60% of total) expresses the mononuclear phagocyte markers mMGL (ER-MP23), F4/80 and CD11b. In addition, these cells show avid phagocytic capacity and thus are identified as dermal macrophages. Different subpopulations can be defined using markers such as sialoadhesin, ER-HR3 and mSIGN-R1 (ER-TR9). Interestingly, MHC class II expression differs significantly between dermal cells from ear versus back skin. Moreover, we have identified small populations of dermal DC and migrating Langerhans cells (together Mononuclear phagocytes, i.e. macrophages and dendritic cells (DC), are crucial initiators and regulators of both innate and adaptive host defense responses. As a consequence, especially tissues in close contact with the environment possess sizeable populations of mononuclear phagocytes. In the skin, different populations of such sentinel cells have been described: in the suprabasal layer of the epidermis, where Langerhans cells (LC) build up a network with their dendrites, and in the dermis, where interstitial dermal dendritic cells and dermal macrophages reside between extracellular matrix components. Dermal mononuclear phagocytes appear to be important for skin immunity, as they are highly competent in inducing an immune response without involvement of epidermal LC (Streilein, 1989;Allan et al, 2003). Surprisingly enough, dermal mononuclear phagocyte populations have never been investigated in a quantitative manner compared to the total amount of dermal cells. Therefore, we performed immunofluorescence double stainings on mouse ear and back skin sections using different macrophage and DC markers. To be able to compare the amount of stained cells to the total amount of dermal cells, we counterstained the sections with DAPI to identify all nucleated cells. Thereafter, we assessed the phenotype of 200–400 dermal nucleated cells, excluding cells in skin appendages.
Results and Discussion
The starting point in our analyses was the reactivity of the anti-mononuclear phagocyte antibody ER-MP23 (Jansen et al, 1994;Leenen et al, 1994). This antibody recognizes an epitope on mMGL (murine macrophage galactose/N-acetylgalactosamine-specific C-type lectin;Oda et al, 1988;McKnight and Gordon, 1998), also known as DC-ASGPR (dendritic cell asialoglycoprotein receptor,Valladeau et al, 2001), and has been described previously to label connective tissue macrophages strongly (Jansen et al, 1994;Leenen et al, 1994). Staining of skin sections for this marker revealed that a majority of all dermal cells expressed it (55%
3% in back skin and 58%4% in ear skin, resp.). To ascertain that this number did not originate from dermal fibroblasts expressing mMGL, we performed double stainings for mMGL and CD45, the pan leukocyte marker. As shown in Figure 1a, all mMGL-positive cells also stained positively for CD45. In addition, around 14% of all back dermal cells were single positive for CD45, whereas 31% expressed neither of both markers. A similar result was obtained in the ear skin dermis.
Figure 1.
The majority of dermal interstitial cells express mononuclear phagocyte markers. Back skin sections were stained for mMGL / ER-MP23 (red) and CD45 (green) (A) or mMGL (red) and F4/80 (green) (B). Sections were counterstained with DAPI, showing nuclei in blue. (A) About 60% of all dermal cells express mMGL. All mMGL-positive cells also express CD45. About 10% of the CD45-positive cells are mMGL-negative. (B) Staining for mMGL and F4/80 virtually coincided. Note the gradient of increasing F4/80 expression toward the epidermal side, which could be observed occasionally. Scale bar=50 
m for all pictures.
To analyze further the nature of mMGL-positive cells, we performed double stainings for mMGL and other macrophage markers: F4/80, CD11b, MOMA-1/sialoadhesin, ER-HR3, ER-TR9/mSIGN-R1 and MHC class II. The staining of F4/80 and mMGL virtually coincided (Figure 1b); 63% of all dermal cells were counted as double positive cells, 1% as single mMGL-positive cells, 2% single F4/80-positive cells and 34% as double negative cells in the back dermis. CD11b was, as F4/80, expressed on almost all mMGL-positive cells (Fig S1, online; 55% double positive, 1% single mMGL-positive, 4% single CD11b-positive and 40% double negative cells). The 4% single CD11b-positive cells presumably represent dermal mast cells. MOMA-1 was found to bind a subpopulation of dermal phagocytes, in particular those cells laying deeper in the dermis (Fig S1, online; 23% double positive, 39% single mMGL-positive, 2% single MOMA-1-positive, 37% double negative). ER-HR3, with still unknown binding epitope, likewise stained a significant subpopulation of mMGL-positive cells throughout the dermis (Fig S1, online; 42% double positive, 18% single mMGL-positive, 5% single ER-HR3-positive, 35% double negative). ER-TR9, which recognizes mSIGN-R1 (Kang et al, 2003), has been found to stain a small percentage of cells in the dermis (6%–8%; Fig S1, online). For all these markers, similar numbers have been obtained comparing back skin to ear skin dermis. When we compared the staining of dermal cells of the two sites for MHC class II, we found that virtually all mMGL-positive cells in the back dermis expressed this marker (58% double positive, 3% single mMGL-positive, 4% single MHC class II-positive, 35% double negative; Figure 2a). In marked contrast, only a minor subpopulation of all mMGL-positive cells expressed MHC class II in the ear dermis (20% double positive, 40% single mMGL-positive, 2% single MHC class II-positive, 38% double negative; Figure 2b). To explain this divergence, we speculate that differences in the structure of the dermis at different locations (subdermal adipose tissue vs cartilage) create different microenvironments that differentially influence the expression of the MHC class II molecules.
