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The Journal of Immunology, 2006, 177: 3398-3405.
Copyright © 2006 by The American Association of Immunologists, Inc.

Role of FcRs in Animal Model of Autoimmune Bullous Pemphigoid1

Minglang Zhao2,*, Mary E. Trimbeger2,*, Ning Li*, Luis A. Diaz*, Steven D. Shapiro{ddagger} and Zhi Liu3,*,{dagger}

* Department of Dermatology and {dagger} Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and {ddagger} Department of Internal Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bullous pemphigoid (BP) is a bullous dermatosis associated with autoantibodies directed against the hemidesmosomal Ags BP180 and BP230. Lesional skin is characterized by detachment of the epidermis from the dermis with an intense inflammatory cell infiltrate in the upper dermis. In experimental BP, subepidermal blistering is triggered by rabbit anti-murine BP180 (mBP180) IgG and depends upon complement activation, mast cell degranulation, and neutrophil infiltration. In this study, we determined the role of Fc{gamma}Rs on neutrophils in experimental BP. Mice deficient in Fc{gamma}RIII (Fc{gamma}RIII–/–) and those deficient in both Fc{gamma}RI and Fc{gamma}RIII (Fc{gamma}RI&III–/–) but not in Fc{gamma}RII (Fc{gamma}RII–/–) were resistant to BP. Pathogenic IgG activated wild-type neutrophils, but not Fc{gamma}RIII-deficient neutrophils, to secrete proteolytic enzymes. The function of anti-mBP180 IgG depended entirely on its Fc domain; F(ab')2 of IgG had no pathogenic activities. In wild-type mice injected with pathogenic IgG, an Fc{gamma}R blocker abolished the BP phenotype and inhibited activation of wild-type neutrophils stimulated by pathogenic IgG. Results from this study establish that Fc{gamma}RIII plays a critical role in the activation of infiltrating neutrophils and the subsequent blistering in experimental BP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bullous pemphigoid (BP)4 is a subepidermal bullous dermatosis seen primarily in the elderly (1). BP patients exhibit circulating and tissue-bound autoantibodies directed against the hemidesmosomal Ags BP180 and BP230, located within the basement membrane zone (BMZ) (2, 3). BP230 (also referred to as BPAG1) is an intracellular protein that localizes to the hemidesmosomal plaque, while BP180 (BPAG2 or type XVII collagen) is a transmembrane protein with a type II orientation (3). The N-terminal region of BP180 localizes to the intracellular hemidesmosomal plaque, while its C-terminal portion projects into the extracellular milieu of the BMZ (4, 5, 6). BP autoantibodies react with at least four distinct antigenic sites on the BP180 ectodomain, all of which are clustered within a 45-aa noncollagenous stretch adjacent to the membrane-spanning domain (called NC16A) (7, 8). Lesional skin is characterized by detachment of the epidermis from the dermis with an intense inflammatory cell infiltrate in the upper dermis (1). A variety of cellular lineages has been identified in lesional inflammatory infiltrates, including neutrophils, eosinophils, lymphocytes, mast cells, and monocytes/macrophages (3, 9, 10, 11, 12, 13, 14).

An experimental model for BP uses the passive transfer of rabbit anti-murine BP180 (mBP180) Abs in neonatal BALB/c mice. This model reproduces key immunopathological features of human BP, including IgG and complement deposition at the dermal-epidermal junction (DEJ), inflammatory infiltration of the upper dermis, and subepidermal blistering (15). Subepidermal blistering in the experimental mice requires complement activation (16), mast cell degranulation (17), and neutrophil infiltration (18). Infiltrating neutrophils at the skin lesional site are activated to release neutrophil elastase (NE), matrix metalloproteinase (MMP)-9 (19), and other proteolytic enzymes that are directly responsible for splitting the epidermis from the dermis (19). The molecular mechanism for the activation of infiltrating neutrophils has previously remained unknown.

FcRs serve a key function in the activation and down-regulation of immune responses (20, 21, 22). They exist for each class of Ab molecules (Fc{gamma}Rs for IgG, Fc{alpha}Rs for IgA, Fc{epsilon}Rs for IgE, FcµRs for IgM, and Fc{delta}Rs for IgD) (20). FcRs for IgG, IgE, and IgA are generally located on the cell surface of effector cells. These FcRs serve as receptors for the constant, Fc portion of an Ig molecule. Following the Fc-FcR molecular interaction, FcRs are cross-linked and resulting cellular responses may occur in effector cells. Cellular responses include the induction of phagocytosis, Ab-dependent cellular cytotoxicity, and the release of inflammatory mediators and reactive oxygen species (22, 23). High- and low-affinity FcRs undergo the molecular interaction with distinctive orders of events. However, both lead to cellular responses with equivalent efficiency. High-affinity FcRs bind monomeric Igs before they are complexed to their specific cellular or soluble Ag (20). These high-affinity receptors include Fc{gamma}RI, Fc{epsilon}RI, and Fc{alpha}RI (22, 24). In contrast, low-affinity FcRs bind Abs following aggregation or complexation with a specific Ag (20). Fc{gamma}RII, Fc{gamma}RIII, and Fc{epsilon}RII are each classified as low-affinity FcRs (24).

