Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions
2004; Elsevier BV; Volume: 165; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63238-5
ISSN1525-2191
AutoresGianni Gerlini, Adrian Tun-Kyi, Christa Dudli, Günter Burg, Nicola Pimpinelli, Frank O. Nestlé,
Tópico(s)Immunotherapy and Immune Responses
ResumoCD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor. CD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor. CD1 molecules are cell surface glycoproteins and represent a non-classical, non-polymorphic antigen-presenting system which is distantly related to major histocompatibility complex (MHC) class I and class II molecules. 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Group 2 consists exclusively of CD1d which is expressed on cells of the hematopoietic lineage.8Dutronc Y Porcelli SA The CD1 family and T cell recognition of lipid antigens.Tissue Antigens. 2002; 60: 337-353Crossref PubMed Scopus (55) Google Scholar DC are professional antigen-presenting cells which express high levels of MHC molecules, the classical antigen-presenting system, but also the entire repertoire of CD1 molecules, thus being able to initiate immune responses to lipid/glycolipid antigens.9Banchereau J Steinman RM Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (12389) Google Scholar, 10Porcelli SA Modlin RL The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids.Annu Rev Immunol. 1999; 17: 297-329Crossref PubMed Scopus (603) Google Scholar, 11Cella M Sallusto F Lanzavecchia A Origin, maturation, and antigen-presenting function of dendritic cells.Curr Opin Immunol. 1997; 9: 10-16Crossref PubMed Scopus (1163) Google Scholar In contrast to MHC-restricted T cells, CD1-restricted T cells recognize foreign lipids which are highly expressed on the surface of bacteria and eukaryotic parasites. Several studies have demonstrated that CD1-restricted T cells play an important role in the immune responses directed against Mycobacterium tuberculosis by mediating cytotoxicity against CD1-positive target cells presenting mycobacterial glycolipids.12Fortsch D Rollinghoff M Stenger S IL-10 converts human dendritic cells into macrophage-like cells with increased antibacterial activity against virulent Mycobacterium tuberculosis.J Immunol. 2000; 165: 978-987PubMed Google Scholar, 13Stenger S Modlin RL T cell-mediated immunity to Mycobacterium tuberculosis.Curr Opin Microbiol. 1999; 2: 89-93Crossref PubMed Scopus (141) Google Scholar, 14Sieling PA Jullien D Dahlem M Tedder TF Rea TH Modlin RL Porcelli SA CD1 expression by dendritic cells in human leprosy lesions: correlation with effective host immunity.J Immunol. 1999; 162: 1851-1858PubMed Google Scholar In addition, CD1 molecules have been shown to be involved in immune responses directed against tumors which express neuraminic acid containing glycolipids called gangliosides.15Toura I Kawano T Akutsu Y Nakayama T Ochiai T Taniguchi M Cutting edge: inhibition of experimental tumor metastasis by dendritic cells pulsed with alpha-galactosylceramide.J Immunol. 1999; 163: 2387-2391PubMed Google Scholar, 16Kawano T Cui J Koezuka Y Toura I Kaneko Y Sato H Kondo E Harada M Koseki H Nakayama T Tanaka Y Taniguchi M Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Valpha14 NKT cells.Proc Natl Acad Sci USA. 1998; 95: 5690-5693Crossref PubMed Scopus (451) Google Scholar, 17Takahashi T Johnson TD Nishinaka Y Morton DL Irie RF IgM anti-ganglioside antibodies induced by melanoma cell vaccine correlate with survival of melanoma patients.J Invest Dermatol. 1999; 112: 205-209Crossref PubMed Scopus (80) Google Scholar, 18Livingston P Ganglioside vaccines with emphasis on GM2.Semin Oncol. 1998; 25: 636-645PubMed Google Scholar Immunotherapy approaches using ganglioside vaccines were therefore used in several cancers, including melanoma.19Livingston PO Ragupathi G Carbohydrate vaccines that induce antibodies against cancer: 2. 