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The Journal of Immunology, 2006, 176: 7232-7242.
Copyright © 2006 by The American Association of Immunologists

A Postmigrational Switch among Skin-Derived Dendritic Cells to a Macrophage-Like Phenotype Is Predetermined by the Intracutaneous Cytokine Balance1

Tanja D. de Gruijl2,*, Claudia C. Sombroek{dagger}, Sinéad M. Lougheed*, Dinja Oosterhoff*, Jan Buter*, Alfons J. M. van den Eertwegh*, Rik J. Scheper{dagger} and Herbert M. Pinedo*

* Department of Medical Oncology, Division of Immunotherapy, and {dagger} Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Migration of dendritic cells (DC) to secondary lymphoid organs under proinflammatory conditions coincides with their maturation and acquisition of T cell stimulatory abilities. In contrast, impaired activation of DC, e.g., in tumor-conditioned environments, may hamper their activation and possibly their subsequent migration to lymph nodes, leading to either immunological tolerance or ignorance, respectively. In this study, the influence of cytokines in the peripheral skin microenvironment on the activation state of migrating cutaneous DC was assessed using an ex vivo human skin explant model. We observed a phenotypic shift from mature CD83+ DC to immature CD14+ macrophage-like cells within 7 days subsequent to migration from unconditioned skin. These macrophage-like cells displayed a poor T cell stimulatory ability and lacked expression of CCR7, thus precluding their migration to paracortical T cell areas in the lymph nodes. The balance of suppressive and stimulatory cytokines during the initiation of migration decided the postmigrational fate of DC with IL-10 accelerating and GM-CSF and IL-4 preventing the phenotypic switch, which proved irreversible once established. These observations indicate that, in immunosuppressed environments, a postmigrational DC-to-macrophage shift may hinder T cell activation, but also that it may be prevented by prior conditioning of the tissue microenvironment by GM-CSF and/or IL-4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Myeloid dendritic cells (DC) are key players in the regulation of Ag-specific immunity through their ability to link the innate to the adaptive immune system. In providing this link, DC control the balance between Ag-specific T cell tolerance and immunity. Unraveling the processes by which DC determine these opposite immunological outcomes will provide clues on how to curb T cell responses to self Ag in autoimmune diseases, but also how to generate them in tumor-conditioned environments (1, 2, 3). Previous studies have shown that the activation (i.e., maturation) state of the DC at the time of their encounter with Ag-specific T cells is a main determinant in this regard (4).

Myeloid DC precursors circulate in peripheral blood, from where they seed peripheral tissues as immature DC (iDC). In this state, the DC’s prime function is to constantly screen its environment for signs of infection through specialized molecular pattern recognition receptors (5). Under appropriate proinflammatory conditions, DC are activated, start to express maturation markers (6), and migrate to secondary lymphoid tissues (5), where they activate T cells upon specific Ag recognition. Migration of DC to the paracortical T cell areas in draining lymph nodes (LN) takes 24–48 h and depends on the expression of the chemokine receptor CCR7 (7, 8, 9). It is generally accepted that DC maturation represents end-stage differentiation, and that mature DC do not leave the LN but are eliminated there through apoptotic processes (3, 10). Low-rate steady-state migratory DC are now believed not to mature but to nevertheless acquire the ability to home to secondary lymphoid tissues (3, 4, 11). These iDC with LN-homing ability may normally be instrumental in maintaining peripheral tolerance to self-Ags (3) and in immunosuppressed tumor environments may actually preclude the generation of effective T cell responses to tumor Ags (3). IL-10 has been implicated as the most prominent cytokine suppressing DC maturation in tumor-conditioned environments (4, 12, 13). To study the activation state and functional properties of DC migrating from peripheral tissues under immunologically suppressed conditions, we made use of IL-10-conditioned human skin explants, which allow the study of migrating DC under near-physiological conditions (14, 15).

CD14+ dermally resident cells in skin explants were previously identified as direct tissue precursors of human Langerhans cells (LC) (16). In this study, we present evidence to suggest that CD14+ cells migrating from human skin explants may actually represent an alternative end-stage differentiation for skin-derived DC, which during the process of migration acquire a macrophage-like phenotype. Intradermal (i.d.) injection of IL-10 accelerated this process, whereas maturational factors like GM-CSF and IL-4 were able to lock the migrating DC in a mature state, even in the presence of suppressive IL-10 concentrations. The prevailing cytokine balance in peripheral tissues during the initiation of migration may thus determine the stability of the activation state of the DC subsequent to migration. Because the CD14+ cells no longer expressed CCR7, indicating a disability to home to the paracortical T cell areas of LN, we propose that a phenotypic DC-to-macrophage shift under tumor conditions with high IL-10 levels may be an effective mechanism to ensure immunological ignorance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Skin explant cultures and DC migration

The culture medium used was IMDM (Invitrogen Life Technologies) supplemented with 50 U/ml penicillin-streptomycin, 1.6 mM L-glutamine, 0.01 mM 2-ME, and 5% complement-inactivated, normal human pooled serum (HSP; Centraal Laboratorium van de Bloedtransfusiedienst). GM-CSF (Schering-Plough), IL-10, and IL-4 (Centraal Laboratorium van de Bloedtransfusiedienst), or an E1-deleted, replication-deficient adenovirus type-5 encoding the green fluorescent reporter protein (Ad-GFP; provided by Dr. D. Curiel, University of Alabama, Birmingham, AL), all diluted in serum-free IMDM without supplements (i.e., plain medium), were used for i.d. injection as described previously (15). Human skin specimens were obtained after informed consent and from patients undergoing corrective breast or abdominal plastic surgery. Cytokines or viruses were injected into the dermis with a BD Micro-Fine insulin syringe (0.33-mm (29G) x 12.7-mm needle) in the indicated amounts and in a total volume of 20 µl. At the site of injection, a 5-mm urtica appeared, and an exact punch biopsy of 6 mm was taken. The biopsy was lifted from the specimen with sterile forceps, and with sterile scissors the dermis was cut at a depth of 2–3 mm to obtain skin explants. To study the migration and phenotypic development of epidermal and dermal DC separately, the epidermal and dermal layers were separated through dispase digestion for 1 h at 37°C (50 mg/ml Dispase grade II; Roche Diagnostics), followed by their culture as described below.

