Date Published: January 13, 2009
Publisher: Public Library of Science
Author(s): Frances Humby, Michele Bombardieri, Antonio Manzo, Stephen Kelly, Mark C Blades, Bruce Kirkham, Jo Spencer, Costantino Pitzalis, Tom Huizinga
Abstract: BackgroundFollicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.Methods and FindingsUsing immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.ConclusionsOur demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.
Partial Text: Rheumatoid arthritis (RA) is a chronic inflammatory erosive polyarthritis with an associated significant morbidity and mortality . One of the hallmarks of the disease is the presence of circulating autoantibodies, such as rheumatoid factor (RF)  and anti-citrullinated protein/peptide antibodies (ACPA) [3–8], that have prompted the notion of an autoimmune pathogenesis. The presence of such antibodies, particularly ACPA, has been shown to be a poor prognostic factor linked with a higher erosive burden [9,10], while ACPA titres have been reported to fall in line with clinical response to biological therapies . Additional support for a pathogenic role of ACPA antibodies comes from recent work in a mouse model of RA that indicated a direct role for ACPA antibodies in tissue destruction .
In this report we provide strong evidence that lymphoid structures in the target organs of a human autoimmune disease are functional by demonstrating that within the synovial membrane of patients with RA, ectopic lymphoid aggregates characterized by FDC networks invariably express AID and are surrounded by ACPA producing plasma cells. In addition, using the human RA-SCID mouse chimera model, we demonstrate that transplanted RA synovial grafts containing ectopic lymphoid structures support B cell survival and proliferation, maintain AID expression, continuously promote ongoing CSR and produce human IgG ACPA. This phenomenon, characterized by the sustained high expression levels of genes regulating ectopic lymphoneogenesis and in the absence of new influx of immune cells, indicates that the establishment of follicular structures in the synovium can lead to self-perpetuating autoimmunity. The self-sustained survival of B cell niches, within rheumatoid synovium, may also have a critical impact on the capacity of B cell–depleting biologics to modulate chronic inflammation and autoimmunity.