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• W2065685732 abstract "The CMRF-44 and CD83 (HB15) antigens are associated with functional maturation and activation of blood dendritic cells (DC). We describe the expression of these antigens on freshly isolated epidermal Langerhans cells and dermal DC as well as the distribution of CD83+/CMRF-44++-activated DC within sections of normal human skin. Fresh Langerhans cells were prepared by standard techniques and large numbers of enriched (25%–55%), viable dermal DC were obtained using an improved collagenase treatment protocol with density gradient enrichment. Freshly isolated Langerhans cells and dermal DC had similar costimulator and activation antigen expression, and both stimulated moderate levels of allogeneic T lymphocyte proliferation as determined in the 7 d mixed leukocyte reaction. In situ labeling of DC within skin sections revealed a population of CD83 and CMRF-44 positive dermal cells of which most (≈75%) were in intimate contact with CD3+ T lymphocytes, especially in the adnexal regions. In contrast, only 25%–30% of the more numerous CD1a++ dermal DC population were directly apposed to T lymphocytes. The CMRF-44++ dermal DC population stimulated an allogeneic mixed leukocyte reaction, confirming their identity as DC. These data, plus comparative data obtained for migratory dermal DC, suggest that only a small proportion of dermal DC have been triggered to a more advanced state of differentiation or activation. The striking association of the activated dermal DC population with T lymphocytes suggests that communication between these two cell types in situ may occur early in the immune response to cutaneous antigen. The CMRF-44 and CD83 (HB15) antigens are associated with functional maturation and activation of blood dendritic cells (DC). We describe the expression of these antigens on freshly isolated epidermal Langerhans cells and dermal DC as well as the distribution of CD83+/CMRF-44++-activated DC within sections of normal human skin. Fresh Langerhans cells were prepared by standard techniques and large numbers of enriched (25%–55%), viable dermal DC were obtained using an improved collagenase treatment protocol with density gradient enrichment. Freshly isolated Langerhans cells and dermal DC had similar costimulator and activation antigen expression, and both stimulated moderate levels of allogeneic T lymphocyte proliferation as determined in the 7 d mixed leukocyte reaction. In situ labeling of DC within skin sections revealed a population of CD83 and CMRF-44 positive dermal cells of which most (≈75%) were in intimate contact with CD3+ T lymphocytes, especially in the adnexal regions. In contrast, only 25%–30% of the more numerous CD1a++ dermal DC population were directly apposed to T lymphocytes. The CMRF-44++ dermal DC population stimulated an allogeneic mixed leukocyte reaction, confirming their identity as DC. These data, plus comparative data obtained for migratory dermal DC, suggest that only a small proportion of dermal DC have been triggered to a more advanced state of differentiation or activation. The striking association of the activated dermal DC population with T lymphocytes suggests that communication between these two cell types in situ may occur early in the immune response to cutaneous antigen. dendritic cell Immunologic surveillance of the skin epithelial surfaces is maintained in part by the presence of large numbers of professional antigen-presenting cells at these sites. Langerhans cells are specialist epidermal antigen-presenting cells with an exceptional ability to stimulate naïve T lymphocytes (Schuler and Steinman, 1985Schuler G. Steinman R.M. Murine epidermal Langerhans cells mature into potent allostimulatory dendritic cells in vitro.J Exp Med. 1985; 161: 526-546Crossref PubMed Scopus (847) Google Scholar) and to present recall antigens to previously activated T lymphocytes (Cohen and Katz, 1992Cohen P.J. Katz S.I. Cultured human Langerhans cells process and present intact protein antigens.J Invest Dermatol. 