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Interleukin-1α Stimulates the Differentiation of Melanocytes but Inhibits theProliferation of Melanoblasts from Neonatal Mouse EpidermisAuthor(s): Tomohisa Hirobe and Haruki OotakaSource: Zoological Science, 24(10):959-970. 2007.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.24.959URL: http://www.bioone.org/doi/full/10.2108/zsj.24.959

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© 2007 Zoological Society of JapanZOOLOGICAL SCIENCE 24: 959–970 (2007)

Interleukin-1α Stimulates the Differentiation of Melanocytesbut Inhibits the Proliferation of Melanoblasts from

Neonatal Mouse Epidermis

Tomohisa Hirobe1,2* and Haruki Ootaka2

1Radiation Effect Mechanism Research Group, National Institute of Radiological Sciences,Chiba 263-8555, Japan

2Graduate School of Science, Chiba University, Chiba 263-8522, Japan

Interleukin (IL)-1α is one of the important cytokines involved in regulating immunological reactions in the mouse skin. However, it is not known whether IL-1α regulates the proliferation and differen-tiation of mouse epidermal melanocytes. In this study, to investigate the role of IL-1α in the regulation of the proliferation and differentiation of mouse epidermal melanocytes, IL-1α was supplemented to serum-free primary cultures of epidermal cell suspensions from the initiation of the primary culture (keratinocytes and melanoblasts-melanocytes) as well as to pure cultures of melanoblasts-melanocytes (keratinocyte-depleted cultures, after 14 days), and its effect was tested. IL-1α inhibited the proliferation of undifferentiated melanoblasts irrespective of the presence or absence of kera-tinocytes, whereas the cytokine inhibited the proliferation of differentiated melanocytes only in the presence of keratinocytes. Moreover, IL-1α induced the differentiation of melanocytes and, in addi-tion, stimulated tyrosinase activity, melanin synthesis, and dendritogenesis of melanocytes irre-spective of the presence or absence of keratinocytes. These results suggest that IL-1α is involved in inhibiting the proliferation of neonatal murine epidermal melanoblasts and in stimulating the dif-ferentiation, melanogenesis, and dendritogenesis of melanocytes. The results also suggest that IL-1α inhibits the proliferation of differentiated melanocytes in cooperation with keratinocyte-derived factors.

Key words: melanocyte, melanoblast, keratinocyte, epidermis, serum-free primary culture, IL-1α

INTRODUCTION

The pigment-producing cells, melanocytes, are derived from the neural crest, a pluripotent embryonic cell population (Rawles, 1947). Their precursors, melanoblasts, invade the epidermis (Mayer, 1973) to colonize the epidermis. Mouse epidermal melanocytes are known to differentiate from mel-anoblasts around the time of birth (Hirobe, 1984). Melano-blasts and melanocytes migrate from epidermis to growing hair follicles and colonize hair bulbs, where they secrete mature melanosomes into surrounding keratinocytes, giving rise to melanized hairs (Mann, 1962; Hirobe, 1992a; Peters et al., 2002). The proliferation and differentiation of mouse epidermal melanocytes are regulated by numerous coat color genes (Silvers, 1979; Ozeki et al., 1995; Prota et al., 1995; Hirobe et al., 1998; Hirobe and Abe, 1999; Lamoreux et al., 2001; Bennett and Lamoreux, 2003; Mouse Genome Database 2004) as well as by surrounding tissue environ-ment, especially keratinocytes (Hirobe, 1994, 2005; Kunisada et al., 1998, 2000).

Melanin synthesis is controlled mainly by tyrosinase

(Tyr) (Hearing, 2000; Ito, 2003). Tyr initiates melanin synthe-sis by catalyzing the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and oxidation of dopa to dopaquinone (Hearing and Tsukamoto, 1991). It has also been suggested that Tyr acts on L-tyrosine to generate dopaquinone, with L-dopa as co-factor (Land et al., 2001). Melanocytes produce two types of melanin: brownish-black eumelanin and reddish-yellow pheomelanin (Prota, 1980; Ito, 2003). Although differences exist in molecular size and general properties, these melanins arise from a common metabolic pathway in which dopaquinone is a key interme-diate (Hearing and Tsukamoto, 1991; Ito, 2003; Land et al., 2001; Prota, 1980).

The proliferation and differentiation of mouse epidermal melanocytes is thought to be regulated by many growth fac-tors and cytokines from keratinocytes (Imokawa, 2004; Hirobe 2005). Interleukin 1 (IL-1) is an important cytokine involved in regulating inflammation, immunological reac-tions, and injury (Dinarello, 1984). IL-1 was first discovered as a substance that increases fever in animals, and subse-quently this cytokine has been shown to possess a wide variety of functions in addition to immunological reactions (Kampschmidt, 1984). IL-1 is produced mainly from mono-cytes and macrophages (Kampschmidt, 1984), though it is also produced from another type of cells (Kupper, 1990). IL-1 possesses two forms, IL-1α and IL-1β (Auron et al., 1984;

* Corresponding author. Phone: +81-43-206-3253/3133;Fax : +81-43-206-4638;E-mail: thirobe@nirs.go.jp

doi:10.2108/zsj.24.959

T. Hirobe and H. Ootaka960

Furutani et al., 1985; March et al., 1985). IL-1α and IL-1βare synthesized as 31-KDa molecules lacking signal pep-tides. Whereas IL-1α remains primarily intracellular in its precursor form, IL-1β is predominantly secreted, following cleavage to a 17-KDa molecule by the cysteine protease caspase-1 at the cell surface (Singer et al., 1995).

The expression patterns of IL-1α and IL-1β are different among cells (March et al., 1985). Keratinocyes have been shown to synthesize both IL-1α and -1β molecules in vitro(Kupper, 1990) in addition to IL-1α receptor type I (IL-1RI) and II (IL-1RII) (Groves et al., 1994). Release of keratinocyte-derived IL-1α may be sufficient to trigger skin inflammation in humans (Groves et al., 1992) and mice (Groves et al., 1995). IL-1α released from keratinocytes has been shown to regulate the homeostasis of mammalian skin (Groves et al., 1992, 1995; Ye et al., 2002; Barland et al., 2004). However, it is not known whether the proliferation and differentiation of mouse epidermal melanocytes are regulated by IL-1α.These circumstances prompted us to investigate in detail the role of IL-1α in the regulation of the proliferation and dif-ferentiation of neonatal mouse epidermal melanocytes. IL-1α was added to serum-free culture media from the initiation of the primary culture (keratinocytes, melanoblasts-melano-cytes) as well as from the keratinocyte-depleted stage (after 14 days) and tested for its effect on the proliferation and dif-ferentiation of primary epidermal melanoblasts and melano-cytes.

