- Email: [email protected]
Human beta defensin-1 regulates the development of tight junctions in cultured human epidermal keratinocytes
Letters to the Editor / Journal of Dermatological Science 71 (2013) 138–152
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focused on the role of MIA and integrins. The presence of MIA in nonmalignant melanocytes was unexpected, as normal melanocytes do not express this molecule. We indicate a novel pathogenetic mechanism for NSV (Fig. 1b). The normal attachment of melanocytes to the basal membrane mediated by a5b1ints is perturbed by MIA. This first pathogenetic step is mandatory for disease development (the ‘‘priming factor’’) and subsequently secondary pathogenetic stimuli, notably physical trauma, oxidative stress or autoantibodies, may lead to exfoliation of pigmented cells. This model may explain the lack of clinical signs of inflammation in vitiligoid lesions and the prevalence of achromic patches in well-defined anatomic sites. According to our hypothesis, melanocytes are not destroyed by the immune system, but they simply leave the scene silently. Melanocytes detach from the basal membrane toward the stratum corneum and exfoliate together with the surrounding keratinocytes. This process is consistent with the absence of an inflammatory response. Our findings demonstrate that MIA protein is present in non-segmental vitiligo skin and may cause the detachment of melanocytes, leading to the formation of achromic patches in response to various stimuli and without promoting any tissue inflammation. As observed in malignant melanoma, MIA target in vitiligo is represented by a5b1int, determining the breaking and/or weakening of connections among melanocytes and basal membrane. Therefore, MIA could represent a plausible molecule for NSV targeted therapies.
References
Acknowledgements
*Corresponding author at: Dermatology Unit, University of Padua, Via C. Battisti 206, 35124 Padova, Italy. Tel.: +39 349 4777195; fax: +39 0424 228369 E-mail address: [email protected] (M. Bordignon)
The authors thank Mrs E. Baliello and A. Dubrovich for skillful technical assistance and Dr. A.P. De Cata e Mr. V.D.P. Bordignon for supporting our work. This work was supported by a Research Grant of University of Padua 60A07-2538/07, 60A07-4234/08 and CPDR099073, CPDA108809.
[1] Alikhan A, Felsten LM, Daly M, Petronic-Rosic V. Vitiligo: a comprehensive overview Part I. Introduction, epidemiology, quality of life, diagnosis, differential diagnosis, associations, histopathology, etiology, and work-up. J Am Acad Dermatol 2011;65:473–91. [2] Ezzedine K, Lim HW, Suzuki T, Katayama I, Hamzavi I, Lan CC, et al. Revised classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference. Pigment Cell Melanoma Res 2012;25:E1–3. [3] Cario-Andre’ M, Pain C, Gauthier Y, Taı¨eb A. The melanocytorrhagic hypothesis of vitiligo tested on pigmented, stressed, reconstructed epidermis. Pigment Cell Melanoma Res 2007;20:385–93. [4] Gauthier Y, Cario-Andre M, Lepreux S, Pain C, Taieb A. Melanocyte detachment after skin friction in non lesional skin of patients with generalized vitiligo. Br J Dermatol 2003;148:95–101. [5] Fukunaga-Kalabis M, Martinez G, Liu ZJ, Kalabis J, Mrass P, Weninger W, et al. CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1. J Cell Biol 2006;175:563–9. [6] Hara M, Yaar M, Tang A, Eller MS, Reenstra W, Gilchrest BA. Role of integrins in melanocyte attachment and dendricity. J Cell Sci 1994;107:2739–48. [7] Bosserhoff AK, Stoll R, Sleeman JP, Bataille F, Buettner R, Holak TA. Active detachment involves inhibition of cell-matrix contacts of malignant melanoma cells by secretion of melanoma inhibitory activity. Lab Invest 2003;83:1583–94. [8] Bauer R, Humphries M, Fassler R, Winklmeier A, Craig SE, Bosserhoff AK. Regulation of integrin activity by MIA. J Biol Chem 2006;281:11669–77.
Matteo Bordignona,*, Chiara Castellanib, Marny Fedrigob, Gaetano Thieneb, Andrea Pesericoa, Mauro Alaibaca, Annalisa Angelinib a Dermatology Unit, University of Padua, Padova, Italy; b Pathology Unit, University of Padua, Padova, Italy
19 October 2012 http://dx.doi.org/10.1016/j.jdermsci.2013.04.005
Letter to the Editor Human beta defensin-1 regulates the development of tight junctions in cultured human epidermal keratinocytes The skin barrier is the body’s defense against infections and environmental insults. Antimicrobial peptides (AMPs) expressed in human skin, including defensins and cathelicidin, play an
important role in host defense against pathogens by killing microbes, but may also affect inflammation, angiogenesis, and wound healing [1]. Aberg et al. reported that the epidermal permeability barrier was abnormal in CRAMP (murine homolog of human cathelicidin LL-37) knock out mice, suggesting that AMPs may affect epidermal permeability [2]. As tight junctions (TJs) are a key component of the permeability barrier [3,4], we hypothesized
Fig. 1. Human beta defensin-1 (hBD-1) promotes the impermeability barrier in normal human epidermal keratinocytes (NHEKs). (A) Transepithelial electrical resistance (TER) of NHEK monolayers following hBD-1 (10 and 20 mg/ml) treatment for 24 h. (B) Quantity of permeated FITC-dextran through the NHEK monolayer exposed to hBD-1 (10 and 20 mg/ml) for 24 h. Values represent mean + SD, n = 3. *p < 0.05, vs. control.
