The B-lymphocyte Component of Inflammation

Abstract

B-lymphocytes have been implicated as key mediators of inflammation and autoimmune disease. However, the mechanisms involved have yet to be fully elucidated. What is known is summarised in Chapter 1. The overall aim of the work reported in this thesis was to identify and expand the current knowledge of genes and molecular pathways involving B-lymphocytes in inflammation using Rheumatoid arthritis as a model. Toll-like receptor 9 (TLR9) contributes to the innate immune receptor repertoire of B-lymphocytes. To date, five isoforms of human TLR9 have been reported (TLR9-A-E). In Chapter 2, gene expression analyses extend these observations to demonstrate tissue dependent expression of TLR9-A and TLR9-C transcript. Expression of the TLR9-A isoform was ~3-fold higher in inflamed synovia, than subcutaneous rheumatoid nodules. In contrast, nodules expressed ~4-fold more TLR9-C transcript. These results reflect differences in the inflammatory cells that dominate the lesion, particularly the macrophages prominent in nodules. Expression of TLR9-A was ~100-fold higher in B-lymphocytes than macrophages or T-lymphocytes, which predominantly expressed the TLR9-C transcript. Prior laboratory work has established the expression of CD21L and interleukin-17A (IL-17A) genes as biomarkers for classifying inflamed synovium. The biological phenomena underlying this classification are unknown. This is considered in Chapter 3. Using the CD21L/IL-17A definition and maximum radial cell count, it is shown that CD21L+/IL-17A+ synovia have larger aggregates containing B-lymphocytes, compared to those with expression of one or neither gene. Similarly, expression of TLR9-A, BAFF and RGS13 were significantly higher in CD21L+/IL-17A+ synovia. These results form the basis of a model, supported by published data, that incorporates the contribution of CD21L and IL-17A to the aggregation of B-lymphocytes in inflammatory tissue. The CD21L/IL-17A classification of synovia appears to reflect different organisation of the inflammatory cells in inflamed synovia. Chapter 4 describes the use of microarrays, to compare the gene expression profiles of CD21L+/IL-17A+ and CD21L-/IL-17A- synovia with the aim of identifying genes and molecular pathways unique to each of these two subtypes. CD21L-/IL-17A- synovia display genes associated with tissue remodelling and repair, including osteoprotegerin and fibroblast growth factor 18. In contrast, CD21L+/IL-17A+ synovia reveal a specific up-regulation of immune-related genes, including immunoglobulins and human leukocyte antigens. Included amongst these up-regulated genes were those encoding members of the signalling lymphocyte activation molecules (SLAM) family that are considered further in Chapter 5. Quantitation of expression levels show ~10-fold more SLAMF3 and ~5-fold more SLAMF7 expression in CD21L+/IL-17A+ synovia, compared to CD21L-/IL-17A- synovia. Previous reports demonstrate SLAMF3 in B- and T-lymphocyte interactions. Data presented herein suggests that SLAMF7 is involved in cellular interactions between DCs and T- or B-lymphocytes. Interestingly, subcutaneous rheumatoid nodules show ~100-fold more SLAMF9 expression than synovia. It is likely that SLAMF9 is associated with macrophages, which dominate nodule inflammation. Collectively, this study highlights the differential expression of genes that contribute to B-lymphocyte involvement in rheumatoid inflammation. In Chapter 6 it is concluded that combined CD21L and IL-17A gene expression defines a subset of synovia that are more highly inflamed. These synovia are characterised by greater organisation and gene expression involving B-lymphocytes. The implications for tissue destruction and patient treatment are discussed.