MATERIALS AND METHODS

MiceAll animals used in this study were of strain C57BL/10JHir

(nonagouti black) of the house mouse, Mus musculus. They were given water and a commercial diet, OA-2 (Clea Japan, Tokyo, Japan) ad libitum. They were maintained at 24±1°C with 40–60% relative humidity; 12 h of fluorescent light was provided daily. The present study was approved by the ethics committee of the National Institute of Radiological Sciences in accordance with the guidelines of the National Institute of Health.

Melanocyte primary cultureThe source of tissue for the culture of melanoblasts and mel-

anocytes was dorsal skin of 0.5-day-old mice. Unless stated other-

wise, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The method for obtaining epidermal cell suspen-sions was reported previously (Hirobe, 1992b, c). Disaggregated epidermal cell suspensions were pelleted by centrifugation and sus-pended in Ham’s F-10 medium (Gibco, Grand Island, NY, USA). The cell pellet after centrifugation was resuspended in melanoblast-defined medium (MDM), consisting of Ham’s F-10 plus 10 μg/ml insulin (bovine), 0.5 mg/ml bovine serum albumin (Fraction V), 1 μM ethanolamine, 1 μM phosphoethanolamine, 10 nM sodium selenite, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, 50 μg/ml gen-tamycin sulfate, and 0.25 μg/ml amphotericin B. The cell pellet was also resuspended in melanocyte-differentiation medium (MDMM) consisting of MDM supplemented with 100 nM α-melanocyte-stimulating hormone (MSH), melanocyte-proliferation medium (MDMD) consisting of MDM supplemented with 0.5 mM dibutyryl adenosine 3’:5’-cyclic monophosphate (DBcAMP), and melano-blast-proliferation medium (MDMDF) consisting of MDM supple-mented with 0.5 mM DBcAMP plus 2.5 ng/ml basic fibroblast growth factor (bFGF; Gibco). The four kinds of culture media are shown in Table 1. The same lots of these supplements were used in this study. Cells in each epidermal cell suspension were counted in a hemocytometer chamber and plated onto type I collagen (Becton Dickinson, Bedford, MA, USA)-coated dishes at an initial density of 1×106 cells/35 mm dish (1.04×105 cells/cm2). Cultures were incu-bated at 37°C in a humidified atmosphere composed of 5% CO2

and 95% air (pH 7.2). Medium was replaced by fresh medium four times a week. After 14 days, almost pure cultures of melanoblasts and/or melanocytes were obtained. IL-1α (PeproTech EC, London, UK) was added to MDM, MDMM, MDMD, and MDMDF at three dif-ferent concentrations of 0.1, 1, and 10 ng/ml from the initiation of the primary culture to 14 days (depletion of keratinocytes). In some cases, IL-1α at the three different concentrations was added to MDM and MDMM from 14 days and cultured for another 7 days. Moreover, endothelin (ET)-1 was added from 14 days to MDMD and MDMDF to induce the proliferation of melanoblasts and melano-cytes, and simultaneously IL-1α at the three different concentrations was added, and the cultures were continued for another 7 days.

Assays for proliferation and differentiationThe number of melanoblasts and melanocytes was determined

per dish by phase-contrast and by bright-field microscopy, and the calculation was based on the average number of cells from 10 ran-domly chosen microscopic fields covering an area of 0.581 mm2. The number of cells estimated by this method corresponds well with the number of cells disaggregated from dishes using trypsin and

Table 1. The four kinds of culture media used in this study

Culture media Name Component

Melanoblasts or melanocytes at 14 days

Cell yield /35 mm dish ×104 at 14 days

MDM Melanoblast- defined medium

Ins,BSA,EA,PEA,SE Pure melanoblasts 3–5

MDMM Melanocyte- differentiation medium

Ins,BSA,EA,PEA,SE,α-MSH Pure melanocytes 3–5

MDMD Melanocyte- proliferation medium

Ins,BSA,EA,PEA,SE,DBcAMP Pure melanocytes 10–20

MDMDF Melanoblast- proliferation medium

Ins,BSA,EA,PEA,SE,DBcAMP,bFGF Pure melanoblasts (90%)and melanocytes (10%)

30–60

Abbreviation used: Ins, insulin; BSA, bovine serum albumin; EA, ethanolamine; PEA, phosphoethanolamine; SE, sodium selenite; α-MSH, α-melanocyte-stimulating hormone; DBcAMP, dibutyryl adenosine 3’:5’-cyclic monophosphate; bFGF, basic fibroblast growth factor

Effect of Interleukin-1α on Melanocytes 961

ethylenediaminetetraacetate. Bipolar, tripolar, dendritic, polygonal, or epithelioid cells, as seen by phase-contrast, that contained brown or black pigment granules, as observed by bright-field microscopy,

were scored as differentiated melanocytes. The number of differen-tiated melanocytes was comparable to the number of dopa-positive melanocytes (Hirobe, 1992c). In contrast, bipolar, tripolar, dendritic,

Fig. 1. Melanoblasts and melanocytes in pure cultures derived from epidermal cell suspensions of 0.5-day-old mice cultured for 14 days in MDM (A, B), MDMM (C, D), MDMD (E, F), and MDMDF (G, H) with (B, D, F, H) or without (A, C, E, G) 10 ng/ml IL-1α. Undifferentiated melano-blasts (A, B, G, H) are shown. Differentiated melanocytes (C, D, E, F) are dendritic, polygonal, or epithelioid in morphology. Few melanocytes (arrow) are observed in MDM supplemented with IL-1α. Melanocytes cultured with IL-1α (D, F; arrows) are more dendritic and more pigmented than those of control cultures (C, E). The number of melanoblasts and melanocytes in IL-1α-treated cultures (F, H) are less than in control cul-tures (E, G). Phase-contrast microscopy. Bar, 100 μm.

