The Journal of Immunology

The Alternative Pathway of Complement Activation Is Critical for 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 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 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 , 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. OD at 460 nm resulting from decomposition of H2O2 in the presence of Induction of blistering in vivo and phenotype assessment o-dianisidine (Sigma-Aldrich). A standard reference curve was established using known concentrations of purified MPO (Sigma-Aldrich). MPO con- Passive transfer studies followed published protocols with minor modifi- tent was expressed as units of MPO activity per mg of protein. Protein cations (9). Briefly, mice received six injections of 7.5 mg of rabbit IgG. concentrations were determined by the Bradford dye-binding assay Blisters or erosions were counted and the extent of skin disease was scored (Bio-Rad). 6516 COMPLEMENT IN EXPERIMENTAL EBA

FIGURE 2. Time course and disease severity in C1q-deficient and control mice. a, C1qaϪ/Ϫand WT mice (n ϭ 10/group) were injected with 7.5 mg of purified rabbit IgG to murine type VII collagen every second day, over a period of 12 days, and evaluated for skin lesions as described in Materials and Significant difference of disease activity (p Ͻ 0.05) between the two groups. b, Serum levels of Abs to type VII collagen in C1qaϪ/Ϫ and WT ,ء .Methods mice (n ϭ 10/group) injected with rabbit Abs against type VII collagen (antiCVII) or normal rabbit IgG (NRIgG) as detected by ELISA using recombinant Ag. All values are mean Ϯ SEM.

For the in vivo reconstitution with leukocytes, murine granulocytes were isolated from peripheral blood and bone marrow of donor mice by 3% dextran sedimentation followed by density gradient centrifugation using Ficoll-Paque (GE Healthcare Bio-Sciences) and hypotonic lysis in 0.2% NaCl. Only cell preparations with viability above 95% as assessed by trypan blue exclusion were used. These consisted of Ͼ90% granulocytes as revealed by Giemsa staining and by flow cytometry as described previously (8). Granulocytes were defined as Gr-1highCD11bhigh using mAb specific to murine Gr-1 (RB6-8C5) and to CD11b (M1/70) (both from BD Bio- sciences). Mice were injected with 5 ϫ 106 cells in 50 ␮l of medium intradermally in the ears. The animals were examined clinically after 12 and 24 h, subsequently killed, and samples prepared and analyzed as de- scribed above. Detection of Ab levels by ELISA ELISA using recombinant murine type VII collagen was performed at room temperature on 96-well microtiter plates as previously reported (11), with minor modifications. Briefly, each well was coated with 500 ng of the recombinant protein GST-mCOL7C or with an equimolar amount of GST in 0.1 M bicarbonate buffer (pH 9.6) and incubated with 200-fold dilutions of mouse serum for 60 min. Bound Abs were detected using a 10,000-fold dilution of a HRP-labeled goat anti-rabbit IgG Ab (DakoCytomation) and o-phenylenediamine (Sigma-Aldrich). The color reaction was read at 490 nm using a multilabel counter (Victor 3; PerkinElmer). To evaluate reac- tivity against the epidermal basement membrane, for each serum, the mean OD reading obtained with GST was subtracted from the mean reading with GST-mCOL7C. Statistical analysis We used OpenStat2 free software for Linux (http://www.agrivisser.com/ cgibin/English/OpenStat2.htm). Differences in disease severity and in MPO activity were calculated using the ␹2 and Student t test, respectively. The Mann-Whitney U test was used to compare values for semiquantitative scoring of immunohistochemistry. Means are presented Ϯ SEM; p Ͻ 0.05 was considered to be statistically significant. Results C1q-deficient mice are susceptible to skin blistering induced by Abs against type VII collagen To examine the role of the C1q complement component for sub- epidermal blister formation in experimental EBA, we injected FIGURE 3. Abs against type VII collagen induce subepidermal blisters C1qaϪ/Ϫ (n ϭ 10) and wild-type (WT; n ϭ 10) mice with rabbit in MBL-null mice. Skin lesions, including blisters and erosions covered by IgG against murine type VII collagen. All mice injected with Abs crusts on the ear and front leg, and alopecia of the snout and around the against type VII collagen developed single blisters 4 days after the eyes developed in both MBL-sufficient (a) and MBL-null (b) mice that received a total dose of 45 mg of rabbit IgG against murine type VII first injection. Widespread lesions, including blisters, erosions, and collagen (day 12). IF analysis of perilesional skin revealed deposition of crusts, occurred 6 days after the first injection (Fig. 1, a and b). c d e f Ϫ/Ϫ rabbit IgG ( and ) and murine C3 ( and ) at the dermal-epidermal Only at the end of the observation period, C1qa mice demon- junction of both MBL-sufficient and MBL-null mice. Histological analysis strated significantly less extensive skin disease compared with of lesional skin revealed extensive dermal-epidermal separation and a neu- control mice (Fig. 2a). The levels of rabbit Abs against type VII trophil-rich inflammatory infiltrate in both MBL-sufficient (g) and MBL- collagen in serum of mice of both groups were similar (Fig. 2b). null (h) mice. The Journal of Immunology 6517

FIGURE 4. Time course and disease severity in MBL-null and control mice. a, MBL-null and control mice (WT) (n ϭ 10/group) were injected with 7.5 mg of purified rabbit IgG to murine-type VII collagen every second day, over a period of 12 days, and evaluated for skin lesions as described in Materials and Methods. b, Serum levels of Abs to type VII collagen in MBL-null and WT mice (n ϭ 10/group) injected with rabbit Abs against type VII collagen (antiCVII) or normal rabbit IgG (NRIgG) as detected by ELISA using recombinant Ag. All values are mean Ϯ SEM.