For the migration assay, the skin explants (12–20 samples per condition) were placed directly in 1 ml of culture medium (floating with the epidermal side up) in a 48-well culture plate (Costar). At day 2 (unless otherwise indicated) the explants were discarded, and the migrated cells were harvested (plastic-adherent cells were detached by incubation with 0.5-mM EDTA) and pooled per test condition or cultured for an additional 5 days before harvesting. Absolute numbers of migrated DC were counted in hemocytometers using trypan blue exclusion, or with Flow-Count fluorospheres (Beckman Coulter) following the manufacturer’s instructions and recalculated to migrated DC number per skin explant. FACS analyses, cytokine release assays, or MLR were subsequently performed.

Separation of epidermis from dermis and dissociation of the separate layers

Cytokine- or medium-injected 6-mm biopsies (10 per condition) were either immediately processed or removed from culture on day 2 and placed in 10-cm diameter culture dishes containing 15 ml of 0.05% trypsin (Invitrogen Life Technologies) for 4–5 h at 37°C, 5% CO2. The epidermis and dermis were separated with tweezers, washed with IMDM-10% FCS, and single-cell suspensions were made of each by pushing through 100-µm pore nylon cell strainers (Falcon; BD Biosciences) with the plunger of a 2-ml syringe. The cell suspensions were resuspended in 5 ml of IMDM and counted before flow cytometric analysis.

Flow cytometry

Cells were incubated on ice for 30 min in PBS with 0.1% BSA and 0.01% NaN3, in the presence of appropriate dilutions of FITC- or PE-labeled mouse mAbs to CD83, Langerin (Immunotech), CD64 (Medarex), CD1a, CD1b, CD86, CD40, DC-SIGN (BD Pharmingen), BDCA-1 (CD1c)/BDCA-3/-4 (Miltenyi Biotec), CD14, and HLA-DR (BD Biosciences), with a CY5-labeled Ab to CD14 (Immunotech), or with unconjugated mAbs to CMRF-44 (a gift from Dr. D. Hart, Mater Medical Research Institute, Queensland, Australia) or to CCR7 (BD Pharmingen). A second incubation step was performed for the unconjugated mAbs with FITC-labeled Goat-anti-Mouse (GaM) Abs (Centraal Laboratorium van de Bloedtransfusiedienst) or with PE-labeled GaM Fab (DakoCytomation). The cells were subsequently analyzed, using a FACStarPlus and CellQuest FACS analysis software (BD Biosciences). For CFSE labeling, day 2-migrated cells were washed with PBS, incubated with 10 µM CFSE (Molecular Probes) for 10 min at 37°C, and washed twice with cold PBS. Labeled cells were cultured for 5 days, before CFSE levels were determined in relation to CD14 expression by FACS analysis. Apoptosis measurements were performed by AnnexinV-FITC and propidium iodide (PI) staining using a kit according to the manufacturer’s instructions (Bender MedSystems).

Immunocytochemistry

Cytospin preparations of migrated cutaneous DC were acetone-fixed for 10 min, preincubated with normal rabbit serum (1:50; CLB) for 10 min, and incubated for 1 h with a primary mAb against CD68 (1:400; DakoCytomation), or with an appropriate isotype control. Subsequent incubation with rabbit anti-mouse-biotin conjugate (1:300; DakoCytomation) for 30 min was followed by incubation with HRP-streptavidin complexes (1:500; DakoCytomation). Staining was then visualized with 3-amino-9-ethyl-carbazol (ICN Biochemicals) in the presence of hydrogen peroxide. Slides were counterstained with hematoxylin and mounted.

Dextran-FITC uptake

Dextran-FITC (molecular mass 42,000; Sigma-Aldrich) was diluted to a concentration of 1 mg/ml in culture medium. DC were incubated with Dextran-FITC at this concentration for 1 hour either on ice or at 37°C. Free Dextran-FITC was washed away, and uptake at both temperatures was determined by flow cytometry. The fluorescence intensity of the DC incubated at 37°C was compared with the corresponding fluorescence intensity of the DC incubated on ice as a measure of active Dextran-FITC uptake.

Mixed lymphocyte reaction

MLR was performed with the migrated skin DC. DC were added as stimulator cells to round-bottom 96-well tissue culture plates (Costar) at graded doses, reflecting the indicated responder-stimulator ratios. Allogeneic plastic nonadherent PBL (prepared as described previously; Ref. 17) were used as a source of responder cells, and 1 x 105 lymphocytes/well were added to the migrated skin DC. Stimulations were performed in duplicate or triplicate. The cells were cultured for 5 days in medium containing 10% FCS (Invitrogen Life Technologies). During the last 18 h of culture, [3H]TdR was added (0.4 µCi/well) (Amersham), after which the cells were harvested onto fiberglass filters, and [3H]TdR incorporation was determined using a flatbed scintillation counter.

