Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris

Abstract

Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell–cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.

Tacrolimus reverses pemphigus vulgaris serum-enhanced expression of desmoglein in HaCaT cells via inhibition of heat shock protein-27 phosphorylation

“…It has been reported that Dsg1-dependent Ca 2+ in ux plays a central role in the epidermal pathology of pemphigus. Inhibition of Ca 2+ signaling blocked pemphigus Dsg IgG-induced loss of keratinocyte adhesion in vitro and blistering of human skin ex vivo [31]. Though we are not sure whether FK506 inhibits calcium in ux in cell membranes.…”

“…The dispase-based keratinocyte dissociation assay is currently the main tool for the analysis of antibodyinduced acantholysis in in vitro models of PV [35]. This assay was performed as previously described [31,36,37]. Brie y, HaCaT cells were seeded in 24-well plates and grown to 60-70% con uency and exposed to different conditions as mentioned above.…”

Tacrolimus reverses pemphigus vulgaris serum-enhanced expression of desmoglein in HaCaT cells via inhibition of heat shock protein-27 phosphorylation

“…It has been reported that Dsg1-dependent Ca 2+ influx plays a central role in the epidermal pathology of pemphigus. Inhibition of Ca 2+ signaling blocked pemphigus Dsg IgG-induced loss of keratinocyte adhesion in vitro and blistering of human skin ex vivo [2]. Though we are not sure whether FK506 inhibits calcium influx in cell membranes.…”

“…The two main pemphigus variants are pemphigus vulgaris(PV), which often originates with painful oral erosions, and pemphigus foliaceus (PF), which is characterized by exclusive skin lesions [1]. IgG antibodies against epidermal desmosomes, including desmoglein (Dsg) 1 and Dsg3, two major cell-cell adhesive structure proteins, cause loss of intercellular adhesion, acantholysis, and blister formation [2,3]. Furthermore, both anti-Dsg1 and -Dsg3 antibodies are involved in the activation of signaling pathways including Ca 2+ , p38 mitogen activated protein kinases (p38MAPK), protein kinase C (PKC), Src, EGFR/Erk, and several others, resulting in modulation of keratinocyte cell adhesion properties [4].…”

In Vitro, Ex Vivo, and In Vivo Models for the Study of Pemphigus

“…DDA is a commonly used assay whose major flaw is inter-and intra-experimental comparability. Very recently, a standardized protocol has been proposed to perform DDA under optimal conditions [23].…”