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Caspase-1−Independent IL-1 Release Mediates Blister Formation in Autoantibody-Induced Tissue Injury through Modulation of Endothelial Adhesion Molecules
J Immunol 2015; 194:3656-3663; Prepublished online 20 March 2015; doi: 10.4049/jimmunol.1402688 http://www.jimmunol.org/content/194/8/3656
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Hengameh Sadeghi, Anike Lockmann, Anna-Carina Hund, Unni K. S. R. L. Samavedam, Elena Pipi, Katerina Vafia, Eva Hauenschild, Kathrin Kalies, Hendri H. Pas, Marcel F. Jonkman, Hiroaki Iwata, Andreas Recke, Michael P. Schön, Detlef Zillikens, Enno Schmidt and Ralf J. Ludwig
The Journal of Immunology
Caspase-1–Independent IL-1 Release Mediates Blister Formation in Autoantibody-Induced Tissue Injury through Modulation of Endothelial Adhesion Molecules Hengameh Sadeghi,* Anike Lockmann,† Anna-Carina Hund,† Unni K. S. R. L. Samavedam,*,‡ Elena Pipi,*,‡ Katerina Vafia,* Eva Hauenschild,x Kathrin Kalies,x Hendri H. Pas,{ Marcel F. Jonkman,{ Hiroaki Iwata,*,1 Andreas Recke,*,‡ Michael P. Scho¨n,† Detlef Zillikens,*,‡ Enno Schmidt,*,‡,2 and Ralf J. Ludwig*,‡,2
A
utoimmune bullous dermatoses (AIBDs) are characterized by autoantibodies against structural proteins of the skin and mucosal tissues. Patients suffer from considerable morbidity and increased mortality. Based on the targeted Ags, AIBDs can be divided into pemphigus and pemphigoid diseases. In pemphigus disease, autoantibodies to desmosomal proteins directly cause blister formation, whereas in pemphigoid
*Department of Dermatology, University of L€ubeck, 23538 L€ubeck, Germany; † Department of Dermatology, Venereology, and Allergology, University Medical Center, 37075 Go¨ttingen, Germany; ‡L€ubeck Institute of Experimental Dermatology, University of L€ ubeck, 23538 L€ubeck, Germany; xInstitute of Anatomy, University of L€ubeck, 23562 L€ ubeck, Germany; and {Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, the Netherlands 1 Current address: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan. 2
E.S. and R.J.L. were equal contributors.
Received for publication October 22, 2014. Accepted for publication February 15, 2015. This work was supported by the Deutsche Forschungsgemeinschaft (Excellence Cluster Inflammation at Interfaces Grant DFG EXC 306/2, Research Training Grant for Modulation of Autoimmunity DFG GRK1727/1, and Grant DFG LU877/5-1), the Dr. Robert Pfleger Foundation, the B. Braun Foundation, the Focus Program on Autoimmunity of the University of L€ ubeck, and a research and development grant from Novartis. Address correspondence and reprint requests to Prof. Ralf J. Ludwig, L€ubeck Institute of Experimental Dermatogy, University of L€ubeck, Ratzeburger Allee 160, D-23538 L€ ubeck, Germany. E-mail address: [email protected] Abbreviations used in this article: AIBD, autoimmune bullous dermatosis; AUC, area under the curve; COL7, type VII collagen; EBA, epidermolysis bullosa acquisita; IF, immunofluorescence; RT, room temperature; WT, wild-type. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402688
disease, blister formation requires the activation of immune mechanisms through the Fc portion of the autoantibodies (1, 2). Despite improved therapeutic options, the mortality of patients with pemphigus and pemphigoid remains high (3); this can be attributed, in part, to the extent of immunosuppressive therapy (4). Therefore, and because of the increasing incidence (3), there is a clear and unmet medical need for the development of effective and safe therapies for these patients. In other autoimmune and chronic inflammatory diseases, cytokine-modulating therapies have greatly improved the therapeutic armamentarium (e.g., TNF-a inhibition in rheumatoid arthritis and psoriasis) (5). In AIBD, increased cytokine expression in several compartments, including serum, blister fluid, and skin, has been well documented (6). However, with the exception of two studies, one indicating the involvement of IL-1 and TNF-a in the pathogenesis of pemphigus vulgaris (7) and the other indicating an anti-inflammatory role for IL-6 in epidermolyis bullosa acquisita (EBA) (8), no functional data are available. Expression profiling identified IL-1ra as 1 of 33 gene products expressed differentially in the skin of BALB/c mice with experimental EBA. IL-1ra expression in diseased mice was closely linked to IL-1b (9), and its local and systemic expression were controlled by IL-6 (8). Furthermore, we recently observed increased IL-1a and IL-1b serum levels in C57BL/6 mice after the induction of experimental EBA induced by transfer of anti–type VII collagen (COL7) IgG. In addition, IL-1ra administration, which blocked IL-1 function, in a preventive experimental setting impaired skin blistering in a model of autoantibody transfer–induced EBA (8). Moreover, elevated levels of IL-1b, but not IL-1a, were detected in blister
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Although reports documented aberrant cytokine expression in autoimmune bullous dermatoses (AIBDs), cytokine-targeting therapies have not been established in these disorders. We showed previously that IL-6 treatment protected against tissue destruction in experimental epidermolysis bullosa acquisita (EBA), an AIBD caused by autoantibodies to type VII collagen (COL7). The antiinflammatory effects of IL-6 were mediated by induction of IL-1ra, and prophylactic IL-1ra administration prevented blistering. In this article, we demonstrate elevated serum concentrations of IL-1b in both mice with experimental EBA induced by injection of anti-COL7 IgG and in EBA patients. Increased IL-1a and IL-1b expression also was observed in the skin of anti-COL7 IgGinjected wild-type mice compared with the significantly less diseased IL-1R–deficient or wild-type mice treated with the IL-1R antagonist anakinra or anti–IL-1b. These findings suggested that IL-1 contributed to recruitment of inflammatory cells into the skin. Accordingly, the expression of ICAM-1 was decreased in IL-1R–deficient and anakinra-treated mice injected with antiCOL7. This effect appeared to be specifically attributable to IL-1 because anakinra blocked the upregulation of different endothelial adhesion molecules on IL-1–stimulated, but not on TNF-a–stimulated, cultured endothelial cells. Interestingly, injection of caspase-1/11–deficient mice with anti-COL7 IgG led to the same extent of skin lesions as in wild-type mice. Collectively, our data suggest that IL-1, independently of caspase-1, contributes to the pathogenesis of EBA. Because anti–IL-1b in a prophylactic setting and anakinra in a quasi-therapeutic setting (i.e., when skin lesions had already developed) improved experimental EBA, IL-1 appears to be a potential therapeutic target for EBA and related AIBDs. The Journal of Immunology, 2015, 194: 3656–3663.
The Journal of Immunology
with experimental EBA and in normal rabbit IgG-injected controls at the indicated time points using Bio-Plex (Bioglobe). IL-1 expression in skin was determined using immunohistochemistry. In brief, for both IL-1a and IL-1b staining, 6 mm-thick sections were incubated with the prediluted primary Ab—goat anti-mouse IL-1a or goat anti-mouse IL-1b Ab, respectively (both R&D Systems, Wiesbaden, Germany)—at room temperature (RT) for 1 h. Nonspecific binding was blocked using 5% rabbit serum. Rabbit anti-goat Ab (Dako, Hamburg, Germany) was used as secondary Ab. Incubation with HistoGreen (LINARIS, Germany), counterstaining with hematoxylin, and dehydrating were performed before mounting on slides. The intensity of IL-1 expression was scored semiquantitatively by an observer who was unaware of the nature of the specimen. The detection of ICAM-1 expression was performed similarly (i.e., Armenian hamster anti-mouse served as the primary Ab; BD Biosciences, Heidelberg, Germany). Rabbit anti-Armenian hamster (Abcam, Cambridge, U.K.) was used as a secondary Ab. For Gr-1 staining, rat antimouse Ly-6G (clone RB6-8C5) was used as primary Ab, and goat anti-rat IgG (both from Abcam) was used as secondary Ab.
RT-PCR
Materials and Methods
For RT-PCR analysis, lesional (n = 4) and nonlesional (n = 5) skin was obtained from mice on day 6 after injection with rabbit anti-murine COL7 IgG and normal rabbit IgG, respectively. RT-PCR of mouse skin was performed as described (19) using the following sets of primers: MLN51 forward (59-CCAAGCCAGCCTTCATTCTTG-39) and MLN51 reverse (59-TAACGCTTAGCTCGACCACTCTG-39) and IL-1b forward (59CTTCCAGGATGAGGACATGAG-39) and IL-1b reverse (59-CACACCAGCAGGTTATCATC-39).