T. Hirobe and H. Ootaka962

or polygonal cells, as seen by phase-contrast, that contained no pigment and were negative for dopa staining, as observed by bright-field microscopy, were scored as melanoblasts. These cells were stained by the combined dopa-premelanin reaction (combined

dopa-ammoniacal silver nitrate staining) (Mishima, 1960; Hirobe, 1982; Hirobe et al., 2002a). This preferential staining reveals undif-ferentiated melanoblasts cultured in MDMDF that contain stage I and II melanosomes in addition to Tyr-containing differentiated mel-

Fig. 2. Kinetics of the proliferation (A, C) and differentiation (B, D) of epidermal melanoblasts and melanocytes cultured in MDM (A, B) and MDMM (C, D) with or without IL-1α [0 (○), 0.1 (●), 1 (△), and 10 (▲) ng/ml]. Epidermal cell suspensions were cultured for 14 days. The num- ber of melanoblasts and melanocytes was counted at 1, 7, and 14 days. The numbers of melanoblasts and melanocytes cultured in MDM and MDMM supplemented with IL-1α did not differ from those of control cultures (A, C), whereas the percentage of melanocytes in the melano-blast-melanocyte population cultured in MDM (B) was increased by IL-1α at 1 and 10 ng/ml compared with that of control cultures. The data are the averages of results from three experiments. Each experiment was performed with different litters of mice. Bars indicate standard error of the mean (SEM) and are shown only when they are larger than the symbols. * Statistically significant differences (P<0.05).

Effect of Interleukin-1α on Melanocytes 963

anocytes. The ammoniacal silver nitrate reaction specifically reveals unmelanized melanosomes as well as melanized melanosomes in melanocytes, the metallic silver particles being deposited with a high degree of selectivity (Mishima, 1964; Hirobe, 1982b; Hirobe et al., 2002b). A “melanoblast” is defined here as an unpigmented cell that possesses no Tyr activity.

The statistical significance of differences in the number of mel-

anoblasts and melanocytes or in the percentage of melanocytes in the melanoblast-melanocyte population was determined by Stu-dent’s t-test for comparisons of groups of equal size.

Mesurement of Tyr activity and melanin contentCultured melanocytes at 7 (MDMD), 14 (MDMD), and 21

(MDMD+ET-1) days in the primary culture were washed with Ca2+-

Fig. 3. Kinetics of the proliferation (A, C) and differentiation (B, D) of epidermal melanoblasts and melanocytes in MDMD (A, B) and MDMDF (C, D) with or without IL-1α. The numbers of melanoblasts and melanocytes cultured in MDMD and MDMDF supplemented with IL-1α at doses of 1 (△) and 10 (▲) ng/ml were decreased compared with those of control cultures (○), whereas their numbers cultured with 0.1 ng/ml (●) IL- 1α did not differ from control cultures (A, C). In contrast, the percentages of melanocytes in the melanoblast-melanocyte population did not dif-fer between control cultures and IL-1α-treated cultures in both MDMD (B) and MDMDF (D). The protocols are as detailed for Fig. 2. * P<0.05.

T. Hirobe and H. Ootaka964

Fig. 4. Effects of IL-1α on the proliferation and differentiation of pure cultured melanocytes. Epidermal cell suspensions were cultured in MDM for 14 days. Pure melanoblasts (MDM) were obtained. They were further cultured in MDM (A) or MDM plus 10 ng/ml IL-1α (B). After 7 days of treatment (21 days in pri-mary culture), many melanocytes (arrows) with well-developed dendrites were observed in IL-1α-treated cultures (B), whereas the control culture showed no change (A). Epidermal cell suspensions were also cultured in MDMM for 14 days. Pure melanocytes were obtained. They were further cultured in MDMM (C) or MDMM plus 10 ng/ml IL-1α (D). After 7 days, melanocytes did not increase in number in IL-1α-treated cultures, but they possessed well-developed dendrites (D; arrows). In contrast, control cultures showed no change (C). Epidermal cell suspensions were also cultured in MDMD for 14 days. Pure mel-anocytes were obtained. They were further cultured in MDMD plus 10 nM ET-1 (E) or MDMD plus ET-1 and 10 ng/ml IL-1α (F). After 7 days, melanocytes with well-developed dendrites were observed in IL-1α-treated cultures (F; arrows), whereas control cultures showed no change (E). However, melanocytes did not increase in number (E, F). Epidermal cell suspensions were also cultured in MDMDF for 14 days. Pure melanoblasts (90%) and melanocytes (10%) were obtained. They were further cultured in MDMDF plus 10 nM ET-1 (G) or MDMDF plus ET-1 and 10 ng/ml IL-1α (H). After 7 days, the number of mel-anoblasts and melanocytes in IL-1α-treated cultures (H) were less than in control cultures (G). Phase-contrast microscopy. Bar, 100 μm.

Effect of Interleukin-1α on Melanocytes 965

and Mg2+-free phosphate buffered saline (CMF-PBS) three times, and 1 ml 1% Triton-X (Nacalai tesque, Inc., Kyoto, Japan) in CMF-PBS was added to them. Cells were incubated at room temperature for 10 minutes while pipetting. Cells were then completely dissolved and 100 μl of 10 mM L-dopa (Wako, Osaka, Japan) in CMF-PBS was added to them, and OD475 was measured. Cells were then incubated at 37°C for 1 hour and OD475 was measured. The increase in OD475 per hour was estimated as Tyr activity. Cultured melanocytes at 7 (MDMD), 14 (MDMD), and 21 (MDMD+ET-1) days in the primary culture were also treated with 1 ml 1 N NaOH for 10 min while pipetting and OD475 was measured.

RESULTS

Effects of IL-1α on the proliferation and differentiation of melanocytes

Within 1–2 days after the initiation of the primary culture in MDM, undifferentiated melanoblasts that were bipolar or tripolar in morphology were in contact with adjacent kerati-nocyte colonies via dendrite processes. After 3 days, the keratinocyte colonies increased in size, and the melano-blasts gradually increased in number. After 8–9 days, the keratinocyte colonies had decreased, and by 14 days, the cultures contained mostly melanoblasts (Fig. 1A). In the case of MDM supplemented with IL-1α at concentrations of 0.1, 1, and 10 ng/ml, similar proliferations of melanoblasts (Figs. 1B and 2A) were observed. However, some pigment-producing differentiated melanocytes with dendrites were observed in the case of IL-1α (Figs. 1B and 2B). The per-centage of differentiated melanocytes in the melanoblast-melanocyte population increased significantly at 7 days at concentrations of 1 (0.51×104 melanocytes/dish versus con-trol, 0.15×104 melanocytes/dish) and 10 (0.74×104 melano-

cytes/dish versus control, 0.15×104 melanocytes/dish) ng/ml IL-1α, and at 14 days at a concentration of 10 (0.65×104

melanocytes/dish versus control, 0.19×104 melanocytes/dish) ng/ml (Fig. 2B; P<0.05).

