Artigo Acesso aberto Revisado por pares

Interleukin-10 Secretion Differentiates Dendritic Cells from Human Liver and Skin

2004; Elsevier BV; Volume: 164; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63141-0

ISSN

1525-2191

Autores

Sarah Goddard, Janine Youster, Emma Morgan, David H. Adams,

Tópico(s)

T-cell and B-cell Immunology

Resumo

Liver dendritic cells (DCs), which may orchestrate the liver's unique immunoregulatory functions, remain poorly characterized. We used a technique of overnight migration from pieces of normal human liver and skin to obtain tissue-derived DCs with minimal culture and no additional cytokine treatment. Liver and skin DCs had a monocyte-like morphology and a partially mature phenotype, expressing myeloid markers, MHCII, and co-stimulatory molecules; but only the skin DCs contained a population of CD1a+ cells. Overnight-migrated liver DCs activated naïve cord blood T cells efficiently. Liver DCs produced interleukin (IL)-10 whereas skin DCs failed to secrete IL-10 even after stimulation and neither skin nor liver-derived DCs secreted significant amounts of IL-12p70. Compared with skin DCs, liver DCs were less effective at stimulating T-cell proliferation and stimulated T cells to produce IL-10 and IL-4 whereas skin DCs were more potent stimulators of interferon-γ and IL-4. Monocyte-derived DCs were down-regulated after culture with liver-conditioned media, suggesting that local microenvironmental factors may be important. Thus we show for the first time clear tissue-specific differences in nonlymphoid DCs. Although it is not possible to conclude from our data whether liver DCs are more regulatory, or skin DCs more proimmunogenic, the ability of liver DCs to secrete IL-10 may be important for regulating local immune responses within the liver in the face of constant exposure to gut antigens. Liver dendritic cells (DCs), which may orchestrate the liver's unique immunoregulatory functions, remain poorly characterized. We used a technique of overnight migration from pieces of normal human liver and skin to obtain tissue-derived DCs with minimal culture and no additional cytokine treatment. Liver and skin DCs had a monocyte-like morphology and a partially mature phenotype, expressing myeloid markers, MHCII, and co-stimulatory molecules; but only the skin DCs contained a population of CD1a+ cells. Overnight-migrated liver DCs activated naïve cord blood T cells efficiently. Liver DCs produced interleukin (IL)-10 whereas skin DCs failed to secrete IL-10 even after stimulation and neither skin nor liver-derived DCs secreted significant amounts of IL-12p70. Compared with skin DCs, liver DCs were less effective at stimulating T-cell proliferation and stimulated T cells to produce IL-10 and IL-4 whereas skin DCs were more potent stimulators of interferon-γ and IL-4. Monocyte-derived DCs were down-regulated after culture with liver-conditioned media, suggesting that local microenvironmental factors may be important. Thus we show for the first time clear tissue-specific differences in nonlymphoid DCs. Although it is not possible to conclude from our data whether liver DCs are more regulatory, or skin DCs more proimmunogenic, the ability of liver DCs to secrete IL-10 may be important for regulating local immune responses within the liver in the face of constant exposure to gut antigens. The unique properties of dendritic cells (DCs) allow them to take up and process antigen from nonlymphoid tissues such as the liver, migrate to lymph nodes, and initiate primary immune responses.1Steinman RM The dendritic cell system and its role in immunogenicity.Annu Rev Immunol. 