No clinical lesions were observed in C1qaϪ/Ϫ (n ϭ 2) and WT lesional mouse skin revealed linear deposition of rabbit IgG at the mice (n ϭ 2) that received normal rabbit IgG at any time during dermal-epidermal junction in all mice that received IgG specific to the observation period (Fig. 1c). IF microscopy analysis of peri- murine type VII collagen (Fig. 1, d and e). No IgG deposits were

FIGURE 5. Factor B-deficient mice are relatively resistant to the induction of subepidermal blisters by Abs against type VII collagen. a, Erosions covered by crusts on the back and hind leg of a WT mouse that received a total dose of 22.5 mg of rabbit IgG against murine type VII collagen, but not in a BfϪ/Ϫ (b) mouse challenged with the same dose of pathogenic IgG (day 7). c, Crusted erosions on the hind leg of a BfϪ/Ϫ mouse injected with 45 mg of pathogenic IgG (day 12). IF micros- copy analysis of mouse skin revealed deposition of rabbit IgG in WT (d) and BfϪ/Ϫ mice on days 7 (e) and 12 (f)of the experiment. Staining for murine C3 revealed similar deposits in the skin of WT and BfϪ/Ϫ (g) mice on days 7 (h) and 12 (i) of the experiment. Deposits of mouse C5 along the dermal-epider- mal junction were strong in the skin of a WT mouse (j) and absent in the skin of BfϪ/Ϫ mice on days 7 (k) and 12 (l) of the experiment. Histo- logical analysis of murine skin re- vealed extensive dermal-epidermal separation and a rich inflammatory infiltrate consisting mainly of neutro- phils in WT (m), but not in BfϪ/Ϫ (n) mice on day 7. o,Onday12,aBfϪ/Ϫ mouse also demonstrated subepidermal cleavage associated with granulocyte infiltration. 6518 COMPLEMENT IN EXPERIMENTAL EBA

control mice (n ϭ 2/group) treated with normal rabbit IgG did not develop skin disease (data not shown). IF microscopy revealed linear deposition of rabbit IgG (Fig. 3, c and d) and murine C3 (Fig. 3, e and f) at the dermal-epidermal junction of mice from both groups. Histological analysis of lesional skin showed sub- epidermal cleavage and a neutrophil-rich inflammatory infiltrate in all mice injected with rabbit IgG to murine type VII collagen (Fig. 3, g and h).

