Intracellular ionic changes induced by bullous pemphigoid IgG subclasses

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 IgG(1) BP antibodies. However, no transient increase of Fluo-3 fluorescence intensity was revealed when the cells were incubated with IgG(2) and IgG(4) BP antibodies. A transient increase of Fluo-3 fluorescence intensity was revealed in DJM-1 cells incubated with 3 of seven IgG(1) and 1 of four IgG(2) 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 IgG(1) BP antibodies, but was not revealed in the cells incubated with IgG(2) or IgG(4) of BP antibodies. Study of complement activation by BP IgG(1) showed a transient increase of Fluo-3 fluorescence intensity of with 3 of five IgG(1) 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 IgG(1) 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 IgG(1) induces Ca2+ release from intracellular storage sites, however, the complement activated by BP IgG(1) does not induce cell lysis. It could not be confirmed that anti-180 kD BP antigen antibody induced Ca2+ release from intracellular storage sites.