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International Immunology Advance Access originally published online on September 5, 2007
International Immunology 2007 19(11):1271-1279; doi:10.1093/intimm/dxm096
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD14+ antigen-presenting cells in human dermis are less mature than their CD1a+ counterparts

Catherine E. Angel1, Aisha Lala1, Chun-Jen J. Chen1, Stephen G. Edgar2, Lena L. Ostrovsky1 and P. Rod Dunbar1

1 School of Biological Sciences
2 Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand

Correspondence to: P. R. Dunbar; E-mail: r.dunbar{at}auckland.ac.nz


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
We recently demonstrated that three antigen-presenting cell (APC) subsets exist in the healthy human dermis, CD14+ and CD1a+ dermal APCs and migratory dermal Langerhans cells. Here, we extend these findings by defining CD208 as an exclusive marker of migratory dermal Langerhans cells, confirming that migratory dermal Langerhans cells (CD1ahigh CD207+ CD208+) and CD1a+ dermal APCs (CD1amid CD207– CD208–) are two distinct APC populations. Using flow cytometry and multicolor fluorescence immunohistochemistry, we demonstrated that there were striking differences between CD1a+ and CD14+ dermal APCs in their expression of pattern recognition receptors and maturation markers. Expression of Toll-like receptor (TLR) 2, CD206 and CD209 was largely restricted to CD14+ dermal APCs. Consistent with these observations, most CD14+ dermal APCs expressed an immature phenotype when compared with CD1a+ dermal APCs, which expressed high levels of the maturation marker CD83 on their cell surface. However, a subset of CD14+ dermal APCs also expressed cell-surface CD83, associated with a loss of cell-surface TLR2, suggesting that they have the capacity to mature. CD14+ dermal APCs are therefore the dominant cutaneous APC population capable of sensing ligands recognized by CD206, CD209 and TLR2 and subsequently may have the potential to mature. CD68 expression was largely restricted to a subset of CD14+ dermal APCs, while both CD14+ and CD1a+ dermal APCs expressed CD11b and CD11c. These findings have important implications for understanding cutaneous immune responses in humans and for the optimization of vaccine delivery via the skin.

Keywords: Dendritic cells, human, monocytes/macrophages, skin


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
The initiation of immune responses to antigens in the human skin has often been attributed to epidermal Langerhans cells, although functional antigen-presenting cells (APCs) have also been detected in the dermis of both mice and humans (1–3). Recent studies using murine models have highlighted the importance of dermal APCs in initiating immune responses in the skin (4), with some dermal APC subsets homing to compartments in the lymph nodes distinct from those colonized by Langerhans cells (5). We recently demonstrated that a human CD1a+ dermal APC population distinct from Langerhans cells had the capacity to migrate in response to lymph node homing chemokines, and was able to prime naive T lymphocytes due to its surface expression of a wide range of molecules involved in antigen presentation and co-stimulation (1). We therefore concluded that CD1a+ dermal APCs represent the dermal APC population capable of initiating immune responses in human skin. In contrast, a CD14+ CD1a– subset of dermal APCs was unable to respond to the chemokines driving lymph node homing, and did not possess a potent stimulatory capacity for naive T lymphocytes (1). This CD14+ subset may represent a cutaneous macrophage population or a precursor for other APC subsets in the skin as previously suggested (6); however, the exact function of these dermal APCs remains unresolved, as does the precise relationship between the subsets.

The expression of pattern recognition receptors (PRRs) and maturation markers can provide important information about APC function. Immature APCs tend to express PRRs that allow sensing and uptake of pathogens and their molecular products. Mature APCs tend to down-regulate these receptors and up-regulate maturation markers and molecules involved in antigen presentation. To further clarify the potential roles of CD1a+ and CD14+ APCs in human dermis and their relationship to Langerhans cells, we analyzed their expression of key PRRs and maturation markers. Flow cytometric studies were complemented by multicolor fluorescence immunohistochemistry to ensure all molecular phenotypes were confirmed on cellular subsets in situ.

