Published online before print June 3, 2004
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* Institute of Molecular Biology and Biochemistry, Charité-Universitätsmedizin Berlin, Free University, Germany; and
Department of Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Humboldt-University, Germany
1 Correspondence: Department of Molecular Biology and Biochemistry, Free University of Berlin, Arnimallee 22, D-14195 Berlin/Germany. E-mail: matthias.peiser{at}medizin.fu-berlin.de
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Key Words: LC • CD40 ligand • CD83
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After encounter with foreign antigen, phagocytosis, and processing, LCs solve adhesion from neighboring keratinocytes by tumor necrosis factor 
(TNF-)- and interleukin (IL)-1ß-induced down-regulation of E-cadherin [3
] and migrate in response to secondary lymphoid-tissue chemokine–chemokine receptor 7 interactions to regional lymph nodes. Danger signals seem to be important for induction of an effective immunity by maturing antigen-presenting cells (APCs) in a self-nonself independent way [4
]. In this study, we analyzed the roles of the T cell costimulatory signal CD40 ligand (CD40L) and of danger signals in LC activation. We used lipopolysaccharide (LPS) from Escherichia coli, peptidoglycan from Staphylococcus aureus, and flagellin from Salmonella typhimurium to engage the Toll-like receptors (TLR)-4, -2, and -5, respectively.
The CD40 ligation on immature DCs plays a crucial role in maturation and survival of the cells. Numerous in vitro studies have shown that CD40 triggering leads to up-regulation of major histocompatibility complex (MHC) class II, costimulatory, and adhesion molecules and to induction of the maturation marker CD83. In addition, CD40L induces the high-level production and secretion of the T helper cell type 1 (Th1)-polarizing cytokine IL-12 [5 6 7 8 ]. There is increasing evidence that the effects of CD40 stimulation depend on the cytokine microenvironment, the use of additional stimuli, and most importantly, on the DC subset and its activation state [9 10 11 12 13 ].
The few studies about CD40 cross-linking on epidermal LCs were performed with rather impure human or murine cell preparations, which have not maintained their immature state as a result of the isolation methods or culture conditions used [14
15
16
]. In the detailed analyses of Peguet-Navarro et al. [14
], for example, the epidermal cell (EC) suspensions consisted of only 75–90% human LCs, and most of the experiments were performed in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF), which promotes LC maturation. Some other studies have used ex vivo-generated monocyte- or CD34+ progenitor cell-derived LCs [8
, 17
, 18
], which are now known to show a more pronounced heterogeneity and plasticity of maturation and differentiation than their counterparts in normal epidermis [19
20
21
]. We have recently developed an effective magnetic-activated, cell-sorting method for the positive selection of human LCs, which ensures the isolation of pure and viable cells with the ultrastructural features, surface antigen expression, cytokine profile, and stimulatory capacity characteristic for epidermis-resident, immature LCs [22
]. In the present study, the effects of CD40 ligation were investigated in these LCs and compared with ex vivo-generated, monocyte-derived LCs, which can be generated in vitro from progenitors in cord blood or bone marrow with GM-CSF and TNF- [23
] and from adherent monocytes with GM-CSF, IL-4, and transforming growth factor (TGF)-ß1. The latter cytokine seems to be essential for induction of BGs [24
].
Monocyte-derived LCs (MoLCs) in many aspects behave like immature epidermis-resident LCs and show a more synchronized maturation as compared with LCs generated from CD34+ hematopoietic progenitor cells [18 , 24 , 25 ]. This is the first report comparing the phenotype and cytokine release of stimulated LCs, isolated from epidermis with LCs, generated by cytokines. MoLCs and CD1c-isolated epidermal LCs show equal CD40 expressions. They all have surface expression of CD1a and human leukocyte antigen (HLA)-DR and contain intracellular BGs. The total population of epidermal LCs neoexpressed CD83 and increased the capacity to stimulate T cells but failed to express CD80 after CD40 triggering. Expression of CD80 together with CD86 and CD54 was found in the CD1c-positive-selected fraction of MoLCs. Only MoLCs could be induced to release high levels of IL-12p70 after activation with LPS and CD40L. IL-10 was detected in MoLCs and LCs after triggering TLR2, -4, and -5.
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(rhIFN-) from BD PharMingen (Heidelberg, Germany), rhTNF- and TGF-ß1 from R&D Systems (Wiesbaden-Nordenstadt, Germany), and soluble rhCD40L trimer from Bender (Vienna, Austria).