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Here, we show down-regulation of CD1 molecules in metastatic but not primary melanoma. We further demonstrate a key role of melanoma secreted IL-10 in mediating this effect. Freshly isolated melanoma cells were isolated as previously described and cultured until they reached confluence (first passage).27Yue FY Dummer R Geertsen R Hofbauer G Laine E Manolio S Burg G Interleukin-10 is a growth factor for human melanoma cells and down-regulates HLA class-I, HLA class-II, and ICAM-1 molecules.Int J Cancer. 1997; 71: 630-637Crossref PubMed Scopus (222) Google Scholar Cells were then detached using trypsin and subcultured (second passage). Melanoma cells were grown in 75-cm2 culture flasks (Becton Dickinson, Basel, Switzerland) as monolayer cultures in complete RPMI containing 5 mmol/L glutamine, 1 mmol/L sodium pyruvate, 1% penicillin/streptomycin, and 10% heat-inactivated fetal calf serum (FCS) (all from Gibco, Basel, Switzerland). Monocyte-derived DC were generated from peripheral blood mononuclear cells (PBMC) isolated from buffy coats by density gradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) as previously described with minor modifications.28Jonuleit H Kuhn U Muller G Steinbrink K Paragnik L Schmitt E Knop J Enk AH Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.Eur J Immunol. 1997; 27: 3135-3142Crossref PubMed Scopus (1012) Google Scholar, 29Nestle FO Alijagic S Gilliet M Sun Y Grabbe S Dummer R Burg G Schadendorf D Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells.Nat Med. 1998; 4: 328-332Crossref PubMed Scopus (2694) Google Scholar Briefly, 50 × 106 PBMC were incubated in 10-cm Petri dishes (Becton Dickinson) in complete RPMI at 37°C and 5% CO2 for 45 minutes. Non-adherent cells were removed by washing twice with phosphate-buffered saline (PBS) and the adherent cells were further cultured in complete RPMI containing 800 IU/ml GM-CSF (Leucomax, Sandoz-Wander Pharma SA, Bern, Switzerland) and 1000 IU/ml IL-4 (R&D Systems, Abingdon, United Kingdom). Fresh medium supplemented with 800 IU/ml GM-CSF and 1000 IU/ml IL-4 was added after 3 and 5 days. The purity of monocytes was assessed by flow cytometry by measuring the percentage of CD14+ cells using a monoclonal antibody (Becton Dickinson) and ranged from 79% to 94% (mean 84.5%) of total cells in culture. Cells were analyzed at different maturation days by flow cytometry as indicated. Representative 7-μm serial cryostat sections from tissue samples of normal skin (n = 10), primary (n = 10), and metastatic (n = 10) melanoma lesions were examined for the expression of CD1 molecules, HLA-DR, and IL-10 using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique as previously described.30Nestle FO Burg G Fah J Wrone-Smith T Nickoloff BJ Human sunlight-induced basal cell carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells.Am J Pathol. 1997; 150: 641-651PubMed Google Scholar Each sample was obtained from a different patient. The numbers of CD1a, b, c, d- and HLA-DR-positive DC in serial sections was assessed by calculating the mean cell number counted in five different fields (original magnification, ×400). Thereafter, the ratio between CD1- and HLA-DR-positive cells was calculated to correct for the density of infiltrating dendritic cells. DC were defined as large mononuclear cells with dendrites being strongly HLA-DR positive as previously described.14Sieling PA Jullien D Dahlem M Tedder TF Rea TH Modlin RL Porcelli SA CD1 expression by dendritic cells in human leprosy lesions: correlation with effective host immunity.J Immunol. 1999; 162: 1851-1858PubMed Google Scholar, 30Nestle FO Burg G Fah J Wrone-Smith T Nickoloff BJ Human sunlight-induced basal cell carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells.Am J Pathol. 