Letters to the Editor / Journal of Dermatological Science 78 (2015) 76–85
dilution of patient sera and different manners in which the ELISA assays were performed possibly caused the discrepancies in these studies [7,8]. With regards to the disease severity of BP, correlations of the following clinical items, namely controlling dosage of prednisone, duration for remission and area of skin lesions, have been investigated with the presence or levels of IgE anti-BP180 autoantibody in four previously reported studies, while the results were not consistent [2,3,5,6]. In our present study, we did not get any positive correlation between the presence or levels of IgE antiBP180 autoantibody with the above items, though, to some extent, patients with higher IgE anti-BP180 autoantibody levels required a larger prednisone dosage, a longer duration for effective control, and had a broader area of skin lesion (Table 1). On the other hand, there was a significant difference in IgG anti-BP180 autoantibody levels between patients with positive IgE antiBP180 autoantibody and those with negative values (t = 2.734, P = 0.009), which suggests that increased IgE anti-BP180 autoantibody levels may also associate with a higher titer of IgG anti-BP180 autoantibody. Regarding correlation of IgE anti-BP180 autoantibody with laboratorial parameters, the circulating eosinophil count may be the most putative one. Two recent related studies have revealed that neither the presence of IgE anti-BP180 autoantibody nor its increased levels correlated with the circulating eosinophil count [5,6]. In this study, we observed a similar result (r = 0.297, P = 0.06 and r = 0.089, P = 0.807, respectively), though the eosinophil counts of patients with BP positive for IgE anti-BP180 autoantibody were indeed higher than those of the other patients with BP (1.08 0.94 vs. 0.74 1.09, Table 1). In addition, the increased IgE anti-BP180 autoantibody levels did not associate with eosinophils infiltration around edematous erythemas and blisterings of skin biopsy specimens (r = 0.283, P = 0.461). To clarify the possible relationship between increased circulating eosinophil count and IgE autoantibody in BP, some studies also focused on another major target antigenic site in BMZ, namely BP230. No positive correlation was found between circulating eosinophil count and IgE anti-BP230 autoantibody [5,6], though a significant correlation with circulating eosinophil count was observed in IgG anti-BP230 autoantibody [6]. So whether there is a positive relationship between circulating eosinophil count and IgE autoantibody specific for BMZ antigen need further research. In conclusion, the present study demonstrates that IgE antiBP180 autoantibody level is increased in some Chinese patients with BP and may associate with their higher titers of IgG antiBP180 autoantibody, although no significant correlations of this IgE autoantibody with various clinical and laboratorial aspects were observed.
77
Acknowledgments This work was funded by Natural Science Foundation of China (grant numbers: 81130030 and 81000694) and the Program for Changjiang Scholars and Innovative Research Team in University (grant number: LRT1003). References [1] Provost TT, Tomasi Jr TB. Immunopathology of bullous pemphigoid: basement membrane deposition of IgE, alternate pathway components and fibrin. Clin Exp Immunol 1974;18:193–200. [2] Delaporte E, Dubost-Brama A, Ghohestani R, Nicolas JF, Neyrinck JL, Bergoend H, et al. IgE autoantibodies directed against the major bullous pemphigoid antigen in patients with a severe form of pemphigoid. J Immunol 1996;157: 3642–7. [3] Hofmann S, Thoma-Uszynski S, Hunziker T, Bernard P, Koebnick C, Stauber A, et al. Severity and phenotype of bullous pemphigoid relate to autoantibody profile against the NH2- and COOH-terminal regions of the BP180 ectodomain. J Invest Dermatol 2002;119:1065–73. [4] Do¨pp R, Schmidt E, Chimanovitch I, Leverkus M, Brocker EB, Zillikens D. IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in Bullous pemphigoid: serum levels of these immunoglobulins reflect disease activity. J Am Acad Dermatol 2000;42:577–83. [5] Iwata Y, Komura K, Kodera M, Usuda T, Yokoyama Y, Hara T, et al. Correlation of IgE autoantibody to BP180 with severe form of bullous pemphigoid. Arch Dermatol 2008;144:41–8. [6] Ishiura N, Fujimoto M, Watanabe R, Nakashima H, Kuwano Y, Yazawa N, et al. Serum levels of IgE anti-BP180 and anti- BP230 autoantibodies in patients with bullous pemphigoid. J Dermatol Sci 2008;49:153–61. [7] Messingham KA, Noe MH, Chapman MA, Giudice GJ, Fairley JA. A novel ELISA reveals high frequencies of BP180-specific IgE production in bullous pemphigoid. J Immunol Methods 2009;346:18–25. [8] Sittampalam GS, Smith WC, Miyakawa TW, Smith DR, McMorris C. Application of experimental design techniques to optimize a competitive ELISA. J Immunol Methods 1996;190:151–61.
