β₂-microglobulin–deficient Mice Are Resistant to Bullous Pemphigoid

Breeding pairs of BALB/ByJ and C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). β₂m^−/− MRL/MpJ–B2m^tm1Unc/Dcr and C57BL/6J–B2m^tm1Unc/ Dcr mice were produced as described (19). β₂m^−/− neonates were produced from an F1 cross of MRL/MpJ–B2m^tm1Unc × C57BL/6J–B2m^tm1Unc mice. Genetically matched β₂m^+/− (wild-type) mice were produced from an F1 cross of MRL/MpJ– B2m^tm1Unc × C57BL/6J–B2m^+/+ mice. The animals were bred at The Jackson Laboratory and litters were delivered at the Medical College of Wisconsin Animal Resource Center. Neonatal mice (24–36 h old with body weights between 1.4 and 1.6 g) were used for passive transfer experiments.

The preparation of recombinant mBP180 and the immunization of rabbits were performed as previously described (29, 32). In brief, a segment of the mBP180 antigen containing the pathogenic epitope was expressed, purified to homogeneity by affinity chromatography (33), and used to immunize New Zealand White rabbits. The IgG fraction from the sera (referred to as R621) was purified as previously described (29). The titer of rabbit anti-mBP180 antibodies was assayed by indirect IF using mouse skin cryosections as substrate (29). The pathogenicity of the IgG preparations was tested by passive transfer experiments as described below.

A 50 μl dose of sterile IgG in PBS was administered to neonatal mice by intraperitoneal injection (2.5 mg IgG/g body weight) or intradermal (2.5 mg IgG/g body weight). The skin of neonatal mice from the test and control groups was examined 12 or 24 h after the IgG injection. The extent of cutaneous disease was scored as follows: minus, no detectable skin disease; 1+, mild erythematous reaction with no evidence of the epidermal detachment sign elicited by gentle friction of the mouse skin, which, when positive, produced fine, persistent wrinkling of the epidermis; 2+, intense erythema and epidermal detachment involving 10–50% of the epidermis in localized areas; and 3+, intense erythema with frank epidermal detachment involving more than 50% of the epidermis. The animals were then sacrificed and skin sections were taken for light microscopy (hematoxylin and eosin, H/E) and for direct immunofluorescence (IF) to detect rabbit IgG and mouse C3 deposition at the BMZ. Sera of injected animals were assayed by indirect IF to determine the circulating titers of anti-mBP180 IgG (29). Monospecific FITC-conjugated goat anti–rabbit IgG was obtained commercially (Kirkeggard & Perry Laboratories, Inc., Gaithersburg, MD). Monospecific goat anti–mouse C3 was purchased from Cappel Laboratories (Durham, NC). Both anti-rabbit IgG and anti-C3 antibodies were used at a 1:100 dilution.

Tissue MPO activity in skin sites of the injected animals was assayed as described (34, 35). A standard reference curve was first established using known concentrations of purified myeloperoxidase (MPO). The skin samples were extracted by homogenization in an extraction buffer containing 0.1 M Tris–HCl, pH 7.6, 0.1 M NaCl, 0.5% hexadecyltrimethylammonium bromide. MPO activity in the supernatant fraction was measured by the change in optical density at OD 460_nm resulting from decomposition of H₂O₂ in the presence of O-dianisidine. MPO content was expressed as units of MPO activity/mg protein. These numbers were subtracted from background MPO activity of skin of mice injected with PBS alone and sacrificed at the same timepoints as the test mice. Protein concentrations were determined by the Bio-Rad dye binding assay using BSA as a standard.

The concentration of serum rabbit IgG was measured by ELISA relative to purified rabbit IgG (Sigma Chemical Co., St. Louis, MO). Microtiter plates were coated with polyclonal goat anti–rabbit IgG Fc antibody (Cappel Laboratories, Durham, NC), incubated with dilutions of serum, and then developed with horseradish peroxidase– conjugated goat antibodies specific for rabbit IgG F(ab′)₂ (Cappel Laboratories, Durham, NC) and read at OD_492nm against a standard curve (Bio-Rad EIA reader, model 2550).

