Autoantibodies against the epidermal desmosomal cadherins desmoglein 1 (Dsg1) and Dsg3 have been shown to cause severe to lethal skin blistering clinically defined as pemphigus foliaceus (PF) and pemphigus vulgaris (PV). It is unknown whether antibody-induced dissociation of keratinocytes is caused by direct inhibition of Dsg1 transinteraction or by secondary cellular responses. Here we show in an in vitro system that IgGs purified from PF patient sera caused cellular dissociation of cultured human keratinocytes as well as significant release of Dsg1-coated microbeads attached to Dsg-containing sites on the keratinocyte cellular surface. However, cell dissociation and bead release induced by PF-IgGs was not caused by direct steric hindrance of Dsg1 transinteraction, as demonstrated by single molecule atomic force measurements and by laser trapping of surface-bound Dsg1-coated microbeads. Rather, our experiments strongly indicate that PF-IgG–mediated dissociation events must involve autoantibody-triggered cellular signaling pathways, resulting in destabilization of Dsg1-based adhesive sites and desmosomes.
Jens Waschke, Paola Bruggeman, Werner Baumgartner, Detlef Zillikens, Detlev Drenckhahn
Submitter: Jens Waschke | firstname.lastname@example.org
JULIUS - MAXIMILIANS - UNIVERSITÄT WÜRZBURG
Published May 8, 2006
In a letter to the editor, Nicola Cirillo and colleagues raised some points of critique concerning the conclusion drawn from our data published. Specifically, the authors are not convinced that pemphigus foliaceus IgG did reduce Dsg 1-mediated binding by mechanisms different from steric hindrance. We applied micromechanical approaches like laser tweezers and atomic force microscopy to study the effects of PF-IgG on Dsg 1 binding under conditions where antibodies induced keratinocyte dissociation in culture. We found that PF-IgG reduced Dsg 1 binding when cells with bound beads were post-incubated with PF-IgG or when cells were pre-incubated with PF-IgG before beads were added. When atomic force measurements were carried out in a cell-free set up and when laser tweezer experiments were performed by pre-incubation of beads with the antibodies, patients IgG were ineffective to reduce Dsg 1 adhesion. In contrast, a Dsg 1-specific monoclonal antibody was equally effective under cell-free conditions also. We concluded that PF-IgG, in contrast to the monoclonal antibody, did not block Dsg 1 transinteraction by steric hindrance and that keratinocyte dissociation must be caused by mechanisms other than steric hindrance.
The following points were raised by Nicola Cirillo and colleagues:
1.The authors mentioned that Dsg 3 in contrast to desmoplakin would not be a good marker to detect desmosomes by immunostaining because it is possible that some Dsg 3 molecules were not incorporated into desmosomes. We showed that Dsg 3 immunoreactivity was lost at margins of intercellular gaps but was still present in cellular processes connecting neighboring keratinocytes. It is possible that these Dsg 3 molecules were not part of desmosomes at these sites. However, the point of these experiments was to demonstrate that antibodies exerted visible biologic effects under conditions used to study Dsg 1 binding.
2.The authors seem to make the point that Dsg 1 molecules on beads were in large excess compared to the amount of PF-IgG in pre-incubation experiments which could explain why PF-IgG were not sufficient to reduce binding of beads to keratinocytes afterwards. However, it has to be noted that the same amount of IgG and of Dsg 1-coated beads was used for post- incubation experiments in which PF-IgG resulted in a substantial loss of bound beads. If this reduction of bead binding would have been due to a steric hindrance mechanism we would expect no difference to pre-incubation experiments.
3.We do not understand why the post-incubation experiments in which PF-IgG reduced Dsg 1-binding should be indicative for steric hindrance. Loss of binding as the final outcome of antibody action does not allow any conclusion on direct (steric hindrance) vs. indirect (signaling) mechanisms responsible for dissociation. Any mechanism finally leading to impaired binding properties (for example EGTA) results in loss of bead binding.
Taken together, we do not see substantial criticism on the methods used or the interpretation of our results. The goal of our study was to provide evidence that mechanisms different from steric hindrance must be involved in acantholysis in PF. We agree with the authors that elucidation of these mechanisms is important to improve strategies in pemphigus therapy.
Submitter: Nicola Cirillo | email@example.com
Second University of Naples, - Naples, Italy
Published May 8, 2006
TO THE EDITORS:
Further elucidation of the molecular phenomena underlying pemphigus foliaceus (PF) represents a prerequisite to come to a better therapy. In the November issue of the Journal of Clinical Investigation, Waschke et al. (1) shed light on the mechanisms of pemphigus antibody-induced acantholysis, providing evidence that PF IgG disrupts desmoglein 1 (Dsg1)- containing desmosomes without preventing Dsg1 transcellular interactions. Although indirectly, the results of this study suggest that "PF IgG reduces Dsg1-mediated adhesion not by steric hindrance but involves secondary cellular mechanisms".
Apart from signaling that PF is caused by antibodies against Dsg1, and not Dsg3 as reported in the Introduction, we would like to consider some issues prior to accept the above hypothesis proposed in the published paper:
1. Dsg3 was used to immunostain desmosome, but it is not clear at all whether the stain represents cell surface Dsg3 assembled into adhering complexes or Dsg3 clusters on the nondesmosomal plasma membrane. Thus, the choise of Dsg3 as desmosomal marker appears at least questionable. Desmoplakin (and not plakoglobin, for the same reasons of Dsg3) could be more useful.
2. Waschke et al. reported that preincubation of Dsg1-coated beads with PF-IgG did not reduce beads binding on HaCaT cell surface: this finding, representing the only evidence that should refutes the steric hindrance hypothesis, allowed Authors to speculate that PF IgG did not inhibit Dsg1-mediated binding to HaCaT cells. However, unless Authors clarify the amount of Dsg1-coated beads preincubated with PF-IgG, it is reasonable to suppose that beads were present in large excess.
3. Waschke et al. reported that, when cells with surface-bound beads were postincubated with PF IgG, the number of bound beads dropped significantly within 30 min. This finding seems to demonstrate that PF IgG are able to detach Dsg1 from one another through steric hindrance.
Substantially, Authors drew their conclusion on the basis of single molecule atomic force measurements in a cell-free system. However, we believe that data reported on the published paper do not permit to extend the above Authors' considerations on cultured keratinocytes and do not definitively demonstrate that mechanisms other than steric hindrance are involved in PF acantholysis.
REFERENCES: 1. Waschke, J., Bruggeman, P., Baumgartner, W., Zillikens, D., and Drenckhahn., D. 2005. Pemphigus foliaceus IgG causes dissociation of desmoglein 1 containing junctions without blocking desmoglein 1 transinteraction. J. Clin. Invest. 115:3157-3165