Figure 1.
Integrated scanning electron microscopy (SEM), immunofluorescence (IF) and AFM imaging of intercellular adhesion structures.
A: Correlation of SEM imaging with AFM imaging. Identically cultured plates of confluent HaCaT cells were fixed and imaged by SEM and Multimode AFM in increasing magnifications. The lower magnification images (SEM: A1, A2; AFM: A5, A6) show the cells with clear boundaries between neighboring cells where cell-cell adhesion occurs. The higher magnification images (SEM: A3, A4; AFM: A7, A8) show details of the adhesion junction with strand-shaped structures in parallel distribution between two cells. B: Correlation of IF imaging with AFM imaging. The same area on a confluent slide of HaCaT cells was captured simultaneously by IF and AFM after fixation of the cells. For IF imaging, HaCaT cells were labeled with anti-cytokeratin antibodies (red) and anti-desmoplakin antibodies (green). AFM images were captured by Bioscope AFM at increasing resolution with scan sizes of 100 µm (B2), 50 µm (B3) and 20 µm (B4). C: Correlation of IF imaging (C1) with AFM imaging [scan sizes of 100 µm (C2), 50 µm (C3) and 20 µm (C4)] after treatment with 10 µg/ml of the pathogenic anti-Dsg3 antibody Px4-3 for 24 h.
Figure 2.
AFM imaging of autoantibody mediated desmosomal disruption in fixed and living cells.
In a first set of experiments, HaCaT cells were grown to confluence, and left with no antibody treatment, or were treated for 24 h with (A) the pathogenic anti-Dsg3 antibody Px4-3, (B) non-pathogenic antibody Px4-4, (C) non-binding irrelevant control antibody goat anti-mouse Ig, or (D) irrelevant control antibody mouse anti-human HLA A, B, C (all antibodies at 10 µg/ml). Images were captured in high resolution (range 6–8 µm) by Multimode AFM after fixation of the cells. In a second set of experiments, confluent HaCaT cells were imaged live before antibody treatment or treated with the antibodies listed above (A–D) for 30 min, 1 h, 2 h, 3 h, and 4 h.
Figure 3.
AFM-guided elasticity measurements on HaCaT cells before and after treatment with anti-Dsg3 and control antibodies.
HaCaT cells were grown to confluence and elasticity was measured in the center of individual cells with an indentation speed of 1.8 µm/s (corresponding to a frequency of 0.3 Hz) over a 4 h time interval (A). At each time point, between 20 and 40 HaCaT cells were tested, and the force curves obtained from these measurements were processed to generate the corresponding Young's modulus as a measure of cellular elasticity. In parallel experiments, measurement were obtained before antibody treatment and after treatment with 10 µg/ml of the pathogenic anti-Dsg3 antibody Px4-3 (B), the non-pathogenic antibody Px4-4 (C), the non-binding irrelevant control antibody goat anti-mouse Ig (D), or the binding irrelevant control antibody mouse anti-human HLA A, B, C (E). Each bar represents the average and standard deviation of repeated measurements obtained over a 1 h period. Each graph represents the average of a minimum of 3 separate experiments for each antibody. The differences in cellular elasticity between conditions within 1 h after antibody treatment compared pre-antibody treatment, and 1–2 h, 2–3 h, and 3–4 h after antibody treatment, and were assessed by Student's t-test and are depicted as * = p≤0.05.
Figure 4.
Decreased cell stiffness after dissociation of intercellular adhesion.
HaCaT cells were cultured in calcium-containing medium for 4 days until reaching confluence (A1, B1, C1), and then calcium-depleted for 2 days (A2, B2, C2), or calcium-depleted for 2 days and then reconstituted in calcium-containing medium for another 2 days (A3, B3, C3). At each interval, we obtained (A) microscopic images (original magnification: 100x), (B) AFM topography images (scan size B1, 50 µm; B2, 20 µm; B3, 33.3 µm), and (C) height measurements at the cross section of an individual cell. In parallel, AFM-guided cell stiffness measurements were obtained by AFM nanomechanical measurement of 50 different cells under each of the conditions described above (D); Each bar represents the average and standard error of the mean (SEM) of repeated measurements for each condition. In a second set of experiments, cell adhesion structures in confluent HaCaT cell cultures grown under standard conditions were visualized by AFM prior to (E1) and after (E2) dissection of cell adhesion structures by nanorobot-guided surgery (traveling distance of AFM tip ∼8 µm; scan size: 19.2 µm; upper panel). The corresponding cross section height measurements show a height decrease by slightly less than 100 nm (from ∼10 nm to −100 nm in the encircled area; lower panel); the arrow in the AFM images points to an intact intercellular connection before cutting (E1) and the disappearance thereof after cutting (E2). F. Cell stiffness was measured before and after the nanorobotic surgery and decreases from 30.0±1.5 kPa to 20.5±2.8 kPa, respectively. The bars represent the mean and standard error of the mean (SEM). E and F. The differences in cellular elasticity between conditions before and after Ca+-depletion and recovery and before and after nano-dissection were assessed by Student's t-test and are depicted as * = p≤0.05.
