characterization and lymphocyte stimulatory capacity. Phenotypic follow-up of DC differentiation (A) with GM-CSF and IL-13 at day 6 (imDC) and day 9 (mDC). DC were pulsed with LPS to allow maturation (n=8). Similar staining was observed for IL-4 DC (n=8, data not shown). (B) Fc{gamma}R membrane expression by DC. First two plots: Fc{gamma}RIII and Fc{gamma}RI expression on imIL-13 DC at day 6 (n=8). Similar results were observed for IL-4 imDC and mDC (n=8, data not shown). Last two plots: comparative analysis of Fc{gamma}RII staining with 2E1 antibody for DC from the same donor according to the differentiation stage and the maturation protocol (n=8). (C) Lymphocyte proliferation as measured in autologous MLR when using IL-4 and IL-13 DC from the same donor pulsed with a soluble antigen (sol Ag) or with IC. Data presented are the means ± SEM of eight independent experiments. MLR were also performed in parallel without antigen (w/o Ag) or with antitetanus toxin IgG (IgG) as negative controls. dpm, Disintegrations per minute.

 
We next assessed the ability of IL-4 and IL-13 DC to initiate autologous lymphocyte proliferation. Eight MLR were performed using the two differentiation protocols in parallel for each donor. As shown in Figure 1C , the lymphocyte proliferative response was similar for both types of DC when soluble antigen was used; it was comparable after pulsing with IC (P=0.05, Fig. 1C ).

We also investigated DC lifetime by measuring the percentage of apoptotic DC at days 10, 12, and 14. IL-4 and IL-13 DC from the same donor were assessed by flow cytometry analysis of Annexin V-positive cells (n=8). No change in lifetime was noticed further to the use of IL-13 instead of IL-4 during the differentiation process (Fig. 2 ). Additionally, the use of LPS or IC as maturation agent resulted in similar lifetimes (Fig. 2) .



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  		Figure 2. Comparative analysis of IL-4 and IL-13 DC apoptosis. day 6 nonadherent cells, differentiated using GM-CSF with IL-4 or IL-13, were pulsed with IC or LPS. Values represent the means ± SEM of eight independent experiments.

 
Monocyte-derived DC and BDCA-1+ DC express Fc{gamma}RIIA, -B1, and -B2 receptors but not sFc{gamma}RIIA
To further characterize Fc{gamma}RII isoforms expressed by human DC, BDCA-1+ myeloid blood DC, CD1a+ imDC, and CD83+ mDC were sorted out and purity-checked by flow cytometry. As shown in Figure 3 after sorting, purity reached 98% CD1a+ cells for imDC and CD83+ cells for mDC. Immunophenotyping for costimulation and presentation molecules also confirmed that these cells were imDC and mDC, respectively (Fig. 3A and 3B) .



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  		Figure 3. Immunophenotype of pure DC populations. Cells were sorted using a positive selection for CD1a at day 6 to purify imDC (A) and a positive selection for CD83 at day 9 to sort mDC (B). Flow cytometry analysis was performed for eight donors. Fc{gamma}RII expression was analyzed using the 2E1 antibody. Results presented here are for IL-13 DC. Similar results were observed for IL-4 DC (n=8, data not shown).

 
Immunoprecipitation and RT-PCR analysis with primer pairs amplifying Fc{gamma}RIIA, -B1, and -B2 were then performed on pure populations (n=8, Fig. 4 ). These experiments showed the expression of Fc{gamma}RIIA, -B1, and -B2 receptors by human monocytes, BDCA-1+ blood DC, and DC. Moreover, DC differentiation with IL-4 or IL-13 resulted in a significant increase of Fc{gamma}RIIB1 and -B2 expression (P≤0.05), whereas minor changes were observed for Fc{gamma}RIIA. Consistently, with flow cytometry analysis (Fig. 1) , we found that Fc{gamma}RIIB expression on IL-13 DC was slightly higher than on IL-4 (P=0.05), whereas Fc{gamma}RIIA expression was similar (Fig. 4) .



