A novel method to investigate pemphigus-induced keratinocyte dysmorphisms through living cell immunofluorescence microscopy

Abstract  Pemphigus vulgaris (PV) blistering occurs as a result of the disruption of intercellular contacts among keratinocytes, or acantholysis. The hallmark of PV acantholysis in vitro is considered to be the retraction of keratin intermediate filaments (KIF) onto the nucleus, which parallels with loss of cell–cell adhesion and rounding up of keratinocytes. However, the fine morphological changes of keratinocytes as well as the fate of cell adhesion structures cannot be appreciated on immunofluorescence by the simple cytokeratin staining. In this paper, we show that acantholytic dysmorphisms are sharply investigated by using PV IgG as a primary antibody on metabolically quiescent living cells. Indeed, PV IgG recognise a wide spectrum of molecules and enabled us to monitor the main changes occurring in acantholytic keratinocytes, including cell shrinkage with the appearance of prickle-like processes, detachment of keratinocytes from one another and collapse of cytoskeleton-bound proteins along nuclear periphery. This method has wider applications as it could be useful for staining cell periphery of keratinocytes and changes in cell shape. Furthermore, images displayed clear and sharp contours because living cell microscopy allows to avoid antigen distortion due to cell manipulation, which usually precedes the immunolabelling.