Preparation of EC suspensions
Normal human skin was obtained as discarded material from plastic surgery (reduction mammoplasty) after approval of the ethics commission of the Charité (Berlin, Germany). To avoid spontaneous migration of DCs from the explants [26
], the skin was kept on ice during transport and prepared immediately. EC suspensions were prepared as described previously [27
] with slight modifications. Briefly, thin skin stripes were incubated in phosphate-buffered saline (PBS) containing three RMB units (1 RMB unit corresponds to 1 µmol/min amino acid or peptide liberated from casein) dispase I (Roche, Mannheim, Germany) for 18 h at 4°C. Epidermal sheets were then stripped off the dermal layer using forceps, and dissociated in PBS containing 0.25% trypsin (Biochrom) and 0.01% DNase (Roche) for 15 min at 37°C. Single-cell suspensions were obtained by vigorous pipetting and passaging through a 40-µm cell strainer (BD, Heidelberg, Germany). Cell numbers, debris, and clumps of aggregated cells were determined using a CASY 1 cell counter (Schaerfe System, Reutlingen, Germany).
Isolation and culture of LCs
LCs were isolated from EC suspensions by positive selection using a biotinylated mouse anti-human CD1c antibody [clone AD5-8E7, immunoglobulin G (IgG)2a, Miltenyi Biotech] and an anti-biotin antibody conjugated to microbeads (clone Bio3-18E7, IgG1, Miltenyi Biotech). Magnetic-activated cell sorting (MACS) was performed according to our recent protocol [22
] by double-passing the cells over large cell separation columns (Miltenyi Biotech). The isolated LCs were cultured for up to 6 days in 24-well tissue-culture plates (Greiner, Frickenhausen, Germany). On alternate days, one-third of the medium was renewed (cell loss a result of change of medium was <1% per well).
Generation of LC-like cells from monocytes (MoLCs)
MoLCs were generated from plastic-adherent human monocytes as recently described [25
]. Briefly, extensively washed, adherent cells were differentiated by 6-day culture with GM-CSF (100 ng/ml), IL-4 (10 ng/ml, only at day 0 added), and TGF-ß1 (10 ng/ml, all from R&D Systems) with complete medium/cytokine exchange at days 2 and 4. Where indicated, MoLCs were additionally sorted by CD1c, as described above for ECs.
Coculture and harvesting
CD40L+ cells were seeded on 24-well tissue-culture plates and precultured for 4 h. After two washing steps with PBS, removing dead and nonadhering fibroblasts, LCs or MoLCs were added (106/ml, ratio 2:1) and cultured for 18 h in RPMI/10% FCS. LCs and MoLCs were recovered by gentle pipetting under microscopical control showing no lacks in the layers of CD40L+ cells.
Flow cytometry
The cell-surface expression of various proteins was analyzed by three-color flow cytometry. Each 2 x 105 cell was labeled with a F(ab')2 fragment of rabbit anti-mouse Ig [clone C0090, RPE-Cy5 dye 3; recognizes the CD1c-MACS-monoclonal antibody (mAb)] and anti-HLA-DR-(clone B8.12.2.PE, Beckman Coulter, Krefeld, Germany). Antigens were detected by the following fluorescein isothiocyanate-coupled mAb: anti-CD54 (84H10) and anti-207 (langerin, DCGM4, Beckman Coulter) and anti-CD1a (HI149), anti-CD40 (5C3), anti-CD80 (L307.4), anti-CD83 (HB15e), anti-CD86 (FUN-1), and isotype control MOPC-21 (all from BD PharMingen) using an Epics XL-II flow cytometer (Beckman Coulter). Dead cells and debris were excluded by scatter gates and propidium iodide staining (1 µg/ml; Sigma).
Electron microscopy (EM)
The methods used have been described in detail elsewhere [28
]. Briefly, the cell preparations were fixed in half-strength Karnovsky’s solution (0.1 M cacodylate buffer, pH 7.4) for 30 min at 4°C, postfixed in 1.33% osmic acid (0.05 M PBS, pH 7.4, Fluka) for 2 h, dehydrated in a graded ethanol series, and embedded in Araldite (Poersch, Frankfurt, Germany). Ultrathin sections were counterstained with uranyl acetate and lead citrate and were examined with a Zeiss electron microscope (EM 906, Zeiss, Oberkochen, Germany).