Pigment-producing differentiated melanocytes appeared around keratinocyte colonies within 2–3 days after the initi-ation of the primary culture of epidermal cells suspended in MDMM, and almost all cells differentiated around 7–9 days. After 14 days, almost all keratinocytes died, and pure cul-tures of melanocytes were obtained (Fig. 1C), though the number of melanocytes (Fig. 2C) did not differ from that of melanoblasts cultured in MDM (Fig. 2A). In the case of MDMM supplemented with IL-1α at all the concentrations, a similar tendency of melanocyte proliferation was observed (Figs. 1D and 2C). Moreover, the percentage of differenti-atied melanocytes in IL-1α-treated cultures was similar to that in control cultures (Fig. 2D). However, the pigmentation and dendritegenisis in IL-1α-treated melanocytes (Fig. 1D) was greater than in control melanocytes (Fig. 1C).

In the case of MDMD, differentiated melanocytes appeared around keratinocyte colonies at 2–3 days and rap-idly increased in number, with almost all cells differentiated around 7–9 days. After 14 days, almost all keratinocytes died, and pure cultures of differentiated melanocytes were obtained (Fig. 1E). In the case of MDMD supplemented with IL-1α, the proliferation of melanocytes was greatly inhibited at concentrations of 1 and 10 ng/ml (Figs. 1F and 3A; P<0.05). On the other hand, the percentage of differentiatied melanocytes in the melanoblast-melanocyte population in IL-1α-treated cultures did not differ from that in control cul-tures (Fig. 3B). However, the pigmentation and dendritoge-

Fig. 5. Effects of IL-1α on the proliferation (A) and differentiation (B) of melanoblasts and melanocytes in MDM. Epidermal cell suspensions were cultured in MDM for 14 days. Pure melanoblasts were obtained. They were further cultured with or without IL-1α at concentrations of 0 (○), 0.1 (●), 1 (△), and 10 (▲) ng/ml. Although the number of melanoblasts showed no change (A), the percentage of melanocytes dramati- cally increased at all the concentrations tested (B). The protocols are as detailed for Fig. 2. * P<0.05.

T. Hirobe and H. Ootaka966

nesis of melanocytes treated with IL-1α at all the concentra-tions tested (Fig. 1F) were greater than in control melanocytes (Fig. 1E).

In the case of MDMDF, undifferentiated melanoblasts were observed around keratinocyte colonies within 1 day and dramatically increased after 3–4 days. After 14 days, almost all keratinocytes died, and pure cultures of numerous melanoblasts (ca. 90%) and a small number of melanocytes (ca. 10%) were obtained (Fig. 1G). In the case of MDMDF supplemented with IL-1α, the proliferation of melanoblasts was greatly inhibited at concentrations of 1 and 10 ng/ml (Figs. 1H and 3C; P<0.05). In contrast, the percentage of differentiated melanocytes (5–10%) in cultures treated with IL-1α at all the concentrations tested was similar to that in control cultures (Fig. 3D).

Effects of IL-1α on the proliferation and differentiation of melanocytes in the absence of keratinocytes

To investigate whether IL-1α controls the proliferation and differentiation of mouse epidermal melanocytes in the absence of keratinocytes, pure primary melanoblasts cul-

tured in MDM for 14 days were cultured in MDM with or with-out IL-1α for another 7 days. IL-1α failed to increase the number of melanoblasts (Figs. 4A, B and 5A). However, IL-1α at all the concentrations tested induced the differentiation of melanocytes (Figs. 4A, B and 5B). Many pigmented mel-anocytes with dendrites were observed (Fig. 4B). The per-centage of differentiated melanocytes increased significantly as early as 1 day after addition of IL-1α (Fig. 5B; P<0.05).

Pure primary melanocytes cultured in MDMM for 14 days were cultured in MDMM with or without IL-1α for another 7 days. IL-1α elicited no further increase in the num-ber of melanocytes (Table 2). However, IL-1α increased the percentage of differentiated melanocytes in the melano-blast-melanocyte population (Table 3A). The differences between cultures treated with IL-1α and control cultures were statistically significant (Table 3A). Moreover, IL-1αmarkedly stimulated the pigmentation and dendritogenesis of melanocytes (Fig. 4C, D).

When the pure primary melanocytes cultured in MDMD for 14 days were further cultured in MDMD, no further increase in the number of melanocytes was observed

Table 2. Effects of IL-1α on the proliferation of mouse epidermal melanocytes in culture

Medium from 0 day

Medium from 14days

IL-1α (ng/ml)

Number of melanoblasts and melanocytes /35 mm dish × 104 (percentage of control)

14 days 15 days 17 days 21 days

MDMM MDMM 0 100±0 95.87±14.79 96.36±21.60 97.10±12.160.1 100±0 102.77± 2.15 102.64±15.91 101.10± 7.851 100±0 90.12± 1.10 94.63± 1.43 95.59± 3.2810 100±0 86.87± 5.11 98.97± 1.79 80.75± 6.86

Epidermal cell suspensions were cultured in MDMM for 14 days. Pure melanocytes were obtained. Melanocytes were further cultured in MDMM and IL-1α at concentrations of 0 (control), 0.1, 1, and 10 ng/ml for another 7 days. The num-bers of melanocytes are not different between control cultures and IL-1α (0.1, 1, 10 ng/ml)-treated cultures at 15, 17, and 21 days. The data are the averages of results from three experiments±standard error of the mean (SEM). Each experiment was performed with different litters of mice.