Figure 5.
A. Induction of apoptotic processes by pathogenic anti-Dsg3 antibodies.
Confluent HaCaT cell cultures were treated with 10 µg/ml of either the pathogenic anti-Dsg3 antibody Px4-3 or the non-pathogenic anti-Dsg3 antibody Px4-4 for 4, 8, 12, 16, 20 and 24 h. Induction of apoptosis was assessed by measuring the expression of both Annexin V and PI positivity by FACS. Each experiment was performed in triplicate. Values are presented as percent induction when compared to baseline apoptosis levels in untreated cells harvested in parallel. The levels of annexin V and/or PI positivity induced by pathogenic anti-Dsg3 antibodies are significantly elevated at 12, 16 and 20 h when compared to untreated cells (p≤0.05, Student’s t-test). B and C. Fas ligand-induced apoptosis correlates with a marked increase in keratinocyte stiffness. Subconfluent HaCaT cell cultures were left untreated (B1), treated with 50 ng/ml FasL (B2) or pretreated with FasL neutralizing antibody (0.5 µg/ml) for 30 min before addition of 50 ng/ml FasL (B3). In untreated HaCaT cells, 4.52±0.32% of all cells are Annexin V- and PI positive as measured by flow cytometry after 44 h of culture. Addition of FasL significantly increases the number of apoptotic cells to 14.63±0.11% (p<0.01, FasL vs. untreated). Pretreatment of FasL-treated cultures with FasL neutralizing antibody significantly reduces FasL-induced apoptosis back to baseline levels 5.95±1.28% (p<0.01, FasL + FasL inhibitor vs. FasL alone). In parallel experiments, 100 cells were randomly selected to collect AFM force-displacement curves to generate a Young's modulus value for each cell. The combined Young’s modulus values for each condition were plotted as Gaussian distribution curves. (C1) Normal cell stiffness distribution in untreated cells (Gaussian fit with peaks around 31.2 kPa and 77.8 kPa). (C2) Cell stiffness distribution in Fas ligand-treated cells (Gaussian fit with peaks around 31.5 kPa and 81.7 kPa). (C3) Cell stiffness distribution in Fas ligand treated cells pretreated with Fas ligand neutralizer (Gaussian fit with single peak around 29.1 kPa). Data are representative of three independent experiments performed in duplicate.
Figure 6.
Blocking of pathogenic anti-Dsg3 antibody-induced increase in keratinocyte stiffness by caspase inhibition, but not Fas ligand neutralization.
HaCaT keratinocytes were grown to confluence and cell stiffness was measured by AFM (expressed as percent of cells measured at a given Young’s Modulus). Cell stiffness distribution in (A) untreated cells (Gaussian fit with peaks around 32.0 kPa and 75.2 kPa), (B) cells treated with the pathogenic anti-Dsg3 antibody Px4-3 (10 µg/ml) alone for 8 h (peaks around 34.8 kPa and 83.5 kPa), (C) cells pretreated with caspase inhibitor (20 µM) 30 min before addition of pathogenic antibody (single peak around 17.7 kPa), (D) cells pretreated with Fas ligand neutralizing antibody (0.5 µg/ml) 30 min before addition of pathogenic antibody (peaks around 29.1 kPa and 73.0 kPa), (E) treated with caspase inhibitor (20 µM) alone for 8.5 h (single peak 30.1 kPa), or (F) treated with Fas ligand neutralizing antibody (0.5 µg/ml) alone for 8.5 h (single peak 32.3 kPa. The single peak of cells of low stiffness observed for treatment with caspase inhibitor (E) and FasL neutralizing antibody (F) alone indicates that the spontaneous apoptosis seen in untreated cells can be completely abrogated by these two blockers.
Figure 7.
Two-Hit hypothesis for antibody-mediated damage in the skin.
Pathogenic and non-pathogenic antibodies have distinct effects on keratinocyte biology: “Hit 1” reflects structural changes in the keratinocyte that are induced both by pathogenic and non-pathogenic antibodies within 1 hour with the pathophysiological correlate of an initial, but incomplete cellular dissociation. “Hit 2” involves a functional change in keratinocyte biology that is promoted by pathogenic, but not non-pathogenic antibodies within 1–4 h with the pathophysiological correlate of an initiation of apoptosis-related signaling pathways. Since the non-pathogenic antibody leads to an initial cellular dissociation that is also seen with pathogenic antibody binding, but does not alter the overall shape of the cell and does not induce the later cell structural changes seen with pathogenic antibodies, it follows logically that “Hit 2” is needed to progress to clinical disease, while “Hit 1” may be a reversible phenomenon.