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  		Figure 4. Fc{gamma}RIIs expressed by human myeloid DC. RT-PCR and immunoprecipitation (IP) experiments (A) were performed on pure DC populations from the same donor (n=8). KB61 and AT10 antibodies were used to immunoprecipitate Fc{gamma}RIIs from postnuclear extracts and 2E1 antibody for Western blotting revelation. Fc{gamma}RIIA, -B1, and -B2 expressions are the results of the same exposure and were measured by densitometry (graphs). (B) Conditioned media of imDC at day 6 and mDC at day 9 were examined for sFc{gamma}RIIA expression (n=12). Immunoprecipitations were performed for analysis of membrane expression of Fc{gamma}RIIs. Monocyte and BDCA-1+ were incubated for 24 h in RPMI with 1% IgG-depleted, autologous plasma after purification, and the conditioned media were collected.

 
Murine CD1a+ Langerhans cells are known to produce sFc{gamma}RIIA, resulting from an alternative splicing of the Fc{gamma}RIIA gene. sFc{gamma}RIIA acts as an inhibitory receptor by competing with membrane Fc{gamma}RIIs for IC binding and severely reduces Langerhans cell antigen-presenting capacity [19 , 20 ]. We carried out immunoprecipitation experiments with DC conditioned media (normal and tenfold-concentrated) and cellular extracts from BDCA-1+ blood DC, sorted imDC, and mDC (n=12). The human K562 cell line and its conditioned medium were used as positive controls [18 , 19 ]. None of these experiments allowed us to detect the sFc{gamma}RIIA protein (Fig. 4) .

Fc{gamma}RIIB deletion increases IC-pulsed–DC lymphocyte-stimulating capacity
To further investigate the role of Fc{gamma}RIIB in DC presentation function, we applied a knock-down strategy. Transfection of a Fc{gamma}RIIB antisense along with hygromycin selection allowed us to yield Fc{gamma}RIIB-deleted DC (DEL). Undeleted DC [wild-type (WT)] were obtained by using a sense construct. Fifteen to 20 percent of the cells initially plated were transfected efficiently and survived antibiotic selection as described previously [23 ]. Flow cytometry analysis of costimulation molecules after transfection indicated no transfection-induced maturation (n=6, Fig. 5A ). Immunoprecipitation experiments confirmed that the antisense construct allowed a significant decrease in expression of both Fc{gamma}RIIBs, with no effect on Fc{gamma}RIIA as compared with the sense construct (n=6, Fig. 5B ). Fc{gamma}RIIB expression was reduced by ~75% for IL-13 DC and 85% for IL-4 (Fig. 5B) . Autologous, primary MLR showed that lymphocyte proliferation was enhanced whenever activating Fc{gamma}RIIA had been selectively engaged through the use of Fc{gamma}RIIB-deleted DC (n=6, Fig. 5C ).



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  		Figure 5. DC deficient in Fc{gamma}RIIB expression are better lymphocyte stimulators when IC-pulsed. IL-4 or IL-13 differentiation was performed in parallel for each donor (n=6). Cell surface expression of costimulation molecules (A) revealed lack of maturation after transfection. Data presented here are for IL-4 DC, but similar results were obtained for IL-13 DC (n=6, data not shown). Immunoprecipitations (B) evidenced efficiency and specificity of the Fc{gamma}RIIB antisense construct (n=6). Densitometric analyses were performed for all experiments and are summarized in the graphic portion of B. Transfected IL-4 and IL-13 DC from the same donor were pulsed with IC in autologous MLR (C). Means ± SEM of six independent experiments are shown. Incorporated counts are plotted as a function of the stimulatory cell number, with IC amounts being constant. As controls, MLR were also performed with antitetanus toxin IgG, no DC (data not shown) or with soluble antigen (sol AG).