Lei Maa,b,c,1, Mingyue Wanga,b,1, Xue Wanga,b, Xixue Chena,b, Xuejun Zhua,b,* a Department of Dermatology, Peking University First Hospital, Beijing, China; bBeijing Key Laboratory of Molecular Diagnosis of Dermatoses, Beijing, China; cDepartment of Dermatology, Binzhou Medical University Hospital, Binzhou, Shandong, China *Corresponding author at: Department of Dermatology, Peking University First Hospital, 8 Xishiku Street, Xicheng District, Beijing 100034, China. Tel.: +86 10 83573075 E-mail address: [email protected] (X. Zhu). 1
These authors contributed equally to this work.
Received 12 February 2015 http://dx.doi.org/10.1016/j.jdermsci.2015.02.015
Letter to the Editor Possible correlation of IgE autoantibody to BP180 with disease activity in bullous pemphigoid
Keywords: Bullous pemphigoid; BP180 antibody; IgE; BPDAI
To the Editor, Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by circulating serum immunoglobulin G (IgG)
antibodies against type XVII collagen/BP180 (BP180), forming tense subepidermal blisters [1]. While IgG BP180 antibodies play an essential role for the development of BP, serum IgE BP180 antibodies are also detected in some BP cases [2]. The involvement of the IgE class of anti-BP180 antibodies remains to be elucidated in the pathogenesis of BP. Here, we report a refractory BP case having a high level of IgE BP180 antibodies. The IgE BP180 antibody level appeared to fluctuate in association with the disease severity of BP. A 47-year-old Japanese man with refractory BP, who had been treated with 60 mg of oral prednisolone and 200 mg of cyclosporine per day for one month, was referred and admitted to our
78
Letters to the Editor / Journal of Dermatological Science 78 (2015) 76–85
Fig. 1. Skin eruptions and clinical course. (a) Scattered erosions and blisters over the patient’s whole body and oral mucosa on admission (Day 1). Bullous pemphigoid disease area index (BPDAI) was 34 (erosions/blisters 33, erythema 3). (b) Recurring exacerbation (Day 68), showing urticarial erythema over the whole body. BPDAI was 67 (erosions/ blisters 39, erythema 49). (c) During the clinical course, IgE BP180 antibody levels correlated with BPDAI especially for erythema.
hospital. More than 20% of the patient’s body was covered with crust, erosions and tense blisters (Fig. 1a). Histopathology showed subepidermal blisters and direct immunofluorescence microscopy revealed IgG deposits at basement membrane zone. The serum level of IgG BP180 antibody was 2620, confirming the definitive diagnosis of BP. The patient was successfully treated with oral prednisolone, cyclosporine, nicotinamide, and intravenous immunoglobulin (IVIG) therapy. However, the eruption was exacerbated as the doses of prednisolone and cyclosporine were reduced (Fig. 1b). He was treated with two courses of methylprednisolone (mPSL) mini-pulse therapy (500 mg/day for 3 consecutive days), followed by plasma exchange (PE). The doses of prednisolone and immunosuppresants were also increased. These treatments improved his condition. During the patient’s clinical course, we frequently monitored the disease severity with a consensus bullous pemphigoid disease area index (BPDAI) scoring, including BPDAI for erosions/blisters and BPDAI for erythema [3], peripheral eosinophil counts, serum total IgE, CCL17/TARC, and eotaxin levels (Fig. 1c). Upon exacerbation of the skin lesions, BPDAI for erythema was markedly elevated, while BPDAI for erosions/blisters was slightly increased. The serum IgE levels were persistently high during the active phase. The peripheral eosinophil counts and TARC levels were increased prior to the exacerbation, but eotaxin was not substantially changed. Because of the elevation of eosinophils, IgE, and TARC, we hypothesized that Th2 cells were involved in the exacerbation of the patient’s BP and IgE BP180 antibodies might participate in the pathogenesis. To quantify IgE BP180 antibodies, we performed BP180 ELISA (MBL, Nagoya, Japan) using horseradish peroxidaselabeled anti-human IgE antibody (1:5000 dilution; DAKO, Tokyo,
