Insights into the pathogenesis of systemic sclerosis based on the gene expression profile of progenitor‐derived endothelial cells

Objective

To determine the gene expression profile of endothelial cells derived from the endothelial progenitor cells (EPCs) of patients with systemic sclerosis (SSc).

Methods

Microarray experiments were performed on Affymetrix GeneChip Human Exon 1.0 ST Arrays in unstimulated and hypoxia‐stimulated EPC‐derived cells from patients with SSc and control subjects. Followup of the raised hypotheses was performed ex vivo by immunohistochemical analysis of skin tissue.

Results

Signals from 92 probe sets and 188 probe sets were different in unstimulated and hypoxia‐stimulated cells, respectively, from patients with SSc compared with controls. Within the largest groups of genes related to cell–cell interaction and vascular remodeling, down‐regulation of tumor necrosis factor ligand superfamily member 10 (TNFSF10) and homeobox A9 (HOX‐A9) was confirmed by real‐time polymerase chain reaction and Western blots in EPC‐derived cells and by immunohistochemistry in SSc skin tissue. Signals from 221 and 307 probe sets were different in unstimulated and hypoxia‐stimulated cells, respectively, from patients with diffuse cutaneous SSc compared with patients with limited cutaneous SSc. Within the largest group of genes related to the inflammatory response, differential expression of TNFα‐induced protein 3 and prostaglandin‐endoperoxide synthase 2 was observed in EPC‐derived cells and skin tissue from patients with SSc.

Conclusion

Our data revealed important gene expression changes in EPC‐derived endothelial cells from patients with SSc, characterized by a proadhesive, proinflammatory, and activated phenotype. Differential expression in lesional SSc skin tissue of new targets, such as TNF family members and HOX‐A9, may contribute to the pathogenesis of SSc and deserves more in‐depth exploration.

     

Identification of the epidermal growth factor–TM7 receptor EMR2 and its ligand dermatan sulfate in rheumatoid synovial tissue

Objective

EMR2 and CD97 are closely related members of the epidermal growth factor (EGF)–TM7 family of adhesion class 7‐span transmembrane (TM7) receptors. Chondroitin sulfates (CS) have recently been identified as ligands for EMR2 and CD97. CS have been implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the expression of EMR2 and the distribution of EMR2 and CD97 ligands within RA synovial tissue (ST).

Methods

ST samples were obtained by arthroscopy from 19 patients with RA, 13 patients with inflammatory osteoarthritis (OA), and 13 patients with reactive arthritis (ReA). Immunohistochemistry was performed with a monoclonal antibody against EMR2, and stained STs were analyzed by digital image analysis. Coexpression of EMR2 with cell lineage– and activation‐specific markers was determined by double immunofluorescence microscopy. To evaluate the expression of EMR2 and CD97 ligands in RA synovium, binding assays were performed using EMR2‐ and CD97‐specific multivalent fluorescent probes.

Results

EMR2 expression in the synovial sublining was found to be significantly higher in RA patients compared with OA and ReA control patients. Most EMR2+ cells were macrophages and dendritic cells expressing costimulatory molecules and tumor necrosis factor α. Dermatan sulfate was shown to be the ligand of the largest isoforms of EMR2 and CD97 in rheumatoid synovium. In addition, the smaller isoforms of CD97, but not those of EMR2, bound CD55 on fibroblast‐like synoviocytes.

Conclusion

The EGF‐TM7 receptors EMR2 and CD97 are abundantly expressed on myeloid cells in ST of RA patients where their cognate ligands dermatan sulfate and CD55 are detected. These results suggest that these interactions may facilitate the retention of activated macrophages in the synovium.

     

ADAM‐10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis

Objective

To examine the expression of ADAM‐10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis.

Methods

ADAM‐10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM‐10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM‐10 small interfering RNA (siRNA). To determine whether ADAM‐10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts.

Results

Endothelial cells and lining cells within RA ST expressed high levels of ADAM‐10 compared with cells within osteoarthritis ST and normal ST. ADAM‐10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM‐10 siRNA–treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA–treated HMVECs. In addition, ADAM‐10 siRNA–treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA–treated HMVECs.