Table 3. Effects of IL-1α on the differentiation of mouse epidermal melanocytes in pure culture

Medium from 0 day

Medium from 14 days

IL-1α (ng/ml)

Percentage of melanocytes in the melanoblast-melanocyte population

14 days 15 days 17 days 21 days

(A) MDMM MDMM 0 83.80±2.90 77.54±1.40 81.88±3.28 85.22±2.030.1 82.04±4.40 86.97±1.87* 89.03±2.02 92.51±2.511 85.04±1.52 87.00±2.41* 92.73±0.37* 92.84±1.9710 86.40±2.55 87.28±2.39* 95.34±0.89* 94.37±1.44*

(B) MDMD MDMD 0 96.11±1.27 97.04±1.08 92.44±1.16 95.71±1.34+ ET-1 0.1 96.68±0.51 97.25±0.68 91.76±1.56 95.81±0.53

1 95.15±0.99 97.98±0.33 94.09±1.52 95.89±0.4210 95.84±0.32 97.22±0.71 93.31±1.60 94.56±1.40

(C) MDMDF MDMDF 0 12.60±2.12 10.56±2.52 10.44±1.97 12.39±3.47+ ET-1 0.1 14.28±3.61 8.76±1.35 9.83±2.69 11.86±2.99

1 11.70±2.13 9.87±2.18 8.77±1.38 11.40±2.2710 10.46±1.67 6.80±1.56 7.39±1.46 10.51±1.68

Epidermal cell suspensions were cultured in MDMM (A), MDMD (B), and MDMDF (C) for 14 days. Pure melano-blasts (ca. 90%) and melanocytes (ca. 10%) were obtained in MDMDF. In contrast, pure melanocytes were obtained in MDMM and MDMD. Melanoblasts and melanocytes in pure culture were further cultured in MDMDF plus ET-1 (10 nM) and IL-1α at concentrations of 0, 0.1, 1, and 10 ng/ml (C) Melanocytes in pure culture were also cultured in MDMM (A) for another 7 days. Melanocytes in pure culture were also cultured in MDMD plus ET-1 (10 nM) and IL-1αat concentrations of 0, 0.1, 1, and 10 ng/ml (B) for another 7 days. Other protocols are same as Table 2.

Effect of Interleukin-1α on Melanocytes 967

(Hirobe 1992c). However, ET-1 supplemented to MDMD induced the proliferation of melanocytes, as reported previ-ously (Hirobe, 2001). To test the proliferation-inhibitory effect of IL-1α on melanocytes, we cultured melanocytes in MDMD plus ET-1 with or without IL-1α for another 7 days. IL-1α at all the concentrations tested elicited no change in the number of melanocytes (Figs. 4E, F and 6A) nor in the percentage of melanocytes in the melanoblast-melanocyte population (Fig. 4E, F and Table 3B). However, IL-1α stim-ulated expansion, pigmentation, and dendritogenesis of mel-anocytes (Fig. 4E, F), and its effect continued for 7 days.

When the pure primary melanoblasts and melanocytes cultured in MDMDF for 14 days were further cultured in MDMDF, no further increase in the number of melanoblasts and melanocytes was observed (Hirobe, 1992c). However, ET-1 supplemented to MDMDF induced the proliferation of

melanoblasts and melanocytes as reported previously (Hirobe, 2001). To test the proliferation-inhibitory effect of IL-1α on melanoblasts and melanocytes, we cultured melano-blasts and melanocytes in MDMDF plus ET-1 with or without IL-1α. IL-1α at all the concentrations tested greatly inhibited the increase in the number of melanoblasts and melano-cytes induced by ET-1 (Figs. 4G, H and 6B). The differences in the number of melanoblasts and melanocytes between control cultures and IL-1α-treated cultures at 17 and 21 days were statistically significant (Fig. 6B; P<0.05). However, the percentage of melanocytes in the melanoblast-melanocyte population did not differ significantly between control cul-tures and IL-1α-treated cultures (Table 3C).

Effects of IL-1α on Tyr activity and melanin contentIL-1α increased Tyr activity in melanocytes cultured with

Table 4. Effects of IL-1α on Tyr activity and melanin content of mouse epidermal melanocytes in culture

Medium from 0 day

Medium from 14 days

Days in culture

Tyr activity Melanin content

Control IL-1α Control IL-1a

MDMD — 7 100±0 137.58± 6.93* 100±0 130.67±5.93*MDMD — 14 100±0 216.67±36.1* 100±0 122.12±7.84*MDMD MDMD+ET-1 21 100±0 128.88±9.58* 100±0 118.02±1.13*

Epidermal cell suspensions were cultured in MDMD for 14 days. Pure melanocytes were obtained. Melanocytes were further cultured in MDMD plus ET-1 (10 nM) with or without 10 ng/ml IL-1α for another 7 days. Tyr activity and melanin content were measured as described in Materials and methods. The data are averages of results from three experiments±SEM. Each experiment was performed with different litters of mice. * P<0.05.

Fig. 6. Effects of IL-1α on the proliferation of melanocytes (A) or melanoblasts plus melanocytes (B) in MDMD (A) or MDMDF (B). Epidermal cell suspensions were cultured in MDMD (A) or MDMDF (B) for 14 days. Pure melanocytes (A) or melanoblasts plus melanocytes (B) were obtained. They were further cultured in MDMD (A) or MDMDF (B) supplemented with 10 nM ET-1 and IL-1α at concentrations of 0 (○), 0.1 (●), 1 (△), and 10 (▲) ng/ml. Although the number of melanocytes cultured in MDMD (A) showed no change, the number of melanoblasts and melanocytes cultured in MDMDF (B) dramatically decreased in IL-1α-treated cultures. The protocols are as detailed for Fig. 2. * P<0.05.

T. Hirobe and H. Ootaka968

10 ng/ml IL-1α at 7, 14, and 21 days (Table 4; P<0.05). Sim-ilarly, melanin content was increased at 7, 14, and 21 days by IL-1α (Table 4; P<0.05). These results are well consistent with the visual observations (Figs. 1F and 4F).

DISCUSSION

This study showed that IL-1α in the presence of DBcAMP and bFGF inhibited the proliferation of melanoblastsirrespective of the presence or absence of keratinocytes, whereas IL-1α inhibited the proliferation of melanocytes in the presence of DBcAMP and keratinocytes. These results suggest the possibility that IL-1α alone can inhibit the prolif-eration of undifferentiated epidermal melanoblasts, while IL-1α can inhibit the proliferation of differentiated melanocytes only in the presence of keratinocytes. In contrast, IL-1αfailed to inhibit the proliferation of melanoblasts in MDM or melanocytes in MDMM. Since the proliferation of melano-blasts in MDM and melanocytes in MDMM is very limited (Hirobe, 1992b), the inhibition of the proliferation of melano-blasts or melanocytes by IL-1α may not be observed using these media. The proliferation of mouse epidermal melano-cytes seems to be inhibited by IL-1α in cooperation with unknown factors derived from keratinocytes.

