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Immunology |
Unité Cytokine et Cancer, Institut National de la Santé et de la Recherche Médicale U-453, Centre Léon Bérard, 69373 Lyon Cedex 08, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain expression on differentiating CD34+ cells. Consistent with these observations, the differentiation of DCs from monocytes cultured with GM-CSF and IL-4 was also impaired by RCC CM, but the minimal inhibitory concentrations of RCC CM were 10-fold higher than for CD34+ cells. In these conditions, monocytes cultured with GM-CSF and IL-4 also exhibited profound phenotypic changes (CD14+CD32+CD86+HLA-DR+CD115lowCD23lowCD1a-) and a poor APC function. These alterations were overcome in a dose-dependent manner by IL-4 (5–500 IU/ml), although not beyond a 40% final concentration of RCC CM. The capacity of RCC CM to block DC differentiation from monocytes strongly correlated with IL-6 and M-CSF concentrations in medium. Taken together, these results demonstrate that IL-4 and IL-13 reverse the inhibitory effect of tumor cells on DC differentiation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor-specific antigens have been reported in RCC, but tumor-specific cytotoxic T cell clones have only rarely been identified (14, 15, 16, 17, 18)
, suggesting a potential impairment of the differentiation of CTLs in patients with RCC. Furthermore, although IL-2 and/or IFN, is an efficient treatment for patients with metastatic RCC, still a majority of patients will also experience progressive disease (13)
, suggesting the existence of a defective immune response in a majority of patients.
In vitro, RCC cell lines produce a wide range of cytokines including TGFß, IL-6, fibroblast growth factor basic, GM-CSF (19, 20, 21) , VEGF, and M-CSF (22) . Several of these cytokines have been reported to interfere at different steps of the antitumor immune response, as follows: (a) blockade of DC differentiation from precursors by VEGF in the murine mode (23) or by IL-6 and M-CSF in man (22) ; (b) blockade of the initiation of the IL-12 signal transduction in T lymphocytes (TGF-ß; Ref. 24 ); and (c) blockade of CTL differentiation (reviewed in Ref. 25 ).
IL-4 blocks the production of proinflammatory cytokines such IL-6, TNF (26)
, and M-CSF (27
, 28)
and improves the differentiation of immature DCs from CD34+ progenitors when added during the differentiation period (day 6 to day 12) period (29
, 30)
. The aim of the present study was to investigate the effect of IL-4 on the blockade of DC differentiation from CD34+ progenitors by RCC CM. The results presented here demonstrate that IL-4 and IL-13 are capable of reversing the inhibitory effects of RCC CM on DC differentiation by inhibiting the expression of the IL-6 receptor transducing chain (gp130) and the M-CSF receptors and preventing the loss of GM-CSF receptor chain receptor expression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(specific activity, 5 x 106 units/mg; Cetus, Amsterdam, the Netherlands) at 2.5 ng/ml (50 units/ml), rhSCF (specific activity, 4 x 105 units/mg; R&D Systems, Abingdon, United Kingdom) at 25 ng/ml (specific activity, 5 x 106 units/mg; Cetus), M-CSF (specific activity, 2 x 106 units/mg, R&D Systems), IL-6 (specific activity, 106 units/mg; Sandoz, Basel, Switzerland), and VEGF165 and IL-10 (R&D Systems) at 40ng/ml.
