title: Intracellular ionic changes induced by bullous pemphigoid IgG subclasses.
abstract: To ascertain whether membrane signal transduction is induced by bullous pemphigoid (BP) antibody and whether cell lysis is induced by its complement activation, we assessed the intracellular Ca2+ concentration ([Ca2+]i), intracellular pH, membrane potential and morphology of living cells by following the time course of fluorescence intensity of Fluo-3/AM, Snaff-1/AM, Dioc-5 and Luciffer yellow, respectively. A transient increase of Fluo-3 fluorescence intensity in DJM-1 cells (a squamous cell carcinoma line) was revealed when the cells were incubated with 2 of five IgG1 BP antibodies. However, no transient increase of Fluo-3 fluorescence intensity was revealed when the cells were incubated with IgG2 and IgG4 BP antibodies. A transient increase of Fluo-3 fluorescence intensity was revealed in DJM-1 cells incubated with 3 of seven IgG1 and 1 of four IgG2 BP antibodies in an EGTA-containing low-Ca2+ medium. On the other hand, the Dioc-5 fluorescence intensity did not change significantly, though the increase of Fluo-3 fluorescence intensity was observed. The increase of Snarf-1 fluorescence intensity was revealed in DJM-1 cells incubated with 2 of five IgG1 BP antibodies, but was not revealed in the cells incubated with IgG2 or IgG4 of BP antibodies. Study of complement activation by BP IgG1 showed a transient increase of Fluo-3 fluorescence intensity of with 3 of five IgG1 BP antibodies when DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. At the same time, however, endocytosis and cell lysis were not observed with 2 IgG1 BP antibodies which did induce an increase of Fluo-3 fluorescence intensity when Lucifer-yellow-loaded DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. We examined next whether anti-180 kD BP antigen monoclonal antibodies (mAbs R-223 and 233) induce an increase of Fluo-3 fluorescence intensity. MAb R-223 did not induce any increase of Fluo-3 fluorescence intensity in DJM-1 cells, when incubated with normal- and low-Ca2+ media However, mAb R-223 induced a transient increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with complement-supplemented normal-Ca2+ medium. MAb 233 did not induced an increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with normal- and low-Ca2+ media. These results suggest that the BP IgG1 induces Ca2+ release from intracellular storage sites, however, the complement activated by BP IgG1 does not induce cell lysis. It could not be confirmed that anti-180 kD BP antigen antibody induced Ca2+ release from intracellular storage sites.
authors.affiliation: Department of Dermatology Jichi Medical School, Tochigi, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Suzuki
authors.forename: S
authors.initials: S
authors.lastname: Harada
authors.forename: K
authors.initials: K
authors.lastname: Owaribe
authors.forename: H
authors.initials: H
authors.lastname: Yaoita
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D007132:Immunoglobulin Isotypes / Q000276:immunology*; D017136:Ion Transport / Q000276:immunology; D008856:Microscopy, Fluorescence / Q000379:methods; D010391:Pemphigoid, Bullous / Q000276:immunology*; D014407:Tumor Cells, Cultured
chemical_list: D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D002118:Calcium