1997; 150: 641-651PubMed Google Scholar The antibodies used were against CD1a (IgG2a) (Dako, Zug, Switzerland), CD1b (BCD1b3.1.6, IgG1) kindly provided by Dr. S. A. Porcelli (Harvard Medical School, Boston, MA, USA), CD1c (IgG1) (Immunotech, Marseilles, France), CD1d (IgG1) (Biosource International, Camarillo, CA, USA), CD1d (51.1.3, IgG2b), kindly provided by Dr. S. P. Balk (Harvard Medical School), HLA-DR (Becton Dickinson) (all at 10 μg/ml), and IL-10 (5 μg/ml) (R&D Systems). Sections were counterstained using hematoxylin. Cryosections were fixed in 2% paraformaldehyde (Sigma, Buchs, Switzerland) and then washed with PBS, pH 7.4. Sections were pre-incubated with normal rabbit serum (Dako). Slides were thereafter incubated with an antibody against any of the CD1 molecules at 10 μg/ml for 2 hours at room temperature in the dark. After washing with PBS, sections were incubated with a fluorescein isothiocynate (FITC)-conjugated rabbit anti-mouse antibody (dilution 1:30) (Dako). After washing with PBS, slides were incubated with normal mouse serum (5% in PBS) for 20 minutes and thereafter incubated with a phycoerythrin (PE)-conjugated antibody against HLA-DR (dilution 1:50) for 2 hours at room temperature in the dark. All steps were performed at room temperature. For examination of CD1/HLA-DR co-expression, confocal microscopy was performed using a Leica DM-IRBE microscope (Leica, Glattbrugg, Switzerland). 105 monocyte-derived DC or DC isolated from the tumor were double-stained with PE-conjugated antibodies against CD1a (IgG2a) (Dako), CD1b (IgG1) (Becton Dickinson), CD1c (IgG1) (Immunotech), CD1d (51.1.3, IgG2b) kindly provided by Dr. S. P. Balk (Harvard Medical School), and HLA-DR (FITC-conjugated) (Becton Dickinson). In some experiments DC were also stained with the following PE or FITC-conjugated monoclonal antibodies: anti-CD11a (IgG2a), anti-CD11b (IgG1), anti-CD11c (IgG1), anti-CD14 (IgG2a), anti-CD80 (IgG1), anti-CD83 (IgG1), and anti-CD86 (IgG1) (all from Becton Dickinson). Each antibody was diluted in PBS containing 2% FCS at a concentration of 10 μg/ml and cells were stained at 4°C for 30 minutes in the dark. The corresponding isotype-matched antibodies were used as negative controls (all from Becton Dickinson). Flow cytometry was performed using a FACSCalibur, and data were subsequently analyzed using CellQuest (both from Becton Dickinson). Supernatants were prepared by culturing 106 melanoma cells or normal fibroblasts in 25-cm2 flasks (Becton Dickinson) with 10 ml of complete RPMI. Supernatants were collected after 24 hours, centrifuged to remove cells and debris, and then stored at −80°C as previously described.31Kiertscher SM Luo J Dubinett SM Roth MD Tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells.J Immunol. 2000; 164: 1269-1276PubMed Google Scholar Cell culture supernatants from primary and metastatic melanoma cell lines, as well as from melanoma cells 12 hours after isolation from the tumor were pre-incubated for 1 hour at 4°C with 0, 0.1, 1, and 10 μg/ml of a monoclonal neutralizing anti-IL-10 antibody (R&D Systems). An irrelevant antibody of the same isotype (IgG2b) (R&D Systems) was used as negative control at 10 μg/ml. Cell culture supernatants were prepared by culturing 106 melanoma cells or normal fibroblasts in 25-cm2 flasks with 10 ml of complete RPMI. IL-10 in the supernatants was measured using a Quantikine HS Human IL-10 ELISA kit from R&D Systems following the manufacturer's guidelines. The detection limit was less than 3.9 pg/ml. Data were statistically analyzed using the Student's t-test and results were considered significant when P < 0.05. Expression of CD1 molecules and HLA-DR was studied by immunohistochemical staining of serial cryosections of primary (n = 10) and metastatic (n = 10) melanoma biopsies as well as normal skin samples (n = 10) from healthy donors. Each sample was obtained from a different donor. In normal skin, CD1a-positive epidermal Langerhans cells and some scattered CD1a, b, c, d-positive dermal DC were observed mainly in the papillary dermis and in a perivascular localization as previously described (data not shown).32Gerlini G Hefti HP Kleinhans M Nickoloff BJ Burg G Nestle FO Cd1d is expressed on dermal dendritic cells and monocyte-derived dendritic cells.J Invest Dermatol. 