| To whom correspondence should be addressed: Mineko Shimohira-Yamasaki, Department of Pathology & Biodefence, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan. Tel: +81–952–31–6511, Fax: +81–952–34–2055 E-mail: yamasaki@med.saga-u.ac.jp |
Merkel cells in the skin contribute to touch perception. Merkel cells are localized in the basal layer of the epidermis, more specifically at the bottom of the rete ridges (Fradette et al., 1995; Moll et al., 1984). This specific cell type has long been assumed to guide sensory nerve fibers to the epidermis and hairs under their cell-cell interaction (Tachibana et al., 1995; Mearow et al., 1988). In fact, our previous study and others have immunohistochemically suggested that Merkel cells in vivo are closely associated with nerve terminals (Kim et al., 2001; Narisawa et al., 1991; Pasche et al., 1990). However, there has been little in vitro evidence that supports this hypothesis, because there is no suitable culture system for investigating Merkel cell-nerve cell interaction (Fradette et al., 2003; Fukuda et al., 1996; Yamashita et al., 1993; Vos et al., 1991).
The aim of our current study was to establish a simple primary culture system of Merkel cells and to study interaction between Merkel cells and nerve cells. In this study a new culture system of Merkel cells with keratinocytes, which were all isolated from rat footpad skin, was developed. In this system, Merkel cells were easily differentiated from coexisting keratinocytes by the Merkel cell-specific marker cytokeratin 20 (Fradette et al., 1995; Moll et al., 1995; Moll et al., 1994) and by their neuroendocrine granules (Kim et al., 2001; Narisawa et al., 1991; Vos et al., 1991). After nerve cells (NG108-15 or PC12) were added to the culture system, Merkel cell-nerve cell interaction was analyzed by histochemistry, immunohistochemistry, and scanning and transmission electron microscopy.
Here we have described a simple culture method that allows Merkel cells to stably interact with nerve cells. In this system, we have shown that outgrowing nerve fibers and cytoplasmic processes of Merkel cells cooperatively organize synapse-like structures at their direct contact points. These results suggest that Merkel cells may guide nerve fibers to the skin.
Culture procedures for Merkel cells, which were isolated from footpad skin, were performed as shown schematically in Fig. 1. Skin samples were dissected from the footpads of Wistar rats (4 week old-male), and incubated overnight in 20 ml of Dispase I solution (Bacterial neutral protease: 1,000 protease units ml–1, Goudoh-Shusei Co. Ltd., Tokyo, Japan) at 4°C. Dispase I was less harmful to cells than collagenase, as previously described (Toda et al., 1990). Epidermis and dermis became easily separable. The detached epidermal sheets were shaken in a flask with 0.1% trypsin solution (Wako-Junyaku, Osaka, Japan) for 3 minutes at 37°C. The suspension was then filtered with mesh (90 μm), and centrifuged at 900 rpm for 5 minutes. Both Merkel cells and keratinocytes were obtained as a cell pellet. These cell types were incubated in culture dishes in a CO2 incubator at 37°C. Complete medium consisted of Ham’s F-12 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), 20 ng ml–1 nerve growth factor (NGF) (Wako-Junyaku), 20 ng ml–1 neurotrophin 3 (NT-3) (Chemicon International, Temecula, CA, USA) and 25 μg ml–1 gentamicin.
 View Details | Fig. 1. The primary culture method of Merkel cells from footpad of rat skin. Skin samples are dissected from the footpad of Wistar rats and incubated in Dispase solution. The detached epidermal sheets, including CK20-positive Merkel cells, are treated with trypsin solution. After filtration and centrifugation, Merkel cells and keratinocytes are obtained as sediment. These two cell types are cocultured with complete medium supplemented with NT-3 and NGF. |
To examine Merkel cell-nerve cell interaction, we used the following nerve cell types. The nerve cell line NG108-15 (Nierenberg et al., 1983) was a kind gift of Prof. H. Higashida (Kanazawa University, Kanazawa, Japan). PC12 was obtained from American Type Culture Collection (ATCC, CRL-1721). NG108-15 cells were maintained in Dulbecco’s modified eagle medium (DMEM, Nissui Pharmaceutical) supplemented with 10% FBS, HT (100 μM hypoxanthine and 16 μM thymidine, Nissui Pharmaceutical), 20 ng ml–1 NGF and 20 ng ml–1 NT3. PC12 cells were maintained in DMEM supplemented with 20% FBS, 20 ng ml–1 NGF, 20 ng ml–1 NT3 and 50 IU ml–1 penicillin.