Mouse keratinocytes are known to produce numerous factors involved in regulating the proliferation of epidermal melanocytes (Hirobe, 2005). ET-1 (Hirobe, 2001), ET-2 (Hirobe, 2001), ET-3 (Hirobe, 2001), leukemia inhibitory fac-tor (LIF) (Hirobe, 2002), steel factor (SLF) or stem cell factor (SCF) (Hirobe et al., 2003), hepatocyte growth factor (HGF) (Hirobe et al., 2004a), and granulocyte-macrophage colony stimulating factor (GMCSF) (Hirobe et al., 2004b) are keratinocyte-derived factors, since these factors induce the proliferation of mouse epidermal melanocytes in cooperation with DBcAMP in the absence of keratinocytes. The prolifer-ation of mouse epidermal melanocytes seems to be regu-lated by numerous factors other than ET-1, ET-2, ET-3, LIF, SLF, HGF, and GMCSF. It is unknown at present what aspects of these known factors or what undefined factors are involved in inhibiting the proliferation of mouse epider-mal melanocytes in cooperation with IL-1α.

In this study, IL-1α at a concentration of 0.1 ng/ml inhib-ited the proliferation of melanoblasts in the absence of kera-tinocytes, but IL-1α at a concentration of 0.1 ng/ml failed to inhibit the proliferation of melanoblasts in the presence of keratinocytes. These results suggest the possibility that some unknown IL-1α-inhibiting factors are released from keratinocytes. These factors remain to be identified in a future study.

This study also showed that IL-1α induced or stimulated the differentiation of mouse epidermal melanocytes as well as the pigmentation, Tyr activity, melanin synthesis, and dendritogenesis of melanocytes, suggesting that IL-1α is a potent inducer or stimulator of melanogenesis and dendrito-genesis. It has been reported that the cAMP elevator (Hirobe and Takeuchi, 1977a, b, 1978; Hirobe, 1978; Herlyn et al., 1988; Halaban et al., 1993), nerve growth factor (NGF; Yaar et al., 1991), ET-1 (Imokawa et al., 1996a; Hirobe, 2001), ET-2 (Hirobe, 2001), ET-3 (Hirobe, 2001), LIF (Hirobe, 2002), SLF (Hirobe et al., 2003; Kunisada et al., 1998), HGF (Matsumoto et al., 1991; Halaban et al., 1992; Hirobe et al., 2004a), and GMCSF (Imokawa et al., 1996b;

Hirobe et al., 2004b) are potent stimulators of melanogene-sis and dendritogenesis of mammalian melanocytes. Keratinocyte growth factor (KGF) is also known to promote melanosome transfer from melanocytes to keratinocytes (Cardinali et al., 2005). However, IL-1α has strong stimula-tory effects on the melanogenesis and dendritogenesis of melanocytes, despite the inhibition of the proliferation of melanoblasts as revealed in this study. Thus, IL-1α seems to inhibit the proliferation of melanoblasts and then induce the differentiation of melanocytes as well as dendritogene-sis. The action of IL-1α is an example of the classical notion that the inhibition of cell proliferation is followed by the induction of cell differentiation.

There may be a complex network in cells constituting the epidermis for secreting and responding to autocrine or paracrine growth factors or cytokines by keratinocytes and melanocytes, respectively (Halaban et al., 1988; Hirobe, 1992a; Abdel-Malek et al., 1995; Imokawa et al., 1997; Tada et al., 1998; Halaban, 2000; Imokawa, 2004; Hirobe, 2005). This is completed via corresponding receptors that are mod-ulated by various growth factors and cytokines, and there is a cross-talk in signaling pathways triggered by these growth factors or cytokines to support the regulation of the prolifer-ation and differentiation of melanocytes. In this study, IL-1αinhibited the proliferation of melanocytes in the presence of ET-1 and keratinocytes, in addition to inducing or stimulating the differentiation, melanogenesis, and dendritogenesis of melanocytes in cooperation with the cAMP elevator. The inhibition of melanocyte proliferation and the stimulation of melanocyte differentiation by IL-1α may be due to inhibition of the synthesis of proteins required for the proliferation of melanocytes, as well as stimulation of the synthesis of pro-teins required for differentiation via signal-transduction pathways, such as protein kinase A (PKA; Hirobe, 1992a) by cAMP elevator and protein kinase C (PKC) by ET-1 (Imokawa et al., 1997). Similarly, the result that IL-1α inhib-ited the proliferation of mouse epidermal melanoblasts in cooperation with the cAMP elevator, bFGF, and ET-1 sug-gests that IL-1α performs cross-talk between the pathways of PKA by cAMP, MAP kinase (MK; Coughlin et al., 1988) by bFGF, and PKC by ET-1. It remains to be investigated in a future study how the signaling pathway of IL-1α performs cross-talk between PKA, PKC, and MK, and how IL-1α trig-gers the inhibition of the proliferation of melanoblasts or mel-anocytes as well as the differentiation of melanocytes.

In human melanocytes, IL-1α has been reported to stim-ulate both proliferation and differentiation (Swope et al., 1991). Although the discrepancy between the results of this previous study and the present findings cannot be well explained at present, it might be due to species differences (human versus mouse).

It has been reported that radiation protection in mice is increased by pretreatment with IL-1 (Neta et al., 1986). These previous results suggest the possibility that IL-1 can serve as a signal that initiates radioprotective events in vivo. Our results suggest the possibility that IL-1α inhibits the production of abnormal melanoblasts by inhibiting the proliferation of melanoblasts and stimulating melanocyte differentiation, and, in consequence, IL-1α contributes to decreasing the dangerous effects of external stimuli.

Effect of Interleukin-1α on Melanocytes 969

ACKNOWLEDGMENTS

This work was supported in part by a Grant-in-Aid for Scientific Research (15591204) to T. H. from the Ministry of Education, Sci-ence, Sports and Culture of Japan.

