Do Hair Bulb Melanocytes Undergo Apotosis During Hair Follicle Regression (Catagen)?

Do Hair Bulb Melanocytes Undergo Apotosis During Hair Follicle Regression (Catagen)? Desmond J. Tobin, Evelin Hagen,* Vladimir A. Botchkarev,* and Ralf Paus* Department of Biomedical Sciences, University of Bradford, Bradford, U.K.; *Department of Dermatology, Charite´, Humboldt University, Berlin, Germany

The fate of the hair follicle pigmentary unit during the cyclical involution of anagen hair follicles is unknown. Using the C57BL/6 mouse model for hair research, hair follicle melanocytes were examined during the anagen– catagen transformation, comparing spontaneous and pharmacologically induced catagen development. This study shows that both spontaneous catagen and dexamethasone-induced catagen display similar changes in the pigmentary unit. Catagen hair follicles exhibited pigment incontinence in the dermal papilla and in selected outer root sheath keratinocytes. Melanocytes deleted by apoptosis were detected in spontaneous catagen and, more commonly, in dexamethasone-induced catagen, and were identified using transmission electron microscopy by the presence of free premelanosomes in affected cells lacking epithelial specializations, and by the colocalization of TUNEL positivity and tyrosinase-related protein-1 immunoreactivity. By contrast, cyclophos-

phamide-induced catagen was characterized by the initial retention of melanogenic and dendritic melanocytes in the presence of widespread keratinocyte apoptosis. Melanocyte incontinence and the ectopic distribution of melanin were more severe than in the other forms of catagen. Whereas much of this melanin was extruded, via the hair canal, to the skin surface, hair follicle-derived pigment was also detected within the epidermis, probably derived from pigment-carrying migrating outer root sheath keratinocytes from the proximal hair follicle. Thus, apoptosis may account, at least in part, for the loss of melanogenic melanocytes during spontaneous catagen. Although dexamethasone-induced catagen may provide a useful model for general hair pigmentation research, catagen induced by cyclophosphamide offers an interesting model for studying the response, and relative resistance, of melanocytes to chemical injury. Key words: cell death/cyclophosphamide/dexamethasone/differentiation/electron microscopy/melanosomes. J Invest Dermatol 111:941–947, 1998

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only in the hair bulb matrix of anagen HF, where they are distributed in close contact with the basal lamina separating the matrix and the dermal papilla (DP). These cells express tyrosinase mRNA and activity, and actively produce pigment during anagen III–VI (Slominski et al, 1991; Slominski and Paus, 1993; Ortonne and Prota, 1993). A second HF MC subpopulation is amelanotic and is distributed widely in the outer root sheath (ORS) during anagen, and may, in part, form a MC reservoir (Horikawa et al, 1996). Strikingly, towards the end of anagen, retraction of MC dendrites and the suppression of melanogenesis (i.e., gene and protein activity) are the earliest signs of imminent HF regression even before structural changes are apparent in the hair bulb. Subsequently, melanogenically active MC ‘‘disappear’’ from the follicular epithelium during catagen and telogen, whereas remnants of melanin granules generated during the preceding anagen may occasionally be seen in the DP of catagen and telogen follicles (Chase, 1954; Sugiyama, 1979; Slominski and Paus, 1993; Slominski et al, 1994). The fate of the hair bulb MC during catagen has remained elusive and has long provided one of the fascinating enigmas of both HF and pigment biology. Where do these MC go during catagen and telogen, and where do they originate from, when follicular melanogenesis is resumed during the next anagen phase? Answering these questions may provide new insights into the physiologic control of MC migration, differentiation, melanogenesis, proliferation, and death in situ. Furthermore, this may provide new clues as to what goes wrong during hair pigmentation disorders such as poliosis, in alopecia areata and vitiligo, in premature canities, and in hair shaft dyschromia after chemotherapy (Dawber, 1997). At least three possible scenarios can be envisaged for the pigmentary