1992; 99: 331-336Crossref PubMed Scopus (24) Google Scholar). In addition, human and murine dermis contain dendritic cells (DC) that may contribute to the maintenance of cutaneous immunity (Davis et al., 1988Davis A.L. McKenzie J.L. Hart DnJ HLA-DR positive leukocyte subpopulations in human skin include dendritic cells, macrophages and CD7-negative T cells.Immunology. 1988; 65: 573-581PubMed Google Scholar;Lenz et al., 1993Lenz A. Heine M. Schuler G. Romani N. Human and murine dermis contain dendritic cells: isolation by means of a novel method and phenotypical and functional characterization.J Clin Invest. 1993; 92: 2587-2596Crossref PubMed Scopus (251) Google Scholar;Nestle et al., 1993Nestle F.O. Zheng X. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.J Immunol. 1993; 151: 6535-6545PubMed Google Scholar). Dermal DC may be identified by their intense HLA class II expression, morphology, and in vitro migratory capacity, but show heterogenous expression of CD1a, CD14, CD36, Lag antigens, and factor XIIIa (Davis et al., 1988Davis A.L. McKenzie J.L. Hart DnJ HLA-DR positive leukocyte subpopulations in human skin include dendritic cells, macrophages and CD7-negative T cells.Immunology. 1988; 65: 573-581PubMed Google Scholar;Cerio et al., 1989Cerio R. Griffiths Cem Cooper K.D. Nickoloff B.J. Headington J.T. Characterization of factor XIIIa positive dermal dendritic cells in normal and inflamed skin.Br J Dermatol. 1989; 121: 421-431Crossref PubMed Scopus (307) Google Scholar;Lenz et al., 1993Lenz A. Heine M. Schuler G. Romani N. Human and murine dermis contain dendritic cells: isolation by means of a novel method and phenotypical and functional characterization.J Clin Invest. 1993; 92: 2587-2596Crossref PubMed Scopus (251) Google Scholar;Nestle et al., 1993Nestle F.O. Zheng X. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.J Immunol. 1993; 151: 6535-6545PubMed Google Scholar;Richters et al., 1994Richters C.D. Hoekstra M.J. van Baare J. Du Pont J.S. Hoefsmit Ecm Kamperdijk EwA Isolation and characterization of migratory human skin dendritic cells.Clin Exp Immunol. 1994; 98: 330-336Crossref PubMed Scopus (38) Google Scholar;Pope et al., 1995Pope M. Betjes Mgh Hirmand H. Hoffman L. Steinman R.M. Both dendritic cells and memory T lymphocytes emigrate from organ cultures of human skin and form distinctive dendritic-T-cell conjugates.J Invest Dermatol. 1995; 104: 11-17Crossref PubMed Scopus (89) Google Scholar;Lukas et al., 1996Lukas M. Stossel H. Hefel L. et al.Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model.J Invest Dermatol. 1996; 106: 1293-1299Crossref PubMed Scopus (98) Google Scholar). Clinically, dermal DC appear to be involved in the pathology of a number of skin conditions. For example, dermal DC isolated from psoriatic skin show increased autostimulation of T lymphocytes (Nestle et al., 1994Nestle F.O. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells in psoriasis.J Clin Invest. 1994; 94: 202-209Crossref PubMed Google Scholar), and dermal DC, but not Langerhans cells, were reported to be important in the clearance of the Lyme disease causative organism Borellia burgdorferi from human skin (Filgueria et al., 1996Filgueria L. Nestle F.O. Rittig M. Joller H.I. Groscurth P. Human dendritic cells phagocytose and process Borrelia burgdorferi..J Immunol. 1996; 157: 2998-3005Google Scholar). Dermal DC may also contribute to HIV transmission (Pope et al., 1994Pope M. Betjes Mgh Romani N. et al.Conjugates of dendritic cells and memory T lymphocytes from skin facilitate productive infection with HIV-1.Cell. 1994; 78: 389-398Abstract Full Text PDF PubMed Scopus (429) Google Scholar). We have analyzed the distribution and activation status of Langerhans cells and dermal DC in normal skin using the monoclonal antibodies (MoAb) CMRF-44 and CD83. These two MoAb recognize different antigens, which are expressed during the functional maturation of fresh DC into potent allostimulatory antigen-presenting cells (Zhou et al., 1992Zhou L. Schwarting R. Smith H.M. Tedder T.F. A novel cell-surface molecule expressed by human interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of the Ig superfamily.J Immunol. 1992; 149: 735-742PubMed Google Scholar;Hock et al., 1994Hock B.D. Starling G.C. Daniel P.B. Hart DnJ Characterisation of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.Immunology. 