Experiments with human samples
Treatment protocols
For the determination of IL-1a and IL-1b concentrations, serum from EBA patients (n = 26) fulfilling the following criteria were used: presentation with skin lesions resembling EBA; linear IgG and/or IgA deposition by direct immunofluorescence (IF) microscopy; and an u-serrated pattern by direct IF microscopy and/or detection of Abs against COL7. Serum from blood donors (n = 52) served as a reference. All of the experiments using human samples were approved by the local ethics committee (University of L€ ubeck, L€ ubeck, Germany) and were performed according to the Declaration of Helsinki. Blood donors and patients provided their written informed consent prior to study participation.
Kineret (anakinra; Biovitrum, Stockholm, Sweden) was injected i.p. into mice at a dosage of either 100 or 200 mg/kg/d. PBS served as a control. Anakinra or PBS treatment was initiated on day 0 and maintained throughout the experiments in the Ab-transfer model. In immunizationinduced EBA, anakinra was administered to mice when $2% of the body surface area was affected by skin lesions; it was continued for a total of 2 wk. Anti-IL-1b (clone ACZ885; Novartis, N€urnberg, Germany) was injected at 200 mg/mouse 1 d before and 2 and 4 d after the first anti-COL7 IgG injection.
Mice SJL/J, C57BL/6, IL-1R-deficient (2/2), and caspase-1/11–deficient mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice aged 6–8 wk were used for the experiments. The mice were kept in specific pathogen–free conditions and fed standard mouse chow and acidified drinking water ad libitum. All of the clinical examinations, biopsies, and blood collections were performed under anesthesia using i.p. administration of a mixture of ketamine (100 mg/g) and xylazine (15 mg/g). The animal experiments were approved by the local authority of the Animal Care and Use Committee (Kiel, Germany) and were performed by certified personnel.
Induction of experimental EBA Rabbit anti-murine COL7 IgG was prepared as previously described (15). In brief, rabbits were immunized with recombinant proteins of the NC1 domain of murine COL7. IgG from immune and normal rabbit sera were purified by affinity chromatography using protein G affinity. Passivetransfer studies into mice followed the published protocols (15, 16). Each experiment was performed twice, using different anti-COL7 IgG preparations. Disease severity was expressed as the percentage of body surface area affected by skin lesions and was determined at three time points (days 4, 8, and 12). From these time points, the area under the curve (AUC) was calculated. Blood and tissue samples were collected on day 12. EBA was induced by immunization with an immunodominant fragment located within murine COL7, as previously reported (17), with minor modifications described elsewhere (18). After immunization, the extent of clinical EBA manifestation was determined weekly. If EBA skin lesions affected 2% or more of the body surface area of individual mice, they were allocated to either anakinra or PBS (see below). The treatments were administered over a 2-wk period, and EBA severity (percentage of affected body surface area) was monitored weekly.
Determination of IL-1 levels in serum and skin Serum from EBA patients and normal controls were analyzed for the expression of IL-1a and IL-1b using Bio-Plex (Bioglobe, Hamburg, Germany). Serum concentrations of IL-1a and IL-1b were determined in mice
Western blotting with extracts of mouse skin Ear specimens from mice were excised, immediately snap-frozen in liquid nitrogen, and stored at 280˚C until further analysis. To prepare total protein skin extracts, frozen ears were ground in liquid nitrogen and homogenized in T-PER reagent (Thermo Scientific, Darmstadt, Germany; 1:6, w/v), with the addition of protease inhibitor mixture set III (Calbiochem, Darmstadt, Germany; 1:100, v/v). Tissue homogenates were centrifuged at 10,000 3 g for 10 min at 4˚C to pellet debris, and the supernatants were collected. Next, the samples were incubated with protein A/G PLUS–Agarose beads (Santa Cruz Biotechnology, Heidelberg, Germany; 5:1, v/v) at 4˚C on a rotating device overnight. The beads were pelleted by centrifugation at 1000 3 g for 5 min at 4˚C, and the supernatants were collected and stored at 280˚C until use. The total protein concentration was determined according to the BCA method (Thermo Scientific). Protein samples were separated by SDS-PAGE under reducing conditions on 15% polyacrylamide gel and transferred to a 0.2-mm pore size nitrocellulose membrane for Western blotting. The membranes were blocked with 5% w/v skim milk powder in TBST for 1 h at RT. Before each step, the membranes were washed five times for 10 min in TBST. All of the Abs were diluted in blocking buffer. Goat polyclonal IgG against murine IL-1b (R&D Systems; cat. no. AF-401-NA) and mouse monoclonal IgG against actin (Santa Cruz Biotechnology; cat. no. sc-47778) were used at 1:400 or 1:3000 dilution, respectively, overnight at 4˚C. HRPlabeled secondary Abs for goat (Dako; cat. no. P0449) and mouse (Jackson ImmunoResearch, Suffolk, U.K.; cat. no. 715-035-150) were applied at 1:20,000 and 1:50,000 dilution, respectively, for 1 h at RT and detected using Amersham ECL Prime reagent (GE Healthcare).
Culture and in vitro activation of human endothelial cells HUVECs (PromoCell, Heidelberg, Germany) were cultured in Endothelial Cell Growth Medium (PromoCell) at 37˚C in a humidified atmosphere containing 5% CO2. Four hours prior to activation, the medium was replaced by Endothelial Cell Basal Medium (PromoCell) supplemented with 10% FCS. To induce adhesion molecule expression, endothelial cells were activated with recombinant human IL-1b (5 ng/ml) or TNF-a (25 ng/ml) for 4 h. These experiments were performed in the presence or absence
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fluid from bullous pemphigoid patients, an AIBD caused by autoantibodies against type XVII collagen, and were correlated with the magnitude of skin lesions (10, 11). Given that some cytokines have potent anti-inflammatory activities (12) or trigger the release of chemokine antagonists (13), we investigated the contribution of IL-1 to the prototypical AIBD EBA in which the autoimmune response is directed against COL7 (14). Based on the observation of reduced skin blistering in autoantibody transfer–induced EBA upon prophylactic anakinra application (8), we hypothesized that IL-1 would exert proinflammatory activity in EBA. However, it remained unclear how IL-1 modulates blister formation in experimental EBA; whether this effect is mediated by IL-1a, IL-1b, or both; whether treatment with IL-1–modulating compounds can improve already established experimental EBA; how IL-1 expression is controlled; and whether these findings are also applicable to EBA patients. We addressed these potentially clinically relevant questions using several animal models of EBA, as well as a large collection of EBA sera. Given that blockade of IL-1 has therapeutic effects in established EBA, and there is increased IL-1 expression in EBA patients, blockade of IL-1 might be a promising therapeutic option for EBA patients.
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3658 of anakinra (5 mg/ml), which was added to the cell cultures 1 h before treatment. Nontreated cells served as negative controls.
Histology and IF microscopy
Statistics The data were analyzed using SigmaPlot software, version 12 (Systat Software, Chicago, IL), and applied tests and 95% confidence intervals are indicated in the respective text and figure legends. For analysis of rankscaled or semiquantitative data, we used ordinal logistic regression (polr) and mosaic plots, provided by GNU R open source statistical software (http://www.r-project.org), together with the “MASS” package. Ordinal logistic regression models were evaluated first by likelihood ratio testing. In the second step, models with a significant likelihood ratio testing were evaluated further; p values are given for the difference between each group and the controls. A p value , 0.05 was considered statistically significant after appropriate adjustment for multiple testing.