Abs against type VII collagen induce a delayed and milder FIGURE 6. Factor B-deficient mice developed a delayed and signifi- blistering in factor B-deficient mice cantly less severe blistering disease compared with control mice. WT and To assess the contribution of the alternative pathway, we injected BfϪ/Ϫmice were injected with 7.5 mg of purified rabbit IgG to murine type Abs against murine type VII collagen in mice lacking factor B. WT VII collagen every second day, over a period of 12 days, and evaluated for ϭ skin lesions as described in Materials and Methods. Disease activity is mice (n 10) developed initial blisters 4 days after the first in- -Significant jection of rabbit IgG to type VII collagen, while widespread dis ,ء .represented as mean Ϯ SEM in 10 WT and BfϪ/Ϫ mice difference of disease activity between the two groups (p Ͻ 0.05). ease was observed 6 days after the first injection (Fig. 5a). Impor- tantly, BfϪ/Ϫ mice (n ϭ 10) treated with Abs against type VII collagen developed a delayed and significantly less severe blister- observed in the skin of mice injected with normal rabbit IgG (Fig. ing phenotype compared with control mice (Figs. 5, b and c, and Ϫ/Ϫ 1f). Staining for murine complement C3 was bright in the skin of 6). WT (n ϭ 2) and Bf (n ϭ 2) mice treated with normal rabbit WT mice (Fig. 1g) and significantly less intense or absent in the IgG did not develop skin disease (data not shown). By histopa- skin of C1qaϪ/Ϫ mice (Fig. 1h) (C1qaϪ/Ϫ vs WT mice: 1.3 Ϯ 0.15 thology, subepidermal blisters and neutrophil infiltration were ob- vs 2.0 Ϯ 0.24; p Ͻ 0.05) injected with Abs against type VII col- served in biopsies of lesional skin obtained from WT mice (Fig. lagen. No C3 deposits were observed in the skin of mice injected 5m), while no or reduced dermal-epidermal separation and neu- Ϫ/Ϫ with normal rabbit IgG (Fig. 1i). However, staining for murine trophil influx were detected in the skin of Bf mice (Fig. 5, n complement C5 was similar in the skin of WT (Fig. 1j) and and o). IF microscopy of skin biopsies showed linear deposition of Ϫ/Ϫ C1qaϪ/Ϫ mice (Fig. 1k) injected with Abs against type VII colla- rabbit IgG at the dermal-epidermal junction of both WT and Bf gen, as shown by grading intensity of C5 staining in perilesional mice (Fig. 5, d– f). Staining for murine complement C3 was similar Ϫ/Ϫ skin biopsies obtained from the two groups of mice (C1qaϪ/Ϫ vs in the skin of Bf (Fig. 5, h and i), compared with WT mice WT mice: 2.6 Ϯ 0.22 vs 2.8 Ϯ 0.26; p Ͼ 0.05). No C5 deposits (Fig. 5g), as shown by grading intensity of C3 staining in the were observed in the skin of mice injected with normal rabbit IgG perilesional skin biopsies obtained from the two groups of mice Ϫ/Ϫ (Fig. 1l). Histological examination of lesional skin biopsies from (Bf vs WT mice: 1.11 Ϯ 0.11 vs 1.22 Ϯ 0.15; p Ͼ 0.05). In diseased WT (Fig. 1m) and C1qaϪ/Ϫ (Fig. 1n) mice injected with contrast, staining for murine complement C5 was bright in the skin IgG against type VII collagen revealed extensive dermal-epider- of WT mice (Fig. 5j) and significantly less intense or absent in the Ϫ/Ϫ Ϫ/Ϫ mal separation accompanied by dense inflammatory infiltrates that skin of Bf mice (Fig. 5, k and l) (Bf vs WT mice: 0.33 Ϯ were dominated by neutrophils. No histological alterations were 0.24 vs 1.33 Ϯ 0.33; p Ͻ 0.05). The levels of rabbit Abs against Ϫ/Ϫ observed in mice injected with normal rabbit IgG (Fig. 1o). type VII collagen were comparable in serum of Bf and WT mice (Fig. 7a). Interestingly, granulocytes were significantly less MBL-null mice develop subepidermal blisters when injected with recruited into the of the BfϪ/Ϫ mice injected with IgG Abs against type VII collagen against type VII collagen compared with the WT mice as demon- In additional experiments, we analyzed the contribution of MBL strated by histological analysis and measurements of the MPO ac- for complement activation and blister formation in experimental tivity in skin biopsies (Fig. 7b). EBA. MBL-null (n ϭ 10) and WT (n ϭ 10) mice were treated with Abs against murine type VII collagen. Both MBL-null and control Reconstitution with granulocytes renders factor B-deficient mice mice developed widespread blistering disease (Fig. 3, a and b). readily susceptible to blistering by Abs against type VII collagen Levels of circulating Abs against type VII collagen and disease To verify whether a lower recruitment of granulocytes is respon- severity were similar in MBL-null and control mice at any time sible for the inhibition of blistering, BfϪ/Ϫ mice were treated with during the observation period (Fig. 4, a and b). MBL-null and Abs against murine type VII collagen and locally injected on day

FIGURE 7. Injection of Abs against type VII collagen results in less neutrophil infiltration in fac- tor B-deficient compared with WT mice. a, Serum levels of Abs to type VII collagen in BfϪ/Ϫ and WT mice (n ϭ 10/group) treated with 7.5 mg per injection of rabbit Abs against type VII collagen (antiCVII) or normal rabbit IgG (NRIgG) as detected by ELISA using recombinant Ag. b, Neutrophil infil- trates in the skin of WT and BfϪ/Ϫ mice treated with the Abs against type VII collagen or control Ab as assessed by measuring MPO activity in skin biopsies significant ,ء) as described in Materials and Methods difference, p Ͻ 0.01). All values are means Ϯ SEM. The Journal of Immunology 6519

FIGURE 8. Local reconstitution with granulocytes renders factor B-deficient mice susceptible to the Ab-induced skin blistering. Mice were treated with a total dose of 22.5 mg of rabbit IgG s.c. into the back and, subsequently, ears were injected with 5 ϫ 106 murine granulocytes in 50 ␮l of medium intradermally. Both a WT mouse (a) injected with rabbit Abs to murine type VII collagen and a BfϪ/Ϫ mouse (b) injected with pathogenic rabbit IgG and locally reconstituted with granulocytes developed blisters and erosions on their ears. c,ABfϪ/Ϫ mouse treated with control rabbit IgG and reconstituted with granulocytes did not show skin alterations. d–f, Histological analysis revealed an infiltrate of neutrophils in the dermis of all mice. Dermal-epidermal separation was observed in the skin of both the WT mouse (d) injected with rabbit Abs to murine type VII collagen and the BfϪ/Ϫ mouse (e) injected with pathogenic rabbit IgG and locally reconstituted with granulocytes, but not in the skin of the BfϪ/Ϫ mouse (f) treated with control rabbit IgG and reconstituted with granulocytes.