Allogeneic stimulation
Naive CD4+ T cells were purified from buffy coats using a CD4+ T cell isolation kit (Miltenyi Biotech). LCs and MoLCs, recovered from the CD40L+ cell plates were irradiated (30 Gy) and added in graded doses as stimulator cells to 105 allogeneic CD4+ T cells in 96-well round-bottom plates in RPMI medium in a 5% CO2 incubator. Cultures were incubated for 5 days at 37°C before being pulsed with [3H]thymidine (TdR; 1 µCi/well, Amersham, Freiburg, Germany) for the last 16 h. Cells were harvested onto glass fiber filters, and [3H]TdR incorporation was determined in a liquid scintillation counter (LS 6500, Beckman Coulter, Unterschleissheim-Lohhof, Germany).
Detection of IL-10 and IL-12p70 by enzyme-linked immunosorbent assay (ELISA)
The cytokines were analyzed immediately after harvesting in the supernatant of 106 LCs/well/ml medium after 18 h culture at 37°C in 5% CO2 using standard Quantikine IL-10 and ultrasensitive Quantiglo IL-12p70 ELISA. IFN- was measured in the supernatant of cocultured, allogeneic CD4+ T cells by Quantikine ELISA (all from R&D Systems).
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Figure 1. Comparison of CD1a, HLA-DR, CD40 surface expression and scatter profiles of epidermal LCs and MoLCs. CD1c sorting was performed with human ECs and with MoLCs, generated from monocytes after 6 days of culture with GM-CSF, IL-4, and TGF-ß1 (CD1c+ LCs, CD1c+ MoLCs, black lines; isotype, gray lines). Isolated LCs and MoLCs were seeded on a layer of adherent hCD40L-transfected mouse L cells or cultured directly on plastic for 18 h. Scatter plots show flow cytometric analyses of 5000 events without gating (panel below).
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Figure 2. Transmission EM analysis showing formation of bicellular clusters with interdigitation and membrane apposition. Epidermal LCs were cocultured with preadhered CD40L+ cells. (A–F) Micrographs show a bicellular cluster at various magnifications. (A) Complete view of the cell contact between LC, identified by numerous BGs, and CD40L+ fibrocytic cell (original x4400). Regions in rectangles are shown at higher magnification in panels with corresponding letters. (B) Higher magnification of the cell contact demonstrates closely apposed, interdigitating plasma membranes, intercellular vesicles, and cytoplasmic protrusions of the LC (upper cell) and the CD40L+ cell (cell below; original x20,000). (C) Ultrastructural organization of the LC with prominent Golgi area, mitochondria, and BGs (original x50,000). (D) Upper part of the intercellular gap with prominent endoplasmic membranes and mitochondria in the LC cytoplasm (original x20,000). (E) Close membrane apposition in the center of the cell contact (original x20,000). (F) Three rod-shaped BGs with prominent trilamellar structure (original x250,000). (G and H) Low and high magnification of a bicellular cluster with a rather small cell contact. The LC extension adjusts to the membrane invagination of the fibrocytic cell (originals x4400, G; x20,000, H). (I and K) Broad cell contact showing numerous interdigitations within the intercellular gap, coated pits (arrowheads), and prominent, rough endoplasmic membranes in the cytoplasma of both cells (originals x4400, I; x20,000, K).
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. After stimulation with LPS, peptidoglycan, or flagellin, MoLCs and LCs secreted IL-10 at comparable levels (Fig. 3B)
.
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Figure 3. CD40L in combination with LPS induces high amounts of IL-12p70 in MoLCs but low cytokine secretion in epidermal LCs. Detection of IL-12p70 by ultrasensitive ELISA after 18 h in the supernatant of epidermal LCs and MoLCs costimulated by TLR ligands LPS, peptidoglycan (PGN), or flagellin (Flag) or by cytokines (A). IL-10 is secreted by LCs and MoLCs after stimulation with LPS, PGN, or Flag; Wo, without stimulation (B). Data represent four and three independent experiments as mean values ± SD.
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and IL-1ß [22
]. On epidermal LCs, LPS also up-regulated CD83 (Fig. 4
), a molecule that is associated with DC maturation [29
]. In contrast, IFN- or CD40L trimer failed to induce CD83 on CD1c-sorted LCs (not shown). Only when cocultured with adherent CD40L+ cells, LCs strongly elevated the surface expression of CD83. After 18 h of stimulation, nearly all LCs were CD83-positive (94%; Fig. 4
). After coculture with CD40L-transfected J558L cells, which grow in suspension, 70% of LCs neoexpressed CD83 (Fig. 4)
. Unstimulated LCs matured only moderately (not shown).