Results Increased IL-1a and IL-1b serum concentrations and correlations with disease severity in experimental and human EBA The induction of experimental EBA in BALB/c mice, by injection of anti-COL7 IgG, led to increases in serum levels of IL-1b, but not IL-1a, compared with untreated control mice (Fig. 1A). The extent of skin lesions correlated with IL-1b, but not with IL-1a, serum levels (Fig. 1B, 1C). Compared with healthy controls, 6and 4-fold increases in IL-1a and IL-1b serum levels were found in EBA patients (Fig. 1A). IL-1b mRNA expression also was significantly increased in lesional skin compared with healthy mouse skin. Six days after the first IgG injection, IL-1b mRNA expression was significantly increased in anti-COL7 IgG-injected animals compared with mice injected with normal rabbit IgG. Median relative (in relation to MLN51) IL-1b expression in mice injected with normal rabbit IgG was 0.16 (0.12–0.57, 25/75 percentiles), whereas it was 34.6 (21.1–194; p = 0.0159, Mann– Whitney rank-sum test) in lesional skin of mice injected with antiCOL7 IgG. Furthermore, we detected increased protein expression of IL-1a and IL-1b in the skin of mice after EBA induction compared with IL-1R2/2 mice and anakinra-treated wild-type (WT) animals (Fig. 1D–F). Induction of experimental EBA was impaired in mice with either genetic or prophylactic pharmacologic inhibition of IL-1 To test whether the increased local and/or systemic IL-1a and IL-1b levels were of functional relevance, IL-1 function was inhibited. EBA was induced in IL-1R2/2 and WT C57BL/6 control mice. Mice of both strains developed experimental EBA; however, in IL-1R2/2 mice, the overall extent of skin blistering
was reduced to 52% of that in control mice (p , 0.001; Fig. 2A, 2B). Comparisons of clinical scores between the two strains at 4, 8, and 12 d showed that the affected body surface area was significantly lower in IL-1R2/2 mice at all time points (day 4: 1.6 6 0.4% versus 0.6 6 0.2% [p = 0.008]; day 8: 7.4 6 0.7% versus 5.2 6 0.7% [p = 0.04]; day 12: 20.7 6 1.8% versus 10.1 6 1.7% [p , 0.001] in WT and IL-1R2/2 mice, respectively). The clinical effects were accompanied by reduced dermal inflammatory infiltrates in IL-1R2/2 treated mice compared with controls (Fig. 2C). Staining for Gr-1+ cells revealed similar results, demonstrating a reduced expression of GR-1 in both IL-1R2/2 and anakinratreated mice (Fig. 2D). Treatment with a function-blocking Ab directed against IL-1b fully recapitulated and even exceeded the effects of total IL-1 inhibition. Compared with C57BL/6 mice injected with isotypecontrol Abs, anti–IL-1b reduced blister formation to 44% (p = 0.001, Fig. 2E, 2F). Blockade of IL-1 function also was associated with decreased expression of both IL-1a and IL-1b in mouse skin (Fig. 1). In contrast, levels of tissue-bound IgG and C3 were similar in all of the mice (Fig. 2B), indicating that the clinical effects were not due to altered IgG metabolism and/or effects on complement activation. Therapeutic application of anakinra led to improvement of immunization-induced EBA Following the prophylactic pharmacological inhibition of IL-1, an IL-1–targeting treatment was applied in a quasitherapeutic setting (i.e., anakinra administration was initiated when mice already had established EBA skin lesions). For this purpose, the immunization-induced EBA mouse model was used. At the initiation of treatment, the clinical disease severity in mice randomly allocated to control and anakinra treatments was identical (i.e., 2.6 6 0.2% affected body surface area). Clinical EBA manifestations continuously progressed in PBS-injected mice (3.2 6 0.2% at week 1 and 3.9 6 0.1% at week 2), whereas it declined in anakinra-treated mice (2.1 6 0.2% at week 1 and 2.0 6 0.2% at week 2, Fig. 3). Corresponding disease activity scores were significantly lower in the anakinra group compared with the PBS group (week 1, p = 0.004; week 2, p , 0.001; AUC, p , 0.001). Inhibition of IL-1 function led to reduced expression of endothelial adhesion molecules Because the induction of skin lesions in experimental EBA depends on CD18 (23) and because IL-1 regulates the expression of the respective endothelial ligands for LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and integrin CD11c/CD18 (24), the expression of ICAM-1 (CD54) in the skin was evaluated in experimental EBA, with and without impaired IL-1 function. In mice with experimental EBA, ICAM-1 expression was detected both on endothelial cells and on basal keratinocytes (Fig. 4A, 4B). In contrast, ICAM-1 expression was almost absent in mice injected with normal rabbit IgG (Fig. 4A). Similarly, blockade of IL-1 function in mice injected with anti-COL7 IgG reduced ICAM-1 expression (Fig. 4A, 4B). To test whether this inhibitory effect of IL-1 blockade also affected other endothelial adhesion molecules, such as VCAM-1 (CD106) and E-selectin (CD62E), the expression of these molecules was assessed on HUVECs stimulated in the absence or presence of anakinra. In line with the in vivo findings, IL-1 induced ICAM-1 expression, which could specifically be inhibited by anakinra treatment (Fig. 4C). Endothelial VCAM-1 and E-selectin expression paralleled ICAM-1 expression (Fig. 4C).
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Murine specimens were subjected to H&E staining, according to a standard protocol. The extent of leukocyte infiltration was scored semiquantitatively in a blinded fashion, as described previously (20). Direct IF microscopy for the detection of rabbit IgG and murine C3 in experimental EBA was performed on 6-mm cryosections, as described, using goat anti-rabbit IgG (Dako) and goat anti-murine C3 (MP Biomedicals, Kaysersberg, France), both labeled with FITC. IF microscopy on cultured HUVECs was performed as reported (21, 22). In brief, cells were fixed in 220˚C cold methanol for 5 min and washed carefully with PBS before blocking with 5% FCS in PBS for 1 h. Anti-VCAM Ab (clone STA; Immunotools, Friesoythe, Germany), anti-ICAM Ab (clone 15.2; FITC-labeled; Immunotools), and anti–E-selectin Ab (clone 1-2B6; PE-labeled; Abs-online, Aachen, Germany) were diluted in PBS containing 2.5% FCS and incubated overnight before washing with PBS. Anti-VCAM reactivity was visualized by goat anti-mouse IgG conjugated with Alexa Fluor 488 (Cell Signaling Technology, Danvers, MA). Mounting medium containing 1% DAPI was added to each culture slide. Stained cells were analyzed using an Axio Imager M1 microscope (Carl Zeiss Meditec, Jena, Germany). Standardized exposure times were used for all of the images.
IL-1 IN EBA
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FIGURE 1. Induction of experimental EBA leads to increased IL-1a and IL-1b expression in serum and skin. (A) Induction of experimental EBA by injections of anti-COL7 IgG increased serum concentrations of IL-1b, but not IL-1a, in BALB/c mice. The data were based on five mice injected with normal rabbit (NR) IgG and eight mice injected with anti-COL7 IgG (mean 6 SEM). In line with this observation in mice, increased serum concentrations of IL-1a and IL-1b were observed in EBA patients compared with controls. The data were based on 52 control subjects’ and 26 EBA patients’ sera (mean 6 SEM). *p , 0.05, rank-sum test. (B) In experimental EBA, IL-1a serum levels did not correlate with the body surface area affected by EBA (p = not significant [ns]). The data were based on four mice injected with normal rabbit IgG (day 12) and three and four mice injected with anti-COL7 IgG on days 6 and 12, respectively. The dashed line is the regression line, and the dotted lines indicate the confidence intervals. (C) In contrast to IL-1a, IL-1b serum levels correlated with the body surface area affected by EBA (p = 0.006, r = +0.763). The data were based on four mice injected with normal rabbit IgG (day 12) and three and four mice injected with anti-COL7 IgG on days 6 and 12, respectively. The dashed line is the regression line, and the dotted lines indicate the confidence intervals. (D) Representative cutaneous expression of IL-1a and IL-1b in the indicated treatment groups. Scale bar, 100 mm. (E) Semiquantitative staining intensities of cutaneous IL-1a expression. All of the mice were injected with anti-COL7 IgG. Compared with C57BL/6 control mice, no significant effect on IL-1a expression was observed (likelihood ratio test on ordered logistic regression model: p = 0.06). Column height represents the number of mice with staining intensities ranging from 0 to 3, and column width represents the number of mice in the different treatment groups. (F) Corresponding results for cutaneous IL-1b expression. Compared with C57BL/6 control mice (n = 17), a significant reduction in IL-1b expression was noted in IL-1R2/2 mice (n = 14; p = 0.018) and in anakinra-treated mice (n = 19; p = 0.02).
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IL-1 IN EBA
Increased IL-1 expression in experimental EBA was independent of caspase-1 expression Because caspase-1 is an important regulator of IL-1b release (25), we next analyzed whether blockade of caspase-1 had an impact on the development of skin lesions in experimental EBA. At the same time, we assessed whether the increased IL-1b expression in this mouse model was controlled by caspase-1. Unexpectedly, com-
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FIGURE 2. Blister formation in experimental EBA is impaired in IL-1R-deficient mice and in mice treated with an anti-IL-1b Ab. (A) Disease severity in the indicated groups is expressed as AUC calculated from the affected body surface area 4, 8 and 12 d after the initial anti-COL7 IgG injection. The data were based on 11–16 mice/group and are presented as mean 6 SEM. *p , 0.05 (t test). (B) Representative clinical pictures, H&E-stained skin biopsies, IgG and C3 deposition at the dermal–epidermal junction 12 d after the initial anti-COL7 IgG injection in the indicated groups. Scale bar, 100 mm. (C) Semiquantitative determination of dermal leukocyte infiltrates in H&E-stained sections. All of the mice were injected with anti-COL7 IgG. Compared with C57BL/6 control mice (n = 16), a decrease in dermal infiltration was noted in IL-1R2/2 mice (n = 8; p = 0.02). Column height represents the number of mice with staining intensities ranging from 0 to 3, and column width corresponds to the number of mice in the different treatment groups. (D) In line, infiltration with Gr-1+ myeloid cells was increased in mice with experimental EBA (p , 0.001, ANOVA on Ranks with Holm-Sidak posttest), as opposed to mice injected with control IgG. Genetic (p , 0.001) or pharmacological (p = 0.002) blockade of IL-1 function significantly reduced the infiltration of the skin with Gr-1+ cells. Scale bar, 200 mm. (E) Disease severity in the indicated groups is expressed as the area under the curve (AUC) calculated from the affected body surface area 4, 8 and 12 d after the initial anti-COL7 IgG injection. The data were based on a minimum of eight mice/group and are mean 6 SEM. *p , 0.05 (t test). (F) Representative clinical images 12 d after the initial anti-COL7 IgG injection in the indicated treatment groups.
pared with WT animals, caspase-1/112/2 mice had similar clinical phenotypes after injections of anti-COL7 IgG. When data from two independent experiments were considered together, overall disease severity (expressed as AUC) was not affected; however, on day 8, a significantly smaller body surface area was affected in caspase-1/112/2 mice compared with WT mice (Fig. 5A, 5B). This finding pointed to noncaspase-1 extracellular processing of
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the IL-1b precursor (e.g., elastase) or cathepsin G in EBA. To test this assumption, IL-1b protein expression was determined in C57BL/6 WT and caspase-1/112/2 mice after injections of antiCOL7 IgG. In both strains, similar IL-1b protein expression was observed (Fig. 5C).