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Fig. 2. hBD-1 promotes the expression and organization of TJ-related proteins in NHEKs. (A) Quantitative real-time PCR analysis of mRNA expression of occludin and claudin-1 in NHEKs following hBD-1 (10 and 20 mg/ml) treatment for 24 h. Values represent means + SD, n = 4. *p < 0.05, vs. control. (B) Immunofluorescence microscopy showing localization of occludin (green) and claudin-1 (purple) in NHEKs exposed to hBD-1 (10 and 20 mg/ml) for 24 h. Co-localization of occludin and claudin-1 is shown in white in the merged image (merge). Arrows indicate co-localization of claudin-1 with occludin, and arrowheads indicate claudin-1 that did not co-localized with occludin. (C) Quantitative analysis of the immunofluorescence intensity of occludin and occludin/claudin-1 merged image in NHEKs following hBD-1 (10 and 20 mg/ml) treatment for 24 h. The intensity of occludin immunofluorescence and co-localization of occludin and claudin-1 are expressed in the number of green (occludin) and white (merge) pixels, respectively. Values represent means + SD, n = 8–10 representative fields (area, 225 mm 225 mm, 262,144 pixels). *p < 0.05, vs. control.
Letters to the Editor / Journal of Dermatological Science 71 (2013) 138–152
that AMPs regulate the permeability barrier by affecting TJs. Thus, we investigated whether human beta defensin-1 (hBD-1) – an AMP expressed ubiquitously in the epidermis that is known to affect keratinocyte function – regulates the development of TJs and the formation of the epidermal permeability barrier. Cultured normal human epidermal keratinocytes (NHEKs) (Kurabo, Osaka, Japan) were cultured in low calcium (0.15 mM) keratinocyte medium (Humedia-KG2 culture medium, Kurabo) for 1 day after passing into the culture plate followed by increasing the calcium concentration to 1.45 mM. After culture in a high calcium environment for 3 days, keratinocytes were exposed to hBD-1 (Peptide Institute, Osaka, Japan) at 0, 10 or 20 mg/ml for 24 h. Transepithelial electrical resistance (TER), a measure of the permeability of water-soluble ions, of NHEKs on Transwell membranes (Corning Inc., NY, USA) was measured using a Millicell ERS volt-ohm meter (Millipore, MA, USA) after exposure to hBD-1 to investigate the effect of hBD-1 on epithelial permeability [5]. As shown in Fig. 1A, keratinocytes exposed to 20 mg/ml of hBD-1 had significantly higher TER compared with cells exposed to vehicle control. Next, paracellular permeability was assessed by examining the diffusion of FITC-dextran (Sigma–Aldrich, MO, USA) through the epithelial layer. In this assay, keratinocyte monolayers on Transwell membranes were exposed to hBD-1 for 24 h, followed by the addition of PBS with or without 1 mg/ml FITC-dextran, to the upper and lower chambers, respectively. After 3 h, the concentration of FITC-dextran in the lower chamber was quantified. Results showed that leakage of FITC-dextran was significantly lower in cell cultures treated with 20 mg/ml hBD-1 compared with those exposed to vehicle control (Fig. 1B), suggesting that hBD-1 decreases paracellular permeability in NHEKs. TER and paracellular permeability assay are routinely used to evaluate TJ-associated barrier function. To determine whether hBD-1 affects the expression of major TJ-related proteins, occludin and claudin-1, in NHEKs, the mRNA expression of these proteins in NHEKs was analyzed by quantitative real-time PCR after exposure to hBD-1. Results showed that occludin and claudin-1 mRNA expression were elevated in cells treated with 20 mg/ml hBD-1 relative to those treated with vehicle control (Fig. 2A), suggesting that the impermeability of NHEK monolayers after exposure to hBD-1 may be due to increased expression of TJ-related proteins. To confirm the relative increase in protein expression and distribution of occludin and claudin-1 in NHEKs after exposure to hBD-1, indirect immunofluorescent staining was performed using the following primary and secondary antibodies: mouse antioccludin: 1:200, rabbit anti-claudin-1: 1:60, goat anti-mouse IgG: 1:100, and goat anti-rabbit IgG: 1:125 (all purchased from Invitrogen, CA, USA). Immunostaining revealed that keratinocyte monolayers exposed to hBD-1 had a more developed network of occludin along the intercellular borders. Claudin-1 was colocalized with occludin in cell monolayers exposed to 20 mg/ml of hBD-1, but claudin-1 only partially co-localized with occludin in monolayers exposed to vehicle control (Fig. 2B). This was also supported by quantitative analysis (Fig. 2C). The intensity of the fluorescence of occludin and claudin-1 in NHEKs treated with or without hBD-1 was determined by using ImageJ 1.46 software, which can quantify the intensity of the signal as the count of pixel [6]. The results demonstrated that hBD-1 increased the intensity of occludin and occludin/claudin-1 merged linear arrangements in NHEKs (Fig. 2C), although that of claudin-1 was not significantly altered by hBD-1 treatment (data not shown). These findings suggest that hBD-1 promotes the organization of TJ-related proteins in NHEKs. AMPs such as defensins and cathelicidins are multifunctional molecules with a central role in infection and inflammation. Despite