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Figure 4. CD40L induces strong up-regulation of CD83 in epidermal LCs. Isolated LCs and MoLCs were activated with hCD40L-transfected mouse L cells (adh., adherent; 2:1), with J558 L cells (susp., suspension; 1:2), or with 1 µg/ml LPS. After activation (18 h), all cells were harvested and stained by anti-HLA-DR and anti-CD83. Histograms show CD83 expression of DR-gated cells. Fluorescein-activated cell sorter (FACS) analyses of 10,000 cells from one donor are representative of five independent experiments; CD1c+, MACS-selected cells; CD1c–, MACS-negative fraction; stimulated by CD40L, black lines; wt, gray lines; isotype, dotted lines.
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Epidermal LCs and MoLCs differentially express CD80, CD54, and HLA-DR after CD40 cross-linking
LCs freshly isolated from human skin and MoLCs at day 6 of generation were sorted by CD1c-MACS. The separated fractions were stimulated with CD40L+ cells or for control, were cocultured with hCD40L-nonexpressing murine L cells (wt cells). After stimulation, only the CD1c+ MoLCs shifted toward high-level CD80 expression (Fig. 5
). CD86 was acquired by CD1c+ MoLCs and LCs (Fig. 5)
. Major populations of CD1c+ MoLCs and of LCs but only a minor fraction of CD1c– MoLCs were induced to express CD54 at high levels (Fig. 5)
. CD40 stimulation increased HLA-DR expression strongly in a subpopulation of LCs, moderately in CD1c+ MoLCs, and not in CD1c– MoLCs (Fig. 5)
.
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Figure 5. CD40L induces different level of antigen expression in epidermal LCs and MoLCs. CD1c isolation was performed with ECs at day 0 and MoLCs after 6 days of culture with GM-CSF, IL-4, and TGF-ß1. Cells were seeded on a layer of adherent CD40L+ cells and cocultured for 18 h. Histograms show phenotype of DR-gated (CD86, CD80, and CD54) and CD1c-gated (HLA-DR) cells after harvesting. FACS analyses of 10,000 cells from one donor are representative of five independent experiments; stimulated by CD40L, black lines; wt, gray lines; isotype, dotted lines.
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(Fig. 7
).
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Figure 6. Epidermal LCs and MoLCs up-regulate CD4+ T cell proliferation in a mixed leukocyte reaction (MLR) after CD40L cross-linking. LCs and MoLCs were isolated by CD1c and cocultured with CD40L+ cells for 18 h. After harvesting of the nonadherent LCs, graded doses of LCs as stimulators were cocultured with 5 x 104/well allogeneic T cells (TC) for 5 days. CD4+ T cells were enriched from peripheral blood mononuclear cells of different donors by MACS depletion (mean of 94% CD4+; 99% CD3+) and were stimulated by LCs or MoLCs. Results of TdR incorporation are presented as mean counts per minute (cpm) of triplicates and are representative of three independent experiments.
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Figure 7. Epidermal LCs induce significant release of IFN- in CD4+ T cells. Graded doses of isolated LCs as stimulators were cocultured with 5 x 104/well allogeneic CD4+ T cells for 18 h. The release of IFN- by T cells was measured by ELISA. Graph shows secretion of IFN- by T cells from one donor and represents three independent experiments with similar results (ratio LC:T cell).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After CD40 ligation, the LCs were characterized by retaining HLA-DR and by strong up-regulation of the adhesion molecule CD54 and the costimulatory molecule CD86. Moreover, nearly all epidermal LCs neoexpressed CD83 upon CD40 triggering. This phenotypic maturation of the cells was, however, not associated with increased CD80 expression nor with secretion of a high level of bioactive IL-12p70. In a former report, induction of CD80 expression was only observed in human LCs that were stimulated in the presence of GM-CSF [14
]. The low-level secretion of IL-12 by LCs, even after additional stimulation with IFN-, LPS, or other TLR ligands, is in accordance with the findings from Nagakawa et al. [30
], who revealed up-regulation of the mRNA for IL-12 p35 and IL-12 p40 but no secretion of bioactive IL-12. In one of the rare reports concerning IL-12 in LCs, its synthesis was found in CD1a-enriched human epidermal LCs, but the level of IL-12p40 mRNA increased spontaneously [31
]. In contrast, murine LCs were found to secrete IL-12 after CD40 ligation, but this effect is most probably a result of the preactivation of the cells by their isolation via HLA-DR binding [15
, 16
]. Coincident with CD1c+ peripheral blood DCs [32
], LCs induced T cell proliferation and IFN- secretion, despite lacking IL-12 production. This might suggest that natural LCs produce an as-yet unidentified T cell activating factor.
