Bullous Pemphigoid Autoantibodies Directly Induce Blister Formation without Complement Activation (1)
Studies to map the epitopes on COL17 have revealed that the juxtamembranous extracellular noncollagenous 16A domain (NC16A) is preferentially recognized by autoantibodies in the sera of BP patients. An IgG passive transfer neonatal mouse model has demonstrated that the binding of Abs to murine COL17 triggers immune reactions, including complement activation, mast cell degranulation, and neutrophil infiltration. The infiltrating neutrophils were shown to be activated via FcγRIII and to release neutrophil elastase and matrix metalloproteinase-9, which are responsible for the dermal/epidermal separation.
rhIgG1 and rhIgG4 mAbs against hNC16A were established by combining a Fab-B4 fragment that reacts to hNC16A with Fc portions of hIgG1 or hIgG4. The reactivity of the rhIgGs against hNC16A was confirmed by IF study using skin samples and human COL17-expressing mammalian cells (HEK293) and ELISA. To detect the epitope of rhIgG1 and rhIgG4 in hNC16A (Glu490 to Arg566), dot blotting was performed by using four chemically synthesized peptides conjugated with BSA: R4 (Glu490 to Ile509), R5 (Arg506 to Gln525), R7 (Asp522 to Gln545), and R8 (Ser542 to Arg566). Epitope mapping of the rhIgGs and Fab-B4 by using four small synthesized peptides of hNC16A revealed both rhIgGs bound to the R5 (Arg506 to Gln525) peptide that was also recognized by Fab-B4.
At lower doses of rhIgGs, blister formation was more frequently induced by rhIgG1 (18 μg, 87.5%; 9 μg, 75%) than by rhIgG4 (30 μg, 50%; 15 μg, 62.5%). IgG binding to hNC16A does not affect the function of murine COL17 in vivo. rhIgGs were injected into neonatal C3-deficient COL17-humanized mice. Both rhIgG1 (30 μg) and rhIgG4 (50 μg) induced blister formation in all C3−/−/COL17-humanized mice associated with linear deposition of hIgG but not mC3 at the DEJ of the skin. Monoclonal hIgGs against hNC16A induce blister formation in mice regardless of complement activation. Six of seven neonatal COL17-humanized mice pretreated with the FcγR blocker developed blisters. Intraperitoneal injection of MG-132, a very potent proteasome inhibitor, prior to the injection of rhIgG4 did not prevent the induction of blister formation in the treated mice
Hhybridomas that produce monoclonal mIgG Abs against hNC16A were developed.
Wild-type mice (C57BL/6) were immunized by grafting skin from human COL17-expressing mice (COL17m+/+,h+) to their backs. Five weeks after the skin graft, spleen cells were removed from the immunized mice and were fused with P3U1 myeloma cells by a polyethylene glycol 400 procedure. Hybridoma supernatants were screened on 96-well plates coated with recombinant hNC16A protein.
Three mIgG1 clones (TS39-3, TS21-1, and TI66-1) and one mIgG2c clone (TS4-2) against hNC16A that strongly reacted to NHS. The hybridomas were derived from C57BL/6 mice; therefore, TS4-2 should be regarded as IgG2c instead of as IgG2a. The reactivity of TS4-2 to COL17-humanized mouse skin was quite weak.
Epitope mapping of those monoclonal mIgGs using the four small synthesized peptides of hNC16A revealed that all the Abs bound to R7 (Asp522 to Gln545) peptide. The mIgG2 and mIgG3 subclasses are potent activators of complements, whereas mIgG1 is a weak activator. C1q-binding activities were high in IgG2c (TS4-2) and low in IgG1 (TS39-3, TS21-1, and TI66-1).
A low dose of monoclonal IgG1 Abs (TS39-3, TS21-1, and TI66-1, 25 μg) induced blisters in all the injected mice that were associated with weak deposition of mC3 at the DEJ (three of three), whereas a high dose of IgG2c (TS4-2, 500 μg) caused weak deposition of IgG and distinct deposition of mC3 at the DEJ but showed a low potential to induce blister formation in the injected mice (positive: two of six). Low doses of monoclonal mIgG1 Abs (TS39-3, TS21-1, and TI66-1, 25 μg) induced blisters in all of the neonatal C3-deficient COL17-humanized mice.
Four weeks after the transfer, each hybridoma formed a large mass on the back. Mild skin changes develop on the ear and snout in the mIgG2c (TS4-2) hybridoma-transferred mice, which are associated with distinct mC3 deposition and with weak mIgG deposition. Meanwhile, significantly severer skin changes, that is, erythema, erosions with crusts, and hair loss, were observed on the snout and ears and around the eyes, legs, and tail in the mIgG1 (TS39-3, TS21-1, or TI66-1) hybridoma-transferred mice, which are associated with strong deposition of mIgG but not mC3. Blister formation was not apparent. Ear samples from each group showed similar extents of inflammatory cell infiltration of neutrophils, lymphocytes, and histiocytes.
Although the IgG4 subclass accounts for only ∼5% of total IgG in normal human sera, IgG4 autoantibodies have been reported to predominate in BP. The capacity of the predominant IgG4 to induce tissue damage has been demonstrated by cryosection assay.