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DFS70, a Major Autoantigen of Atopic Dermatitis, Is a Component of Keratohyalin Granules
ORIGINAL ARTICLE
LEDGF/DFS70, a Major Autoantigen of Atopic Dermatitis, Is a Component of Keratohyalin Granules Kazumitsu Sugiura1, Yoshinao Muro1, Yuji Nishizawa2, Miyako Okamoto1, Toshimichi Shinohara3, Yasushi Tomita1 and Jiro Usukura2 Lens epithelium-derived growth factor/dense fine speckles 70 kDa protein (LEDGF/DFS70) is a transcriptional cofactor, a transcriptional activator, survival factor, and HIV-1 transporter. It is also a major autoantigen in patients with atopic dermatitis (AD), because autoantibodies to this protein are found in approximately 30% of AD patients. To better understand the role of autoantibodies and autoantigens in the pathogenesis of AD, we examined the distribution of LEDGF/DFS70 in the epidermis of normal human skin by light and electron microscopic immunocytochemistry. Increased amounts of LEDGF/DFS70 were located in the nuclei of cells in the basal layer, whereas the cytoplasm of cells in the granular layer stained for LEDGF/DFS70 by light microscopy. Using immunoelectron microscopy, we observed the accumulation of LEDGF/DFS70 in keratohyalin granules (KGs) in the cytoplasm of cells in the granular layer. In addition, Ig heavy chain-binding protein/glucose-regulated protein, 78-kDa (Bip/GRP78), a stress sensing protein in the endoplasmic reticulum, colocalized with LEDGF/DFS70 in the KGs. These results suggest that LEDGF/DFS70 is predominantly located in the nucleus of the basal epidermal cells and translocates into the cytoplasm during differentiation. Once in the cytoplasm, LEDGF/DFS70 accumulates in the KGs in the granular layer. Finally, LEDGF/DFS70, a ‘‘nuclear’’ autoantigen in AD, may play a functional role in the KGs. Journal of Investigative Dermatology (2007) 127, 75–80. doi:10.1038/sj.jid.5700487; published online 20 July 2006
INTRODUCTION There is strong evidence that autoimmunity plays a role in the pathogenesis of atopic dermatitis (AD) in combination with immune hypersensitivity to innocuous environmental antigens such as pollen and household dust (Hanifin, 1996; Valenta et al., 2000). Many patients suffering from AD have higher levels of IgG and IgE autoantibodies, which react to various human proteins (Muro, 2001; Mittermann et al., 2004). Indeed, serum levels of IgG anti-nuclear antibodies are higher in AD patients with severe facial lesions (Sanda et al., 1992; Taniguchi et al., 1992; Tada et al., 1994; Ohkouchi et al., 1999). We isolated a dense fine speckles 70 kDa (DFS70) autoantigen by immunoscreening the serum of a patient with interstitial cystitis, and DFS70 is a major autoantigen found in 1
Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Japan; 2Department of Anatomy, Nagoya University Graduate School of Medicine, Nagoya, Japan and 3Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska Correspondence: Dr Kazumitsu Sugiura, Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showaku, Nagoya 466-8550, Japan. E-mail: [email protected] Abbreviations: AD, atopic dermatitis; Bip, Ig heavy chain-binding protein; DFS70, dense fine speckles 70 kDa protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein, 78-kDa; KG, keratohyalin granule; LEDGF, lens epithelium-derived growth factor; PBS, phosphate-buffered saline
Received 13 February 2006; revised 8 May 2006; accepted 6 June 2006; published online 20 July 2006
& 2006 The Society for Investigative Dermatology
about 30% of AD patients (Ochs et al., 2000; U94319). DFS70 is one of several proteins producing anti-nuclear antibodies staining, and it is the same protein as p75 (Ge et al., 1998) and lens epithelium-derived growth factor (LEDGF) (Singh et al., 1999). This protein was isolated and characterized independently by three different laboratories. P75 was initially isolated as a transcriptional cofactor (Ge et al., 1998), and LEDGF was identified as a survival factor for lens epithelial cells and named LEDGF (Singh et al., 1999; Shinohara et al., 2002). LEDGF exhibits (Shinohara et al., 2002) DNA-binding and transcriptional activity (Ge et al., 1998; Singh et al., 1999; 2000), and it activates heat-shock protein 27, aB-crystalline, and involucrin in lens epithelial cells and keratinocytes in vitro (Singh et al., 2001; Kubo et al., 2002). Additionally, it promotes cell survival, enhances stress resistance in different cell lines (Shinohara et al., 2002), and induces the disassembly of gap junctions of lens epithelial cells via the activation of protein C gamma (Nguyen et al., 2003). LEDGF also interacts with HIV-1 integrase and mediates its chromatin targeting (Maertens et al., 2003; Ciuffi et al., 2005). Finally, high levels of LEDGF/ DFS70 autoantibodies are seen in AD patients (Ochs et al., 2000; Ayaki et al., 2002). Accumulating evidence suggests that LEDGF/DFS70 is involved in the pathogenesis of skin epithelial lesions, and autoantibodies targeting LEDGF/DFS70 are also frequently found in alopecia areata patients (Okamoto et al., 2004). Interestingly, expression of LEDGF/DFS70 is increased by www.jidonline.org
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endoplasmic reticulum (ER) stress (our unpublished data), suggesting that Bip/GRP78, a central sensor of ER stress, is also involved in the terminal differentiation of epidermal cells. We wished to determine the exact cellular location(s) of LEDGF/DFS70 and related proteins, including Bip/GRP78, in normal epidermis, and examine the role of LEDGF/DFS70 in the pathogenesis of the lesions induced by the autoantibodies to LEDGF/DFS70. RESULTS Specificity of antibodies to LEDGF/DFS70 in HeLa and HaCaT cell extracts
Our research goals required specific reagents for detecting LEDGF/DFS70, and we first examined the specificity of the obtained anti-LEDGF/DFS70 reagents by Western blotting. When we examined whole-cell extracts from both HeLa and HaCaT cells with the two polyclonal antisera (S-S and No. 6369) and a commercially available monoclonal antibody targeting LEDGF/DFS70, we observed a band migrating at the
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Figure 1. Specificity of the LEDGF/DFS70 IDPS70 antisera and immunocytochemistry of thin sections of the epidermis with antisera to LEDGF/DFS70. (a) Protein blot analyses of (a) HeLa cell extracts and (b) HaCaT cell extracts stained with: lane 1, commercial antibodies to LEDGF/ DFS70; lane 2, polyclonal S-S antiserum to LEDGF/DFS70; lane 3, polyclonal No. 6369 antiserum to LEDGF/DFS70; and lane 4, preimmune serum of No. 6369. Numbers on the left are molecular weight markers. Thin sections of the epidermis immunostained with (c) anti-LEDGF antiserum No. 6369 or (d) control preimmune antiserum No. 6369. LEDGF/DFS70 was visualized with horseradish peroxidase. Bar ¼ 50 mm.
