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The Journal of Immunology, 2007, 178: 6514-6521.
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Alternative Pathway of Complement Activation Is Critical for Blister Induction in Experimental Epidermolysis Bullosa Acquisita1

Sidonia Mihai*, Mircea T. Chiriac*, Kazue Takahashi{dagger}, Joshua M. Thurman{ddagger},§, V. Michael Holers{ddagger},§, Detlef Zillikens*, Marina Botto and Cassian Sitaru2,*

* Department of Dermatology, University of Lübeck, Lübeck, Germany; {dagger} Department of Pediatrics, Laboratory of Developmental Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115; {ddagger} Department of Medicine and § Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80218; and Rheumatology Section, Imperial College School of Medicine, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epidermolysis bullosa acquisita is a subepidermal blistering disease associated with tissue-bound and circulating autoantibodies 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epidermolysis bullosa acquisita (EBA),3 a severe chronic subepidermal blistering disease of skin and mucous membranes, is characterized by tissue-bound and circulating IgG Abs to the dermal-epidermal junction (1). Patients’ serum autoantibodies bind to the 290-kDa type VII collagen, the major component of anchoring fibrils (2, 3). Epitopes recognized by the majority of EBA sera were mapped to the non-collagenous 1 domain of type VII collagen (4, 5, 6). The pathogenic relevance of Abs against type VII collagen is supported by compelling evidence: 1) EBA autoantibodies were shown to recruit and activate leukocytes ex vivo, resulting in dermal-epidermal separation in cryosections of human skin (7, 8). 2) Abs against type VII collagen induce subepidermal blisters when passively transferred into mice (9, 10). 3) Immunization with recombinant autologous type VII collagen induces an autoimmune response to this protein, resulting in a blistering phenotype closely resembling human EBA (11).

Autoantibodies against type VII collagen are able to activate the complement system in vivo and in vitro. In the skin of EBA patients, deposition of different complement components, including C3, C5b, and membrane attack complex, are found with an incidence ranging from ~40 to 100% (12, 13, 14). In addition, deposition of C3 at the dermal-epidermal junction of murine skin is a constant feature in different passive transfer mouse models of EBA (9, 10) and in experimental EBA induced by immunization of mice with autologous type VII collagen (11). These observations suggested that the complement system is involved in the autoimmune tissue injury in EBA. Indeed, this hypothesis was confirmed by our recent studies showing that C5-deficient mice are resistant to blister induction by passive transfer of Abs against type VII collagen (9). The initiators and processes that result in complement activation can be assigned to three different pathways: classical, alternative, and lectin. The classical pathway is activated primarily by Ag-Ab complexes binding to C1q (15). The lectin pathway is initiated by ficolins and mannan-binding lectin (MBL) (16). The alternative pathway is continuously activated by factor B through a process called "tickover" of C3 and requires active control mechanisms to prevent autologous injury (17). The relevance of the different complement components and pathways of the complement cascade in experimental EBA, which could represent targets for treatment, have remained unknown.

In the present study, we examined the relative contribution of the different complement activation pathways for blister formation in experimental EBA. Our findings provide the first direct evidence for the involvement of the alternative pathway in an autoantibody-induced blistering disease and a conceptual framework for developing rational therapeutic strategies for EBA and related diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C1qa–/– and Bf–/– mice, backcrossed to BALB/c mice (for 10 and 7 generations, respectively), and MBL-null mice backcrossed to C57BL/6J mice (for 7 generations), were previously described (18, 19, 20, 21). Age- and sex-matched BALB/c and C57BL/6J mice were obtained from Charles River. All injections and bleedings were performed on mice narcotized by inhalation of isoflurane or i.p. administration of a mixture of ketamine (100 µg/g) and xylazine (15 µg/g). The experiments were approved by the local authorities of the Animal Care and Use Committee (reference number: 6/g/04) and performed by certified personnel.

Affinity purification of Abs

Rabbits were immunized with recombinant forms of murine type VII collagen as described elsewhere (9). IgG from immune and preimmune rabbit sera was purified by affinity chromatography using protein G affinity as previously reported (9). Reactivity of IgG fractions was analyzed by immunofluorescence (IF) microscopy on murine skin.