There are three classes of FcRs for IgG: Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII. Fc{gamma}RI is high affinity, activating FcR; Fc{gamma}RII is low affinity, inhibiting FcR; and Fc{gamma}RIII is low affinity, activating FcR (20, 21, 22, 25, 26, 27). Fc{gamma}RI and Fc{gamma}RIII are trimeric complexes containing an IgG-binding {alpha} subunit and a signaling subunit, the ITAM-containing {gamma}-chain. IgG-immune complex binds to Fc{gamma}RIII, leading to cellular activation. In contrast, Fc{gamma}RII is a single-chain receptor; its extracellular domain binds to IgG and its cytoplasmic domain contains the ITIM and inhibits ITAM-medicated cellular activation (20, 21, 22).

Fc{gamma}Rs have been implicated in numerous autoimmune diseases. FcR{gamma} chain-deficient mice are resistant to the induction and/or spontaneous onset of autoimmune hemolytic anemia, alveolitis, glomerulonephritis, and vasculitis (28, 29, 30, 31, 32, 33) and associated with attenuated Arthus reaction and IgE-induced cutaneous anaphylaxis (34, 35). In contrast, Fc{gamma}RIIb deficiency is associated with enhanced Arthus reaction (36), enhanced IgG- and IgE-induced local and/or systemic anaphylaxis, and increased susceptibility and severity to organ-specific and systemic autoimmune diseases, such as ITP, glomerulonephritis, arthritis, and lupus (29, 31, 36, 37, 38, 39, 40, 41, 42, 43, 44).

BP is generally characterized by the IgG class of autoantibodies (45, 46). Thus, Fc{gamma}Rs on the cell surface of effector cells likely play a significant role in the immunopathogenesis. In the present study, we investigate the role of different Fc{gamma}Rs in experimental BP and compare their relative contributions in subepidermal blistering using Fc{gamma}R-deficient mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Laboratory animals

Breeding pairs of wild-type (WT) C57BL/6J, mice deficient in Fc{gamma}RIII (Fc{gamma}RIII knockout (KO)), mice deficient in both Fc{gamma}RI and III (Fc{gamma}RI&III KO) were purchased from The Jackson Laboratory. Mice deficient in Fc{gamma}RIIB (Fc{gamma}RIIB KO) were obtained from Taconic Farms. Mice deficient in NE (NE KO) were described previously (47). All deficient mice were in C57BL/6J background. The animals were maintained at the University of North Carolina (Chapel Hill, NC) Animal Facility. Neonatal mice (24–36 h old with body weights between 1.4 and 1.6 g) were used for passive transfer experiments. Animal care and animal experiments were approved by the Animal Care Committee at the University of North Carolina (Chapel Hill, NC) and were in accordance with National Institutes of Health guidelines.

Preparation of pathogenic anti-BP180 IgG

The preparation of recombinant murine BP180 and the immunization of rabbits were performed as previously described (15). Briefly, a segment of the ectodomain of the murine BP180 Ag (48) was expressed as a GST fusion protein using the pGEX prokaryotic expression system (Pharmacia LKB Biotechnology). The murine BP180 fusion protein, designated GST-mBP180ABC, was purified to homogeneity by affinity chromatography using glutathione agarose beads (Sigma-Aldrich). New Zealand White rabbits were immunized with the purified mBP180 fusion protein and the IgG fraction collected from their serum (designated R530) was purified as previously described (15). The IgG fractions were concentrated, sterilized by ultrafiltration, and their protein concentrations were determined by OD280 (E (1%, 1 cm) = 13.6). The titers of anti-murine BP180 Abs were assayed by indirect immunofluorescence (IF) using mouse skin cryosections as substrate. The Ab preparations were also tested by immunoblotting against the GST-mBP180ABC fusion protein. The IF and immunoblotting techniques have been reported elsewhere (15). The pathogenicity of these IgG preparations was tested by passive transfer experiments as described below. A nonpathogenic anti-mBP180 IgG preparation (designated R50) was used as a control (49). F(ab')2 of pathogenic and control IgG were prepared by pepsin digestion (16).