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expected molecular weight of 75 kDa (Figure 1a, b). No additional proteins were detected under these experimental conditions suggesting that these antibodies were highly specific for LEDGF/DFS70. Light microscopic immunocytochemistry
LEDGF/DFS70 was localized exclusively in the nucleus in cultured NRK, Madin–Darby canine kidney, and Chinese hamster ovary-K1 cell lines (Nishizawa et al., 2001), but when we examined thin sections of epidermal tissue immunocytochemically both the nucleus and cytoplasm were labeled (Figure 1c). Both anti-LEDGF/DFS70 anti-sera, No. 6369 and S-S, gave identical staining patterns (data not shown). Under higher magnification, however, the nuclei of progenitor or mitotic cells in the basal layer were more strongly labeled than the cytoplasm. Conversely, in the more differentiated epidermal cells, LEDGF/DFS70 staining was substantially reduced in the nucleus and increased in the cytoplasm. In particular, intense LEDGF/DFS70 staining was seen in the cytoplasm of cells in the granular layer. We could not determine whether the overall increased LEDGF/DFS70 staining resulted from keratinocyte differentiation, or whether the relative protein concentration was increased by the dehydration of cells in the granular layer and the stratum corneum in vivo. However, LEDGF/DFS70 messenger RNA levels did not change in proliferating HaCaT cells compared to differentiated HaCaT cells by real-time PCR (data not shown) in vitro. The LabCyte EPI-Model system consists of normal humanderived epidermal keratinocytes cultured to form a multilayered, highly differentiated model of the human epidermis. This model is structurally similar to human epidermal tissue as shown by hematoxylin and eosin staining of thin sections (Figure 2a). When EPI-Model samples were examined immunocytochemically with the LEDGF/DFS70 antisera No. 6369 and S-S, both the nucleus and cytoplasm were labeled (Figure 2b). Nuclear staining was seen both in the basal and granular layers, and the staining intensity appeared similar in these two layers. However, cytoplasmic staining of cells in the granular layer was more prominent. Bip/GRP78 is an ER resident protein that acts a chaperone to facilitate protein folding, but it is also essential for sensing conditions of ER stress (Shen et al., 2002). We have observed protein–protein interactions between LEDGF/DFS70 and Bip/GRP78 (our unpublished data), suggesting a role for Bip/GRP78 in the biosynthesis of LEDGF/DFS70. When we examined the localization of Bip/GRP78, it was primarily found in a diffusely staining pattern consistent with an ER distribution. Surprisingly, few cells were stained in the basal layers, but staining increased substantially upon epidermal cell differentiation (Figure 3). Immunoelectron microscopy
Immunocytochemistry and light microscopy are useful for grossly examining the cellular distribution of a particular protein, but to precisely determine changes in the localization of LEDGF/DFS70 with keratinocyte differentiation we used immuno-electron microscopy. We previously observed
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Figure 2. Immunocytochemistry of thin sections of multilayered cultured keratinocytes with an antiserum to LEDGF/DFS70. Thin sections of LabCyte EPI-Model were stained with (a) hematoxylin and eosin, and were immunostained with (b) antiserum No. 6369, and with (c) preimmune serum of No. 6369. Asterisk shows the layer corresponding to the granular layer. Bar ¼ 50 mm.
Figure 3. Immunocytochemistry of thin sections of the epidermis with anti-Bip/GRP78 antibodies. Thin sections of the epidermis were immunostained with polyclonal anti-Bip/GRP78 antibodies. Arrow points to the basement membrane. Bip/GRP78 was visualized with horseradish peroxidase. Bar ¼ 50 mm.
LEDGF/DFS70 predominantly around the chromatin in the nuclei of cultured cells (Nishizawa et al., 2001), and LEDGF/ DFS70 was also seen in the nuclei of keratinocytes in the granular layer (Figure 4e). Additionally, within the cytoplasm, LEDGF/DFS70 was found exclusively in multivesicular bodylike structures surrounded by bundles of keratin, in the cells of the granular layer. LEDGF/DFS70 was also seen in the cells in the stratum corneum (Figure 4a–c), and these signals were eliminated by pre-incubating antiserum No. 6369 with the peptides used for immunization demonstrating the specificity of this staining (compare Figure 4b and d). These multivesicular body-like structures likely represent keratohyalin
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Figure 4. Localization of LEDGF/DFS70 in the KGs by immunoelectron microscopy analysis. The subcellular localization of LEDGF/DGS70 in the keratinocytes of the granular layer and stratum corneum were determined by immunoelectron microscopy. LEDGF/DFS70 was found in KGs, (a–c) in the granular layers, and (e) in the nuclei. (d) In controls, the antiserum No. 6369 pre-incubated with the immunizing peptide was used for immunoelectron microscopy. (b) Magnified image of the inset in (a). Arrow indicates the irregularly shaped and multivesicular structures of the KGs. An arrowhead points to a keratin bundle. The K4M embedding method was used. Goat anti-rabbit antiserum coupled to 10 nm immunogold particles was used for detection. Bar ¼ 200 nm.