Induction of blistering in vivo and phenotype assessment

Passive transfer studies followed published protocols with minor modifications (9). Briefly, mice received six injections of 7.5 mg of rabbit IgG. Blisters or erosions were counted and the extent of skin disease was scored as follows: 0, no lesions; 1, <10 lesions or <1% of the skin surface; 2, >10 lesions or 1–5% of the skin surface; 3, 5–10%; 4, 10–20%; and 5, >20% involvement of the skin surface. Biopsies of lesional and perilesional skin were obtained 2 days after the last injection of IgG and prepared for examination by histopathology and IF microscopy as described previously (9, 11). Tissue-bound murine C5 was detected by incubation of the frozen sections prepared from tissue biopsies with a mAb specific to murine C5 (BB5.1) (22) and, finally, with a FITC-labeled Ab specific to mouse IgG (DakoCytomation). The staining intensity of immunoreactants in the skin of immunized mice was assessed semiquantitatively using a score comprising 0, for no staining; 1, faint staining; 2, medium; and 3, intense staining (11).

Neutrophil infiltration of murine skin was assayed as described previously (23), with minor modifications. Briefly, in both clinically diseased and not diseased mice, the left ear was removed after killing the mice and skin samples (~10 x 5 mm in size) were extracted by homogenization in a buffer containing 0.1 M Tris-Cl (pH 7.6), 0.15 M NaCl, and 0.5% hexadecyl trimethylammonium bromide (Sigma-Aldrich). Myeloperoxidase (MPO) activity in the supernatant fraction was measured by the change in OD at 460 nm resulting from decomposition of H2O2 in the presence of o-dianisidine (Sigma-Aldrich). A standard reference curve was established using known concentrations of purified MPO (Sigma-Aldrich). MPO content was expressed as units of MPO activity per mg of protein. Protein concentrations were determined by the Bradford dye-binding assay (Bio-Rad).

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 >90% 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 Biosciences). Mice were injected with 5 x 106 cells in 50 µl of medium intradermally in the ears. The animals were examined clinically after 12 and 24 h, subsequently killed, and samples prepared and analyzed as described 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 reactivity 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 {chi}2 and Student t test, respectively. The Mann-Whitney U test was used to compare values for semiquantitative scoring of immunohistochemistry. Means are presented ± SEM; p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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 subepidermal blister formation in experimental EBA, we injected C1qa–/– (n = 10) and wild-type (WT; n = 10) mice with rabbit IgG against murine type VII collagen. All mice injected with Abs against type VII collagen developed single blisters 4 days after the first injection. Widespread lesions, including blisters, erosions, and crusts, occurred 6 days after the first injection (Fig. 1, a and b). Only at the end of the observation period, C1qa–/– mice demonstrated significantly less extensive skin disease compared with control mice (Fig. 2a). The levels of rabbit Abs against type VII collagen in serum of mice of both groups were similar (Fig. 2b). No clinical lesions were observed in C1qa–/– (n = 2) and WT mice (n = 2) that received normal rabbit IgG at any time during the observation period (Fig. 1c). IF microscopy analysis of perilesional mouse skin revealed linear deposition of rabbit IgG at the dermal-epidermal junction in all mice that received IgG specific to murine type VII collagen (Fig. 1, d and e). No IgG deposits were observed in the skin of mice injected with normal rabbit IgG (Fig. 1f). Staining for murine complement C3 was bright in the skin of WT mice (Fig. 1g) and significantly less intense or absent in the skin of C1qa–/– mice (Fig. 1h) (C1qa–/– vs WT mice: 1.3 ± 0.15 vs 2.0 ± 0.24; p < 0.05) injected with Abs against type VII collagen. No C3 deposits were observed in the skin of mice injected with normal rabbit IgG (Fig. 1i). However, staining for murine complement C5 was similar in the skin of WT (Fig. 1j) and C1qa–/– mice (Fig. 1k) injected with Abs against type VII collagen, as shown by grading intensity of C5 staining in perilesional skin biopsies obtained from the two groups of mice (C1qa–/– vs WT mice: 2.6 ± 0.22 vs 2.8 ± 0.26; p > 0.05). No C5 deposits were observed in the skin of mice injected with normal rabbit IgG (Fig. 1l). Histological examination of lesional skin biopsies from diseased WT (Fig. 1m) and C1qa–/– (Fig. 1n) mice injected with IgG against type VII collagen revealed extensive dermal-epidermal separation accompanied by dense inflammatory infiltrates that were dominated by neutrophils. No histological alterations were observed in mice injected with normal rabbit IgG (Fig. 1o).