Discussion To our knowledge, this study provides the first experimental evidence for the potential therapeutic use of IL-1 inhibition in AIBD. Serum concentrations of IL-1a and IL-1b were increased and correlated with disease severity in the passive transfer mouse model of EBA, a prototypic AIBD characterized by autoimmunity to COL7. Elevated serum levels of IL-1b were described previously in blister fluid, but not sera, and correlated with disease activity in patients with bullous pemphigoid, another subepidermal AIBD characterized by autoimmunity against COL17 (1, 10, 11). Similarly, in a recently established model of bullous pemphigoid that was induced by the passive transfer of rabbit antimurine COL17 IgG in adult mice (26), serum IL-1b levels correlated with the extent of skin lesions (F.S. Schulze and E. Schmidt, unpublished observations). We found that blockade of IL-1b impaired the induction of experimental EBA, and blockade of IL-1 function improved already established skin lesions. These data support the notion that inhibition of IL-1, in particular of IL-1b, might be a novel therapeutic modality for EBA and bullous pemphigoid. So far, three IL-1–targeted agents have been approved: the recombinant form of the naturally occurring IL-1ra anakinra; the soluble decoy receptor rilonacept, which blocks both IL-1a and IL-1b; and the anti–IL-1b neutralizing mAb canakinumab (27). These drugs have been used successfully in various inflammatory disorders, including the autoimmune diseases rheumatoid arthritis and relapsing polychondritis (27). In fact, IL-1b inhibition would be the first therapeutic regimen without unspecific immunosuppressive/
immunomodulative action for AIBDs, such as EBA and bullous pemphigoid (1, 28). In EBA, blister formation depends on and is initiated by the binding of autoantibodies to COL7, located at the dermal–epidermal junction (29, 30). Subsequently, Fc-dependent mechanisms result in the formation of a proinflammatory milieu in the skin (31). Complement activation significantly (15, 32, 33), but not exclusively (34), contributes to the formation of this proinflammatory milieu. Finally, proteases, such as those released from skin-infiltrated inflammatory cells were seen in this study, and the induction of EBA led to increased endothelial ICAM-1 expression, which was absent if IL-1 function was blocked. In vitro experiments extended these findings to additional endothelial adhesion molecules, such as VCAM-1 and E-selectin. Dependence on adhesion molecule expression in the pathogenesis of EBA was demonstrated by the observed absence of skin lesions in CD18deficient mice after the injection of anti-COL7 IgG (23). Collectively, these data suggest that interactions of leukocyte b2 integrins (CD18) with endothelial adhesion molecules of the Ig superfamily are indispensable for blister formation in experimental EBA. Similar findings were reported in neonatal mice with experimental bullous pemphigoid: blockade of CD11a, CD11b, or CD18 protected mice from blistering after injection of Abs to COL17. In this bullous pemphigoid model, detailed analysis of the neutrophil inflammatory response revealed that CD11a was necessary for neutrophil recruitment, whereas CD11b mediated late neutrophil accumulation and neutrophil apoptosis (35). In addition, neutrophil recruitment into the skin also was shown to depend on additional adhesion molecules, including selectins (36), and on junctional adhesion molecules (20). Hence, it is tempting to speculate that blockade of adhesion molecules might also protect mice from blister formation in experimental EBA. Furthermore, our data demonstrated that IL-1b expression in the skin and IL-1b secretion into the serum were, at least to a major
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FIGURE 3. Anakinra treatment leads to improvement of already established skin blistering in immunization-induced experimental EBA. SJL/J mice with already established skin blistering after immunization with COL7 were treated with anakinra (100 mg/kg, n = 11) for 2 wk. Control mice received PBS (n = 10). (A) Analysis of the extent of blistering over time showed an increase in skin disease during the observation period in PBS-injected mice. In contrast, disease severity decreased in mice treated with anakinra. (B) Cumulative disease severity, expressed as AUC during the 2-wk treatment period, was reduced significantly in anakinratreated mice. Compared with PBS-injected mice, animals treated with anakinra had significantly reduced overall disease activity. Representative clinical disease manifestation at the beginning of treatment (Week 0) and at the end of the treatment period (Week 2) in PBStreated (C) and anakinra-treated (D) animals. *p , 0.001, t test.
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IL-1 IN EBA
extent, not mediated by caspase-1. This finding indicates an alternative scenario for the processing of the extracellular IL-1b precursor. Because blockade of IL-1 function in both EBA models led to incomplete inhibition of skin blistering, and lack of caspase1/11 expression in mice showed little to no impact on blister formation, it is tempting to speculate that neutrophil-derived serine proteases (such as neutrophil elastase, proteinase-3, and cathepsin G) (37, 38) cleave to the IL-1b precursor and control IL-1b protein expression. After extravasation into the skin, Gr-1+ leukocytes bind to immune complexes located at the dermal–epidermal junction in an FcgRIV-dependent (mice) or FcgRIIA- and FcgRIIIB-dependent
(humans) manner. Interestingly, these extravasated cells are indispensable for blister formation by the release of reactive oxygen species and proteolytic enzymes (30), and they express IL-1R after migration. This increased IL-1R expression is functionally relevant because the stimulation of neutrophils with IL-1 resulted in the transcription of NF-kB and the release of a number of downstream chemokines (39). Therefore, the inhibition of IL-1 might also block this increased IL-1R expression on neutrophils, including downstream neutrophil activation. In summary, this study provides novel insight into the contributions of IL-1 to the pathogenesis of autoantibody-induced tissue injury in a prototypical organ-specific autoimmune dis-
FIGURE 5. Increased IL-1 expression in experimental EBA is independent of caspase-1 expression. (A) EBA-affected body surface area (%) in caspase 1/1–deficient (Casp KO, n = 9) and control C57BL/6 mice (n = 10) during the 12-d observation period. In both strains, the onset of blistering was observed at day 4, and it was slightly, but significantly, delayed in Casp KO mice. (B) At the end of the experiment and with regard to overall disease severity, expressed as the AUC from (A), both strains of mice showed comparable magnitudes of blistering. (C) On day 12, ear samples were obtained, and IL-1b expression was quantified by Western blotting of the tissue extracts. An equal amount of IL-1b was observed in WT and Casp KO mice. Five representative experiments of n = 6/group are shown.
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FIGURE 4. Induction of experimental EBA leads to increased cutaneous expression of ICAM-1, which is sensitive to blockade of IL-1 function. (A) Representative ICAM-1 expression 12 d after the first injection of anti-COL7 IgG into mice of the indicated treatment groups. ICAM-1 was almost completely absent in mice injected with normal rabbit (NR) IgG, whereas ICAM-1 was expressed on keratinocytes and around the vasculature in mice with experimental EBA. (B) Overall, visual assessment of ICAM-1 staining intensity in mice with experimental EBA showed a significant decrease if IL-1 function was blocked by knockout (n = 15; p = 0.011) or anakinra (n = 16; p = 0.014) compared with control WT mice (n = 10). Column height represents the number of mice with staining intensities ranging from 0 to 3, and column width represents the number of mice in the different treatment groups. (C) Stimulation of HUVECs with either IL-1b or TNF-a led to increased expression of ICAM-1, VCAM-1, and E-selectin. Anakinra (Ana) selectively blocked the IL-1b–induced, but not the TNF-a–induced, upregulation of adhesion molecules. Nuclei are shown in blue, and the expression of the indicated adhesion molecules is shown in green or pink. Original magnification 3 400. The data shown are representative of three experiments.
The Journal of Immunology ease. We showed a proinflammatory contribution of IL-1, which was independent of caspase-1 expression. This proinflammatory effect of IL-1 was associated with the induction of endothelial adhesion molecule expression. This proinflammatory role for IL-1 in experimental EBA could be considered when therapeutic decisions must be made in patients with treatment-refractory and/or relapsing EBA and other subepidermal autoimmune skin blistering diseases with a similar pathogenesis, such as bullous pemphigoid.
Acknowledgments The anti–IL-1b Ab was kindly provided by Novartis (Basel, Switzerland).
Disclosures The authors have no financial conflicts of interest.