Cells in culture dishes were fixed with 4% paraformaldehyde solution, air-dried, and processed by standard methods. All procedures were carried out in culture dishes. Merkel cells and nerve cells were identified with monoclonal cytokeratin 20 (CK20) (Progen Biotechnik, Heidelberg, Germany) and synaptophysin (Chemicon International) antibodies, respectively. To examine the topographical relationship between Merkel cells and nerve cells, a double immunohistochemistry with peroxidase (for CK20) and alkaline phosphatase (for synaptophysin) was performed, as previously described (Toda et al., 1997).
For scanning electron microscopy (SEM), the skin and cultured cells were fixed in 1.5% glutaraldehyde (in 0.05 M cacodylate buffer, pH 7.2), dehydrated in alcohol, critical-point dried with CO2, and sputter coated with gold for 40 seconds. The specimens were examined using a Hitachi S700 SEM. For the transmission electron microscopy (TEM), the skin or cultured cells were fixed with 2.5% glutaraldehyde followed by 1% osmic acid, dehydrated with alcohol, and embedded in epoxy resin. The sections were examined with a JME-1210 electron microscopy. When we examined synapse-like structures organized by the interaction between Merkel cells and nerve cells, we were able to easily determine both cell types at semi-sections, because the cell size of NG108-15 and PC12 nerve cell types were clearly larger than that of Merkel cells.
To estimate the proliferation of Merkel cells, we used a double immunostaining with bromodeoxyuridine (BrdU) and CK20 according to the method described in our previous study (Toda et al., 1997). First, we carried out immunohistochemistry for BrdU (Cell Proliferation Kit, Amersham, Arlington Heights, IL), after a 24 h-incubation with 3 μg ml–1 BrdU, as described elsewhere (Toda et al., 1990). Then, immunohistochemistry for CK20 was performed to identify Merkel cells. To obtain the rate of nuclear BrdU intake of CK20-expressing Merkel cells, all cells in a culture dish were examined, and BrdU- and CK20-positive cells were scored. Their frequency was calculated. We also investigated effects of some cytokines on Merkel cell proliferation, because Merkel cells tend to be seen more numerously in inflammatory lesions (Tachibana et al., 1998). Merkel cells with or without nerve cells were stimulated every day with 10 ng ml–1 interleukin-6 (IL-6, Chemicon International) or 100 ng ml–1 tumor necrosis factor-α (TNF-α, Chemicon International). The effect of IL-6 and TNF-α on Merkel cell proliferation was analyzed as described above.
As Merkel cells disappeared after 7 days in culture, we examined apoptosis of Merkel cells at 2 days in culture before we were able to determine their apoptosis in morphology by light microscopy. To investigate apoptosis of Merkel cells, we used a mouse monoclonal single-stranded DNA (ss-DNA) antibody (Dako, Glostrup, Denmark) that detects apoptosis. In this study, we carried out a double immunohistochemistry with CK 20 (Merkel cell marker) and ss-DNA (apoptosis marker), as previously described (Toda et al., 1997). To obtain the rate of apoptosis of Merkel cells, all cells in a culture dish were examined, and ss-DNA- and CK20-positive cells were scored.
To determine the number of Merkel cells in a culture dish (9.1 cm2), immunohistochemistry with CK20 was carried out. Then, CK20-expressing Merkel cells were counted in a dish at 2 days in culture by light microscopy.
Data obtained from three independent experiments were analyzed by Mann-Whitney’s U-test. Results were expressed as means±SD and were considered significant with P values of 0.05 or less.
CK20-expressing Merkel cells were detected in the basal layer of footpad skin (Fig. 2A). The cells were specifically located in the apices of rete ridges, as described by Merkel (Merkel, 1875). Ultrastructurally, characteristic features of Merkel cells were large lobulated nucleus and cytoplasm that contained many dense-core granules (50–110 nm in diameter) (Fig. 2B, C).