 
IC uptake, surface expression of maturation markers, cytokine release
To further characterize the Fc{gamma}RIIB mechanism of action, we next assessed IC uptake by pure populations of WT and DEL imDC. IL-4 imDC and IL-13 imDC were pulsed with IC, and uptake was evidenced at t = 24 h by intracellular staining for human IgG as described previously [5 , 9 ]. Fc{gamma}RIIB DEL imDC demonstrated a greater ability to internalize IC than WT DC (n=6, Fig. 6A ). DEL IL-4 and DEL IL-13 imDC also displayed similar phagocytic capacities (Fig. 6A) .



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  		Figure 6. Fc{gamma}RIIB mechanism of action. Pure populations of DEL and WT IL-4 and IL-13 imDC from the same donor were obtained at day 6 and used for all experiments (n=6). IC internalization (A) was evidenced by intracellular staining for human IgG after 24 h incubation. The first four plots show controls without saponin with IC (open) and without saponin without IC (solid). The next two plots show intracellular staining for WT (shaded), DEL (solid line) DC, and control without IC (solid peak). Means ± SEM of fluorescence intensity (MFI) and percentages of positive cells (means±SEM) are indicated. One representative experiment of six is shown. (B) Surface expression of costimulation and presentation molecules on transfected IL-13 mDC. Incubation with IC was performed with pure populations of DC. Isotypic control fluorescence is shown as a solid peak; those of WT DC as a thin line; and DEL DC as a bold line. Similar results were observed for IL-4 DC. One representative experiment of six is shown. The graph summarizes variations of means of fluorescence intensity at day 9. Means of fluorescence intensity for WT IL-4 DC were taken as 1 for each staining. Data are shown as means ± SEM. Statistically significant differences between staining on WT and DEL DC are marked (*, P≤0.01). IL-10, p70 IL-12, and IFN-{gamma} release (C) measured by ELISA in DC conditioned media at day 9. LPS was used to generate positive controls. Values represent means ± SEM of six experiments. No Ag, No antigen.

 
Moreover, DC maturation results in an enhanced surface expression of CD80, CD83, CD86, CD40, CD54, and HLA class I and II molecules, which are believed to be important for antigen presentation and lymphocyte stimulation [1 2 3 4 ]. Pure populations of transfected cells were matured with IC. DEL DC displayed a marked increase in staining for costimulatory molecules (CD80, -83, -86, -40, and -54), whereas no change was detected for presentation molecules (HLA class I and II) nor for CD11c (n=6, Fig. 6B ). These effects were observed for IL-4 and IL-13 DC. Means of fluorescence intensities were also alike for both types of DEL DC (Fig. 6B) . Fc{gamma}RIIB deletion had no significant impact on the expression levels of presentation and costimulation molecules by LPS mDC (n=6, data not shown).

According to literature data, DC maturation leads to the secretion of cytokines such as IL-10, p70 IL-12, and IFN-{gamma}, which are known to play a key role in the regulation of immune response [1 2 3 4 ]. We therefore used LPS or IC to pulse pure populations of WT/DEL IL-4 and WT/DEL IL-13 DC from the same donor (n=6). Measurement of cytokine amounts was performed at day 9 by ELISA. In the presence of LPS, WT/DEL IL-4 and IL-13 DC behaved similarly and displayed similar cytokine secretion levels (Fig. 6C) . Conversely, this was not observed when pulsing with IC. No difference between WT and DEL DC was found for IL-10 secretion (Fig. 6C) . However, Fc{gamma}RIIB deletion resulted in a greater secretion of p70 IL-12 and IFN-{gamma} for both types of DC (n=6, Fig. 6C ).

Altogether, our results indicate that Fc{gamma}RIIB prevents DC maturation and lymphocyte proliferation by hampering IC uptake and limiting the surface expression of costimulation molecules and release of p70 IL-12 and IFN-{gamma}.