Japan) as secondary antibody. Sera were diluted 1:100. To subtract the background level, we collected 46 normal human sera following informed consent and used them to set a positive cut-off value. The mean SD of OD450 value from the control samples was 0.166 0.106. We therefore set the positive cut-off value of 0.484 (mean + 3SD) for this analysis. It was noted that during the clinical course, both IgG and IgE BP180 antibody levels fluctuated in parallel with BPDAI (Fig. 1c). Intriguingly, IgE BP180 antibody levels correlated with BPDAI for erythema rather than BPDAI for erosions/blisters. Tense blisters and urticarial erythema are typically observed on the trunk and extremities of BP patients [1]. Although both IgG and IgE BP180 antibody levels were reported to correlate with the disease activity of BP [4], erosions/blisters and erythema have not been assessed separately. In this study, we carefully and frequently monitored these two eruption elements with the other biomarkers. It is known that tense blisters are induced by the binding of IgG antibodies to BP180, followed by complement-dependent and independent inflammatory pathways [1]. Accordingly, IgG BP180 antibody levels correlated with BPDAI for erosions/blisters in our case. In contrast, it was reported that urticarial erythema was not evoked by IgG BP180 antibodies in BP model mice [5]. Furthermore, IgE antibodies from BP patients induced urticarial erythema in human skin grafted onto athymic nude mice [6]. In consistent with these findings, the IgE BP180 antibody levels correlated with BPDAI for erythema in our case. Although the in vivo pathogenic role of IgE BP180 antibodies remains unclear, neutralization of IgE activity with omalizumab, which blocks the binding of IgE to its receptors, improved the BP disease activity [7]. These observations support the idea that IgE BP180 antibodies contribute to the occurrence of urticarial erythema in BP patients.
Letters to the Editor / Journal of Dermatological Science 78 (2015) 76–85
Recently developed methods allowed IgE BP180 antibodies to be easily detected in BP patients [8,9]. Considering that IgG and IgE BP180 antibodies induce the different clinical symptoms, the evaluation of both classes of the antibodies may be useful to understand the treatment efficacy in relation to the individual symptoms. Further studies are necessary to explain the detailed mechanism underlying the induction of erythema by IgE BP180 antibodies.
79
[6] Fairley JA, Burnett CT, Fu CL, Larson DL, Fleming MG, Giudice GJ. A pathogenic role for IgE in autoimmunity: bullous pemphigoid IgE reproduces the early phase of lesion development in human skin grafted to nu/nu mice. J Invest Dermatol 2007;127:2605–11. [7] Yu KK, Crew AB, Messingham KA, Fairley JA, Woodley DT. Omalizumab therapy for bullous pemphigoid. J Am Acad Dermatol 2014;71:468–74. [8] Pomponi D, Di Zenzo G, Zennaro D, Calabresi V, Eming R, Zuzzi S, et al. Detection of IgG and IgE reactivity to BP180 using the ISAC(R) microarray system. Br J Dermatol 2013;168:1205–14. [9] Messingham KA, Noe MH, Chapman MA, Giudice GJ, Fairley JA. A novel ELISA reveals high frequencies of BP180-specific IgE production in bullous pemphigoid. J Immunol Methods 2009;346:18–25.
Funding This work was supported by a grant from the Ministry of Health, Labour and Welfare (Research for Intractable Diseases), and a Ministry of Education, Culture, Sports, Science and Technology. References [1] Nishie W. Update on the pathogenesis of bullous pemphigoid: an autoantibodymediated blistering disease targeting collagen XVII. J Dermatol Sci 2014;73: 179–86. [2] Ishiura N, Fujimoto M, Watanabe R, Nakashima H, Kuwano Y, Yazawa N, et al. Serum levels of IgE anti-BP180 and anti-BP230 autoantibodies in patients with bullous pemphigoid. J Dermatol Sci 2008;49:153–61. [3] Levy-Sitbon C, Barbe C, Plee J, Goeldel AL, Antonicelli F, Reguiai Z, et al. Assessment of bullous pemphigoid disease area index during treatment: a prospective study of 30 patients. Dermatology 2014;229:116–22. [4] Iwata Y, Komura K, Kodera M, Usuda T, Yokoyama Y, Hara T, et al. Correlation of IgE autoantibody to BP180 with a severe form of bullous pemphigoid. Arch Dermatol 2008;144:41–8. [5] Nishie W, Sawamura D, Goto M, Ito K, Shibaki A, McMillan JR, et al. Humanization of autoantigen. Nat Med 2007;13:378–83.
