The Journal of Immunology The Alternative Pathway of Complement Activation Is Critical for Blister Induction in Experimental Epidermolysis Bullosa Acquisita1 Sidonia Mihai,* Mircea T. Chiriac,* Kazue Takahashi,† Joshua M. Thurman,‡§ V. Michael Holers,‡§ Detlef Zillikens,* Marina Botto,¶ and Cassian Sitaru2* Epidermolysis bullosa acquisita is a subepidermal blistering disease associated with tissue-bound and circulating autoanti- bodies against type VII collagen, a major constituent of the dermal-epidermal junction. The passive transfer of Abs against type VII collagen into mice induces a subepidermal blistering disease dependent upon activation of terminal complement components. To further dissect the role of the different complement activation pathways in this model, we injected C1q- deficient, mannan-binding lectin-deficient, and factor B-deficient mice with rabbit Abs against murine type VII collagen. The development and evolution of blistering had a similar pattern in mannan-binding lectin-deficient and control mice and was initially only marginally less extensive in C1q-deficient mice compared with controls. Importantly, factor B-deficient mice developed a delayed and significantly less severe blistering disease compared with factor B-sufficient mice. A significantly lower neutrophilic infiltration was observed in factor B-deficient mice compared with controls and local reconstitution with granulocytes restored the blistering disease in factor B-deficient mice. Our study provides the first direct evidence for the involvement of the alternative pathway in an autoantibody-induced blistering disease and should facilitate the development of new therapeutic strategies for epidermolysis bullosa acquisita and related autoimmune diseases. The Journal of Immu- nology, 2007, 178: 6514–6521. pidermolysis bullosa acquisita (EBA),3 a severe chronic with recombinant autologous type VII collagen induces an au- subepidermal blistering disease of skin and mucous toimmune response to this protein, resulting in a blistering phe- E membranes, is characterized by tissue-bound and cir- notype closely resembling human EBA (11). culating IgG Abs to the dermal-epidermal junction (1). Patients’ Autoantibodies against type VII collagen are able to activate the serum autoantibodies bind to the 290-kDa type VII collagen, the complement system in vivo and in vitro. In the skin of EBA pa- major component of anchoring fibrils (2, 3). Epitopes recog- tients, deposition of different complement components, including nized by the majority of EBA sera were mapped to the non- C3, C5b, and membrane attack complex, are found with an inci- collagenous 1 domain of type VII collagen (4–6). The patho- dence ranging from ϳ40 to 100% (12–14). In addition, deposition genic relevance of Abs against type VII collagen is supported of C3 at the dermal-epidermal junction of murine skin is a constant by compelling evidence: 1) EBA autoantibodies were shown to feature in different passive transfer mouse models of EBA (9, 10) recruit and activate leukocytes ex vivo, resulting in dermal- and in experimental EBA induced by immunization of mice with epidermal separation in cryosections of human skin (7, 8). 2) autologous type VII collagen (11). These observations suggested Abs against type VII collagen induce subepidermal blisters that the complement system is involved in the autoimmune tissue when passively transferred into mice (9, 10). 3) Immunization injury in EBA. Indeed, this hypothesis was confirmed by our re- cent studies showing that C5-deficient mice are resistant to blister *Department of Dermatology, University of Lu¨beck, Lu¨beck, Germany; †Department induction by passive transfer of Abs against type VII collagen (9). of Pediatrics, Laboratory of Developmental Immunology, Massachusetts General The initiators and processes that result in complement activation ‡ Hospital, Harvard Medical School, Boston, MA 02115; Department of Medicine and can be assigned to three different pathways: classical, alternative, §Department of Immunology, University of Colorado Health Sciences Center, Den- ver, CO 80218; and ¶Rheumatology Section, Imperial College School of Medicine, and lectin. The classical pathway is activated primarily by Ag-Ab London, United Kingdom complexes binding to C1q (15). The lectin pathway is initiated by Received for publication December 19, 2006. Accepted for publication March ficolins and mannan-binding lectin (MBL) (16). The alternative 2, 2007. pathway is continuously activated by factor B through a process The costs of publication of this article were defrayed in part by the payment of page called “tickover” of C3 and requires active control mechanisms to charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. prevent autologous injury (17). The relevance of the different com- 1 This work was supported by Grants Zi 439/6-2 and SI 1281/1-1 from the Deutsche plement components and pathways of the complement cascade in Forschungsgemeinschaft (to C.S. and D.Z.) and National Institutes of Health Grants experimental EBA, which could represent targets for treatment, R01 AI-31105 (to V.M.H.) and K08 DK64790 (to J.M.T.). have remained unknown. 