References
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Caspase-1−Independent IL-1 Release Mediates Blister Formation in Autoantibody-Induced Tissue Injury through Modulation of Endothelial Adhesion Molecules
J Immunol 2015; 194:3656-3663; Prepublished online 20 March 2015; doi: 10.4049/jimmunol.1402688 http://www.jimmunol.org/content/194/8/3656
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Hengameh Sadeghi, Anike Lockmann, Anna-Carina Hund, Unni K. S. R. L. Samavedam, Elena Pipi, Katerina Vafia, Eva Hauenschild, Kathrin Kalies, Hendri H. Pas, Marcel F. Jonkman, Hiroaki Iwata, Andreas Recke, Michael P. Schön, Detlef Zillikens, Enno Schmidt and Ralf J. Ludwig
The Journal of Immunology
Caspase-1–Independent IL-1 Release Mediates Blister Formation in Autoantibody-Induced Tissue Injury through Modulation of Endothelial Adhesion Molecules Hengameh Sadeghi,* Anike Lockmann,† Anna-Carina Hund,† Unni K. S. R. L. Samavedam,*,‡ Elena Pipi,*,‡ Katerina Vafia,* Eva Hauenschild,x Kathrin Kalies,x Hendri H. Pas,{ Marcel F. Jonkman,{ Hiroaki Iwata,*,1 Andreas Recke,*,‡ Michael P. Scho¨n,† Detlef Zillikens,*,‡ Enno Schmidt,*,‡,2 and Ralf J. Ludwig*,‡,2
A
utoimmune bullous dermatoses (AIBDs) are characterized by autoantibodies against structural proteins of the skin and mucosal tissues. Patients suffer from considerable morbidity and increased mortality. Based on the targeted Ags, AIBDs can be divided into pemphigus and pemphigoid diseases. In pemphigus disease, autoantibodies to desmosomal proteins directly cause blister formation, whereas in pemphigoid
*Department of Dermatology, University of L€ubeck, 23538 L€ubeck, Germany; † Department of Dermatology, Venereology, and Allergology, University Medical Center, 37075 Go¨ttingen, Germany; ‡L€ubeck Institute of Experimental Dermatology, University of L€ ubeck, 23538 L€ubeck, Germany; xInstitute of Anatomy, University of L€ubeck, 23562 L€ ubeck, Germany; and {Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, the Netherlands 1 Current address: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan. 2
E.S. and R.J.L. were equal contributors.
Received for publication October 22, 2014. Accepted for publication February 15, 2015. This work was supported by the Deutsche Forschungsgemeinschaft (Excellence Cluster Inflammation at Interfaces Grant DFG EXC 306/2, Research Training Grant for Modulation of Autoimmunity DFG GRK1727/1, and Grant DFG LU877/5-1), the Dr. Robert Pfleger Foundation, the B. Braun Foundation, the Focus Program on Autoimmunity of the University of L€ ubeck, and a research and development grant from Novartis. Address correspondence and reprint requests to Prof. Ralf J. Ludwig, L€ubeck Institute of Experimental Dermatogy, University of L€ubeck, Ratzeburger Allee 160, D-23538 L€ ubeck, Germany. E-mail address: [email protected] Abbreviations used in this article: AIBD, autoimmune bullous dermatosis; AUC, area under the curve; COL7, type VII collagen; EBA, epidermolysis bullosa acquisita; IF, immunofluorescence; RT, room temperature; WT, wild-type. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402688
disease, blister formation requires the activation of immune mechanisms through the Fc portion of the autoantibodies (1, 2). Despite improved therapeutic options, the mortality of patients with pemphigus and pemphigoid remains high (3); this can be attributed, in part, to the extent of immunosuppressive therapy (4). Therefore, and because of the increasing incidence (3), there is a clear and unmet medical need for the development of effective and safe therapies for these patients. In other autoimmune and chronic inflammatory diseases, cytokine-modulating therapies have greatly improved the therapeutic armamentarium (e.g., TNF-a inhibition in rheumatoid arthritis and psoriasis) (5). In AIBD, increased cytokine expression in several compartments, including serum, blister fluid, and skin, has been well documented (6). However, with the exception of two studies, one indicating the involvement of IL-1 and TNF-a in the pathogenesis of pemphigus vulgaris (7) and the other indicating an anti-inflammatory role for IL-6 in epidermolyis bullosa acquisita (EBA) (8), no functional data are available. Expression profiling identified IL-1ra as 1 of 33 gene products expressed differentially in the skin of BALB/c mice with experimental EBA. IL-1ra expression in diseased mice was closely linked to IL-1b (9), and its local and systemic expression were controlled by IL-6 (8). Furthermore, we recently observed increased IL-1a and IL-1b serum levels in C57BL/6 mice after the induction of experimental EBA induced by transfer of anti–type VII collagen (COL7) IgG. In addition, IL-1ra administration, which blocked IL-1 function, in a preventive experimental setting impaired skin blistering in a model of autoantibody transfer–induced EBA (8). Moreover, elevated levels of IL-1b, but not IL-1a, were detected in blister
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Although reports documented aberrant cytokine expression in autoimmune bullous dermatoses (AIBDs), cytokine-targeting therapies have not been established in these disorders. We showed previously that IL-6 treatment protected against tissue destruction in experimental epidermolysis bullosa acquisita (EBA), an AIBD caused by autoantibodies to type VII collagen (COL7). The antiinflammatory effects of IL-6 were mediated by induction of IL-1ra, and prophylactic IL-1ra administration prevented blistering. In this article, we demonstrate elevated serum concentrations of IL-1b in both mice with experimental EBA induced by injection of anti-COL7 IgG and in EBA patients. Increased IL-1a and IL-1b expression also was observed in the skin of anti-COL7 IgGinjected wild-type mice compared with the significantly less diseased IL-1R–deficient or wild-type mice treated with the IL-1R antagonist anakinra or anti–IL-1b. These findings suggested that IL-1 contributed to recruitment of inflammatory cells into the skin. Accordingly, the expression of ICAM-1 was decreased in IL-1R–deficient and anakinra-treated mice injected with antiCOL7. This effect appeared to be specifically attributable to IL-1 because anakinra blocked the upregulation of different endothelial adhesion molecules on IL-1–stimulated, but not on TNF-a–stimulated, cultured endothelial cells. Interestingly, injection of caspase-1/11–deficient mice with anti-COL7 IgG led to the same extent of skin lesions as in wild-type mice. Collectively, our data suggest that IL-1, independently of caspase-1, contributes to the pathogenesis of EBA. Because anti–IL-1b in a prophylactic setting and anakinra in a quasi-therapeutic setting (i.e., when skin lesions had already developed) improved experimental EBA, IL-1 appears to be a potential therapeutic target for EBA and related AIBDs. The Journal of Immunology, 2015, 194: 3656–3663.
The Journal of Immunology
with experimental EBA and in normal rabbit IgG-injected controls at the indicated time points using Bio-Plex (Bioglobe). IL-1 expression in skin was determined using immunohistochemistry. In brief, for both IL-1a and IL-1b staining, 6 mm-thick sections were incubated with the prediluted primary Ab—goat anti-mouse IL-1a or goat anti-mouse IL-1b Ab, respectively (both R&D Systems, Wiesbaden, Germany)—at room temperature (RT) for 1 h. Nonspecific binding was blocked using 5% rabbit serum. Rabbit anti-goat Ab (Dako, Hamburg, Germany) was used as secondary Ab. Incubation with HistoGreen (LINARIS, Germany), counterstaining with hematoxylin, and dehydrating were performed before mounting on slides. The intensity of IL-1 expression was scored semiquantitatively by an observer who was unaware of the nature of the specimen. The detection of ICAM-1 expression was performed similarly (i.e., Armenian hamster anti-mouse served as the primary Ab; BD Biosciences, Heidelberg, Germany). Rabbit anti-Armenian hamster (Abcam, Cambridge, U.K.) was used as a secondary Ab. For Gr-1 staining, rat antimouse Ly-6G (clone RB6-8C5) was used as primary Ab, and goat anti-rat IgG (both from Abcam) was used as secondary Ab.
RT-PCR
Materials and Methods
For RT-PCR analysis, lesional (n = 4) and nonlesional (n = 5) skin was obtained from mice on day 6 after injection with rabbit anti-murine COL7 IgG and normal rabbit IgG, respectively. RT-PCR of mouse skin was performed as described (19) using the following sets of primers: MLN51 forward (59-CCAAGCCAGCCTTCATTCTTG-39) and MLN51 reverse (59-TAACGCTTAGCTCGACCACTCTG-39) and IL-1b forward (59CTTCCAGGATGAGGACATGAG-39) and IL-1b reverse (59-CACACCAGCAGGTTATCATC-39).