1994; 83: 573-581PubMed Google Scholar;Zhou and Tedder, 1995Zhou L.J. Tedder T.F. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily.J Immunol. 1995; 154: 3821-3835PubMed Google Scholar;Fearnley et al., 1997Fearnley D.B. McLellan A.D. Mannering S.I. Hock B.D. Hart DnJ Isolation of human blood dendritic cells using the CMRF-44 monoclonal antibody: implications for studies on antigen presenting cell function and immunotherapy.Blood. 1997; 89: 3708-3716Crossref PubMed Google Scholar). Fresh blood DC do not express CD83 and only a subpopulation is weakly positive for CMRF-44; however, the expression of both these DC-associated antigens is rapidly upregulated on blood DC following a period of in vitro culture (Fearnley et al., 1997Fearnley D.B. McLellan A.D. Mannering S.I. Hock B.D. Hart DnJ Isolation of human blood dendritic cells using the CMRF-44 monoclonal antibody: implications for studies on antigen presenting cell function and immunotherapy.Blood. 1997; 89: 3708-3716Crossref PubMed Google Scholar;Zhou et al., 1992Zhou L. Schwarting R. Smith H.M. Tedder T.F. A novel cell-surface molecule expressed by human interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of the Ig superfamily.J Immunol. 1992; 149: 735-742PubMed Google Scholar;Zhou and Tedder, 1995Zhou L.J. Tedder T.F. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily.J Immunol. 1995; 154: 3821-3835PubMed Google Scholar). Low density expression of the CD83 and CMRF-44 antigens can be induced on blood monocytes by stimuli such as interferon or ionophore treatment and also on lymphocytes by mitogens or Ig cross-linking (Zhou et al., 1992Zhou L. Schwarting R. Smith H.M. Tedder T.F. A novel cell-surface molecule expressed by human interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of the Ig superfamily.J Immunol. 1992; 149: 735-742PubMed Google Scholar;Hock et al., 1994Hock B.D. Starling G.C. Daniel P.B. Hart DnJ Characterisation of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.Immunology. 1994; 83: 573-581PubMed Google Scholar;Czerniecki et al., 1997Czerniecki B.J. Carter C. Rivoltini L. et al.Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells.J Immunol. 1997; 159: 3823-3837PubMed Google Scholar; and our unpublished data). High density expression of CD83 and CMRF-44 is found on cultured blood DC and these markers have proved to be valuable for identifying activated/differentiated DC in kidney (Troy et al., 1998aTroy A.J. Summers K.L. Davidson Pjt Atkinson C.A. Hart DnJ The dendritic cell infiltrate in human renal cell carcinoma: a probable mechanism for tumor escape from immune surveillance.Clin Cancer Res. 1998; 4: 585-593PubMed Google Scholar) and prostate (Troy et al., 1998bTroy A.J. Davidson P. Atkinson C. Hart DnJ Phenotypic characterization of the dendritic cell infiltrate in prostate cancer.J Urol. 1998; 160: 214-219Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) cancers. Although the upregulation of the CMRF-44 and CD83 antigens on blood DC correlates with increased levels of the costimulatory antigens CD40, CD80, and CD86 (McLellan et al., 1995aMcLellan A.D. Starling G.C. Williams L.A. Hock B.D. Hart DnJ Activation of human peripheral blood dendritic cells induces the CD86 costimulatory molecule.Eur J Immunol. 1995; 25: 2064-2068Crossref PubMed Scopus (114) Google Scholar,McLellan et al., 1996McLellan A.D. Sorg R.V. Williams L.A. Hart DnJ Human dendritic cells activate T lymphocytes via a CD40, CD40 ligand–dependent pathway.Eur J Immunol. 1996; 26: 1204-1210Crossref PubMed Google Scholar), neither the CMRF-44 nor the CD83 MoAb influence T lymphocyte activation and at present the function of these molecules is unknown (Zhou and Tedder, 1995Zhou L.J. Tedder T.F. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily.J Immunol. 1995; 154: 3821-3835PubMed Google Scholar;Fearnley et al., 1997Fearnley D.B. McLellan A.D. Mannering S.I. Hock B.D. Hart DnJ Isolation of human blood dendritic cells using the CMRF-44 monoclonal antibody: implications for studies on antigen presenting cell function and immunotherapy.Blood. 