Statistical analysis

Percentages of phenotypically distinguishable DC populations and absolute numbers of migrated DC were compared between conditions using the paired or unpaired Student’s t test (two-sided). Differences were considered significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
i.d. cytokines modulate the postmigrational phenotype of cutaneous DC

To investigate the influence of cytokines in the skin milieu on the phenotype of migrating DC, skin explants were i.d. injected with serum-free medium containing IL-10 (10 ng/explant), or GM-CSF (100 ng/explant) with or without IL-4 (1000 U/explant), and subsequently cultured, while floating in medium, with the epidermal side up. No cytokines were added to the culture medium. The bulk of skin-derived DC (discernable by their characteristic morphology) were found to migrate from the explants between 24 and 48 h of culture, by which time migration was complete. Explants were therefore removed after 48 h, and the migrated DC were harvested, counted, and analyzed by flow cytometry. Mean absolute numbers of migrated DC (per explant) for the tested conditions were as follows: medium control, 4,454 (range 875–14,500; n = 14); IL-10, 4,193 (range 344–9,961; n = 10); GM-CSF, 4,318 (range 1,333–16,000; n = 12); GM-CSF + IL-4, 8,366 (range 3,125–21,000; n = 11). Of these, only GM-CSF + IL-4 significantly increased DC migration in comparison to medium (p < 0.005). For flow cytometric analysis, DC were gated as a separate population by their characteristic scatter properties (high forward and side scatter values (FSC and SSc, respectively) as described (15, 16); see Fig. 1A). Phenotypic assessment showed a mature CD83+CD1a+ phenotype for DC migrated from explants that were injected with GM-CSF ± IL-4 with high levels of the costimulatory and adhesion molecules CD40, CD54, and CD86, of HLA-DR, and of the maturation marker CMRF-44 (Fig. 1A). In contrast, DC from medium- and IL-10-injected explants displayed lower expression levels of these molecules and, of note, a considerable proportion (particularly from the IL-10-conditioned explants) expressed the monocyte/macrophage marker CD14 (Fig. 1A). Double-staining experiments further revealed coexpression of CD14 and DC markers on cells migrated from medium- and IL-10-injected explants (Fig. 1B): many of the CD14+ cells also expressed the LC-associated marker CD1a, whereas under the same conditions almost all cells expressed CMRF-44, an early maturation marker previously identified on DC migrating from skin (6). Of note, cells positive for the DC maturation marker CD83 expressed CMRF-44 at high levels, while they completely lacked CD14 expression (Fig. 1B).


Figure 1
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  		FIGURE 1. The i.d. injection of cytokines into human skin explants before culture determines the phenotype of migrated DC at day 2 of explant culture. Before culture, medium, IL-10 (10 ng/explant), and GM-CSF (100 ng/explant) with or without IL-4 (1000 U/explant) were i.d. injected into skin explants. After 2 days in vitro culture the explants were discarded, and the migrated cells were harvested and analyzed by FACS analysis. A, SSc/FSC scatters and histograms showing expression of the indicated markers. The used DC gate is shown in the SSc/FSC dot plots, and the mean fluorescence intensities are indicated for each of the histograms. The markers in the histograms denote the fluorescence levels obtained with appropriate isotype controls. B, Double-staining FACS analyses of the migrated DC for the indicated conditions and markers. FACS data are representative of 3–10 independent experiments.

 
De novo CD14 expression on LC in cultured skin explants

To ascertain the origins of the skin-emigrated DC and further determine the phenotype of unmigrated DC remaining in situ, single-cell suspensions from separated epidermal and dermal layers of uncultured or 2-day-cultured explants were analyzed by flow cytometry. DC from fresh, uncultured explants displayed a predominantly immature LC phenotype (CD1ahigh/Langerin+/CD83–; see Fig. 2A), and constituted 1–3% of both the epidermal and the dermal fractions. No CD1a+CD14+ double-positive DC could be detected at this stage (Fig. 2A). Indeed, CD14+ cells made up only 0.01–0.09% of the total epidermal and dermal cell populations and could not account for the numbers of CD14+ cells found among the emigrated cells from skin explants by day 2 of culture. At day 2 subsequent to i.d. injection of either medium or IL-10, de novo expression of CD14 was apparent on skin-resident CD1ahighLangerin+ LC (at an average of 25%), as shown for the epidermis upon IL-10 injection in Fig. 2B. In contrast, LC in explants injected with GM-CSF and IL-4 remained completely negative for CD14 (Fig. 2B). Conversely, the majority of CD1a+ LC in explants injected with GM-CSF and IL-4 expressed CD83 at that time (71% on average), whereas in the IL-10-injected explants (and in the medium-injected explants; data not shown) the majority of LC remained immature (CD83– at an average of 74%; see Fig. 2B). Similar observations were made for the epidermal and dermal fractions (summarized in Fig. 2C), with this one exception, that both pre- and postculture CD83 expression was higher on dermal LC in the medium and IL-10 conditions, consistent with their migratory state, in transit through the dermis.


Figure 2
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  		FIGURE 2. De novo expression of CD14 on LC after in vitro culture of skin explants. Expression of Langerin, CD83, and CD14 in relation to CD1a in cell suspensions from separated epidermal and dermal fractions of skin explants before culture (day 0) (A) or the epidermal layer of skin explants injected on day 0 with the indicated cytokines and subsequently cultured for 2 days (B). C, Percentages of CD14- and CD83-expressing (CD1ahighLangerin+) LC in the epidermis and the dermis on day 0 (d.0 pre) and day 2 (d.2) of culture after prior i.d. injection of medium, IL-10, or GM-CSF+IL-4. *, Denotes significant differences with the d.2 medium condition in a 2-sided paired Student’s t test (p < 0.05; n = 3). D, Langerin expression in relation to CD1a on LC in cell suspensions from separated epidermal and dermal fractions from GM-CSF- and IL-4-injected and 2-day-cultured skin explants, and expression of the LC markers Langerin and E-cadherin and the dermal DC markers DC-SIGN and CD1b in relation to CD1a on corresponding skin-emigrated cells at day 2 of explant culture. All results are representative of at least three experiments.

 
In contrast to the epidermis, the dermis of 2-day-cultured skin explants contained a Langerin-negative population of DC with intermediate levels of CD1a expression, closely resembling the majority of skin-emigrated DC at that time (shown in Fig. 2D for GM-CSF/IL-4-conditioned skin explants). This observation is consistent with the reported down-regulation of both Langerin and CD1a upon migration of LC (16, 18). Further analysis of 2-day-migrated DC revealed expression of the LC-specific markers Langerin and E-cadherin on the small population of LC that maintained high expression levels of CD1a. In contrast, expression of DC-SIGN, a dermal DC marker, was mostly restricted to a minority of CD1a– DC (Fig. 2D). Of note, CD1b, a marker commonly associated with dermal DC, was clearly expressed on the subpopulation of the skin-emigrated DC with intermediate CD1a expression levels, but to a lesser extent also on subpopulations of CD1ahigh LC and CD1a– dermal DC, leaving the assignment of the skin-derived DC with intermediate CD1a expression levels to either the LC or dermal DC subset ambiguous.