granules (KGs) because of their structural characteristics, for example, tufted outlines and tendency to form vacuoles (Steven et al., 1990). Indeed, profilaggrin, a KGs marker, was located in these structures with LEDGF/DFS70 (Figure 5a). Thus, LEDGF/DFS70 colocalized with filaggrin in the stratum corneum (Figure 5b). We further examined whether LEDGF/DFS70 and Bip/ GRP78 colocalized in the KGs. Indeed, both LEDGF/DFS70 and Bip/GRP78 were observed in the KGs of cells in the granular layer of human epidermal cells (Figure 6a). Finally, Bip/GRP78 also localized in the stratum corneum (Figure 6b). www.jidonline.org
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Figure 5. Colocalization of LEDGF/DFS70 and profilaggrin in the KGs of cells in the granular layer. A double immunostaining method was used with anti-LEDGF/DFS70 atniserum and anti-filaggrin antibody. Profilaggrin, the precursor of filaggrin, is a marker of KGs. Five nanometer immunogold-conjugated secondary antibody was used for LEDGF/DFS70 detection and 10 nm gold for filaggrin immunostaining. (a) The inset picture is a magnified picture of square. (b) Photograph showing the same pattern of immunostaining in the stratum corneum. Bar ¼ (a) 200 nm and (b) 500 nm.
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Figure 6. Bip/GRP78 localizes in KGs and in the stratum corneum. (a, b) Double immunostaining method was used with anti-LEDGF/DFS70 antiserum and anti-Bip/GRP78 antibody. Five nanometer immunogold conjugated second antiserum was used for LEDGF/DFS70 and 10 nm for Bip/ GRP78. (a) A photograph showing KGs, and (b) a photograph showing the staining pattern in the stratum corneum. Bar ¼ (a) 200 nm and (b) 500 nm.
DISCUSSION LEDGF/DFS70 is a major autoantigen in AD, and, in order to better understand the role of LEDGF/DFS70 in health and disease, we wished to precisely characterize its subcellular distribution. We observed LEDGF/DFS70 not only in the nucleus but also in the cytoplasm of human epidermal cells, and the chaperone Bip/GRP78 was diffusely distributed in cells from the suprabasal layer to the stratum corneum but not the basal layer. Higher levels of LEDGF/DFS70 were seen in the KGs of the cells in the granular layer of the epidermis, and light and electron microscopic immunocytochemistry confirmed that the KGs first appears in cells of the granular layer that also contain LEDGF/DFS70 and Bip/GRP78. The intensity of LEDGF/DFS70 staining in the nucleus and cytoplasm of epidermal cells varied considerably. This variation may arise from the heterogeneity of the cell populations in the epidermis. The epidermis is comprised of 78
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cells at different developmental stages, such as progenitor, mitotic, post-mitotic, differentiated cells, and anuclear cells. Progenitor and mitotic cells expressed higher levels of LEDGF/DFS70 in the nucleus than in the cytoplasm, consistent with reports of human ocular tissues, fetal human brain, and human prostate cancer cells (Kubo et al., 2003; Chylack et al., 2004; Daniels et al., 2005). Transformed cultured cells are predominantly mitotic cells, which contain higher levels of LEDGF/DSF70 in the nucleus (Muro, 2001; Nishizawa et al., 2001; Maertens et al., 2004). In contrast, post-mitotic, differentiated, and terminally differentiated cells are predominantly found in the granular layer, and these cells contain high levels of LEDGF/DFS70 in the KGs. Enucleated cells in the stratum corneum, also contain high levels of LEDGF/DSF70. Because LEDGF/DFS70 is a transcriptional coactivator (Ge et al., 1998; Singh et al., 2000) and survival factor (Shinohara et al., 2002), the intensely stained cells might represent progenitor cells or mitotic cells in the basal layer. This is consistent with a number of studies showing that LEDGF/DFS70 is localized in the nucleus of many cultured cells including fetal rat epidermal cells (Muro, 2001; Nishizawa et al., 2001; Maertens et al., 2004). In contrast, differentiated, proapoptotic, and apoptotic cells in lens epithelial cells lose LEDGF/DFS70 from the nucleus (Kubo et al., 2003). Thus, in the skin, LEDGF/ DFS70 is transported from the nucleus to the cytoplasm and is accumulated in the KGs as the keratinocytes differentiate. The inclusion of LEDGF/DFS70 and Bip/GRP78 in the KGs in the cells of the granular layer may be functionally important and we propose that the KGs may act as an epidermal cell-specific protein transport system or protein disposal organelle in vivo. The KGs contain keratin-associated proteins, including profilaggrin, repetin, and caspase 14 (Steven et al., 1990; Alibardi et al., 2004; Huber et al., 2005), but not keratin (Warhol et al., 1985). Profilaggrin is cleaved into the bioactive filaggrin molecule (McKinleyGrant et al., 1989), and it aggregates with keratin filaments in the lower layers of the stratum corneum (Dale et al., 1978). It is later degraded to form natural moisturizing factors in the upper layers of the stratum corneum (Kashima et al., 1988). Caspase 14 plays an important role in the terminal differentiation of keratinocytes; however, no substrate for this protease has been found (Eckhart et al., 2000). LEDGF/DFS70 is a substrate of caspase 3 and caspase 7 in apoptotic cells (Wu et al., 2002), and we observed that the partial colocalization of LEDGF/DFS70 with caspase 14 by immunoelectron microscopy (our unpublished data). Protein compartmentalization into KGs may be important for proper keratinocyte apoptosis. Alternatively, the KGs may serve as a means to collect proteins for elimination by enucleation, a characteristic feature of keratinocytes. Further studies are clearly needed to identify the functional roles of the KGs in cells of the granular layer. The transcription of involucrin, the most abundant protein of the crosslinked keratinocyte envelope is regulated by LEDGF/DFS70 in cultured keratinocytes, and the skin of AD patients contains lower levels of involucrin (Eckert and Green, 1986; Seguchi et al., 1996; Kubo et al., 2002; Jensen et al.,
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2004). IgG deposits have been seen in the stratum corneum of AD patients (Hodgkinson et al., 1977), and these autoantibodies may target LEDGF/DFS70 in the upper layers of epidermis and interfere with its function in the nucleus as well as the KGs. This may suppress involucrin transcription in the keratinocytes of the granular layer. Thus, autoantibodies targeting LEDGF/ DFS70 in the cells of the granular layer and stratum corneum in patients with AD may alter epidermis homeostasis. In conclusion, LEDGF/DFS70, a major autoantigen in patients with AD, is a component of the KGs in the cells of the granular layer of the skin. The unique relocation of LEDGF/ DFS70 from the nucleus to the KGs coincides with the terminal differentiation of keratinocytes and suggests a possible role for DFS70/LEDGF in the KGs. Thus, autoantibodies targeting LEDGF/DFS70 may affect LEDGF/DFS70 function and promote the development of different skin diseases. MATERIALS AND METHODS Normal human skin Normal human skin for electron microscopic immunocytochemistry was obtained from the left anterior arm of a 35-year-old man and from a finger of a 74-year-old man after obtaining informed written consent. Normal human skin for light microscopic immunocytochemistry was obtained from the scalp of a patient with benign scalp tumor after obtaining informed written consent as described (Okamoto et al., 2004). The use of human materials was approved by the Ethics Committee of Nagoya University, Graduate School of Medicine. The study was conducted according to the Declaration of Helsinki Principles.