air grows in a cyclical manner, characterized by a finite period of hair fiber production (anagen), a brief regression phase resulting in the loss of up to 70% of the hair follicle (HF) (catagen), and a resting period of minimal activity (telogen) (Chase, 1954; Paus, 1996). This sequence of events occurs in HF producing either pigmented or unpigmented hair fibers, although recent evidence suggests that growth rate and fiber caliber may relate to pigmentation status (Nagl, 1995), and that the transfer of melanosomes to keratinocytes may stimulate keratinocyte differentiation (Slominski et al, 1993). A most striking feature of pigmented hair growth is the observation that the activity of hair melanocytes (MC) located in the hair matrix, unlike those in the epidermis, is under cyclical control and that melanogenesis and anagen are tightly coupled (Slominski et al, 1993). The factors that control both hair growth and pigmentation are currently unknown, although apoptosis of hair bulb keratinocytes is responsible for catagenassociated tissue regression at the end of anagen (Weedon and Strutton, 1981; Lindner et al, 1997). The HF contains at least two distinct subpopulations of MC; one melanogenic, the other not (Tobin et al, 1995; Horikawa et al, 1996; Tobin and Bystryn, 1996). Melanogenically active MC are located

Manuscript received December 10, 1997; revised August 3, 1998; accepted for publication August 12, 1998. Reprint requests to: Dr. R. Paus, Hautklinik, Charite´, Humboldt-Universita¨t zu Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. Abbreviations: CYP, cyclophosphamide; DEX, dexamethasone; DP, dermal papilla; HF, hair follicle; MC, melanocyte.

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0022-202X/98/$10.50 Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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unit during the hair cycle. One is that pigmented hair bulb MC do survive successive hair cycles (Sugiyama, 1979), during which they would have to avoid the extensive apoptosis-driven regression of the hair bulb (Weedon and Strutton, 1981; Lindner et al, 1997) by actively suppressing apoptosis. Another is that functional hair bulb MC are terminally differentiated and die in early catagen by an as yet unknown mode of cell death, possibly apoptosis. It is feasible that MC loss during catagen could be replenished from the undifferentiated MC pool in the permanent portion of the HF or from another, as yet unidentified precursor pool. Given the strict dependence of mature MC on keratinocyte-derived signals like FGF-2 and neurotrophins (Yaar et al, 1994), it is also conceivable, though less likely, that melanogenically active MC leave the regressing follicular epithelium and migrate into the DP or the perifollicular mesenchyme after having switched off melanogenesis. These cells may then re-enter the follicular epithelium only after keratinocytes of the newly developing anagen hair bulb secrete appropriate inductive signals. Study of the life cycle of the HF MC in situ has been hindered because the brevity of catagen permits access to only a very small proportion of HF in this stage at any one time. This important limitation to hair research was, in part, alleviated by studying fairly well-synchronized, spontaneous catagen development in mice (Straile et al, 1961), and by artificially inducing massive premature, but apparently histologically normal, catagen in C57BL/6 mice by topical dexamethasone (DEX) (Paus et al, 1994a). This assay provided the rapid, reproducible, and predictable pharmacologic induction of catagen in a large proportion of HF. Subsequently, it was found that HF pigmentation in this model could be disrupted pharmacologically (Paus et al, 1994b) in a manner that imitates chemotherapy-induced human hair pigmentation disorder, thus providing an attractive model for studying the MC response to and recovery from drug damage in situ (Slominski et al, 1996). In an attempt to further study the fate of HF MC and the disintegration of the ‘‘HF pigmentary unit’’ during early catagen, we have adapted these models (Paus et al, 1994a, b; Slominski et al, 1994, 1996) to assess the MC status after spontaneous catagen and after catagen induction by either topical DEX or intraperitoneal cyclophosphamide (CYP). The effect of these drugs on HF MC status was examined by both light and electron microscopy, and by TUNEL/TRP-1 double staining, and compared with MC changes during normal spontaneous catagen. MATERIALS AND METHODS Animals Six to nine week old, female C57BL/6 mice (Charles River, Sulzfeld, Germany) were housed in community cages at the Charite´/Virchow animal facilities, and were fed water and mouse chow ad libitum. In vivo manipulations Anagen was induced by depilation in the back skin of mice with all HF in telogen as described (Paus et al, 1990). Seventeen to nineteen days after anagen induction, catagen develops spontaneously and moderately well synchronized, starting in the neck region (Straile et al, 1961), which allows one to perform systematic, sequential studies of the reorganization of the HF pigmentary unit during the anagen–catagen–telogen transformation of the hair cycle (Slominski et al, 1994). In order to induce massive, premature, but light microscopically normal catagen development, depilation-induced anagen skin was treated once daily with topical DEX-21-acetate (0.1%) from days 9–13 after depilation as described (Paus et al, 1994a). In order to induce chemotherapy-induced HF dystrophy, associated with a massive disruption of the HF pigmentary unit, depilation-induced anagen mice were given a single injection of 150 mg CYP per kg i.p. as described (Paus et al, 1994b). This not only induces HF dystrophy, severe pigmentation disorders, and alopecia, but also dramatically upregulates keratinocyte apoptosis in the hair bulb (yet not the DP) (Lindner et al, 1997) and forces anagen VI HF to enter the dystrophic anagen or dystrophic catagen pathways in response to chemical damage (Paus et al, 1994b, 1996). Transmission electron microscopy (TEM) Representative tissue samples of spontaneous catagen and CYP-treated or DEX-treated and vehicle controls were fixed in Karnovsky’s fixative (Karnovsky, 1965), post-fixed in 2% osmium tetroxide and uranyl acetate, and embedded in resin as previously described (Tobin et al, 1991). Semi-thin and ultra-thin sections were cut with a ReichartJung microtome; the former were stained with the metachromatic stain,