1997; 89: 3708-3716Crossref PubMed Google Scholar). We show here that the majority of activated (CMRF-44++ or CD83+) dermal DC are in intimate contact with T lymphocytes in normal skin. This finding suggests that communication between these two cell types in situ may occur early in the immune response to cutaneous antigen. The CMRF-44 (IgM;Hock et al., 1994Hock B.D. Starling G.C. Daniel P.B. Hart DnJ Characterisation of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.Immunology. 1994; 83: 573-581PubMed Google Scholar), CD14 (CMRF-31; IgG2a), and negative control MoAb CMRF-75 (IgM) were produced in this laboratory. CD1a (Na1/34; IgG2a) was a gift from Prof. A. McMichael (Oxford, U.K.). CD3 (OKT3; IgG2a), CD8 (OKT8; IgG2a), CD25 (7G7; IgG2a), CD45RO (UCHL1; IgG2a), and anti-HLA-DQ (L227; IgG1) supernatants were produced from hybridomas obtained from the American Type Culture Collection (Manassas, VA). Fluoresceinated HLA-DQ MoAb (L227) was prepared by incubating 1 mg of protein-A (Sigma, St Louis, MO) purified MoAb at 1 mg per ml in 0.1 M borate (pH 8.5) with 30 μl of 10 mg fluorescein-isothiocyanate (FITC; Isomer I; Sigma) per ml in dimethylsulfoxide for 1 h at room temperature. Excess FITC was removed by Centricon-100 (Amicon, Beverly, MA) dialysis. CD80 (L307; IgG1), phycoerythrin (PE)-conjugated anti-HLA-DR (IgG2a), CD86-PE (BU63; IgG1), and PE-conjugated isotype controls were purchased from Becton Dickinson (San Jose, CA). Quantum Red (QR) conjugated HLA-DR was from Sigma. Unconjugated BU63 (CD86; IgG1) was kindly provided by Dr. D. Hardie (University of Birmingham, Birmingham, GB), and HB15a (CD83; IgG2b) was kindly provided by Prof. T.F. Tedder (Duke University, Durham, NC) and from Coulter (Hialeah, FL). The isotype control MoAb X63 (IgG1) producing hybridoma was obtained from the American Type Culture Collection and Sal-4 (IgG2b) was from Dr. Leonie Ashman (IMVS, Adelaide, Australia). Fluorescein isothiocyanate-conjugated sheep anti-mouse-immunoglobulin (FITC-SAM-Ig) was obtained from Silenus Laboratories (Hawthorn, Australia). Human skin was obtained from reduction mammoplasty specimens with ethical permission from the Southern Regional Health Authority Ethical Committee. Cryostat cut en face sections (7 μm) of the epidermis and upper dermis of normal human breast skin mounted in Tissue-Tek OCT (Miles, Elkhart, IN) were placed onto gelatin-coated glass slides and allowed to dry overnight. The sections were fixed for 10 min in ice cold acetone and air dried for 30 min. Sections were then blocked with 10% human serum/phosphate-buffered saline (PBS) prior to reaction with primary MoAb followed by biotinylated goat anti-mouse Ig (1/200 dilution; DAKO, Carpinteria, CA) and then addition of 1/200 streptavidin-alkaline phosphatase (Sigma). Slides were washed three times with TBS between each 30 min incubation. Enzymatic activity was revealed with Fast Blue substrate solution: 2 mg sodium AS phosphate dissolved in 100 μl dimethyl formamide, 10 mg fast blue salt, 6 μl 1 M levamisole (all from Sigma), and 10 ml 0.1 M Tris pH 8.2. For double immunoenzyme labeling, the sections were reacted with the second MoAb, followed by peroxidase conjugated goat anti-mouse Ig (1/100; DAKO). The substrate used for detection of the second MoAb was 3-amino-9-ethyl cabazole (AEC) (10 mg AEC; Sigma, first dissolved in 100 μl of DMF, then in 10 ml of 0.05 M acetate pH 5.1 with 6 μl of H2O2). Cell density was estimated by counting labeled cells in a tissue section area of normally at least 2 mm2, with an eyepiece graticule of known area at 200× magnification. For in situ analysis of the percentage of dermal DC apposition to T lymphocytes, dermal DC were scored as clustered if part of the stained dermal DC membrane contacted at least one T lymphocyte. Slides were photographed on Kodak Gold 100 ASA film using an Olympus BX-50 equipped with a PM-30 camera system. Cell culture medium used throughout the study was RPMI-1640 supplemented with 100 U penicillin per ml, 100 μg streptomycin per ml, 2 mM L-glutamine, 25 mM HEPES (pH 7.4), and either 10% heat inactivated fetal calf serum for DC isolation and culture (Life Technologies, Auckland, NZ), or 10% pooled AB serum for