Mechanisms of Innate and Adaptive Immune Responsiveness to Bacterial Infection in Cornea

Abstract

Purpose. The dysfunctional tear syndrome of Sjogren’s syndrome (SjS) is associated with focal CD4+ cell infiltration of the lacrimal glands and serum autoantibodies to intracellular proteins. To learn how lacrimal acinar cells (AC) expose or present autoantigen epitopes, we wish to identify autoantigens in amenable experimental models. Methods. Subcellular fractions from rat lacrimal glands were analyzed by SDS-PAGE. Western blots were probed with sera from NOD mice with diagnosed SjS-like disease, and IgG1 heavy chain was detected with isotype specific secondary antibody. Labeled bands were analyzed using matrix-associated laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry. Tryptic peptides were identified by Mascot Matrix Science search software. Compartmental distributions of candidate B cell autoantigens were determined by Western blotting of isolated membrane fractions from rabbit AC cultured with and without chronic carbachol (CCh) stimulation. Results. Subcellular fractionation analysis indicated that the candidate autoantigens were primarily associated with the membrane phase; most were primarily in compartments of the biosynthetic and lysosomal pathways. Some also were in secretory vesicle-related compartments of control cells but re-distributed to the endosomes after chronic CCh stimulation. The proteomic analysis identified Grp 78, rab3D, and ribosomal protein L7 as candidate autoantigens. Western blotting with monoclonal antibodies confirmed the presence and compartmental distributions of Grp 78 and rab3D. Protein sequence analysis with TEPITOPE confirmed that an identified B cell epitope at the N-terminal end of L7 also is a likely T cell epitope. Conclusion Grp78 and L7 are known to be antigens in other autoimmune diseases but have not previously been known to be associated with SjS. Rab3D has not previously been recognized as a disease-associated autoantigen. These and other AC autoantigens are present in intracellular compartments where they may be subject to aberrant proteolytic processing that we theorize generates otherwise cryptic epitopes. Commercial Relationship(s): None; Support: EY13720, EY05801, DK48522