The receptors we analyzed included members of the Toll-like receptor (TLR) family and the C-type lectin family, both of which have important functions in determining the immune responses to pathogens and vaccines. TLR expression varies greatly between cell subsets, and the TLR profile of an APC plays a large part in determining the molecular constituents of pathogens or vaccine adjuvants to which it responds (7, 8). Here, we have studied the expression of TLR2 and TLR4, which jointly control APC responses to bacterial lipopeptides and lipopolysaccharides (8). C-type lectins recognize particular carbohydrate structures in a Ca2+-dependent manner, enabling them to selectively bind and internalize glycosylated micro-organisms or their molecular constituents (9, 10). Unlike TLRs, there is only limited evidence that C-type lectins are capable of initiating signaling in APCs (11), but there is now plenty of data supporting a role for these molecules in directing bound antigen into antigen-processing pathways within APCs (12, 13). CD206 is the classical mannose receptor, and is well known for its ability to enhance uptake and processing of mannosylated antigens, although recent data support roles other than pathogen processing (14). CD209 (DC-SIGN) was originally defined as an intercellular adhesion receptor, facilitating dendritic cell (DC) adhesion to T lymphocytes (10, 15), but it has also been shown to bind carbohydrate structures expressed by micro-organisms (16, 17). CD206 and CD209 are of particular interest in the uptake of HIV through binding of viral gp120 (18, 19). Hence, definition of the APC subsets carrying TLR2, TLR4, CD206 and CD209 has wide implications for the biology of pathogen recognition in the skin, as well as vaccine design.

To assess the maturation status of the dermal APCs, we used two markers CD83 and CD208. Preformed CD83 exists in intracellular compartments within monocytes and immature DCs, but following activation is rapidly shuttled to the cell surface where it can be detected as a surface marker of maturation (20). CD83 is a member of the Ig superfamily and although its precise function remains unclear, it is believed to be involved in enhancing immune responses (21). Following DC activation, expression of CD208 or DC-LAMP is up-regulated; hence, this molecule is also used as a marker of maturation and to detect interdigitating DC in lymphoid tissue (22). The role of this molecule also remains unresolved, however, it is believed to be involved in lysosomal function (22). In addition to these maturation markers, we screened the dermal APCs for expression of the lysosomal membrane protein CD68 which is often regarded as the classical macrophage marker.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Preparation of dermal and epidermal cell suspensions
Fresh skin samples were obtained from healthy patients undergoing breast reduction surgery. Patients gave written informed consent, under a protocol approved by the Auckland Ethics Committee and the Clinical Board of the Counties-Manukau District Health Board. Samples were refrigerated and processed no longer than 4 h after surgery.

Subcutaneous tissue was excised and discarded. The trimmed skin was cut into strips and washed with RPMI 1640 (GIBCO-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (RF10). The dermal layer was scored with a scalpel and digested with 1 mg ml–1 collagenase (Type I) (316 U mg–1) (Gibco-BRL) and 1 mg ml–1 dispase (1.17 U mg–1) (Gibco-BRL) in RF10 for 2 h with gentle stirring, at 37°C with 5% CO2. The epidermis was peeled off the dermis using forceps. The dermis and epidermis were incubated separately at 37°C for a further 16 h in RF10 alone, before mechanical disruption by pipetting and filtration through 70-µm cell strainers (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. Single-cell preparations were cryopreserved in 50% RPMI 1640, 40% FBS and 10% dimethyl sulfoxide. Cryopreservation and subsequent thawing did not influence cell-surface phenotype by flow cytometry when compared with fresh cells (1).

Enrichment of dermal APCs and peripheral blood monocytes
Prior to CD1a+ dermal APC enrichment, CD207+ dermal Langerhans cells were depleted from the dermal single-cell suspensions using anti-CD207–PE (DCGM4) (Beckman Coulter, Miami, FL, USA), followed by anti-PE-conjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Thereafter, CD1a+ APCs were positively selected using anti-CD1a–FITC (NA1/34-HLK) (Serotec, Raleigh, NC, USA) followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec). CD14+ APCs were positively selected from dermal single-cell suspensions using anti-CD14-conjugated magnetic beads (Miltenyi Biotec). Each preparation was passed sequentially through two LS columns (Miltenyi Biotec) to improve purity.

Peripheral blood monocytes were enriched from buffy coats from healthy donors. CD14+ monocytes were positively selected using anti-CD14-conjugated magnetic beads.

Scanning electron microscopy
Enriched cells were briefly cultured on a poly-L-lysine-coated cover slip in RPMI, fixed with 2.5% glutaraldehyde followed by 1% OsO4 and 1% U acetate in water, dehydrated in ascending concentrations of ethanol, transferred to amyl acetate and critical point dried with CO2. The dried specimens were sputter coated with gold and examined using a Quanta 200 FEG Environmental Scanning Electron Microscope (FEI, Hillsboro, OR, USA) under vacuum with an accelerating voltage of 4 kV.