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the prominence of hBD-1 in the human epidermis, relatively little is known about its non-antimicrobial activities. In this study, we found that hBD-1 promoted the impermeability of keratinocyte monolayers by regulating the maturation of TJs. Permeability of the epidermis is not only regulated by the intercellular lipid layer of the stratum corneum (SC), but also by TJs – intercellular junctions that regulate the flux of water, solutes and pathogens [3,4]. Recent evidence suggests that TJs are related to the process of SC maturation by regulating lamellar body (LB) secretory system, calcium ion gradient and differentiation [7,8]. Several studies, on the other hand, suggest that AMPs and epidermal permeability barrier are closely linked [2,9,10]. Aberg et al. showed that CRAMP knock out mice display a significant delay in skin barrier recovery after tape-stripping, as well as abnormalities in the LB secretory system [2]. Our previous study also suggested that secretion of LB is dependent upon functional TJs [7]. Taking these results into consideration, the development of TJs might be disturbed in CRAMP knock out mice, involving in the abnormalities in skin barrier recovery and LB secretory system. Ahrens et al. also reported that expression of murine beta defensins (mBDs) was increased following barrier disruption, and artificial barrier repair by occlusion canceled the increase in mBDs expression [9]. Based on these studies and our present study, we speculate that the upregulation of mBDs, which are well known to have defense activities against pathogens, in the epidermis following barrier disruption might repair the disrupted barrier by promoting the development of TJs. Further studies are needed to clarify the significance of our findings in which hBD-1 promote the development of TJs. In conclusion, we found that hBD-1 increased epithelial impermeability of cultured NHEK monolayers by promoting the development of TJs. Our findings proved new insights into the relationship between AMPs and the epidermal permeability barrier. Funding source This work was funded by POLA Chemical Industries, Inc. Acknowledgements We thank Kazuko Nishikawa for her technical assistance. References [1] Nakatsuji T, Gallo RL. Antimicrobial peptides: old molecules with new ideas. J Invest Dermatol 2012;132:887–95. [2] Aberg KM, Man MQ, Gallo RL, Ganz T, Crumrine D, Brown BE, et al. Coregulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers. J Invest Dermatol 2008;128:917–75. [3] Kirschner N, Houdek P, Fromm M, Moll I, Brandner JM. Tight junctions form a barrier in human epidermis. Eur J Cell Biol 2010;89:839–42. [4] Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, et al. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 2002;156:1099–111. [5] Chen YH, Lu Q, Goodenough DA, Jeansonne B. Nonreceptor tyrosine kinase cYes interacts with occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 2002;13:1227–37. [6] Bronkhorst IH, Ly LV, Jordanova ES, Vrolijk J, Versluis M, Luyten GP, Jager MJ. Detection of M2-macrophages in uveal melanoma and relation with survival. Invest Ophthalmol Vis Sci 2011;52:643–50. [7] Kuroda S, Kurasawa M, Mizukoshi K, Maeda T, Yamamoto T, Oba A, et al. Perturbation of lamellar granule secretion by sodium caprate implicates epidermal tight junctions in lamellar granule function. J Dermatol Sci 2010;59:107–14. [8] Kurasawa M, Maeda T, Oba A, Yamamoto T, Sasaki H. Tight junction regulates epidermal calcium ion gradient and differentiation. Biochem Biophys Res Commun 2011;406:506–11. [9] Ahrens K, Schunck M, Podda GF, Meingassner J, Stuetz A, Schro¨der JM, et al. Mechanical and metabolic injury to the skin barrier leads to increased expression of murine b-defensin-1, -3, and -14. J Invest Dermatol 2011; 131:443–52.
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[10] Aberg KM, Radek KA, Choi EH, Kim DK, Demerjian M, Hupe M, et al. Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice. J Clin Invest 2007; 117:3339–49.
Haruka Gotoa,1,*, Maya Hongoa,1, Hiroshi Ohshimaa, Masumi Kurasawaa, Satoshi Hirakawaa, Yasuo Kitajimab a
Skin Research Department, POLA Chemical Industries, Inc., Yokohama, Japan; bKizawa Memorial Hospital, Gifu, Japan
*Corresponding author at: Skin Research Department, POLA Chemical Industries, Inc., 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812, Japan. Tel.: +81 45 826 7134; fax: +81 45 826 7249 E-mail address: [email protected] (H. Goto) 1
These authors contributed equally to this work.