Koji Kamiyaa,b,*, Yumi Aoyamab, Kazuyo Nodab, Tomoko Miyakeb, Mari Yamaguchib, Toshihisa Hamadab, Yoshiki Tokuraa, Keiji Iwatsukib a Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan; b Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan *Corresponding author. Tel.: +81 53 435 2303; fax: +81 53 435 2368 E-mail address: [email protected] (K. Kamiya).
Received 3, February 2015 Revised 12, February, 2015 Accepted 18, February 2015 http://dx.doi.org/10.1016/j.jdermsci.2015.02.009
Letter to the Editor A mouse model of skin aging: Fragmentation of dermal collagen fibrils and reduced fibroblast spreading due to expression of human matrix metalloproteinase-1
Keywords: MMP-1; Collagen; Aging; Transgenic mice; Fibroblast; Skin
Dear Editor, Fragmentation of dermal collagen fibrils, the major structural proteins in skin, is a prominent feature of human skin aging [1,2]. Elevated collagenase (MMP-1) is largely responsible for initiation of collagen fragmentation in aging human skin [1,3,4]. We previously reported that expression of mutant human MMP-1 (hMMP-1/V94G), which undergoes auto-activation in either young human skin in organ culture or fibroblasts cultured in 3D collagen lattices, causes collagen fibril fragmentation similar to that in aged human skin [5]. To further explore MMP-1 function in skin aging, we generated transgenic mice that express mutant auto-activating human hMMP-1/V94G, under control of an epithelial-specific keratin-5 promoter [6]. hMMP-1/V94G contains a valine to glycine mutation at amino acid 94 within the N-terminal inhibitory domain. This mutation alters the interaction between the inhibitory and catalytic domains, thereby allowing auto-cleavage of the inhibitory domain. The cleaved N-terminal is released thereby converting full length 54 kDa MMP-1 to 44 kDa catalytically active form [5].
Fig. 1A shows immunostaining of hMMP-1/V94G in the epidermis of transgenic mice (K5-hMMP-1/V94GTg). Immunostaining in non-transgenic (non-Tg) littermate mice skin was negative. As expected, hMMP-1/V94G mRNA was readily detected in skin of K5-hMMP-1/V94GTg mice, but absent in non-Tg littermate mice skin (Fig. 1B). Western blot analysis revealed the presence of both full length and cleaved active forms of hMMP1/V94G, at the expected molecular weights of 54 kDa and 44 kDa, respectively, in K5-hMMP-1/V94GTg mice skin (Fig. 1C). Full length hMMP-1/V94G was the predominant form of the protein. The relative low level of cleaved hMMP-1/V94G may have been due to a relatively high rate of turnover, or reflect inefficient conversion. In vitro, cleaved hMMP-1/V94G undergoes further degradation [5], suggesting that the former possibility is more likely. Skin of seven months old K5-hMMP-1/V94GTg mice had normal gross appearance (data not shown). However, Masson’s trichrome staining of skin sections revealed reduced density and disorganization of collagen fibrils (Fig. 1D), compared to non-Tg littermate mice skin. Atomic force microscopy (AFM) was used to assess nanoscale structure of dermal collagen fibrils [7]. Collagen fibrils in non-Tg littermate mice skin were intact, densely packed and wellorganized (Fig. 2A, upper left panel). Characteristic D-band striations, representing periodic alignment of microfibrils that assemble to form larger fibrils, were readily apparent. Collagen fibrils in non-Tg littermate mice skin closely resemble collagen fibrils in young human upper inner arm (underarm) skin (Fig. 2A, lower left panel). In contrast, collagen fibrils in K5-hMMP-1/ V94GTg mice skin were less densely packed, disorganized, and appeared to be fragmented (Fig. 2A, upper right panel). These alterations closely resemble disorganization and fragmentation of collagen fibrils that are observed in aged human forearm skin
