Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid

BACKGROUND: Anti-laminin 5 cicatricial pemphigoid (CP) is a mucosal-dominant subepithelial blistering disease characterized by IgG anti-basement membrane zone autoantibodies, that bind to dermal side of 1 M NaCl split skin and immunoprecipitate laminin 5. Laminin 5 is an epidermis-specific extracellular matrix consisting of alpha3, beta3 and gamma2 subunits. Recent studies have suggested that autoantibodies of anti-laminin 5 CP recognize the G domains of alpha3 subunit. OBJECTIVE: We examined the reactivity of anti-laminin 5 CP by immunoblotting using purified laminin 5 and recombinant proteins of alpha3 subunit. METHOD: We first examined the reactivity of anti-laminin 5 CP by immunoblotting using purified laminin 5. To further investigate the epitopes in the G domains of alpha3 subunit, we produced recombinant proteins of G1-2, G1-3, G2-3, G3-5 domains, that covered entire G domain, and examined the reactivity of anti-laminin 5 CP sera with these recombinant proteins by immunoblotting. RESULTS: By immunoblotting using purified laminin 5, 7 of 21 anti-laminin 5 CP sera reacted with alpha3 subunit, while 8 sera reacted with beta3 subunit and one serum reacted with gamma2 subunit. Two sera reacted with both alpha3 and beta3 subunits, while seven sera did not show positive reactivity. This result indicates that the reactivity of anti-laminin 5 CP sera is much more heterogeneous, although the previous studies suggested that most sera reacted with alpha3 subunit. However, in the studies using recombinant proteins of G domains of alpha3 subunit, none of the CP sera, including the sera reactive with alpha3 subunit in purified laminin 5, reacted with any recombinant proteins. The reason for this negative reactivity with the recombinant proteins is not clear. CONCLUSION: The immunoblotting using purified laminin 5 should be useful technique for the diagnosis of anti-laminin 5 CP, although the sensitivity was less than conventional immunoprecipitation analysis.     

Crescentic glomerulonephritis and subepidermal blisters with autoantibodies to alpha5 and alpha6 chains of type IV collagen

We describe a novel autoimmune disease characterized by severe subepidermal bullous eruption and crescentic glomerulonephritis with autoantibodies directed against the noncollagenous domain of the alpha5 and alpha6 chains of type IV collagen. Biopsy of perilesional skin revealed a subepidermal blister with marked polymorphonuclear infiltrate with linear deposits of IgA and C3. Light microscopy of a kidney biopsy specimen revealed a crescentic glomerulonephritis, and immunofluorescence microscopy showed linear basement membrane staining for IgA (3+), C3 (1+), and IgG (1+). No electron-dense deposits were observed by transmission electron microscopy. The patient's autoantibodies reacted with normal human skin and kidney: IgA (3+) and IgG (1+) antibodies stained the basement membrane zones of skin, renal glomerulus, and some tubules. The identity of the target antigen was determined by immunochemical analyses of candidate antigens using the patient's autoantibodies. The patient's IgA and IgG autoantibodies reacted with a 185- to 190-kDa antigen from a human dermal extract that was distinguished from the other dermal or epidermal antigens, including the 145- to 290-kDa (type VII collagen) epidermolysis bullosa acquisita antigen, the 165- to 200-kDa alpha3 laminin mucous membrane cicatricial pemphigoid antigen, and the 230-kDa and the 180-kDa bullous pemphigoid antigens. Patient's IgA and IgG autoantibodies further reacted with the alpha5(IV) and weakly with the alpha6(IV) chains of type IV collagen by Western blot and ELISA. This report expands the repertoire of bullous skin disorders and provides an explanation for the association of anti-type IV collagen autoantibodies and glomerulonephritis with subepidermal blisters.     

Desmosomes and their autoimmune pathologies

Desmosomes guarantee the integrity of the epidermis, by functioning both as an adhesive complex and as a cell-surface attachment site for the keratin intermediate filaments of the cytoskeleton. Considerable progress has been made in our knowledge of desmosomes and their components. The structure and function of many of the desmosomal molecules have been determined, and a number of the molecular interactions between desmosomal proteins have been elucidated. Desmosomal proteins are major antigens in pemphigus. Each type of pemphigus has its own antigenic targets, but in the last few years it has been shown that certain autoantibody populations are not restricted to just one form of pemphigus. The production of autoantibodies against multiple intracellular and extracellular desmosomal proteins, whose pathogenic role remains to be elucidated, suggests an overlapping distribution of antibody specificities among different forms of pemphigus.     

Desmoglein-3 is a target autoantigen in spontaneous canine pemphigus vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease of humans and companion animals. In human patients, PV is associated with the production of IgG autoantibodies specific for keratinocyte desmosomal glycoproteins of the cadherin family. The purpose of this study was to determine whether antikeratinocyte IgG autoantibodies were present in the skin and serum of dogs with PV, and also to identify the canine PV autoantigen(s) targeted by circulating autoantibodies. Eleven dogs were selected because of the microscopic demonstration of suprabasal epithelial acantholysis. Direct immunofluorescence revealed the presence of IgG autoantibodies bound to the membrane of keratinocytes in skin biopsy specimens of 8/9 dogs (89%). Using indirect immunofluorescence, serum-circulating IgG autoantibodies were found in 10/11 (91%) and 5/11 (45%) dogs, using normal canine gingiva and cultured canine oral keratinocytes, respectively. By immunoblotting using cultured canine oral keratinocyte protein lysates, IgG autoantibodies from 7/9 (78%) tested dogs recognized a 130-kDa antigen that comigrated with that identified by rabbit polyclonal antibodies raised against desmoglein-3. This 130 kDa antigen was confirmed to represent the canine equivalent of human desmoglein-3 by immunoprecipitation-immunoblotting. The results of these studies provide evidence that the canine desmoglein-3 homologue is a major autoantigen in dogs with PV. These observations further establish spontaneous canine PV as a natural model for research on pathogenesis, etiology and novel therapeutic approaches for this disease of humans.