IgG was purified from PV sera using Melon Gel IgG Purification Resins and Kits (Thermo Fisher Scientific, Rockford, IL) according to the manufacturers protocol. chase period at 37C. The results indicate that the AK23-biotin labeling procedure used in this study did not induce changes in Dsg3 distribution as no detectable change in pixel number between peaks could be detected over the 6 hr time course.(TIF) pone.0050696.s003.tif (3.6M) GUID:?EDD369F2-3878-4DBB-8605-7AA424FA952C Figure S4: PV IgG directed against the Dsg3 EC1 domain are not required to cause blistering em in vivo /em . Clinical presentation of lower back epidermal blisters (A) and histopathology (B) of patient PV IgG(b). Note that this patient lacks IgG directed against the Dsg3 EC1 domain (see Figure 7).(TIF) pone.0050696.s004.tif (5.7M) Vinflunine Tartrate GUID:?D126983B-E3F5-49E9-9269-5FCB1D1BA18A Figure S5: p38MAPK inhibition prevents Dsg3 clustering induced by PV IgG lacking EC1 antibodies. Cell surface Dsg3 was monitored using biotinylated AK23 followed by the addition of NH IgG, PV IgG and PV IgG (a). Cells were treated with the p38MAPK inhibitor SB202190 prior and during the addition of IgG. SB202190 prevented Dsg3 clustering induced by both PV IgG and PV IgG (a), the latter which only contains antibodies directed against domains EC3C4.(TIF) pone.0050696.s005.tif (4.2M) GUID:?1E2613D2-29CF-4AE0-88CB-B793EC9E2064 Abstract Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 Vinflunine Tartrate antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions. Introduction Desmosomes are adhesive intercellular junctions which are anchored to the keratin intermediate filament cytoskeleton [1]C[5]. These robust intercellular junctions are prominent in tissues that experience substantial mechanical stress, such as the skin and heart. Desmosomes are composed Vinflunine Tartrate primarily of desmosomal cadherins, desmogleins and desmocollins, armadillo proteins such as plakoglobin and the plakophilins, and a plakin family member, desmoplakin. Together, these proteins couple calcium-dependent adhesive interactions mediated by the desmosomal cadherins to Vinflunine Tartrate the intermediate filament cytoskeleton, thereby mechanically coupling adjacent cells [1]C[3]. Although essential for tissue integrity, desmosomes are highly dynamic complexes that are often remodeled during various cellular processes, such as development and wound healing [1], [6]. Pemphigus is a family of potentially fatal autoimmune CDC42BPA blistering skin diseases caused by autoantibodies directed against desmosomal cadherins desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) [7]C[12]. The major forms of.