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toluidine blue/borax, examined by light microscopy and photographed (Leitz). Loss of DP metachromasia is a marker for early catagen (Young, 1980). The latter were stained with uranyl acetate and lead citrate (Tobin et al, 1991). These ultrathin sections were then examined and photographed using a Joel 100CX electron microscope. Twenty HF from spontaneous catagen were examined by light microscopy from each of four mice, and 10 follicles from each mouse were examined by TEM. Twenty HF from DEX-induced catagen in each of six study and three control mice were examined by light microscopy and 10 follicles from each mouse were examined by TEM. Similarly, 20 HF from CYP-induced catagen were examined by light microscopy from each of six study and three control mice and 10 HF from each mouse by TEM. A total of 22 blocks of skin were examined by light microscopy and TEM. Double immunodetection of TUNEL and tyrosinase-related protein 1 (TRP-1) positive cells To evaluate apoptotic cells, we used an established, commercially available TUNEL kit (ApopTag, Oncor, Gaithesburg, MD) as described before (Lindner et al, 1997). Briefly, fixed cryostat sections (8 µm) of C57BL/6 back skin were incubated with TdT solution, blocked with 10% normal goat serum, and incubated overnight with rabbit αPEP1-anti-serum (1:200; a gift from Dr. V. Hearing, NIH), which recognizes murine TRP-1 protein (Jimene´z et al, 1988, 1989). Sections were incubated with antidigoxigenin FITC-conjugated F(ab)2 fragments for the detection of digoxigenin-dUTPlabeled DNA compounds according to the manufacturer’s guidelines. Secondary goat-antirabbit TRITC-conjugated antibody was applied and the sections countered-stained by Hoechst 33342. Negative controls for the TUNEL staining were made omitting TdT, according to the manufacturer’s protocol, and positive TUNEL controls tissue sections from the thymus of young mice were used, as this tissue displays a high degree of spontaneous thymocyte apoptosis (BulfonePaus et al, 1997). After washing in Tris-buffered saline, all sections were mounted with immunomount medium (Shandon, Pittsburgh, PA). Sections were examined under a Zeiss Axioscope microscope, using the appropriate excitation-emission filter systems for studying of the fluorescence, induced by Hoechst 33342, FITC, or TRITC. Photodocumentation was done with the help of a digital image analysis system (ISIS Metasystems, Altlussheim, Germany).

RESULTS Spontaneous catagen development in C57BL/6 mice was recognized grossly by the characteristic change in skin color from black to gray around day 17–19 after anagen induction by depilation due to reduction in melanogenesis at the late anagen–early catagen transition (Slominski et al, 1991). The staging of catagen was performed as previously described (Slominski et al, 1994, 1996). The major morphologic features associated with early catagen included the thinning of matrix volume, a marked reduction in keratinocyte proliferation, and a lengthening of the DP with loss of extracellular matrix material. These DP changes coincided with the loss of metachromasia of the DP (Young, 1980) and with the marked reduction in intercellular spaces. Only catagen I–IV follicles were examined in this study. Distribution of MC in spontaneous catagen The most striking feature of spontaneously developed catagen HF was the scarcity of melanogenic MC in the early regressing hair bulb, and the fact that melanin-granules were distributed in complexes within precortical keratinocytes (Fig 1a). Earliest catagen was identified by the absence of metachromasia of the DP (Young, 1980). In catagen II–III, melanotic MC, when present, were located high in the bulb around the upper spire of the DP and were ultrastructurally distinguishable from adjacent keratinocytes by their increased heterochromatism and irregular nuclear profiles, by the lack of desmosomal junctions and tonofilaments, and by their tendency to align their long axis with the basal lamina that separates the DP from the hair matrix (not shown). These cells, which were no longer dendritic at this stage of the anagen–catagen transformation, commonly contained some stage IV melanosomes (Fitzpatrick et al, 1971) of variable size, but exhibited few if any premelanosomes. By catagen IV, HF were devoid of melanogenic hairbulb MC (not shown). Although functionally active MC were very rare in spontaneous catagen stage II–III hair bulbs, low levels of melanin incontinence were common. Melanin granules outside the hair matrix were usually restricted to the DP, where they were distributed either singly or as clusters of stage IV granules within these fibroblast-like cells (not shown). Rare examples of stage III/IV melanosomes clustered within