Figure 1
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  		FIGURE 1. C1q-deficient mice are susceptible to the induction of subepidermal 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 developed in both WT (a) and C1qa–/– mice (b) that received a total dose of 45 mg of rabbit IgG against type VII collagen (day 12). c, A C1qa–/– mouse receiving the same amount of control rabbit IgG did not develop skin lesions. IF microscopy analysis of perilesional skin revealed deposition of rabbit IgG at the dermal-epidermal junction of both WT (d) and C1qa–/– (e) mice injected with Abs against type VII collagen, but not in the C1qa–/– (f) mouse treated with control rabbit IgG. Deposits of mouse C3 along the dermal-epidermal junction were strong in the skin of WT (g) and weak in the skin of C1qa–/– (h) mice injected with Abs against type VII collagen and absent in the C1qa–/– mouse (i) treated with control rabbit IgG. Staining for murine C5 revealed similar deposits in the skin of the WT (j) and of the C1qa–/– (k) mice treated with Abs against type VII collagen, but was absent in the C1qa–/– (l) mouse injected with control rabbit IgG. Histological analysis of lesional skin revealed extensive dermal-epidermal separation and a neutrophil-rich inflammatory infiltrate in both WT (m) and C1qa–/– (n) mice injected with Abs against type VII collagen. o, Normal histological appearance in the skin of the C1qa–/– mouse injected with control rabbit IgG.

 

Figure 2
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  		FIGURE 2. Time course and disease severity in C1q-deficient and control mice. a, C1qa–/–and WT mice (n = 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. *, Significant difference of disease activity (p < 0.05) between the two groups. b, Serum levels of Abs to type VII collagen in C1qa–/– and WT mice (n = 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 ± SEM.

 
MBL-null mice develop subepidermal blisters when injected with Abs against type VII collagen

In additional experiments, we analyzed the contribution of MBL for complement activation and blister formation in experimental EBA. MBL-null (n = 10) and WT (n = 10) mice were treated with Abs against murine type VII collagen. Both MBL-null and control mice developed widespread blistering disease (Fig. 3, a and b). Levels of circulating Abs against type VII collagen and disease severity were similar in MBL-null and control mice at any time during the observation period (Fig. 4, a and b). MBL-null and control mice (n = 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 subepidermal cleavage and a neutrophil-rich inflammatory infiltrate in all mice injected with rabbit IgG to murine type VII collagen (Fig. 3, g and h).


Figure 3
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  		FIGURE 3. Abs against type VII collagen induce subepidermal blisters in MBL-null mice. Skin lesions, including blisters and erosions covered by crusts on the ear and front leg, and alopecia of the snout and around 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 collagen (day 12). IF analysis of perilesional skin revealed deposition of rabbit IgG (c and d) and murine C3 (e and f) at the dermal-epidermal junction of both MBL-sufficient and MBL-null mice. Histological analysis of lesional skin revealed extensive dermal-epidermal separation and a neutrophil-rich inflammatory infiltrate in both MBL-sufficient (g) and MBL-null (h) mice.

 

Figure 4
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  		FIGURE 4. Time course and disease severity in MBL-null and control mice. a, MBL-null and control mice (WT) (n = 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 = 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 ± SEM.

 
Abs against type VII collagen induce a delayed and milder blistering in factor B-deficient mice