The data were expressed as mean ± SEM and were analyzed using the Student's t-test. A P value <0.05 was considered significant.

Pathogenic anti-mBP180 antibodies administered intraperitoneally do not induce experimental BP in β₂m^−/− mice. When neonatal BALB/c (n = 5), C57BL/6J (n = 5), and β₂m^+/− (n = 5) mice were injected intraperitoneally with pathogenic anti-mBP180 IgG (2.5 mg/g body weight), as expected, these animals developed extensive blisters 24 h after injection (Fig. 1,A; Table 1). The skin of these animals was markedly erythematous and, upon gentle friction, developed persistent epidermal wrinkling due to epidermal detachment from underlying dermis. Direct IF of cryosections of lesional and peri-lesional skin showed in vivo deposition of rabbit IgG and mouse C3 at the dermal– epidermal junction (Fig. 1,B). H/E stained sections from these mice showed dermal–epidermal separation with neutrophilic infiltration (Fig. 1,C). In contrast, β₂m^−/− mice (n = 5) exhibited no blisters 24 h after injection with an identical dose of pathogenic anti-mBP180 IgG (Fig. 1,D). Direct IF also showed a significant reduction in in situ deposition of rabbit IgG and mouse C3 at the BMZ (Fig. 1,E). H/E staining of the skin sections from injected mice exhibited no epidermal detachment from the dermis and a neutrophilic infiltrate milder (Fig. 1,F) than positive control mice. Quantitation of neutrophilic infiltration with the MPO assay showed significant changes in the extractable enzyme activity at the injected site at 24 h after injection, with 0.06 ± 0.01 in β₂m^−/− mice versus 0.41 ± 0.04 in BALB/c, 0.38 ± 0.04 in C57BL/6J, and 0.38 ± 0.03 in β₂m^+/− mice (P <0.001) (Fig. 2). However, there was no difference in tissue MPO activity in both β₂m^−/− and control mice 4 h after intraperitoneal administration of anti-mBP180 IgG (Fig. 2), suggesting that lack of β₂m expression did not interfere with neutrophil migration from circulation into the site of tissue inflammation.

Indirect IF showed a lower level of circulating rabbit IgG in β₂m^−/− mice (titer = 1:1,280) than control groups (titer >1:5,120 for all three groups of mice). ELISA further demonstrated significant changes in circulating R621 IgG in β₂m^−/− mice at different timepoints after injection (Fig. 3). R621 IgG levels in β₂m^+/− mice were 1.18 ± 0.09, 2.32 ± 0.06, 1.83 ± 0.19, at 6 h, 12 h, 24 h, respectively, after intraperitoneal injection. In contrast, R621 IgG levels in β₂m^−/− mice were 0.89 ± 0.08, 1.40 ± 0.14, 1.04 ± 0.10, at 6 h, 12 h, 24 h, respectively, after intraperitoneal IgG injection. There were significant reductions of circulating R621 IgG between β₂m^−/− and β₂m^+/− at 12 h (40%; P <0.001) and 24 h (43%; P <0.001) after injection.

Intradermal injection of pathogenic anti-mBP180 IgG induced subepidermal blisters in β₂m^−/− mice. To demonstrate that anti-mBP180 IgG is pathogenic in β₂m^−/− mice if it binds to its target antigen sufficiently, neonatal β₂m^−/− (n = 5), β₂m^+/− (n = 5), BALB/c (n = 5), and C57BL/6J (n = 5) mice were administered intradermally pathogenic anti-mBP180 IgG (5 mg/g body weight). After 12-h incubation, like normal control mice, β₂m^−/− mice developed subepidermal blisters, along with in vivo deposition of rabbit IgG and murine C3 at BMZ and neutrophilic infiltration (see Table 1). However, clinical examination showed that disease activity in β₂m^−/− mice (2+) was less severe than in control mice (3+) (Table 1). Direct IF revealed a less intense staining of BMZ in β₂m^−/− than in control mice (data not shown). Quantitation of neutrophilic infiltration by the MPO assay also showed a similar trend, with 0.77 ± 0.08 in β₂m^−/− mice versus 0.89 ± 0.09 in β₂m^+/− mice (Fig. 4). At 12 h after injection, circulating anti-mBP180 IgG levels in β₂m^−/− were 77% of those in β₂m^+/− mice (Fig. 5). Indirect IF of sections of skin from uninjected β₂m^−/− and normal control mice indicated that the pathogenic rabbit antibody bound the basement membrane zone with equal intensity and pattern (data not shown), indicating that the absence of β₂m did not prevent binding of the pathogenic antibody to its target.