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Figure 1. MC status in early catagen. (a) Hair follicle in spontaneous catagen I/II. Note the presence of melanin in precortical keratinocytes, but the absence of detectable MC in the bulb, despite the presence of a full DP profile. (b) Melanin incontinence within lysosomes in distal ORS keratinocytes. (c) MC apoptosis in spontaneous early catagen. Note typical morphologic features of apoptosis including cell shrinkage and nuclear condensation. (d) High magnification of inset in (c). Image is turned 180° from that in (c). The affected cells contain MC-specific premelanosomes. ly, lysosome; dp, dermal papilla; am, apoptotic MC; p, premelanosomes; m, melanosome; k, keratinocyte. Scale bars: (a) 20 µm, (b) 2 µm (inset, 5 µm), (c) 2 µm, (d) 0.5 µm (inset, 0.25 µm).

lysosomes of keratinocytes, however, were also observed in the mid and upper ORS of catagen HF (Fig 1b). These cells were usually located adjacent to the CTS and did not show any association with the hair canal. Evidence of MC death during spontaneous catagen In line with previous reports (Weedon and Strutton, 1981; Lindner et al, 1997) keratinocyte apoptosis most commonly affected individual scattered cells restricted to the hair bulb of catagen II–VI HF. These cells were detected first in the peripheral outer hair bulb of catagen II HF, and thereafter throughout the hair bulb and ORS. These cells displayed the characteristic features of apoptosis (Wyllie, 1981; Tobin et al, 1991), including condensation of nuclear chromatin and cytoplasm, followed by nuclear segregation and fragmentation. Intact organelles, particularly mitochondria, were observed in many apoptotic keratinocytes (not shown, cf. Tobin et al, 1991). Importantly, MC were also affected by apoptosis (Fig 1c, d). These cells were identified as MC by the presence of premelanosomes and melanosomes distributed free in the cytoplasm (Fig 1d), and by the concomitant absence of epithelial cell specializations, including tonofilaments. Only mature melanosomes (stage IV) are transferred to precortical keratinocytes (Cesarini, 1990), where they are located within phagolysosomes. Further support of MC identity was provided by the double immunodetection of TUNEL and TRP1 positive cells in the hair bulbs of spontaneous by regressing HF (Fig 4). TRP-1 staining was located primarily in the precortex region of the early catagen HF, although in late anagen (anagen VI) staining was also seen more proximally. This reflected the changing distribution of MC during the transition from anagen to catagen. Indeed, there is some evidence that MC may even be lost via the precortex in murine early catagen (Sweet and Quevedo, 1968); however, by catagen III– IV TRP-1 staining was completely absent from the regressing HF (not

shown). In addition, TUNEL-positive keratinocytes were seen first in the precortex region, whereas maximal TUNEL positivity was observed in the hair matrix only during catagen III (not shown). Distribution of MC in DEX-induced catagen DEX-induced catagen was examined because it provides a far greater yield of normalappearing (by light microscopy) early catagen HF than are ever available in skin with spontaneously developing follicle regression (Paus et al, 1994a). HF were treated topically daily from day 9–13, after anagen induction by depilation, with 0.1 DEX-12-acetate in propylene glycol or with vehicle alone (control). Skin was harvested at days 13, 14, or 15 post-depilation. HF treated with vehicle alone did not exhibit catagen development or detectable apoptosis and were all in normal anagen VI (not shown). The ultrastructural features of hair bulb MC in the treated HF were, in the main, very similar to those observed in HF after induction of spontaneous catagen. Occasionally, melanogenic MC were detected in the hair bulb of catagen II HF, but not catagen III or IV HF (not shown). The majority of melanin was seen to be distributed within precortical keratinocytes, as described before (Chase, 1954). Low levels of melanin incontinence were observed in these specimens, even in catagen I or II HF: mature melanosomes (stage IV) were usually distributed singly or in clusters within DP fibroblast-like cells (Fig 2a). Rare examples of melanosome complexes within the lysosomes of keratinocytes (some of which were apoptotic), however, were detected in the ORS of the catagen stage III/IV HF (not shown). As in spontaneous catagen, melanin-containing ORS keratinocytes were located adjacent to the CTS and did not exhibit any direct association with the hair canal. Evidence of MC apoptosis in DEX-induced catagen Apoptosis affected individual, scattered cells or small groups of neighboring cells

