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Intracellular ionic changes induced by bullous pemphigoid IgG subclasses
Insights
Bullous pemphigoid (BP) IgG1 antibodies trigger intracellular calcium release in DJM-1 cells, but complement activation by BP IgG1 does not lead to cell lysis. Anti-180 kD BP antigen antibodies
Area of Science:
- Immunodermatology
- Cellular Signaling
- Complement Biology
Background:
- Bullous pemphigoid (BP) is an autoimmune blistering disease.
- Understanding the role of BP antibodies and complement activation in cellular responses is crucial.
Purpose of the Study:
- To investigate if bullous pemphigoid (BP) antibodies induce membrane signal transduction.
- To determine if complement activation by BP antibodies leads to cell lysis.
Main Methods:
- Assessed intracellular Ca2+ concentration ([Ca2+]i), intracellular pH, membrane potential, and cell morphology using fluorescence microscopy.
- Incubated DJM-1 cells (squamous cell carcinoma line) with various BP antibodies (IgG1, IgG2, IgG4) and complement.
Main Results:
- Certain IgG1 BP antibodies induced a transient increase in intracellular Ca2+ ([Ca2+]i) in DJM-1 cells.
- Complement activation by BP IgG1 antibodies did not result in observed endocytosis or cell lysis.
- Monoclonal antibodies against the 180 kD BP antigen showed varied effects on Ca2+ release.
Conclusions:
- BP IgG1 antibodies can induce Ca2+ release from intracellular stores.
- Complement activation by BP IgG1 does not appear to cause cell lysis in this model.
- The role of anti-180 kD BP antigen antibodies in Ca2+ release requires further investigation.
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.