dilution of patient sera and different manners in which the ELISA assays were performed possibly caused the discrepancies in these studies [7,8]. With regards to the disease severity of BP, correlations of the following clinical items, namely controlling dosage of prednisone, duration for remission and area of skin lesions, have been investigated with the presence or levels of IgE anti-BP180 autoantibody in four previously reported studies, while the results were not consistent [2,3,5,6]. In our present study, we did not get any positive correlation between the presence or levels of IgE antiBP180 autoantibody with the above items, though, to some extent, patients with higher IgE anti-BP180 autoantibody levels required a larger prednisone dosage, a longer duration for effective control, and had a broader area of skin lesion (Table 1). On the other hand, there was a significant difference in IgG anti-BP180 autoantibody levels between patients with positive IgE antiBP180 autoantibody and those with negative values (t = 2.734, P = 0.009), which suggests that increased IgE anti-BP180 autoantibody levels may also associate with a higher titer of IgG anti-BP180 autoantibody. Regarding correlation of IgE anti-BP180 autoantibody with laboratorial parameters, the circulating eosinophil count may be the most putative one. Two recent related studies have revealed that neither the presence of IgE anti-BP180 autoantibody nor its increased levels correlated with the circulating eosinophil count [5,6]. In this study, we observed a similar result (r = 0.297, P = 0.06 and r = 0.089, P = 0.807, respectively), though the eosinophil counts of patients with BP positive for IgE anti-BP180 autoantibody were indeed higher than those of the other patients with BP (1.08 0.94 vs. 0.74 1.09, Table 1). In addition, the increased IgE anti-BP180 autoantibody levels did not associate with eosinophils infiltration around edematous erythemas and blisterings of skin biopsy specimens (r = 0.283, P = 0.461). To clarify the possible relationship between increased circulating eosinophil count and IgE autoantibody in BP, some studies also focused on another major target antigenic site in BMZ, namely BP230. No positive correlation was found between circulating eosinophil count and IgE anti-BP230 autoantibody [5,6], though a significant correlation with circulating eosinophil count was observed in IgG anti-BP230 autoantibody [6]. So whether there is a positive relationship between circulating eosinophil count and IgE autoantibody specific for BMZ antigen need further research. In conclusion, the present study demonstrates that IgE antiBP180 autoantibody level is increased in some Chinese patients with BP and may associate with their higher titers of IgG antiBP180 autoantibody, although no significant correlations of this IgE autoantibody with various clinical and laboratorial aspects were observed.
77
Acknowledgments This work was funded by Natural Science Foundation of China (grant numbers: 81130030 and 81000694) and the Program for Changjiang Scholars and Innovative Research Team in University (grant number: LRT1003). References [1] Provost TT, Tomasi Jr TB. Immunopathology of bullous pemphigoid: basement membrane deposition of IgE, alternate pathway components and fibrin. Clin Exp Immunol 1974;18:193–200. [2] Delaporte E, Dubost-Brama A, Ghohestani R, Nicolas JF, Neyrinck JL, Bergoend H, et al. IgE autoantibodies directed against the major bullous pemphigoid antigen in patients with a severe form of pemphigoid. J Immunol 1996;157: 3642–7. [3] Hofmann S, Thoma-Uszynski S, Hunziker T, Bernard P, Koebnick C, Stauber A, et al. Severity and phenotype of bullous pemphigoid relate to autoantibody profile against the NH2- and COOH-terminal regions of the BP180 ectodomain. J Invest Dermatol 2002;119:1065–73. [4] Do¨pp R, Schmidt E, Chimanovitch I, Leverkus M, Brocker EB, Zillikens D. IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in Bullous pemphigoid: serum levels of these immunoglobulins reflect disease activity. J Am Acad Dermatol 2000;42:577–83. [5] Iwata Y, Komura K, Kodera M, Usuda T, Yokoyama Y, Hara T, et al. Correlation of IgE autoantibody to BP180 with severe form of bullous pemphigoid. Arch Dermatol 2008;144:41–8. [6] Ishiura N, Fujimoto M, Watanabe R, Nakashima H, Kuwano Y, Yazawa N, et al. Serum levels of IgE anti-BP180 and anti- BP230 autoantibodies in patients with bullous pemphigoid. J Dermatol Sci 2008;49:153–61. [7] Messingham KA, Noe MH, Chapman MA, Giudice GJ, Fairley JA. A novel ELISA reveals high frequencies of BP180-specific IgE production in bullous pemphigoid. J Immunol Methods 2009;346:18–25. [8] Sittampalam GS, Smith WC, Miyakawa TW, Smith DR, McMorris C. Application of experimental design techniques to optimize a competitive ELISA. J Immunol Methods 1996;190:151–61.