Conclusion

These data show that ADAM‐10 is overexpressed in RA and suggest that ADAM‐10 may play a role in RA angiogenesis. ADAM‐10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.

     

Cadherin 11 promotes invasive behavior of fibroblast‐like synoviocytes

Objective

To define the expression pattern of cadherin 11 in the destructive pannus tissue of patients with rheumatoid arthritis, and to determine whether cadherin 11 expression in fibroblast‐like synoviocytes controls their invasive capacity.

Methods

Cadherin 11 expression in rheumatoid synovial tissue was evaluated using immunohistochemistry. To examine the role of cadherin 11 in regulating the invasive behavior of fibroblast‐like synoviocytes, we generated L cell clones expressing wild‐type cadherin 11, mutant cadherin 11, and empty vector–transfected controls. The invasive capacity of L cell transfectants and cultured fibroblast‐like synoviocytes treated with a blocking cadherin 11–Fc fusion protein or control immunoglobulin was determined in Matrigel invasion assays.

Results

Immunohistochemical analysis revealed that cadherin 11 is abundantly expressed in cells at the cartilage–pannus junction in rheumatoid synovitis. Assays to determine invasion demonstrated a 2‐fold increased invasive capacity of cadherin 11–transfected L cells compared with L cells transfected with E‐cadherin or control vector. The invasive behavior of L cells stably transfected with a cadherin 11 construct that lacked the juxtamembrane cytoplasmic domain was diminished to the level of vector control L cells. Furthermore, treatment with the cadherin 11–Fc fusion protein diminished the invasive capacity of fibroblast‐like synoviocytes.

Conclusion

The results of these in vitro studies implicate a role for cadherin 11 in promoting cell invasion and contribute insight into the invasive nature of fibroblast‐like synoviocytes in chronic synovitis and rheumatoid arthritis.

     

Regulation of angiogenesis by the C‐X‐C chemokines interleukin‐8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint

Objective

Angiogenesis, the growth of new blood vessels, is vital to the ingress of inflammatory leukocytes in rheumatoid arthritis (RA) synovial tissue and to the growth and proliferation of RA pannus. The factors that mediate the growth of new blood vessels have not been completely defined. This study examined the ability of Glu‐Leu‐Arg (ELR)–containing chemokines to induce angiogenesis in the RA joint.

Methods

To reflect angiogenic activity in vivo, we selected a model using whole human synovial tissue rather than isolated cells. Tissues were examined by immunohistochemistry and enzyme‐linked immunosorbent assay, and tissue homogenates were immunoneutralized and assayed for their ability to induce endothelial cell chemotaxis and rat corneal neovascularization.

Results

Cells expressing interleukin‐8 (IL‐8) and epithelial neutrophil activating peptide 78 (ENA‐78) were located in proximity to factor VIII–related antigen–immunopositive endothelial cells. RA homogenates produced more IL‐8 and ENA‐78 compared with normal synovial tissue homogenates. Moreover, homogenates from RA synovial tissue produced significantly more chemotactic activity for endothelial cells in vitro and angiogenic activity in the rat cornea in vivo than did normal synovial tissue homogenates. The effects of IL‐8 and ENA‐78 accounted for a significant proportion of the chemotactic activity of endothelial cells and angiogenic activity found in RA synovial tissue homogenates.

Conclusion

These results indicate that the ELR‐containing chemokines IL‐8 and ENA‐78 are important contributors to the angiogenic activity found in the inflamed RA joint. It is possible that efforts aimed at down‐regulating these chemokines offer a novel targeted therapy for the treatment of RA.

     

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

Objective

A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen‐degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1‐MMP), in synovial pannus invasiveness.

Methods

The expression and localization of MT1‐MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1‐MMP was analyzed by 3‐dimensional (3‐D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP‐1), TIMP‐2, or GM6001. The effect of adenoviral expression of a dominant‐negative MT1‐MMP construct lacking a catalytic domain was also examined.