6 with granulocytes purified from WT and BfϪ/Ϫ mice. Both WT genic effects of autoantibodies in other autoimmune diseases such mice (n ϭ 4) treated with IgG against type VII collagen alone (Fig. as (30) are mediated strictly by binding to their target Ϫ Ϫ 8a) and Bf / mice treated with Abs against murine type VII col- Ag and do not involve the complement network. An overall remark Ϫ Ϫ lagen and intradermally injected with WT (Fig. 8b) and Bf / with regard to the role of complement in tissue injury in autoim- (data not shown) granulocytes (n ϭ 4/group) developed blisters mune diseases is that its contribution varies depending on the par- Ϫ Ϫ and erosions on their ears. In contrast, Bf / mice (n ϭ 2) treated ticular tissue involved and the genetic background. Examining with control rabbit IgG and reconstituted with WT granulocytes complement activation pathways in different autoimmune diseases did not show any skin alterations (Fig. 8c). Histological analysis in patients and experimental animals is therefore mandatory to revealed an infiltrate of neutrophils in the dermis of all groups of identify key molecular effectors of inflammatory tissue injury for mice (Fig. 8, d–f). Recruitment of granulocytes to the dermal-epi- therapeutic interventions. dermal junction and subepidermal cleavage was found in the skin It is textbook knowledge that complement-fixing (auto)antibod- of control mice injected with Abs against murine type VII collagen ies bind to the C1q and trigger the complement cascade by the Ϫ/Ϫ (Fig. 8d) and of Bf mice injected with Abs to murine type VII classical pathway. It has been therefore proposed that Ab-induced collagen and locally reconstituted with WT (Fig. 8e) and BfϪ/Ϫ Ϫ/Ϫ tissue injury essentially depends on complement activation by the (data not shown) granulocytes, but not in the skin of Bf mice classical pathway. In the present study, we found the complement treated with control rabbit IgG and reconstituted with WT granu- component C1q to be dispensable for the induction of blisters by locytes (Fig. 8f). Abs against type VII collagen. In line with the present observa- tions, C1q deficiency does not protect from autoantibody-induced Discussion tissue injury in cryoglobulin-induced immune complex glomeru- We made the unanticipated observation that C1q deficiency and lonephritis (26). In addition, the blockade of the classical pathway blockade of complement activation by the classical pathway does Ϫ Ϫ in C4 / mice does not abolish the pathogenic effects of K/BxN- not inhibit tissue injury induced by Abs against type VII collagen derived anti-GPI serum or of anti-collagen II Abs in a passive in mice. Instead, we found that the activation of the alternative transfer model of rheumatoid arthritis (24, 31). Fetal loss and complement pathway played a pivotal role for the Ab-induced blis- growth restriction triggered by anti-cardiolipin Abs in mice, main tering. The passive transfer of Abs against type VII collagen into mice provokes within days a subepidermal blistering disease re- features of the antiphospholipid syndrome, and blistering induced producing the clinical, histopathological, immunopathological, by Abs against BP180 in experimental also and electron microscopic findings in patients with EBA. A likely depend on complement activation (28, 32, 33). However, in these Ϫ/Ϫ scenario is that, after binding to their target at the dermal-epider- experimental models, the resistance of C4 mice to tissue injury mal junction, Abs against type VII collagen trigger a cascade of demonstrates an important role of an intact classical complement events that includes the activation of the complement network activation pathway (29, 33). The differences with regard to the and/or engagement of FcRs on leukocytes recruited into the skin. classical pathway contribution to pathology in different experimen- Potent inflammatory mediators, including cytokines, reactive ox- tal disease models have not yet been explained. One may only ygen species, and proteases released by granulocytes, most prob- speculate that differences of targeted Ags and binding of Abs as ably amplify local inflammation and are