Biphasic activation of p38MAPK suggests that apoptosis is a downstream event in pemphigus acantholysis

Abstract

In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.

FIGURE 1.

Phospho-p38MAPK activity is detected in epidermal keratinocytes of PF IgG-treated mice at 4, 8, and 21 h but not at 6 h, suggesting biphasic activation of p38MAPK. Phospho-p38MAPK immunoreactivity is seen in skin biopsies of mice treated with PF IgG and precedes blistering. Neonatal C57BL/6J mice were injected intradermally with PF IgG for the indicated times. A, skin biopsies examined by routine hematoxylin and eosin (×20). Blister formation is not seen within the first 8 h after injection of PF IgG but is readily apparent at 21 h; the intraepithelial cleavage plane is marked by the black arrow. B, antibodies to phospho-p38MAPK were used to stain frozen sections of skin biopsies of mice treated with PF IgG for the indicated times and examined by confocal immunofluorescence microscopy. A PBS-treated control (CON) is shown for comparison. Immunofluorescent staining for phospho-p38MAPK is apparent in biopsies of mice treated with PF IgG for 4 h, markedly diminished at 6 h, but increased again at 8 and 21 h. The asterisk marks the stratum corneum; the white arrow marks the epidermal keratinocytes magnified in the inset.

FIGURE 2.

Time course of p38MAPK activation in mice treated with PF IgG. Two peaks of phospho-p38MAPK (P-P38MAPK) activity are detected by Western blot of skin extracts from PF IgG-treated mice. The first peak of p38MAPK activity is observed at 4 h, and the second peak is observed at 8-21 h. Neonatal C57BL/6J mice were injected intradermally with PF IgG (0.1 mg/g of body weight) or control (Con) IgG for the indicated times. A, Western blots of three independent time course experiments. Extracts (20 μg of protein/lane) from skin biopsies of mice were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and immunoblotted with antibodies to phospho-p38MAPK and total p38MAPK. B, signal intensity from the ECL reaction for each band was quantified with a GeneGnome HR scanner and GeneSnap software. Each time course (TC) was individually plotted. Biphasic activation of p38MAPK was induced by PF IgG in vivo.

FIGURE 3.

Time course of p38MAPK phosphorylation in cultured normal human keratinocytes treated with PV IgG. Normal human keratinocytes cultured to 80-90% confluence were treated with PV IgG (3.0 mg/ml) for the indicated times, and extracts (15 μg of protein/lane) were separated by 10% SDS-PAGE. Blots were probed with antibodies to phospho-p38MAPK (P-P38MAPK), stripped, and then reprobed with antibodies to p38MAPK. Similar to the in vivo experiments with PF IgG, biphasic activation of p38MAPK is observed in keratinocytes treated with PV IgG.

FIGURE 4.

Increased apoptotic activity is observed subsequent to activation of p38MAPK in PF passive transfer mice. Neonatal C57BL/6J mice were injected intradermally with PF IgG for various times from 1 to 48 h, and skin biopsies were obtained for biochemical analysis and for TUNEL. A, immunoblots of murine skin biopsy extracts (50 μg/lane) using antibodies to cleaved PARP and cleaved caspase 3, two markers of apoptosis. B, marked increases in TUNEL-positive keratinocytes were not observed until mice were treated with PF IgG for periods exceeding 21 h. Controls consisted of omitting the terminal deoxynucleotidyltransferase enzyme from the labeling solution (negative control (Negative)) or pretreating sections for 30 min with DNase to induce DNA strand breaks (positive control (Positive)).

FIGURE 5.

Detection of apoptosis in PV-IgG-treated normal human keratinocyte. Normal human keratinocytes were grown to 80-90% confluence and exposed to control IgG or PV IgG (3 mg/ml) for the indicated times. A, treated cells were analyzed for the presence of cleaved-PARP, a marker of ongoing apoptosis, by immunofluorescence microscopy. Actin filaments were labeled with Alexa Fluor 488-conjugated phalloidin (green), and nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). B, TUNEL showed an increase in TUNEL-positive cells when cells were incubated with PV IgG for 10 h, but not at 1 or 6 h, when compared with control IgG-treated cells. Controls for TUNEL staining consisted of omitting the terminal deoxynucleotidyl-transferase enzyme form the labeling solution (negative control (Negative)) or pretreating sections for 30 min with DNase to induce DNA strand breaks (positive control (Positive)). C, TUNEL staining at different time points was quantified by counting the TUNEL-positive cells in each sample. For each slide, three fields were randomly chosen using a defined rectangular area (×20 objective); assays were performed in triplicate. S.D. is shown by error bars; the asterisk indicates p = 0.046 for comparison of control and PV IgG-treated cells at the 10-h time point. p values were determined using the Mann-Whitney U test.