Results

MT1‐MMP was highly expressed at the pannus–cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3‐D collagen matrix in an MT1‐MMP–dependent manner. Invasion was blocked by TIMP‐2 and GM6001 but not by TIMP‐1. Invasion was also inhibited by the overexpression of a dominant‐negative MT1‐MMP, which inhibits collagenolytic activity and proMMP‐2 activation by MT1‐MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1‐MMP–dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.

Conclusion

MT1‐MMP serves as an essential collagen‐degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1‐MMP–dependent invasion may represent a novel therapeutic strategy for RA.

     

Identification of immunodominant CD4+ T cell epitopes in patients with Yersinia‐induced reactive arthritis by cytometric cytokine secretion assay

Objective

In reactive arthritis (ReA), a bacteria‐specific T cell response to the triggering microbe is detected in synovial fluid. So far, direct characterization of bacteria‐specific T cells and identification of the immunodominant fine specificities remain difficult due to the lack of appropriate techniques. The aim of the present study was to directly determine the fine specificity of CD4+ T cells specific to ReA‐associated bacteria‐derived protein.

Methods

In 2 patients with Yersinia‐induced ReA, live Yersinia Hsp60–specific CD4+ T cells were directly isolated from synovial fluid after stimulation with Yersinia‐derived protein Hsp60 using a cytometric cytokine secretion assay. Generated short‐term T cell lines were then tested in vitro for their peptide epitope specificity. Also, direct cross‐reactivity of one line with Chlamydia‐ and human‐derived Hsp60 was assessed.

Results

Generated short‐term CD4+ T cell lines were highly antigen‐specific and revealed single immunodominant peptide epitopes that were confirmed by direct testing with single peptides in both peripheral blood and synovial fluid cells. Yersinia Hsp60–specific T cells of one patient cross‐reacted directly with human Hsp60.

Conclusion

Our results demonstrate the feasibility of direct assessment of live, potentially pathogenic, antigen‐specific interferon‐γ+ CD4+ T cells taken from inflammatory lesions of patients with rheumatic diseases such as ReA. This might have implications not only regarding pathogenesis, but also in the design of new immunotherapies.

     

Interleukin‐1α expression in capillaries and major histocompatibility complex class I expression in type II muscle fibers from polymyositis and dermatomyositis patients: Important pathogenic features independent of inflammatory cell clusters in muscle tissue

Objective

To address the hypothesis that endothelial cells and/or muscle fibers are primary targets in the disease process by analysis of muscle tissue from patients with polymyositis (PM) and dermatomyositis (DM).

Methods

We included patients with laboratory signs and clinical symptoms typical of myositis, but without detectable infiltration of clusters of inflammatory cells in their muscle biopsy samples. An immunohistochemical technique was applied to identify CD3, CD68, lymphocyte function–associated antigen 1α, CD11b, very late activation antigen 4, endothelium 4, interleukin‐1α (IL‐1α), intercellular adhesion molecule 1, vascular cell adhesion molecule 1, IgG, IgM, IgA, and HLA–A/B/C in muscle tissue. Fiber type was defined by ATPase staining.

Results

IL‐1α expression was detected in endothelial cells of capillaries to a greater extent in patients than in controls, and class I major histocompatibility complex (MHC) expression was significantly increased in muscle fibers. We also observed that class I MHC expression was mainly confined to type II muscle fibers.

Conclusion

Our findings imply that defined molecular changes of blood vessels and muscle fibers are both independent of adjacent inflammatory infiltrates and could thus be primary events in the development of myositis. Moreover, both IL‐1α and class I MHC molecules might be important for the development of clinical symptoms in PM and DM patients.

     

Protective role of systemic furin in immune response–induced arthritis

Objective

Rheumatoid arthritis (RA) is an autoimmune joint disease associated with chronic inflammation of the synovium that causes profound damage of joints. Inflammation results in part from the influx of immune cells secreting inflammatory cytokines and the reduction in the number of Treg cells. We undertook this study to assess the effect of furin, a proteinase implicated in the proteolytic activity of various precursor proteins and involved in the regulation of both proteinase maturation and immune cells, in an experimental model of RA.