13 December 2012 http://dx.doi.org/10.1016/j.jdermsci.2013.04.017
Letter to the Editor Genetic polymorphisms in the IL22 gene are associated with psoriasis vulgaris in a Japanese population
Keywords: Genetic polymorphisms; IL22 gene; Psoriasis vulgaris; Japanese population
Jikei University School of Medicine. Genomic DNA was prepared in accordance with standard protocols. We resequenced the IL22 gene regions with genomic DNA from 36 individuals and identified a total of 32 polymorphisms (Table 1). We next examined the linkage disequilibrium (LD) between identified SNPs (Fig. S1). Pairwise LD coefficients D0 and r2 were calculated among the 24 SNPs with minor allele frequencies (MAF) of greater than 5% using Haploview 4.2 (http://www.broad. mit.edu/mpg/haploview/). We selected a total of 11 tag SNPs for association studies using tagger in Haploview 4.2, and the 11 tag
To the Editor, Psoriasis vulgaris (PsV) is an inflammatory skin disease histologically characterized by epidermal hyperplasia, inflammatory cell infiltration and vascular changes in which T-lymphocytes and associated cytokines play a central role [1]. A dysregulated cutaneous immune response occurs in genetically susceptible individuals and the features of inflammation are characterized by tumor necrosis factor (TNF)-a dependence and exaggerated helper T cell 1 (Th1) and 17 (Th17) activation. Interleukin (IL)-22 is an IL10 family cytokine member produced by Th17 cells and plays a role in the promotion of inflammation and tissue repair at barrier surfaces [2]. IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation [3], and circulating IL-22 levels are significantly higher in psoriatic patients than in normal subjects [4,5]. Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease that is basically considered to be a Th-2 type disease. However, a recent study suggests a possible role of Th17 cells in AD [6]. The study has shown that the number of Th17 cells is increased in the peripheral blood and acute lesional skin of AD and that IL-17 and IL-22 synergistically enhance the production of IL-8 from keratinocytes [6]. Since there are few genetic studies of the polymorphisms of IL22 in populations of Asian and European ancestry, we conducted association studies to assess whether IL22 gene variants contribute to the susceptibility to PsV or AD in a Japanese population. We recruited a total of 236 patients with PsV (mean age 53, 11– 85 years, male:female ratio = 1.0:2.8), and all subjects were diagnosed by clinical and histopathological findings. A total of 916 patients with AD (mean age 30, 3–77 years, male:female ratio = 1.0:2.2) and 844 controls (mean age 50, 20–75 years, male:female ratio = 1.0:1.3) were recruited as described [7]. Patients with AD were diagnosed according to the criteria of Hanifin and Rajka, and control subjects were never diagnosed with AD or PsV. All individuals were unrelated Japanese and gave written informed consent to participate in the study. The study was approved by the ethical committees at the Institute of Physical and Chemical Research (RIKEN), the University of Tokyo and the
Table 1 Frequencies of polymorphisms of the IL22 gene in a Japanese population.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 a
SNPa
Allele
Location
2479 2378 2375 2161 1905 1810 1588 1536 1394 1114 1113 1089 1075 948 701 485 201 393 708 1254 1366 1945 2178 2385 2449 2611 3270 3531 3635 5301 5433 5697
T/C C/T T/C G/A A/G G/A T/A C/T T/C C/T C/T AT/del AT repeat T/A C/T C/T A/G T/A A/G A/C G/T G/C G/C T/C C/A T/A C/A A/G T/C A/T CT/del A/T
50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking Intron 1 Intron 2 Intron 3 Intron 3 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 30 -Flanking 30 -Flanking 30 -Flanking
region region region region region region region region region region region region region region region region region
region region region
MAFb
NCBIc
0.319 0.278 0.014 0.319 0.278 0.319 0.292 0.028 0.431 0.111 0.278 0.000 0.000 0.292 0.111 0.278 0.014 0.264 0.278 0.375 0.028 0.014 0.361 0.097 0.097 0.278 0.278 0.278 0.014 0.097 0.014 0.444
rs57947370 rs11177135 rs77156535 rs7139027 rs2227472 rs2227473d rs2227476d rs2227477 rs2227478d rs2227480 rs2227481 rs35774195 rs10699698 rs2227483 rs2227484d rs2227485d rs141972126 rs17224704d rs2227491 rs2046068d rs3782552 rs1179251d rs1179250d rs1179249 rs1012356 rs2227501 rs2227503 rs976748 rs2227508d rs1182844d
Numbering according to the genomic sequence of IL-22 (NC_000012.11). Position 1 is the A of the initiation codon. b MAF (minor allele frequencies) in the screening population (N = 36). c NCBI, number from the dbSNP of NCBI (http://www.ncbi.nlm.nih.gov/SNP/). d SNPs were genotyped in this study. Genotyping of the 11 SNPs in IL-22 were performed by the TaqManTM allele-specific amplification (TaqMan-ASA) method (Applied Biosystems) and multiplex-PCR based Invader assay (Third Wave Technologies).
