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Baraniak AP, Lasda EL, Wagner EJ, Garcia-Blanco MA: A stem structure in fib- Munkvad S, Jorgensen M: Resistance to activated protein C: A common anti- roblast growth factor receptor 2 transcripts mediates cell-type-specific coagulant deficiency in patients with venous leg ulceration. J Dermatol splicing by approximating intronic control elements. Mol Cell Biol 134:296–298, 1996 23:9327–9337, 2003 Oldridge M, Zackai EH, McDonald-McGinn DM, et al: De novo Alu-element in- Bauters C, Six I, Meurice T, Van Belle E: Growth factors and endothelial dys- sertions in FGFR-2 identify a distinct pathological basis for Apert syn- function. Drugs 59:11–15, 1999 drome. Am J Hum Genet 64:471–711, 1999 Di Paola R, Frittitta L, Miscio G, et al: A variation in 30 UTR of hPTP1B increases Raccah D, Coste TC, Vague P: Genetics of diabetic complications: Peripheral specific gene expression and associates with insulin resistance. Am J neuropathy. Ann Endocrinol 65 (Suppl. 1):S5–S9, 2004 Hum Genet 70:806–812, 2002 Shan S, Robson ND, Cao Y, et al: Responses of vascular endothelial cells to Kan SH, Elanko N, Johnson D, et al: Genomic screening of fibroblast growth- angiogenic signaling are important for tumor cell survival. FASEB J factor receptor 2 reveals a wide spectrum of mutations in patients with 18:326–328, 2004 syndromic craniosynostosis. Am J Hum Genet 70:472–486, 2002 Stucker M, Harke K, Rudolph T, Altmeyer P: Pathogenesis of therapy refractory Loot MA, Kenter SB, Au FL, van Galen WJ, Middelkoop E, Bos JD, Mekkes JR: ulcus cruris. Hautarzt 54:750–755, 2003 Fibroblasts derived from chronic diabetic ulcers differ in their response to Vasilopoulos Y, Cork MJ, Murphy R, et al: Genetic association between an stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls. AACC insertion in the 30UTR of the stratum corneum chymotryptic Eur J Cell Biol 81:153–160, 2002 enzyme gene and atopic dermatitis. J Invest Dermatol 123:62–66, Maas-Szabowski N, Szabowski A, Stark HJ, et al: Organotypic cocultures with 2004 genetically modified mouse fibroblasts as a tool to dissect molecular Yeh BK, Igarashi M, Eliseenkova AV, et al: Structural basis by which alternative mechanisms regulating keratinocyte growth and differentiation. J Invest splicing confers specificity in fibroblast growth factor receptors. Proc Natl Dermatol 116:816–820, 2001 Acad Sci USA 100:2266–2271, 2003 McIntosh I, Bellus GA, Jab EW: The pleiotropic effects of fibroblast growth factor receptors in mammalian development. Cell Struct Funct 25:85–96, 2000

Increased Serum CCL28 Levels in Patients with Atopic Dermatitis, Psoriasis Vulgaris and Bullous Pemphigoid

To the Editor: homologous to CTACK/CCL27, displaying about 40% iden- tity, and is most abundant in the salivary gland, with strong Atopic dermatitis is an inflammatory skin disease that is expression in other tissues associated with mucosal epi- characterized by pruritic and eczematous lesions persisting thelial surfaces, including colon, trachea, and mammary chronically. Histopathologically, the skin lesions in atopic gland. MEC/CCL28 attracts subsets of memory lymph- dermatitis show infiltration of T lymphocytes, especially cu- ocytes and eosinophils (Pan et al, 2000). Wang et al (2000) taneous lymphocyte antigen-positive memory T cells, as showed that MEC/CCL28 mRNA was expressed predom- well as eosinophils and macrophages (Novak et al, 2003). inantly in psoriasis patient skin samples and to a lesser Previous studies revealed that increased amounts of thy- degree in healthy human skin. But MEC/CCL28 production mus and activation-regulated chemokine (CCL17) and cu- at protein levels in skin is totally unknown. Furthermore, taneous T cell-attracting chemokine (CTACK)/CCL27 were there has been no report of serum MEC/CCL28 levels in detected in the serum and lesional keratinocytes of patients patients with skin diseases. This prompted us to investigate with atopic dermatitis (Kakinuma et al, 2001, 2003). These the relationship between MEC/CCL28 and skin diseases. results suggest involvement of these chemokines in the Therefore, we