Experiments with human samples
Treatment protocols
For the determination of IL-1a and IL-1b concentrations, serum from EBA patients (n = 26) fulfilling the following criteria were used: presentation with skin lesions resembling EBA; linear IgG and/or IgA deposition by direct immunofluorescence (IF) microscopy; and an u-serrated pattern by direct IF microscopy and/or detection of Abs against COL7. Serum from blood donors (n = 52) served as a reference. All of the experiments using human samples were approved by the local ethics committee (University of L€ ubeck, L€ ubeck, Germany) and were performed according to the Declaration of Helsinki. Blood donors and patients provided their written informed consent prior to study participation.
Kineret (anakinra; Biovitrum, Stockholm, Sweden) was injected i.p. into mice at a dosage of either 100 or 200 mg/kg/d. PBS served as a control. Anakinra or PBS treatment was initiated on day 0 and maintained throughout the experiments in the Ab-transfer model. In immunizationinduced EBA, anakinra was administered to mice when $2% of the body surface area was affected by skin lesions; it was continued for a total of 2 wk. Anti-IL-1b (clone ACZ885; Novartis, N€urnberg, Germany) was injected at 200 mg/mouse 1 d before and 2 and 4 d after the first anti-COL7 IgG injection.
Mice SJL/J, C57BL/6, IL-1R-deficient (2/2), and caspase-1/11–deficient mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice aged 6–8 wk were used for the experiments. The mice were kept in specific pathogen–free conditions and fed standard mouse chow and acidified drinking water ad libitum. All of the clinical examinations, biopsies, and blood collections were performed under anesthesia using i.p. administration of a mixture of ketamine (100 mg/g) and xylazine (15 mg/g). The animal experiments were approved by the local authority of the Animal Care and Use Committee (Kiel, Germany) and were performed by certified personnel.
Induction of experimental EBA Rabbit anti-murine COL7 IgG was prepared as previously described (15). In brief, rabbits were immunized with recombinant proteins of the NC1 domain of murine COL7. IgG from immune and normal rabbit sera were purified by affinity chromatography using protein G affinity. Passivetransfer studies into mice followed the published protocols (15, 16). Each experiment was performed twice, using different anti-COL7 IgG preparations. Disease severity was expressed as the percentage of body surface area affected by skin lesions and was determined at three time points (days 4, 8, and 12). From these time points, the area under the curve (AUC) was calculated. Blood and tissue samples were collected on day 12. EBA was induced by immunization with an immunodominant fragment located within murine COL7, as previously reported (17), with minor modifications described elsewhere (18). After immunization, the extent of clinical EBA manifestation was determined weekly. If EBA skin lesions affected 2% or more of the body surface area of individual mice, they were allocated to either anakinra or PBS (see below). The treatments were administered over a 2-wk period, and EBA severity (percentage of affected body surface area) was monitored weekly.
Determination of IL-1 levels in serum and skin Serum from EBA patients and normal controls were analyzed for the expression of IL-1a and IL-1b using Bio-Plex (Bioglobe, Hamburg, Germany). Serum concentrations of IL-1a and IL-1b were determined in mice
Western blotting with extracts of mouse skin Ear specimens from mice were excised, immediately snap-frozen in liquid nitrogen, and stored at 280˚C until further analysis. To prepare total protein skin extracts, frozen ears were ground in liquid nitrogen and homogenized in T-PER reagent (Thermo Scientific, Darmstadt, Germany; 1:6, w/v), with the addition of protease inhibitor mixture set III (Calbiochem, Darmstadt, Germany; 1:100, v/v). Tissue homogenates were centrifuged at 10,000 3 g for 10 min at 4˚C to pellet debris, and the supernatants were collected. Next, the samples were incubated with protein A/G PLUS–Agarose beads (Santa Cruz Biotechnology, Heidelberg, Germany; 5:1, v/v) at 4˚C on a rotating device overnight. The beads were pelleted by centrifugation at 1000 3 g for 5 min at 4˚C, and the supernatants were collected and stored at 280˚C until use. The total protein concentration was determined according to the BCA method (Thermo Scientific). Protein samples were separated by SDS-PAGE under reducing conditions on 15% polyacrylamide gel and transferred to a 0.2-mm pore size nitrocellulose membrane for Western blotting. The membranes were blocked with 5% w/v skim milk powder in TBST for 1 h at RT. Before each step, the membranes were washed five times for 10 min in TBST. All of the Abs were diluted in blocking buffer. Goat polyclonal IgG against murine IL-1b (R&D Systems; cat. no. AF-401-NA) and mouse monoclonal IgG against actin (Santa Cruz Biotechnology; cat. no. sc-47778) were used at 1:400 or 1:3000 dilution, respectively, overnight at 4˚C. HRPlabeled secondary Abs for goat (Dako; cat. no. P0449) and mouse (Jackson ImmunoResearch, Suffolk, U.K.; cat. no. 715-035-150) were applied at 1:20,000 and 1:50,000 dilution, respectively, for 1 h at RT and detected using Amersham ECL Prime reagent (GE Healthcare).
Culture and in vitro activation of human endothelial cells HUVECs (PromoCell, Heidelberg, Germany) were cultured in Endothelial Cell Growth Medium (PromoCell) at 37˚C in a humidified atmosphere containing 5% CO2. Four hours prior to activation, the medium was replaced by Endothelial Cell Basal Medium (PromoCell) supplemented with 10% FCS. To induce adhesion molecule expression, endothelial cells were activated with recombinant human IL-1b (5 ng/ml) or TNF-a (25 ng/ml) for 4 h. These experiments were performed in the presence or absence
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fluid from bullous pemphigoid patients, an AIBD caused by autoantibodies against type XVII collagen, and were correlated with the magnitude of skin lesions (10, 11). Given that some cytokines have potent anti-inflammatory activities (12) or trigger the release of chemokine antagonists (13), we investigated the contribution of IL-1 to the prototypical AIBD EBA in which the autoimmune response is directed against COL7 (14). Based on the observation of reduced skin blistering in autoantibody transfer–induced EBA upon prophylactic anakinra application (8), we hypothesized that IL-1 would exert proinflammatory activity in EBA. However, it remained unclear how IL-1 modulates blister formation in experimental EBA; whether this effect is mediated by IL-1a, IL-1b, or both; whether treatment with IL-1–modulating compounds can improve already established experimental EBA; how IL-1 expression is controlled; and whether these findings are also applicable to EBA patients. We addressed these potentially clinically relevant questions using several animal models of EBA, as well as a large collection of EBA sera. Given that blockade of IL-1 has therapeutic effects in established EBA, and there is increased IL-1 expression in EBA patients, blockade of IL-1 might be a promising therapeutic option for EBA patients.
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3658 of anakinra (5 mg/ml), which was added to the cell cultures 1 h before treatment. Nontreated cells served as negative controls.
Histology and IF microscopy
Statistics The data were analyzed using SigmaPlot software, version 12 (Systat Software, Chicago, IL), and applied tests and 95% confidence intervals are indicated in the respective text and figure legends. For analysis of rankscaled or semiquantitative data, we used ordinal logistic regression (polr) and mosaic plots, provided by GNU R open source statistical software (http://www.r-project.org), together with the “MASS” package. Ordinal logistic regression models were evaluated first by likelihood ratio testing. In the second step, models with a significant likelihood ratio testing were evaluated further; p values are given for the difference between each group and the controls. A p value , 0.05 was considered statistically significant after appropriate adjustment for multiple testing.
