Unconjugated
TNF-α plays an important role in the pathogenesis of salivary inflammatory diseases. Salivary dysfunction, which leads to impaired saliva secretion, can be caused by TNF-α-induced disrupted epithelial barrier. However, the signaling mechanism involved in TNF-α-modulated tight junction barrier in salivary gland remains unclear. Here, we found that TNF-α reduced transepithelial resistance (TER) and increased FITC-dextran flux in a rat submandibular cell line SMG-C6. Claudin (Cln)-3 was selectively downregulated and disrupted by TNF-α, whereas Cln-1, Cln-4, and β-catenin were not affected. Overexpression of Cln-3 retained and Cln-3 knockdown abolished the TNF-α-induced alterations. Moreover, TNF-α increased extracellular signal-regulated kinase (ERK1/2) phosphorylation and the expression of transcriptional factor slug. ERK1/2 kinase inhibitor PD98059 abrogated TNF-α-induced increase in paracellular permeability, alterations of Cln-3, and elevation of slug. Overexpression of slug decreased and slug knockdown increased Cln-3 expression. In addition, slug bind to the E-box elements of Cln-3 promoter in TNF-α-treated cells, and this response was blocked by PD98059. Furthermore, TNF-α decreased Cln-3 expression and increased slug content in cultured human submandibular gland. Taken together, our data suggest that Cln-3 plays a vital role in TNF-α-modulated paracellular permeability in submandibular epithelium and ERK1/2/slug signaling axis is involved in alteration of Cln-3 redistribution and downregulation.
We report on a novel, high-dimensional method to detect autoantibodies that are complexed with their natural autoantigens. Specifically, autoantibody-autoantigen complexes in serum or plasma are directly incubated onto a high-density antibody microarray. Detection of the bound autoantibody-antigen complex is made via fluorescently labeled antihuman immunoglobulin G or other immunoglobulin isotype secondary antibodies and quantification in a microarray scanner. Uncomplexed antibodies do not interfere with this assay. The whole process is very rapid and applicable for high-throughput screening without the need for production of proteins or immunoglobulin purification from the samples. Using these methods, we found that plasma from healthy individuals contains hundreds of autoantibodies complexed with cellular proteins. Thus, this highly sensitive, multiplex method is capable of discovering new autoantibody-antigen or circulating immune complexes, many of which will likely be useful for disease detection and characterization.