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Table I. Melanocyte behavior in response to spontaneous, DEX-, and CYP-induced catagen MC location Spontaneous DEX CYP

MC apoptosisa 1 11 1/2

Distal matrix, early catagen Distal matrix, early catagen Distal and proximal matrix, early and late catagen

Pigment fate Pre-cortex, DP, ORS Pre-cortex, DP, ORS Pre-cortex, DP, ORS, inner root sheath CTS, cuticle, epidermis

1, Low level MC apoptosis; 11, moderate MC apoptosis; 1/2, rare unconfirmed MC apoptosis.

Figure 2. MC status in DEX-induced early catagen. (a) Melanin incontinence was observed in the DP. (b) MC apoptosis. Note nuclear and cytoplasmic condensation and the presence of numerous MC-specific premelanosomes in addition to many fully mature, stage IV, melanosomes. am, apoptotic MC; k, keratinocyte; p, premelanosomes; m, melanosomes; dp, DP; pc, precortex. Scale bars: (a) 10 µm, (b) 1 µm (inset, 0.3 µm).

in the lower hair bulb of catagen II–IV HF. Interestingly, and more commonly than in spontaneous catagen, these apoptotic cells did not display any ultrastructural features of epithelial cells (e.g., tonofilaments), and contained melanosomes, many of which were small and poorly melanized (stage II premelanosomes). Some of these cells were observed in stages of apoptosis prior to phagocytosis by neighboring keratinocytes, facilitating their positive identification. Furthermore, melanosomecontaining apoptotic bodies located within keratinocyte phagolysosomes did not themselves exhibit epithelial cell features (e.g., tonofilaments), again indicative of a melanocytic origin (Fig 2b). Distribution of MC in CYP-induced catagen Mice were treated intraperitoneally with 150 mg CYP per kg or with vehicle (0.9% NaCl) on day 9 after anagen induction by depilation. The skin was harvested 8 h, 24 h, and 6 d after CYP treatment, i.e., on days 10 or 15 after anagen induction by depilation. As described before in detail, CYP treatment massively disrupts the HF pigmentary unit and induces severe follicle dystrophy along two pathways; dystrophic anagen and dystrophic catagen (Paus et al, 1994a, 1996; Slominski et al, 1996). For the purpose of this study, only CYP-induced dystrophic catagen follicles were analyzed. The status of MC in CYP-treated HF was quite unlike that in either spontaneous catagen or DEX-treated HF (Table I). Melanogenic hairbulb MC were observed in catagen II/III HF at 8 h after CYP injection, when considerable keratinocyte apoptosis was evident by ultrastructural criteria below Auber’s line. The same keratinocytes had previously been demonstrated to stain strongly positive in the TUNEL reaction (Lindner et al, 1997). Hair bulb MC, however, were histologically normal-appearing in these catagen follicles, and were not only distributed in the matrix around the mid to upper DP, but were also observed in the most proximal hair bulb epithelium (Fig 3a, b). A normal distribution of melanosomes was observed in these cells; i.e., mature melanosomes located in the cell periphery and small, poorly melanized granules in the perikaryon (Fig 3b). The restriction of melanin to precortical keratinocytes in HF 8 h

Figure 3. MC status in CYP-induced early catagen. (a, b) Re-distribution of melanogenic MC to the most proximal ORS. Note also the presence of widespread keratinocyte apoptosis, some in very early stages of programmed cell death 10 d after CYP-treatment initiation. (c) Ectopic distribution of melanin granules within epidermal keratinocytes. (d) Presumptive MC apoptosis in CYP-induced catagen. Some apoptotic cells contain premelanosome-like granules. me, MC; a, apoptosis; m, melanin; cts, connective tissue sheath; k, keratinocytes; s, stratum corneum; p, premelanosome-like granules. Scale bars: (a) 10 µm, (b) 5 µm (inset, 1 µm), (c) 2 µm, (d) 1 µm.