Antibodies Two different polyclonal antisera against LEDGF/DFS70 were used; one was from Dr T. Shinohara’s laboratory (S-S antiserum; Singh et al., 2000), and the other was from Dr Y Muro’s laboratory, named No. 6369 antiserum (Okamoto et al., 2004). The following mAbs were purchased from commercial sources; anti-LEDGF, anti-Bip/ GRP78 (BD Biosciences, San Jose, CA), and anti-profilaggrin (Abcam Ltd. Cambridge, UK). Polyclonal anti-Bip/GRP78 (Santa Cruz Biotecnology, Inc., Santa Cruz, CA) was also purchased.
Immunoblotting HeLa and HaCaT cell extracts The immortalized keratinocyte cell line HaCaT was kindly provided by Dr N. Fusenig (Boukamp et al., 1988). Whole-cell extracts of HeLa and HaCaT cells were obtained as previously described (Watanabe et al., 2004). Cell lysates were separated by 10% SDSPAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) as described (Sugiura and Muro, 1999). Strips of membrane were incubated with the monoclonal antiLEDGF antibodies and polyclonal anti-LEDGF antisera (S-S and No. 6369) at dilutions of 1:100, 1:100, and 1:1,000, respectively. The antibody–antigen complexes were detected with horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG (Dako Co. Ltd, Glostrup, Denmark) at 1:1,000 dilutions and visualized with Konica immunostaining (Konica, Tokyo, Japan).
Immunocytochemistry of human skin and multilayered cultured keratinocytes The LabCyte EPI-Model (J-Tec Co Ltd, Gamagori, Japan), similar to the EpiDermTM System (MatTek Co, Ashland, MA), consists of normal
human-derived epidermal keratinocytes cultured to form a multilayered and represents a highly differentiated model of the human epidermis. Immunocytochemistry on human skin and the Labcyte EPI-Model were performed as described with slight modifications (Okamoto et al., 2004). Briefly, the VECTASTAIN Elite ABC-PO (rabbit IgG) kit (Vector Laboratories Inc., Burlingame, CA) was used for staining. Thin sections (6 mm) were cut from paraffin blocks of normal human scalp skin and the LabCyte EPI-Model. The sections were immersed in 0.4% pepsin solution for 30 minutes, and soaked for 20 minutes at room temperature in 0.3% H2O2/methanol to block endogenous peroxidase activity. After washing in phosphatebuffered saline (PBS) with 0.01% Triton X-100, the sections were incubated for 30 minutes in PBS buffer with 4% BSA and followed by the anti-LEDGF antisera S-S or No. 6369 (1:1,000 dilutions) or antiBip/GRP (1:100) in PBS buffer containing 1% BSA overnight at 41C. After washing in PBS buffer, the thin sections were exposed to avidin-conjugated goat anti-rabbit Ig antibodies for 1 hour and washed again in PBS buffer. After washing, the tissue sections were immersed in VECTASTAIN Elite ABC Reagent for 30 minutes and washed again in PBS buffer. The antibody complexes were visualized by the addition 1% H2O2. Pre-immune No. 6369 serum diluted at 1:1,000 was used as a negative control.
