ABSTRACT
In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG-/-) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG-/- cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG-/- keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.
Subject(s)
Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Drosophila Proteins , Pemphigus/immunology , Pemphigus/metabolism , Trans-Activators , Animals , Armadillo Domain Proteins , Autoantibodies/pharmacology , Cell Adhesion/immunology , Cell Differentiation/physiology , Cell Membrane/immunology , Cell Membrane/metabolism , Cells, Cultured , Desmogleins , Desmoplakins , Desmosomes/immunology , Desmosomes/metabolism , Fetus/cytology , Immunoglobulin G/pharmacology , Insect Proteins , Keratinocytes/cytology , Keratinocytes/immunology , Keratinocytes/metabolism , Keratins/metabolism , Mice , Mice, Knockout , Pemphigus/pathology , Protein Binding/immunology , Signal Transduction/immunology , gamma CateninABSTRACT
The mechanisms leading to mucus accumulation in equine inflammatory airway disease (IAD) and recurrent airway obstruction (RAO) are unclear. In airways of human patients with asthma and/or chronic obstructive pulmonary disease as well as in animal models of these diseases, associations of mucus hyperproduction with increased calcium-activated chloride channel 1 (CLCA1), epidermal growth factor receptor (EGFR), mucin 5AC (MUC5AC), B-cell lymphoma 2 (Bcl-2), interleukin (IL)-13 and interferon (IFN)-gamma expression have been reported. We hypothesized that increased mucus accumulation in RAO and IAD are associated with alterations in inflammatory cytokine (IL-13 and IFN-gamma) and epithelial gene (CLCA1, EGFR, Bcl-2 and MUC5AC) profiles. Therefore, mRNA expression of these genes in cell pellets extracted from bronchoalveolar lavage fluid (BALF) and bronchial epithelial brushing (BEB) was compared between 11 clinically healthy (Control group), 7 IAD- and 12 RAO-affected horses by reverse transcription polymerase chain reaction. We also performed arterial blood gas analysis, endoscopic scoring of mucus accumulation in the trachea and cytology of tracheo-bronchial secretions (TBS) and of BALF. Tracheal mucus accumulation, along with TBS and BALF neutrophils were significantly increased and arterial pO(2) was decreased in RAO-affected horses compared to the Control group. IL-13 in BALF samples was significantly lower in the RAO group. None of the other genes' relative mRNA levels displayed significant differences between groups. Our findings suggest that mucus production in equine RAO is induced by pathways independent of IL-13, CLCA1, EGFR, MUC5AC and Bcl-2 up-regulation.