Induction of experimental BP and clinical evaluation of animals

Neonates were given one intradermal (i.d.) injection of a sterile solution of either control IgG or anti-BP180 IgG in PBS (50-µl volume; 2.5 mg of IgG/g body weight), as described (15). The skin of neonatal mice from the test and control groups were examined 2, 4, 12, or 24 h after injection. The extent of cutaneous disease was scored as follows: –, no detectable skin disease; 1+, mild erythematous reaction with no evidence of the "epidermal detachment sign" (this sign was elicited by gentle friction of the mouse skin which, when positive, produced fine, persistent wrinkling of the epidermis); 2+, intense erythema and "epidermal detachment" sign involving 10–50% of the epidermis in localized areas; and 3+, intense erythema with frank "epidermal detachment" sign involving >50% of the epidermis in the injection site.

After clinical examination, the animals were terminated, and skin and serum specimens were obtained. The skin samples were used for routine histological examination by light microscopy (H&E staining) and direct IF assays to detect rabbit IgG and mouse C3 deposition at the BMZ. Other skin samples were used for the enzymatic assays described below. The sera of injected animals were tested by indirect IF techniques to determine the titers of rabbit anti-murine BP180 Abs. Direct and indirect IF studies were performed as previously described (15) using commercially available FITC-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories). Monospecific goat anti-mouse C3 serum was purchased from Cappel Laboratories.

Quantification of polymorphonuclear leukocyte (PMN) accumulation at Ab injection sites

Tissue myeloperoxidase (MPO) activity was used as an indicator of PMNs within skin samples of experimental animals, as described (18, 50). A standard reference curve was first established by obtaining activity levels on aliquots of known amounts of purified MPO (Athens Research and Technology). The mouse skin samples were extracted by homogenization in a buffer containing 0.1 M Tris-Cl (pH 7.6), 0.15 M NaCl, 0.5% hexadecyltrimethylammonium bromide. MPO activity levels in supernatant fractions were determined by the change in OD460 nm resulting from decomposition of H2O2 in the presence of o-dianisidine. MPO content was expressed as relative MPO activity (OD460 nm reading/mg protein). Protein concentrations were determined by the Bio-Rad dye binding assay using BSA as a standard.

Neutrophil isolation

Mouse neutrophils were isolated from heparinized blood by dextran sedimentation, followed by separation on a density gradient as described (51). PMN purity of the final cell preparation was consistently >96% as determined by cell cytospin and LeukoStat staining (Fisher Diagnostics). The viability of the PMN was >96% as determined by trypan blue exclusion. PMN was kept in PBS/10 mM glucose at 4°C before use.

In vivo reconstitution of neutrophils in Fc{gamma}R-deficient mice

Neonatal Fc{gamma}R-deficient mice were injected i.d. with pathogenic anti-mBP180 IgG (2.5 mg/g body weight). Two hours later, 5 x 105 neutrophils from WT, Fc{gamma}RIII KO, or Fc{gamma}RI&III KO mice were injected into IgG injection site (19). The animals were then examined 24 h after IgG injection as described above.

Quantification of mast cells (MCs) and MC degranulation

MCs and MC degranulation in skin samples were quantified according to Wershil et al. (52) with modification (17, 52). Briefly, lesional and nonlesional skin sections of IgG-injected mice were fixed in 10% formalin. Paraffin sections (5-µm thick) were prepared and stained with toluidine blue and H&E. MCs were counted and classified as "degranulated" (>10% of the granules exhibiting fusion or discharge) or "normal," in five fields under a light microscope as described previously. The results were expressed as percentage of MC degranulating.

Inhibition of Fc{gamma}Rs in vitro and in vivo

Rat anti-mouse mAb 2.4G2 (specific for Fc{gamma}RII and Fc{gamma}RIII) and matched isotype control rat IgG2b were purchased from BD Pharmingen. F(ab')2 of 2.4G2 and control rat IgG were prepared by pepsin digestion (16). Undigested IgG and Fc fragments were removed by affinity chromatography using a Protein G column (Sigma-Aldrich). To inhibit Fc{gamma}R-mediated neutrophil activation in vitro, purified neutrophils (1 x 106 cells/ml) from WT and Fc{gamma}R-deficient mice were incubated with rabbit anti-mBP180 IgG (5 µg/ml) and mBP180 Ag (5 µg/ml) in the presence or absence of F(ab')2 of 2.4G2 or with control rat IgG2b for 15 min at 37°C. The supernatant was then analyzed by enzyme assays for NE and MMP-9 activities. To inhibit Fc{gamma}R in vivo, neonatal mice were pretreated i.d. with 2.4G2 or control rat IgG2b (10 µg/g body weight) and 2 h later were injected i.d. with pathogenic IgG. The animals were then examined 24 h after IgG injection as described above.

In vitro neutrophil degranulation

In vitro neutrophil degranulation assays were performed as described (53). Briefly, purified neutrophils from WT and Fc{gamma}R-deficient mice were suspended in HBSS (Invitrogen Life Technologies) at a final concentration of 106 cells/ml and triggered with rabbit anti-mBP180 IgG (5 µg/ml) and mBP180 Ag (5 µg/ml) for 15 min at 37°C. The cells were then palletized by centrifugation (1000 x g, 5 min) and the supernatant was analyzed by casein and gelatin gel zymography for NE and MMP-9, respectively, as described below.