Figure 2.
Dermal macrophages from different sites show differential MHC class II expression. Back skin sections (A) and ear skin sections (B) were stained for mMGL (red) and for MHC class II (green). Sections were counterstained with DAPI. (A) In the back skin, virtually all mMGL-positive cells coexpress MHC class II. (B) Similar to the back skin, the ear dermis contains about 60% interstitial cells expressing mMGL. However, only about one third of these cells coexpresses MHC class II molecules. These mMGL/MHC class II double-positive cells are arranged in clusters. Scale bar=50 m for all pictures.
To seek further confirmation regarding the nature of mMGL-positive dermal cells, we made use of the fact that macrophages are the only connective tissue cells showing avid phagocytic capacities. Therefore we injected DiI-labeled liposomes, which previously have been shown to target phagocytic cells exclusively (Buiting et al, 1993), into the ear dermis. Thereafter we killed the mice, made sections of the treated ears and stained for mMGL. As shown in Figure 3, we indeed found that the vast majority of mMGL-positive cells had taken up DiI-labeled liposomes. However, a few mMGL-positive cells as well as all mMGL-negative cells remained free of DiI-fluorescence, indicating the specificity of this procedure. Therefore, we conclude that mMGL-positive cells not only express mononuclear phagocyte markers but also possess phagocytic capacity, thus identifying them as dermal macrophages unequivocally.
Figure 3.
mMGL-positive dermal cells are phagocytic. DiI-labeled liposomes (red label) were injected into ear dermis. Four hours later, animals were euthanized, ears cut off, frozen, sectioned, and stained for mMGL (green). Note that the vast majority, but not all mMGL-positive cells, have taken up liposomes, whereas mMGL-negative cells and keratinocytes remained free of labeling. Scale bar=50 m for all pictures.
To evaluate the presence of DC in the dermis, we stained back and ear sections for CD11c. To distinguish dermal DC from migrating LC that are on their way from the epidermis to skin-draining lymph nodes, we performed a double labeling for Langerin, which specifically stains LC (Valladeau et al, 2002). As shown in Figure 4, we indeed found a subpopulation of CD11c+ Langerin- dermal DC (7%), while even fewer cells represented migrating LC, which all expressed CD11c (4% CD11c+ Langerin+). DC-SIGN is a specific marker for dermal DC in the human situation (Turville et al, 2002). Therefore, we wondered whether ER-TR9 (mSIGN-R1) would recognize dermal DC specifically in the mouse. Double stainings for ER-TR9 and CD11c revealed that ER-TR9 recognized a small subset of dermal APC (6%–8%), but that it did not uniquely stain CD11c+ cells (Fig S1, online and data not shown).
Figure 4.
Dermal DC constitute a small, distinct mononuclear phagocyte population. Back skin sections were stained for CD11c (red) and Langerin (green) (A) or CD11c (red) and mMGL (green) (B). Sections were counterstained with DAPI. (A) About 7% of all dermal cells express CD11c but does not express Langerin. These cells thus represent genuine DDC (marked with thin arrows). Note that the CD11c staining is too weak to let CD11c/Langerin-double-positive mLC (marked with thick arrows) appear yellow. (B) Double staining for CD11c and mMGL shows that most CD11c-positive cells coexpress mMGL (arrow). Thus, most if not all DDC express mMGL. The red labeled cell marked with an asterisk is part of a sebaceous gland and displays autofluorescence rather than specific labeling. Scale bar=50 m for all pictures.
To summarize, we have shown that mononuclear phagocytes constitute the major population of nucleated cells in the mouse dermis where they amount to about 60% of all dermal cells (Table I). Previous studies have quantified dermal mononuclear phagocyte subsets for mice (Duraiswamy et al, 1994) and for humans (Meunier et al, 1993) by flowcytometric analyses of dermal cell suspensions. In contrast to our findings, these authors concluded that dermal macrophages and dermal DC only composed around 5% of all dermal cells. The discrepancy between their findings and ours lies most probably in the difference of the used methods, as we stained and counted cells in situ in the non-denaturated dermis. Clearly, during the digestion procedure needed to obtain a dermal cell suspension, markers either may have been destroyed by enzymatic digestion, as is known to happen with the CD1 molecules, or the procedure may have released fibroblasts preferentially. We further tried to strengthen our findings by staining and counting fibroblasts specifically, but were not successful due to the unavailability of suitable markers. However, the concept that most dermal interstitial cells are indeed mononuclear phagocytes is well supported as we found similar percentages for dermal mononuclear phagocytes using several markers (F4/80, CD11b, mMGL, CD68 (data not shown) and MHC class II in the back dermis). Furthermore, this high frequency is confirmed by the CD45 pan-leukocyte labeling. Taken together, we feel that the size of the dermal mononuclear phagocyte population has been clearly underestimated so far. These cells probably play crucial roles in skin inflammatory and immune responses and they should be taken into account when the role of the dermis is studied in steady state and inflammatory conditions.