the mixed lymphocyte reaction (MLR). Epidermal sheets were prepared from split thickness human breast skin by overnight Dispase II (0.5% in PBS; Boehringer, Mannheim, Germany) digestion at 4°C for cell labeling studies, or for 1 h at 37°C for some of the functional studies. The epidermis was minced by scissor action with scalpels and digested for 20 min with 0.25% trypsin (Sigma) and DNase I (5 U per ml, Sigma) in PBS at 37°C. Epidermal fragments were then disaggregated by vigorous pipetting and passage through a cell dissociation cup (grade 40 mesh; Sigma). The cells were immediately collected into HEPES buffered RPMI/fetal calf serum. Following nylon mesh filtration (80 μm; Millipore, Bedford, MA), cell suspensions were washed twice in RPMI/10% fetal calf serum/DNaseI with DNase I, resuspended in RPMI/10% fetal calf serum/DNaseI and Langerhans cells enriched by up to three sequential Lymphoprep gradients (Pharmacia, Auckland, NZ) as described byPeguet-Navarro et al., 1995Peguet-Navarro J. Dalbiez-Gauthier C. Rattis F.M. Van Kooten C. Banchereau J. Schmitt D. Functional expression of CD40 antigen on human epidermal Langerhans cells.J Immunol. 1995; 155: 4241-4247PubMed Google Scholar). Langerhans cells were typically enriched from 1% to 2% (following trypsinization) to 4%–11% (as determined by HLA-DR labeling) after the first Lymphoprep gradient purification. Cell suspensions obtained following the second (22%–32% Langerhans cells) or third (28%–45% Langerhans cells) Lymphoprep were used for most experiments. In some experiments, Langerhans cells enriched epidermal cell suspensions were cultured in medium at 5 × 106 cells per ml in 24 well plates (Nunc, Life Technologies, Auckland, NZ) for 40 h prior to immunofluorescence analysis. For reverse transcriptase-polymerase chain reaction analysis, fresh Lymphoprep enriched Langerhans cells were further purified by sorting of the HLA-DR+++ fraction on a FACS Vantage (Becton Dickinson). Reverse transcriptase-polymerase chain reaction was performed as previously described (McLellan et al., 1995aMcLellan A.D. Starling G.C. Williams L.A. Hock B.D. Hart DnJ Activation of human peripheral blood dendritic cells induces the CD86 costimulatory molecule.Eur J Immunol. 1995; 25: 2064-2068Crossref PubMed Scopus (114) Google Scholar). All Langerhans cell enriched populations used in this study for reverse transcriptase-polymerase chain reaction were greater than 75% HLA-DR+++. Fresh dermal DC were prepared from split thickness normal human breast skin following overnight Dispase digestion at 4°C and meticulous removal of all epidermis. The split thickness dermal sheets were minced by scissor action with scalpels into 0.5–2 mm2 pieces and partially digested with 1.0 mg collagenase (trypsin free, type D, Boehringer) per ml and DNase I, in 20–30 ml of RPMI-1640/fetal calf serum with 25 mM HEPES (pH 7.3) in a Petri dish with gentle swirling for 1 h at 37°C. Shorter collagenase digestion times (30 min) failed to release detectable leukocytes, and longer collagenase treatment (2–3 h) resulted in contamination of dermal cell preparations with many CD45– cells (data not shown). Undigested tissue was removed by 80 μm screen filtration and a large wash volume (10–20 volumes of bovine serum albumin/DNase I/PBS) was required to pellet the cells from the viscous cell suspension. Following a second wash in 100 ml and 60 μm mesh filtration, dead cells, debris and some contaminating fibroblasts were removed by Lymphoprep gradient separation (ρ = 1.077 g per cm3, 10 min, 500 × g). Migratory DC were isolated from cultured dermis using standard techniques (Lenz et al., 1993Lenz A. Heine M. Schuler G. Romani N. Human and murine dermis contain dendritic cells: isolation by means of a novel method and phenotypical and functional characterization.J Clin Invest. 1993; 92: 2587-2596Crossref PubMed Scopus (251) Google Scholar;Nestle et al., 1993Nestle F.O. Zheng X. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.J Immunol. 