2001; 117: 576-582Crossref PubMed Scopus (51) Google Scholar Primary melanoma lesions showed numerous large dendritic mononuclear cells staining positive for CD1 a, b, c, d located in the dermal intra- and peritumoral infiltrate. Figure 1A shows CD1c and is representative for CD1 molecule expression in primary melanoma sections examined. Staining of serial primary melanoma sections showed HLA-DR expression by large mononuclear dendritic cells (Figure 1B). In metastatic melanoma sections, CD1a, b, c, d expression by DC was consistently reduced and often undetectable. Figure 1C shows a representative CD1c staining of metastatic melanoma which is representative for CD1 molecule expression in metastatic lesions. In contrast, HLA-DR was still strongly expressed by large mononuclear cells with dendritic processes (Figure 1D). To further support these data, cell suspensions from metastatic melanoma lesions and a primary melanoma sample were prepared. Flow cytometry analysis showed that the percentages of CD1a, b, c, d/HLA-DRbright double-positive cells in the primary melanoma was higher than in the metastatic melanoma tested (data not shown). To further demonstrate in situ CD1 down-regulation on HLA-DR-positive cells in metastatic but not in primary melanoma, confocal microscopy was performed. Figure 1E shows representative CD1b/HLA-DR double-immunofluorescence stainings of primary and metastatic melanoma cryosections. CD1b-positive (green) and CD1b/HLA-DR double-positive cells (yellow) could be detected in primary melanomas (top row) whereas CD1b was down-regulated in metastatic melanoma lesions (bottom row). HLA-DR expression levels were comparable in both primary and metastatic melanoma (red). These data support the immunohistochemical results showing that CD1 molecules, but not HLA-DR, are down-regulated in metastatic melanoma lesions compared to primary melanoma. To quantify CD1a, b, c, d and HLA-DR down-regulation in primary (n = 10) and metastatic (n = 10) melanoma lesions, the numbers of CD1 and HLA-DR expressing cells in serial sections were assessed by counting the positive cells in five different microscopic fields (original magnification, ×400). The mean values were thereafter calculated as previously described.14Sieling PA Jullien D Dahlem M Tedder TF Rea TH Modlin RL Porcelli SA CD1 expression by dendritic cells in human leprosy lesions: correlation with effective host immunity.J Immunol. 1999; 162: 1851-1858PubMed Google Scholar To correct for the density of infiltrating DC, the ratios between CD1a, b, c- and HLA-DR-positive cells were thereafter calculated for each sample. A significantly lower mean number of CD1a, b, c, d-positive cells relative to HLA-DR-positive cells was observed in metastatic compared to primary lesions. The CD1/HLA-DR ratios in metastatic compared to primary melanomas were as follows: for CD1a, 0.02 ± 0.03 versus 0.4 ± 0.1 (P < 0.001); for CD1b, 0.04 ± 0.05 versus 0.3 ± 0.1 (P < 0.001); for CD1c, 0.1 ± 0.1 versus 0.5 ± 0.2 (P < 0.001); and for CD1d, 0.1 ± 0.1 versus 0.2 ± 0.05 (P < 0.001) (Figure 2). The mean numbers of HLA-DR-positive cells in primary (35.1 ± 5.4) and metastatic (31 ± 4.5) melanoma did not differ significantly (P = 0.335) (data not shown). IL-10 is believed to be involved in the suppression of an anti-tumor immune response and could be detected at high levels in the blood of metastatic melanoma patients compared to primary melanoma patients and healthy individuals.26Nemunaitis J Fong T Shabe P Martineau D Ando D Comparison of serum interleukin-10 (IL-10) levels between normal volunteers and patients with advanced melanoma.Cancer Invest. 