Production of RCC CM
Renal carcinoma cell lines obtained from the American Type Culture Collection (CAKI-1, ACHN, and A-704) or established in the laboratory (CLB-CHA, CLB-GUI, CLB-MET, CLB-TUG, CLB-TUT, and CLB-VER; Ref. 31
) were plated in 100 mm-diameter dishes at a density of 5 x 105 cells/ml in RPMI 1640 supplemented with 2 mM glutamine, 200 IU/ml penicillin, 200 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY), and 10% FCS (Biowittaker, Verviers, Belgium). After 2 days of culture, supernatants were harvested, filtered, aliquoted, and stored at -20°C for later use. Conditioned media were prepared under the same conditions in specific media (Table 1)
supplemented with 2 mM glutamine, 200 IU/ml penicillin, 200 µg/ml streptomycin, and 10% FCS for the following other tumoral cell lines: breast carcinoma (T47-D, BT-20, and CLB-SAV), prostate carcinoma (LnCaP and HTB-81), melanoma (CLB-DOR), Burkitt’s lymphoma (Daudi, Raji and BJAB), colon carcinoma (SW620 and HCT-116), osteosarcoma (SAOS), and fibrosarcoma (HT-1080), obtained from the American Type Culture Collection or generated in the laboratory.
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Naive T lymphocytes (CD45RA+) were purified from the total lymphocyte population by immunomagnetic depletion using a cocktail of MAbs (33) . After two rounds of depletion with beads, the purity of CD45RA+ was routinely >95%. The T lymphocyte population was also cryopreserved in 10% DMSO for additional MLR assays.
DC Development
From CD34+ Progenitors.
Umbilical cord blood samples were obtained according to institutional guidelines. Cells bearing CD34 antigen were isolated from mononuclear fractions through positive selection by mini MACS (Miltenyi Biotec, Gmbh) using an anti-CD34 MAb (Immu 133.3; Immunotech, Marseille, France) and goat antimouse IgG-coated microbeads (Miltenyi Biotec, Paris, France; Ref. 33
). In all experiments, 80–99% isolated cells were CD34+. CD34+ progenitors were seeded at 5 x 103 to 104 cells/ml in 24-well plates in the presence of GM-CSF (100 ng/ml), TNF (2.5 ng/ml), SCF (20ng/ml), and 2% human sAB+, as previously described, and left to expand for 6 days (33
, 34)
. Cells were then harvested, numbered, phenotyped, and seeded in the absence of sAB+ but in the presence of GM-CSF and TNF at 5 x 104 cells/ml for an additional 6-day expansion period. A last medium change was performed at day 10, then cells (CD34+-DC) were collected at day 12. Eventually, adherent cells were recovered using a 5-mM EDTA solution.
From Peripheral Blood Monocytes.
Total peripheral blood mononuclear cells were isolated from citrate phosphate dextran blood obtained from healthy volunteers using Ficoll Hypaque density gradient centrifugation (Eurobio). Enriched monocyte preparations were additionally purified on a multistep percoll gradient, as described by Sallusto and Lanzavecchia (32)
. Purified monocytes were seeded at 4 x 105 monocytes/ml, and, after an adherence step (2 h at 37°C and removal of nonadherent cells), cultured for 6–7 days in the presence of GM-CSF (100ng/ml) and IL-4 (500 IU/ml) in complete RPMI 1640. This procedure allows the differentiation of monocytes into MO-DCS.
MLR Assay
After culture, MO-DC or CD34+-DC were collected, irradiated (30 Gy), then used as stimulator cells for allogeneic adult naive T lymphocytes. Ten to 104 stimulator cells were added to the T cells (2 x 104 cells/well) in 96-well round-bottomed microtest culture plates (Nunc, Rockilde, Denmark). Cultures were performed in RPMI 1640 supplemented with 10% FCS. After 5 days of incubation, cells were pulsed with 0.5 µCi of [3H]thymidine per well (specific activity, 5mCi/mMol) for the last 18 h, then harvested and counted. Tests were carried out in triplicates and results are expressed as mean cpm (cpm ± SD). The levels of [3H]thymidine uptake by stimulator cells alone were always <100 cpm.