Identification of NE and MMP-9 in blister fluids

A total of 100 µl of PBS were injected and withdrawn 1 min later into both the nonlesional sites and the skin blisters that formed 24 h following pathogenic IgG injection. The PBS "washout" was centrifuged at low speed (1,000 x g) for 5 min to remove cells and then at high speed (12,000 x g) for 5 min to remove cell debris (54). The supernatant was analyzed for NE and MMP-9 by zymography (54, 55). Protein extracts of samples from IgG-injected animals were subjected to SDS-PAGE on casein-containing acrylamide gels (12% acrylamide and 1% casein) or gelatin-containing acrylamide gels (8% acrylamide and 1% gelatin) under nonreducing conditions. After electrophoresis, gels were washed twice with 2.5% Triton X-100 for 30 min to remove SDS. They were then rinsed briefly with H2O and then incubated overnight at 37°C in reaction buffer (50 mM Tris (pH 7.4), 150 mM NaCl, and 5 mM CaCl2). The gels were stained with 0.125% Coomassie Brilliant blue. Caseinolytic and gelatinolytic activity appeared as colorless zones against a blue background.

Treatment of mouse skin in organ culture

Mouse skin sections were obtained from neonatal C57BL/6J mice (36–48 h old) and cut into 2 x 2-mm strips with a razor blade. The skin strips were then incubated in MEM alone, MEM with 100 µg/ml human NE, or neutrophil culture supernatants at 37°C for 24 h. At the end of the incubation, the skin strips were rinsed in fresh MEM, fixed in 10% formalin, and embedded in paraffin, after which sections were cut and stained with H&E (54).

Statistical analysis

The data were expressed as mean ± SEM and were analyzed using Student’s t test. A p value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mice lacking Fc{gamma}RIII are resistant to experimental BP

To evaluate the role of activating Fc{gamma}Rs in experimental BP, neonatal WT mice and mice deficient in Fc{gamma}RIII (Fc{gamma}RIII KO) or deficient in both Fc{gamma}RI and Fc{gamma}RIII (Fc{gamma}RI&III KO) were injected i.d. with pathogenic anti-mBP180 IgG. Twenty-four hours following IgG injection, WT mice developed clinical blisters (Fig. 1a; also see Table I). Direct IF showed deposition of rabbit IgG and murine complement component 3 (C3) at the cutaneous BMZ (Fig. 1, b and c). Histological examination of diseased mouse skin revealed DEJ separation with neutrophil infiltration in the dermis (Fig. 1d). In contrast, the Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice were resistant to experimental BP, as evidenced by lack of blister formation (Fig. 1, e and i). There was no separation at the DEJ in the deficient mice (Fig. 1, h and l), despite the presence of IgG and murine C3 at the BMZ when examined by direct IF (Fig. 1, f, g, j, and k). The positive direct IF staining in the experimental mice rules out the possibility that deficiency of Fc{gamma}Rs impairs binding of IgG to its target or the subsequent complement activation. These results demonstrate that activating Fc{gamma}RIII is required for experimental BP.


Figure 1
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  		FIGURE 1. Fc{gamma}R-deficient mice are resistant to experimental BP. WT, Fc{gamma}RIII-deficient (Fc{gamma}RIII KO), and Fc{gamma}RI&III KO mice were injected i.d. with pathogenic anti-mBP180 IgG R530 and examined 24 h postinjection. The WT mice developed clinical (a) and histologic (d) BP blisters and showed in situ deposition of rabbit IgG (b) and mouse C3 (c) at the BMZ. In contrast, Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice showed no clinical (e and i) and histological (h and l) skin lesions, although IgG (f and j) and C3 (g and k) were seen at the BMZ. Site of Ab labeling and basal keratinocytes (arrowhead), dermis (D), epidermis (E), vesicle (V) (x200); n = 9.

 

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  		Table I. Relative contribution of Fc{gamma}Rs to experimental bullous pemphigoida

 
Fc{gamma}RIII plays a major role in experimental BP

To determine the relative contribution of each of these Fc{gamma}Rs to experimental BP, we assessed the disease severity of these mice by quantifying infiltrating neutrophils at the skin site (Fig. 2A). There was a significant reduction in the number of infiltrated neutrophils in the Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice compared with WT mice. There were no significant differences in neutrophil infiltration between Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice.