2001; 19: 239-247Crossref PubMed Scopus (111) Google Scholar, 33Dummer W Becker JC Schwaaf A Leverkus M Moll T Brocker EB Elevated serum levels of interleukin-10 in patients with metastatic malignant melanoma.Melanoma Res. 1995; 5: 67-68Crossref PubMed Scopus (127) Google Scholar To study IL-10 expression in melanoma lesions in situ, immunohistochemistry was performed on primary as well as metastatic melanoma cryosections. Faint IL-10 staining was detected in suprabasal keratinocytes but not in melanoma cells of primary melanoma sections (n = 10). In contrast, metastatic lesions (n = 10) showed strong IL-10 staining in most of the melanoma cells. Figure 3, A and B, show representative IL-10 staining in primary and metastatic melanoma lesions, respectively. Some melanin-producing melanoma cells can be seen in Figure A (brown color). To further compare IL-10 protein secretion by primary and metastatic melanoma cells, we performed enzyme-linked immunosorbent assays (ELISA) using supernatants from primary (n = 3) and metastatic (n = 21) melanoma cell lines, as well as from fibroblast cultures (n = 3). Metastatic melanoma cell lines were divided into first passage metastatic melanoma cell cultures (n = 3), short-term (two passages, n = 7), and long-term cultures (> two passages, n = 11). Primary melanoma and fibroblast cultures were in continuous culture for two passages. The highest IL-10 concentrations could be detected in metastatic melanoma supernatants of first passage cultures (91 to 306 pg/ml, mean 199.6 ± 87.8 pg/ml) and were significantly higher (P < 0.05) than in second passage metastatic melanoma cultures (0.1 to 31.6 pg/ml, mean 14.6 ± 11.2 pg/ml), and in long-term metastatic melanoma cultures (0 to 0.6 pg/ml, mean 0.1 ± 0.2 pg/ml, Figure 4). No IL-10 could be detected in the supernatants of primary melanoma cell lines. The difference in IL-10 levels between short-term primary and both short-term and long-term metastatic melanoma cell line supernatants were statistically significant (P < 0.05). The significant decrease of IL-10 from passage one to passage two (P < 0.05) and from passage two to long-term cultures (P < 0.05) suggests that cultured melanoma cells gradually loose the capacity to produce IL-10. Only low amounts of IL-10 were found in the supernatants from fibroblast cell cultures (0 to 0.2 pg/ml, mean 0.1 ± 0.1 pg/ml) (Figure 4). Dendritic cells in the blood of tumor patients have been shown to be reduced in numbers and function.34Almand B Resser JR Lindman B Nadaf S Clark JI Kwon ED Carbone DP Gabrilovich DI Clinical significance of defective dendritic cell differentiation in cancer.Clin Cancer Res. 2000; 6: 1755-1766PubMed Google Scholar To rule out the possibility that monocyte-derived DC from patients suffering from metastatic melanoma have inherently altered CD1 expression, monocyte-derived DC were generated from PBMC of melanoma patients (n = 3) as well as healthy individuals (n = 3) and CD1 expression was compared after 5 days using flow cytometry. No significant difference in CD1a, b and c expression levels could be observed (data not shown). Down-regulation of CD1d molecules by IL-10 treatment was not assessed since FCS-cultured monocyte-derived DC bear little or no CD1d, as previously described.32Gerlini G Hefti HP Kleinhans M Nickoloff BJ Burg G Nestle FO Cd1d is expressed on dermal dendritic cells and monocyte-derived dendritic cells.J Invest Dermatol. 