1991; 9: 271-296Crossref PubMed Scopus (4254) Google Scholar Since a method for generating DCs from human monocytes was established,2Sallusto F Lanzavecchia A Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4475) Google Scholar the ability of these cells to initiate strong immune responses has been appreciated and exploited for instance in the treatment of malignancy.3Nestle FO Alijagic S Gilliet M Sun Y Grabbe S Dummer R Burg G Schadendorf D Vaccination of melanoma patients with peptide or tumour lysate-pulsed dendritic cells.Nat Med. 1998; 4: 328-333Crossref PubMed Scopus (2680) Google Scholar However it has been difficult to study DCs from normal human tissue where they are likely to play a crucial role in regulating local immune responses. This may be particularly true for the liver where there is a need to tightly control immune responses to the many antigens entering the liver from the gut via the portal vein.4Knolle PA Gerken G Local control of the immune response in the liver.Immunol Rev. 2000; 174: 21-34Crossref PubMed Scopus (488) Google ScholarDCs freshly isolated from nonlymphoid organs are immature.5Austyn JM Hankins DF Larsen CP Morris PJ Rao AS Roake JA Isolation and characterization of dendritic cells from mouse heart and kidney.J Immunol. 1994; 152: 2401-2410PubMed Google Scholar They are unable to act as antigen-presenting cells and lack cell surface class II, which is found within the cell but not at the cell membrane.6Pierre P Turley SJ Gatti E Hull M Meltzer J Mirza A Inaba K Steinman RM Mellman I Developmental regulation of MHC class II transport in mouse dendritic cells [see comments].Nature. 1997; 388: 787-792Crossref PubMed Scopus (641) Google Scholar They do however display good antigen-capturing abilities, with high levels of expression of toll-like receptors, Fc receptors, complement and mannose receptors, and the ability to take up antigen by a number of mechanisms.7Kadowaki N Antonenko S Liu YJ Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c(−) type 2 dendritic cell precursors and CD11c(+) dendritic cells to produce type I IFN.J Immunol. 2001; 66: 2291-2295Google Scholar In the normal state there is a steady movement of DCs from peripheral tissue via the lymph to the secondary lymphoid tissue,8Huang FP Platt N Wykes M Major JR Powell TJ Jenkins CD MacPherson GG A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes.J Exp Med. 2000; 191: 435-444Crossref PubMed Scopus (777) Google Scholar and this trafficking is markedly increased in the presence of an immunogenic stimulus.9Macatonia SE Knight SC Edwards AJ Griffiths S Fryer P Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional and morphological studies.J Exp Med. 1987; 166: 1654-1667Crossref PubMed Scopus (518) Google Scholar Migration of DCs from the liver to draining lymph nodes has been demonstrated in a rat model by following the movement of cells that take up colloidal carbon in the hepatic sinusoids and migrate via the portal tract to the lymphatics and draining nodes.10Matsuno K Ezaki T Kudo S Uehara Y A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph [see comments].J Exp Med. 1996; 183: 1865-1878Crossref PubMed Scopus (240) Google Scholar It is clear from this elegant work, that immature liver DCs enter the liver from the circulation via the sinusoids where they take up antigens and express many pattern recognition receptors in common with macrophages, eg, CD14. On maturation they express chemokine receptors that direct them to the lymph vessels of the portal tracts, where they express more typical markers of DCs. These migratory cells when isolated from lymph have an intermediate phenotype, lacking phagocytic function but with less than maximum levels of class II expression. Similar cells have been identified migrating from sections of skin in culture.11Larsen CP Steinman RM Witmer-Pack M Hankins DF Morris PJ Austyn JM Migration and maturation of Langerhans cells in skin transplants and explants.J Exp Med. 1990; 172: 1483-1493Crossref PubMed Scopus (581) Google ScholarIt is difficult to examine the function of normal tissue DCs in the steady state, but these cells, which continually traffic to the lymph nodes, may play a critical role in maintaining local tolerance. There is evidence that CD4 T-cell tolerance requires the presentation of antigen by a bone marrow-derived cell,12Adler AJ Marsh DW Yochum GS Guzzo JL Nigam A Nelson WG Pardoll DM CD4+ T cell tolerance to parenchymal self-antigens requires presentation by bone marrow-derived antigen-presenting cells.J Exp Med. 1998; 187: 1555-1564Crossref PubMed Scopus (244) Google Scholar and DCs have been identified as agents in the generation of T-cell tolerance in vivo.13Kurts C Kosaka H Carbone FR Miller JF Heath WR Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.J Exp Med. 1997; 186: 239-245Crossref PubMed Scopus (607) Google Scholar, 14Dhodapkar MV Steinman RM Krasovsky J Munz C Bhardwaj N Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells.J Exp Med. 2001; 193: 233-238Crossref PubMed Scopus (1203) Google Scholar, 15Martin E O'Sullivan B Low P Thomas R Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10.Immunity. 