Electron microscopic immunocytochemistry Human skin, biopsied with a dermal punch (Maruho Inc., Tokyo, Japan), was fixed in 2% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer adjusted to pH 7.4. The fixed samples were dehydrated in an ascending ethanol series up to 99.5% and embedded in Lowicryl K4M resin (Polysciences, Warrington, PV) according to the manufacturer’s protocol. Ultrathin sections (95 nm) were mounted on nickel grids coated with Formvar, and immunolabeling was carried out as described in detail (Kachi et al., 1999). Briefly, after blocking for 10 minutes with 4% bovine serum albumin in PBS, the sections were incubated with the anti-LEDGF antisera S-S or No. 6369, each diluted 1:100 (10 mg/ml). For control, we used No. 6369 (10 mg/ml ) preincubated with immunized peptides (40 mg/ml ). Anti-filaggrin and anti-Bip/GRP78 antisera were used diluted 1:20. After washing seven times with a droplet of PBS, the sections were incubated with 10 nm or 5 nm gold conjugated-goat anti-rabbit IgG or goat anti-mouse IgG (1:30 dilution: GE Healthcare, Chalfont Saint Giles, UK). After washing, the sections were fixed in 2% glutarardehyde followed by a droplet of water eight times. All sections were stained for 5 minutes with uranyl acetate before observation. For control, pre-immune No. 6369 serum diluted 1:100 was used. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS Dr K. Sugiura was supported by Grants 16790635 and 18790783, and Dr Y. Muro was supported by Grant 15591174, from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Maertens G, Cherepanov P, Debyser Z, Engelborghs Y, Engelman A (2004) Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75. J Biol Chem 279:33421–9
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Maertens G, Cherepanov P, Pluymers W, Busschots K, De Clercq E, Debyser Z et al. (2003) LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells. J Biol Chem 278:33528–39
Watanabe A, Kodera M, Sugiura K, Usuda T, Tan EM, Takasaki Y et al. (2004) Anti-DFS70 antibodies in 597 healthy hospital workers. Arthritis Rheum 50:892–900
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LEDGF/DFS70, a Major Autoantigen of Atopic Dermatitis, Is a Component of Keratohyalin Granules Kazumitsu Sugiura1, Yoshinao Muro1, Yuji Nishizawa2, Miyako Okamoto1, Toshimichi Shinohara3, Yasushi Tomita1 and Jiro Usukura2 Lens epithelium-derived growth factor/dense fine speckles 70 kDa protein (LEDGF/DFS70) is a transcriptional cofactor, a transcriptional activator, survival factor, and HIV-1 transporter. It is also a major autoantigen in patients with atopic dermatitis (AD), because autoantibodies to this protein are found in approximately 30% of AD patients. To better understand the role of autoantibodies and autoantigens in the pathogenesis of AD, we examined the distribution of LEDGF/DFS70 in the epidermis of normal human skin by light and electron microscopic immunocytochemistry. Increased amounts of LEDGF/DFS70 were located in the nuclei of cells in the basal layer, whereas the cytoplasm of cells in the granular layer stained for LEDGF/DFS70 by light microscopy. Using immunoelectron microscopy, we observed the accumulation of LEDGF/DFS70 in keratohyalin granules (KGs) in the cytoplasm of cells in the granular layer. In addition, Ig heavy chain-binding protein/glucose-regulated protein, 78-kDa (Bip/GRP78), a stress sensing protein in the endoplasmic reticulum, colocalized with LEDGF/DFS70 in the KGs. These results suggest that LEDGF/DFS70 is predominantly located in the nucleus of the basal epidermal cells and translocates into the cytoplasm during differentiation. Once in the cytoplasm, LEDGF/DFS70 accumulates in the KGs in the granular layer. Finally, LEDGF/DFS70, a ‘‘nuclear’’ autoantigen in AD, may play a functional role in the KGs. Journal of Investigative Dermatology (2007) 127, 75–80. doi:10.1038/sj.jid.5700487; published online 20 July 2006
INTRODUCTION There is strong evidence that autoimmunity plays a role in the pathogenesis of atopic dermatitis (AD) in combination with immune hypersensitivity to innocuous environmental antigens such as pollen and household dust (Hanifin, 1996; Valenta et al., 2000). Many patients suffering from AD have higher levels of IgG and IgE autoantibodies, which react to various human proteins (Muro, 2001; Mittermann et al., 2004). Indeed, serum levels of IgG anti-nuclear antibodies are higher in AD patients with severe facial lesions (Sanda et al., 1992; Taniguchi et al., 1992; Tada et al., 1994; Ohkouchi et al., 1999). We isolated a dense fine speckles 70 kDa (DFS70) autoantigen by immunoscreening the serum of a patient with interstitial cystitis, and DFS70 is a major autoantigen found in 1
Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Japan; 2Department of Anatomy, Nagoya University Graduate School of Medicine, Nagoya, Japan and 3Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska Correspondence: Dr Kazumitsu Sugiura, Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showaku, Nagoya 466-8550, Japan. E-mail: [email protected] Abbreviations: AD, atopic dermatitis; Bip, Ig heavy chain-binding protein; DFS70, dense fine speckles 70 kDa protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein, 78-kDa; KG, keratohyalin granule; LEDGF, lens epithelium-derived growth factor; PBS, phosphate-buffered saline
Received 13 February 2006; revised 8 May 2006; accepted 6 June 2006; published online 20 July 2006
& 2006 The Society for Investigative Dermatology
about 30% of AD patients (Ochs et al., 2000; U94319). DFS70 is one of several proteins producing anti-nuclear antibodies staining, and it is the same protein as p75 (Ge et al., 1998) and lens epithelium-derived growth factor (LEDGF) (Singh et al., 1999). This protein was isolated and characterized independently by three different laboratories. P75 was initially isolated as a transcriptional cofactor (Ge et al., 1998), and LEDGF was identified as a survival factor for lens epithelial cells and named LEDGF (Singh et al., 1999; Shinohara et al., 2002). LEDGF exhibits (Shinohara et al., 2002) DNA-binding and transcriptional activity (Ge et al., 1998; Singh et al., 1999; 2000), and it activates heat-shock protein 27, aB-crystalline, and involucrin in lens epithelial cells and keratinocytes in vitro (Singh et al., 2001; Kubo et al., 2002). Additionally, it promotes cell survival, enhances stress resistance in different cell lines (Shinohara et al., 2002), and induces the disassembly of gap junctions of lens epithelial cells via the activation of protein C gamma (Nguyen et al., 2003). LEDGF also interacts with HIV-1 integrase and mediates its chromatin targeting (Maertens et al., 2003; Ciuffi et al., 2005). Finally, high levels of LEDGF/ DFS70 autoantibodies are seen in AD patients (Ochs et al., 2000; Ayaki et al., 2002). Accumulating evidence suggests that LEDGF/DFS70 is involved in the pathogenesis of skin epithelial lesions, and autoantibodies targeting LEDGF/DFS70 are also frequently found in alopecia areata patients (Okamoto et al., 2004). Interestingly, expression of LEDGF/DFS70 is increased by www.jidonline.org
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endoplasmic reticulum (ER) stress (our unpublished data), suggesting that Bip/GRP78, a central sensor of ER stress, is also involved in the terminal differentiation of epidermal cells. We wished to determine the exact cellular location(s) of LEDGF/DFS70 and related proteins, including Bip/GRP78, in normal epidermis, and examine the role of LEDGF/DFS70 in the pathogenesis of the lesions induced by the autoantibodies to LEDGF/DFS70. RESULTS Specificity of antibodies to LEDGF/DFS70 in HeLa and HaCaT cell extracts
Our research goals required specific reagents for detecting LEDGF/DFS70, and we first examined the specificity of the obtained anti-LEDGF/DFS70 reagents by Western blotting. When we examined whole-cell extracts from both HeLa and HaCaT cells with the two polyclonal antisera (S-S and No. 6369) and a commercially available monoclonal antibody targeting LEDGF/DFS70, we observed a band migrating at the
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Figure 1. Specificity of the LEDGF/DFS70 IDPS70 antisera and immunocytochemistry of thin sections of the epidermis with antisera to LEDGF/DFS70. (a) Protein blot analyses of (a) HeLa cell extracts and (b) HaCaT cell extracts stained with: lane 1, commercial antibodies to LEDGF/ DFS70; lane 2, polyclonal S-S antiserum to LEDGF/DFS70; lane 3, polyclonal No. 6369 antiserum to LEDGF/DFS70; and lane 4, preimmune serum of No. 6369. Numbers on the left are molecular weight markers. Thin sections of the epidermis immunostained with (c) anti-LEDGF antiserum No. 6369 or (d) control preimmune antiserum No. 6369. LEDGF/DFS70 was visualized with horseradish peroxidase. Bar ¼ 50 mm.