examined serum MEC/CCL28 levels of pathogenesis of this disease. patients with atopic dermatitis, psoriasis vulgaris, and Psoriasis vulgaris is also a chronic and relapsing inflam- bullous pemphigoid. Furthermore, in order to determine matory skin disease characterized as a T cell-mediated au- which cells produced MEC/CCL28 in skin, we performed toimmune disorder (Chang, 1992). CTACK/CCL27 was strongly detected in the serum and lesional keratinocytes of patients with psoriasis vulgaris (Kakinuma et al, 2003). It has been proposed that interaction between T cells and epidermal keratinocytes plays a central role in the patho- genesis of psoriasis vulgaris (Chang, 1992). Bullous pemphigoid is a blistering autoimmune skin dis- ease. It often provokes lesional eosinophil infiltration (Pier- ard et al, 1961). Eotaxin/CCL11 was strongly detected in blister fluid of bullous pemphigoid and strongly expressed in keratinocytes around blisters of this disease (Wakugawa et al, 2000). Mucosae-associated epithelial chemokine (MEC)/CCL28 is a novel chemokine ligand for CC chemokine receptor (CCR) 10 and CCR3 (Pan et al, 2000). MEC/CCL28 is most Figure 1 Serum CCL28 levels. Serum mucosae-associated epithelial chemo- Abbreviations: CCR, CC chemokine receptor; CTACK, cutaneous T kine (MEC)/CCL28 levels in patients with atopic dermatitis (AD), pso- cell-attracting chemokine; MEC, Mucosae-associated epithelial riasis vulgaris (PsV), bullous pemphigoid (BP), and healthy controls chemokine (Control). Data are presented as mean standard error. 124:5 MAY 2005 LETTERS TO THE EDITOR 1089

Figure 2 Expression of CCL28 in the epidermis. Mucosae-associated epithelial chemo- kine (CCL28) was strongly expressed in the lesional epidermal keratinocytes of patients with atopic dermatitis (a) and psoriasis vulgaris (b). It was weakly ex- pressed in patients with bullous pe- mphigoid (c) and healthy controls (d). It was hardly expressed in patients with in- sect bite (e) and non-lesional skin of pa- tients with psoriasis vulgaris (f). When an isotype-matched control was used, no reactivity was observed in patients with atopic dermatitis (g)(scale bar ¼ 200 mm).

immunohistochemical staining of this chemokine in the les- not correlate with disease activity of these three conditions ional skin of patients with these diseases. (data not shown). The elevated serum MEC/CCL28 levels of Forty-eight patients with atopic dermatitis (mean patients with atopic dermatitis tended to decrease after standard deviation age: 28.7 6.7 y), 20 patients with treatments (n ¼ 6, before: 169.8 75.9 pg per mL, after: psoriasis vulgaris (28.6 6.3 y), 28 patients with bullous 65.8 22.7 pg per mL), although the change was not sig- pemphigoid (68.2 20.1 y), and 20 healthy controls nificant (data not shown). (28.5 8.4 y) were enrolled in this study. The medical eth- MEC/CCL28 was strongly expressed in epidermal kera- ical committee of the University of Tokyo approved all de- tinocytes of patients with atopic dermatitis and psoriasis scribed studies and the study was conducted according to vulgaris, and weakly expressed in epidermal keratinocytes Declaration of Helsinki Principles. Informed consent was of patients with bullous pemphigoid, insect bite, healthy obtained to use the sera and skin tissues from the patients controls, and non-lesional skin of patients with psoriasis and healthy controls. The 20 healthy controls had no history vulgaris (Fig 2). Thus, MEC/CCL28 was strongly expressed of allergy, psoriasis or bullous disease. All sera were stored only in the lesional skin of systemic skin diseases, such as at 201C until use. ELISA was performed using MEC/ atopic dermatitis and psoriasis vulgaris. CCL28 immunoassay kits obtained from R&D Systems This is the first report describing MEC/CCL28 expression (Minneapolis, Minnesota). Serum levels of MEC/CCL28 by epidermal keratinocytes in skin and elevated levels of were measured according to the manufacturer’s instruc- this chemokine in the serum of patients with some inflam- tions. Optical densities were measured at 450 nm with a matory skin diseases. Bio-Rad Model 550 microplate reader (Bio-Rad Laborato- Both CTACK/CCL27 and MEC/CCL28 attract CCR10 þ ries, Hercules, California). The concentrations were calcu- lymphocytes. The CCR10 þ lymphocytes invading the skin lated from the standard curve generated by a curve-fitting in atopic dermatitis and psoriasis have different Th1/Th2 program. Data obtained from the ELISA are presented as profiles (Vestergaard et al, 2000). Therefore, CTACK/CCL27 mean standard error. Data were analyzed using Mann– and MEC/CCL28 may contribute to the pathogenesis of Whitney’s U test and paired t test. A p-value less than 0.05 both a Th2-dominant disease like atopic dermatitis and a was considered to be statistically significant. Th1-dominant disease like psoriasis vulgaris. Biopsy specimens obtained from patients and healthy MEC/CCL28, but not CTACK/CCL27, stimulates migra- controls were used for immunohistochemical staining. To tion of CCR3 transfectants as well as eosinophils (Pan et al, detect MEC/CCL28, affinity-purified goat polyclonal anti- 2000). In this context, MEC/CCL28 may play a role, in con- human MEC/CCL28 (Dako Cytomation, Glostrup, Denmark) junction with other regulatory elements, in the recruitment of was used. Normal goat IgG1 (Santa Cruz Biotechnology, eosinophils and potentially, of rare CCR3 þ T cells into epi- Santa Cruz, California) was used as an isotype-matched dermis. MEC/CCL28 seems to be one of the important control. chemokines in the lesional formation of eosinophil infiltrat- The serum MEC/CCL28 levels of patients with atopic ing diseases like atopic dermatitis and bullous pemphigoid. dermatitis (148.9 13.6 pg per mL), psoriasis vulgaris These results suggest that MEC/CCL28 is involved in the (167.8 34.2 pg per mL), and bullous pemphigoid pathogenesis of these choronic inflammatory skin diseases (89.2 45.3 pg per mL) were significantly higher than those in cooperation with other chemokines. in healthy controls (44.7 5.9 pg per mL) (Fig 1). Because à à à patients with bullous pemphigoid were much older than the Shinji Kagami, Takashi Kakinuma, Hidehisa Saeki, Yuichiro à à à w other cohorts, it will be necessary to compare them with Tsunemi, Hideki Fujita, Kiyo Sasaki, Koichiro Nakamura, Tomonori Takekoshi,à Megumi Kishimoto,à Hiroshi Mitsui,à Mayumi Komine,à much older healthy controls in a future study in order to Akihiko Asahina,à and Kunihiko Tamakià confirm the conclusion. The serum MEC/CCL28 levels did ÃDepartment of Dermatology, Faculty of Medicine, University of Tokyo, 1090 LETTERS TO THE EDITOR THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Tokyo, Japan; wDepartment of Dermatology, Fukushima Medical Uni- Kakinuma T, Nakamura K, Wakugawa M, et al: Thymus and activation-regulated versity School of Medicine, Fukushima, Japan chemokine in atopic dermatitis: Serum thymus and activation-regulated chemokine level is closely related with disease activity. J Allergy Clin Immunol 107:535–541, 2001 This work was supported by Health Science Research Grants from the Kakinuma T, Saeki H, Tsunemi Y, et al: Increased serum cutaneous T cell-at- Ministry of Health, Labor and Welfare of Japan and by grants from tracting chemokine (CCL27) levels in patients with atopic dermatitis and the Ministry of Education, Culture, Sports, Science and Technology of psoriasis vulgaris. J Allergy Clin Immunol 111:592–597, 2003 Japan. Novak N, Bieber T, Leung DYM: Immune mechanisms leading to atopic derma- titis. J Allergy Clin Immunol 112:S128–S139, 2003 DOI: 10.1111/j.0022-202X.2005.23700.x Pan J, Kunkel EJ, Gosslar U, et al: A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues. J Immunol Manuscript received October 26, 2004; revised December 27, 2004; 165:2943–2949, 2000 accepted for publication January 3, 2005 Pierard J, Whimster I: The histological diagnosis of dermatitis herpetiformis, bullous pemphigoid and erythema multiforme. Br J Dermatol 73:253–266, Address correspondence to: Shinji Kagami, MD, Department of Der- 1961 matology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Vestergaard C, Deleuran M, Gesser B, Gronhoj Larsen C: Expression of the T- Bunkyo-ku, Tokyo 113-8655, Japan. Email: [email