Results Increased IL-1a and IL-1b serum concentrations and correlations with disease severity in experimental and human EBA The induction of experimental EBA in BALB/c mice, by injection of anti-COL7 IgG, led to increases in serum levels of IL-1b, but not IL-1a, compared with untreated control mice (Fig. 1A). The extent of skin lesions correlated with IL-1b, but not with IL-1a, serum levels (Fig. 1B, 1C). Compared with healthy controls, 6and 4-fold increases in IL-1a and IL-1b serum levels were found in EBA patients (Fig. 1A). IL-1b mRNA expression also was significantly increased in lesional skin compared with healthy mouse skin. Six days after the first IgG injection, IL-1b mRNA expression was significantly increased in anti-COL7 IgG-injected animals compared with mice injected with normal rabbit IgG. Median relative (in relation to MLN51) IL-1b expression in mice injected with normal rabbit IgG was 0.16 (0.12–0.57, 25/75 percentiles), whereas it was 34.6 (21.1–194; p = 0.0159, Mann– Whitney rank-sum test) in lesional skin of mice injected with antiCOL7 IgG. Furthermore, we detected increased protein expression of IL-1a and IL-1b in the skin of mice after EBA induction compared with IL-1R2/2 mice and anakinra-treated wild-type (WT) animals (Fig. 1D–F). Induction of experimental EBA was impaired in mice with either genetic or prophylactic pharmacologic inhibition of IL-1 To test whether the increased local and/or systemic IL-1a and IL-1b levels were of functional relevance, IL-1 function was inhibited. EBA was induced in IL-1R2/2 and WT C57BL/6 control mice. Mice of both strains developed experimental EBA; however, in IL-1R2/2 mice, the overall extent of skin blistering
was reduced to 52% of that in control mice (p , 0.001; Fig. 2A, 2B). Comparisons of clinical scores between the two strains at 4, 8, and 12 d showed that the affected body surface area was significantly lower in IL-1R2/2 mice at all time points (day 4: 1.6 6 0.4% versus 0.6 6 0.2% [p = 0.008]; day 8: 7.4 6 0.7% versus 5.2 6 0.7% [p = 0.04]; day 12: 20.7 6 1.8% versus 10.1 6 1.7% [p , 0.001] in WT and IL-1R2/2 mice, respectively). The clinical effects were accompanied by reduced dermal inflammatory infiltrates in IL-1R2/2 treated mice compared with controls (Fig. 2C). Staining for Gr-1+ cells revealed similar results, demonstrating a reduced expression of GR-1 in both IL-1R2/2 and anakinratreated mice (Fig. 2D). Treatment with a function-blocking Ab directed against IL-1b fully recapitulated and even exceeded the effects of total IL-1 inhibition. Compared with C57BL/6 mice injected with isotypecontrol Abs, anti–IL-1b reduced blister formation to 44% (p = 0.001, Fig. 2E, 2F). Blockade of IL-1 function also was associated with decreased expression of both IL-1a and IL-1b in mouse skin (Fig. 1). In contrast, levels of tissue-bound IgG and C3 were similar in all of the mice (Fig. 2B), indicating that the clinical effects were not due to altered IgG metabolism and/or effects on complement activation. Therapeutic application of anakinra led to improvement of immunization-induced EBA Following the prophylactic pharmacological inhibition of IL-1, an IL-1–targeting treatment was applied in a quasitherapeutic setting (i.e., anakinra administration was initiated when mice already had established EBA skin lesions). For this purpose, the immunization-induced EBA mouse model was used. At the initiation of treatment, the clinical disease severity in mice randomly allocated to control and anakinra treatments was identical (i.e., 2.6 6 0.2% affected body surface area). Clinical EBA manifestations continuously progressed in PBS-injected mice (3.2 6 0.2% at week 1 and 3.9 6 0.1% at week 2), whereas it declined in anakinra-treated mice (2.1 6 0.2% at week 1 and 2.0 6 0.2% at week 2, Fig. 3). Corresponding disease activity scores were significantly lower in the anakinra group compared with the PBS group (week 1, p = 0.004; week 2, p , 0.001; AUC, p , 0.001). Inhibition of IL-1 function led to reduced expression of endothelial adhesion molecules Because the induction of skin lesions in experimental EBA depends on CD18 (23) and because IL-1 regulates the expression of the respective endothelial ligands for LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and integrin CD11c/CD18 (24), the expression of ICAM-1 (CD54) in the skin was evaluated in experimental EBA, with and without impaired IL-1 function. In mice with experimental EBA, ICAM-1 expression was detected both on endothelial cells and on basal keratinocytes (Fig. 4A, 4B). In contrast, ICAM-1 expression was almost absent in mice injected with normal rabbit IgG (Fig. 4A). Similarly, blockade of IL-1 function in mice injected with anti-COL7 IgG reduced ICAM-1 expression (Fig. 4A, 4B). To test whether this inhibitory effect of IL-1 blockade also affected other endothelial adhesion molecules, such as VCAM-1 (CD106) and E-selectin (CD62E), the expression of these molecules was assessed on HUVECs stimulated in the absence or presence of anakinra. In line with the in vivo findings, IL-1 induced ICAM-1 expression, which could specifically be inhibited by anakinra treatment (Fig. 4C). Endothelial VCAM-1 and E-selectin expression paralleled ICAM-1 expression (Fig. 4C).
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Murine specimens were subjected to H&E staining, according to a standard protocol. The extent of leukocyte infiltration was scored semiquantitatively in a blinded fashion, as described previously (20). Direct IF microscopy for the detection of rabbit IgG and murine C3 in experimental EBA was performed on 6-mm cryosections, as described, using goat anti-rabbit IgG (Dako) and goat anti-murine C3 (MP Biomedicals, Kaysersberg, France), both labeled with FITC. IF microscopy on cultured HUVECs was performed as reported (21, 22). In brief, cells were fixed in 220˚C cold methanol for 5 min and washed carefully with PBS before blocking with 5% FCS in PBS for 1 h. Anti-VCAM Ab (clone STA; Immunotools, Friesoythe, Germany), anti-ICAM Ab (clone 15.2; FITC-labeled; Immunotools), and anti–E-selectin Ab (clone 1-2B6; PE-labeled; Abs-online, Aachen, Germany) were diluted in PBS containing 2.5% FCS and incubated overnight before washing with PBS. Anti-VCAM reactivity was visualized by goat anti-mouse IgG conjugated with Alexa Fluor 488 (Cell Signaling Technology, Danvers, MA). Mounting medium containing 1% DAPI was added to each culture slide. Stained cells were analyzed using an Axio Imager M1 microscope (Carl Zeiss Meditec, Jena, Germany). Standardized exposure times were used for all of the images.
IL-1 IN EBA
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FIGURE 1. Induction of experimental EBA leads to increased IL-1a and IL-1b expression in serum and skin. (A) Induction of experimental EBA by injections of anti-COL7 IgG increased serum concentrations of IL-1b, but not IL-1a, in BALB/c mice. The data were based on five mice injected with normal rabbit (NR) IgG and eight mice injected with anti-COL7 IgG (mean 6 SEM). In line with this observation in mice, increased serum concentrations of IL-1a and IL-1b were observed in EBA patients compared with controls. The data were based on 52 control subjects’ and 26 EBA patients’ sera (mean 6 SEM). *p , 0.05, rank-sum test. (B) In experimental EBA, IL-1a serum levels did not correlate with the body surface area affected by EBA (p = not significant [ns]). The data were based on four mice injected with normal rabbit IgG (day 12) and three and four mice injected with anti-COL7 IgG on days 6 and 12, respectively. The dashed line is the regression line, and the dotted lines indicate the confidence intervals. (C) In contrast to IL-1a, IL-1b serum levels correlated with the body surface area affected by EBA (p = 0.006, r = +0.763). The data were based on four mice injected with normal rabbit IgG (day 12) and three and four mice injected with anti-COL7 IgG on days 6 and 12, respectively. The dashed line is the regression line, and the dotted lines indicate the confidence intervals. (D) Representative cutaneous expression of IL-1a and IL-1b in the indicated treatment groups. Scale bar, 100 mm. (E) Semiquantitative staining intensities of cutaneous IL-1a expression. All of the mice were injected with anti-COL7 IgG. Compared with C57BL/6 control mice, no significant effect on IL-1a expression was observed (likelihood ratio test on ordered logistic regression model: p = 0.06). Column height represents the number of mice with staining intensities ranging from 0 to 3, and column width represents the number of mice in the different treatment groups. (F) Corresponding results for cutaneous IL-1b expression. Compared with C57BL/6 control mice (n = 17), a significant reduction in IL-1b expression was noted in IL-1R2/2 mice (n = 14; p = 0.018) and in anakinra-treated mice (n = 19; p = 0.02).
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IL-1 IN EBA
Increased IL-1 expression in experimental EBA was independent of caspase-1 expression Because caspase-1 is an important regulator of IL-1b release (25), we next analyzed whether blockade of caspase-1 had an impact on the development of skin lesions in experimental EBA. At the same time, we assessed whether the increased IL-1b expression in this mouse model was controlled by caspase-1. Unexpectedly, com-
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FIGURE 2. Blister formation in experimental EBA is impaired in IL-1R-deficient mice and in mice treated with an anti-IL-1b Ab. (A) Disease severity in the indicated groups is expressed as AUC calculated from the affected body surface area 4, 8 and 12 d after the initial anti-COL7 IgG injection. The data were based on 11–16 mice/group and are presented as mean 6 SEM. *p , 0.05 (t test). (B) Representative clinical pictures, H&E-stained skin biopsies, IgG and C3 deposition at the dermal–epidermal junction 12 d after the initial anti-COL7 IgG injection in the indicated groups. Scale bar, 100 mm. (C) Semiquantitative determination of dermal leukocyte infiltrates in H&E-stained sections. All of the mice were injected with anti-COL7 IgG. Compared with C57BL/6 control mice (n = 16), a decrease in dermal infiltration was noted in IL-1R2/2 mice (n = 8; p = 0.02). Column height represents the number of mice with staining intensities ranging from 0 to 3, and column width corresponds to the number of mice in the different treatment groups. (D) In line, infiltration with Gr-1+ myeloid cells was increased in mice with experimental EBA (p , 0.001, ANOVA on Ranks with Holm-Sidak posttest), as opposed to mice injected with control IgG. Genetic (p , 0.001) or pharmacological (p = 0.002) blockade of IL-1 function significantly reduced the infiltration of the skin with Gr-1+ cells. Scale bar, 200 mm. (E) Disease severity in the indicated groups is expressed as the area under the curve (AUC) calculated from the affected body surface area 4, 8 and 12 d after the initial anti-COL7 IgG injection. The data were based on a minimum of eight mice/group and are mean 6 SEM. *p , 0.05 (t test). (F) Representative clinical images 12 d after the initial anti-COL7 IgG injection in the indicated treatment groups.
pared with WT animals, caspase-1/112/2 mice had similar clinical phenotypes after injections of anti-COL7 IgG. When data from two independent experiments were considered together, overall disease severity (expressed as AUC) was not affected; however, on day 8, a significantly smaller body surface area was affected in caspase-1/112/2 mice compared with WT mice (Fig. 5A, 5B). This finding pointed to noncaspase-1 extracellular processing of
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the IL-1b precursor (e.g., elastase) or cathepsin G in EBA. To test this assumption, IL-1b protein expression was determined in C57BL/6 WT and caspase-1/112/2 mice after injections of antiCOL7 IgG. In both strains, similar IL-1b protein expression was observed (Fig. 5C).