145
focused on the role of MIA and integrins. The presence of MIA in nonmalignant melanocytes was unexpected, as normal melanocytes do not express this molecule. We indicate a novel pathogenetic mechanism for NSV (Fig. 1b). The normal attachment of melanocytes to the basal membrane mediated by a5b1ints is perturbed by MIA. This first pathogenetic step is mandatory for disease development (the ‘‘priming factor’’) and subsequently secondary pathogenetic stimuli, notably physical trauma, oxidative stress or autoantibodies, may lead to exfoliation of pigmented cells. This model may explain the lack of clinical signs of inflammation in vitiligoid lesions and the prevalence of achromic patches in well-defined anatomic sites. According to our hypothesis, melanocytes are not destroyed by the immune system, but they simply leave the scene silently. Melanocytes detach from the basal membrane toward the stratum corneum and exfoliate together with the surrounding keratinocytes. This process is consistent with the absence of an inflammatory response. Our findings demonstrate that MIA protein is present in non-segmental vitiligo skin and may cause the detachment of melanocytes, leading to the formation of achromic patches in response to various stimuli and without promoting any tissue inflammation. As observed in malignant melanoma, MIA target in vitiligo is represented by a5b1int, determining the breaking and/or weakening of connections among melanocytes and basal membrane. Therefore, MIA could represent a plausible molecule for NSV targeted therapies.
References
Acknowledgements
*Corresponding author at: Dermatology Unit, University of Padua, Via C. Battisti 206, 35124 Padova, Italy. Tel.: +39 349 4777195; fax: +39 0424 228369 E-mail address: [email protected] (M. Bordignon)
The authors thank Mrs E. Baliello and A. Dubrovich for skillful technical assistance and Dr. A.P. De Cata e Mr. V.D.P. Bordignon for supporting our work. This work was supported by a Research Grant of University of Padua 60A07-2538/07, 60A07-4234/08 and CPDR099073, CPDA108809.
[1] Alikhan A, Felsten LM, Daly M, Petronic-Rosic V. Vitiligo: a comprehensive overview Part I. Introduction, epidemiology, quality of life, diagnosis, differential diagnosis, associations, histopathology, etiology, and work-up. J Am Acad Dermatol 2011;65:473–91. [2] Ezzedine K, Lim HW, Suzuki T, Katayama I, Hamzavi I, Lan CC, et al. Revised classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference. Pigment Cell Melanoma Res 2012;25:E1–3. [3] Cario-Andre’ M, Pain C, Gauthier Y, Taı¨eb A. The melanocytorrhagic hypothesis of vitiligo tested on pigmented, stressed, reconstructed epidermis. Pigment Cell Melanoma Res 2007;20:385–93. [4] Gauthier Y, Cario-Andre M, Lepreux S, Pain C, Taieb A. Melanocyte detachment after skin friction in non lesional skin of patients with generalized vitiligo. Br J Dermatol 2003;148:95–101. [5] Fukunaga-Kalabis M, Martinez G, Liu ZJ, Kalabis J, Mrass P, Weninger W, et al. CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1. J Cell Biol 2006;175:563–9. [6] Hara M, Yaar M, Tang A, Eller MS, Reenstra W, Gilchrest BA. Role of integrins in melanocyte attachment and dendricity. J Cell Sci 1994;107:2739–48. [7] Bosserhoff AK, Stoll R, Sleeman JP, Bataille F, Buettner R, Holak TA. Active detachment involves inhibition of cell-matrix contacts of malignant melanoma cells by secretion of melanoma inhibitory activity. Lab Invest 2003;83:1583–94. [8] Bauer R, Humphries M, Fassler R, Winklmeier A, Craig SE, Bosserhoff AK. Regulation of integrin activity by MIA. J Biol Chem 2006;281:11669–77.
Matteo Bordignona,*, Chiara Castellanib, Marny Fedrigob, Gaetano Thieneb, Andrea Pesericoa, Mauro Alaibaca, Annalisa Angelinib a Dermatology Unit, University of Padua, Padova, Italy; b Pathology Unit, University of Padua, Padova, Italy
19 October 2012 http://dx.doi.org/10.1016/j.jdermsci.2013.04.005
Letter to the Editor Human beta defensin-1 regulates the development of tight junctions in cultured human epidermal keratinocytes The skin barrier is the body’s defense against infections and environmental insults. Antimicrobial peptides (AMPs) expressed in human skin, including defensins and cathelicidin, play an
important role in host defense against pathogens by killing microbes, but may also affect inflammation, angiogenesis, and wound healing [1]. Aberg et al. reported that the epidermal permeability barrier was abnormal in CRAMP (murine homolog of human cathelicidin LL-37) knock out mice, suggesting that AMPs may affect epidermal permeability [2]. As tight junctions (TJs) are a key component of the permeability barrier [3,4], we hypothesized
Fig. 1. Human beta defensin-1 (hBD-1) promotes the impermeability barrier in normal human epidermal keratinocytes (NHEKs). (A) Transepithelial electrical resistance (TER) of NHEK monolayers following hBD-1 (10 and 20 mg/ml) treatment for 24 h. (B) Quantity of permeated FITC-dextran through the NHEK monolayer exposed to hBD-1 (10 and 20 mg/ml) for 24 h. Values represent mean + SD, n = 3. *p < 0.05, vs. control.