Flow cytometric analysis
Cells suspensions were stained with the following mouse mAbs on ice for 45 min: CD207 (DCGM4) (Beckman Coulter); CD1a (HI149) and CD11b (ICRF44) (BD Biosciences) and CD1a (NA1/34-HLK), CD11c (BU15), CD14 (UCHM1), CD83 (HB15e), TLR2 (TLR2.3) and TLR4 (HTA125) (Serotec). The nuclear stain 7-aminoactinomycin-D (7AAD) (BD Biosciences) was included with each stain, and 7AAD+ cells were gated out of all analyses to exclude non-viable cells. Stained cells were analyzed using a four-color FACSCalibur flow cytometer (BD Biosciences). Four peripheral blood monocyte and dermal samples were assessed. Previous reports have shown that certain cell-surface markers on APCs are susceptible to enzyme cleavage during tissue digestion (18, 23), so we used positive control cells expressing each marker analyzed (monocytes and monocyte-derived DCs) to confirm that the enzyme cocktail used for skin digestion did not cleave any cell-surface marker assessed using flow cytometry.

Two-color immunofluorescence staining
Fresh skin was embedded in TissueTek OCT compound (Sakura Finetek, Torrance, CA, USA), snap frozen in liquid nitrogen and sectioned using a cryostat. Sections 5-µm thick were fixed with ice-cold acetone and blocked with serum-free protein block (DAKO, Glostrup, Denmark). Fixed sections were probed with the following mouse mAbs: podoplanin (clone18H5) (Abcam, Cambridge, UK); CD207 (DCGM4) and CD208 (104.G4) (Beckman Coulter); CD1a (HI149), CD144 (55-7Hh1), CD206 (19.2) and CD209 (DCN46) (BD Biosciences) and CD1a (NA1/34), CD14 (MEM18), CD14 (UCHM1), CD68 (514H12) and TLR2 (TL2.1) (Serotec). The primary antibodies were detected with the corresponding isotype-specific goat anti-mouse or goat anti-FITC secondary antibodies conjugated to a fluorochrome (Alexa 488 or 555, FITC or TRITC) (Southern Biotech, Birmingham, AL, USA and Molecular Probes, Eugene, OR, USA). The specificity of each secondary antibody was confirmed using an isotype mismatched primary antibody. When two primary antibodies of the same isotype were applied to the same section, they were either tagged with Zenon Alexa 568 or 488 (Molecular Probes) or applied sequentially; following application of the first primary antibody and detecting isotype-specific secondary antibody, the section was blocked with 1% mouse IgG and the second FITC-conjugated primary antibody was then applied and detected using anti-FITC–Alexa 488 (Molecular Probes).

The slides were mounted using Vectashield-containing 4,6-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, CA, USA). Sections were visualized with a Leica DMRE fluorescent microscope equipped with the following epi fluorescent filters: UV, 470–490 µm and 515–560 µm (Leica Microsystems, Heerbrugg, Switzerland). Images were obtained using a Leica DC500 digital camera and processed using Photoshop (Adobe, San Jose, CA, USA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Identification of APC subsets in human skin
As we previously reported (1), Langerhans cells migrating through the dermis to the afferent lymphatic vessels could be distinguished from other APCs by their expression of CD207, as well as their high expression of CD1a (Fig. 1). While a few CD1a+ CD207+ migratory Langerhans cells were detected in the upper dermis, sometimes associating with cellular clumps, most CD1a+ cells in the dermis did not express CD207 (Fig. 1A). All CD1a+ epidermal cells expressed CD207 (Fig. 1A and B), confirming the only APCs in epidermis were Langerhans cells. Close inspection of epidermal Langerhans cells revealed co-localization of CD1a and CD207 in the same subcellular compartments (Fig. 1B), a feature not previously documented in human Langerhans cells.


Figure 1
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  		Fig. 1. Rare migratory Langerhans cells can be detected in healthy human dermis. Immunohistochemistry illustrated that rare CD207+ CD1a+ migratory Langerhans cells were present in the dermis (A). All CD1a+ epidermal cells expressed the Langerhans cell marker CD207 (A and B). Close inspection of CD207+ CD1a+ epidermal Langerhans cells revealed that CD1a and CD207 appear to be located in the same subcellular compartments (B). Epidermis is orientated upward and blue represents DAPI staining of cell nuclei. Scale bars represent 50 µm (A) and 20 µm (B). Data are representative of four independent experiments.

 
CD14+ and CD1a+ dermal APCs were identified in dermal cell suspensions using flow cytometry by their lack of CD207 expression and the presence of surface CD14 and CD1a, respectively (Fig. 2). CD14+ and CD1a+ dermal APCs both expressed high levels of the adhesion molecule CD11b (Fig. 2) by flow cytometry, similar to that observed on monocytes. Immunohistochemistry confirmed that CD11b was only expressed by cells in the dermis, but was absent from the epidermis (data not shown). Both populations of dermal APCs also expressed CD11c, though its expression level was slightly higher on CD1a+ dermal APCs than either CD14+ dermal APCs or monocytes (Fig. 2). These data confirm that CD11b and CD11c are not helpful in distinguishing between subsets of APCs in human skin, beyond the discrimination afforded by CD207, CD1a and CD14.