Modulation of Fc{gamma}RII expression and lymphocyte-proliferative response
The combined action of pro- and anti-inflammatory compounds is thought to maintain immune homeostasis. Among proinflammation mediators, PGE-2, IL-1ß, IFN-{gamma}, TNF-{alpha}, ODN, and polyIC are known to increase DC activating receptor expression or to improve full DC maturation [25 26 27 , 32 , 33 ]. Anti-inflammatory context or tumor development is often associated with increased levels of vEGF, TGF-ß, IL-6, and IL-10, which tend to counteract the effects of proinflammatory molecules [28 29 30 31 ]. We investigated the effects of these mediators, alone or in combination, on a Fc{gamma}RII expression pattern on DC.

First, we observed that none of the tested compounds induced sFc{gamma}RIIA release by imDC or mDC (IL-4 or IL-13 DC, n=8, data not shown). Fc{gamma}RIIA, -B1, and -B2 expression on IL-4 or IL-13 mDC also remained unaffected (n=8, data not shown). Second, we failed to detect any secretion of sFc{gamma}RIIA by imDC or mDC or variation of Fc{gamma}RIIA, -B1, and -B2 expression on IL-4 or IL-13 mDC after incubation with mixtures (30 combinations were tested, n=8, data not shown). Some changes were, however, noticed on imDC.

IL-6 treatment, at concentrations beyond 100 ng/ml, resulted in overexpression of Fc{gamma}RIIB on IL-13 DC without affecting Fc{gamma}RIIA (Fig. 7 ). Conversely, no effect was observed for IL-4 DC. Similar variations in the Fc{gamma}RII expression pattern were also found on both DC types with a high IL-10 concentration (100 ng/ml, Fig. 7 ). Together, IL-10 and IL-6 up-regulated Fc{gamma}RIIB1 and -B2 expression on IL-4 DC, whereas Fc{gamma}RIIA expression was unaffected (n=6, Fig. 7 ). Further addition of TGF-ß and vEGF resulted in an additional up-regulation of both forms of Fc{gamma}RIIBs, whereas Fc{gamma}RIIA expression remained constant (n=6, Fig. 7 ).



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  		Figure 7. Regulation of Fc{gamma}RII expression by anti-inflammatory compounds. Membrane expression of Fc{gamma}RIIA, -B1, and -B2 proteins on imDC, as evidenced by immunoprecipitation after 24 h culture with mixtures of anti-inflammatory molecules and further sorting. Fc{gamma}RIIA, -B1, and -B2 expression is the result from the same exposure. Values represent means ± SEM of six independent experiments. Significant variations between control and treated cells are marked (*, P≤0.05).

 
Associating PGE-2 and IL-1ß had opposite effects on IL-4 and IL-13 DC (Fig. 8 ): Indeed, expression of Fc{gamma}RIIA was slightly increased, whereas Fc{gamma}RIIB1 and -B2 were strongly decreased, and that of Fc{gamma}RIIB2 became undetectable, and DC remained CD83-negative. The selective reduction of Fc{gamma}RIIB expression observed for IL-13 DC but not for IL-4 DC with PGE-2 alone is worth being noticed (Fig. 8) . TNF-{alpha} could also efficiently improve the ratio between activating Fc{gamma}RIIA and inhibitory Fc{gamma}RIIB (B1+B2), as down-regulation of Fc{gamma}RIIBs was more important than that of Fc{gamma}RIIA (Fig. 8) . CD83 expression was observed when the TNF-{alpha} concentration was beyond 500 U/ml, and the percentage of CD83-positive cells increased in a TNF-{alpha} concentration-dependent manner (data not shown). IL-1ß and PGE-2 potentialized TNF-{alpha} action, and cells remained CD83-negative as long as concentrations were kept low (100 U/ml TNF-{alpha}, 2 ng/ml IL-1ß, 1 ng/ml PGE-2, Fig. 8 ). The simultaneous addition of these compounds led to CD83 expression and simultaneous down-regulation of all Fc{gamma}RIIs, which suggests the initiation of the maturation process (Fig. 8) . IFN-{gamma} was unable to regulate expression of any Fc{gamma}RIIs at concentrations ranging from 10 to 500 U/ml (data not shown). Moreover, the down-regulation of Fc{gamma}RIIA and Fc{gamma}RIIB was observed after treatment of both DC types with ODN 2006, ODN 2216, and poly IC at concentrations beyond 5 and 50 µg/ml, respectively (Fig. 8) . It was also accompanied with CD83 expression when compound concentrations were beyond 5 µg/ml ODN and 50 µg/ml polyIC (Fig. 8) . The activating Fc{gamma}RIIA/inhibitory Fc{gamma}RIIB (B1+B2) ratio was thus not improved with those molecules.



