Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Molecular and Cellular Evolution of Functional Tertiary Lymphoid Structures in Salivary Glands of NOD Mice.
Astorri3, Elisa, Bombardieri3, Michele, Corsiero3, Elisa, Barone1, Francesca, Proctor2, Gordon, Pitzalis3, Costantino
Tertiary Lymphoid Structures (TLSs) formation is a common feature of chronic inflammatory diseases including Sjogren's syndrome (SS). We recently showed that these ectopic structures acquire functional properties typical of secondary lymphoid organs and are capable of supporting autoreactive B cell activation and autoantibody production as demonstrated by expression of activation-induced cytidine deaminase (AID) and Ig class switching. Dissecting TLSs dynamics in humans is technically and ethically challenging. Thus, we used the NOD mouse model, a spontaneous model of autoimmune sialoadenitis, to characterize the cellular and molecular basis of autoreactive B cell activation and evolution of functional Ectopic Lymphoid Structures (ELS) in the chronically inflamed NOD salivary glands.
Submandibolar (SUBM) glands from 110 female NOD mice from 4 to 35 weeks of age were collected. Paired snap-frozen, OCT-embedded samples were analysed by immunohistochemistry (IHC) for T and B lymphocytes (CD3/CD20) in order to evaluate immune cell infiltration and the degree of B/T cell segregation. ELS were detected by staining for FDC-M1 (follicular dendritic cell networks), GL7 (germinal centre B cells) and AID (marker for ELS functionality). Characterization of B cell subsets within the infiltrates was carried out by immunostaining and by FACS analysis with CD19, CD21, CD23, B220, IgD, IgM, CD1d and CXCR5 antibodies. Quantitative TaqMan real-time PCR was performed to investigate the mRNA expression of ELS related genes. The same analysis was performed in sex/age matched Balb/c mice as controls.
NOD infiltrates in SUBM glands displayed progressive features of ELS from week 8, with 75% of mice developing B/T cell segregation, FDC networks and GL7+ ectopic germinal centers (GCs) from week 20. Formation of ectopic GCs was always associated with B/T segregation. Evolution of TLSs was closely associated with mRNA upregulation of genes regulating ectopic lymphoid tissue organization and function such as lymphoid chemokines CXCL13/CCL19 and their specific receptors CXCR5/CCR7, lymphotoxins and B cell survival factors BAFF and APRIL. In agreement with CXCL13/CXCR5 mRNA expression, B cells infiltrating NOD SUBM glands display strong CXCR5 expression and were mostly characterised by a follicular phenotype (B220+/IgD+/IgMlow/CD23+/CD21low) as demonstrated by both IHC and FACS analysis on isolated cells. Finally, functionality of ELS was demonstrated by expression of AID mRNA and protein within FDC networks, which paralleled the detection of circulating SS-related autoantibodies.
This work provided the first in depth characterization of the cellular and molecular mechanisms underlying the evolution of functional TLSs within SUBM infiltrates of NOD mice. These data strongly support the hypothesis that B cells can be activated within TLSs in the target organ and promote an in situ autoantibody response. Overall, these data support the critical importance of ELS formation in chronic autoimmune inflammation and identified NOD mice as a suitable model to test therapeutic strategies aimed at modulating B cell functionality.
To cite this abstract, please use the following information:
Astorri, Elisa, Bombardieri, Michele, Corsiero, Elisa, Barone, Francesca, Proctor, Gordon, Pitzalis, Costantino; Molecular and Cellular Evolution of Functional Tertiary Lymphoid Structures in Salivary Glands of NOD Mice. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1906