Methods

The effect of furin and its inhibitor α1‐PDX was tested in mice with collagen‐induced arthritis (CIA). Joints were processed for histology and protein expression. Levels of cytokines were measured in joint tissue, and Treg cell numbers were measured in spleens.

Results

Furin expression and activity were high in the synovial pannus in RA patients and mice with CIA. Systemic administration of furin prevented increases in the arthritis score, joint destruction, and bone loss, in contrast to systemic administration of the furin inhibitor α1‐PDX, which enhanced these parameters. By preventing the development of synovial pannus, furin reduced the expression of metalloproteinases in the joints. In contrast, α1‐PDX enhanced synovial proliferation and the expression and activity of matrix metalloproteinases. Furthermore, furin reversed the local Th1/Th2 balance and restored the number of Treg cells in the spleen, indicating mediation by immune cells.

Conclusion

These findings show the protective role of exogenous furin against RA, mediated by an immune response. The data suggest the potential therapeutic use of furin or its derivatives in autoimmune diseases including RA.

     

Interleukin‐15 inhibits sodium nitroprusside–induced apoptosis of synovial fibroblasts and vascular endothelial cells

Objective

One of the pathologic hallmarks of juvenile rheumatoid arthritis (JRA) is a tumor‐like expansion of inflamed synovial tissue, or pannus, which causes much of the joint damage in this disease. The expansion of pannus is supported by extensive formation of new blood vessels. We have previously shown that revascularization of minced JRA synovial tissues engrafted into SCID mice correlated with the intensity of inflammatory activity in the tissues and with interleukin‐15 (IL‐15) expression. Since synovial vascular endothelial cells (VECs) expressed IL‐15 receptors, the present study was undertaken to investigate the hypothesis that IL‐15 might play a role in neovascularization of the pannus.

Methods

To evaluate IL‐15 for possible angiogenic activity, we assessed the ability of recombinant human IL‐15 (rHuIL‐15) to induce VEC growth directly and to stimulate synovial cells to produce endothelial growth factors. Since IL‐15 had been shown to inhibit apoptosis of certain immune cells, we were also interested in whether it might have similar effects on VECs. Apoptosis was induced by addition of sodium nitroprusside (SNP) at 1–2 mM to >80% confluent primary VECs, and numbers of apoptotic cells were determined by annexin V assay.

Results

Addition of rHuIL‐15 at 10–100 ng/ml to primary synovial fibroblast cultures failed to up‐regulate expression of vascular endothelial growth factor and angiopoietin 1 by these cells. Although rHuIL‐15 failed to induce a mitogenic response of VECs, it promoted survival of these cells on Matrigel. Preincubation of VECs with rHuIL‐15 at 50 ng/ml significantly reduced the proportion of VECs undergoing apoptosis.

Conclusion

IL‐15 promotes survival of VECs on Matrigel and inhibits SNP‐induced apoptosis of endothelial cells. We hypothesize that this mechanism may be relevant to the stabilization of newly formed vascular structures in JRA synovium.

     

Differential survival of leukocyte subsets mediated by synovial,bone marrow,and skin fibroblasts: Site‐specific versus activation‐dependent survival of T cells and neutrophils

Objective

Synovial fibroblasts share a number of phenotype markers with fibroblasts derived from bone marrow. In this study we investigated the role of matched fibroblasts obtained from 3 different sources (bone marrow, synovium, and skin) to test the hypothesis that synovial fibroblasts share similarities with bone marrow–derived fibroblasts in terms of their ability to support survival of T cells and neutrophils.

Methods

Matched synovial, bone marrow, and skin fibroblasts were established from 8 different patients with rheumatoid arthritis who were undergoing knee or hip surgery. Resting or activated fibroblasts were cocultured with either CD4 T cells or neutrophils, and the degree of leukocyte survival, apoptosis, and proliferation were measured.