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Fig. 2. hBD-1 promotes the expression and organization of TJ-related proteins in NHEKs. (A) Quantitative real-time PCR analysis of mRNA expression of occludin and claudin-1 in NHEKs following hBD-1 (10 and 20 mg/ml) treatment for 24 h. Values represent means + SD, n = 4. *p < 0.05, vs. control. (B) Immunofluorescence microscopy showing localization of occludin (green) and claudin-1 (purple) in NHEKs exposed to hBD-1 (10 and 20 mg/ml) for 24 h. Co-localization of occludin and claudin-1 is shown in white in the merged image (merge). Arrows indicate co-localization of claudin-1 with occludin, and arrowheads indicate claudin-1 that did not co-localized with occludin. (C) Quantitative analysis of the immunofluorescence intensity of occludin and occludin/claudin-1 merged image in NHEKs following hBD-1 (10 and 20 mg/ml) treatment for 24 h. The intensity of occludin immunofluorescence and co-localization of occludin and claudin-1 are expressed in the number of green (occludin) and white (merge) pixels, respectively. Values represent means + SD, n = 8–10 representative fields (area, 225 mm 225 mm, 262,144 pixels). *p < 0.05, vs. control.
Letters to the Editor / Journal of Dermatological Science 71 (2013) 138–152
that AMPs regulate the permeability barrier by affecting TJs. Thus, we investigated whether human beta defensin-1 (hBD-1) – an AMP expressed ubiquitously in the epidermis that is known to affect keratinocyte function – regulates the development of TJs and the formation of the epidermal permeability barrier. Cultured normal human epidermal keratinocytes (NHEKs) (Kurabo, Osaka, Japan) were cultured in low calcium (0.15 mM) keratinocyte medium (Humedia-KG2 culture medium, Kurabo) for 1 day after passing into the culture plate followed by increasing the calcium concentration to 1.45 mM. After culture in a high calcium environment for 3 days, keratinocytes were exposed to hBD-1 (Peptide Institute, Osaka, Japan) at 0, 10 or 20 mg/ml for 24 h. Transepithelial electrical resistance (TER), a measure of the permeability of water-soluble ions, of NHEKs on Transwell membranes (Corning Inc., NY, USA) was measured using a Millicell ERS volt-ohm meter (Millipore, MA, USA) after exposure to hBD-1 to investigate the effect of hBD-1 on epithelial permeability [5]. As shown in Fig. 1A, keratinocytes exposed to 20 mg/ml of hBD-1 had significantly higher TER compared with cells exposed to vehicle control. Next, paracellular permeability was assessed by examining the diffusion of FITC-dextran (Sigma–Aldrich, MO, USA) through the epithelial layer. In this assay, keratinocyte monolayers on Transwell membranes were exposed to hBD-1 for 24 h, followed by the addition of PBS with or without 1 mg/ml FITC-dextran, to the upper and lower chambers, respectively. After 3 h, the concentration of FITC-dextran in the lower chamber was quantified. Results showed that leakage of FITC-dextran was significantly lower in cell cultures treated with 20 mg/ml hBD-1 compared with those exposed to vehicle control (Fig. 1B), suggesting that hBD-1 decreases paracellular permeability in NHEKs. TER and paracellular permeability assay are routinely used to evaluate TJ-associated barrier function. To determine whether hBD-1 affects the expression of major TJ-related proteins, occludin and claudin-1, in NHEKs, the mRNA expression of these proteins in NHEKs was analyzed by quantitative real-time PCR after exposure to hBD-1. Results showed that occludin and claudin-1 mRNA expression were elevated in cells treated with 20 mg/ml hBD-1 relative to those treated with vehicle control (Fig. 2A), suggesting that the impermeability of NHEK monolayers after exposure to hBD-1 may be due to increased expression of TJ-related proteins. To confirm the relative increase in protein expression and distribution of occludin and claudin-1 in NHEKs after exposure to hBD-1, indirect immunofluorescent staining was performed using the following primary and secondary antibodies: mouse antioccludin: 1:200, rabbit anti-claudin-1: 1:60, goat anti-mouse IgG: 1:100, and goat anti-rabbit IgG: 1:125 (all purchased from Invitrogen, CA, USA). Immunostaining revealed that keratinocyte monolayers exposed to hBD-1 had a more developed network of occludin along the intercellular borders. Claudin-1 was colocalized with occludin in cell monolayers exposed to 20 mg/ml of hBD-1, but claudin-1 only partially co-localized with occludin in monolayers exposed to vehicle control (Fig. 2B). This was also supported by quantitative analysis (Fig. 2C). The intensity of the fluorescence of occludin and claudin-1 in NHEKs treated with or without hBD-1 was determined by using ImageJ 1.46 software, which can quantify the intensity of the signal as the count of pixel [6]. The results demonstrated that hBD-1 increased the intensity of occludin and occludin/claudin-1 merged linear arrangements in NHEKs (Fig. 2C), although that of claudin-1 was not significantly altered by hBD-1 treatment (data not shown). These findings suggest that hBD-1 promotes the organization of TJ-related proteins in NHEKs. AMPs such as defensins and cathelicidins are multifunctional molecules with a central role in infection and inflammation. Despite