REFERENCES

Abdel-Malek Z, Swope VB, Suzuki I, Akcali C, Harriger MD, Boyce ST, Urabe K, Hearing VJ (1995) Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic pep-tides. Proc Natl Acad Sci USA 92: 1789–1793

Auron PE, Webb AC, Rosenwasser LJ, Mucci SF, Rich A, Wolff SM, Dinarello CA (1984) Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc Natl Acad Sci USA 81: 7907–7911

Barland CO, Zettersten E, Brown BS, Ye J, Elias PM, Ghadially R (2004) Imiquimod-induced interleukin-1a stimulation improves barrier homeostasis in aged murine epidermis. J Invest Derma-tol 122: 330–336, 2004

Bennett DC, Lamoreux ML (2003) The color loci of mice – a genetic century. Pigment Cell Res 16: 333–344

Cardinali G, Ceccarelli S, Kovacs D, Aspite N, Lotti LV, Torrisi MR, Picardo M (2005) Keratinocyte growth factor promotes melano-some transfer to keratinocytes. J Invest Dermatol 125: 1190–1199

Coughlin SR, Barr PJ, Cousens LS, Fretto LJ, Williams LT (1988) Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo. J Biol Chem 263: 988–993

Dinarello CA (1984) Interleukin-1. Rev Infect Dis 6: 51–95Furutani Y, Notake M, Yamayoshi M, Yamagishi J-I, Monuma H,

Ohue M, Furuta R, Fukui T, Yamada M, Nakamura S (1985) Cloning and characterization of the cDNAs for human and rab-bit interleukin-1 precursor. Nucleic Acids Res 13: 5869–5882

Groves RW, Ross E, Baker JNWN, Ross JS, Camp RDR, MacDonald DM (1992) Effect of in vivo interleukin- on adhesion molecule expression in normal human skin. J Invest Dermatol 98: 384–387

Groves RW, Sherman L, Mizutani H, Dower SK, Kupper TS (1994) Detection of interleukin-1 receptors in human epidermis. Induc-tion of the type II receptor after organ culture and in psoriasis. Am J Pathol 145: 1048–1056

Groves RW, Mizutani H, Kieffer JD, Kupper TS (1995) Inflammatory skin disease in transgenic mice that express high levels of inter-leukin 1a in basal epidermis. Proc Natl Acad Sci USA 92: 11874–11878

Halaban R (2000) The regulation of normal melanocyte proliferation. Pigment Cell Res 13: 4–14

Halaban R, Langdon R, Birchall N, Cuono C, Baird A, Scott G, Moellmann G, McGuire J (1988) Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol 107: 1611–1619

Halaban R, Rubin JS, Funasaka Y, Cobb M, Boulton T, Faletto D, Rosen E, Chan A, Yoko K, White W, Cook C, Moellmann G (1992) Met and hepatocyte growth factor/scatter factor signal transduction in normal melanocytes and melanoma cells. Onco-gene 7: 2195–2206

Halaban R, Tyrrell L, Longley J, Yarden Y, Rubin J (1993) Pigmen-tation and proliferation of human melanocytes and the effects of melanocyte-stimulating hormone and ultraviolet B light. Ann NY Acad Sci 680: 290–301

Hearing VJ (2000) The melanosome: the perfect model for cellular response to the environment. Pigment Cell Res 13 (Suppl 8): 23–34

Hearing VJ, Tsukamoto K (1991) Enzymatic control of pigmentation in mammals. FASEB J 5: 2902–2909

Herlyn M, Mancianti ML, Jambrosic J, Bolen JS, Koprowski H (1988)

Regulatory factors that determine growth and phenotype of nor-mal human melanocytes. Exp Cell Res 179: 322–321

Hirobe T (1978) Stimulation of dendritogenesis in the epidermal melanocytes of newborn mice by melanocyte-stimulating hor-mone. J Cell Sci 33: 371–383

Hirobe T (1982a) Genes involved in regulating the melanocyte and melanoblast-melanocyte populations in the epidermis of new-born mouse skin. J Exp Zool 223: 257–264

Hirobe T (1982b) Origin of melanosome structures and cytochemi-cal localizations of tyrosinase activity in differentiating epider-mal melanocytes of newborn mouse skin. J Exp Zool 224: 355–363

Hirobe T (1984) Histochemical survey of the distribution of the epi-dermal melanoblasts and melanocytes in the mouse during fetal and postnatal periods. Anat Rec 208: 589–594

Hirobe T (1992a) Control of melanocyte proliferation and differentia-tion in the mouse epidermis. Pigment Cell Res 5: 1–11

Hirobe T (1992b) Melanocyte stimulating hormone induces the dif-ferentiation of mouse epidermal melanocytes in serum-free cul-ture. J Cell Physiol 152: 337–345

Hirobe T (1992c) Basic fibroblast growth factor stimulates the sustained proliferation of mouse epidermal melanoblasts in a serum-free medium in the presence of dibutyryl cyclic AMP and keratinocytes. Development 114: 435–445

Hirobe T (1994) Keratinocytes are involved in regulating the devel-opmental changes in the proliferative activity of mouse epider-mal melanoblasts in serum-free culture. Dev Biol 161: 59–69

Hirobe T (2001) Endothelins are involved in regulating the prolifera-tion and differentiation of mouse epidermal melanocytes in serum-free primary culture. J Invest Dermatol Symp Proc 6: 25–31

Hirobe T (2002) Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epider-mal melanocytes in culture. J Cell Physiol 192: 315–326

Hirobe T (2005) Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes. Pigment Cell Res 18: 2–12

Hirobe T, Abe H (1999) Genetic and epigenetic control of the prolif-eration and differentiation of mouse epidermal melanocytes in culture. Pigment Cell Res 12: 147–163

Hirobe T, Takeuchi T (1977a) Induction of melanogensis in the epidermal melanoblasts of newborn mouse skin by MSH. J Embryol Exp Morphol 37: 79–90

Hirobe T, Takeuchi T (1977b) Induction of melanogenesis in vitro in the epidermal melanoblasts of newborn mouse skin by MSH. In Vitro 13: 311–315

Hirobe T, Takeuchi T (1978) Changes of organelles associated with the differentiation of epidermal melanocytes in the mouse. J Embryol Exp Morph 43: 107–121

Hirobe T, Wakamatsu K, Ito S (1998) Effects of genic substitution at the agouti, brown, albino, dilute, and pink-eyed dilution loci on the proliferation and differentiation of mouse epidermal melano-cytes in serum-free culture. Eur J Cell Biol 75: 184–191

Hirobe T, Furuya R, Akiu S, Ifuku O, Fukuda M (2002a) Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice. Pigment Cell Res 15: 391–399