Figure 2
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  		Fig. 2. Adhesion molecule expression by human dermal APCs compared with peripheral blood monocytes. Flow cytometry data showing surface expression of CD11b and CD11c by monocytes and dermal APCs. Dermal single-cell suspensions were labeled with the distinguishing cell-surface markers CD14 and CD1a to identify each APC subset. CD14+ CD1a– and CD1amid CD14– were classified as CD14+ dermal APCs and CD1a+ dermal APCs, respectively, while CD1ahigh CD14– dermal Langerhans cells were excluded from the analysis. Data are representative of three independent experiments.

 
Morphology of dermal APCs
CD14+ and CD1a+ dermal APCs were enriched from dermal single-cell suspensions using magnetic bead separation and imaged by scanning electron microscopy in parallel with monocytes purified from human blood. CD14+ (Fig. 3C) and CD1a+ dermal APCs (Fig. 3E) were both larger than monocytes (Fig. 3A) with CD1a+ dermal APCs been the largest; these size differences were confirmed using flow cytometry (Fig. 3B, D and F). Flow cytometry also indicated that the dermal APC subsets were more granular than monocytes. The surface morphology of each population differed substantially. CD14+ dermal APCs had a more ruffled surface than monocytes (Fig. 3C) but they did not exhibit the prominent cell membrane veils typical of DCs that were present on CD1a+ dermal APCs (Fig. 3E).


Figure 3
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  		Fig. 3. Human monocytes and dermal APC populations each have a distinct cell-surface morphology and forward scatter (FSC)/side scatter (SSC) profile. Monocytes and CD14+ and CD1a+ dermal APCs were enriched from healthy human buffy coats and dermal cell suspensions, respectively, using magnetic beads and their size and surface morphology was assessed using scanning electron microscopy. Flow cytometry was used to determine the forward and side scatter profile of monocytes and CD14+ and CD1a+ dermal APCs. Monocytes (A and B) were smaller and had a smoother surface than the dermal APC subsets (C–F). CD14+ dermal APCs had a ruffled surface and were slightly smaller (C and D) than CD1a+ dermal APCs that possessed numerous veils some of which extended into dendrites (E and F). Scale bars represent 5 µm. Scanning electron microscopy data are representative of two independent experiments while the flow cytometry data are representative of four independent experiments.

 
Expression of TLR2 and TLR4 by APC subsets in human skin
CD14+ and CD1a+ dermal APCs were identified in dermal cell suspensions using flow cytometry by their lack of CD207 expression and the presence of surface CD14 and CD1a, respectively (Fig. 4A-C). TLR4 was detected on both CD1a+ and CD14+ dermal APCs at a similar level to that on monocytes (Fig. 4A). However, TLR2 was not detected on the surface of CD1a+ dermal APCs and was only present on a subset of CD14+ dermal APCs (Fig. 4C and D). Expression of TLR2 by CD14+ dermal APCs in situ was confirmed using immunohistochemistry (Fig. 4E). The CD14+ dermal APCs located in the deeper dermal regions expressed TLR2, while the more superficial members of this subset just below the epidermis appeared to be devoid of this receptor (Fig. 4E). Consistent with flow cytometry data, TLR2 expression was not detected on CD1a+ cells in the dermis using immunohistochemistry (Fig. 4F). TLR2 expression was also absent from the epidermis (Fig. 4E and F).


Figure 4
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  		Fig. 4. PRR and CD83 expression by human dermal APCs compared with peripheral blood monocytes. Flow cytometry data showing expression of TLR2 and TLR4 (A) and the maturation marker CD83 (B) by monocytes and dermal APCs. Dermal single cell suspensions were labeled with the distinguishing cell-surface markers CD14 and CD1a to identify each APC subset. CD14+ CD1a– and CD1amid CD14– were classified as CD14+ dermal APCs and CD1a+ dermal APCs, respectively, while CD1ahigh CD14– dermal Langerhans cells were excluded from the analysis (A–C). Staining patterns of CD83 and TLR2 on CD14+ dermal APCs in different preparations revealed two populations; some cell suspensions predominantly consisted of immature CD83– CD14+ dermal APC that expressed low-level TLR2 while other cell preparations primarily contained mature CD83+ TLR2– CD14+ dermal APC (C). Simultaneous staining of dermal cell suspensions with CD14, CD83 and TLR2 antibodies confirmed that CD14+ dermal APC identified by surface expression of CD14 consisted of both the CD83– TLR2+ and CD83+ TLR2– subsets (D). Two-color fluorescence immunohistochemistry was used to confirm that a subset of CD14+ dermal APCs expressed TLR2 (E) and CD1a+ dermal cells lacked this PRR in situ (F). Epidermis is orientated upward and blue represents DAPI staining of cell nuclei. Scale bars represent 50 µm (E) and 30 µm (F). Data are representative of three independent experiments.