2003; 18: 155-167Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar However, the study of such mechanisms is hindered by the difficulties in isolating cells in sufficient numbers from tissue and the comparative lack of specific markers to positively identify immature DCs in the human. Furthermore cells isolated directly from tissue require maturation before developing the ability to present antigen.16Lu L Woo J Rao AS Guzzo JL Nigam A Nelson WG Pardoll DM Watkins SC Qian S Starzl TE Demetris AJ Thomson AW Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen.J Exp Med. 1994; 179: 1823-1834Crossref PubMed Scopus (223) Google Scholar, 17Woo J Lu L Rao AS Li Y Subbotin V Starzl TE Thomson AW Isolation, phenotype, and allostimulatory activity of mouse liver dendritic cells.Transplantation. 1994; 58: 484-491Crossref PubMed Scopus (94) Google Scholar To overcome some of these problems we have adapted a technique used for skin to isolate DCs migrating out of human liver tissue.To determine whether differences in tissue-specific DCs contribute to organ-specific immune responses we compared the ability of DCs isolated using similar techniques from human skin and liver tissue to secrete cytokines and stimulate lymphocyte responses.Materials and MethodsHuman Liver and Skin TissueNormal liver tissue was obtained from liver available after reduction of donor liver for transplantation into children, after partial hepatectomy for colorectal metastases and benign focal disease: cystadenoma (n = 2), hemangioma (n = 1), follicular nodular hyperplasia (n = 2). Tissue used was at least 3 cm from tumor. Normal skin was obtained from tissue removed during plastic surgery. In some cases peripheral blood was collected during surgery and peripheral blood mononuclear cells (PBMCs) were isolated over Ficoll (Lymphoprep) gradients and cryopreserved. All tissue and blood samples were obtained with the informed consent of patients and after approval from the local ethics committee.Migration and Isolation of CellsTissue (1- to 2-mm-thick wafers) were collected from the liver, and cut into 0.5-cm2 squares. At least 12 g of tissue were collected from each liver and after washing in phosphate-buffered saline were incubated in six-well plates and cultured overnight in complete media (RPMI 1640, 10% fetal calf serum, 2 mmol/L glutamine, 100 μg/ml streptomycin, and 100 U/ml penicillin). Human skin DCs were isolated as previously described18Richters CD Hoekstra MJ van Baare J Du PJ Hoefsmit EC Kamperdijk EW Isolation and characterization of migratory human skin dendritic cells.Clin Exp Immunol. 1994; 98: 330-336Crossref PubMed Scopus (39) Google Scholar by floating full thickness skin pieces on complete media. The skin tissue was cultured overnight in the same way as the liver DCs. Nonadherent cells that migrated out of the tissue into the media were collected. To study the result of further culture on maturation, the migrated cells were purified by isolation over a Ficoll (Lymphoprep metrizamide, Nygaard, Oslo, Norway) gradient, and cultured in smaller wells at 2 × 106Pierre P Turley SJ Gatti E Hull M Meltzer J Mirza A Inaba K Steinman RM Mellman I Developmental regulation of MHC class II transport in mouse dendritic cells [see comments].Nature. 1997; 388: 787-792Crossref PubMed Scopus (641) Google Scholar cells/ml, in complete media for a further 2 to 3 days. In all cases DCs were finally isolated by centrifugation over a 14.5% metrizamide gradient. Twelve percent Optiprep (Nycomed UK Ltd, Oxford, UK) in complete media was used for experiments at later time points.19McLellan AD Starling GC Hart DN Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation.J Immunol Methods. 