Cell Surface Phenotyping
Extensive phenotyping was performed at day 6 for MO-DCs and at day 12 for CD34+-DC. Flow cytometry was carried out by incubating 5 x 104-105 cells for 20 min on ice under 50 µl with optimal concentrations of the appropriate antibodies coupled to PE (except when specified). Antibodies were purchased from Becton Dickinson (Pont de Claix, France; CD14, CD15, HLA-DR, CD80, and CD16), PharMingen (San Diego, CA; CD86), Immunotech (CD23, CD83, CD116, CD40, CD54, and CD58), Caltag Laboratories (Burlingame, CA; CD32 and CD64), Ortho Diagnostic (Raritan, NJ; CD1a-FITC), Diaclone (Besancon, France; CD126-FITC and CD130-FITC), and Oncogene Science [Cambridge, MA; rat control isotype and CD115 (M-CSF receptor)]. Fluorescence analysis was performed on a FACScan flow cytometer after acquisition of 5000 events (Becton Dickinson). Negative controls were performed with unrelated murine MAbs or MAbs-PE purchased from Becton Dickinson.
Neutralizing Antibodies
Anti IL-6 (BE-8) and anti IL-6R (BR-6) Mabs were purchased from Diaclone (Besançon, France). Anti-VEGF165 antibody was purchased from R&D Systems; rabbit and mouse immunoglobulins used as control antibodies were purchased from R&D Systems, and polyclonal anti-M-CSF was purchased from Genzyme (Paris, France). Polyclonal anti-IL-10 antibody was kindly provided by the Schering Plough Corporation. All antibodies were used at optimal concentrations (10–20 µg/ml).
Cytokine Detection
Cytokines were detected in conditioned media and culture supernatants using commercial quantitative sandwich immunoassay kits from Immunotech (Beckmann-Coulter; IL-6 and IL-10) and R&D Systems (M-CSF, VEGF, and IL-6sR). The detection limits of these immunoassays were respectively 3 pg/ml, 15 pg/ml, 18 pg/ml, 5 pg/ml, and 7 pg/ml.
Phagocytosis
Peripheral blood monocyte cells were cultured for 6 days in the presence of GM-CSF+IL-4 alone or with 40% RCC CM or breast CM or IL-6+M-CSF (40ng/ml). During the last 6 h of the culture, 0.5-µm latex beads coupled to FITC (1:400; Polysciences, Warrington, PA) were added to the culture. To analyze the phagocytic capacity, cells were recovered and washed three times in cold PBS, and bead(s) incorporation was evaluated on a FACScan analyzer (histograms) or observed on a fluorescence microscope after cytocentrifugation and May Grünwald Giemsa coloration.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and poor APC function (Fig. 2)
; this inhibition correlates with the presence of IL-6 and M-CSF in tumor cell medium and recombinant IL-6 and/or M-CSF induced similar phenotypic and functional alterations (Ref. 22
; Fig. 1
). In the present report, IL-4 (500 IU/ml) was found capable to prevent the blockade of DC differentiation induced by RCC CM (10%) or IL-6+M-CSF (10ng/ml) despite the presence of RCC CM, CD34+ cells cultured with IL-4 were similar to those obtained in the absence of RCC CM, as evaluated by morphology (not shown) and phenotype (Fig. 1)
. Moreover, the addition of IL-4 prevented the functional inhibition induced by RCC CM: cells cultured in the presence of RCC CM and IL-4 displayed a significant APC capacity, as evidenced by their ability to stimulate naive T cell proliferation (Fig. 2)
, and displayed decreased phagocytic capacity (Fig. 3)
. Of note, in the absence of RCC CM, the percentage of cells with a typical DC morphology (not shown), and phenotype (i.e., expressing a CD14-CD1a+ phenotype with a definitive loss of CD14 Ag; Fig. 1
) was higher when IL-4 (500 IU/ml) was added to the standard cytokine combination (GM-CSF+TNF) than in GM-CSF+TNF alone (Fig. 1)
; in these experiments, IL-4 prevented the maturation of DCs, which was in agreement with previous reports (29
, 30
, 35 , 36)
.