FIGURE 6.

Inhibition of the first peak of p38MAPK activity blocked blister formation, whereas inhibition of the second peak failed to block blistering in vivo. Neonatal mice (n = 3 per treatment group) were injected intradermally with 1) PF IgG only, 2) the p38MAPK inhibitor SB202190 2 h before PF IgG injection, or 3) the p38MAPK inhibitor SB202190 4 h after PF IgG injection. After 18 h, the skin of the three different groups was examined. A, skin biopsies examined by routine hematoxylin and eosin (H&E, ×20) and by direct immunofluorescence (IF) using phospho-p38MAPK (P-P38MAPK) antibodies. Blistering is observed in PF IgG-treated mice but not in inhibitor-pretreated mice. In contrast, blistering is observed when the inhibitor is administered 4 h after PF IgG. The arrow shows increased phospho-p38MAPK IF signal in PF IgG-treated mice. B, extracts from skin biopsies of three mice (lanes 1-3) from each treatment group were probed by immunoblot with antibodies to phospho-p38MAPK and total p38MAPK. C, signal intensity from the ECL reaction for each band was quantified with a GeneGnome HR scanner (Syngene) using GeneSnap software (n = 3, S.D. shown by error bars). The phospho-p38MAPK signal was normalized to total p38MAPK for each sample. p values (^*, p < 0.001; ^**, p < 0.001) were calculated using the Student's t test. D, neonatal mice were treated with 1) control (Con) IgG, 2) PF IgG, or 3) the p38MAPK inhibitor SB202190 4 h after PF IgG injection to block the second peak of p38MAPK activation. After 30 h, extracts from skin biopsies were probed with antibodies to phospho-p38MAPK, total p38MAPK, or cleaved caspase 3. In the mice treated with the inhibitor 4 h after PF IgG, the second peak of p38MAPK activity and the increase in caspase 3 cleavage are blocked.

FIGURE 7.

Inhibition of the first peak of p38MAPK activity blocked PV IgG-triggered cytokeratin retraction, whereas inhibition of the second peak did not. Primary human keratinocytes were treated with (A) vehicle (DMSO) control, (B) vehicle control 2 h prior to PV IgG, (C) SB202190 2 h prior to PV IgG treatment, or (D) SB202190 3 h after PV IgG treatment. Six hours after the addition of PV IgG, cells were fixed and stained for cytokeratin 5/8 and examined by confocal microscopy. Increased cytokeratin retraction is observed over time in keratinocytes treated with PV IgG (B). In cells pretreated with the p38MAPK inhibitor (prior to the first peak), cytokeratin retraction is blocked. In contrast, treating keratinocytes with the p38MAPK inhibitor after the first peak of p38 activity, but prior to the second peak, failed to block PV IgG-triggered cytokeratin retraction (D).

FIGURE 8.

Model for the temporal relationship of pemphigus IgG-mediated activation of p38MAPK to blistering and apoptosis. Two peaks of p38MAPK phosphorylation occur subsequent to treatment with pemphigus IgG. Inhibiting the first, but not second, peak of p38MAPK activity blocks blistering. Markers of apoptosis, including caspase 3 cleavage, PARP cleavage, and TUNEL-positive staining, occur subsequent to the second peak of p38MAPK phosphorylation. Blocking the first peak of p38MAPK phosphorylation blocks blistering, indicating a role for the first peak of p38MAPK activation in loss of cell-cell adhesion. In contrast, blocking this second peak of p38MAPK phosphorylation fails to block blistering but blocks increases in apoptotic markers (e.g. caspase 3 cleavage). The second peak of p38MAPK phosphorylation is not part of the mechanism of acantholysis but may represent stress response signaling secondary to acantholysis.