To assess the contribution of the alternative pathway, we injected Abs against murine type VII collagen in mice lacking factor B. WT mice (n = 10) developed initial blisters 4 days after the first injection of rabbit IgG to type VII collagen, while widespread disease was observed 6 days after the first injection (Fig. 5a). Importantly, Bf–/– mice (n = 10) treated with Abs against type VII collagen developed a delayed and significantly less severe blistering phenotype compared with control mice (Figs. 5, b and c, and 6). WT (n = 2) and Bf–/– (n = 2) mice treated with normal rabbit IgG did not develop skin disease (data not shown). By histopathology, subepidermal blisters and neutrophil infiltration were observed in biopsies of lesional skin obtained from WT mice (Fig. 5m), while no or reduced dermal-epidermal separation and neutrophil influx were detected in the skin of Bf–/– mice (Fig. 5, n and o). IF microscopy of skin biopsies showed linear deposition of rabbit IgG at the dermal-epidermal junction of both WT and Bf–/– mice (Fig. 5, d– f). Staining for murine complement C3 was similar in the skin of Bf–/– (Fig. 5, h and i), compared with WT mice (Fig. 5g), as shown by grading intensity of C3 staining in the perilesional skin biopsies obtained from the two groups of mice (Bf–/– vs WT mice: 1.11 ± 0.11 vs 1.22 ± 0.15; p > 0.05). In contrast, staining for murine complement C5 was bright in the skin of WT mice (Fig. 5j) and significantly less intense or absent in the skin of Bf–/– mice (Fig. 5, k and l) (Bf–/– vs WT mice: 0.33 ± 0.24 vs 1.33 ± 0.33; p < 0.05). The levels of rabbit Abs against type VII collagen were comparable in serum of Bf–/– and WT mice (Fig. 7a). Interestingly, granulocytes were significantly less recruited into the dermis of the Bf–/– mice injected with IgG against type VII collagen compared with the WT mice as demonstrated by histological analysis and measurements of the MPO activity in skin biopsies (Fig. 7b).


Figure 5
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  		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 Bf–/– (b) mouse challenged with the same dose of pathogenic IgG (day 7). c, Crusted erosions on the hind leg of a Bf–/– mouse injected with 45 mg of pathogenic IgG (day 12). IF microscopy analysis of mouse skin revealed deposition of rabbit IgG in WT (d) and Bf–/– mice on days 7 (e) and 12 (f) of the experiment. Staining for murine C3 revealed similar deposits in the skin of WT and Bf–/– (g) mice on days 7 (h) and 12 (i) of the experiment. Deposits of mouse C5 along the dermal-epidermal junction were strong in the skin of a WT mouse (j) and absent in the skin of Bf–/– mice on days 7 (k) and 12 (l) of the experiment. Histological analysis of murine skin revealed extensive dermal-epidermal separation and a rich inflammatory infiltrate consisting mainly of neutrophils in WT (m), but not in Bf–/– (n) mice on day 7. o, On day 12, a Bf–/– mouse also demonstrated subepidermal cleavage associated with granulocyte infiltration.

 

Figure 6
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  		FIGURE 6. Factor B-deficient mice developed a delayed and significantly less severe blistering disease compared with control mice. WT and Bf–/–mice 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. Disease activity is represented as mean ± SEM in 10 WT and Bf–/– mice. *, Significant difference of disease activity between the two groups (p < 0.05).

 

Figure 7
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  		FIGURE 7. Injection of Abs against type VII collagen results in less neutrophil infiltration in factor B-deficient compared with WT mice. a, Serum levels of Abs to type VII collagen in Bf–/– and WT mice (n = 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 infiltrates in the skin of WT and Bf–/– mice treated with the Abs against type VII collagen or control Ab as assessed by measuring MPO activity in skin biopsies as described in Materials and Methods (*, significant difference, p < 0.01). All values are means ± SEM.

 
Reconstitution with granulocytes renders factor B-deficient mice readily susceptible to blistering by Abs against type VII collagen

To verify whether a lower recruitment of granulocytes is responsible for the inhibition of blistering, Bf–/– mice were treated with Abs against murine type VII collagen and locally injected on day 6 with granulocytes purified from WT and Bf–/– mice. Both WT mice (n = 4) treated with IgG against type VII collagen alone (Fig. 8a) and Bf–/–mice treated with Abs against murine type VII collagen and intradermally injected with WT (Fig. 8b) and Bf–/– (data not shown) granulocytes (n = 4/group) developed blisters and erosions on their ears. In contrast, Bf–/– mice (n = 2) treated with control rabbit IgG and reconstituted with WT granulocytes did not show any skin alterations (Fig. 8c). Histological analysis revealed an infiltrate of neutrophils in the dermis of all groups of mice (Fig. 8, d–f). Recruitment of granulocytes to the dermal-epidermal junction and subepidermal cleavage was found in the skin of control mice injected with Abs against murine type VII collagen (Fig. 8d) and of Bf–/– mice injected with Abs to murine type VII collagen and locally reconstituted with WT (Fig. 8e) and Bf–/– (data not shown) granulocytes, but not in the skin of Bf–/– mice treated with control rabbit IgG and reconstituted with WT granulocytes (Fig. 8f).