1993; 151: 6535-6545PubMed Google Scholar), originally described for murine split skin explants byLarsen et al., 1990Larsen C.P. Steinman R.M. Witmer-Pack M. Hankins D.F. Morris P.J. Austyn J.M. Migration and maturation of Langerhans cells in skin transplants and explants.J Exp Med. 1990; 172: 1483-1493Crossref PubMed Scopus (568) Google Scholar. Briefly, the epidermis was removed from Dispase treated split thickness skin and the dermis chopped into ≈1–2 mm3 fragments and cultured in RPMI/10% fetal calf serum with 25 mM HEPES (pH 7.4) in Petri dishes at 37°C, 5% CO2. Medium acidification was minimized by replacing up to half of the medium after 24 h. Migratory dermal cells were obtained at day 2 as a cell suspension following removal of the dermal fragments by 80 μm nylon gauze filtration. In most experiments, dermal DC were enriched to 80%–90% using a hypertonic Nycodenz gradient (Nycoprep;ρ = 1.068; generous gift from Nycomed-Pharma, Oslo, Norway), as described for the isolation of mature blood DC (McLellan et al., 1995bMcLellan A.D. Starling G.C. Hart DnJ Isolation of human blood dendritic cells by Nycodenz discontinuous gradient centrifugation.J Immunol Meth. 1995; 184: 81-89Crossref PubMed Scopus (52) Google Scholar). Cells were labeled with saturating concentrations of primary MoAb for 30 min on ice and incubation with FITC-SAM-Ig. Following primary staining, cells were incubated in 10% mouse serum/PBS for 5 min, labeled with FITC-, PE-, or QR-conjugated MoAb for 30 min, and then analyzed on a FACS Vantage using Cellquest software. For flow cytometric analysis or sorts of both Langerhans cells and dermal DC, gates were set to include cells with high forward scatter and moderate to high side scatter. These gates excluded small lymphocytes and debris as well as dead cells as confirmed by propidium iodide labeling. Graded doses of mitomycin C treated dermal DC or Langerhans cells were cultured with 105 allogeneic ER+ T lymphocytes (prepared by ammonium chloride lysis of neuraminidase treated sheep red blood cell rosettes of peripheral blood mononuclear cells) in a final volume of 200 μl of HEPES buffered RPMI-1640 with 10% heat inactivated AB serum in round bottom 96 well plates (Falcon/Becton Dickinson, Franklin Lakes, NJ). After 5 or 7 d of culture at 37°C in 5% CO2, cultures were pulsed with 1 μCi [3H]thymidine (Amersham International, Arlington Heights, IL) per well for the final 19 h before harvesting and liquid scintillation counting. All assays were performed in triplicate. Flow cytometric analysis of Lymphoprep enriched Langerhans cells (n = 4) indicated weak to moderate expression of the CMRF-44, CD83, and CD86 antigens on either a subpopulation or all of the strongly HLA-DR+++/CD1a+++ positive Langerhans cells Figure 1a. CD80 expression on Langerhans cells was either very weak or absent, although we were able to detect CD80, as well as CD86 mRNA transcripts, in sorted fresh Langerhans cells and culture of fresh Langerhans cell-induced CD80 surface expression (data not shown). We found that 65%–90% of the Langerhans cells present in the pregradient trypsinised cell suspension were lost into the pellet fraction during the gradient isolation procedure. To ensure that the activation antigen expression on the gradient enriched Langerhans cell preparations was representative of total Langerhans cells, we also labeled unfractionated trypsinised epidermal cell suspensions. The results were similar: 22%–55% of HLA-DR positive cells expressed the CMRF-44 antigen prior to gradient isolation, versus 40%–75% after gradient isolation (n = 2). Dermal DC were prepared using low tryptic activity collagenase digestion for 1 h prior to a single density gradient enrichment of dermal DC. The forward and side scatter plots of dermal cell suspensions (with CD45+ cells backgated) obtained after collagenase treatment and following a single Lymphoprep density cut are shown in Figure 2. The typical positioning of myeloid and lymphoid gates used in this study for flow cytometric analysis of dermal cell suspensions are also shown in Figure 2. The resulting cell suspensions (5.4 × 106 cells ± 4.9 × 106 SD, range 1–12 × 106 per experiment, n = 20) were enriched in HLA-DR+++/CD1a++ dermal DC (Figure 1b; 41% ± 10% SD, range 25%–55%, n = 15) and effectively depleted of strongly CD14 positive tissue macrophages, despite an apparent abundance of these cells in the dermis enumerated by immunohistologic staining (see later); however, a small HLA-DR+++/CD1a–/+ subpopulation (<10%) of dermal cells showed very weak CD14 staining, similar to migratory dermal DC populations (Nestle et al., 1993Nestle F.O. Zheng X. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.J Immunol. 1993; 151: 6535-6545PubMed Google Scholar, and data not shown). Control studies showed that CD14 antigen expression was not altered by 1 h collagenase treatment of peripheral blood mononuclear cells (data not shown). Although ≈50% of dermal cells were lost into the pellet during the Lymphoprep gradient step, further labeling analyses confirmed that the gradient isolated dermal DC showed a surface phenotype representative of the whole population prior to this step (data not shown). Some lymphocytes were also included in these preparations (≈10% of dermal cells) and flow cytometry analysis showed these to be mainly CD4+ or CD8+ T lymphocytes (data not shown). DC:T lymphocyte conjugates, although numerous in cultured migratory dermal DC preparations (Pope et al., 1995Pope M. Betjes Mgh Hirmand H. Hoffman L. Steinman R.M. Both dendritic cells and memory T lymphocytes emigrate from organ cultures of human skin and form distinctive dendritic-T-cell conjugates.J Invest Dermatol. 1995; 104: 11-17Crossref PubMed Scopus (89) Google Scholar, and data not shown), were not observed in the hemocytometer directly following collagenase isolation. Morphologically, many of the dermal DC had myeloid features with oval or slightly indented nuclei, moderate basophilic cytoplasm, minimal or no granulation, and a ruffled cell surface Figure 3a, typical of short-term cultured blood DC (Hart, 1997Hart DnJ Dendritic cells: unique leucocyte populations which control the primary immune response.Blood. 1997; 90: 3245-3287Crossref PubMed Google Scholar). Larger, more vacuolated dermal cells with typical macrophage morphology were also present Figure 3b, some of which contained ingested melanin granules (Figure 3c; arrow). Numerous small lymphocytes found in fresh collagenase-released dermal cell preparations are also shown Figure 3d. Using this isolation method, we were able to obtain consistent yields of 2–5 × 105 leukocytes per cm2 of dermis (n = 15). The ability of freshly isolated mitomycin C-treated dermal DC to stimulate the proliferation of allogeneic peripheral blood T lymphocytes was next determined. Only minimal T lymphocyte proliferation occurred at day 5 and in five of six experiments an additional 2 d of MLR coculture led to large increases (relative maximal CPM increase; 83% ± 85% SD, n = 6) in allogeneic T lymphocyte proliferation Figure 4a. Similar results were obtained using Langerhans cells isolated from the same donors (relative maximal CPM increase; 88% ± 95% SD, n = 6, Figure 4b). In contrast, migratory dermal DC, isolated after 2 d culture of dermis by Nycodenz gradient separation, stimulated very high levels of allogeneic T lymphocyte proliferation at both day 5 and day 7 (Figure 4c, n = 3). Control studies showed that similar handling and collagenase treatment of peripheral blood mononuclear cell suspensions did not affect their allostimulatory function in the MLR (data not shown). Dermal DC-enriched suspensions were labeled for the CMRF-44 or CD83 DC differentiation/activation antigens or the CD80 and CD86 costimulatory antigens (FITC) followed by double labeling with HLA-DR-PE to identify dermal DC. Isolated dermal DC showed an activated phenotype as determined by their weak to moderate expression of the CD83 and CMRF-44 differentiation/activation antigens. The CMRF-44++ subset expressed slightly higher levels of HLA-DR antigens than did the CMRF-44–" @default.
• W2065685732 created "2016-06-24" @default.
• W2065685732 creator A5000309083 @default.
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• W2065685732 date "1998-11-01" @default.
• W2065685732 modified "2023-10-14" @default.
• W2065685732 title "Dermal Dendritic Cells Associated with T Lymphocytes in Normal Human Skin Display an Activated Phenotype" @default.
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