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from CD34+ progenitors.
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. As observed for IL-4, IL-13 reversed the inhibitory effects of recombinant IL-6+M-CSF and low concentrations of IL-13 (5ng/ml) were sufficient to reverse the inhibitory effect of RCC CM (not shown).
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(CD116) is lost on cells generated in the presence of 10% RCC CM as compared with cells generated in the presence of cytokines (GM-CSF+TNF) alone (Table 3
; Ref. 22
). The addition of IL-4 during the differentiation process with RCC CM strongly increased CD116 expression, which then reached levels similar to those observed without RCC CM (Table 3)
. This induction of CD116 expression was also associated with a decrease of M-CSF receptor (CD115) expression (Table 3)
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(CD126/gp80) was undetectable at day 12 in all culture conditions (not shown), whereas gp130 was detectable on cells generated in presence of RCC CM only (Table 3)
. Addition of IL-4 in the culture abrogated the effect of RCC CM on gp130 expression (Table 3)
. Soluble IL-6R (sgp80) is an agonist of IL-6 (41
, 42)
, forming an IL-6/sgp80 complex which binds with high affinity to the gp130 transducing chain (43
, 44)
. sIL-6R/sgp80 was produced during DC differentiation from CD34+ progenitors (Fig. 5)
in agreement with previous reports (45)
. In the presence of RCC CM, the content of sIL-6R/sgp80 in DC culture supernatant decreased, although the magnitude of this decrease varied according to the RCC CM used and its content in IL-6; for CLB-TUT, a high IL-6-producer, the level of sIL-6R/sgp80 was reduced by 50%, whereas for CLB-VER, a low IL-6-producer, sIL-6R/sgp80 production decreased more slightly. Of note, the addition of exogenous recombinant IL-6 (not shown) as well as IL-6+M-CSF (Fig. 5)
at a concentration close to that observed in the RCC CM also induced a decrease of sIL-6R/sgp80 content in the supernatant. The addition of IL-4 to the culture (Fig. 5)
was associated with a dose-dependent decrease of sIL-6R/sgp80 production by cells cultured in GM-CSF+ TNF without RCC CM. In the presence of IL-4, the addition of RCC CM (10%) no longer modulated sIL-6R/sgp80 content in the culture medium (Fig. 5)
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; 1907 pg/ml and 1020 pg/ml, respectively) and was comparable with that of DC cultured without RCC CM (1016 pg/ml in the medium alone versus 1020 pg/ml for RCC CM+IL-4). Taken together, these results suggest that IL-4 reverses the inhibitory effects of RCC CM on DC differentiation in this model through a restoration of CD116 expression, a decrease of the expression of M-CSF receptor expression and M-CSF production as well as a down-regulation of the IL-6 receptor transducing chain (gp130) membrane expression.
High Concentrations of RCC CM Block the Differentiation of Monocytes into DC.
In view of the antagonistic effect of IL-4 on the blockade of DC differentiation by RCC CM in the CD34+ model, the effects of RCC CM on the differentiation of DC from peripheral blood monocytes cultured for 6 days with GM-CSF (100 ng/ml) and IL-4 (500 IU/ml; Refs. 32
and 46
) were investigated. In the presence of high (500 IU/ml) concentrations of IL-4 (32
, 42
, 46)
, high concentrations (20–40%) of RCC CM were required to block MO-DC differentiation (Fig. 6)
; the minimum inhibitory concentrations were therefore 10-fold higher than in the CD34+ model (Ref. 22
; Fig. 1A
). The phenotype of cells generated in the presence of 40% RCC CM is shown in Fig. 7
. The addition of RCC CM reduced CD1a expression (42.2%, MFI = 153 versus 97.4%, MFI = 1268), increased CD54 and CD58 expression and maintained CD32 expression, which is normally lost during DC differentiation (39.5%, MFI = 69 versus 3.8%, MFI = 26), as compared with cells cultured in GM-CSF+IL-4 alone (Fig. 7)
. In addition, whereas CD86 was expressed only at low levels on immature MO-DCs (47)
, high levels of CD86 (96.3%, MFI = 531 versus 64.5%, MFI = 317) and HLA-DR expressions were observed in the presence of RCC CM. The induction of CD23 (Fc
RII) expression, a specific property of IL-4 (48)
, was partly inhibited by the presence of high concentrations of RCC CM. Finally, incubation in the presence of RCC CM induced a decrease of CD115 (M-CSF-R) expression as compared with cells cultured with cytokines alone.