Postmigrational phenotypic development of cutaneous DC is predetermined by the intracutaneous cytokine balance

The observed coexpression of the monocyte marker CD14 and the DC markers CD1a and CMRF-44 on cells migrated from medium- or IL-10-injected explants indicated an intermediate state of DC differentiation. To further assess the phenotypic development of the emigrated DC over time, they were cultured for up to 7 days after the start of migration in cytokine-free medium. In the cultures of DC, migrated from medium- and IL-10-injected skin explants, a gradual shift toward a CD14+ state (via a CD1a+CD14+ phenotype) was observed with a concomitant loss of CD1a and CD83 expression over the course of 1–7 days subsequent to migration (shown for the IL-10 condition in Fig. 3A). Together with the expression of CD14, also expression levels of the pan-myeloid marker CD11c were elevated (Fig. 3A). Day 2 vs day 7 comparisons of the skin-emigrated DC revealed these phenotypic changes to be accompanied by a down-regulation of the LN-homing chemokine receptor CCR7 and costimulatory markers such as CD40 and CD86 (Fig. 3B). Although this shift toward the immature CD14+ state appeared to be accelerated and was significantly more pronounced by day 2 after i.d. injection of IL-10, it was completely prevented by i.d. administration of GM-CSF ± IL-4 (see Fig. 3, B and C).


Figure 3
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  		FIGURE 3. Study of the postmigrational phenotypic development of skin DC up to day 7 of culture reveals a switch to a CD14+ immature state following i.d. injection of medium or IL-10 and the persistence of a CD83+ mature state after i.d. injection of GM-CSF+IL-4. A, Skin explants were injected with IL-10 and removed from culture at day 1. Migrated cells were harvested at days 1, 2, 4, or 7 and analyzed by flow cytometry for CD1a, CD14, CD11c, and C D83 expression. Percentages of positive cells are indicated in the quadrants of the FACS plots. B, FACS dot plots of CD1a/CD14, CD83/CCR7, and CD40/CD86 double stainings of migrated DC at day 2 and day 7 after the start of explant culture (explants were removed on day 2). Before culture, medium, IL-10, and GM-CSF+IL-4 were i.d. injected into skin explants. Results from one representative experiment of five are shown. C, Shifts between the mature CD83+CD1a+CD14– DC, and the immature CD83–CD1a+CD14+ DC and CD83–CD1a–CD14+ macrophage-like cells from day 2 to day 7 after start of migration. *, Denotes significant shifts in populations between day 2 and 7 in a paired Student’s t test at p < 0.005 (medium, n = 8; IL-10, n = 6; GM-CSF, n = 6; GM-CSF+IL-4, n = 6). Differences between the percentages of mature DC at day 2 were also significant between the medium and each of the cytokine conditions and at day 7 between the medium and the GM-CSF and GM-CSF+IL-4 conditions (p < 0.05).

 
To also ascertain the effects of IL-10 and GM-CSF/IL-4 on the selectively for LC from the epidermis and for DC from the dermis, skin explants were separated into epidermal and dermal layers. The dermal layers were injected with cytokines and cultured conforming to procedures applied to full-thickness skin explants, while the epidermal layers were cultured floating on medium to which either IL-10 (50 ng/ml) or GM-CSF (100 ng/ml) and IL-4 (1000 U/ml) were added. Results from a representative experiment are shown in Fig. 4A and summarized for four experiments in Fig. 4B. LC migrating from the epidermis displayed typical high CD1a expression on day 2, accompanied by CD83 expression (data not shown), both of which were lost over the next 5 days unless GM-CSF and IL-4 had been added to the medium, in which case CD1a and CD83 expression levels were preserved, albeit at lower levels for CD1a (Fig. 4A). Of note, in only one of four of the experiments was the switch to a CD14+ phenotype observed between days 2 and 7 after migration of LC from the separated epidermis; while no CD14 positivity was observed at day 2, 5 days later 5.4% of cells migrated from medium control cultures were CD14+ vs 21.9% from IL-10-conditioned cultures. In contrast to epidermis-emigrated LC, DC migrated from the dermal layer displayed mostly intermediate CD1a expression levels and/or CD14 expression. Over the next 5 days, CD1a expression was completely lost in DC migrated from medium- or IL-10-injected dermal explants, whereas a near complete transition to the CD14+ phenotype was observed. Of note, CD1a expression was not conserved in the DC migrated from GM-CSF- and IL-4-injected dermal explants, but the transition to the CD14+ phenotype was significantly inhibited (Fig. 4B). Thus, the observed CD14-skewing effect in DC after migration from full-thickness skin explants seems to be mostly determined by conditions in the dermal environment, whereas the GM-CSF/IL-4-induced conservation of the CD1a+CD83+ mature DC phenotype involves LC and/or epidermis-derived maturing factors.


Figure 4
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  		FIGURE 4. The GM-CSF/IL-4-induced postmigrational preservation of the mature DC phenotype is predominant in epidermis cultures, whereas the medium- and IL-10-associated postmigrational switch to a CD14+ phenotype is predominant in isolated dermis cultures. Epidermal and dermal layers were separated by dispase treatment and separately cultured for 7 days side by side with full-thickness skin explants. Medium with or without the indicated cytokines was i.d. injected or (in case of the epidermal cultures) added to the medium at the start of culture. Skin explants were removed on day 2, and migrated cells were harvested for flow cytometric analysis at the same time (day 2) or 5 days later (day 7). A, FACS dot plots of CD1a/CD14 double stainings of migrated DC at day 2 and day 7 after the start of culture. Results from one representative experiment of four are shown. B, Shifts between the mature CD83+CD1a+CD14– DC, and the immature CD83–CD1a+CD14+ DC and CD83–CD1a–CD14+ macrophage-like cells from day 2 to day 7 after start of migration. *, Denotes significant differences with the corresponding medium conditions in a paired Student’s t test at p < 0.05 (n = 4).