instrumental for tissue well as different isotypes and glycosylation of Abs may influence destruction (1). the interaction of Ag-Ab complexes with various components of This scenario bears similarities to the effector phase of other the complement network. autoantibody-induced inflammatory diseases, including arthritis To evaluate the contribution of factor B for Ab-induced blister- (24), vitiligo (25), cryoglobulinemia (26, 27), bullous pemphigoid ing, we analyzed disease expression in BfϪ/Ϫ mice (19). Induction (28), and antiphospholipid syndrome (29). In contrast, the patho- of blistering was delayed and the extent of cutaneous disease was 6520 COMPLEMENT IN EXPERIMENTAL EBA significantly lower in BfϪ/Ϫ compared with WT mice. This inter- nancy loss in mice (17). To avoid a global inhibition of comple- esting finding raised the question of what activates the alternative ment activation, more specific approaches were explored to target pathway. A classical explanation is that the alternative pathway is inhibitors of complement activation to the site of inflammation by initiated as “an amplification loop” by fixed C3b generated by linking inhibitors to the complement receptor (CR) 2 (46, 47). C3 classical or lectin pathways. However, blockade of complement breakdown products deposited at sites of complement activation activation by the classical pathway in C1q-deficient mice or by the are natural ligands for CR2. Thus, blockers of the alternative path- lectin pathway in MBL-null mice did not alter the Ab-induced way could be directed to the dermal-epidermal junction by using blistering. These findings suggest that initial complement activa- fusion proteins containing CR2 and single-chain variable frag- tion occurs by both classical and lectin pathways, which can thus ments of mAb or peptides, which are capable of inhibiting com- compensate for each other. An additional possibility is that the plement activation. activation and amplification of the alternative pathway is due to a In conclusion, our results demonstrate that an intact alternative breakdown of the active control of the alternative pathway in the complement activation pathway is required for blistering induced skin caused by binding of Abs, which may act as activating sur- by Abs against type VII collagen. Thus, selectively blocking this faces. Although it has not been reported that ficolins bind Igs, our pathway may offer therapeutic benefit in patients with EBA and study cannot rule out a possible involvement of ficolins that are related autoantibody-mediated diseases. also able to initiate the lectin pathway (34, 35). Granulocyte recruitment to the dermal-epidermal junction by Acknowledgment Abs is a prerequisite for blister induction in the animal model used We are grateful to Dr. Yi Wang for providing the Ab specific to murine in this study (36) and in the ex vivo cryosection model of EBA (7). C5 (BB5.1). Our present findings show that the absence of factor B impairs the recruitment of granulocytes into the skin of mice injected with Abs Disclosures against type VII collagen. In line with these results, inhibition of The authors have no financial conflict of interest. neutrophil recruitment into the joints has been observed in factor B-deficient mice passively transferred with K/BxN-derived anti- References GPI serum (24). The relevance of granulocyte recruitment into the 1. Sitaru, C., and D. Zillikens. 2005. Mechanisms of blister induction by autoanti- bodies. Exp. Dermatol. 14: 861–875. skin is further supported by the fact that local reconstitution of 2. Woodley, D. T., R. A. Briggaman, E. J. O’Keefe, A. O. Inman, L. L. Queen, and factor B-deficient mice with granulocytes following injection of W. R. Gammon. 1984. Identification of the skin basement-membrane autoantigen Abs against type VII collagen resulted in blister formation. Gran- in epidermolysis bullosa acquisita. N. Engl. J. Med. 310: 1007–1013. 3. Stanley, J. R., N. Rubinstein, and V. Klaus-Kovtun. 1985. Epidermolysis bullosa ulocytes were shown to produce factors of the alternative pathway acquisita antigen is synthesized by both human keratinocytes and human dermal (37, 38). The finding that BfϪ/Ϫ granulocytes, similar to WT cells, fibroblasts. J. Invest. Dermatol. 