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, BT-20, and CLB-SAV], Burkitt’s lymphoma, osteosarcoma, as well as prostate carcinoma (Table 1)
, or from primary cultures from non-tumoral renal epithelial cells (CLB-CANnor; Fig. 6
). The presence of high (>400 pg/ml) concentrations of IL-6 and M-CSF in tumor cell supernatant was strongly correlated with the inhibitory capacity of tumor cell medium on MO-DC differentiation (Fisher’s exact test; P < 0.001), whereas no correlation was found for VEGF (Table 1)
.
In addition to these phenotypic alterations, monocytes cultured with GM-CSF+IL-4 and in the presence of RCC CM displayed a strong phagocytic capacity, as evaluated by the ingestion of 0.5-µm latex beads coupled to FITC compared with MO-DCs (Fig. 8A)
. The capacity of cultured MO-DCs to induce the proliferation of purified naive (CD45RA+) allogeneic CD4+ T was also affected in the presence of 40% RCC CM, although not completely abolished; 10-fold more cells are required to reach levels of T cell proliferation comparable with those induced by MO-DC (Fig. 8B)
.
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). Using a dose range of both IL-4 and RCC CM, concentrations of RCC CM required to induce the blockade of MO-DC differentiation were found to be directly dependent on IL-4 concentration in the culture medium (Table 4)
. A concentration of 40% RCC CM was required to block MO-DC differentiation in the presence of high concentrations of IL-4 (250 IU/ml), whereas, in the presence of 100-fold lower doses of IL-4 (2.5 IU/ml), the minimum inhibitory concentration of RCC CM was only 4% (Table 4)
. In contrast, lower doses of GM-CSF (1–100ng/ml) did not affect the concentration of RCC CM required to block MO-DC differentiation in the presence of high doses of IL-4 (data not shown). The capacity of monocyte to differentiate into dendritic cells in vitro is therefore dependent on the ratio of inhibitory cytokines produced by tumor cells and stimulatory cytokines such as IL-4 or IL-13 in the culture medium.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It had been reported that IL-4 added to GM-CSF and TNF affects the process of CD34+ cell differentiation into DCs in a complex manner according to the timing of IL-4 addition (29
, 30
, 49
, 50) as follows. IL-4 (a) reduces the cell yield by 50% (day 0 to day 6; Ref. 49
); (b) favors the differentiation of immature DCs (after day 7) but blocks TGFß-mediated differentiation into Langerhans cells (30
, 36
, 51, 52, 53)
; and (c) promotes the maturation of DCs (days 12–20; Ref. 54
). Similar results have been obtained with IL-13 in the MO-DC model (55)
.