Figure 8
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  		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 x 106 murine granulocytes in 50 µl of medium intradermally. Both a WT mouse (a) injected with rabbit Abs to murine type VII collagen and a Bf–/– mouse (b) injected with pathogenic rabbit IgG and locally reconstituted with granulocytes developed blisters and erosions on their ears. c, A Bf–/– 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 Bf–/– mouse (e) injected with pathogenic rabbit IgG and locally reconstituted with granulocytes, but not in the skin of the Bf–/– mouse (f) treated with control rabbit IgG and reconstituted with granulocytes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We made the unanticipated observation that C1q deficiency and blockade of complement activation by the classical pathway does not inhibit tissue injury induced by Abs against type VII collagen in mice. Instead, we found that the activation of the alternative complement pathway played a pivotal role for the Ab-induced blistering. The passive transfer of Abs against type VII collagen into mice provokes within days a subepidermal blistering disease reproducing the clinical, histopathological, immunopathological, and electron microscopic findings in patients with EBA. A likely scenario is that, after binding to their target at the dermal-epidermal junction, Abs against type VII collagen trigger a cascade of events that includes the activation of the complement network and/or engagement of FcRs on leukocytes recruited into the skin. Potent inflammatory mediators, including cytokines, reactive oxygen species, and proteases released by granulocytes, most probably amplify local inflammation and are instrumental for tissue destruction (1).

This scenario bears similarities to the effector phase of other autoantibody-induced inflammatory diseases, including arthritis (24), vitiligo (25), cryoglobulinemia (26, 27), bullous pemphigoid (28), and antiphospholipid syndrome (29). In contrast, the pathogenic effects of autoantibodies in other autoimmune diseases such as pemphigus (30) are mediated strictly by binding to their target Ag and do not involve the complement network. An overall remark with regard to the role of complement in tissue injury in autoimmune diseases is that its contribution varies depending on the particular tissue involved and the genetic background. Examining complement activation pathways in different autoimmune diseases in patients and experimental animals is therefore mandatory to identify key molecular effectors of inflammatory tissue injury for therapeutic interventions.

It is textbook knowledge that complement-fixing (auto)antibodies bind to the C1q and trigger the complement cascade by the classical pathway. It has been therefore proposed that Ab-induced tissue injury essentially depends on complement activation by the classical pathway. In the present study, we found the complement component C1q to be dispensable for the induction of blisters by Abs against type VII collagen. In line with the present observations, C1q deficiency does not protect from autoantibody-induced tissue injury in cryoglobulin-induced immune complex glomerulonephritis (26). In addition, the blockade of the classical pathway in C4–/– mice does not abolish the pathogenic effects of K/BxN-derived anti-GPI serum or of anti-collagen II Abs in a passive transfer model of rheumatoid arthritis (24, 31). Fetal loss and growth restriction triggered by anti-cardiolipin Abs in mice, main features of the antiphospholipid syndrome, and blistering induced by Abs against BP180 in experimental bullous pemphigoid also depend on complement activation (28, 32, 33). However, in these experimental models, the resistance of C4–/– mice to tissue injury demonstrates an important role of an intact classical complement activation pathway (29, 33). The differences with regard to the classical pathway contribution to pathology in different experimental disease models have not yet been explained. One may only speculate that differences of targeted Ags and binding of Abs as well as different isotypes and glycosylation of Abs may influence the interaction of Ag-Ab complexes with various components of the complement network.

To evaluate the contribution of factor B for Ab-induced blistering, we analyzed disease expression in Bf–/– mice (19). Induction of blistering was delayed and the extent of cutaneous disease was significantly lower in Bf–/– compared with WT mice. This interesting finding raised the question of what activates the alternative pathway. A classical explanation is that the alternative pathway is initiated as "an amplification loop" by fixed C3b generated by classical or lectin pathways. However, blockade of complement activation by the classical pathway in C1q-deficient mice or by the lectin pathway in MBL-null mice did not alter the Ab-induced blistering. These findings suggest that initial complement activation occurs by both classical and lectin pathways, which can thus compensate for each other. An additional possibility is that the activation and amplification of the alternative pathway is due to a breakdown of the active control of the alternative pathway in the skin caused by binding of Abs, which may act as activating surfaces. Although it has not been reported that ficolins bind Igs, our study cannot rule out a possible involvement of ficolins that are also able to initiate the lectin pathway (34, 35).