Figure 2
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  		FIGURE 2. Fc{gamma}RIII is crucial in experimental BP. A, WT, Fc{gamma}RIII KO, and Fc{gamma}RI and III KO mice were injected i.d. with pathogenic anti-mBP180 IgG R530. Neutrophil infiltration in the skin of injected mice was quantified by MPO assay 24 h postinjection. There was a significant reduction in the number of neutrophils in Fc{gamma}RIII KO (bar 3) and Fc{gamma}RI&III KO (bar 4) mice compared with WT mice (bar 2); n = 9, p < 0.01. There were no significant differences between Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice (p = 0.59). Fc{gamma}RIII KO (B) and Fc{gamma}RI&III KO (C) mice were injected i.d. with pathogenic IgG and then locally reconstituted with 5 x 105 neutrophils from WT, Fc{gamma}RIII KO, or Fc{gamma}RI&III KO mice. The mice were examined for BP blisters at 24 h postinjection. WT but not Fc{gamma}RIII KO and Fc{gamma}RI&III KO neutrophils restored BP in Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice; n = 6.

 
To further demonstrate that Fc{gamma}RIII plays a major role in experimental BP, we performed neutrophil reconstitution experiments. Fc{gamma}RI&III KO mice reconstituted locally with 0.5 x 106 neutrophils from WT but not from Fc{gamma}RIII KO or Fc{gamma}RI&III KO mice developed BP (Fig. 2B; also see Table I). Similarly, BP disease was restored in Fc{gamma}RIII KO mice reconstituted with 0.5 x 106 neutrophils from WT but not from Fc{gamma}RIII KO or Fc{gamma}RI&III KO mice (Fig. 2C). Thus, Fc{gamma}RIII is crucial in the development of experimental BP.

MC degranulation and neutrophil infiltration in Fc{gamma}RIII-deficient mice is normal

Experimental BP depends on mast cell activation and neutrophil infiltration (17, 18). To determine whether these functions are impaired in Fc{gamma}RIII-deficient mice, we evaluated the pathogenic IgG-injected WT, Fc{gamma}RIII KO, and Fc{gamma}RI&III KO mice for neutrophil recruitment and MC number and degranulation. At 2 h post-IgG injection, when MC degranulation peaks, toluidine blue staining revealed that both the total number of mast cells and the percentage of degranulating mast cells in the Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice were comparable to those in diseased WT animals (Figs. 3, A and B). Neutrophil infiltration time course study showed that at early time points (2 and 4 h), there was no difference in the number of infiltrating neutrophils between WT and Fc{gamma}R-deficient mice (Fig. 3C). At the later time points (8, 12, and 24 h), the lesional skin of the diseased WT mice had a significantly higher number of neutrophils relative to the skin of Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice (Fig. 3C). These results demonstrated that MC activation and the early phase of neutrophil infiltration were not impaired in Fc{gamma}RIII-deficient mice.


Figure 3
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  		FIGURE 3. Pathogenic anti-mBP180 IgG-induced MC and neutrophil activation in Fc{gamma}R-deficient mice. WT, Fc{gamma}RIII KO, and Fc{gamma}RI&III KO mice were injected i.d. with pathogenic anti-mBP180 IgG R530. MC degranulation and neutrophil infiltration were analyzed by toluidine blue staining and MPO activity assay, respectively. At 2 h postinjection when MC degranulation peaks, skin sections were stained with toluidine blue. MCs in the dermis were then counted and classified as "degranulated" (>10% of the granules exhibiting fusion or discharge) or "normal" as previously described (21 ). A, Toluidine blue staining showed similar degrees of MC degranulation (arrows) in pathogenic IgG-injected WT (b) and Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice (c and d). WT mice injected with control IgG showed a minimal degree of MC degranulation (a). B, Quantitation of degranulated MCs revealed no significant difference in mast cell degranulation between the R530 IgG-injected WT and KO mice (expressed as percent MC degranulation, mean ± SE). C, MPO activity assay showed that at the early phase of neutrophil recruitment (time points 0–4 h), there was no difference in skin neutrophil numbers between R530 IgG-injected WT and KO mice. At the late (amplification) stage of neutrophil recruitment (time points 8–24 h), neutrophils in the diseased WT mice were significantly higher that in the KO mice; n = 6, p < 0.01.

 
The inhibitory Fc{gamma}RII plays a minimal role in experimental BP

To determine whether the inhibitory Fc{gamma}RII negatively regulates Fc{gamma}RIII-mediated functions, we induced experimental BP in WT and Fc{gamma}RII KO mice with different amounts of pathogenic Ab. As shown previously (56), pathogenic anti-mBP180 Abs triggered a similar degree of clinical BP disease in both WT and Fc{gamma}RII KO mice (Fig. 4A; also see Table I). MPO activity assay revealed slightly more infiltrating neutrophils in Fc{gamma}RII KO than in WT mice; however, this increase was not statistically significant (Fig. 4B). Therefore, Fc{gamma}RII is not an important regulator of Fc{gamma}RIII in experimental BP.