 View Details | Fig. 2. Localization and fine structures of Merkel cells in footpad of rat skin. (A) CK20-positive Merkel cells (arrow) localize in the apices of rete ridges. (B) Spindle-shaped Merkel cell (14 μm in the long axis) is situated among basal cells of the epidermis. (C) Merkel cell has numerous dense core granules (50–110 nm in diameter). Scale bars: 100 μm (A), 1 μm (B) and 100 nm (C). |
In our culture system, we were able to maintain Merkel cells that were very similar to those in vivo. The primary cultured cells derived from the epidermis attached well to the surface of culture dishes. A total of about 90% of CK20-expressing Merkel cells (inset of Fig. 3A) were usually observed as single cells and located among keratinocyte colonies; but about 10% of Merkel cells were seen within keratinocyte colonies. Merkel cells exhibited polygonal shapes with multiple cytoplasmic processes (Fig. 3A), and their size ranged from 3 to 5 μm in the shorter diameter and from 6 to 20 μm in the longer diameter. Several cytoplasmic processes protruded at various angles from the cell surface (Fig. 3A, B). The Merkel cells possessed large lobulated nucleus, and their cytoplasm contained many dense-core granules (60–100 nm in diameter) that were accumulated in the apical protrusions (Fig. 3B). These granules were compatible with Merkel cell granules in vivo (inset of Fig. 3B). Merkel cells bore spike-like protrusions which interdigitated with the surrounding keratinocytes that had tonofilaments in their cytoplasm as in vivo (Fig. 3C). In contrast, round keratinocytes were clearly larger than Merkel cells. Keratinocytes presented an extensive network of keratin filaments. Along with increasing culture time, the morphology of Merkel cells changed from a roundish shape on culture days 1/2–1 to a more flattened one on days 1–3. On culture day 2, the number of Merkel cells was 500–900 in a 35 mm culture dish. About 10% of the Merkel cells interacted with the keratinocytes in the dish.
 View Details | Fig. 3. Scanning (A) and transmission electron microscopy (B–C), and CK20 expression (inset in A) of Merkel cells in primary culture. (A) Merkel cell (6–20 μm in the long axis) is polygonal in shape and has multiple cytoplasmic processes. The cytoplasmic processes are 4–7 μm in length. CK20-positive Merkel cells are seen around CK20-negative keratinocytes (left lower area) on culture day 3 (inset of A). (B) Merkel cell is characterized by both lobular nucleus and cytoplasmic dense core granules. Inset of B (higher magnification of asterisk (*) in B) indicates dense core granules (60–100 nm in diameter). (C) Merkel cell (MC) protrudes cytoplasmic process (arrows) into cytoplasm of surrounding keratinocyte (KC), suggesting that both cell types interdigitate with each other. Scale bars: 5 μm (A, inset of A and B), 100 nm (inset of B) and 1 μm (C). |
When Merkel cells were cocultured with nerve cells, nerve fibers actively extended to Merkel cells, and contacted the cytoplasmic processes of about 70% of the Merkel cells. The nerve fiber’s extension onto Merkel cells is demonstrated in Fig. 4A. The double immunostaining with CK20 for Merkel cells and synaptophysin for nerve cells indicated that synapse-like structures appeared at their direct contact points (Fig. 4B). Note the synapse-like contacts between cell membranes of Merkel cells and nerve terminals (Fig. 4C). The number of cytoplasmic processes of Merkel cells with nerve cells was 2 times that of Merkel cells without nerve cells. The length of cytoplasmic processes of Merkel cells with nerve cells was 1.5 times that of Merkel cells without nerve cells. Interestingly, the number of Merkel cells with coculture of nerve cells (1692.3±23.3) was greater than that of Merkel cells without coculture of nerve cells (629.7±248.2) (p<0.05) at 2 days in culture.
 View Details | Fig. 4. Phase contrast microscopy (A), double immunohistochemistry with CK20 and synaptophisin (B) and transmission electron microscopy (C) of Merkel cell-nerve cell interaction at 3 days in coculture. (A) A nerve cell (NC; NG108-15) characteristically extends a nerve fiber toward a Merkel cell (MC). Merkel cell (MC) also extends cytoplasmic processes to the nerve fiber. Both nerve fiver and cytoplasmic processes of Merkel cell clearly contact each other (arrowheads). (B) Nerve cell (NC; NG108-15) expresses synaptophisin (red in color), and Merkel cell (MC) displays CK20 (brown in color). (C) Asterisk (*) indicates synapse-like structure that is organized cooperatively by both cell types (MC and NC; NG108-15) at their contact points. Scale bars: 10 μm (A and B) and 500 nm (C). |
Since Merkel cells tend to be seen more numerously in inflammatory lesions (Tachibana et al., 1998), we examined the effect of some cytokines on Merkel cell proliferation. IL-6 and TNF-α tested here did not induce an increase in the number of Merkel cells or an increase in BrdU intake. In Merkel cell cultures with and without nerve cells, no intranuclear BrdU-intake was detected in CK20-expressing Merkel cells. In addition, Merkel cells disappeared after 7 days in our culture system with or without nerve cells and cytokines above. This suggests that Merkel cells have no proliferative ability in vitro.