 
To determine the relative abilities of GM-CSF and IL-4 to counteract the loss of the mature DC phenotype subsequent to migration, head-to-head comparisons were made between them in full-thickness skin explants, and no significant differences were observed. Of note, i.d. administration of either of these cytokines, or a combination of both together with IL-10 overruled the suppressive effect of IL-10 and locked the DC in a mature CD83+CD14– phenotype up to day 7 after the start of migration (Fig. 5A) and preserved high-level expression of costimulatory molecules (Fig. 5B). Accordingly, the otherwise lost T cell stimulatory ability of the skin-emigrated DC was preserved, as established in an allogeneic MLR (shown for GM-CSF in Fig. 5C).


Figure 5
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  		FIGURE 5. GM-CSF and IL-4, injected simultaneously with IL-10 and before skin explant culture, are equally effective at preserving a mature, T cell-stimulatory phenotype of skin-emigrated DC. IL-10 was i.d. injected, alone or in combination with either GM-CSF, IL-4, or GM-CSF+IL-4, before skin explant culture. Explants were removed from culture at day 2, and migrated cells were harvested 5 days later and stained with mAbs against the indicated markers. A, CD83 expression in relation to CD14. Percentages of positive cells are indicated in the corresponding quadrants. B, CD86 and CD40 expression, after i.d. injection of IL-10 alone (open histograms) or in combination with the indicated cytokines (closed histograms). The marker denotes fluorescence intensity levels obtained with the isotype control mAb. C, Allogeneic T cell stimulation in an MLR by migrated DC at day 2 vs day 7 for the indicated test conditions. Representative data from three experiments are shown.

 
Skin-emigrated CD14+ cells express DC precursor markers but do not differentiate into DC in the presence of GM-CSF and IL-4

The skin-emigrated CD14+ cells, gaining in predominance over time in the medium and IL-10 conditions, were further characterized and distinct from the CD14-negative DC by their expression of the previously identified DC precursor markers BDCA-3 and BDCA-4 (i.e., neuropilin-1), both at day 2 and at day 7 after the start of explant culture (19, 20) (Fig. 6A). In contrast, CD1c (i.e., BDCA-1) was expressed on the CD14+ cells as well as on the CD14-negative DC (as was also previously reported (Ref. 16 ; see Fig. 6A). The expression of these BDCA/DC precursor markers seemed to suggest a capacity for DC differentiation of the CD14+ skin-emigrated cells. However, as shown in Fig. 6, B and C, addition of GM-CSF (100 ng/ml) and IL-4 (1000 U/ml), subsequent to their migration from IL-10-injected skin explants, did not induce their differentiation to DC. Although it did preserve CD1a positivity in the CD1a single-positive DC, CD1a+CD14+ DC still reverted to a CD1a–CD14+ state over the course of the next 5 days (Fig. 6B). In addition, whereas combined i.d. administration of GM-CSF/IL-4 and IL-10 before migration preserved CD83 and CCR7 expression up to day 7 postmigration, administration of GM-CSF and IL-4 to DC subsequent to their migration from IL-10-conditioned explants could not prevent the progressive loss of these markers (Fig. 6B). The profound suppressive effect of IL-10 became apparent from the proportionate balance between the CD14-negative and CD14+ skin-emigrated cells in culture at day 7; whereas the postmigrational addition of GM-CSF and IL-4 at day 2 of culture resulted in a more equal distribution between these two populations in the i.d. medium condition, the balance was quite clearly tipped in favor of the CD14+ population in the i.d. IL-10 condition (Fig. 6C).


Figure 6
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  		FIGURE 6. Skin-emigrated CD14+ cells express DC lineage-associated precursor markers but cannot differentiate into DC upon culture with GM-CSF and IL-4. A, Skin explants were i.d. injected with medium, IL-10, or GM-CSF+IL-4 and cultured for 2 days, at which time they were removed from the cultures. Expression of CD1c (BDCA-1), BDCA-3, and BDCA-4 in relation to CD14 on skin-emigrated cells at day 2 vs day 7 after the start of explant culture. B, Skin explants were i.d. injected with IL-10, both with and without simultaneous injection of GM-CSF+IL-4, and cultured for 2 days. On day 2 the explants were removed, and in parallel cultures either nothing was added or GM-CSF+IL-4 (at 100 ng/ml and 1000 IU/ml, respectively), as indicated. Migrated cells were harvested at day 2 and day 7 and analyzed by FACS. FACS results of CD1a/CD14 or CD83/CCR7 double stainings for the indicated test conditions are shown. Percentages of positive cells are listed in the corresponding quadrants. C, Mean percentages ± SD of CD14– ({blacksquare}) or CD14+ DC ({square}) are shown on day 7 for the indicated test conditions (i.d. injection of medium or IL-10 at day 0, with either GM-CSF+IL-4 coinjected or added to the DC cultures at day 2; n = 3).

 
Postmigrational DC-to-macrophage switch

At day 2, most cells that had emigrated from medium-injected explants had a typical DC morphology with lobulated nuclei and a veiled appearance. By day 7, these cells were larger with more rounded nuclei and lacking the spikes and veils typical for migrating DC. Of note, this change in morphology was already apparent by day 2 of migration in DC from IL-10-injected explants (Fig. 7A). By contrast, DC that migrated from explants injected with GM-CSF and IL-4, mostly retained their typical DC morphology (Fig. 7A). The loss of DC morphology was accompanied by an increase in plastic adherence and a macrophage-like appearance in culture. In keeping with this macrophage-like morphology, a strong and diffuse cytoplasmic CD68 expression was observed, whereas the cells with preserved DC morphology (after injection of GM-CSF+IL-4) displayed a perinuclear expression pattern of CD68, previously reported as typical for LC (16) (Fig. 7A). In accordance with their immature phenotype, macrophage-like CD14+ cells appeared to have a greater capacity for endocytic processes as evidenced by their more efficient uptake of Dextran-FITC and higher expression levels of Fc{gamma}RI CD64 (shown in Fig. 7B for 7-day cultured DC, migrated from IL-10-injected skin explants) and displayed a reduced T cell stimulatory ability in allogeneic MLRs (see Fig. 5C). In keeping with this latter observation, the CD14+ macrophage-like cells displayed lower expression levels than the CD1a+ DC of the costimulatory molecules CD40, CD80, CD86, of CD54, and in particular of HLA-ABC and HLA-DR (shown for the GM-CSF/IL-4 and IL-10 conditions in Fig. 7C).