Figure 4
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  		FIGURE 4. Role of the inhibitory receptor Fc{gamma}RII in experimental BP. WT and Fc{gamma}RII-deficient (Fc{gamma}RII KO) mice were injected i.d. with 1.5 (bars 1 and 2), 2.0 (bars 3 and 4), and 2.5 (bars 5 and 6) mg/g body weight of pathogenic anti-mBP180 R530. Mice were examined 24 h post-IgG-injection. The WT and Fc{gamma}RII KO mice developed a similar degree of clinical BP disease (A) and similar levels of neutrophil infiltration (B) as determined by MPO activity assay at these pathogenic IgG doses; n = 6, p = 0.27–0.62.

 
Neutrophil activation in experimental BP is dependent upon the interaction between the Fc of the pathogenic IgG and the Fc{gamma}RIII

If activating Fc{gamma}RIII is required for neutrophil activation in experimental BP, then F(ab')2 of the pathogenic anti-mBP180 Abs would not be pathogenic. The following results confirm what we expected. WT mice injected with intact pathogenic IgG but not F(ab')2 developed BP (Fig. 5A; also see Table I). Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice injected with whole pathogenic IgG, followed by local reconstitution with 0.5 x 106 WT neutrophils, developed BP blisters (Fig. 5A). In contrast, Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice injected with F(ab')2 of the pathogenic IgG, then reconstituted with 0.5 x 106 WT neutrophils, did not develop BP skin lesions (Fig. 5A). Without the Fc domain of pathogenic IgG, the F(ab')2 were unable to initiate clinical disease, even in the presence of sufficient neutrophils. These results suggest that the Fc-Fc{gamma}RIII interaction is a prerequisite for activation of infiltrating neutrophils


Figure 5
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  		FIGURE 5. Neutrophil activation in experimental BP is dependent upon the interaction between the Fc domain of IgG and the Fc{gamma}R. WT, Fc{gamma}RIII KO, and Fc{gamma}RI&III KO mice were injected with intact pathogenic IgG R530 (2.5 mg/g body weight) or R530 F(ab')2 (2.5 mg/g body weight) and 2 h later, reconstituted with buffer control or 5 x 105 WT neutrophils. The animals were examined 24 h postinjection. A, Intact R530 IgG-injected WT (bar 1), Fc{gamma}RIII KO (bar 4), and Fc{gamma}RI&III KO (bar 6) mice developed extensive BP blisters, while the R530 F(ab')2-injected WT (bars 2 and 3), Fc{gamma}RIII KO (bar 5), and Fc{gamma}RI&III KO (bar 7) mice showed no or minimal degree of skin lesions. B, Casein gel zymograph revealed NE present in the lesional skin of intact R530 IgG-injected WT (lane 1), Fc{gamma}RIII KO (lane 4), and Fc{gamma}RI&III KO (lane 6) mice, but not in the skin of the R530 F(ab')2-injected WT (lanes 2 and 3), Fc{gamma}RIII KO (lane 5), and Fc{gamma}RI&III KO (lane 7). C, Gelatin gel zymograph revealed MMP-9 present in the lesional skin of intact R530 IgG-injected WT (lane 1), Fc{gamma}RIII KO (lane 4), and Fc{gamma}RI&III KO (lane 6) mice, but not in the skin of the R530 F(ab')2-injected WT (lanes 2 and 3), Fc{gamma}RIII KO (lane 5), and Fc{gamma}RI&III KO (lane 7); n = 6.

 
To directly demonstrate that neutrophil activation via the Fc-Fc{gamma}RIII interaction indeed takes place in the inflamed skin site, we assayed NE and MMP-9 in the blister fluids of the diseased mice. As shown previously (19, 54, 55), the blister fluid of diseased WT mice injected with the intact anti-mBP180 IgG displayed significantly higher levels of NE and MMP-9 activities than that of mice injected with the F(ab')2. Similarly, the blister fluids of Fc{gamma}RIII KO and Fc{gamma}RI&III KO mice injected with 5 x 105 WT neutrophils plus the intact pathogenic IgG contained high levels of NE and MMP-9 activities (Fig. 5, B and C). In contrast, the PBS "washout" of these Fc{gamma}R-deficient mice injected with 5 x 105 WT neutrophils plus the F(ab')2 had minimal levels of NE and MMP-9. These results demonstrate that infiltrating neutrophils are activated by the molecular interaction between the Fc of the pathogenic IgG and Fc{gamma}RIII.