Results

Fibroblasts derived from all 3 sites supported increased survival of CD4 T cells, mediated principally by interferon‐β. However, synovial and bone marrow fibroblasts shared an enhanced site‐specific ability to maintain CD4 T cell survival in the absence of proliferation, an effect that was independent of fibroblast activation or proliferation but required direct T cell–fibroblast cell contact. In contrast, fibroblast‐mediated neutrophil survival was less efficient, being independent of the site of origin of the fibroblast but dependent on prior fibroblast activation, and mediated solely by soluble factors, principally granulocyte–macrophage colony‐stimulating factor.

Conclusion

These results suggest an important functional role for fibroblasts in the differential accumulation of leukocyte subsets in a variety of tissue microenvironments. The findings also provide a potential explanation for site‐specific differences in the pattern of T cell and neutrophil accumulation observed in chronic inflammatory diseases.

     

Rheumatoid fibroblast‐like synoviocytes overexpress the chemokine stromal cell–derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within synovial tissue

Objective

A characteristic feature of the inflammatory infiltrate in rheumatoid arthritis is the segregation of CD4 and CD8 T lymphocyte subsets into distinct microdomains within the inflamed synovium. The aim of this study was to test the hypothesis that chemokines in general and stromal cell–derived factor 1 (SDF‐1; CXCL12) in particular are responsible for generating this distinctive microcompartmentalization.

Methods

We examined how synovial CD4/CD8 T cell subsets interacted in coculture assays with fibroblasts derived from chronic inflammatory synovial lesions and normal synovial tissue as well as from fetal lung and adult skin. We used the ability of T cells to migrate beneath fibroblasts (a process called pseudoemperipolesis) as an in vitro marker of T cell accumulation within synovial tissue.

Results

Rheumatoid fibroblast‐like synoviocytes (FLS) displayed a unique ability to support high levels of CD4 and CD8 T cell pseudoemperipolesis. Nonrheumatoid FLS as well as fetal lung fibroblasts supported low levels of pseudoemperipolesis, while skin‐derived fibroblasts were unable to do so. CD8 T cells migrated under fibroblasts more efficiently and at a higher velocity than CD4 T cells, a feature that was intrinsic to CD8 T cells. Rheumatoid fibroblasts constitutively produced high levels of SDF‐1 (CXCL12), which was functionally important, since blocking studies showed reductions in T cell pseudoemperipolesis to levels seen in nonrheumatoid FLS. Rheumatoid fibroblasts also constitutively produced high levels of vascular cell adhesion molecule 1 (VCAM‐1; CD106), but this did not contribute to T cell pseudoemperipolesis, unlike the case for B cells, which require SDF‐1 (CXCL12)–CXCR4 and CD49d–VCAM‐1 (CD106) interactions. Importantly, only combinations of rheumatoid FLS and rheumatoid‐derived synovial fluid T cells supported pseudoemperipolesis when examined ex vivo, confirming the in vivo relevance of these findings.

Conclusion

These studies demonstrate that features intrinsic to both fibroblasts (the production of SDF‐1) and CD8/CD4 T cells (the expression of CXCR4) are responsible for the characteristic pattern of T lymphocyte accumulation seen in the rheumatoid synovium. These findings suggest that the SDF‐1/CXCR4 ligand/receptor pair is likely to play an important functional role in T lymphocyte accumulation and positioning within the rheumatoid synovium.

     

Chemokine receptor expression in rat adjuvant‐induced arthritis

Objective

Chemokine receptors mediate leukocyte migration into inflamed rheumatoid arthritis (RA) synovial tissue (ST). Knowledge of their distribution is crucial for understanding the evolution of the inflammatory process. In this study, we used rat adjuvant‐induced arthritis (AIA), a model for RA, to define the temporospatial expression of chemokine receptors.

Methods

ST from rats with AIA was immunostained, the percentage of cells expressing each receptor was determined, and findings were correlated with levels of inflammation. Chemokine receptor expression was evaluated on rat macrophages in vitro.