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  		Figure 8. Proinflammation mediators regulate Fc{gamma}RII expression. Immunoprecipitation analysis of membrane expression of Fc{gamma}RIIs following 24 h incubation of imDC with proinflammatory compounds and further sorting (n=6). Fc{gamma}RIIA, -B1, and -B2 expression is the result from the same exposure. Data are displayed as means ± SEM, and statistically significant variations between control and treated DC are indicated (*, P≤0.05).

 
Physiological effects of a modified, activating Fc{gamma}RIIA/inhibitory Fc{gamma}RIIB (B1+B2) balance were next assessed for several combinations using MLR. Treated DC were first washed twice with cell culture medium and then sorted before MLR were performed by pulsing these DC with IC or a soluble antigen. Treatments were active at modulating lymphocyte proliferation, albeit with different efficiencies. In all cases, the effects were more important when pulsing with IC than with a soluble antigen (n=6, Fig. 9 ).



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  		Figure 9. Balance between activatory and inhibitory Fc{gamma}RIIs modulates the ability of DC to stimulate T cells. Some mediator-treated DC were sorted and used in MLR assays with IC or soluble antigen (sol Ag) as control (n=6). Negative controls included reactions with antitetanus toxin IgG or without antigen, respectively (table), and without DC (data not shown). Means ± SEM for six independent experiments are shown. Incorporated counts are plotted as a function of the stimulatory cell number, IC, or soluble antigen amount being constant. Table displays mean values ± SEM for the one-tenth ratio. (ND, Not done.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is now well established that Fc{gamma}RIIs strongly modulate the efficiency of humanized antibodies as well as the action of the phagocyte system [7 , 15 , 36 ]. In addition, DC express activating and inhibitory Fc{gamma}RIIs [10 ], and opsonizing tumor cells with humanized antibodies result in efficient presentation and immune response in vitro and in vivo [6 , 8 , 9 ]. As DC are mainly differentiated in vitro, according to two protocols using GM-CSF with IL-4 or GM-CSF with IL-13, we compared the ability of these two types of DC to initiate an immune response when pulsed with IC.

The lack of Fc{gamma}RI and Fc{gamma}RIII expression previously reported for IL-4 DC [4 , 5 , 25 ] was confirmed by our experiments. We also found that these receptors are missing on IL-13 DC. Expression levels of Fc{gamma}RIIs are still a matter of debate [4 , 5 ]. In agreement with previous data [4 , 5 ], we found Fc{gamma}RII expression to be lower on mDC than on imDC. Shedding has been described to be more pronounced when IL-4 DC were pulsed with IC rather than with LPS [4 , 5 ]. This was also evidenced in our experiments for IL-4 and IL-13 DC.

As expected, pulsing DC with IC resulted in the initiation of immune response. Tetanus toxin IC were preferred to antibody-coated tumor cells, as they allow the use of a fully autologous system [24 ]. Time-course analysis of apoptotic DC evidenced no difference in DC half-time according to the differentiation protocol or the maturation signal (soluble antigen or IC-linked antigen). Moreover, throughout the experiments performed in our study, the differences between IL-4 and IL-13 DC were only barely significant.