The electron microscopical examination of the CD40-stimulated LCs demonstrated that the cells retain their BGs and show the features of cellular and endocytotic activation. Similar changes are typically seen in epidermal LCs after epicutaneous application of contact-sensitizing compounds [33 ], whereas fully matured LCs lose their BGs and resemble veiled DCs [34 ]. Cellular activation without full maturation of the CD40-stimulated LCs is finally strengthened by the results of the MLR analyses showing only a weak increase of their stimulatory capacity. Such a partial activation by CD40 ligation was found in murine epidermal LCs also [35 ].
According to Geissmann et al. [18 , 24 , 25 ], MoLCs behave like immature epidermis-resident LCs in many aspects though little expression of BG. Our analyses revealed that MoLCs represent a less distinct and homogenous cell population than the LCs obtained from human epidermis by CD1c cell sorting. Nevertheless, we, like Geissmann et al. [18 ], observed that MoLCs express functional CD40 and undergo maturation, including up-regulation of CD80 expression after CD40 ligation.
Moreover, CD40 stimulation of the MoLCs resulted in secretion of bioactive IL-12 and IL-10. It is remarkable that the IL-12 secretion could not be inhibited by external addition of TGF-ß1. IL-12 secretion was enhanced by additional stimulation with IFN- and most prominently, with LPS. In spite of this phenotypic and functional maturation, the CD40-stimulated MoLCs did, however, not resemble fully maturated APCs. We, in contrast to Geissmann et al. [18
], observed only a moderate up-regulation of CD83 expression and a rather weak increase of their allostimulatory capacity.
Unexpectedly, the stimulation of MoLCs with LPS resulted in a markedly enhanced IL-12 secretion; stimulation with LPS, peptidoglycan, or flagellin achieved a significant secretion of IL-10. The release of this cytokine is contrary to previous investigations and to the prevalent view that LCs unlike DCs are not able to produce this immunosuppressive cytokine [17 , 18 , 36 ]. We measured the highest IL-12 and IL10 amounts in supernatants of the same culture wells and the same time-point (after 18 h). Thus, in LCs, IL-12p70 and IL-10 are not expressed exclusively. The IL-10 detected cannot derive from contaminating monocyte-derived DCs, as TGF-ß1, which is critically involved in MoLC generation, inhibits monocyte-derived DCs to produce IL-10 [18 , 24 ]. The autocrine IL-10 production of CD40L- and bacteria-stimulated DCs has recently been recognized to limit maturation, migration, and IL-12 secretion, thereby preventing inappropriate Th1 immune responses [37 , 38 ]. Whether such an inhibitory effect also applies to maturing LCs remains to be verified in detail.
The MoLCs were subjected to the CD1c magnetic-activating cell-sorting protocol as used for the epidermal LCs. CD40L induced a distinct population of positive, sorted MoLCs to strongly express HLA-DR, CD54, CD86, and different from epidermal LCs, CD80. In unsorted MoLCs, CD40L induced these phentotypical changes at a weaker level. This indicates that the treatment of monocytes with cytokines does not result in generation of a homogenous DC population.
Except from CD80 and IL-12, LCs and MoLCs display similar phenotypic and functional alterations after CD40 triggering. Considering a plasticity more pronounced for generated than for ex vivo LCs, stimulation of MoLCs may propagate a cell subset that still can achieve the potential to release inflammatory and anti-inflammatory cytokines. It seems more likely that in the epidermis, LCs are sentinels that mainly fulfill their task to capture foreign antigen. For an effective induction of a Th1 response, LCs probably require more than just a danger and a T cell signal. This may reflect a resistance against activation protecting from undue inflammations.
Unequal levels of CD40L-stimulated cytokine production were also found in a recent study that compared isolated CD1c+ peripheral blood DCs with DCs generated from monocytes [32 ]. Analogous to MoLCs and LCs, generated DCs vigorously responded to CD40L, whereas isolated DCs proved to be only low-level cytokine producers [32 ]. In conclusion, these differences illustrate the limitations of monocyte-derived DCs as a model for natural DCs.
Received July 16, 2003; revised April 7, 2004; accepted April 26, 2004.
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