85: 542–545. 4. Lapiere, J. C., D. T. Woodley, M. G. Parente, T. Iwasaki, K. C. Wynn, also mediate blister formation strongly suggests that factor B pro- A. M. Christiano, and J. Uitto. 1993. Epitope mapping of type VII collagen: duced by granulocytes is not essentially required for Ab-induced identification of discrete peptide sequences recognized by sera from patients with blistering in experimental EBA. acquired epidermolysis bullosa. J. Clin. Invest. 92: 1831–1839. 5. Tanaka, T., F. Furukawa, and S. Imamura. 1994. Epitope mapping for epider- Deposition of the complement protein C3 at sites of autoanti- molysis bullosa acquisita autoantibody by molecularly cloned cDNA for type VII body binding in tissues is a hallmark of autoimmune diseases in collagen. J. Invest. Dermatol. 102: 706–709. humans (39). Our present results show that C3 deposition in the 6. Tanaka, H., A. Ishida-Yamamoto, T. Hashimoto, K. Hiramoto, T. Harada, Y. Kawachi, H. Shimizu, T. Tanaka, K. Kishiyama, B. Hopfner, et al. 1997. A skin is not imperiously associated with Ab-induced tissue injury. novel variant of acquired epidermolysis bullosa with autoantibodies against the On one hand, a reduced C3 deposition was found in C1q-deficient central triple-helical domain of type VII collagen. Lab. Invest. 77: 623–632. 7. Sitaru, C., A. Kromminga, T. Hashimoto, E. B. Bro¨cker, and D. Zillikens. 2002. mice susceptible to experimental EBA. On the other hand, C3 Autoantibodies to type VII collagen mediate Fc␥-dependent granulocyte activa- deposits were found in factor B-deficient mice resistant to blister tion and induce dermal-epidermal separation in cryosections of human skin. induction by Abs against type VII collagen. Interestingly, a mark- Am. J. Pathol. 161: 301–311. 8. Shimanovich, I., S. Mihai, G. J. Oostingh, T. T. Ilenchuk, E. B. Brocker, edly reduced C5 deposition was found in factor B-deficient mice G. Opdenakker, D. Zillikens, and C. Sitaru. 2004. Granulocyte-derived elastase compared with controls. Indeed, it has been recently shown that and gelatinase B are required for dermal-epidermal separation induced by auto- generation of C5a does not absolutely require C3 (40). Proteases antibodies from patients with epidermolysis bullosa acquisita and bullous pem- phigoid. J. Pathol. 204: 519–527. released by leukocytes recruited at the dermal-epidermal junction 9. Sitaru, C., S. Mihai, C. Otto, M. T. Chiriac, I. Hau␤er, B. Dotterweich, H. Saito, could also contribute to generation of C5a in our model (41, 42). C. Rose, A. Ishiko, and D. Zillikens. 2005. Induction of dermal-epidermal sep- aration in mice by passive transfer of antibodies to type VII collagen. J. Clin. Why complement activation at the dermal-epidermal junction in Invest. 115: 870–878. C1q-deficient mice is curtailed beyond the C3 activation step is 10. Woodley, D. T., R. Ram, A. Doostan, P. Bandyopadhyay, Y. Huang, unclear, but is reminiscent of the targeted and restricted comple- J. Remington, Y. Hou, D. R. Keene, Z. Liu, and M. Chen. 2006. Induction of epidermolysis bullosa acquisita in mice by passive transfer of autoantibodies ment activation that may occur on modified self-tissues (43). from patients. J. Invest. Dermatol. 126: 1323–1330. Taken together, these findings suggest that generation of C5a in 11. Sitaru, C., M. T. Chiriac, S. Mihai, J. Bu¨ning, A. Gebert, A. Ishiko, and our model is required for disease expression, but partly indepen- D. Zillikens. 2006. Induction of complement-fixing autoantibodies against type VII collagen results in subepidermal blistering in mice. J. Immunol. 177: dent of the classical C5 convertase (C4b2a3b) availability. 3461–3468. New mechanistic insights into the role of complement activation 12. Mooney, E., R. J. Falk, and W. R. Gammon. 1992. Studies on complement de- posits in epidermolysis bullosa acquisita and bullous pemphigoid. Arch. Derma- in the pathogenesis of autoimmune inflammatory diseases facili- tol. 128: 58–60. tate targeting complement pathways for therapeutic drug develop- 13. Smoller, B. R., and D. T. Woodley. 