RT-PCR and immunoprecipitation experiments were performed on pure DC populations including BDCA-1+ circulating blood DC, IL-4, and IL-13 DC. In agreement with previous results, Fc{gamma}RIIA and Fc{gamma}RIIB were found to be expressed on human DC [10 ]. We also evidenced for the first time the expression of Fc{gamma}RIIB1 and -B2 receptors. It should be noted that the ratios between activating Fc{gamma}RIIA and inhibitory Fc{gamma}RIIB (B1+B2) were constant from one donor to another. The relative constancy of Fc{gamma}RIIA expression on IL-4 and IL-13 DC agrees with previous studies about IL-4 DC [5 , 10 ]. However, contrary to a recent report about DC and in agreement with data about monocytes and U937 cells [10 , 16 , 17 ], we found that Fc{gamma}RIIB (B1+B2) expression on DC was high but less than Fc{gamma}RIIA. Differences between the techniques (cytometry vs. immunoprecipitation) used to investigate Fc{gamma}RIIB expression on DC may explain this discrepancy. Moreover, RT-PCR analysis allowed us to detect mRNA for the third Fc{gamma}RII expressed in humans, Fc{gamma}RIIC, where its protein is a hybrid gene product resulting from a crossing-over between Fc{gamma}RIIA and Fc{gamma}RIIB genes [37 ]. Fc{gamma}RIIC extracellular domain is therefore identical with that of Fc{gamma}RIIB, whereas the intracytoplasmic region is the same as for Fc{gamma}RIIA and includes the ITAM-activating signaling motif [37 ]. As a result, Fc{gamma}RIIB and Fc{gamma}RIIC can be detected with antibodies unable to recognize Fc{gamma}RIIA. In our experiments, Fc{gamma}RIIC transcripts and protein levels were low in IL-4 and IL-13 DC as well as in BDCA-1+ DC. Whereas Fc{gamma}RIIA, -B1, and -B2 receptors were readily detectable, different antibodies, extraction, and/or immunoprecipitation buffers may be required for Fc{gamma}RIIC detection. Whether Fc{gamma}RIIC may be functional in human DC also needs to be investigated further.

Furthermore, Fc{gamma}RIIB1 and -B2 were expressed on IL-4 DC. A greater expression of Fc{gamma}RIIB1 and Fc{gamma}RIIB2 on IL-4 DC compared with monocytes is in agreement with the already reported up-regulation of Fc{gamma}RIIB expression by IL-4 [5 , 16 , 17 ]. Expression of Fc{gamma}RIIBs by IL-13 DC could also be expected, as IL-13 has been shown recently to control the activity of the Fc{gamma}RIIB promoter [36 ]. However, the mechanisms underlying the alternative splicing of the Fc{gamma}RIIB gene product need to be explored further. Of interest is also the expression of Fc{gamma}RIIB1 and -B2, where the functional properties have been shown to differ in a B cell line model, especially regarding capping, internalization, and transduction [38 ].

When IC internalization by the phagocytic system through engagement of Fc{gamma}RIIs allows antigen presentation, it requires a sufficient amount of free IC. The secretion of sFc{gamma}RIIA by murine Langherans cells has been demonstrated to limit their ability to capture IC [18 , 19 ]. Our experimental data highlighted a lack of sFc{gamma}RIIA expression by human-circulating BDCA-1+ blood DC and DC. Immunoprecipitation experiments on one- and tenfold concentrated media were confirmed by analysis of cellular extracts to exclude a fast proteolysis of sFc{gamma}RIIA in conditioned media detection. RT-PCR experiments also failed to detect sFc{gamma}RIIA mRNA. Together, these data suggest that unlike their murine counterparts, human BDCA-1+ blood DC and DC cannot escape the maturation process triggered by IC through the secretion of sFc{gamma}RIIA.