1995; 184: 81-89Crossref PubMed Scopus (54) Google Scholar The yield was typically 0.5 × 105Austyn JM Hankins DF Larsen CP Morris PJ Rao AS Roake JA Isolation and characterization of dendritic cells from mouse heart and kidney.J Immunol. 1994; 152: 2401-2410PubMed Google Scholar cells/g of liver tissue. For assessment of function pure populations of DCs were obtained by negative selection. CD4, CD8, and CD15 antibodies were used to remove remaining lymphocytes and neutrophils by depletion using anti-Fc-conjugated Dynabeads (Dynal). Flow cytometry and immunocytochemistry of cytospins was used to confirm purity.Monocyte-derived DCs (mDCs) were produced by isolating PBMCs from human peripheral blood using a Lymphoprep gradient and selecting CD14+ve cells using microbeads (Miltenyi Biotech) and columns. The cells were then cultured for 6 days in GM-CSF (800 U/ml) and interleukin (IL)-4 (500 U/ml) in complete media. The purity of these DCs was >95% by flow cytometry. These cells were used to compare the effects of media from cultured skin and liver on DC function. mDCs were cultured overnight in conditioned tissue culture media derived from 18-hour cultures of skin and liver tissue. Cells and debris were removed from the conditioned media by centrifugation. Fresh complete media was used as a control. The cells were then washed and compared in proliferation assays. The results were tested for significance by analysis of variance.ReagentsThe following unconjugated mouse anti-human antibodies were used to stain cells by flow cytometry and immunocytochemistry: UCHM-1 (CD14) and HB15A17.11 (CD83) from Serotec; 2LPM19c (CD11b), BU63 (CD86), UCHT-1 (CD3), MT310 (CD4), C8/144B (CD8), HD37 (CD19), EBM11 (CD68), DJ130c (CD16), and CR3/43 (HLA-DR) from DAKO; B-Ly6 (CD11c), HI149 (CD1a), and 5C3 (CD40) from Pharmingen; BDCA-2 and CD1c from Miltenyi Biotec; CD57 from Biogenics; 2G5 (CXCR4) and 120507 (DC-sign) from R&D; 7H12 (CCR7) kindly provided by Lijun Wu (Millenium Pharmaceuticals Inc., Cambridge, MA); 2D7 (CCR5) kindly provided by S. Qin (Millenium Pharmaceuticals Inc.), and for dual-color flow cytometry, phycoerythrin-conjugated L243 (HLA-DR) from Pharmingen. Mouse immunoglobulin isotype controls were obtained from Pharmingen.For flow cytometry primary antibody staining was detected using fluorescein isothiocyanate-conjugated rabbit anti-mouse Fab from DAKO. For immunocytochemistry, primary antibodies were detected using rabbit anti-mouse immunoglobulin followed by mouse monoclonal alkaline phosphatase-anti-alkaline phosphatase (APAAP) (DAKO).Immunostaining and Flow CytometryCytospins and frozen sections were prepared and stored at −20°C until use. They were then fixed for 5 minutes in acetone and stained using a standard APAAP technique.20Afford SC Rhandawa S Eliopoulos AG Hubscher SG Young LS Adams DH CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface FasL expression and amplifies Fas mediated hepatocyte death during allograft rejection.J Exp Med. 1999; 189: 441-446Crossref PubMed Scopus (164) Google Scholar Briefly, primary antibody was followed by rabbit anti-mouse monoclonal and mouse monoclonal APAAP (DAKO). The stain was developed with fast red and naphthol AS-MX phosphate substrate (Sigma).Flow cytometry was used to assess phenotype of class II-positive cells. Cells were preincubated in human Ig to block Fc receptors, then stained with mouse anti-human primary antibody, followed by rabbit anti-mouse fluorescein isothiocyanate. Cells were incubated in 10% mouse serum before final incubation in phycoerythrin-class II antibody. For intracellular cytokine staining DCs were stimulated for 5 hours with 5 ng/ml of PMA and 500 ng/ml of ionomycin (Sigma), and cytokine export blocked with monensin (Pharmingen) used according to the manufacturer's instructions. The cells were fixed using 4% paraformaldehyde and permeabilized with 0.5% saponin. Cell surface markers were stained, eg, CD11c followed by rabbit anti-mouse fluorescein