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expected molecular weight of 75 kDa (Figure 1a, b). No additional proteins were detected under these experimental conditions suggesting that these antibodies were highly specific for LEDGF/DFS70. Light microscopic immunocytochemistry
LEDGF/DFS70 was localized exclusively in the nucleus in cultured NRK, Madin–Darby canine kidney, and Chinese hamster ovary-K1 cell lines (Nishizawa et al., 2001), but when we examined thin sections of epidermal tissue immunocytochemically both the nucleus and cytoplasm were labeled (Figure 1c). Both anti-LEDGF/DFS70 anti-sera, No. 6369 and S-S, gave identical staining patterns (data not shown). Under higher magnification, however, the nuclei of progenitor or mitotic cells in the basal layer were more strongly labeled than the cytoplasm. Conversely, in the more differentiated epidermal cells, LEDGF/DFS70 staining was substantially reduced in the nucleus and increased in the cytoplasm. In particular, intense LEDGF/DFS70 staining was seen in the cytoplasm of cells in the granular layer. We could not determine whether the overall increased LEDGF/DFS70 staining resulted from keratinocyte differentiation, or whether the relative protein concentration was increased by the dehydration of cells in the granular layer and the stratum corneum in vivo. However, LEDGF/DFS70 messenger RNA levels did not change in proliferating HaCaT cells compared to differentiated HaCaT cells by real-time PCR (data not shown) in vitro. The LabCyte EPI-Model system consists of normal humanderived epidermal keratinocytes cultured to form a multilayered, highly differentiated model of the human epidermis. This model is structurally similar to human epidermal tissue as shown by hematoxylin and eosin staining of thin sections (Figure 2a). When EPI-Model samples were examined immunocytochemically with the LEDGF/DFS70 antisera No. 6369 and S-S, both the nucleus and cytoplasm were labeled (Figure 2b). Nuclear staining was seen both in the basal and granular layers, and the staining intensity appeared similar in these two layers. However, cytoplasmic staining of cells in the granular layer was more prominent. Bip/GRP78 is an ER resident protein that acts a chaperone to facilitate protein folding, but it is also essential for sensing conditions of ER stress (Shen et al., 2002). We have observed protein–protein interactions between LEDGF/DFS70 and Bip/GRP78 (our unpublished data), suggesting a role for Bip/GRP78 in the biosynthesis of LEDGF/DFS70. When we examined the localization of Bip/GRP78, it was primarily found in a diffusely staining pattern consistent with an ER distribution. Surprisingly, few cells were stained in the basal layers, but staining increased substantially upon epidermal cell differentiation (Figure 3). Immunoelectron microscopy
Immunocytochemistry and light microscopy are useful for grossly examining the cellular distribution of a particular protein, but to precisely determine changes in the localization of LEDGF/DFS70 with keratinocyte differentiation we used immuno-electron microscopy. We previously observed
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Figure 2. Immunocytochemistry of thin sections of multilayered cultured keratinocytes with an antiserum to LEDGF/DFS70. Thin sections of LabCyte EPI-Model were stained with (a) hematoxylin and eosin, and were immunostained with (b) antiserum No. 6369, and with (c) preimmune serum of No. 6369. Asterisk shows the layer corresponding to the granular layer. Bar ¼ 50 mm.
Figure 3. Immunocytochemistry of thin sections of the epidermis with anti-Bip/GRP78 antibodies. Thin sections of the epidermis were immunostained with polyclonal anti-Bip/GRP78 antibodies. Arrow points to the basement membrane. Bip/GRP78 was visualized with horseradish peroxidase. Bar ¼ 50 mm.
LEDGF/DFS70 predominantly around the chromatin in the nuclei of cultured cells (Nishizawa et al., 2001), and LEDGF/ DFS70 was also seen in the nuclei of keratinocytes in the granular layer (Figure 4e). Additionally, within the cytoplasm, LEDGF/DFS70 was found exclusively in multivesicular bodylike structures surrounded by bundles of keratin, in the cells of the granular layer. LEDGF/DFS70 was also seen in the cells in the stratum corneum (Figure 4a–c), and these signals were eliminated by pre-incubating antiserum No. 6369 with the peptides used for immunization demonstrating the specificity of this staining (compare Figure 4b and d). These multivesicular body-like structures likely represent keratohyalin
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Figure 4. Localization of LEDGF/DFS70 in the KGs by immunoelectron microscopy analysis. The subcellular localization of LEDGF/DGS70 in the keratinocytes of the granular layer and stratum corneum were determined by immunoelectron microscopy. LEDGF/DFS70 was found in KGs, (a–c) in the granular layers, and (e) in the nuclei. (d) In controls, the antiserum No. 6369 pre-incubated with the immunizing peptide was used for immunoelectron microscopy. (b) Magnified image of the inset in (a). Arrow indicates the irregularly shaped and multivesicular structures of the KGs. An arrowhead points to a keratin bundle. The K4M embedding method was used. Goat anti-rabbit antiserum coupled to 10 nm immunogold particles was used for detection. Bar ¼ 200 nm.