Table I - Frequencies of different cell populations in the mouse dermis, related to total nucleated cells; the quantification is derived from findings in both ear and back skin.
Full table Materials and Methods
Immunofluorescence double stainings on mouse (C57BL/6) ear and back skin sections have been performed essentially as described (Stoitzner et al, 2003). Details can be obtained from the online supplementary materials.
References
| 1. | Allan RS, Smith CM, Belz GT, van Lint AL, Wakim LM, Heath WR & Carbone FR. Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells. Science (2003) 301: 1925–1928 10.1126/science.1087576. | Article | PubMed | ISI | ChemPort | |
| 2. | Buiting AM, de Rover Z, Claassen E & van Rooijen N. In vivo distribution of particulate antigens and liposomes in murine spleen. A possible role in the humoral immune response. Immunobiology (1993) 188: 13–22. | PubMed | ISI | ChemPort | |
| 3. | Duraiswamy N, Tse Y, Hammerberg C, Kang S & Cooper KD. Distinction of class II MHC+ Langerhans cell-like interstitial dendritic antigen-presenting cells in murine dermis from dermal macrophages. J Invest Dermatol (1994) 103: 678–683 10.1111/1523-1747.ep12398513. | Article | PubMed | ISI | ChemPort | |
| 4. | Jansen A, Homo-Delarche F, Hooijkaas H, Leenen PJ, Dardenne M & Drexhage HA. Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and beta-cell destruction in NOD mice. Diabetes (1994) 43: 667–675. | PubMed | ISI | ChemPort | |
| 5. | Kang YS, Yamazaki S & Iyoda T et al. SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran. Int Immunol (2003) 15: 177–186 10.1093/intimm/dxg019. | Article | PubMed | ISI | ChemPort | |
| 6. | Leenen PJ, de Bruijn MF, Voerman JS, Campbell PA & van Ewijk W. Markers of mouse macrophage development detected by monoclonal antibodies. J Immunol Methods (1994) 174: 5–19 10.1016/0022-1759(94)90005-1. | Article | PubMed | ISI | ChemPort | |
| 7. | McKnight AJ & Gordon S. Membrane molecules as differentiation antigens of murine macrophages. Adv Immunol (1998) 68: 271–314. | PubMed | ISI | ChemPort | |
| 8. | Meunier L, Gonzalez-Ramos A & Cooper KD. Heterogeneous populations of MHC class II+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells. J Immunol (1993) 151: 4067–4080. | PubMed | ISI | ChemPort | |
| 9. | Oda S, Sato M, Toyoshima S & Osawa T. Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galactosamine from tumoricidal macrophages. J Biochem (1988) 104: 600–605. | PubMed | ISI | ChemPort | |
| 10. | Stoitzner P, Holzmann S & McLellan AD et al. Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against Langerin/CD207. J Invest Dermatol (2003) 120: 266–274 10.1046/j.1523-1747.2003.12042.x. | Article | PubMed | ISI | ChemPort | |
| 11. | Streilein JW. Antigen-presenting cells in the induction of contact hypersensitivity in mice: Evidence that Langerhans cells are sufficient but not required. J Invest Dermatol (1989) 93: 443–448 10.1111/1523-1747.ep12284018. | Article | PubMed | ISI | |
| 12. | Turville SG, Cameron PU, Handley A, Lin G, Pohlmann S, Doms RW & Cunningham AL. Diversity of receptors binding HIV on dendritic cell subsets. Nat Immunol (2002) 3: 975–983 10.1038/ni841. | Article | PubMed | ISI | ChemPort | |
| 13. | Valladeau J, Clair-Moninot V & Dezutter-Dambuyant C et al. Identification of mouse Langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues. J Immunol (2002) 168: 782–792. | PubMed | ISI | ChemPort | |
| 14. | Valladeau J, Duvert-Frances V & Pin JJ et al. Immature human dendritic cells express asialoglycoprotein receptor isoforms for efficient receptor-mediated endocytosis. J Immunol (2001) 167: 5767–5774. | PubMed | ISI | ChemPort | |
Acknowledgments
We thank Drs Errol Prens, Leslie van der Fits and Jon Laman for stimulating discussions, Dr Sem Saeland (Schering Plough, Dardilly, France) for the anti-Langerin antibody, Dr Nico van Rooijen (Free University Amsterdam, The Netherlands) for DiI-labeled liposomes and Tar van Os for preparing the figures. This study was supported by the Interuniversitary Research Institute for Radiopathology and Radioprotection (IRS), Leiden, The Netherlands.