1992. Differences in direct immunofluores- ment. In addition to many reports of a beneficial effect of comple- cence staining patterns in epidermolysis bullosa acquisita and bullous pemphi- goid. J. Am. Acad. Dermatol. 27: 674–678. ment blockade in animal models, anti-C5 therapy is well tolerated 14. Cho, H. J., I. J. Lee, and S. C. Kim. 1998. Complement-fixing abilities and IgG and effective in patients with paroxysmal nocturnal hemoglobin- subclasses of autoantibodies in epidermolysis bullosa acquisita. Yonsei. Med. J. uria (44, 45). Our present study identifies factor B as an additional 39: 339–344. 15. Lachmann, P. J., and N. C. Hughes-Jones. 1984. Initiation of complement acti- target of therapy in inflammatory autoimmune blistering diseases. vation. Springer Semin. Immunopathol. 7: 143–162. Selective inhibition of this pathway may not result in side effects 16. Fujita, T., M. Matsushita, and Y. Endo. 2004. The lectin-complement pathway: its role in innate immunity and evolution. Immunol. Rev. 198: 185–202. seen with inhibitors of C3 and C5 convertases. Such an inhibitory 17. Thurman, J. M., and V. M. Holers. 2006. The central role of the alternative mAb against factor B prevents anti-phospholipid Ab-induced preg- complement pathway in human disease. J. Immunol. 176: 1305–1310. The Journal of Immunology 6521

18. Botto, M., C. Dell’Agnola, A. E. Bygrave, E. M. Thompson, H. T. Cook, F. Petry, Complement C3 activation is required for antiphospholipid antibody-induced fe- M. Loos, P. P. Pandolfi, and M. J. Walport. 1998. Homozygous C1q deficiency tal loss. J. Exp. Med. 195: 211–220. causes glomerulonephritis associated with multiple apoptotic bodies. Nat. Genet. 33. Nelson, K. C., M. Zhao, P. R. Schroder, N. Li, R. A. Wetsel, L. A. Diaz, and 19: 56–59. Z. Liu. 2006. Role of different pathways of the complement cascade in experi- 19. Matsumoto, M., W. Fukuda, A. Circolo, J. Goellner, J. Strauss-Schoenberger, mental bullous pemphigoid. J. Clin. Invest. 116: 2892–2900. X. Wang, S. Fujita, T. Hidvegi, D. D. Chaplin, and H. R. Colten. 1997. Abro- 34. Matsushita, M., Y. Endo, and T. Fujita. 2000. Cutting edge: complement-acti- gation of the alternative complement pathway by targeted deletion of murine vating complex of ficolin and mannose-binding lectin-associated serine protease. factor B. Proc. Natl. Acad. Sci. USA 94: 8720–8725. J. Immunol. 164: 2281–2284. 20. Shi, L., K. Takahashi, J. Dundee, S. Shahroor-Karni, S. Thiel, J. C. Jensenius, 35. Lynch, N. J., S. Roscher, T. Hartung, S. Morath, M. Matsushita, D. N. Maennel, F. Gad, M. R. Hamblin, K. N. Sastry, and R. A. Ezekowitz. 2004. Mannose- M. Kuraya, T. Fujita, and W. J. Schwaeble. 2004. L-ficolin specifically binds to binding lectin-deficient mice are susceptible to infection with Staphylococcus lipoteichoic acid, a cell wall constituent of Gram-positive bacteria, and activates aureus. J. Exp. Med. 199: 1379–1390. the lectin pathway of complement. J. Immunol. 172: 1198–1202. 21. Møller-Kristensen, K., M. R. Hamblin, S. Thiel, J. C. Jensenius, and 36. Chiriac, M. T., J. Roesler, A. Sindrilaru, K. Scharffetter-Kochanek, D. Zillikens, K. Takahashi. 2007. Burn injury reveals altered phenotype in mannan-binding and C. Sitaru. 2007. NADPH oxidase is required for neutrophil-dependent au- lectin-deficient mice. J. Invest. Dermatol. In press. toantibody-induced tissue damage. J. Pathol. In press. 22. Wang, Y., S. A. Rollins, J. A. Madri, and L. A. Matis. 1995. Anti-C5 monoclonal 37. Okuda, T. 1991. Murine polymorphonuclear leukocytes synthesize and secrete antibody therapy prevents collagen-induced arthritis and ameliorates established the third component and factor B of complement. Int. Immunol. 3: 293–296. disease. Proc. Natl. Acad. Sci. USA 92: 8955–8959. 38. Wirthmueller, U., B. Dewald, M. Thelen, M. K. Schafer, C. Stover, K. Whaley, 23. Bradley, P. P., D. A. Priebat, R. D. Christensen, and G. Rothstein. 1982. Mea- J. North, P. Eggleton, K. B. Reid, and W. J. Schwaeble. 1997. Properdin, a surement of cutaneous inflammation: estimation of neutrophil content with an positive regulator of complement activation, is released from secondary granules enzyme marker. J. Invest. Dermatol. 