Blocking Fc{gamma}RIII inhibits experimental BP

If Fc{gamma}RIII is the critical receptor for pathogenic IgG binding, then administration of neutralizing Ab against Fc{gamma}RIII should phenocopy the response of Fc{gamma}RIII–/– mice. WT mice injected with pathogenic Ab developed BP disease (Fig. 6Ab; also see Table I). In contrast, WT mice pretreated with the FcR blocker (inhibiting both Fc{gamma}RII and Fc{gamma}RIII) and then injected with pathogenic Ab failed to show BP skin lesions (Fig. 6Ac). MPO activity assay revealed a significant decrease in neutrophil infiltration in the FcR blocker-treated mice relative to the disease mice (Fig. 6B). Consistent with these in vivo findings, in vitro results showed that WT neutrophils incubated with pathogenic anti-mBP180 Ab and mBP180 Ag released significantly increased levels of NE (Fig. 6C) and MMP-9 (Fig. 6C). But, in the presence of the FcR blocker, WT neutrophils were no longer activated by pathogenic Ab and Ag (Fig. 6, C and D).


Figure 6
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  		FIGURE 6. Blocking Fc{gamma}Rs inhibits experimental BP. Neonatal WT mice were injected with control Ig or R530 IgG (2.5 mg/g body weight) with or without FcR blocker (25 µg/g body weight). The animals were examined 24 h later. A, Mice injected with R530 (b), but not control IgG (a) or R530 plus FcR blocker developed BP. B, MPO activity assay revealed a significant decrease in neutrophil infiltration in FcR blocker-treated mice (bar 3) than the disease mice (bar 2); n = 6, p < 0.05. Gelatin (C) and casein (D) gel zymography assays showed that WT neutrophils incubated with pathogenic anti-mBP180 IgG R530 and mBP180 Ag released MMP-9 and NE, respectively (bar 1); but the activation was abolished by the FcR blocker blocking Fc{gamma}RIII (bar 2). As a control, R530 IgG alone did not activate neutrophils to release MMP-9 and NE (bar 3). E, Neutrophils from WT (a, e, and f), Fc{gamma}RIII KO (b), Fc{gamma}RI&III KO (c), and NE KO (d) were stimulated with R530 IgG plus mBP180 in the presence of FcR blocker (e) or buffer control for 30 min. Neonatal mouse skin sections were then incubated with the neutrophil supernatants plus the NE inhibitor {alpha}1-proteinase inhibitor {alpha}1-PI (f) or buffer control at 37°C for 24 h. The skin sections were examined by H&E staining. Only supernatants from stimulated WT neutrophils without FcR inhibition produced DEJ separation. The DEJ separation was blocked by {alpha}1-PI.

 
To further demonstrate that in vitro neutrophil activation depends on the Fc{gamma}RIII interaction with pathogenic IgG, neutrophils from WT, NE KO, Fc{gamma}RIII KO, and Fc{gamma}RI&III KO were incubated with pathogenic anti-mBP180 IgG plus mBP180 Ag. The cell culture supernatants were then assayed for producing dermal-epidermal separation in the skin organ system (54). As shown previously (54), dermal-epidermal separation was seen in the skin sections incubated with supernatant from WT neutrophils (Fig. 6Ea), but not with supernatants from Fc{gamma}RIII KO (Fig. 6Eb), Fc{gamma}RI&III KO (Fig. 6Ec), or NE KO (Fig. 6Ed) neutrophils. As expected, supernatant from WT neutrophils plus FcR blocker (Fig. 6Ee) or WT neutrophil supernatant plus the NE inhibitor {alpha}1-proteinase inhibitor (Fig. 6Ef) were no longer able to produce tissue injury at the BMZ. Taken together, blockage of the Fc{gamma}RIII binding to pathogenic IgG prevented Fc{gamma}RIII-mediated neutrophil activation and the subsequent release of proteolytic enzymes, a critical step in BP blister formation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous research findings have suggested that subepidermal blistering requires a specific sequence of events in the immunopathogenesis of BP. These events are initiated through the binding of pathogenic anti-BP180 IgG to the BP180 Ag at the BMZ (15), followed by complement activation (16), MC degranulation (17), and neutrophil infiltration (18). Infiltrating neutrophils are then activated to release NE, MMP-9, and other proteolytic enzymes that are directly responsible for splitting the epidermis from the dermis (19). The aim of this study was to determine the role of Fc{gamma}Rs in the activation of neutrophils in experimental BP. Our study definitively establishes Fc{gamma}RIII as a key receptor for the pathogenic anti-mBP180 IgG binding to activate infiltrating neutrophils. It also provides direct evidence that Fc{gamma}RIII plays an essential role in the neutrophil activation and subsequent blister formation in experimental BP.