 
Expression of CD206 and CD209 by APC subsets in human skin
Most CD14+ dermal APCs expressed both CD206 (Fig. 5A) and CD209 (Fig. 5D). In contrast, the majority of CD1a+ cells in the dermis did not express CD206, although a few CD206+ CD1a+ dermal cells were detected in the lymphatic vessel-rich upper dermis (Fig. 5B). Similarly, CD209 was rarely detected on CD1a+ cells in the dermis (Fig. 5E). Expression of CD206 and CD209 was not detected in the epidermis (Fig. 5), so epidermal Langerhans cells did not express these receptors. Immunohistochemistry also revealed that migratory Langerhans cells in the dermis did not express CD206 (Fig. 5C) or CD209 (data not shown).


Figure 5
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  		Fig. 5. CD14+ dermal APCs express the C-type lectins CD206 and CD209. Immunohistochemistry illustrated that the majority of CD14+ dermal APCs expressed CD206 and CD209 (A and D). Most CD1a+ dermal cells did not express CD206, although a few CD206+ CD1a+ cells were identified (B). CD206 expression was not detected on epidermal or dermal migratory Langerhans cells (C). CD209 was rarely detected on CD1a+ dermal cells (E). Epidermis is orientated upward and blue represents DAPI staining of cell nuclei. Scale bars represent 50 µm (A, B, D and E) and 20 µm (C). Data are representative of three independent experiments.

 
Expression of activation markers by APC subsets in human skin
CD1a+ dermal APCs expressed a high level of CD83 on their cell surface (Fig. 4B). In contrast, surface CD83 was only weakly detectable on a subset of CD14+ dermal APCs (Fig. 4C and D). This CD83+ subset was TLR2–, while conversely the CD83– subset was TLR2+ (Fig. 4C and D). Hence, the presence of cell-surface CD83 was associated with a lack of TLR2 expression, for both CD14+ and CD1a+ dermal APCs.

Clumps of CD1a+ cells in the upper dermis co-expressed CD208 (Fig. 6A). All CD208+ cells were subsequently shown to be Langerhans cells, co-expressing CD207 (Fig. 6B and C). Langerhans cells in the epidermis did not express CD208 (Fig. 6B and C). It therefore appears that mature Langerhans cells that are either migrating through the dermis toward the lymphatic vessels or which have already migrated into the draining lymphatic vessels can be recognized using CD208 expression. The lack of CD208 on many of the CD1a+ cells visualized in the dermis (Fig. 6A) again confirms the existence of a CD1a+ dermal APC population distinct from migratory Langerhans cells.


Figure 6
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  		Fig. 6. Migratory Langerhans cells in human dermis co-express the interdigitating dendritic cell marker CD208. A proportion of CD1a+ dermal cells co-expressed CD208 (A) and all cells expressing CD208 were CD207+ dermal Langerhans cells (B and C). CD208+ cells were located in the upper dermis and were often associated with structures with similar morphology to lymphatic vessels (A–C). Epidermal Langerhans cells did not co-express CD208 (A–C). Epidermis is orientated upward and blue represents DAPI staining of cell nuclei. Scale bars represent 50 µm (A and C) and 100 µm (B). Data are representative of four independent experiments.

 
Expression of CD68 by APC subsets in human skin
The majority of CD14+ dermal APCs expressed CD68 in both the upper (Fig. 7A) and deeper dermis (Fig. 7B). A few CD68– CD14+ dermal APCs were also detected, and these cells were usually located in the peripheral areas of the dermis just below the basement membrane (Fig. 7A). CD1a+ cells in the dermis did not express CD68 (Fig. 7C). CD68 expression was not detected in the epidermis (Fig. 7A and C).


Figure 7
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  		Fig. 7. CD14+ dermal APCs express the macrophage marker CD68. Immunohistochemistry illustrated that CD14+ dermal APCs located in the upper (A) and deeper dermis (B) expressed CD68. In contrast, CD68 was not detected on CD1a+ dermal or epidermal cells (C). Epidermis is orientated upward and blue represents DAPI staining of cell nuclei. Scale bars represent 50 µm (A), 20 µm (B) and 40 µm (C). Data are representative of three independent experiments.

 
Distribution of blood and lymphatic endothelial structures in the human dermis
As noted above, significant differences were noted in the distribution of some of the molecules studied with respect to dermal depth. To confirm the distribution of blood and lymphatic vessels reported in human dermis by others (24), we used antibodies to CD144/VE-cadherin (25) and podoplanin (26, 27) to locate the blood and lymphatic endothelial cells, respectively, in our samples. Blood vessels appeared as large cellular clumps often with an irregular lumen and were predominantly located in the deeper dermis, though small CD144+ capillaries were also detected in the upper dermis (Fig. 8A). Lymphatic vessels appeared as elongated cellular clumps and were concentrated in the upper dermis (Fig. 8B).