after CYP application indicated that normal pigment transfer was still operative at this stage; however, by 24 h and 10 d these MC exhibited the effects of drug-induced insult as evidenced by a gross pigmentary defect in these HF. Melanosome transfer mechanisms were severely disrupted, resulting in the ectopic distribution of melanin aggregates into keratinocytes destined for the inner root sheath, ORS, and even cuticle, rather than the precortex and medulla only as occurs during normal hair pigmentation (Cesarini, 1990). Further, melanin was observed in dermal melanophages and, surprisingly, in the epidermis (Fig 3c), which is normally completely devoid of pigment in murine truncal skin (Slominski et al, 1994). Melanin was also distributed in the follicular canal from where it left the skin (not shown). Here, melanin was not located within cells, but appeared commonly as small dust-like particles in the hair canal. This melanin

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Figure 4. Apoptosis of hair bulb melanocytes in spontaneous catagen. Cryostat sections of adolescent C57BL/6 skin, with all HF in early stages of spontaneous catagen (day 17 post-depilation), were processed for double immunostaining with TUNEL and TRP-1. Nuclei were counterstained with Hoechst 33342. Large arrowhead, TRP1-IR (red fluorescence) was detected in the keratogenic zone; small arrowheads, TUNEL expression (green fluorescence); arrows, co-localization of TRP1 and TUNEL (yellow fluorescence); DP, dermal papilla; HM, hair matrix; IRS, inner root sheath; ORS, outer root sheath. Scale bar: 50 µm.

become more degraded along their way to the skin surface, as evidenced by the dust-like appearance of the melanin granules at the infundibulum; however, in the DP, melanin was observed only rarely, even in hair bulbs exhibiting ectopic melanin. Histologically normal-appearing MC were also present at 6 d postCYP injection, even though massive keratinocyte apoptosis occurred in their direct vicinity. Unexpectedly, MC apoptosis could not be unequivocally demonstrated during the early stages of CYP-induced dystrophic catagen. Some apoptotic cells were seen that contained vesicles reminiscent of premelanosomes, yet lacked the lamellar matrix characteristic of stage II premelanosomes (Fig 3d). MC remained intact and, remarkably, even retained their dendritic phenotype. Moreover they were still actively engaged in melanogenesis, as evidenced by the presence of many melanosomes in early stages of maturation, as had already been seen 24 h after CYP treatment. This indicates that keratinocyte apoptosis predated MC damage, although the latter may eventually occur, as evidenced by the presence of rare apoptotic cells and apoptotic bodies containing premelanosome-like vesicles (Fig 3d). None of these ultrastructural pigmentary abnormalities described above were observed in vehicle-treated control follicles (not shown). DISCUSSION This study has shown that murine HF MC undergo significant catagenassociated changes that affect their distribution, morphology, and survival. Furthermore, spontaneous and DEX-induced catagen both resulted in similar morphologic changes of HF MC during early catagen development. Thus, DEX-induced catagen, with its maximum yield of catagen follicles, may act as an alternative model for hair pigmentation research. Melanin incontinence was a feature of HF during early catagen and this was moderately increased in DEX-treated mice. Removal of melanin from regressing hair bulbs occurred via the DP and by a previously unreported subpopulation of ORS keratinocytes. The location of these cells in the mid and upper ORS strongly suggests that these cells are migratory. Furthermore, these cells were in most cases closely associated with the CTS, suggesting that these melanincontaining keratinocytes migrated along this route rather than via the hair canal. In this way, this form of melanin incontinence differs from the removal of extracellular melanin ‘‘dust’’ seen in CYP-treated HF, where the hair canal does appear to act as a route for ectopic melanin removal. This was not a feature of either spontaneous or DEXinduced catagen.