isothiocyanate, before fixation and permeabilization, and phycoerythrin-conjugated antibodies to cytokine or the recommended control antibodies from Pharmingen were used to stain permeabilized cells.Viability of cells was assessed using Viaprobe (7-amino-actinomycin D), Pharmingen. Analysis was performed on an Epics XL cytometer (Coulter). Data were processed using WinMDI version 2.4.Electron MicroscopyAfter migration from liver and skin, cells were either cultured for 2 further days in complete media or were purified immediately. Cells were fixed in 2% glutaraldehyde, and sections prepared and viewed by the University of Birmingham Electron Microscopy Centre.Mixed DC and Lymphocyte ResponseThe ability of the cells to activate naïve T cells was measured in a mixed DC/lymphocyte response (MLR). Cord blood was collected and the mononuclear cells isolated by separation over a Lymphoprep gradient. The antigen-presenting function of overnight-migrated DCs from normal liver was compared with PBMCs from the same patient. Graded numbers of irradiated (3000 rads) stimulator cells were added to 2 × 105 allogeneic cord blood mononuclear cells in 100 μl of complete media, in 96-well plates. The cells were cultured for 5 days and 1 μCi of 3H-thymidine was added for the last 18 hours. Thymidine uptake was counted on a β-plate counter and a mean of triplicate wells calculated (n = 3).To compare the function of tissue-specific DCs, pure skin (n = 3) and liver DCs (n = 6) were isolated by negative selection and compared using a MLR; in this case, pure T cells were isolated by negative selection from adult blood, using CD19, CD11b, CD14, and CD56 antibodies and used as responders. DCs cultured overnight in skin or liver tissue conditioned media, were compared with DCs cultured in complete media using the same proliferation assay (n = 4). Data were compared by analysis of variance.Detection of IL-10 and IL-12 by Enzyme-Linked Immunosorbent AssayOvernight-migrated cells were cultured in 96-well plates at 106 cells/ml in complete media supplemented with 50 ng/ml of GM-CSF. In further experiments cells were stimulated with the addition of 10 ng/ml of interferon (IFN)-γ and 10 to 100 ng/ml of lipopolysaccharide (LPS) (Sigma) (higher concentrations were tested in experiments to detect IL-12p70), or IFN-γ and CD40 ligation. CD40L-transfected J558 cells21Lane P Burdet C McConnell F Lanzavecchia A Padovan E CD40 ligand-independent B cell activation revealed by CD40 ligand-deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation.Eur J Immunol. 1995; 25: 1788-1793Crossref PubMed Scopus (64) Google Scholar were paraformaldehyde-fixed and washed before adding to the cells at a ratio of one cell to three DCs, for the IL-12 studies 1 μg/ml of soluble CD40L with enhancer (Alexis) was also tested. Media was saved and stored at −70°C. IL-10 and IL-12p70 were measured in the media using OPTeia kits produced by Pharmingen. The standard enzyme-linked immunosorbent assay protocol recommended by the company was followed. The results were compared by analysis of variance.DC Stimulation of T CellsA DC and T-cell MLR was used to study the T-cell responses generated by the DCs from skin and liver. Pure T cells were co-cultured in complete media for 5 days at 1:20 stimulator:responder ratios and the cells were then restimulated for 5 hours with anti-CD3-coated plates and soluble anti-CD28, in the presence of monensin. Pure T cells without co-culture with DCs were restimulated in the same way, and used as a control. The cells were stained for intracellular cytokine as described before. Statistical significance was assessed for each cytokine using a Mann-Whitney U-test and the difference in expression of all cytokines by each type of DC was assessed by analysis of variance.Detection of IL-12 Expression by Real-Time Polymerase Chain Reaction (PCR)Pure overnight-migrated DCs isolated as described above were stored at −70°C. RNA was isolated later using an RNeasy kit (Qiagen) including a DNase step to remove any contaminating genomic DNA.The RNA was reverse-transcribed using superscript II reverse transcriptase (Invitrogen). The real-time PCR was set up using TaqMan Universal PCR Mastermix, a primer/probe mix specific to the gene of interest, and a primer/probe mix specific to 18S rRNA control reagent. The following primer/probe sequences were kindly designed by David Fitzpatrick, Immunex: IL-12 p35 forward primer 5′-gcc act cca gac cca gga a-3′, IL-12 p35 reverse primer 5′-gac ggc cct cag cag gtt-3′, IL-12 p35 probe ttc cca tgc ctt cac cac tcc ca, IL-12 p40 forward primer 5′-ctt cac cga caa gac ctc agc-3′, IL-12 p40 reverse primer 5′-aga tga gct ata gta gcg gtc ctg-3′, IL-12 p40 probe tca tct gcc gca aaa atg cca gc. The primer/probe mix for 18S was kindly supplied by Kai Toellner, Department of Immunology, University of Birmingham. The plate was run in the Applied Biosystems Prism 7700 sequence detector using Sequence Detection System analysis software. The PCR conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, then 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The software then analyzed the data and output a pair of cycle threshold (ct) values for each sample, one for the gene of interest (labeled FAM) and one for the 18S housekeeping control (labeled VIC). The ct values were then transported to a Microsoft Excel spreadsheet and analyzed to give a value representing the relative mRNA levels present for the gene of interest linearly as per the manufacturer's instructions.ResultsOvernight-Migrated Cells Have a Partially Mature Phenotype and a Proportion Become Fully Mature in CultureAfter a few hours in culture, cells that had migrated out of the tissue were seen in the media. The rate of emigration fell after 24 hours and we therefore used an overnight migration.Cytospin preparations of overnight-migrated cells showed they were monocyte-like with an eccentric oval nucleus and more cytoplasm than lymphocytes (Figure 1A), whereas electron microscopy showed that the cells contained numerous vesicles, some electron opaque (Figure 1B). Using immunocytochemistry, they displayed strong staining for class II, CD86, and CD11b; weaker staining for CD11c, CD40, and ICAM-1; but little detectable CD83. Neither CD68 nor CD16 was present in DCs from normal liver. With further culture in complete media 25 to 50% of class II-positive cells from 3-day cultured samples increased in size and looked like typical mature DCs (Figure 1C), by electron microscopy the cells showed less dense cytoplasm, complex long processes/veils, and an open-lobed nucleus. The cultured cells had increased surface expression for class II and the co-stimulatory molecules compared with the overnight-migrated cells.The cells were analyzed using dual-color flow cytometry (Figure 2, A and B, and Table 1) with DCs identified by surface class II staining. In general the surface staining confirmed the findings noted using immunocytochemistry. Class II-gated cells were CD16-, CD3-, CD19-, and CD57-negative, and they had a myeloid phenotype expressing CD11c, CD1c, low levels of CD123 and DC-SIGN, and no BDCA-2. Flow cytometry of cells cultured further shows the same heterogeneity as that seen with immunocytochemistry, with 25 to 50% of the class II cells developing a mature phenotype. These cells express increased levels of co-stimulatory molecules, CD86, CD40, and also CD83 (Figure 2B). Confirming the process of maturation, CD14 and CD11b, were detected on 50% and 90% of cells, respectively, after overnight migration but were down-regulated on class II high cells after further culture. Chemokine receptor expression by migrated cells was also assessed (Figure 2A). Most overnight-migrated liver DCs expressed CXCR4 whereas only those cells with high class II expression were CCR7+. A low level of CCR5 was detectable.Figure 2Phenotype of liver DCs by flow cytometry. A: Overnight-migrated liver DCs were characterized by dual-color flow cytometry. Cells shown were gated on scatter characteristics and positive staining for class II. The control staining is shown as an unfilled histogram. B: Two-color plots show the emergence of mature high class II-expressing cells after culture in plain media for a further 2 days. The cells were isolated using a gradient only therefore some class II-/CD11b+ neutrophils are present.