Lei Maa,b,c,1, Mingyue Wanga,b,1, Xue Wanga,b, Xixue Chena,b, Xuejun Zhua,b,* a Department of Dermatology, Peking University First Hospital, Beijing, China; bBeijing Key Laboratory of Molecular Diagnosis of Dermatoses, Beijing, China; cDepartment of Dermatology, Binzhou Medical University Hospital, Binzhou, Shandong, China *Corresponding author at: Department of Dermatology, Peking University First Hospital, 8 Xishiku Street, Xicheng District, Beijing 100034, China. Tel.: +86 10 83573075 E-mail address: [email protected] (X. Zhu). 1
These authors contributed equally to this work.
Received 12 February 2015 http://dx.doi.org/10.1016/j.jdermsci.2015.02.015
Letter to the Editor Possible correlation of IgE autoantibody to BP180 with disease activity in bullous pemphigoid
Keywords: Bullous pemphigoid; BP180 antibody; IgE; BPDAI
To the Editor, Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by circulating serum immunoglobulin G (IgG)
antibodies against type XVII collagen/BP180 (BP180), forming tense subepidermal blisters [1]. While IgG BP180 antibodies play an essential role for the development of BP, serum IgE BP180 antibodies are also detected in some BP cases [2]. The involvement of the IgE class of anti-BP180 antibodies remains to be elucidated in the pathogenesis of BP. Here, we report a refractory BP case having a high level of IgE BP180 antibodies. The IgE BP180 antibody level appeared to fluctuate in association with the disease severity of BP. A 47-year-old Japanese man with refractory BP, who had been treated with 60 mg of oral prednisolone and 200 mg of cyclosporine per day for one month, was referred and admitted to our
78
Letters to the Editor / Journal of Dermatological Science 78 (2015) 76–85
Fig. 1. Skin eruptions and clinical course. (a) Scattered erosions and blisters over the patient’s whole body and oral mucosa on admission (Day 1). Bullous pemphigoid disease area index (BPDAI) was 34 (erosions/blisters 33, erythema 3). (b) Recurring exacerbation (Day 68), showing urticarial erythema over the whole body. BPDAI was 67 (erosions/ blisters 39, erythema 49). (c) During the clinical course, IgE BP180 antibody levels correlated with BPDAI especially for erythema.
hospital. More than 20% of the patient’s body was covered with crust, erosions and tense blisters (Fig. 1a). Histopathology showed subepidermal blisters and direct immunofluorescence microscopy revealed IgG deposits at basement membrane zone. The serum level of IgG BP180 antibody was 2620, confirming the definitive diagnosis of BP. The patient was successfully treated with oral prednisolone, cyclosporine, nicotinamide, and intravenous immunoglobulin (IVIG) therapy. However, the eruption was exacerbated as the doses of prednisolone and cyclosporine were reduced (Fig. 1b). He was treated with two courses of methylprednisolone (mPSL) mini-pulse therapy (500 mg/day for 3 consecutive days), followed by plasma exchange (PE). The doses of prednisolone and immunosuppresants were also increased. These treatments improved his condition. During the patient’s clinical course, we frequently monitored the disease severity with a consensus bullous pemphigoid disease area index (BPDAI) scoring, including BPDAI for erosions/blisters and BPDAI for erythema [3], peripheral eosinophil counts, serum total IgE, CCL17/TARC, and eotaxin levels (Fig. 1c). Upon exacerbation of the skin lesions, BPDAI for erythema was markedly elevated, while BPDAI for erosions/blisters was slightly increased. The serum IgE levels were persistently high during the active phase. The peripheral eosinophil counts and TARC levels were increased prior to the exacerbation, but eotaxin was not substantially changed. Because of the elevation of eosinophils, IgE, and TARC, we hypothesized that Th2 cells were involved in the exacerbation of the patient’s BP and IgE BP180 antibodies might participate in the pathogenesis. To quantify IgE BP180 antibodies, we performed BP180 ELISA (MBL, Nagoya, Japan) using horseradish peroxidaselabeled anti-human IgE antibody (1:5000 dilution; DAKO, Tokyo,