2 Address correspondence and reprint requests to Dr. Cassian Sitaru, Department of Dermatology, University of Lu¨beck, Ratzeburger Allee 160, Lu¨beck, Germany. In the present study, we examined the relative contribution of E-mail address: [email protected] the different complement activation pathways for blister formation 3 Abbreviations used in this paper: EBA, epidermolysis bullosa acquisita; IF, immu- in experimental EBA. Our findings provide the first direct evidence nofluorescence; Bf, complement factor B; MBL, mannan-binding lectin; MPO, my- for the involvement of the alternative pathway in an autoantibody- eloperoxidase; CR, complement receptor; WT, wild type. induced blistering disease and a conceptual framework for devel- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 oping rational therapeutic strategies for EBA and related diseases. www.jimmunol.org The Journal of Immunology 6515 FIGURE 1. C1q-deficient mice are susceptible to the induction of subepi- dermal blisters by Abs specific to type VII collagen. Skin lesions, including blisters and erosions covered by crusts on the ear and front leg, and alopecia of the snout and around the eyes devel- oped in both WT (a) and C1qaϪ/Ϫ mice (b) that received a total dose of 45 mg of rabbit IgG against type VII collagen (day 12). c, A C1qaϪ/Ϫ mouse receiv- ing the same amount of control rabbit IgG did not develop skin lesions. IF mi- croscopy analysis of perilesional skin revealed deposition of rabbit IgG at the dermal-epidermal junction of both WT (d) and C1qaϪ/Ϫ (e) mice injected with Abs against type VII collagen, but not in the C1qaϪ/Ϫ (f) mouse treated with control rabbit IgG. Deposits of mouse C3 along the dermal-epidermal junc- tion were strong in the skin of WT (g) and weak in the skin of C1qaϪ/Ϫ (h) mice injected with Abs against type VII collagen and absent in the C1qaϪ/Ϫ mouse (i) treated with control rabbit IgG. Staining for murine C5 revealed similar deposits in the skin of the WT (j) and of the C1qaϪ/Ϫ (k) mice treated with Abs against type VII collagen, but was absent in the C1qaϪ/Ϫ (l) mouse injected with control rabbit IgG. Histological analysis of lesional skin revealed extensive dermal-epidermal separation and a neutrophil-rich inflam- matory infiltrate in both WT (m) and C1qaϪ/Ϫ (n) mice injected with Abs against type VII collagen. o, Normal histological appearance in the skin of the C1qaϪ/Ϫ mouse injected with con- trol rabbit IgG. Materials and Methods as follows: 0, no lesions; 1, Ͻ10 lesions or Ͻ1% of the skin surface; 2, Ͼ Mice 10 lesions or 1–5% of the skin surface; 3, 5–10%; 4, 10–20%; and 5, Ͼ20% involvement of the skin surface. Biopsies of lesional and perile- C1qaϪ/Ϫ and BfϪ/Ϫ mice, backcrossed to BALB/c mice (for 10 and 7 sional skin were obtained 2 days after the last injection of IgG and prepared generations, respectively), and MBL-null mice backcrossed to C57BL/6J for examination by histopathology and IF microscopy as described previ- mice (for 7 generations), were previously described (18–21). Age- and ously (9, 11). Tissue-bound murine C5 was detected by incubation of the sex-matched BALB/c and C57BL/6J mice were obtained from Charles frozen sections prepared from tissue biopsies with a mAb specific to mu- River. All injections and bleedings were performed on mice narcotized by rine C5 (BB5.1) (22) and, finally, with a FITC-labeled Ab specific to inhalation of isoflurane or i.p. administration of a mixture of ketamine (100 mouse IgG (DakoCytomation). The staining intensity of immunoreactants ␮g/g) and xylazine (15 ␮g/g). The experiments were approved by the local in the skin of immunized mice was assessed semiquantitatively using a authorities of the Animal Care and Use Committee (reference number: score comprising 0, for no staining; 1, faint staining; 2, medium; and 3, 6/g/04) and performed by certified personnel. intense staining (11). Neutrophil infiltration of murine skin was assayed as described previ- Affinity purification of Abs ously (23), with minor modifications. Briefly, in both clinically diseased and not diseased mice, the left ear was removed after killing the mice and Rabbits were immunized with recombinant forms of murine type VII col- skin samples (ϳ10 ϫ 5 mm in size) were extracted by homogenization in lagen as described elsewhere (9). IgG from immune and preimmune rabbit a buffer containing 0.1 M Tris-Cl (pH 7.6), 0.15 M NaCl, and 0.5% hexa- sera was purified by affinity chromatography using protein G affinity as decyl trimethylammonium bromide (Sigma-Aldrich). Myeloperoxidase previously reported (9). Reactivity of IgG fractions was analyzed by im- (MPO) activity in the supernatant fraction was measured by the change in munofluorescence (IF) microscopy on murine skin.