Figure 8
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  		Fig. 8. Distribution of vascular and lymphatic endothelial vessels in healthy human skin. Fluorescence immunohistochemistry illustrated that CD144+ blood endothelial vessels were predominantly located in the deeper dermis with occasional capillaries in the upper dermis (A). In contrast, podoplanin+ lymphatic endothelial vessels were concentrated in the upper region of the dermis with less frequent structures in the deeper dermis and appeared as elongated discontinuous cellular clumps (B). Epidermis is orientated upward and blue represents DAPI staining of cell nuclei. Scale bars represent 100 µm. Data are representative of four independent experiments.

 
The location of CD207+ CD208+ dermal migratory Langerhans cells in the upper dermis is therefore consistent with their migration through the dermis to the afferent lymphatic vessels (Fig. 6B and C). Similarly, the subtle differences in distribution of the CD14+ dermal APC subsets correlate with differences in the vasculature at different depths. CD14+ dermal APCs in the deeper dermis nearest the blood vessels consistently expressed CD68 and TLR2, while in the upper dermis, nearer the lymphatic vessels, many CD14+ dermal APCs were devoid of each of these molecules (Fig. 4E, 7A and 7BGo).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
We recently demonstrated that three APC subsets can be distinguished in the healthy human dermis: migratory dermal Langerhans cells and CD14+ and CD1a+ dermal APCs (1). The data presented here confirm our previous findings, but extend them in several important ways. In particular, we demonstrated that CD208 is an exclusive marker for migratory dermal Langerhans cells, confirming that CD1a+ dermal APCs and migratory dermal Langerhans cells are two distinct APC populations. For the first time, we have identified CD14+ dermal APCs as the dominant APC population expressing CD206 and CD209 in situ. Although earlier publications have detected C-type lectin expression by dermal cells, they did not definitively identify the APC subset that expressed these receptors. In addition, we demonstrated that CD14+ dermal APCs are the only cutaneous APC subset expressing cell-surface TLR2. Consistent with their ability to detect pathogens, CD14+ dermal APCs expressed a less mature phenotype than CD1a+ dermal APCs, although a sub-population did appear to have the capacity to mature, probably into CD1a+ dermal APC rather than Langerhans cells. Cumulatively, these data identify CD14+ dermal APCs as the dermal cells most likely to be capable of recognizing pathogens, and subsequently maturing into potent stimulators of T cell responses.

Defining the APC subsets in the healthy human dermis
Several APC populations have previously been identified in human skin (2, 3) and CD14 and CD1a have emerged as key markers of different subsets (1, 19, 28). We recently demonstrated that CD207 can be used to distinguish the small population of Langerhans cells migrating from the epidermis through the dermis to the draining lymphatic vessels (1), consistent with their steady-state migration to the lymph node (29–31). This allowed discrimination of three populations of APCs in healthy human dermis. We have consistently been unable to detect plasmacytoid DCs in healthy human skin (data not shown), supporting the concept that they only infiltrate the skin in high numbers following inflammation (3).

Here, we show that migrating Langerhans cells within the dermis are the only cells in the skin to express the maturation marker CD208. Epidermal Langerhans cells did not express CD208, consistent with their immature state before migration. The lack of CD208 on the CD1amid CD207– dermal APC population confirms that these CD1a+ dermal APCs are different from migrating Langerhans cells. The CD1a+ cells in the dermis can therefore be distinguished using the following phenotypes: migratory Langerhans cells are CD1ahigh CD207+ CD208+ while CD1a+ dermal APCs are CD1amid CD207– CD208–.

CD14+ dermal APCs are less mature than CD1a+ dermal APCs
Striking differences between CD1a+ and CD14+ dermal APCs were apparent. The PRRs TLR2, CD206 and CD209 were largely restricted to CD14+ dermal APCs, and since these receptors were not expressed in the epidermis, these cells may represent the major APC subset in healthy skin capable of sensing and internalizing their ligands. CD14+ dermal APCs were also the only APC subset expressing CD68. All these characteristics suggest that many CD14+ dermal APCs are in an immature state, capable of pathogen sensing and antigen acquisition. In contrast, CD1a+ dermal APCs universally expressed the maturation marker CD83 on their cell surface, and scanning electron microscopy showed a cell-surface morphology typical of DCs. These findings together with the relative lack of PRR expression confirm that CD1a+ dermal APCs are a mature APC subset. Hence, the current findings extend our earlier observations of the superior lymph node homing and T-cell priming capacity of CD1a+ dermal APCs relative to CD14+ dermal APCs (1).