Figure 7
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  		FIGURE 7. Skin explant-emigrated DC acquire phenotypic, morphological, and functional characteristics of macrophages. A, May Grünwald-Giemsa and immunocytochemical CD68 stainings of cytospin preparations of migrated cells at day 2 vs day 7 of skin explant culture (explants removed at day 2) for the indicated test conditions (magnification, x400). Perinuclear localization of CD68 is indicated by arrowheads. B, Fc{gamma}RI (CD64) expression on and Dextran-FITC uptake by CD14+ or CD1a+ skin-emigrated LC on day 7 of culture. Results are shown for LC migrated from IL-10-injected explants (representative of three experiments). Staining with a CD64 mAb (closed histograms) or an isotype control (open histograms) and Dextran-FITC fluorescence after uptake at 37°C (closed histograms) and on ice (open histograms) are shown. C, Expression of the indicated costimulatory, adhesion, and HLA molecules on skin-emigrated CD1a+ DC and CD14+ macrophage-like cells on day 7 (d7) after the start of explant cultures; before culture, explants were injected with either GM-CSF and IL-4 or IL-10. Mean fluorescence intensities are indicated. Results are representative of at least three experiments.

 
Individual cells undergo a postmigrational DC-to-macrophage switch

It was next established whether the observed postmigrational changes were due to phenotypic events in individual skin-emigrated DC or rather due to the outgrowth of CD14+ precursors or the selective death or survival of the more mature CD1a+ DC. Because Ab-mediated isolation of DC, e.g., through binding to CD1a or CD83, may interfere with their activation state (21), we opted for less invasive means to study the possible DC-to-macrophage conversion. To this end, absolute numbers of CD1a+ or CD14+ cells were determined at both day 2 of migration (at which time explants were removed) and 5 days later, and related to cell division, apoptosis, and cell death (Fig. 8, A–C). Although the total absolute numbers of migrated DC remained constant, the numbers of CD1a+-migrated DC decreased and of CD14+ cells increased, from day 2 to day 7 after injection of either medium or IL-10 (Fig. 8A). Coinjection of GM-CSF resulted in constant absolute numbers of CD1a+ LC over time without a shift to CD14 positivity. Day-2-migrated DC were labeled with CFSE and analyzed 5 days later for CD14 expression and CFSE content by flow cytometry. The increase in CD14 positivity among DC migrated from medium- or IL-10-injected explants as compared with their GM-CSF-coinjected counterparts was not accompanied by cell division, as evidenced by the unaltered high levels of CFSE in these cells (Fig. 8B). Although at day 2 of explant culture high percentages of apoptotic AnnexinV+PI– cells were observed among skin-emigrated DC (irrespective of IL-10 or GM-CSF (co)-injection), these percentages were not increased much by day 7, nor were the numbers of dead cells (i.e., AnnexinV+PI+) significantly increased (Fig. 8C). The constant absolute numbers of migrated DC, the absence of cell division, and the unchanged percentages of apoptotic and dead cells, indicate the phenotypic changes occurring among skin-emigrated DC to take place in individual cells. This was further confirmed through the i.d. injection of GFP-encoding adenoviruses (109 viral particles per explant), which, after intracutaneous transduction, resulted in a clearly traceable GFP+ population of emigrated DC. In medium-injected explants, these transduced DC, gated by GFP expression, were found to be mostly CD14 negative and CD83 positive (see Fig. 8D; cells gated by GFP positivity). Upon five additional days of cultures, the same population of GFP+ DC lost CD83 expression and a considerable fraction acquired CD14.


Figure 8
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  		FIGURE 8. Phenotypic shifts among DC subsequent to migration from skin are not related to cell division or cell death. Skin explants were i.d. injected with medium, IL-10, IL-10+GM-CSF, or GM-CSF alone, and cultured for 2 days. At day 2 explants were discarded, and migrated DC were examined immediately thereafter or 5 days later at day 7. A, Absolute counts of migrated DC (recalculated per explant in mean ± SD) are shown in relation to phenotype on day 2 and on day 7: CD1a+ (CD1a) or CD1a–CD14+ (CD14) (n = 3). B, Migrated DC were harvested at day 2 and labeled with CFSE. After 5 days of additional in vitro culture (day 7), CFSE content was determined in relation to CD14 expression by FACS analysis. Percentages of CD14+ cells are listed. Results from one representative experiment of three are shown. C, Migrated DC were stained with Annexin-V (AnnV) and PI, and the percentage of apoptotic (AnnV+/PI–) and dead cells (AnnV+/PI+) were determined by FACS analysis. Bar graphs show means ± SD for the indicated test conditions at day 2 and day 7 (n = 3). D, Skin explants were injected with 109 viral particles of an adenoviral GFP-encoding vector, in plain medium, before culture. Explants were discarded on day 2, and cells were harvested on day 2 or day 7. FACS dot plots are shown of GFP-transduced DC (gated by GFP fluorescence) and their expression of CD14 and CD83 (indicated as percentage positive cells) at the indicated time points of culture.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although the mature CD83+ DC phenotype is generally believed to represent end-stage DC differentiation, our data from in vitro-cultured human skin explants and skin-emigrated DC shows that there may be an alternative end-stage differentiation wherein DC resemble CD14+ macrophages. This macrophage-like state is accompanied by a reduced ability to prime allogeneic T cells and an increased endocytotic capacity, both of which are characteristics fully consistent with a functionally immature phenotype. The stability of the mature phenotype of migrating DC or their switch to the alternative macrophage-like state appears to be determined by the balance of stimulatory and inhibitory signals in the tissue milieu during the initiation of migration. These observations provide new insights into the physiology of cutaneous DC and how maturational changes during or subsequent to their migration may determine immunological outcome in terms of activation, tolerization, or immunological ignorance.