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We also provide the first evidence that HF melanotic MC can be deleted by apoptosis as evaluated using well-described morphologic features (Wyllie, 1981), by TUNEL staining (Lindner et al, 1997), and by cell death without necrosis-associated immune cell infiltrate (Searle et al, 1982). This form of cell death was again more widely seen in DEX-treated HF. The melanocytic identity of the apoptosing cells and apoptotic bodies/fragments was indicated by the presence of melanosomes (in all stages of maturation) distributed free in the cytoplasm, rather than grouped within phagolysosomes or within delimiting membranes as occurs after transfer to keratinocytes, and by the concomitant absence of epithelial specializations in these cells. By contrast, keratinocytes undergoing apoptosis in the regressing bulb commonly contained tonofilament bundles and no premelanosomes. The reduction and loss of MC was confirmed by the observation that the preponderance of cells within catagen hair bulbs was desmosomally linked, indicative of an epithelial origin that contrasted strikingly with the anagen hair matrix, which contains many MC (1:5 ratio to matrix keratinocytes). Also, the presence of melanosomes in early stages of maturation within apoptosing cells is highly indicative that the originating cells were functionally active MC undergoing apoptosis, as pigment donation to precortical keratinocytes is restricted to mature stage III/ IV melanosomes (Prunerias, 1969; Cesarini, 1990). The colocalization of TUNEL and TRP-I positivity in the same cells and the observation that TRP-1 positivity is restricted to stage I/II premelanosomes1 provides further evidence that these apoptosing cells in the regressing hair bulb are indeed MC. Delivery of premelanosomes or unmelanized melanosomes to keratinocytes is restricted to disease states, including albinism (Parakkal, 1967). The MC effects associated with CYP-induced dystrophic catagen differed strikingly from both spontaneous and DEX-induced catagen. Widespread keratinocyte apoptosis occurred prior to any morphologically apparent defects in the MC population. Similar CYP-induced HF keratinocyte death by apoptosis in the C57BL/6 murine model for hair research has been previously reported (Lindner et al, 1997). Although the pigmentary unit was adversely affected, it was somewhat surprising that evidence of MC cell death after CYP treatment was inconclusive suggesting that MC may be more resistant to druginduced damage than HF keratinocytes; however, this may reflect the greater sensitivity of proliferating cells to CYP cytotoxicity. In any case, although keratinocyte loss predated MC loss, it is not clear that MC loss is immediately dependent on the primary loss of keratinocytes. Early catagen HF, however, showed signs of significant melanin incontinence with ectopic deposition of pigment in sites both inside and outside the HF. Interestingly, significant melanin incontinence and clumping is also a feature of alopecia areata, although in this disease melanin is usually restricted to the DP and proximal connective tissue sheath stalk and evidence of MC damage is common (Tobin et al, 1990, 1991). By contrast, the loss of pigmentation in miniaturizing HF in androgenetic alopecia may reflect an androgen-stimulated regression of the HF MC pool. This may occur via cell death, although this is unlikely given the protracted nature of the change in this condition, and instead is more likely to result from reduced MC division due to inadequate keratinocyte support. The striking loss of morphologically identifiable MC from the hair bulb after catagen II (Fig 1) has previously been ascribed to the loss of the differentiated MC phenotype, as only nondendritic, dopanegative MC that lacked melanosomes were observed in the telogen follicle (Sugiyama, 1979). Sugiyama proposed that these cells redifferentiate during the subsequent anagen phase. One of the major problems with this hypothesis is that, to date, no evidence has ever been provided for the de-differentiation in nonmalignant MC in any system. In addition, irradiation studies (Potten, 1972) and electron microscopic studies (Sugiyama and Kukita, 1976) have suggested that resting telogen HF may contain ‘‘precursor’’ MC. The cells were identified as MC on the basis of ultrastructural features including

1Jimbow K, Gomez PF, Chen H, Matsusaka H, Miura S: Involvement of rab 7 and phosphatidyl inositol 3-kinase for vesicular transport of TRP-1 between TGN and melanosome. J Invest Dermatol 110:473, 1998 (abstr.)

