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.

CXCL13 and CCL21 have been functionally implicated in lymphoid tissue organization both in the upstream phases of lymphoid tissue embryogenesis and in ectopic lymphoid neogenesis in transgenic mice. Here, we analyzed the relationship between CXCL13 and CCL21 production and lymphoid tissue organization in rheumatoid synovitis as a model of a naturally occurring ectopic lymphoneogenesis. Through systematic analysis of mRNA and protein expression, we defined the microanatomical relationship between CXCL13 and CCL21 in progressive aggregational and structural phases of synovial inflammatory infiltrate. We provide the first direct in situ evidence that production of CXCL13 and CCL21 (rather than simply protein binding) is associated with inflammatory lymphoid tissue formation and development with the demonstration, in organized aggregates, of a secondary lymphoid organ-like compartmentalization and vascular association. Notably, the presence of CXCL13 and CCL21 (protein and mRNA) was also demonstrated in non-organized clusters and minor aggregational stages, providing evidence that their induction can take place independently and possibly upstream of T-B compartmentalization, CD21 + follicular dendritic cell network differentiation and germinal center formation. Our data support the concept that, under inflammatory conditions, CXCL13 and CCL21 participate in lymphoid tissue microanatomical organization, attempting to recapitulate, in an aberrant lymphoid neogenetic process, their homeostatic and morphogenetic physiologic functions.

Objective. Ectopic lymphoneogenesis can occur in the salivary glands of Sjögren's syndrome (SS) patients and is associated with local antigen-driven B cell responses, autoantibody formation, and potential lymphomatous transformation. CXCL13 and CCL21 have been identified in salivary glands, but their role in ectopic lymphoneogenesis in SS remains unclear. This study aimed to evaluate the microanatomic association between CXCL13 and CCL21 expression and the acquisition of lymphoid features in periductal foci.Methods. Salivary glands from 37 SS patients and 9 chronic sialadenitis patients were analyzed by immunohistochemistry for T cell/B cell segregation, CD21؉ follicular dendritic cell networks, and peripheral lymph node addressin (PNAd)-positive high endothelial venules (HEVs) in relationship to the size of the aggregates and the expression of CXCL13 and CCL21 within infiltrating cells, epithelium, and endothelium.Results. Grade 1 aggregates (10-50 lymphocytes) demonstrated predominance of nonorganized CD3؉ cells, while grade 2 (>50 lymphocytes) and grade 3 (>50 with germinal centers) showed a progressive increase in CD20؉ B cells and T cell/B cell segregation. This higher degree of lymphoid organization was significantly related to an increased expression of CXCL13 within infiltrating cells and PNAd؉ HEV-associated CCL21-producing cells. Conversely, no association between lymphoid organization and lymphoid chemokine expression by epithelial cells was observed.Conclusion. The acquisition of lymphoid features by inflammatory foci in SS is critically associated with the enlargement of the inflammatory foci and with the expression of CXCL13 and CCL21 within the infiltrate, but is not associated with their expression by epithelial cells. These data strongly support an active participation of CXCL13 and CCL21 in regulating the progressive organization and maintenance of periductal foci.

Serum amyloid A (A-SAA), an acute-phase protein with cytokine-like properties, is expressed at sites of inflammation. This study investigated the effects of A-SAA on chemokine-regulated migration and angiogenesis using rheumatoid arthritis (RA) cells and whole-tissue explants in vitro, ex vivo, and in vivo. A-SAA levels were measured by real-time PCR and ELISA. IL-8 and MCP-1 expression was examined in RA synovial fibroblasts, human microvascular endothelial cells, and RA synovial explants by ELISA. Neutrophil transendothelial cell migration, cell adhesion, invasion, and migration were examined using transwell leukocyte/monocyte migration assays, invasion assays, and adhesion assays with or without anti–MCP-1/anti–IL-8. NF-κB was examined using a specific inhibitor and Western blotting. An RA synovial/SCID mouse chimera model was used to examine the effects of A-SAA on cell migration, proliferation, and angiogenesis in vivo. High expression of A-SAA was demonstrated in RA patients (p < 0.05). A-SAA induced chemokine expression in a time- and dose-dependent manner (p < 0.05). Blockade with anti-scavenger receptor class B member 1 and lipoxin A4 (A-SAA receptors) significantly reduced chemokine expression in RA synovial tissue explants (p < 0.05). A-SAA induced cell invasion, neutrophil–transendothelial cell migration, monocyte migration, and adhesion (all p < 0.05), effects that were blocked by anti–IL-8 or anti–MCP-1. A-SAA–induced chemokine expression was mediated through NF-κB in RA explants (p < 0.05). Finally, in the RA synovial/SCID mouse chimera model, we demonstrated for the first time in vivo that A-SAA directly induces monocyte migration from the murine circulation into RA synovial grafts, synovial cell proliferation, and angiogenesis (p < 0.05). A-SAA promotes cell migrational mechanisms and angiogenesis critical to RA pathogenesis.

Objective. The mechanisms by which monocyte/ macrophage cells migrate to the joint involve a series of integrated adhesion and signaling events in which chemokines and their receptors are strongly implicated. This study was undertaken to investigate the hypothesis that stromal cell-derived factor 1 (SDF-1), a CXC chemokine (CXCL12), plays a critical role in monocyte/ macrophage localization to synovium.Methods. SDF-1 and CXC receptor 4 (CXCR4) expression in rheumatoid arthritis (RA) and osteoarthritis synovium and graft SDF-1, tumor necrosis factor ␣ (TNF␣), and human and murine vascular markers were examined by immunohistochemistry and doubleimmunofluorescence. The functional capacity of SDF-1 to modulate monocyte migration into joints was investigated by examining the localization of promyelomonocytic U937 cells into synovial tissue transplanted into SCID mice. SDF-1, TNF␣, or saline was injected into graft sites and response determined by the number of fluorescently labeled U937 cells (injected intravenously) detected in grafts by ultraviolet microscopy.Results. SDF-1 and CXCR4 were highly expressed in CD68؉ cells in the RA synovium. SDF-1 induced U937 cell migration in vitro and in vivo in a dosedependent manner and, in vivo, SDF-1 was more effective than TNF␣. In contrast to TNF␣, SDF-1 did not induce intracellular adhesion molecule 1 in transplant microvasculature. Furthermore, intragraft injection of SDF-1 did not up-regulate TNF␣, or vice versa.Conclusion. This study demonstrates, for the first time, that SDF-1 is functional in vivo when injected into synovial grafts. In addition, SDF-1 is more potent than TNF␣, and its mechanisms of action appear to be autonomous. Therefore, SDF-1 may be an important TNF-independent molecule involved in the migration to and retention of inflammatory effector cells in the joint.