Pemphigus vulgaris immunoglobulin G can recognize a 130 000 MW antigen other than desmoglein 3 on peripheral blood mononuclear cell surface

Abstract

Pemphigus vulgaris (PV) is considered to be an autoimmune disease affecting skin and mucous membranes. Traditionally, PV autoantibodies are thought to recognize antigens located in the intercellular substance (ICS) of keratinocytes; antigens represented mainly by the desmosomal cadherin desmoglein 3 (Dsg3). Accordingly, titres of anti-ICS and anti-Dsg3 immunoglobulin G (IgG) are considered to be major laboratory criteria when making a diagnosis of PV. In this paper, we demonstrated for the first time that PV IgG bind antigen(s) expressed on the surface of peripheral blood mononuclear cells (PBMC), as revealed by immunofluorescence studies. This novel autoantigen is immunoprecipitated by PV IgG as a 130 000 molecular weight protein. However, Western blot analysis of the immunocomplexes failed to show reactivity with anti-Dsg3 monoclonal and polyclonal antibodies. Taken together, our data provide strong evidence that PV autoimmunity targets a 130 000 antigen other than Dsg3 on PBMC. This shifting from epidermis to blood cells may open new perspectives for a better understanding of pemphigus autoimmunity and more rational approaches to its treatment.

Figure 1

(a) PV IgG, but not Nh IgG, bound antigen(s) within the intercellular substance (ICS), describing the typical fishnet-like pattern (b). As revealed by immunofluorescence, cells expressed Dsg1 and Dsg3, whereas Dsg2 was virtually undetectable. Figures are representative of three independent experiments. (c) PV IgG (n = 4), but not control IgG, immunoprecipitated the 130 000 MW Dsg3 (n = 4) and were slightly positive to the 160 000 MW Dsg1. The immunoreactivity of sera was first probed with anti-Dsg3 and anti-Dsg1 IgG separately, in three independent experiments. Then the filter shown in the figure was stripped and incubated with both antibodies to visualize the autoantigenic pattern in the same blot.

Figure 2

Immunofluorescence studies and Western blotting (WB) showed that PBMC lack Dsg1 and Dsg3. Anti-Dsg1 and anti-Dsg3 were first probed separately, then the results were summarized in the same blot by incubating PVDF filters with both antibodies. Keratinocyte lysates (ker) served as a positive control. Con, negative control PBMC obtained by using only FITC-conjugated anti-human antibodies. The figures are representative of three independent experiments.

Figure 3

(a) Ponceau staining of PVDF membranes blotted with immunocomplexes shows a slight 130 000 band immunoprecipitated by PV (n = 4) but not control sera. (b) By increasing the contrast of the surface of interest (adobe Photoshop), the 130 000 band (arrow) clearly appeared in lanes 1–4 (PV sera, asterisks) but not in lane 5 (control). (c) Combined immunoprecipitation (IP)-Western blotting (WB) with PV IgG demonstrated that IgG from PV sera recognized a 130 000 MW protein. (d) Pre-incubation of PV IgG (primary antibody) with gel-pure 130 000 MW protein(s) from keratinocyte extracts strongly reduced the ability of PV IgG to bind the 130 000 PBMC antigen. (e) Immunoprecipitation of keratinocyte extracts with H-145 anti-Dsg3 (positive control), Nh or PV IgG followed by Western blotting against Dsg3 revealed that immunocomplexes other than positive controls did not contain Dsg3. Results were confirmed in at least two experiments carried out independently.

Figure 4

(a) PBMC were fixed and permeabilized in paraformaldehyde solution and then incubated with PV or normal IgG. Binding of the antibodies on PBMC surface was revealed by FITC-conjugated anti-human IgG. Only PV IgG stained all around cell membrane of PBMC. The right-hand panels represent magnifications of the highlighted boxes in the left panels. (b) Pre-incubation of PV serum with purified keratinocyte 130 000 protein(s) abolished the staining of PV IgG on PBMC surface (left panel), whereas preincubation with gel-pure 130 000 protein(s) from PBMC extracts apparently did not impaire the ability of PV IgG to recognize keratinocyte antigens. The figure is representative of at least two independent experiments.