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the prominence of hBD-1 in the human epidermis, relatively little is known about its non-antimicrobial activities. In this study, we found that hBD-1 promoted the impermeability of keratinocyte monolayers by regulating the maturation of TJs. Permeability of the epidermis is not only regulated by the intercellular lipid layer of the stratum corneum (SC), but also by TJs – intercellular junctions that regulate the flux of water, solutes and pathogens [3,4]. Recent evidence suggests that TJs are related to the process of SC maturation by regulating lamellar body (LB) secretory system, calcium ion gradient and differentiation [7,8]. Several studies, on the other hand, suggest that AMPs and epidermal permeability barrier are closely linked [2,9,10]. Aberg et al. showed that CRAMP knock out mice display a significant delay in skin barrier recovery after tape-stripping, as well as abnormalities in the LB secretory system [2]. Our previous study also suggested that secretion of LB is dependent upon functional TJs [7]. Taking these results into consideration, the development of TJs might be disturbed in CRAMP knock out mice, involving in the abnormalities in skin barrier recovery and LB secretory system. Ahrens et al. also reported that expression of murine beta defensins (mBDs) was increased following barrier disruption, and artificial barrier repair by occlusion canceled the increase in mBDs expression [9]. Based on these studies and our present study, we speculate that the upregulation of mBDs, which are well known to have defense activities against pathogens, in the epidermis following barrier disruption might repair the disrupted barrier by promoting the development of TJs. Further studies are needed to clarify the significance of our findings in which hBD-1 promote the development of TJs. In conclusion, we found that hBD-1 increased epithelial impermeability of cultured NHEK monolayers by promoting the development of TJs. Our findings proved new insights into the relationship between AMPs and the epidermal permeability barrier. Funding source This work was funded by POLA Chemical Industries, Inc. Acknowledgements We thank Kazuko Nishikawa for her technical assistance. References [1] Nakatsuji T, Gallo RL. Antimicrobial peptides: old molecules with new ideas. J Invest Dermatol 2012;132:887–95. [2] Aberg KM, Man MQ, Gallo RL, Ganz T, Crumrine D, Brown BE, et al. Coregulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers. J Invest Dermatol 2008;128:917–75. [3] Kirschner N, Houdek P, Fromm M, Moll I, Brandner JM. Tight junctions form a barrier in human epidermis. Eur J Cell Biol 2010;89:839–42. [4] Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, et al. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 2002;156:1099–111. [5] Chen YH, Lu Q, Goodenough DA, Jeansonne B. Nonreceptor tyrosine kinase cYes interacts with occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 2002;13:1227–37. [6] Bronkhorst IH, Ly LV, Jordanova ES, Vrolijk J, Versluis M, Luyten GP, Jager MJ. Detection of M2-macrophages in uveal melanoma and relation with survival. Invest Ophthalmol Vis Sci 2011;52:643–50. [7] Kuroda S, Kurasawa M, Mizukoshi K, Maeda T, Yamamoto T, Oba A, et al. Perturbation of lamellar granule secretion by sodium caprate implicates epidermal tight junctions in lamellar granule function. J Dermatol Sci 2010;59:107–14. [8] Kurasawa M, Maeda T, Oba A, Yamamoto T, Sasaki H. Tight junction regulates epidermal calcium ion gradient and differentiation. Biochem Biophys Res Commun 2011;406:506–11. [9] Ahrens K, Schunck M, Podda GF, Meingassner J, Stuetz A, Schro¨der JM, et al. Mechanical and metabolic injury to the skin barrier leads to increased expression of murine b-defensin-1, -3, and -14. J Invest Dermatol 2011; 131:443–52.
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[10] Aberg KM, Radek KA, Choi EH, Kim DK, Demerjian M, Hupe M, et al. Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice. J Clin Invest 2007; 117:3339–49.
Haruka Gotoa,1,*, Maya Hongoa,1, Hiroshi Ohshimaa, Masumi Kurasawaa, Satoshi Hirakawaa, Yasuo Kitajimab a
Skin Research Department, POLA Chemical Industries, Inc., Yokohama, Japan; bKizawa Memorial Hospital, Gifu, Japan
*Corresponding author at: Skin Research Department, POLA Chemical Industries, Inc., 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812, Japan. Tel.: +81 45 826 7134; fax: +81 45 826 7249 E-mail address: [email protected] (H. Goto) 1
These authors contributed equally to this work.