The results presented here also indicate that IL-4 and IL-13 are both capable of abrogating the capacity of tumor cells and recombinant IL-6 and/or M-CSF to promote the commitment of CD34+ progenitors into the monocyte/macrophage lineage with poor APC capacity. The protecting effect of IL-4 on the process of DC differentiation involves a down-modulation of the expression of M-CSF receptor (CD115) at the surface of DC progenitors early during the differentiation process, a decrease in M-CSF production, and, more importantly, the maintenance of GM-CSF R (CD116) expression. We reported previously (22)
that RCC CM as well as recombinant IL-6 and/or M-CSF induce an early increase of CD115 expression at the surface of DCs that is associated with a down-regulation of the CD116 required for DC differentiation (45)
. Here, we show that concentrations of IL-4 capable of protecting against the inhibitory effects of RCC CM on DC differentiation also antagonized their effects on CD115 and CD116 expression. In addition to the modulation of CD115, the addition of IL-4 at day 6 induced a decrease in M-CSF production measured at day 12. This observation was consistent with an earlier report on activated monocytes showing that the addition of IL-4 strongly inhibited M-CSF production (56)
. The decreased production of endogenous M-CSF is also likely to be an important mechanism of the protective effect of IL-4, because autocrine M-CSF production has been shown to be involved in the blockade of DC differentiation by IL-6 alone (22)
.
Whereas incubation with RCC CM, IL-6, and/or M-CSF induced a rapid (<48 h) down-regulation of GM-CSF R/CD116, in the presence of IL-4, CD116 expression remained detectable at day 9 of the 12-day DC culture from CD34+ progenitors (data not shown). These results are consistent with a previous study showing that a single pulse with IL-4 (48 h) is sufficient to promote DC development (30)
. This is also consistent with the kinetic of the inhibitory effect of RCC CM on CD115 and CD116 expression, which is an early phenomenon (days 8–9; Ref. 22
). The capacity of IL-4 to maintain CD116 expression is likely to be one of the major mechanisms of its antagonistic effect on RCC CM or IL-6/M-CSF effect during the process of DC differentiation.
IL-6R/gp80 is the ligand-binding chain within the IL-6 receptor, active either in its transmembrane form or as a soluble protein; both forms interact with the gp130 transducing chain (43
, 44)
. Membrane bound IL-6R/gp80 was detectable at the surface of day-6 CD34+ progenitors using anti-IL-6R/gp80 and FACS analysis, but was found undetectable at later stages of DC differentiation (data not shown). Day-12 DCs generated from CD34+ cells produced significant levels of sIL-6R/sgp80 (45)
. In the presence of RCC CM, a decrease of the concentration of sIL-6R/sgp80 was also observed in supernatant at day 12; this phenomenon is likely to result from an internalization of IL-6 coupled to its receptor; IL-6 has been shown to deliver an inhibitory signal at this step, which could be blocked by an anti-IL-6 antibody (22)
. In the presence of IL-4, the content of IL-6R/sgp80 in supernatants strongly decreased, and no more modulation was observed with or without RCC CM, suggesting that IL-4 could interfere with the inhibitory signal of IL-6 in these conditions. Importantly, during DC differentiation from CD34+ progenitors, the expression of the gp130 chain is lost rapidly since DC-committed progenitors (simultaneously expressing CD14 and CD1a Ag) have already lost the expression of membrane gp130 (data not shown). In contrast, at day 12, in the presence of RCC CM, the majority of cells generated expressed gp130. The addition of IL-4 abrogated the induction of the gp130 expression induced by RCC CM.
This phenomenon is likely to play a major role in the antagonistic effect of IL-4 on RCC CM and IL-6/M-CSF inhibitory effect on DC differentiation. Indeed, by favoring the differentiation of CD14+CD1a- progenitors into CD14+CD1a+ population (29
, 30)
, IL-4 abrogates the expression of gp130 and, hence, blocked the capacity of RCC CM to act through the IL-6 pathway. Taken together, these results indicate that IL-4 reversed the effects of tumor cell medium or IL-6/M-CSF: (a) by decreasing M-CSF production and the M-CSF receptor expression; (b) by decreasing the expression of gp130 on committed precursors (CD14+CD1a+); and (c) by preventing the loss of GM-CSF receptor chain expression at the surface of differentiating DCs between day 6 and day 12.