granules (KGs) because of their structural characteristics, for example, tufted outlines and tendency to form vacuoles (Steven et al., 1990). Indeed, profilaggrin, a KGs marker, was located in these structures with LEDGF/DFS70 (Figure 5a). Thus, LEDGF/DFS70 colocalized with filaggrin in the stratum corneum (Figure 5b). We further examined whether LEDGF/DFS70 and Bip/ GRP78 colocalized in the KGs. Indeed, both LEDGF/DFS70 and Bip/GRP78 were observed in the KGs of cells in the granular layer of human epidermal cells (Figure 6a). Finally, Bip/GRP78 also localized in the stratum corneum (Figure 6b). www.jidonline.org
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Figure 5. Colocalization of LEDGF/DFS70 and profilaggrin in the KGs of cells in the granular layer. A double immunostaining method was used with anti-LEDGF/DFS70 atniserum and anti-filaggrin antibody. Profilaggrin, the precursor of filaggrin, is a marker of KGs. Five nanometer immunogold-conjugated secondary antibody was used for LEDGF/DFS70 detection and 10 nm gold for filaggrin immunostaining. (a) The inset picture is a magnified picture of square. (b) Photograph showing the same pattern of immunostaining in the stratum corneum. Bar ¼ (a) 200 nm and (b) 500 nm.
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Figure 6. Bip/GRP78 localizes in KGs and in the stratum corneum. (a, b) Double immunostaining method was used with anti-LEDGF/DFS70 antiserum and anti-Bip/GRP78 antibody. Five nanometer immunogold conjugated second antiserum was used for LEDGF/DFS70 and 10 nm for Bip/ GRP78. (a) A photograph showing KGs, and (b) a photograph showing the staining pattern in the stratum corneum. Bar ¼ (a) 200 nm and (b) 500 nm.
DISCUSSION LEDGF/DFS70 is a major autoantigen in AD, and, in order to better understand the role of LEDGF/DFS70 in health and disease, we wished to precisely characterize its subcellular distribution. We observed LEDGF/DFS70 not only in the nucleus but also in the cytoplasm of human epidermal cells, and the chaperone Bip/GRP78 was diffusely distributed in cells from the suprabasal layer to the stratum corneum but not the basal layer. Higher levels of LEDGF/DFS70 were seen in the KGs of the cells in the granular layer of the epidermis, and light and electron microscopic immunocytochemistry confirmed that the KGs first appears in cells of the granular layer that also contain LEDGF/DFS70 and Bip/GRP78. The intensity of LEDGF/DFS70 staining in the nucleus and cytoplasm of epidermal cells varied considerably. This variation may arise from the heterogeneity of the cell populations in the epidermis. The epidermis is comprised of 78
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cells at different developmental stages, such as progenitor, mitotic, post-mitotic, differentiated cells, and anuclear cells. Progenitor and mitotic cells expressed higher levels of LEDGF/DFS70 in the nucleus than in the cytoplasm, consistent with reports of human ocular tissues, fetal human brain, and human prostate cancer cells (Kubo et al., 2003; Chylack et al., 2004; Daniels et al., 2005). Transformed cultured cells are predominantly mitotic cells, which contain higher levels of LEDGF/DSF70 in the nucleus (Muro, 2001; Nishizawa et al., 2001; Maertens et al., 2004). In contrast, post-mitotic, differentiated, and terminally differentiated cells are predominantly found in the granular layer, and these cells contain high levels of LEDGF/DFS70 in the KGs. Enucleated cells in the stratum corneum, also contain high levels of LEDGF/DSF70. Because LEDGF/DFS70 is a transcriptional coactivator (Ge et al., 1998; Singh et al., 2000) and survival factor (Shinohara et al., 2002), the intensely stained cells might represent progenitor cells or mitotic cells in the basal layer. This is consistent with a number of studies showing that LEDGF/DFS70 is localized in the nucleus of many cultured cells including fetal rat epidermal cells (Muro, 2001; Nishizawa et al., 2001; Maertens et al., 2004). In contrast, differentiated, proapoptotic, and apoptotic cells in lens epithelial cells lose LEDGF/DFS70 from the nucleus (Kubo et al., 2003). Thus, in the skin, LEDGF/ DFS70 is transported from the nucleus to the cytoplasm and is accumulated in the KGs as the keratinocytes differentiate. The inclusion of LEDGF/DFS70 and Bip/GRP78 in the KGs in the cells of the granular layer may be functionally important and we propose that the KGs may act as an epidermal cell-specific protein transport system or protein disposal organelle in vivo. The KGs contain keratin-associated proteins, including profilaggrin, repetin, and caspase 14 (Steven et al., 1990; Alibardi et al., 2004; Huber et al., 2005), but not keratin (Warhol et al., 1985). Profilaggrin is cleaved into the bioactive filaggrin molecule (McKinleyGrant et al., 1989), and it aggregates with keratin filaments in the lower layers of the stratum corneum (Dale et al., 1978). It is later degraded to form natural moisturizing factors in the upper layers of the stratum corneum (Kashima et al., 1988). Caspase 14 plays an important role in the terminal differentiation of keratinocytes; however, no substrate for this protease has been found (Eckhart et al., 2000). LEDGF/DFS70 is a substrate of caspase 3 and caspase 7 in apoptotic cells (Wu et al., 2002), and we observed that the partial colocalization of LEDGF/DFS70 with caspase 14 by immunoelectron microscopy (our unpublished data). Protein compartmentalization into KGs may be important for proper keratinocyte apoptosis. Alternatively, the KGs may serve as a means to collect proteins for elimination by enucleation, a characteristic feature of keratinocytes. Further studies are clearly needed to identify the functional roles of the KGs in cells of the granular layer. The transcription of involucrin, the most abundant protein of the crosslinked keratinocyte envelope is regulated by LEDGF/DFS70 in cultured keratinocytes, and the skin of AD patients contains lower levels of involucrin (Eckert and Green, 1986; Seguchi et al., 1996; Kubo et al., 2002; Jensen et al.,
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2004). IgG deposits have been seen in the stratum corneum of AD patients (Hodgkinson et al., 1977), and these autoantibodies may target LEDGF/DFS70 in the upper layers of epidermis and interfere with its function in the nucleus as well as the KGs. This may suppress involucrin transcription in the keratinocytes of the granular layer. Thus, autoantibodies targeting LEDGF/ DFS70 in the cells of the granular layer and stratum corneum in patients with AD may alter epidermis homeostasis. In conclusion, LEDGF/DFS70, a major autoantigen in patients with AD, is a component of the KGs in the cells of the granular layer of the skin. The unique relocation of LEDGF/ DFS70 from the nucleus to the KGs coincides with the terminal differentiation of keratinocytes and suggests a possible role for DFS70/LEDGF in the KGs. Thus, autoantibodies targeting LEDGF/DFS70 may affect LEDGF/DFS70 function and promote the development of different skin diseases. MATERIALS AND METHODS Normal human skin Normal human skin for electron microscopic immunocytochemistry was obtained from the left anterior arm of a 35-year-old man and from a finger of a 74-year-old man after obtaining informed written consent. Normal human skin for light microscopic immunocytochemistry was obtained from the scalp of a patient with benign scalp tumor after obtaining informed written consent as described (Okamoto et al., 2004). The use of human materials was approved by the Ethics Committee of Nagoya University, Graduate School of Medicine. The study was conducted according to the Declaration of Helsinki Principles.