13 December 2012 http://dx.doi.org/10.1016/j.jdermsci.2013.04.017
Letter to the Editor Genetic polymorphisms in the IL22 gene are associated with psoriasis vulgaris in a Japanese population
Keywords: Genetic polymorphisms; IL22 gene; Psoriasis vulgaris; Japanese population
Jikei University School of Medicine. Genomic DNA was prepared in accordance with standard protocols. We resequenced the IL22 gene regions with genomic DNA from 36 individuals and identified a total of 32 polymorphisms (Table 1). We next examined the linkage disequilibrium (LD) between identified SNPs (Fig. S1). Pairwise LD coefficients D0 and r2 were calculated among the 24 SNPs with minor allele frequencies (MAF) of greater than 5% using Haploview 4.2 (http://www.broad. mit.edu/mpg/haploview/). We selected a total of 11 tag SNPs for association studies using tagger in Haploview 4.2, and the 11 tag
To the Editor, Psoriasis vulgaris (PsV) is an inflammatory skin disease histologically characterized by epidermal hyperplasia, inflammatory cell infiltration and vascular changes in which T-lymphocytes and associated cytokines play a central role [1]. A dysregulated cutaneous immune response occurs in genetically susceptible individuals and the features of inflammation are characterized by tumor necrosis factor (TNF)-a dependence and exaggerated helper T cell 1 (Th1) and 17 (Th17) activation. Interleukin (IL)-22 is an IL10 family cytokine member produced by Th17 cells and plays a role in the promotion of inflammation and tissue repair at barrier surfaces [2]. IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation [3], and circulating IL-22 levels are significantly higher in psoriatic patients than in normal subjects [4,5]. Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease that is basically considered to be a Th-2 type disease. However, a recent study suggests a possible role of Th17 cells in AD [6]. The study has shown that the number of Th17 cells is increased in the peripheral blood and acute lesional skin of AD and that IL-17 and IL-22 synergistically enhance the production of IL-8 from keratinocytes [6]. Since there are few genetic studies of the polymorphisms of IL22 in populations of Asian and European ancestry, we conducted association studies to assess whether IL22 gene variants contribute to the susceptibility to PsV or AD in a Japanese population. We recruited a total of 236 patients with PsV (mean age 53, 11– 85 years, male:female ratio = 1.0:2.8), and all subjects were diagnosed by clinical and histopathological findings. A total of 916 patients with AD (mean age 30, 3–77 years, male:female ratio = 1.0:2.2) and 844 controls (mean age 50, 20–75 years, male:female ratio = 1.0:1.3) were recruited as described [7]. Patients with AD were diagnosed according to the criteria of Hanifin and Rajka, and control subjects were never diagnosed with AD or PsV. All individuals were unrelated Japanese and gave written informed consent to participate in the study. The study was approved by the ethical committees at the Institute of Physical and Chemical Research (RIKEN), the University of Tokyo and the
Table 1 Frequencies of polymorphisms of the IL22 gene in a Japanese population.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 a
SNPa
Allele
Location
2479 2378 2375 2161 1905 1810 1588 1536 1394 1114 1113 1089 1075 948 701 485 201 393 708 1254 1366 1945 2178 2385 2449 2611 3270 3531 3635 5301 5433 5697
T/C C/T T/C G/A A/G G/A T/A C/T T/C C/T C/T AT/del AT repeat T/A C/T C/T A/G T/A A/G A/C G/T G/C G/C T/C C/A T/A C/A A/G T/C A/T CT/del A/T
50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking 50 -Flanking Intron 1 Intron 2 Intron 3 Intron 3 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 Intron 4 30 -Flanking 30 -Flanking 30 -Flanking
region region region region region region region region region region region region region region region region region
region region region
MAFb
NCBIc
0.319 0.278 0.014 0.319 0.278 0.319 0.292 0.028 0.431 0.111 0.278 0.000 0.000 0.292 0.111 0.278 0.014 0.264 0.278 0.375 0.028 0.014 0.361 0.097 0.097 0.278 0.278 0.278 0.014 0.097 0.014 0.444
rs57947370 rs11177135 rs77156535 rs7139027 rs2227472 rs2227473d rs2227476d rs2227477 rs2227478d rs2227480 rs2227481 rs35774195 rs10699698 rs2227483 rs2227484d rs2227485d rs141972126 rs17224704d rs2227491 rs2046068d rs3782552 rs1179251d rs1179250d rs1179249 rs1012356 rs2227501 rs2227503 rs976748 rs2227508d rs1182844d
Numbering according to the genomic sequence of IL-22 (NC_000012.11). Position 1 is the A of the initiation codon. b MAF (minor allele frequencies) in the screening population (N = 36). c NCBI, number from the dbSNP of NCBI (http://www.ncbi.nlm.nih.gov/SNP/). d SNPs were genotyped in this study. Genotyping of the 11 SNPs in IL-22 were performed by the TaqManTM allele-specific amplification (TaqMan-ASA) method (Applied Biosystems) and multiplex-PCR based Invader assay (Third Wave Technologies).