78: 206–209. of stimulated peripheral blood neutrophils. J. Immunol. 158: 4444–4451. 24. Ji, H., K. Ohmura, U. Mahmood, D. M. Lee, F. M. Hofhuis, S. A. Boackle, 39. Paul, E., and M. C. Carroll. 2002. Complement and complement receptors in K. Takahashi, V. M. Holers, M. Walport, C. Gerard, et al. 2002. Arthritis criti- . In The Molecular Pathology of Autoimmune Diseases. cally dependent on innate immune system players. Immunity 16: 157–168. A. N. Theophilopoulos and C. A. Bona, eds. Taylor and Francis, New York, pp. 243–258. 25. Trcka, J., Y. Moroi, R. A. Clynes, S. M. Goldberg, A. Bergtold, M. A. Perales, 40. Huber-Lang, M., J. V. Sarma, F. S. Zetoune, D. Rittirsch, T. A. Neff, M. Ma, C. R. Ferrone, M. C. Carroll, J. V. Ravetch, and A. N. Houghton. 2002. S. R. McGuire, J. D. Lambris, R. L. Warner, M. A. Flierl, L. M. Hoesel, et al. Redundant and alternative roles for activating Fc receptors and complement in an 2006. Generation of C5a in the absence of C3: a new complement activation antibody-dependent model of autoimmune vitiligo. Immunity 16: 861–868. pathway. Nat. Med. 12: 682–687. 26. Trendelenburg, M., L. Fossati-Jimack, J. Cortes-Hernandez, D. Turnberg, 41. Ward, P. A., and J. H. Hill. 1970. C5 chemotactic fragments produced by an M. Lewis, S. Izui, H. T. Cook, and M. Botto. 2005. The role of complement in enzyme in lysosomal granules of neutrophils. J. Immunol. 104: 535–543. J. Immunol. cryoglobulin-induced immune complex glomerulonephritis. 175: 42. Huber-Lang, M., E. M. Younkin, J. V. Sarma, N. Riedemann, S. R. McGuire, 6909–6914. K. T. Lu, R. Kunkel, J. G. Younger, F. S. Zetoune, and P. A. Ward. 2002. 27. Izui, S., T. Fulpius, L. Reininger, Y. Pastore, and T. Kobayakawa. 1998. Role of Generation of C5a by phagocytic cells. Am. J. Pathol. 161: 1849–1859. neutrophils in murine cryoglobulinemia. Inflamm. Res. 47(Suppl. 3): S145–S150. 43. Riley-Vargas, R. C., S. Lanzendorf, and J. P. Atkinson. 2005. Targeted and re- 28. Liu, Z., G. J. Giudice, S. J. Swartz, J. A. Fairley, G. O. Till, J. L. Troy, and stricted complement activation on acrosome-reacted spermatozoa. J. Clin. Invest. L. A. Diaz. 1995. The role of complement in experimental bullous pemphigoid. 115: 1241–1249. J. Clin. Invest. 95: 1539–1544. 44. Hillmen, P., C. Hall, J. C. Marsh, M. Elebute, M. P. Bombara, B. E. Petro, 29. Girardi, G., J. Berman, P. Redecha, L. Spruce, J. M. Thurman, D. Kraus, M. J. Cullen, S. J. Richards, S. A. Rollins, C. F. Mojcik, and R. P. Rother. 2004. T. J. Hollmann, P. Casali, M. C. Caroll, R. A. Wetsel, et al. 2003. Complement Effect of eculizumab on hemolysis and transfusion requirements in patients with C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syn- paroxysmal nocturnal hemoglobinuria. N. Engl. J. Med. 350: 552–559. drome. J. Clin. Invest. 112: 1644–1654. 45. Hill, A., P. Hillmen, S. J. Richards, D. Elebute, J. C. Marsh, J. Chan, 30. Anhalt, G. J., G. O. Till, L. A. Diaz, R. S. Labib, H. P. Patel, and N. F. Eaglstein. C. F. Mojcik, and R. P. Rother. 2005. Sustained response and long-term safety of 1986. Defining the role of complement in experimental in eculizumab in paroxysmal nocturnal hemoglobinuria. Blood 106: 2559–2565. mice. J. Immunol. 137: 2835–2840. 46. Atkinson, C., H. Song, B. Lu, F. Qiao, T. A. Burns, V. M. Holers, G. C. Tsokos, 31. Banda, N. K., J. M. Thurman, D. Kraus, A. Wood, M. C. Carroll, W. P. Arend, and S. Tomlinson. 2005. Targeted complement inhibition by C3d recognition and V. M. Holers. 2006. Alternative complement pathway activation is essential ameliorates tissue injury without apparent increase in susceptibility to infection. for inflammation and joint destruction in the passive transfer model of collagen- J. Clin. Invest. 115: 2444–2453. induced arthritis. J. Immunol. 177: 1904–1912. 47. Song, H., C. He, C. Knaak, J. M. Guthridge, V. M. Holers, and S. Tomlinson. 32. Holers, V. M., G. Girardi, L. Mo, J. M. Guthridge, H. Molina, S. S. Pierangeli, 2003. Complement receptor 2-mediated targeting of complement inhibitors to R. Espinola, L. E. Xiaowei, D. Mao, C. G. Vialpando, and J. E. Salmon. 2002. sites of complement activation. J. Clin. Invest. 111: 1875–1885.