A further finding is that CD14+ dermal APCs are diverse. After screening numerous single-cell suspensions, two populations were identified: TLR2+ CD83– and TLR2– CD83+. It seems possible that the latter subset represents a more mature version of the former, since this difference in phenotype resembles the change observed when monocytes are induced in vitro to become mature DCs (20, 32). This change in phenotype might have been induced by the maturation stimuli that are inevitable when preparing single-cell suspensions from human skin (1, 33). However, it is intriguing to note that immunohistochemistry shows TLR2 expression by CD14+ cells declines as the cells become more superficial in the dermis, especially in the areas where the CD1a+ dermal APCs and the lymphatic vessels are concentrated. This suggests that loss of TLR2 from CD14+ dermal APCs may be associated with maturation as the cells move up through the dermis. Loss of CD68 expression by CD14+ dermal APCs follows a similar pattern.

Possible functions of CD14+ dermal APC
Are CD14+ dermal APCs an independent macrophage-like subset or a less mature precursor of the other APC populations present in skin? It has been suggested that human Langerhans cells are replenished by precursors in the dermis and that CD14+ dermal APCs are the main precursor population (6). Similarly, data generated using murine models suggest that under steady-state conditions, Langerhans cells either self-renew or are replenished by a proliferating precursor in the skin (34). It has been demonstrated that under steady-state conditions, the turnover of Langerhans cells in normal skin is slow, with a residence time of weeks to months (3, 33, 35). If the Langerhans cell precursor does reside in the dermis, it seems likely that a small population of precursors could replenish the low number of Langerhans cells that leave the skin under steady-state conditions. The substantial size of the CD14+ dermal APC population and their potential to detect antigen and mature suggests at the very least that replenishing Langerhans cells is not their sole function.

Possible functions of CD1a+ dermal APC
Given the mature phenotype of CD1a+ dermal APCs and their association with the lymphatic vessels in the upper dermis, it seems unlikely that CD1a+ dermal APCs are involved in replenishing epidermal Langerhans cells, despite their expression of CD1a. What then is the function of CD1a+ dermal APCs? In some studies of human atopic dermatitis, a CD1alow CD1blow CD11bhigh inflammatory dendritic epidermal cell (IDEC) population distinct from Langerhans cells was observed infiltrating the epidermis (36, 37). As we have suggested previously (1) and further support with CD11b staining, the phenotype of IDECs is similar to the CD1a+ dermal APCs we have characterized in healthy dermis. There is, however, one distinct difference between IDECs and CD1a+ dermal APCs, IDECs express surface CD206 (38), while the majority of CD1a+ dermal APC lacked expression of this C-type lectin. Therefore, while it does seem possible that CD1a+ dermal APCs may be capable of infiltrating the epidermis in response to inflammatory signals and becoming IDECs, the relationship between these two subsets remains unresolved.

Do CD14+ dermal APCs have the capacity to mature into CD1a+ dermal APCs?
On the basis of the phenotypic data above, and the functional data presented previously, we propose that CD14+ dermal APCs, or at least a sub-population of these cells, are the precursors of CD1a+ dermal APCs. The presence of a CD83+ TLR2– population among the CD14+ APCs in single-cell suspensions strongly suggests a capacity for maturation. It is also notable that in our earlier work (1), as in that of others (19), a minor population of dermal APCs in single-cell suspensions co-expressed intermediate levels of CD14 and CD1a. Given the clear evidence presented here that CD14+ dermal APCs are less mature than CD1a+ dermal APCs, this co-expression suggests that CD14+ cells are acquiring CD1a as they mature, rather than the other way round. It is also striking that CD14+ APCs found in upper layers of the dermis present a more mature phenotype than in the lower layers. Consistent with these observations, CD14+ dermal APCs in the upper dermis express higher levels of HLA-DR than those located in the deeper dermis (39). Similarly, it is possible that the occasional CD1a+ dermal APCs expressing CD206 and CD209 are cells only recently matured from CD14+ dermal APCs, which have yet to down-regulate those receptors. Definitive proof of the capacity of CD14+ dermal APCs to mature into CD1a+ dermal APCs will require further study of purified immature CD14+ dermal APCs. Unfortunately, it is not feasible to use CD83 as a maturation marker in immunohistochemistry, since it is expressed intracellularly by immature APCs (20).

Such a maturation pathway from CD14+ to CD1a+ dermal APCs, without involvement in the Langerhans cell lineage, would also answer an important question about presentation in draining lymph nodes of antigen acquired in skin through CD206 or CD209. If CD14+ dermal APCs are the main immune sentinel population in human skin for ligands of these receptors, how can any antigen acquired by these cells be presented to T cells, when our previous data suggest CD14+ dermal APCs are not likely to migrate to lymph nodes or strongly stimulate naive T cells (1)? If CD14+ dermal APCs mature into CD1a+ dermal APCs, they will gain the capacity to migrate to lymph nodes and strongly stimulate naive T lymphocytes with the antigen they have acquired (1).