Antibodies Two different polyclonal antisera against LEDGF/DFS70 were used; one was from Dr T. Shinohara’s laboratory (S-S antiserum; Singh et al., 2000), and the other was from Dr Y Muro’s laboratory, named No. 6369 antiserum (Okamoto et al., 2004). The following mAbs were purchased from commercial sources; anti-LEDGF, anti-Bip/ GRP78 (BD Biosciences, San Jose, CA), and anti-profilaggrin (Abcam Ltd. Cambridge, UK). Polyclonal anti-Bip/GRP78 (Santa Cruz Biotecnology, Inc., Santa Cruz, CA) was also purchased.
Immunoblotting HeLa and HaCaT cell extracts The immortalized keratinocyte cell line HaCaT was kindly provided by Dr N. Fusenig (Boukamp et al., 1988). Whole-cell extracts of HeLa and HaCaT cells were obtained as previously described (Watanabe et al., 2004). Cell lysates were separated by 10% SDSPAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) as described (Sugiura and Muro, 1999). Strips of membrane were incubated with the monoclonal antiLEDGF antibodies and polyclonal anti-LEDGF antisera (S-S and No. 6369) at dilutions of 1:100, 1:100, and 1:1,000, respectively. The antibody–antigen complexes were detected with horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG (Dako Co. Ltd, Glostrup, Denmark) at 1:1,000 dilutions and visualized with Konica immunostaining (Konica, Tokyo, Japan).
Immunocytochemistry of human skin and multilayered cultured keratinocytes The LabCyte EPI-Model (J-Tec Co Ltd, Gamagori, Japan), similar to the EpiDermTM System (MatTek Co, Ashland, MA), consists of normal
human-derived epidermal keratinocytes cultured to form a multilayered and represents a highly differentiated model of the human epidermis. Immunocytochemistry on human skin and the Labcyte EPI-Model were performed as described with slight modifications (Okamoto et al., 2004). Briefly, the VECTASTAIN Elite ABC-PO (rabbit IgG) kit (Vector Laboratories Inc., Burlingame, CA) was used for staining. Thin sections (6 mm) were cut from paraffin blocks of normal human scalp skin and the LabCyte EPI-Model. The sections were immersed in 0.4% pepsin solution for 30 minutes, and soaked for 20 minutes at room temperature in 0.3% H2O2/methanol to block endogenous peroxidase activity. After washing in phosphatebuffered saline (PBS) with 0.01% Triton X-100, the sections were incubated for 30 minutes in PBS buffer with 4% BSA and followed by the anti-LEDGF antisera S-S or No. 6369 (1:1,000 dilutions) or antiBip/GRP (1:100) in PBS buffer containing 1% BSA overnight at 41C. After washing in PBS buffer, the thin sections were exposed to avidin-conjugated goat anti-rabbit Ig antibodies for 1 hour and washed again in PBS buffer. After washing, the tissue sections were immersed in VECTASTAIN Elite ABC Reagent for 30 minutes and washed again in PBS buffer. The antibody complexes were visualized by the addition 1% H2O2. Pre-immune No. 6369 serum diluted at 1:1,000 was used as a negative control.
Electron microscopic immunocytochemistry Human skin, biopsied with a dermal punch (Maruho Inc., Tokyo, Japan), was fixed in 2% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer adjusted to pH 7.4. The fixed samples were dehydrated in an ascending ethanol series up to 99.5% and embedded in Lowicryl K4M resin (Polysciences, Warrington, PV) according to the manufacturer’s protocol. Ultrathin sections (95 nm) were mounted on nickel grids coated with Formvar, and immunolabeling was carried out as described in detail (Kachi et al., 1999). Briefly, after blocking for 10 minutes with 4% bovine serum albumin in PBS, the sections were incubated with the anti-LEDGF antisera S-S or No. 6369, each diluted 1:100 (10 mg/ml). For control, we used No. 6369 (10 mg/ml ) preincubated with immunized peptides (40 mg/ml ). Anti-filaggrin and anti-Bip/GRP78 antisera were used diluted 1:20. After washing seven times with a droplet of PBS, the sections were incubated with 10 nm or 5 nm gold conjugated-goat anti-rabbit IgG or goat anti-mouse IgG (1:30 dilution: GE Healthcare, Chalfont Saint Giles, UK). After washing, the sections were fixed in 2% glutarardehyde followed by a droplet of water eight times. All sections were stained for 5 minutes with uranyl acetate before observation. For control, pre-immune No. 6369 serum diluted 1:100 was used. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS Dr K. Sugiura was supported by Grants 16790635 and 18790783, and Dr Y. Muro was supported by Grant 15591174, from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Journal of Investigative Dermatology (2007), Volume 127