Fc{gamma}RIII-deficient mice are associated with attenuated Ab-induced vasculitis and autoimmune hemolytic anemia (33, 57). The results from this study are in agreement with these findings. The critical role that Fc{gamma}RIII plays with infiltrating neutrophils is further substantiated by in vitro neutrophil activation experiments. Pathogenic IgG activates WT neutrophils to secrete NE and MMP-9, but fails to stimulate Fc{gamma}RIII-deficient neutrophils to release the same proteolytic enzymes. These results establish that neutrophil activation following exposure to pathogenic IgG requires Fc{gamma}RIII interaction with Fc domain of the anti-mBP180 IgG. Thus, the pathogenic anti-mBP180 IgG dictates tissue injury site at the DEJ by both Fab and Fc domains. While the Fab domain binds to the BP180, the Fc domain activates complement (16), and, in turn, complement activation leads to neutrophil recruitment. Upon activation, these infiltrating neutrophils locally release NE, MMP-9, and other proteolytic enzymes, resulting in subepidermal blistering.

In general, Fc{gamma}RIIB acts as a major inhibitor of the activating Fc{gamma}R-induced effector functions (22, 27, 29). Mice lacking Fc{gamma}RIIB develop more severe collagen-induced arthritis, Goodpasture’s syndrome, Ab-induced glomerulonephritis, and alveolitis (29, 31, 37, 38, 39, 40, 41, 42, 43). These mice also show enhanced Arthus reaction and IgG- and IgE-induced anaphylaxis (36, 37, 44). Interestingly, Fc{gamma}RIIB has no significant effect on Fc{gamma}RIII-mediated neutrophil activation in experimental BP. WT mice and mice lacking Fc{gamma}RII are equally susceptible to experimental BP. Why then does experimental BP differ from some other Ab-mediated animal models in the role of Fc{gamma}RII? One possibility is that the coexpression of Fc{gamma}RII and Fc{gamma}RIII varies between effector cells such as macrophages and neutrophils in different pathological conditions (36). Alternatively, relative contributions of Fc{gamma}RII and Fc{gamma}RIII depend on tissue site. Our current data cannot exclude an involvement of Fc{gamma}RI in experimental BP. More direct approaches such as using Fc{gamma}RI-deficient mice or using Ab against only the Fc{gamma}RI will be able to provide conclusive evidence.

We should note that in experimental BP, pathogenic rabbit anti-mBP180 IgG targets a single pathogenic epitope and belongs to only one isotype, whereas human BP autoantibodies recognize multiple epitopes and belong to IgE and to IgG1, 2, 3, and 4. In experimental BP, subepidermal blistering triggered by rabbit anti-mBP180 IgG depends on neutrophils and neutrophil-released proteolytic enzymes (18, 54, 55). In human BP, several lines of evidence using in vitro skin culture and cryosections systems demonstrated that dermal-epidermal separation induced by anti-BP180 autoantibodies also depends on neutrophils (58, 59). However, another study showed that anti-BP180 autoantibodies are directly pathogenic without the need of neutrophils in the human skin culture system and SCID mice grafted with human skin (60). Thus, it is possible that pathogenic BP180 autoantibodies in BP patients may induce neutrophil-dependent and neutrophil-independent blister formation. In junctional epidermolysis bullosa, mutations in BP180 cause subepidermal blistering without neutrophil infiltration (61, 62). Taken together, both human and mouse BP findings suggest that BP180 plays a critical role in maintaining the integrity of the DEJ. Impairment of BP180 function by direct autoantibody insults, proteolytic cleavage, or through genetic mutations will lead to subepidermal blistering.

In summary, this study demonstrates that Fc{gamma}RIII is essential in the activation of infiltrating neutrophils in experimental BP. These findings give us novel insights into the immunopathology of BP and other IgG- and neutrophil-mediated autoimmune diseases. They also carry significant clinical implications for the future development of innovative therapeutic strategies for these diseases.


   Acknowledgments
 
We thank Dr. Kevin McGovan for his critical reading of the manuscript and Sarah Rice for her excellent editing.


   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 in part by U.S. Public Health Service Grants AI40768 and AI61430 (to Z.L.), AR052109 (to N.L.), and R01 AR-32599 and R37-AR32081 (to. L.A.D.). M.E.T. received a research fellowship grant from the Holderness Foundation. Back

2 M.Z. and M.E.T. made equal contributions to this work. Back

3 Address correspondence and reprint requests to Dr. Zhi Liu, Department of Dermatology, University of North Carolina, 3100 Thurston-Bowles, Chapel Hill, NC 27599. E-mail address: zhiliu{at}med.unc.edu Back

4 Abbreviations used in this paper: BP, bullous pemphigoid; BMZ, basement membrane zone; DEJ, dermal-epidermal junction; NE, neutrophil elastase; MMP, matrix metalloproteinase; mBP180, murine BP180; WT, wild type; KO, knockout; IF, immunofluorescence; i.d., intradermal; PMN, polymorphonuclear leukocyte; MPO, myeloperoxidase; MC, mast cell. Back

Received for publication January 30, 2006. Accepted for publication June 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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