Despite the protecting effect of IL-4 on the tumor cell-mediated inhibition of DC differentiation in the CD34 model, our results demonstrate that RCC CM as well as recombinant exogenous IL-6 and IL-6+M-CSF (not shown) also block the differentiation of DCs from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4. However, in the presence of high IL-4 concentrations (500 IU/ml), the minimum RCC CM concentrations required to block MO-DC generation were 10-fold higher than those required to block CD34+-DC differentiation. Actually, the dose-response study indicates that low concentrations of IL-4 (2.5 to 5 IU/ml) with GM-CSF were sufficient to induce the generation of MO-DC, in agreement with previous reports (57) . In the presence of low concentrations of IL-4, the final concentrations of tumor cell conditioned media required to inhibit the differentiation of monocytes into MO-DC were much lower (4%), similar to those required in the CD34+ model.
It is noteworthy that concentrations of RCC CM inhibiting DC differentiation in this model blocked CD23 induction at the surface of monocyte-derived cells. The induction of CD23 expression is one of the specific biological activities of IL-4 that have been reported on B cells (58, 59, 60) and immature MO-DCs (48 , 61) . RCC CM as well as IL-6 and M-CSF are therefore able to block the effects of IL-4, possibly through a down-modulation of its specific receptors. Of note, neither IL-4 receptor at tumor cell surface, nor sIL-4R production in RCC CM were detectable using immuno-fluorescence and FACS analysis and specific ELISA respectively (data not shown). An inhibitory effect of IL-6+M-CSF and RCC CM involving very low levels of IL-4 R expression at the surface of differentiating progenitors cannot however be ruled out.
In contrast with what is observed in the CD34+ model, although recombinant IL-6 and M-CSF (not shown) induced the same effects as RCC CM in the MO-DC model, anti-IL-6 or anti-M-CSF antibodies were not able to reverse completely the inhibitory effects of high RCC CM concentrations (40%) in the presence of high IL-4 concentration. Of note, neither anti-VEGF, nor anti-IL-10 antibodies nor any combination of the four antibodies, reversed the inhibitory effect of RCC CM (not shown). This may be related to the requirement of high final concentrations (40ng/ml) of IL-6 and M-CSF to inhibit DC differentiation from monocytes; these high levels of IL-6 and M-CSF may be too high to be blocked by the anti-cytokine antibodies used in the present study.
Finally, it must be noted that the capacity of RCC CM to block DC differentiation is directly correlated with their capacity to produce IL-6 and/or M-CSF. RCC cell lines not producing IL-6 or M-CSF do not inhibit DC differentiation from monocytes or from CD34+ progenitors (22) . Of note, Kiertscher et al. (62) recently reported that tumor cell conditioned media issued from a RCC (R11) did not block the MO-DC differentiation but altered their maturation, but the concentration of IL-6 and M-CSF present in the conditioned media of this cell line was not reported.
In both culture systems, IL-13 induced the same biological effects as IL-4 with an increase of CD1a+ cell percentage and a decrease of APC capacity. This is also in agreement with the capacity of IL-13 to block the differentiation of cells from the monocyte/macrophage lineage (63) . These results are consistent with the overlapping effects of these two cytokines on DC differentiation (29) reported previously and further indicate that both cytokines antagonize the effects of RCC CM and their cytokines on DC differentiation.
Taken together, these results indicate a balance between the stimulatory effects of IL-4 and the inhibitory effects of RCC CM or IL-6/M-CSF on DC differentiation. High concentrations of IL-4 are capable of antagonizing the inhibitory effects of relatively high concentrations of RCC CM.
The present findings may be useful in clinical practice. The ex vivo generation of DC for therapeutic use in cancer should ideally be performed in serum-free conditions. However, DC differentiation is strongly decreased under these conditions (64) . Autologous serum could be used instead of FCS in cultures (65 , 66) . However, in RCC patients, high levels of IL-6 (19) , VEGF, and M-CSF5 are detectable; all three cytokines have been described to block DC differentiation from CD34+ progenitors in the murine (23) and human models (22) . IL-4 or IL-13 might therefore be useful for ex vivo DC differentiation in clinical trials of DC therapy when autologous plasma, which frequently contains high levels of inhibitory cytokines (RCC, melanoma, colon carcinoma, etc.; Refs. 19 , 67 , and 68 ) is added to DC cultures.