The human skin environment contains at least two types of DC, i.e., the epidermal LC and the dermal DC. Functional differences between these two cutaneous DC subsets are still largely undefined, and the possible relationship between them is unclear (22, 23). Larregina et al. (24, 25) recently defined CD1a+ skin-emigrated DC as LC and CD1a– DC as dermal DC. In keeping with this classification, DC-SIGN, reportedly expressed by dermal DC but not by LC, was mostly detected on CD1a– DC that had migrated from GM-CSF- and IL-4-injected skin explants. In contrast, expression of the dermal DC-associated marker CD1b was found to be independent of CD1a status: we even observed expression of CD1b on CD1ahighLangerin+E-cadherin+ LC, leaving its significance as a specific marker for dermal DC in doubt. Discrimination between migrating LC and dermal DC is further complicated by the down-modulation of both CD1a and Langerin on LC, following their maturation and migration from the epidermis into the dermis (18). Nevertheless, to assess the specific effects of the tested cytokines on LC in the epidermis and DC in the dermis, we studied DC migration from the separated epidermal and dermal layers. These experiments revealed that addition of GM-CSF and IL-4 to the epidermis cultures preserved the postmigrational mature phenotype of migrated LC. The fact that this effect was equally clear for DC that migrated from full-thickness skin explants, but could not be demonstrated for CD1a+ DC (including CD1ahigh LC) that migrated from the separated dermal layer, indicates that epidermal factors (either secondarily induced or in concert with GM-CSF and IL-4) are instrumental in the stabilization of the postmigrational mature phenotype of both dermal and epidermal DC. In reverse, dermal factors seemed to control the observed postmigrational DC-to-macrophage conversion, because the acquisition of the CD14+ macrophage-like phenotype among epidermis-emigrated LC was only observed in the presence of IL-10 in one of four experiments, but was consistently observed and near-complete among DC migrated from medium- or IL-10-conditiond dermal explants. This process might be determined by suppressive factors produced in the dermal environment, such as endogenous IL-10. In line with this, we have observed low but measurable IL-10 release from medium-injected skin explants (at 5–20 pg/ml). Alternatively, the process of DC transmigration across the dermal lymphatics might provide essential signals needed for the conversion process, in a reverse analogy to the findings of Randolph et al. (26), who observed a monocyte-to-DC conversion upon reverse transmigration over endothelial layers. Of note, acquisition of the CD14+ phenotype was significantly inhibited by injection of the dermal explants with GM-CSF and IL-4 before culture. Thus, the net effect on the postmigrational phenotypic development of cutaneous DC seems determined by a delicate balance and cross-regulation of activating and suppressive factors in both the epidermal and dermal tissue environments. The identification of endogenous factors or tissue components involved in these processes certainly warrants further study.

To directly demonstrate the conversion from a CD1a+ DC to a CD14+ macrophage at the single-cell level, we isolated LC from epidermal sheets but did not observe a DC-to-macrophage switch during their subsequent cytokine-free culture over the period of a week (data not shown). This is in line with the apparent essential role of dermal environmental factors in this process. Nevertheless, the absence of proliferation or massive cell death, in combination with relatively constant absolute cell counts over culture, is a clear indication of an actual postmigrational DC-to-macrophage conversion taking place in individual cells.

The skin-emigrated CD1a–Langerin–CD14+ macrophage-like cells described by us are distinct from previously reported dermal CD14+Langerin+ LC precursors (16), but rather more reminiscent of a previously described CD1a–CD14+ cutaneous cell population with poor T cell stimulatory ability, which, upon migration from the dermis, could not be in vitro differentiated to CD1a+ DC (27). In contrast, the CD1a+Langerin+CD14+ cells we observed both in the dermis and epidermis of 2-day-cultured explants may be more akin to the CD14+ LC precursors identified by Larregina et al. (16), repopulating the epidermis from the dermis, as previously suggested. The i.d. administration of GM-CSF and IL-4 may have resulted in full maturation to CD83+CD1a+ LC of these CD14+ precursors and consequently precluded their intracutaneous detection under these conditions. These CD14+ LC precursors were recently reported not to induce regulatory T cells, nor to suppress Th1 skewing, but rather to gain in stimulatory capacity upon prolonged exposure to T cells and then to induce Th1 effector cells (25). It will be of particular interest to determine how the DC-derived macrophage-like cells described here will perform in comparison, and how they will affect the functionality of effector T cells.