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the lack of epithelial specializations and occasionally premelanosome granules. Possibly these cells were part of the ORS pool of amelanotic, fairly undifferentiated MC rather than being de-differentiated MC originally located in the hair matrix. Teleologically, the loss of terminally differentiated hair bulb MC by apoptosis during catagen or telogen, when much of the HF epithelium undergoes apoptosis-driven involution, would not result in the permanent loss of follicle pigmentary units, if such ‘‘precursor’’ populations were in place. The subsequent anagen bulb would then be re-populated upon the activation, migration, and differentiation of MC from the precursor pool. Support for the existence of a follicular MC reservoir in the lower HF comes from the repigmentation of vitiligo skin after transplantation of hair bulbs only (Arroyo et al, 1994) and the outgrowth of small, bipolar, and amelanotic MC from human hair bulb explants in culture (Tobin et al, 1995; Tobin and Bystryn, 1996). The existence for a MC reservoir in the HF has important implications for dermatology. Indeed, these cells are likely to replenish the epidermis during the repigmentation of vitiligo (Cui and Shen Wang, 1991; Arroyo et al, 1994), after burns and dermabrasion (Montagna and Chase, 1956; Starrico, 1961), and after hair bulb MC destruction in alopecia areata (Tobin et al, 1990, 1991). Furthermore, recent in vitro studies have indicated that the amelanotic MC in the ORS of the anagen HF have considerable proliferative potential and can pigment in culture (Tobin et al, 1995; Tobin and Bystryn, 1996). By contrast, the differentiated hair matrix MC could not be grown successfully using this culture system, suggesting that these cells may be terminally differentiated. Thus, amelanotic ORS MC may serve as a MC reservoir in the HF for both the epidermis and the new anagen bulb. It is likely that the cessation of pigment donation to precortical keratinocytes is a highly controlled event and that residual pigment formed after this point by quiescent hair bulb MC is removed from the HF via the DP, resulting in the ‘‘pigment incontinence’’ seen in normal catagen. There is also some evidence that Langerhans cells may be involved to some extent in the removal of unused melanin from regressing HF matrix to the DP in catagen (Tobin, 1998). A surprising feature of spontaneous catagen and drug-induced catagen was the detection of pigment within the phagolysosomes of a subpopulation of undifferentiated ORS keratinocytes. The distribution of these cells in the mid and upper HF indicates that these cells are migratory as they contained pigment originating in the anagen hair bulb. This finding runs contrary to the current dogma that melanin is transferred only to precortical keratinocytes and provides further insight into ORS keratinocyte movement during the hair growth cycle. The location of these melanin-containing cells is unusual and suggests that the DP is not the only route for the clearance of ‘‘unused’’ pigment from the anagen HF. Although the phagocytic potential of keratinocytes has been described before (Weedon and Strutton, 1981), this usually occurs in the context of the removal of apoptotic cells during catagen. CYP-induced catagen HF exhibited pigment within dermal melanophages and epidermal keratinocytes. The presence of pigment in the epidermis was a unexpected finding given that the epidermis in murine truncal skin is devoid of functioning MC. Thus, pigment derived originally from the hair bulb may be removed from the skin via the epidermis. Melanin not yet incorporated into the hair shaft was also found degraded in the follicular canal from where it was extruded from the skin as melanin ‘‘dust’’. As this melanin was extracellular, however, it appeared to be extruded from cells (e.g., after loss of inner root sheath) into the hair canal. In this way it is different to melanin within the phagolysosomes of migratory ORS keratinocytes or in the basal layer of the epidermis. This suggests that these two methods of melanin removal are distinct. It has long been observed that melanin synthesis in the HF only occurs when a hair fiber is being actively produced, although this coupling of melanogenesis with anagen depends on the differential expression and activity of various melanogenesis-related proteins, it is still unclear how HF melanogenesis is controlled (Fitzpatrick et al, 1971; Ortonne and Prota, 1993; Slominski and Paus, 1993). How the fairly well-synchronized entry of murine anagen VI follicles into catagen is controlled is still basically unknown, but is likely to be the net result of complex interactions between catagen-inducing and

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

catagen-suppressing factors (Paus, 1996). Specifically, FGF5, FGF receptor expression, TGFβ, PTHrp, and a decline in IGF-1 may be important elements in the control of catagen development (Philpott et al, 1994; Seiberg et al, 1995; Rosenquist and Martin, 1996; Paus et al, 1997; Schilli et al, 1997). It now needs to be dissected which of these factors, if any, are responsible for switching off melanogenesis, and for inducing (or failing to suppress) HF MC apoptosis in situ. Again, the C57BL/6 mouse model appears ideally suited to address this problem.

The support of Prof. T.G. Baker, and the excellent technical assistance of R. Pliet are gratefully acknowledged. The authors wish to thank Dr. V. Hearing (NIH, Bethesda) for the generous gift of anti-TRP-1 antibody. This study was supported in part by departmental funds and by a grant from Wella AG, Darmstadt to DT, as well as by grants from Deutsche Forschungsgemeinschaft (Pa 345/6–1) and Deutsche Krebshilfe to RP.

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