Discussion To our knowledge, this study provides the first experimental evidence for the potential therapeutic use of IL-1 inhibition in AIBD. Serum concentrations of IL-1a and IL-1b were increased and correlated with disease severity in the passive transfer mouse model of EBA, a prototypic AIBD characterized by autoimmunity to COL7. Elevated serum levels of IL-1b were described previously in blister fluid, but not sera, and correlated with disease activity in patients with bullous pemphigoid, another subepidermal AIBD characterized by autoimmunity against COL17 (1, 10, 11). Similarly, in a recently established model of bullous pemphigoid that was induced by the passive transfer of rabbit antimurine COL17 IgG in adult mice (26), serum IL-1b levels correlated with the extent of skin lesions (F.S. Schulze and E. Schmidt, unpublished observations). We found that blockade of IL-1b impaired the induction of experimental EBA, and blockade of IL-1 function improved already established skin lesions. These data support the notion that inhibition of IL-1, in particular of IL-1b, might be a novel therapeutic modality for EBA and bullous pemphigoid. So far, three IL-1–targeted agents have been approved: the recombinant form of the naturally occurring IL-1ra anakinra; the soluble decoy receptor rilonacept, which blocks both IL-1a and IL-1b; and the anti–IL-1b neutralizing mAb canakinumab (27). These drugs have been used successfully in various inflammatory disorders, including the autoimmune diseases rheumatoid arthritis and relapsing polychondritis (27). In fact, IL-1b inhibition would be the first therapeutic regimen without unspecific immunosuppressive/
immunomodulative action for AIBDs, such as EBA and bullous pemphigoid (1, 28). In EBA, blister formation depends on and is initiated by the binding of autoantibodies to COL7, located at the dermal–epidermal junction (29, 30). Subsequently, Fc-dependent mechanisms result in the formation of a proinflammatory milieu in the skin (31). Complement activation significantly (15, 32, 33), but not exclusively (34), contributes to the formation of this proinflammatory milieu. Finally, proteases, such as those released from skin-infiltrated inflammatory cells were seen in this study, and the induction of EBA led to increased endothelial ICAM-1 expression, which was absent if IL-1 function was blocked. In vitro experiments extended these findings to additional endothelial adhesion molecules, such as VCAM-1 and E-selectin. Dependence on adhesion molecule expression in the pathogenesis of EBA was demonstrated by the observed absence of skin lesions in CD18deficient mice after the injection of anti-COL7 IgG (23). Collectively, these data suggest that interactions of leukocyte b2 integrins (CD18) with endothelial adhesion molecules of the Ig superfamily are indispensable for blister formation in experimental EBA. Similar findings were reported in neonatal mice with experimental bullous pemphigoid: blockade of CD11a, CD11b, or CD18 protected mice from blistering after injection of Abs to COL17. In this bullous pemphigoid model, detailed analysis of the neutrophil inflammatory response revealed that CD11a was necessary for neutrophil recruitment, whereas CD11b mediated late neutrophil accumulation and neutrophil apoptosis (35). In addition, neutrophil recruitment into the skin also was shown to depend on additional adhesion molecules, including selectins (36), and on junctional adhesion molecules (20). Hence, it is tempting to speculate that blockade of adhesion molecules might also protect mice from blister formation in experimental EBA. Furthermore, our data demonstrated that IL-1b expression in the skin and IL-1b secretion into the serum were, at least to a major
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FIGURE 3. Anakinra treatment leads to improvement of already established skin blistering in immunization-induced experimental EBA. SJL/J mice with already established skin blistering after immunization with COL7 were treated with anakinra (100 mg/kg, n = 11) for 2 wk. Control mice received PBS (n = 10). (A) Analysis of the extent of blistering over time showed an increase in skin disease during the observation period in PBS-injected mice. In contrast, disease severity decreased in mice treated with anakinra. (B) Cumulative disease severity, expressed as AUC during the 2-wk treatment period, was reduced significantly in anakinratreated mice. Compared with PBS-injected mice, animals treated with anakinra had significantly reduced overall disease activity. Representative clinical disease manifestation at the beginning of treatment (Week 0) and at the end of the treatment period (Week 2) in PBStreated (C) and anakinra-treated (D) animals. *p , 0.001, t test.
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extent, not mediated by caspase-1. This finding indicates an alternative scenario for the processing of the extracellular IL-1b precursor. Because blockade of IL-1 function in both EBA models led to incomplete inhibition of skin blistering, and lack of caspase1/11 expression in mice showed little to no impact on blister formation, it is tempting to speculate that neutrophil-derived serine proteases (such as neutrophil elastase, proteinase-3, and cathepsin G) (37, 38) cleave to the IL-1b precursor and control IL-1b protein expression. After extravasation into the skin, Gr-1+ leukocytes bind to immune complexes located at the dermal–epidermal junction in an FcgRIV-dependent (mice) or FcgRIIA- and FcgRIIIB-dependent
(humans) manner. Interestingly, these extravasated cells are indispensable for blister formation by the release of reactive oxygen species and proteolytic enzymes (30), and they express IL-1R after migration. This increased IL-1R expression is functionally relevant because the stimulation of neutrophils with IL-1 resulted in the transcription of NF-kB and the release of a number of downstream chemokines (39). Therefore, the inhibition of IL-1 might also block this increased IL-1R expression on neutrophils, including downstream neutrophil activation. In summary, this study provides novel insight into the contributions of IL-1 to the pathogenesis of autoantibody-induced tissue injury in a prototypical organ-specific autoimmune dis-
FIGURE 5. Increased IL-1 expression in experimental EBA is independent of caspase-1 expression. (A) EBA-affected body surface area (%) in caspase 1/1–deficient (Casp KO, n = 9) and control C57BL/6 mice (n = 10) during the 12-d observation period. In both strains, the onset of blistering was observed at day 4, and it was slightly, but significantly, delayed in Casp KO mice. (B) At the end of the experiment and with regard to overall disease severity, expressed as the AUC from (A), both strains of mice showed comparable magnitudes of blistering. (C) On day 12, ear samples were obtained, and IL-1b expression was quantified by Western blotting of the tissue extracts. An equal amount of IL-1b was observed in WT and Casp KO mice. Five representative experiments of n = 6/group are shown.
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FIGURE 4. Induction of experimental EBA leads to increased cutaneous expression of ICAM-1, which is sensitive to blockade of IL-1 function. (A) Representative ICAM-1 expression 12 d after the first injection of anti-COL7 IgG into mice of the indicated treatment groups. ICAM-1 was almost completely absent in mice injected with normal rabbit (NR) IgG, whereas ICAM-1 was expressed on keratinocytes and around the vasculature in mice with experimental EBA. (B) Overall, visual assessment of ICAM-1 staining intensity in mice with experimental EBA showed a significant decrease if IL-1 function was blocked by knockout (n = 15; p = 0.011) or anakinra (n = 16; p = 0.014) compared with control WT mice (n = 10). Column height represents the number of mice with staining intensities ranging from 0 to 3, and column width represents the number of mice in the different treatment groups. (C) Stimulation of HUVECs with either IL-1b or TNF-a led to increased expression of ICAM-1, VCAM-1, and E-selectin. Anakinra (Ana) selectively blocked the IL-1b–induced, but not the TNF-a–induced, upregulation of adhesion molecules. Nuclei are shown in blue, and the expression of the indicated adhesion molecules is shown in green or pink. Original magnification 3 400. The data shown are representative of three experiments.
The Journal of Immunology ease. We showed a proinflammatory contribution of IL-1, which was independent of caspase-1 expression. This proinflammatory effect of IL-1 was associated with the induction of endothelial adhesion molecule expression. This proinflammatory role for IL-1 in experimental EBA could be considered when therapeutic decisions must be made in patients with treatment-refractory and/or relapsing EBA and other subepidermal autoimmune skin blistering diseases with a similar pathogenesis, such as bullous pemphigoid.
Acknowledgments The anti–IL-1b Ab was kindly provided by Novartis (Basel, Switzerland).
Disclosures The authors have no financial conflicts of interest.
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