BALB/c Mice Produce Blister-Causing Antibodies Upon Immunization with a Recombinant Human Desmoglein 3 (1)
Pemphigus vulgaris (PV) is an Ab-mediated blistering disease of mucocutaneous surfaces. Over 95% of PV patients express HLA-DR4 or HLA-DRw6, placing this in a group of autoimmune diseases with almost an absolute HLA association. The disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. Passive transfer of Ig from PV patients can induce blisters in neonatal BALB/c mice. The recombinant extracellular domain of Dsg 3 (E-Dsg 3) and full-length Dsg 3 can react with and neutralize blister-causing Abs in PV patient sera.
Peptides designated 1 through 28 are derived from the predicted extracellular domain (aa 1–592) of the human Dsg 3. Peptide 00 (aa 593–616) represents the transmembrane region and peptides 29–45 are derived from the predicted intracellular region (aa 617–976). Numbering does not include the first 23 amino acids, which represent the putative signal peptide.
With immunization of Dsg 3 and E-Dsg 3, SJL/J mice exhibited the highest response, with DBA/1 mice showing the lowest titers. BALB/c and HRS/J mice showed moderate responses. In spite of high levels of Abs against E-Dsg 3, none of the adult mice exhibited mucocutaneous lesions.
Although Ab responses against both Dsg 3 and E-Dsg 3 consisted of IgG1, IgG2a, and IgG2b, the Ab response to the E-Dsg 3 was predominantly of the IgG1 subclass.
To check for the presence of Abs against the native Dsg 3, sera were tested by indirect immunofluorescence staining at different dilutions against native Dsg 3 expressed in monkey esophagus. There were considerable amounts of Abs against native Dsg 3 in each strain of mice
Sera from all four strains of mice were capable of reacting with the native mouse Dsg
Sera from all four different strains of mice showed considerable reactivity against the mouse Dsg 3. Moreover, with the exception of HRS/J mice, which had a slightly lower reactivity, the other three strains of mice exhibited very similar titers.
Each strain of mice exhibited its own pattern of epitope preference. All four strains mounted an Ab response against peptide 7, derived from the extracellular domain. SJL/J mice mounted the broadest Ab response and showed reactivity against peptides 1, 7, 10, 12, 23, 30, and 38. In contrast, sera from DBA/1 mice recognized only peptide 7. BALB/c sera reacted strongly and uniformly against peptides 7, 12, and 30, exhibiting the highest titer against peptide 30 derived from the cytoplasmic region. HRS/J mice exhibited only moderate reactivity against peptides 7 and 23.
Only sera from BALB/c mice immunized with Dsg 3 specifically caused acantholysis, whereas sera from the other three strains had no apparent effect.
Sera were pre-incubated either with E-Dsg 3 or a control Ag before testing for their ability to induce acantholysis in human foreskin. The pathogenic Abs were neutralized only by E-Dsg 3 and thus failed to cause acantholysis.
This in vitro finding was further corroborated using the neonatal passive transfer mouse mode. Concentrated Igs obtained from pooled sera of each mouse strain were inoculated into neonatal mice and the mice were observed for blister formation. Only neonatal mice which received Ig from Dsg 3-immunized BALB/c mice developed the lesion.
HRS/J mice were selected because they have a hyperpermeable epidermis and lymphocytic infiltration in the skin; BALB/c mice were selected because they are used in the neonatal passive transfer model of PV and are susceptible to another Ab-mediated autoimmune disease, Graves’ disease. SJL/J mice were selected because they are susceptible to experimental autoimmune encephalomyelitis and have a propensity for cutaneous vasodilation; and DBA/1 mice were selected because they are susceptible to collagen-induced arthritis, an animal model for rheumatoid arthritis. Because PV and rheumatoid arthritis are more prevalent in DR4+ patients, DBA/1 mice might carry a common immunogenetic susceptibility element. Either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. SJL/J mice had the highest titer of Abs against Dsg 3, and HRS/J mice had Ab levels comparable to the levels in BALB/c mice as measured by ELISA. In spite of this, antisera from HRS/J and SJL/J mice were nonpathogenic. Because Dsg 3 is a glycoprotein, it is likely that glycosylation plays a critical role in the formation of B cell epitopes but that the synthetic peptides are devoid of sugars. Alternatively, the pathogenic Abs primarily recognize conformational epitopes formed by either contiguous or noncontiguous amino acids.