Integrin expression by CD14+ and CD1a+ dermal APCs
Our observation that both CD1a+ and CD14+ dermal APCs expressed high levels of the surface integrins CD11b and CD11c has relevance to the fate of CD14+ dermal APCs. In contrast to dermal APCs, CD11b expression on epidermal Langerhans cells was negligible and CD11c expression was low (data not shown). Langerhans cells remain immobilized in the epidermis for long periods of time (3, 33, 35), suggesting the lack of these molecules is associated with a lack of motility. CD11b and CD11c can facilitate cell movement through extracellular matrix and are involved in adhesion of circulatory cells to endothelium prior to chemokine-driven diapedesis (40, 41). It is therefore possible that dermal APCs require integrin expression to enable them to move through the dense network of collagen and elastic fibers that make up the dermis and traverse endothelium. The wide distribution of CD14+ dermal APCs bearing CD11b and CD11c throughout the dermal layers from the blood vessel-rich deeper dermis to the upper dermis where the lymphatic vessels are concentrated suggests that these cells may be in constant flux through the dermis rather than fixed tissue macrophages, and would therefore be capable of migrating to superficial layers to differentiate into CD1a+ dermal APC.

Comparing data with earlier observations
Several of the markers we studied have previously been detected in human skin. However, this is the first study to definitively identify the APC subsets bearing them.

CD206 expression by dermal APC subsets has been demonstrated previously (19) but the data relied on derivation of single-cell suspensions from skin and different preparation methods gave different results. CD206 was present on both CD1ahi and CD14+ CD1alow dermal APCs isolated from the dermis using enzyme digestion, however, it was absent when dermal APCs were allowed to spontaneously migrate from skin explants (19). In the same study, weak CD209 expression was detected on the cell surface of a subgroup of CD14+ CD1alow dermal APCs prepared using enzyme digestion, but was again absent on cells spontaneously migrating from explanted skin (19). By using multicolor immunohistochemistry, we have definitively shown that CD14+ dermal APCs not CD1a+ dermal APCs are the dominant population expressing CD206, while CD209 is strongly expressed by the vast majority of CD14+ dermal APCs in situ. Other work has confirmed the expression of CD209 in dermis by immunohistochemistry but did not definitively identify the APC subset bearing this receptor (23, 42, 43).

Many earlier reports have noted the presence of CD68+ cells in human dermis and have classified these cells as dermal macrophages (44). However, our data suggest that at least a subset of this population may not retain this macrophage-like phenotype but have the capacity to mature into DCs. Again a unique feature of our study is the demonstration of co-localization of CD68 with CD14 but not CD1a.

The only CD208+ cells detected in human skin previously were located in epidermal sheets split from skin ex vivo and since they co-expressed high levels of HLA-DR, these cells were assumed to be mature Langerhans cells (23). Although the anatomical location of these CD208+ HLA-DRhigh cells differed from our CD208+ CD207+ population, it is likely that the Langerhans cells in the epidermal sheet matured in response to the epidermal sheet preparation procedure, hence supporting our in situ observations that mature migratory Langerhans cells express CD208. CD208 up-regulation may be a defining characteristic of Langerhans cells migrating through the dermis into the lymphatic vessels under steady-state conditions, along with a change in chemokine receptor profile to CCR7 (2, 3, 35).

Cumulatively, these findings suggest that many CD14+ dermal APCs have the potential to sense pathogen and acquire antigen, and may subsequently mature into CD1a+ dermal APCs, thereby acquiring the capacity to present antigen to T lymphocytes within lymph nodes. Hence, CD14+ dermal APCs may prove to be an important target for vaccines administered to humans via the skin.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Wellcome Trust (WT066630MA); Auckland Burns Support Group; the Endocore Trust.


   Acknowledgements
 
We gratefully acknowledge technical assistance and advice from Sharleen Power, Beryl Davy, Adrian Turner, Bryony James and Catherine Hobbis. We thank the patients and staff at Middlemore Hospital, the Manukau Super Clinic and the New Zealand Blood Service for donated clinical material.


   Abbreviations
 
APC, antigen-presenting cell
7AAD, 7-aminoactinomycin-D
DC, dendritic cell
FBS, fetal bovine serum
IDEC, inflammatory dendritic epidermal cell
PRR, pattern recognition receptor
TLR, Toll-like receptor

   Notes
 
Transmitting editor: K. Inaba

Received 20 December 2006, accepted 7 August 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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