Japan) as secondary antibody. Sera were diluted 1:100. To subtract the background level, we collected 46 normal human sera following informed consent and used them to set a positive cut-off value. The mean SD of OD450 value from the control samples was 0.166 0.106. We therefore set the positive cut-off value of 0.484 (mean + 3SD) for this analysis. It was noted that during the clinical course, both IgG and IgE BP180 antibody levels fluctuated in parallel with BPDAI (Fig. 1c). Intriguingly, IgE BP180 antibody levels correlated with BPDAI for erythema rather than BPDAI for erosions/blisters. Tense blisters and urticarial erythema are typically observed on the trunk and extremities of BP patients [1]. Although both IgG and IgE BP180 antibody levels were reported to correlate with the disease activity of BP [4], erosions/blisters and erythema have not been assessed separately. In this study, we carefully and frequently monitored these two eruption elements with the other biomarkers. It is known that tense blisters are induced by the binding of IgG antibodies to BP180, followed by complement-dependent and independent inflammatory pathways [1]. Accordingly, IgG BP180 antibody levels correlated with BPDAI for erosions/blisters in our case. In contrast, it was reported that urticarial erythema was not evoked by IgG BP180 antibodies in BP model mice [5]. Furthermore, IgE antibodies from BP patients induced urticarial erythema in human skin grafted onto athymic nude mice [6]. In consistent with these findings, the IgE BP180 antibody levels correlated with BPDAI for erythema in our case. Although the in vivo pathogenic role of IgE BP180 antibodies remains unclear, neutralization of IgE activity with omalizumab, which blocks the binding of IgE to its receptors, improved the BP disease activity [7]. These observations support the idea that IgE BP180 antibodies contribute to the occurrence of urticarial erythema in BP patients.
Letters to the Editor / Journal of Dermatological Science 78 (2015) 76–85
Recently developed methods allowed IgE BP180 antibodies to be easily detected in BP patients [8,9]. Considering that IgG and IgE BP180 antibodies induce the different clinical symptoms, the evaluation of both classes of the antibodies may be useful to understand the treatment efficacy in relation to the individual symptoms. Further studies are necessary to explain the detailed mechanism underlying the induction of erythema by IgE BP180 antibodies.
79
[6] Fairley JA, Burnett CT, Fu CL, Larson DL, Fleming MG, Giudice GJ. A pathogenic role for IgE in autoimmunity: bullous pemphigoid IgE reproduces the early phase of lesion development in human skin grafted to nu/nu mice. J Invest Dermatol 2007;127:2605–11. [7] Yu KK, Crew AB, Messingham KA, Fairley JA, Woodley DT. Omalizumab therapy for bullous pemphigoid. J Am Acad Dermatol 2014;71:468–74. [8] Pomponi D, Di Zenzo G, Zennaro D, Calabresi V, Eming R, Zuzzi S, et al. Detection of IgG and IgE reactivity to BP180 using the ISAC(R) microarray system. Br J Dermatol 2013;168:1205–14. [9] Messingham KA, Noe MH, Chapman MA, Giudice GJ, Fairley JA. A novel ELISA reveals high frequencies of BP180-specific IgE production in bullous pemphigoid. J Immunol Methods 2009;346:18–25.
Funding This work was supported by a grant from the Ministry of Health, Labour and Welfare (Research for Intractable Diseases), and a Ministry of Education, Culture, Sports, Science and Technology. References [1] Nishie W. Update on the pathogenesis of bullous pemphigoid: an autoantibodymediated blistering disease targeting collagen XVII. J Dermatol Sci 2014;73: 179–86. [2] Ishiura N, Fujimoto M, Watanabe R, Nakashima H, Kuwano Y, Yazawa N, et al. Serum levels of IgE anti-BP180 and anti-BP230 autoantibodies in patients with bullous pemphigoid. J Dermatol Sci 2008;49:153–61. [3] Levy-Sitbon C, Barbe C, Plee J, Goeldel AL, Antonicelli F, Reguiai Z, et al. Assessment of bullous pemphigoid disease area index during treatment: a prospective study of 30 patients. Dermatology 2014;229:116–22. [4] Iwata Y, Komura K, Kodera M, Usuda T, Yokoyama Y, Hara T, et al. Correlation of IgE autoantibody to BP180 with a severe form of bullous pemphigoid. Arch Dermatol 2008;144:41–8. [5] Nishie W, Sawamura D, Goto M, Ito K, Shibaki A, McMillan JR, et al. Humanization of autoantigen. Nat Med 2007;13:378–83.