protected] helper 2-specific chemokine receptor CCR4 on CCR10-positive lymph- ocytes in atopic dermatitis skin but not in psoriasis skin. Br J Dermatol 149:457–463, 2000 References Wakugawa M, Nakamura K, Hino H, et al: Elevated levels of eotaxin and inter- leukin-5 in blister fluid of bullous pemphigoid: Correlation with tissue Chang EY, Hammerberg C, Fisher G, Baadsgaard O, Ellis CN, Voorhees JJ, eosinophilia. Br J Dermatol 143:112–116, 2000 Cooper KD: T-cell activation is potentiated by cytokines released by les- Wang W, Soto H, Oldham ER, et al: Identification of a novel CC chemokine ional psoriatic, but not normal, epidermis. Arch Dermatol 128:1479–1485, (CCL28) which binds CCR10 (GPR2). J Biol Chem 275:22313–22323, 1992 2000

Cultured Cells from the Adult Human Hair Follicle Dermis can be Directed Toward Adipogenic and Osteogenic Differentiation

To The Editor: adipogenic and osteogenic differentiation in human hair follicle-derived DP and DS cultures. Human scalp or beard Mounting evidence suggests that adult stem cells have follicles were isolated from skin biopsies from four individ- greater plasticity than previously thought. Among stem cells uals (aged 23,48, 89, and 91 y). DP and DS tissues were derived from mesenchymal tissues (e.g., Toma et al, 2001; dissected from amputated end bulbs as previously de- Zuk et al, 2001; Jiang et al, 2002), one population from the scribed (Reynolds et al, 1999). Briefly, amputated follicle skin dermis has received considerable prominence, owing end bulbs were inverted, and the exposed epithelial matrix to its accessibility and wide spectrum of differentiation ac- was detached. Adherent epithelial cells and debris were tivities in culture (Toma et al, 2001). removed, the DP and DS were separated and explant cul- Within the adult hair follicle dermis, two developmentally tures initiated in MEM plus 20% FBS from a minimum of 10 active cell populations, the dermal papilla (DP) and dermal isolated DP or DS samples from each biopsy. sheath (DS), have well-established instructive powers in re- Cells between passage 3 and 7 (50,000 cells per 35 mm lation to the control of hair follicle cycling and induction. plate) were allowed to adhere overnight in MEM plus 10% Although the stem cell activity of the hair follicle epithelium FBS at 371C and 5% CO2. In control dishes, cells were kept has been studied intensively (Taylor et al, 2000; Morris et al, in this medium. Others were incubated in either osteogenic 2004; Tumbar et al, 2004), until recently the follicle dermis media (MEM plus 10% FBS, 100 nM dexamethasone, 10 mM has been largely overlooked in this context. b-glycerophosphate, supplemented every 3 d with 50 mM We have previously suggested that follicle dermal cells L-ascorbate-2-phosphate) or adipogenic medium (MEM have extensive stem or progenitor cell activities within the plus 15% rabbit serum, 2.07 mM insulin, 100 nM dexa- skin, including an important role in wound healing (Jahoda methasone, supplemented with 9 mM isobutyl-methylxan- and Reynolds, 2001). Using rodent models we have dem- thine after 9 d of culture) (both modified from Zuk et al, onstrated that the follicle dermis harbors hematopoietic 2001). Cultures were maintained for 6–11 d and medium stem cell activity (Lako et al, 2002). We recently directed changed every 3–4 days. clonally derived papilla and sheath cell lines towards ad- For each of the osteogenic and adipogenic differenti- ipocyte and osteocyte phenotypes (Jahoda et al, 2003). For ation experiments, the DP and DS cell lines were estab- follicle dermal stem cells to be useful in any therapeutic lished from two different human samples. All established context, we need, however, to show that human hair follicle cell lines could be directed towards osteogenesis (five of dermal stem cells have similar capabilities. Here, we report five) and, in contrast to a previous report (Iguchi et al, 2001), adipogenesis (five of five). These differentiation events occured irrespective of the origin of the cell line or Abbreviations: DP, dermal papilla; DS, dermal sheath patient age. Interestingly, as repeat experiments were