Hirobe T, Kawa Y, Mizoguchi M, Ito S, Wakamatsu K (2002b) Effects of genic substitution at the pink-eyed dilution locus on the proliferation and differentiation of mouse epidermal melano-cytes in vivo and in vitro. J Exp Zool 292: 351–366

Hirobe T, Osawa M, Nishikawa S-I (2003) Steel factor controls the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture. Pigment Cell Res 16: 644–655

Hirobe T, Osawa M, Nishikawa S-I (2004a) Hepatocyte growth factor controls the proliferation of cultured epidermal melano-

T. Hirobe and H. Ootaka970

blasts and melanocytes from newborn mice. Pigment Cell Res 17: 51–61

Hirobe T, Furuya R, Ifuku O, Osawa M, Nishikawa S-I (2004b) Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the prolifera-tion and differentiation of neonatal mouse epidermal melano-cytes in culture. Exp Cell Res 297: 593–606

Imokawa G (2004) Autocrine and paracrine regulation of melano-cytes in human skin and in pigmentary disorders. Pigment Cell Res 17: 96–110

Imokawa G, Yada Y, Kimura M (1996a) Signaling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes. Biochem J 314: 305–312

Imokawa G, Yada Y, Kimura M, Morisaki N (1996b) Granulocyte/macrophage colony-stimulating factor is an intrinsic keratinocyte-derived growth factor for human melanocytes in UVA-induced melanosis. Biochem J 313: 625–631

Imokawa G, Kobayashi T, Miyagishi M, Higashi K, Yada Y (1997) The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis. Pig-ment Cell Res 10: 218–228

Ito S (2003) A chemist’s view of melanogenesis. Pigment Cell Res 16: 230–236

Kampschmidt RF (1984) The numerous postulated biological mani-festations of interleukin-1. J Leuk Biol 36: 341–355

Kunisada T, Yoshida H, Yamazaki H, Miyamoto A, Hemmi H, Nishimura E, Shultz LD, Nishikawa S-I, Hayashi S-I (1998) Transgene expression of steel factor in the basal layer of epidermis promotes survival, proliferation, differentiation and migration of melanocyte precursors. Development 125: 2915–2923

Kunisada T, Yamazaki H, Hirobe T, Kamei S, Omoteno M, Tagaya H, Hemmi H, Koshimizu U, Nakamura T, Hayashi S-I (2000) Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocy-tosis. Mech Dev 94: 67–78

Kupper TS (1990) Immune and inflammatory processes in cutane-ous tissues: mechanisms and speculations. J Clin Invest 86: 1783–1789

Lamoreux ML, Wakamatsu K, Ito S (2001) Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin. Pigment Cell Res 14: 23–31

Land EJ, Ramsden CA, Riley PA (2001) Pulse radiolysis studies of orthoquinone chemistry relevant to melanogenesis. J Photo-chem Photobiol B Biol 64: 123–135

Mann SJ (1962) Prenatal formation of hair types. Anat Rec 144: 135–141

March CJ, Mosley B, Larsen A, Cerretti DP, Braedt G, Price V, Gillis S, Henney CS, Kronheim SR, Grabstein K, Conlon PJ, Hopp TP, Cosman D (1985) Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs. Nature 315: 641–647

Matsumoto K, Tajima H, Nakamura T (1991) Hepatocyte growth fac-tor is a potent stimulator of human melanocyte DNA synthesis and growth. Biochem Biophys Res Comm 176: 45–51

Mayer TC (1973) The migratory pathway of neural crest cells into the skin of mouse embryos. Dev Biol 34: 39–46

Mishima Y (1960) New technique for comprehensive demonstration of melanin, premelanin, and tyrosinase sites–combined dopa-premelanin reaction. J Invest Dermatol 34: 355–360

Mishima Y (1964) Electron microscopic cytochemistry of melano-somes and mitochondria. J Histochem Cytochem 12: 784–790

Mouse Genome Database, 2004. Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine. http://www. informatics.jax.org/

Neta R, Douches S, Oppenheim JJ (1986) Interleukin 1 is a radio-protector. J Immunol 136: 2483–2485

Ozeki H, Ito S, Wakamatsu K, Hirobe T (1995) Chemical character-ization of hair melanins in various coat-color mutants of mice. J Invest Dermatol 105: 361–364

Peters EMJ, Tobin DJ, Botchkareva N, Maurer M, Paus R (2002) Migration of melanoblasts into the developing murine hair folli-cle is accompanied by transient c-kit expression. J Histochem Cytochem 50: 751–766

Prota G (1980) Recent advances in the chemistry of melanogenesis in mammals. J Invest Dermatol 75: 122–127

Prota G, Lamoreux ML, Muller J, Kobayashi T, Napolitano A, Vincensi MR, Sakai C, Hearing VJ (1995) Comparative analysis of melanins and melanosomes produced by various coat color mutants. Pigment Cell Res 8: 153–163

Rawles ME (1947) Origin of pigment cells from the neural crest in the mouse embryo. Physiol Zool 20: 248–266

Silvers WK (1979) The Coat Colors of Mice. Springer-Verlag, BerlinSinger II, Scott S, Chin J, Bayne EK, Limjuco G, Weidner J, Miller

DK, Chapman K, Kostura MJ (1995) The interleukin-1β-converting enzyme (ICE) is localized on the external cell sur-face membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy. J Exp Med 182: 1447–1459

Swope VB, Abdel-Malek Z, Kassem LM, Nordlund JJ (1991) Inter-leukins 1α and 6 and tumor necrosis factor-α are paracrine inhibitors of human melanocyte proliferation and melanogene-sis. J Invest Dermatol 96: 180–185

Tada A, Suzuki I, Im S, Davis MB, Cornelius J, Babcock G, Nordlund JJ, Abdel-Malek ZA (1998) Endothelin-1 is a paracrine growth factor that modulates melanogenesis of human melanocytes and participates in their responses to ultaraviolet radiation. Cell Growth Differ 9: 575–584

Yaar M, Grossman K, Eller M, Gilchrest BA (1991) Evidence for nerve growth factor-mediated paracrine effects in human epi-dermis. J Cell Biol 115: 821–828

Ye J, Garg A, Calhourn C, Feingold KR, Elias PM, Ghadially R (2002) Alterations in cytokine regulation in aged epidermis: implications for permeability barrier homeostasis and inflamma-tion. I. IL-1 gene family. Exp Dermatol 11: 209–216

(Received May 1, 2007 / Accepted June 11, 2007)