The British journal of dermatology · 2010
Binding of pemphigus vulgaris IgG to antigens in desmosome core domains excludes immune complexes rather than directly splitting desmosomes.
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Aoyama Y, Nagai M, Kitajima Y
Аннотация
BACKGROUND: Pemphigus vulgaris (PV) is characterized by autoantibodies against desmoglein (Dsg) 3 or both Dsg1 and Dsg3, i.e. desmosomal adhesion molecules.
OBJECTIVES: We examined whether or not PV IgG binding to Dsg3 directly impairs the adhesion of desmosomes.
METHODS: For immunofluorescence microscopy, keratinocytes were first incubated with PV IgG for 30 min in low Ca(2+) medium, in which no desmosomes were formed, and then for 1 h in high Ca(2+) medium to generate desmosomes. For immunoelectron microscopy, after a 30-min incubation with PV IgG in low Ca(2+) medium, cells were incubated with antihuman IgG with 5-nm gold particles for 5 min; after washing, cells were further incubated in high Ca(2+) medium for 1 h. For tracing of PV IgG/Dsg3 immune complexes formed in the desmosomal core domain, cells were first incubated with PV IgG for 5 min to allow PV IgG to bind the desmosomal core domain and were further incubated with PV IgG-free medium for different times.
RESULTS: Immunofluorescence microscopy revealed that PV IgG bound in a random-punctate pattern on the cell surface in low Ca(2+) medium was translocated to the cell-cell contacts forming a dotted-linear distribution, suggesting desmosome generation even in the presence of PV IgG. Immunoelectron microscopy revealed that half-desmosome-like structures decorated with gold particles in low Ca(2+) keratinocytes coupled to form desmosomes and gold particles were sandwiched in the desmosomal core domain after Ca(2+) switch, even though their surfaces were covered with PV IgG/antihuman IgG 5-nm gold particles. In the tracing experiments, although PV IgG demonstrated a dotted-linear distribution along the cell-cell contacts colocalized with desmoplakin (DPK) after a 30-min tracing, it disappeared from cell-cell contacts after a 5-h tracing, leaving DPK and desmocollin 3.
CONCLUSIONS: These results suggest that the PV IgG/Dsg3 immune complexes are excluded from the desmosomal core domain rather than directly splitting the desmosome.
OBJECTIVES: We examined whether or not PV IgG binding to Dsg3 directly impairs the adhesion of desmosomes.
METHODS: For immunofluorescence microscopy, keratinocytes were first incubated with PV IgG for 30 min in low Ca(2+) medium, in which no desmosomes were formed, and then for 1 h in high Ca(2+) medium to generate desmosomes. For immunoelectron microscopy, after a 30-min incubation with PV IgG in low Ca(2+) medium, cells were incubated with antihuman IgG with 5-nm gold particles for 5 min; after washing, cells were further incubated in high Ca(2+) medium for 1 h. For tracing of PV IgG/Dsg3 immune complexes formed in the desmosomal core domain, cells were first incubated with PV IgG for 5 min to allow PV IgG to bind the desmosomal core domain and were further incubated with PV IgG-free medium for different times.
RESULTS: Immunofluorescence microscopy revealed that PV IgG bound in a random-punctate pattern on the cell surface in low Ca(2+) medium was translocated to the cell-cell contacts forming a dotted-linear distribution, suggesting desmosome generation even in the presence of PV IgG. Immunoelectron microscopy revealed that half-desmosome-like structures decorated with gold particles in low Ca(2+) keratinocytes coupled to form desmosomes and gold particles were sandwiched in the desmosomal core domain after Ca(2+) switch, even though their surfaces were covered with PV IgG/antihuman IgG 5-nm gold particles. In the tracing experiments, although PV IgG demonstrated a dotted-linear distribution along the cell-cell contacts colocalized with desmoplakin (DPK) after a 30-min tracing, it disappeared from cell-cell contacts after a 5-h tracing, leaving DPK and desmocollin 3.
CONCLUSIONS: These results suggest that the PV IgG/Dsg3 immune complexes are excluded from the desmosomal core domain rather than directly splitting the desmosome.