2001; 117: 576-582Crossref PubMed Scopus (51) Google Scholar It has previously been demonstrated that IL-10 added at the beginning of the culture inhibits expression of CD1 molecules on GMCSF/IL-4-treated monocytes and that IL-10 down-regulates CD1a on monocyte-derived DC.12Fortsch D Rollinghoff M Stenger S IL-10 converts human dendritic cells into macrophage-like cells with increased antibacterial activity against virulent Mycobacterium tuberculosis.J Immunol. 2000; 165: 978-987PubMed Google Scholar, 35Thomssen H Kahan M Londei M Differential effects of interleukin-10 on the expression of HLA class II and CD1 molecules induced by granulocyte/macrophage colony-stimulating factor/interleukin-4.Eur J Immunol. 1995; 25: 2465-2470Crossref PubMed Scopus (37) Google Scholar We therefore studied whether IL-10 can down-regulate CD1a, CD1b, CD1c expression on our monocyte-derived DC preparations. After 3 days in culture, DC exhibited the features of immature DC: the majority of cells was in suspension and showed a dendritic phenotype. Flow cytometry analysis showed high expression levels of HLA-DR (98.3 ± 0.6% of cells; mean fluorescence intensity (MFI), 326.4 ± 35.1), CD11a (97.7 ± 1.3%; MFI, 214.2 ± 31.1), CD11b (95.5 ± 1%; MFI, 69.9 ± 12.1) and CD11c (98.8 ± 0.5%; MFI, 156.8 ± 26.5). CD80 and CD86 expression could be detected on 23.8 ± 9.7% and 48.6 ± 11.5% of DC, respectively. Only 29.4 ± 2.5% of DC expressed CD14, which was a 4.7-fold decrease compared to fresh monocytes (MFI 124.1 ± 18.0 versus 588.3 ± 23.1, respectively). A certain degree of spontaneous maturation was observed on 5.7 ± 2.3% of cells that expressed CD83 molecules. At this time point the majority of the cells stained positive for CD1 molecules: CD1a, 87.8 ± 6.8%; CD1b, 88.0 ± 5.3%; and CD1c, 95.7 ± 2.2% (data not shown). Cells were further cultured for 48 and 72 hours in the presence of 800 IU/ml GM-CSF and 1000 IU/ml IL-4 with or without IL-10 (1, 10, and 100 ng/ml). Flow cytometry analysis of DC cultured in the absence of IL-10 showed that the MFI of HLA-DR (469.7 ± 12.9), CD11a (288.9 ± 8.4), CD11b (81.0 ± 9.8), and CD11c (210.6 ± 11.1) was further up-regulated after 72 hours although no significant changes in the percentage of positive cells was observed. Similarly, CD80 and CD86 expression levels were further increased and could be detected on 33.5 ± 9.5% and 77.8 ± 4.9% of DC, respectively. In contrast, CD14 was no longer detectable. A dose-dependent down-regulation of CD1a, b, c expression on IL-10-treated, monocyte-derived DC could be observed after 48 hours (data not shown) and was even stronger after 72 hours (Figure 5A). Relative to DC cultured in the absence of IL-10, the decrease of the MFI of IL-10-treated, monocyte-derived DC using 1, 10, and 100 ng/ml was as follows: CD1a: 15% (P = 0.260), 48% (P < 0.05), 80% (P < 0.01); CD1b: 34% (P < 0.01), 39% (P < 0.01), 64% (P < 0.01), and CD1c: 32% (P = 0.080), 46% (P < 0.01), 68% (P < 0.01). Previous studies have shown that IL-10 treatment also affects co-stimulatory molecule expression by DC.36Buelens C Willems F Delvaux A Pierard G Delville JP Velu T Goldman M Interleukin-10 differentially regulates B7–1 (CD80) and B7–2 (CD86) expression on human peripheral blood dendritic cells.Eur J Immunol. 1995; 25: 2668-2672Crossref PubMed Scopus (248) Google Scholar, 37Brown RD Pope B Murray A Esdale W Sze DM Gibson J Ho PJ Hart D Joshua D Dendritic cells from patients with myeloma are numerically normal but functionally defective as they fail to up-regulate CD80 (B7–1) expression after huCD40LT stimulation because of inhibition by transforming growth factor-beta1 and interleukin-10.Blood. 2001; 98: 2992-2998Crossref PubMed Scopus (305) Google Scholar, 38Corinti S Albanesi C la Sala A Pastore S Girolomoni G Regulatory activity of autocrine IL-10 on dendritic cell functions.J Immunol. 2001; 166: 4312-4318PubMed Google Scholar We confirmed that IL-10
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