More than 90% of Merkel cells underwent apoptosis in the coculture system of Merkel cells and keratinocytes (Fig. 5). The number of apoptosis of Merkel cells was not affected by coexistence of nerve cells (NG108-15 or PC12) and by stimulation of TNF-α or IL-6.
 View Details | Fig. 5. Double immunohistochemistry with CK20 (Merkel cell marker) and ss-DNA (apoptosis marker). CK20 (red) is presented in cytoplasm of Merkel cells, and ss-DNA (brown) is expressed in their nuclei. Scale bars: 10 μm. |
In this study, we have shown that viable Merkel cells were successfully maintained in a primary culture intermingled with keratinocytes. These Merkel cells also expressed a well-differentiated phenotype similar to that of in vivo Merkel cells, in that they expressed the Merkel cell marker CK20 (Fradette et al., 1995; Moll et al., 1995; Moll et al., 1994) and showed the specific fine structures with numerous neuroendocrine granules (Narisawa et al., 1991; Vos et al., 1991). This suggests that our culture method may open a way to the study of the biological behavior of Merkel cells. In addition, the cell-isolated solution Dispase I, which was less harmful to cells (Toda et al., 1990), may serve to achieve a relatively long term-maintenance of Merkel cells in vitro.
As described in our current study, under Merkel cell-nerve cell interaction, outgrowing nerve fibers and cytoplasmic processes of Merkel cells cooperatively organize synapse-like structures at their direct contact points. This demonstrates the active interaction taking place between Merkel cells and nerve cells in vitro. In addition, our previous study and others have shown that Merkel cells in vivo or in vitro are closely associated with nerve terminals (Kim et al., 2001; Narisawa et al., 1991; Vos et al., 1991; Pasche et al., 1990). These results support the hypothesis that Merkel cells may guide nerve fibers to the skin.
In this study, the proliferative ability of Merkel cells was not detected in all conditions with or without keratinocytes and nerve cells. Several studies have showed that Merkel cells were unable to proliferate in vivo (Tachibana et al., 2000; Moll et al., 1996). Even with stimulation of IL-6 and TNF-α, we did not detect any proliferative ability of Merkel cells. It seems likely that isolated Merkel cells undergo terminal differentiation with the loss of their growth potential; but the other factors should be investigated in terms of induction of Merkel cell proliferation. We may expect the development of optimal culture conditions for the Merkel cell population to shed light on long-standing questions concerning the origin of Merkel cells and their ability to undergo mitosis, with regard to their requirements for proliferation and the mechanisms underlying their generation from precursor cells.
As described herein, the number of Merkel cells with nerve cells was greater than that of Merkel cells with keratinocytes only. IL-6 and TNF-α also did not affect the survival of Merkel cells in vitro. This suggests that nerve cells, but not keratinocytes, may produce some survival factors other than the cytokines above for Merkel cells. However, Merkel cells ultimately disappeared after 7 days in our culture system. Using ss-DNA that detects apoptosis, we demonstrated that Merkel cells extensively underwent apoptosis in all conditions tested here. This suggests that Merkel cells spontaneously undergo apoptosis in our culture system even with coexistence of keratinocytes and nerve cells, and with stimulation of TNF-α and IL-6. In general, the microenvironment of the skin is maintained by numerous cell types, including Langerhans cells, melanocytes, mast cells, fibroblasts, endothelial cells and histiocytes. Therefore, the interaction between Merkel cells and these cell types may be critical for the survival and/or proliferation of Merkel cells (Taira et al., 2002). To address these important items, further studies are needed.
In conclusion, we have developed a simple culture system of Merkel cells isolated from rat footpad skin. Our study has successfully demonstrated that outgrowing nerve fibers and cytoplasmic processes of Merkel cells cooperatively organize synapse-like structures under Merkel cell-nerve cell interaction in vitro. Further optimization of culture conditions for rat Merkel cells will provide a unique in vitro model that will provide insight into the functions and features of the particular cell type.
We thank Prof. H. Higashida, Messrs. H. Ideguchi, S. Nakahara, F. Mutoh, T. Tabata, Ms. M. Fushihara and Mrs. M. Nishida for their helpful technical assistance. We also thank Drs. A. Ootani and S. Aoki for their helpful suggestions.
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