Granulocyte recruitment to the dermal-epidermal junction by Abs is a prerequisite for blister induction in the animal model used in this study (36) and in the ex vivo cryosection model of EBA (7). Our present findings show that the absence of factor B impairs the recruitment of granulocytes into the skin of mice injected with Abs against type VII collagen. In line with these results, inhibition of neutrophil recruitment into the joints has been observed in factor B-deficient mice passively transferred with K/BxN-derived anti-GPI serum (24). The relevance of granulocyte recruitment into the skin is further supported by the fact that local reconstitution of factor B-deficient mice with granulocytes following injection of Abs against type VII collagen resulted in blister formation. Granulocytes were shown to produce factors of the alternative pathway (37, 38). The finding that Bf–/– granulocytes, similar to WT cells, also mediate blister formation strongly suggests that factor B produced by granulocytes is not essentially required for Ab-induced blistering in experimental EBA.

Deposition of the complement protein C3 at sites of autoantibody binding in tissues is a hallmark of autoimmune diseases in humans (39). Our present results show that C3 deposition in the skin is not imperiously associated with Ab-induced tissue injury. On one hand, a reduced C3 deposition was found in C1q-deficient mice susceptible to experimental EBA. On the other hand, C3 deposits were found in factor B-deficient mice resistant to blister induction by Abs against type VII collagen. Interestingly, a markedly reduced C5 deposition was found in factor B-deficient mice compared with controls. Indeed, it has been recently shown that generation of C5a does not absolutely require C3 (40). Proteases released by leukocytes recruited at the dermal-epidermal junction could also contribute to generation of C5a in our model (41, 42). Why complement activation at the dermal-epidermal junction in C1q-deficient mice is curtailed beyond the C3 activation step is unclear, but is reminiscent of the targeted and restricted complement activation that may occur on modified self-tissues (43). Taken together, these findings suggest that generation of C5a in our model is required for disease expression, but partly independent of the classical C5 convertase (C4b2a3b) availability.

New mechanistic insights into the role of complement activation in the pathogenesis of autoimmune inflammatory diseases facilitate targeting complement pathways for therapeutic drug development. In addition to many reports of a beneficial effect of complement blockade in animal models, anti-C5 therapy is well tolerated and effective in patients with paroxysmal nocturnal hemoglobinuria (44, 45). Our present study identifies factor B as an additional target of therapy in inflammatory autoimmune blistering diseases. Selective inhibition of this pathway may not result in side effects seen with inhibitors of C3 and C5 convertases. Such an inhibitory mAb against factor B prevents anti-phospholipid Ab-induced pregnancy loss in mice (17). To avoid a global inhibition of complement activation, more specific approaches were explored to target inhibitors of complement activation to the site of inflammation by linking inhibitors to the complement receptor (CR) 2 (46, 47). C3 breakdown products deposited at sites of complement activation are natural ligands for CR2. Thus, blockers of the alternative pathway could be directed to the dermal-epidermal junction by using fusion proteins containing CR2 and single-chain variable fragments of mAb or peptides, which are capable of inhibiting complement activation.

In conclusion, our results demonstrate that an intact alternative complement activation pathway is required for blistering induced by Abs against type VII collagen. Thus, selectively blocking this pathway may offer therapeutic benefit in patients with EBA and related autoantibody-mediated diseases.


   Acknowledgment
 
We are grateful to Dr. Yi Wang for providing the Ab specific to murine C5 (BB5.1).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants Zi 439/6-2 and SI 1281/1-1 from the Deutsche Forschungsgemeinschaft (to C.S. and D.Z.) and National Institutes of Health Grants R01 AI-31105 (to V.M.H.) and K08 DK64790 (to J.M.T.). Back

2 Address correspondence and reprint requests to Dr. Cassian Sitaru, Department of Dermatology, University of Lübeck, Ratzeburger Allee 160, Lübeck, Germany. E-mail address: csitaru{at}fastmail.fm Back

3 Abbreviations used in this paper: EBA, epidermolysis bullosa acquisita; IF, immunofluorescence; Bf, complement factor B; MBL, mannan-binding lectin; MPO, myeloperoxidase; CR, complement receptor; WT, wild type. Back

Received for publication December 19, 2006. Accepted for publication March 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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