Koji Kamiyaa,b,*, Yumi Aoyamab, Kazuyo Nodab, Tomoko Miyakeb, Mari Yamaguchib, Toshihisa Hamadab, Yoshiki Tokuraa, Keiji Iwatsukib a Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan; b Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan *Corresponding author. Tel.: +81 53 435 2303; fax: +81 53 435 2368 E-mail address: [email protected] (K. Kamiya).
Received 3, February 2015 Revised 12, February, 2015 Accepted 18, February 2015 http://dx.doi.org/10.1016/j.jdermsci.2015.02.009
Letter to the Editor A mouse model of skin aging: Fragmentation of dermal collagen fibrils and reduced fibroblast spreading due to expression of human matrix metalloproteinase-1
Keywords: MMP-1; Collagen; Aging; Transgenic mice; Fibroblast; Skin
Dear Editor, Fragmentation of dermal collagen fibrils, the major structural proteins in skin, is a prominent feature of human skin aging [1,2]. Elevated collagenase (MMP-1) is largely responsible for initiation of collagen fragmentation in aging human skin [1,3,4]. We previously reported that expression of mutant human MMP-1 (hMMP-1/V94G), which undergoes auto-activation in either young human skin in organ culture or fibroblasts cultured in 3D collagen lattices, causes collagen fibril fragmentation similar to that in aged human skin [5]. To further explore MMP-1 function in skin aging, we generated transgenic mice that express mutant auto-activating human hMMP-1/V94G, under control of an epithelial-specific keratin-5 promoter [6]. hMMP-1/V94G contains a valine to glycine mutation at amino acid 94 within the N-terminal inhibitory domain. This mutation alters the interaction between the inhibitory and catalytic domains, thereby allowing auto-cleavage of the inhibitory domain. The cleaved N-terminal is released thereby converting full length 54 kDa MMP-1 to 44 kDa catalytically active form [5].
Fig. 1A shows immunostaining of hMMP-1/V94G in the epidermis of transgenic mice (K5-hMMP-1/V94GTg). Immunostaining in non-transgenic (non-Tg) littermate mice skin was negative. As expected, hMMP-1/V94G mRNA was readily detected in skin of K5-hMMP-1/V94GTg mice, but absent in non-Tg littermate mice skin (Fig. 1B). Western blot analysis revealed the presence of both full length and cleaved active forms of hMMP1/V94G, at the expected molecular weights of 54 kDa and 44 kDa, respectively, in K5-hMMP-1/V94GTg mice skin (Fig. 1C). Full length hMMP-1/V94G was the predominant form of the protein. The relative low level of cleaved hMMP-1/V94G may have been due to a relatively high rate of turnover, or reflect inefficient conversion. In vitro, cleaved hMMP-1/V94G undergoes further degradation [5], suggesting that the former possibility is more likely. Skin of seven months old K5-hMMP-1/V94GTg mice had normal gross appearance (data not shown). However, Masson’s trichrome staining of skin sections revealed reduced density and disorganization of collagen fibrils (Fig. 1D), compared to non-Tg littermate mice skin. Atomic force microscopy (AFM) was used to assess nanoscale structure of dermal collagen fibrils [7]. Collagen fibrils in non-Tg littermate mice skin were intact, densely packed and wellorganized (Fig. 2A, upper left panel). Characteristic D-band striations, representing periodic alignment of microfibrils that assemble to form larger fibrils, were readily apparent. Collagen fibrils in non-Tg littermate mice skin closely resemble collagen fibrils in young human upper inner arm (underarm) skin (Fig. 2A, lower left panel). In contrast, collagen fibrils in K5-hMMP-1/ V94GTg mice skin were less densely packed, disorganized, and appeared to be fragmented (Fig. 2A, upper right panel). These alterations closely resemble disorganization and fragmentation of collagen fibrils that are observed in aged human forearm skin