In conclusion, the present results show a novel biological mechanism by which IL-4 and IL-13 exert antitumoral properties in vitro and possibly in vivo. Low concentrations of IL-4 and IL-13 block the inhibitory effects of tumor cells on the differentiation of DCs from CD34+ or monocyte progenitors. This antagonistic effect of IL-4 involves the inhibition of the production of M-CSF as well as the inhibition of M-CSF and gp130 (IL-6 receptor transducing chain) receptors, and the conservation of CD116 expression, both mechanisms being responsible for the commitment of progenitors toward the monocyte/macrophage lineage instead of the DC lineage.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the Comité de Saône et Loire, the Comité de la Savoie, and the Comité du Rhône de La Ligue Contre le Cancer, the Ligue Nationale Contre le Cancer, and the Lyons Club. Marie Cécile Thomachot was supported by a grant from the Comité départemental de Saône et Loire de la Ligue Contre Le Cancer. 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Unité "Cytokines et Cancers," Inserm U-453, Centre Léon Bérard, 28 rue Laënnec, 69373 Lyon Cedex 08, France. Fax: (33) 4-78-78-27-20. E-mail: blay{at}lyon.fnclcc.fr
4 The abbreviations used are: DC, dendritic cell; APC, antigen-presenting cell; RCC, renal cell carcinoma; IL, interleukin; TNF, tumor necrosis factor; RCC CM, RCC tumor cell conditioned medium; MAb, monoclonal antibody; rh, recombinant human; MLR, mixed lymphocyte reaction; MO-DC, monocyte-derived dendritic cell; sAB+, AB+ serum; PE, phycoerythrin; APC, antigen-presenting cell; MFI, mean fluorescence intensity; GM-CSF, granulocyte macrophage colony stimulating factor; VEGF, vascular endothelial growth factor; M-CSF, macrophage colony stimulating factor, SCF, stem cell factor.
5 Blay et al., manuscript in preparation.
Received 11/14/00. Accepted 2/ 1/01.
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H. Okada, T. Tsugawa, H. Sato, N. Kuwashima, A. Gambotto, K. Okada, J. E. Dusak, W. K. Fellows-Mayle, G. D. Papworth, S. C. Watkins, et al. Delivery of Interferon-{alpha} Transfected Dendritic Cells into Central Nervous System Tumors Enhances the Antitumor Efficacy of Peripheral Peptide-Based Vaccines Cancer Res., August 15, 2004; 64(16): 5830 - 5838. [Abstract] [Full Text] [PDF]
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C. C. Sombroek, A. G. M. Stam, A. J. Masterson, S. M. Lougheed, M. J. A. G. Schakel, C. J. L. M. Meijer, H. M. Pinedo, A. J. M. van den Eertwegh, R. J. Scheper, and T. D. de Gruijl Prostanoids Play a Major Role in the Primary Tumor-Induced Inhibition of Dendritic Cell Differentiation J. Immunol., May 1, 2002; 168(9): 4333 - 4343. [Abstract] [Full Text] [PDF]
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), in the presence of T47-D 10% (breast cell carcinoma CM; 
), CLB-TUT 10% (RCC CM; •) or IL-6 + M-CSF (10 ng/ml; 
; A), alone or associated with IL-4 (500 IU/ml; B). At day 12, cells were harvested, irradiated (30 Gy) and used as stimulator cells for naïve T lymphocytes (2.104 cells/well). The proliferation was evaluated using [3H]thymidine uptake after 5 days of culture. The results are expressed as the mean ± SD of triplicate culture. These results are representative of four different experiments.
) or 50 IU/ml (