We next investigated more thoroughly whether the balance between Fc{gamma}RII activating and inhibitory receptors could be critical for the initiation of an immune response by IC-pulsed DC, which when deleted for Fc{gamma}RIIB expression, allowed us to show that Fc{gamma}RIIB hampered IC uptake by DC. Intracellular staining 24 h after pulsing DC with IC was more pronounced in DEL DC than in WT for IL-4 and IL-13 DC. After a 4- to 8-h coculture of DC with antibody-coated tumor cells, a recent study showed no improvement of uptake by antibody blockade of Fc{gamma}RIIB/C receptors [10 ]. This discrepancy with our own data may result from the use of different strategies to bypass Fc{gamma}RIIBs, different times of analysis, and models. However, in both studies, the selective engagement of activating Fc{gamma}RIIA led to surface remodeling with up-regulation of costimulatory molecules (CD80/80/86) and no alteration of the expression of the presentation molecules (HLA class I and II). We also provided evidence for an up-regulation of CD40 and CD54. In addition, a significant increase in p70IL-12 and IFN-{gamma} amounts was noted in both studies, and we showed IL-10 secretion not to vary significantly. Finally, the thought that a modulation in vitro of the balance between Fc{gamma}RII-activating Fc{gamma}RIIA/inhibitory Fc{gamma}RIIBs (B1+B2) could be of interest for immunotherapy protocols led us to investigate whether this ratio could be affected by a panel of immune mediators, known to alter DC functions. A marked increase of Fc{gamma}RIIB expression was observed after treatment of IL-4 and IL-13 imDC with a mixture of IL-6 and IL-10, in agreement with a previous report that described the regulation of the Fc{gamma}RIIB promoter activity by IL-10 [36 ]. Moreover, the combinations of PGE-2 with IL-1ß or TNF-{alpha} selectively down-regulated Fc{gamma}RIIB expression. These combinations enhanced the proliferation of lymphocytes in vitro. Such in vitro treatments may be of interest for immunotherapy protocols, although further investigations are still needed to assess these effects for potential therapeutic use.

The engagement of ITIM-containing receptors, Ig-like transcript 2 (ILT-2), ILT-4, and paired Ig-like receptor B (PIRB), has recently been described to render DC tolerogenic with the induction of anergic and immunosuppressive T cells [39 40 41 ]. Fc{gamma}RIIB seems unable to act alike, as its activation appears to prevent spontaneous DC activation and "horor toxicus" [10 ]. Moreover, the use of a human polyclonal antibody against tetanus toxin allowed us to generate IC able to bind Fc{gamma}RIIA, -B1, and -B2 as a result of the lack of selection regarding IgG subclasses. As pulsing DC with IC resulted, in our study and in those of others [5 6 7 8 9 10 ], in the initiation of an immune response, we would then not dissociate the effect of Fc{gamma}RIIB from its expression level and that of Fc{gamma}RIIA. We would suggest that Fc{gamma}RIIB expression level and activation may determine the threshold required for DC activation. Consequently, we surmise that Fc{gamma}RIIB activation, by itself, is not sufficient to render DC tolerogenic. However, consequences of a selective engagement of Fc{gamma}RIIB on DC lifetime need further investigations.

Together, the results reported here evidence the ability of Fc{gamma}RIIB to act as an inhibitory receptor in human DC and describe its mechanism of action. They also provide clues for an improved efficiency of DC vaccination protocols. Engineering the Fc region of IgG molecules to preferentially engage activatory Fc{gamma}RIIA but not inhibitory Fc{gamma}RIIBs (B1+B2) could also allow one to turn this inhibition and improve the efficacy of immunization with IC.


   ACKNOWLEDGEMENTS
 
This work was supported by "Région Bretagne" (CPER 2000-2006), Centre Hospitalier Universitaire de Brest, Ligue Contre le Cancer 29, Association Céline et Stéphane. We thank Drs. S. Castellain, J. H. Abalain, Pr. J. L. Carre (Biochemistry and Molecular Biology Laboratory, CHU, Brest), and Dr. L. Corcos (EA 948, Brest) for their support and helpful discussions. We also thank Dr. M. Moser (Brussels University, Belgium) and Dr. T. Martin (Strasbourg University, France) for critical reading of the manuscript.


   FOOTNOTES
 
2 Current address: Institut Curie, service d’oncologie médicale, 26 rue d’Ulm, 75005 Paris, France. Back

Received March 18, 2005; revised July 19, 2005; accepted August 9, 2005.


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