Rather than LC precursors, the skin-emigrated CD14+ macrophage-like cells seem to represent an alternative end-stage differentiation state to the fully mature CD83+ DC phenotype. This observation raises some interesting issues. A large number of DC in LN display a CD83– immature phenotype (28), and the presence of immature Langerin+CD83– LC in the paracortical T cell zones of LN, draining chronically inflamed skin, was reported (11). It was suggested, based on in vitro evidence from monocyte-derived DC cultures, that LC might be able to achieve CCR7 expression without concomitant activation, which would enable them to migrate to LN T cell zones in an immature CD83– state (11, 29). Our data suggest an alternative scenario whereby DC migrate from skin to LN in a mature CD83+CCR7+ state but lose this mature phenotype once they have reached the paracortical areas of the LN. It appears that the strength of the maturational signals in the periphery at the time of migration determines the stability of the mature phenotype of DC as they travel to the LN. Whereas the maturational signals afforded by the physical process of punching and cutting out the skin explants before putting them in culture are not sufficient to secure a stable CD83+CD14– phenotype, additional conditioning of the explants by GM-CSF or IL-4 ensures maturation of the migrated DC for up to 7 days after the start of migration. Because migration of DC to LN may take up to 2 days and DC may persist in LN as long as 2 weeks subsequent to migration (30), the time frame within which the phenotypic shifts in migrated DC were observed in vitro, may well have physiological relevance in vivo (8, 9, 31). It seems appropriate that circumstances in the periphery should thus dictate the stimulatory ability of DC migrating to the LN: a multitude of proinflammatory danger signals would effectively lock DC in a mature state and ensure homing to the paracortical T cell zones of the LN and efficient T cell recognition and stimulation. A lack of maturational signals (as in steady-state conditions) or the presence of suppressive signals (such as IL-10, e.g., in tumor-conditioned environments) would result in the loss of maturation and the acquisition of the immature macrophage-like phenotype lacking CCR7 expression. Consequently, many of the migrating DC may not actually reach the paracortical T cell zones but rather stay in the marginal sinuses of the LN. In effect, this would result in immunological ignorance. Because these iDC closely resemble macrophages, this raises the intriguing possibility that part of the marginal sinus macrophages commonly found in LN may actually be derived from afferent DC. In keeping with this, afferent lymph from normal human skin was shown to contain CD1a+CD14– DC as well as CD1a+CD14+ DC and CD1a–CD14+ macrophage-like cells (32), and marginal macrophages in murine spleen were found to display phenotypic characteristics of both macrophages and DC (33).

Although differentiation toward a macrophage-like state subsequent to presumed end-stage maturation of DC may seem unexpected, a similar observation was recently reported in a murine model system (34). Zhang et al. (34) showed that LPS-matured DC, generated in vitro from murine bone marrow, proliferated and differentiated to a CD11b+ macrophage-like state when cultured on a monolayer of stromal cells derived from the neonatal murine spleen. Notably, these DC-derived macrophages displayed decreased levels of the characteristic DC markers CD11c and MHC class II and suppressed T cell activation. Hypothesizing that at least part of the macrophages present in the spleen may have similarly originated from DC in vivo, they isolated a population of macrophage-like cells from the murine spleen with similar phenotypic and functional properties as the CD11b+ macrophage-like cells derived from mature DC in vitro. In a comment on this work, Shortman and Wu (35) suggested that the used in vitro system of generating DC from bone marrow precursors in the presence of GM-CSF and IL-4 might not be a relevant model for steady-state DC, but rather for a DC type arising after microbial infection or inflammation. The skin explant model used by us does allow for the study of DC subsets involved in steady-state migration and similarly points to differentiation of mature DC to macrophage-like cells under these conditions, with similarly decreased levels of costimulatory and HLA molecules.

IL-10-conditioned iDC were previously shown to be resistant to maturation (36) and to display deregulated chemokine receptor patterns with low CCR7 levels (37, 38). The upshot of these characteristics is the induction of Ag-specific T cell anergy (3). In tumor environments, which generally contain high levels of IL-10, these processes may in part be responsible for immune suppression and tumor progression (13, 36). Interestingly, studies with monocyte-derived DC have reported mature CD83+ DC to be resistant to suppressive effects of IL-10 (3). This is consistent with our observation that the IL-10-induced acceleration of the postmigrational loss of the mature LC phenotype was effected by the administration of IL-10 before migration, at a time when the skin-resident LC were still in an immature state. In vitro studies with bone marrow- and monocyte-derived DC previously showed IL-10 to convert DC to macrophage-like cells and to "decommission" them for T cell activation (39, 40). Interestingly, a recent report indicated that such IL-10-induced macrophage-like DC were characterized by BDCA3 expression (41). In this study, we show similar effects of IL-10 on skin-emigrated DC, which assume a BDCA3+ macrophage-like phenotype.

In conclusion, the postmigrational phenotypic development of DC can be environmentally instructed by the balance of proinflammatory and suppressive cytokines in the periphery, ultimately resulting in either immune activation, tolerance, or ignorance. The finding that the peripheral cytokine balance predetermines the stability of the postmigrational mature DC phenotype is particularly relevant in view of the ongoing discussion on what exactly constitutes end-stage maturation of DC (4, 35). Strong maturational signals may induce a stable ("fully") mature state, whereas weaker signals or signals attenuated by suppressive factors, such as IL-10, may induce a reversible ("semi-") mature state. It is conceivable that during their migration from the skin, interactions of DC with extracellular matrix components or the presence of accessory cell-derived cytokines help preserve their mature phenotype (22, 42). Such factors are clearly absent from the in vitro model used in this study. Further in vivo studies are therefore required to validate our findings as well as those of Zhang et al. (34) observing a similar conversion into macrophages of in vitro-generated mature murine DC (35). Nevertheless, these observations demonstrate an additional level of phenotypic plasticity in DC differentiation and maturation pathways that may constitute a novel mechanism of immunoregulation. For instance, in tumor-conditioned, immunosuppressed tissues, this phenomenon may interfere with effective antitumor immunization. From our observations, local GM-CSF and/or IL-4 administration may tip the balance in favor of a stable DC maturation state and prevent postmigrational conversion of local DC to macrophage-like cells. This would ensure proper homing of mature DC to the LN and subsequent T cell activation.


   Acknowledgments
 
We thank Dr. R. B. Karim (Plastic Surgery Department, Slotervaart Ziekenhuis, Amsterdam) and the Departments of Plastic Surgery and Pathology of the VU University Medical Center for providing us with skin samples, and Drs. Marco Schreurs and Hetty Bontkes for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Netherlands Organization for Scientific Research through Grants 901-10-116 and 917-56-32 (to T.D.d.G.), and the Spinoza award (to H.M.P.). Back

2 Address correspondence and reprint requests to Dr. Tanja D. de Gruijl, Department of Medical Oncology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail address: td.degruijl{at}vumc.nl Back

3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; LN, lymph node; LC, Langerhans cell; i.d., intradermal; PI, propidium iodide; FSC, forward scatter; SSc, side scatter. Back

Received for publication July 28, 2005. Accepted for publication March 24, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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