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Phenotype of Liver Dendritic Cells after Migration and after MaturationOvernight migratedClass II high population after maturationClass II++++++++ (Increased level of expression)CD40+++++CD86++++++++ (Increased level of expression)ICAM++++++++CD11c++++++++CD11b++++++CD14+++CD83++++CD16−−CD68− (Normal liver)−CD1c++++CD1a−−CD123++BDCA-2−−Assessment made using flow cytometry from at least five separate experiments. No expression, −; <20%, +; 20 to 50%, ++; 50 to 75%, +++; 75 to 100%, ++++. Open table in a new tab Skin DCs isolated by migration have been previously characterized and our findings were consistent with published reports.11Larsen CP Steinman RM Witmer-Pack M Hankins DF Morris PJ Austyn JM Migration and maturation of Langerhans cells in skin transplants and explants.J Exp Med. 1990; 172: 1483-1493Crossref PubMed Scopus (581) Google Scholar The only notable difference between liver and skin DCs was in expression of CD1a. In our study 70% of the cells isolated from full thickness skin were CD1a+, whereas none of the liver DCs expressed CD1a. In addition none of the normal liver sections studied by immunohistochemistry contained any CD1a+ cells although very rare CD1a+ cells were detected in liver tissue from patients with inflammatory liver disease (Figure 1D).Overnight-Migrated Cells Are Able to Activate Cord Blood T Cells, but Are Poor Stimulators in Comparison to Skin-Derived CellsTo confirm that the liver DCs were functional we compared the ability of overnight-migrated DCs and syngeneic PBMCs to stimulate T-cell proliferation in a modified MLR (Figure 3A). The stimulator cells were added to 1.5 × 105 allogeneic cord blood mononuclear cells. A peak response was elicited by liver-derived cells at a ratio of 1/10 to 20 stimulator cells/cord blood mononuclear cells in a 5-day assay whereas PBMCs were unable to elicit a proliferative response. However when purified liver DCs were compared with skin DCs in an alloproliferative response using pure adult T cells as responders the liver DCs were markedly less efficient at inducing T-cell proliferation, P < 0.001 (Figure 3B).Figure 3Capacity of liver DCs to induce proliferation of allogeneic T cells. A: Migrated liver-derived DCs stimulate naïve T-cell proliferation. Proliferation of T cells in response to liver-derived DCs was measured in a thymidine incorporation assay. Varying numbers of stimulator cells, liver DCs, or PBMCs, were used to stimulate 2 × 105 cord blood mononuclear cells in a 5-day assay. Mean of triplicate wells is shown. Each line represents a different experiment with different DC preparations (n = 3). B: The ability of DCs from skin (n = 3) and liver (n = 5) to stimulate allogeneic T cells was tested in a modified MLR in which highly purified, negatively selected allogeneic T cells were used as responder cells and DCs that had been purified using negative selection as stimulators. The skin DCs were significantly better stimulators of allogeneic T cells than liver-derived DCs (P < 0.0001). C: The influence of soluble tissue factors to modify the response of DCs after overnight migration was tested using the media from these cultures. mDCs were cultured overnight in tissue-conditioned media or complete media, before comparison in a modified MLR using pure adult allogeneic T cells (n = 4). mDCs cultured overnight in liver tissue-conditioned media were poor stimulators of T-cell proliferation compared to normal mDCs (P < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Soluble Tissue Factors from Liver Reduce the Ability of mDCs to Stimulate T-Cell ProliferationTo assess the factors involved in tissue-specific DC function, pure mDCs were cultured overnight in liver- or skin-conditioned media, or plain complete media. The ability of the DCs to stimulate the proliferation of negatively selected pure adult T cells was compared using the same proliferation assay as before. The ability of mDCs

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