Our data show that mice deficient in β₂m expression were protected from experimental BP when the pathogenic antibody was administered intraperitoneally, whereas the skin of these mice was readily susceptible to the inflammatory effects of the antibody given intradermally. These findings are predicted by the hypothesis that in β₂m-deficient mice, the amount of pathogenic anti-BP antibody reaching the epidermal target site is greatly reduced because the antibody is eliminated rapidly by systemic catabolic mechanisms. A milder pathologic reaction thus ensues. Conversely, the systemic expression of FcRn in β₂m-intact mice protects IgG, including the pathogenic antibody, from catabolism, thereby maintaining an effective pathogenic dose. The cellular mechanism for IgG degradation postulated by Brambell (1) and subsequently elaborated (reviewed in reference 23) sufficiently explains this observation. It is proposed that IgG is pinocytosed nonspecifically into an acidic endosomal compartment where it encounters and binds FcRn at low pH. At this point the transport pathway forks. Those endosomes bearing FcRn complexed with IgG move through the cell to be expressed on the cell surface where at physiologic pH the ligand dissociates from FcRn and is free to circulate in the animal or to be pinocytosed by the cell again for recycling. Pinocytosed IgG that does not bind to the endosomal FcRn (because of receptor saturation or by chance) is moved to lysosomes for enzymatic degradation. It has been calculated that an IgG molecule is degraded every eighth pass through this recycling pathway (21). Which cells might be responsible for IgG degradation is not yet clear. To date, a variety of cells of humans have been shown to express the receptor including endothelium and the syncytiotrophoblast (8, 36) (Sedmak, D.D., manuscript submitted). We know of no data suggesting that our findings on experimental BP could be the result of other functional defects of the β₂m-deficient mice; specifically, those defects resulting from the lack of MHC class I, CD1 (37), HFE (38–40), and perhaps others.

The hypothesis accounts for additional findings. We note that pathogenic antibody given intradermally caused lesions slightly less severe in the β₂m-deficient mice than in normal mice. In the absence of FcRn, such antibody would be expected to be degraded more rapidly, and therefore be less damaging, than when given to normal mice. Considering the findings of others, we would propose that FcRn deficiency accounts for several observations in β₂m-deficient mice; viz, both acute (19) and chronic (18, 26, 27) (Roopenian, D.C., manuscript submitted) forms of the SLE syndrome, the low titers of virus-specific IgG seen after vaccinia inoculation (16), and DNP-specific IgG responses generated to both T-dependent and -independent antigens (Roopenian, D.C., manuscripts submitted).

Although these studies were performed in neonatal mice, there is no reason why the observations should not be applicable to the adult. Certainly, the murine model of experimental BP is valid in adult mice (Liu, Z., unpublished observations); neonates are used experimentally only to conserve pathogenic antibody and to inspect more readily the hairless skin for blisters. Likewise, the IgG catabolic studies have been done only in adult mice, but we can think of no reason why neonates would behave any differently with respect to IgG degradation (20–22).

We would suggest that FcRn, appearing to be a critical molecule in antibody-mediated immunity, might be manipulated pharmacologically to decrease pathogenic levels of IgG in various antibody-mediated human diseases, thereby modulating the inflammatory process.

This work was supported in part by United States Public Health Service grants R29-AI40768 (Z. Liu), RO1-AR32599 (L.A. Diaz), R37-AR32081 (L.A. Diaz), RO1-AI24544 (D.C. Roopenian), RO1-AI32744 (D.D. Sedmak and C.L. Anderson), and RO1-HD35121 (C.L. Anderson) from the National Institutes of Health, a Dermatology Foundation Research Grant (Z. Liu), and a grant from the Pediatrics AIDS Foundation (C.L. Anderson).