Results

CCR1, a receptor for macrophage inflammatory protein 1α (MIP‐1α)/CCL3 and RANTES/CCL5, exhibited high constitutive expression on macrophages in AIA. CCR5, binding MIP‐1α/CCL3 and RANTES/CCL5, was up‐regulated on ST macrophages during the course of AIA, correlating with macrophage expression of CCR2, a receptor for monocyte chemoattractant protein 1/CCL2. Endothelial cell (EC) CCR2 was down‐regulated as arthritis progressed, inversely correlating with inflammation. CCR3, another RANTES/CCL5 receptor, was constitutively high on macrophages in vivo and in vitro, with down‐regulation during AIA. CXCR4, a receptor for stromal cell–derived factor 1/CXCL12), was prominently up‐regulated on ECs, preceding the peak of inflammation.

Conclusion

These findings show that 1) constitutive expression of CCR1 on macrophages remains high during AIA; 2) CCR2 and CCR3 may play a role in initial recruitment of leukocytes to ST in AIA; 3) macrophage expression of CCR2 and CCR5 may be important for sustaining inflammatory changes; and 4) EC CXCR4 may be a harbinger of inflammatory changes. Our results may help guide chemokine receptor blockade–targeting treatment strategies in inflammatory arthritis.

     

Interleukin‐13–producing CD8+ T cells mediate dermal fibrosis in patients with systemic sclerosis

Objective

Fibrosis is a major contributor to morbidity and mortality in systemic sclerosis (SSc). T cells are the predominant inflammatory infiltrate in affected tissue and are thought to produce cytokines that drive the synthesis of extracellular matrix (ECM) proteins by fibroblasts, resulting in excessive fibrosis. We have previously shown that aberrant interleukin‐13 (IL‐13) production by peripheral blood effector CD8+ T cells from SSc patients correlates with the extent of skin fibrosis. The present study was undertaken to investigate the role of IL‐13 production by CD8+ T cells in dermal fibrosis, an early and specific manifestation of SSc.

Methods

ECM protein production by normal dermal fibroblasts cocultured with SSc CD8+ T cell supernatants was determined by quantitative polymerase chain reaction and Western blotting. Skin‐homing receptor expression and IL‐13 production by CD8+ T cells in the peripheral blood of SSc patients were measured by flow cytometry. IL‐13+ and CD8+ cells in sclerotic skin were identified by immunohistochemistry.

Results

IL‐13–producing circulating CD8+ T cells from patients with SSc expressed skin‐homing receptors and induced a profibrotic phenotype in normal dermal fibroblasts, which was inhibited by an anti–IL‐13 antibody. High numbers of CD8+ T cells and IL‐13+ cells were found in the skin lesions of SSc patients, particularly during the early inflammatory phase of the disease.

Conclusion

These findings show that IL‐13–producing CD8+ T cells are directly involved in modulating dermal fibrosis in SSc. The demonstration that CD8+ T cells homing to the skin early in the course of SSc are associated with accumulation of IL‐13 is an important mechanistic contribution to the understanding of the pathogenesis of dermal fibrosis in SSc and may represent a potential target for therapeutic intervention.

     

Activated memory B cells may function as antigen‐presenting cells in the joints of children with juvenile idiopathic arthritis

Objective

B cells impact the perpetuation of chronic inflammatory or autoimmune diseases in multiple ways. A role of B cells as antigen‐presenting cells (APCs) in the pathogenesis of chronic arthritis in humans has been suggested; however, as of yet the presence of such B cells at the site of inflammation has not been demonstrated. This study was undertaken to investigate whether synovial B cells in patients with juvenile idiopathic arthritis (JIA) might display features of APCs.

Methods

The frequency, phenotype, and immunoglobulin repertoire of synovial B cells were studied by flow cytometry and single‐cell polymerase chain reaction (PCR). Cytokine expression by B cells was analyzed by real‐time PCR, and interaction between B cells and T cells was investigated in a mixed lymphocyte culture.

Results

CD27+IgD− and CD27−IgD− B cells accumulated in the joints of JIA patients and displayed an activated phenotype. Both B cell subsets expressed hypermutated and class‐switched immunoglobulins, indicators of memory B cells. The accumulating memory B cells expressed the costimulatory molecules CD80/CD86 and showed a higher capacity to activate allogeneic T cells and prime a Th1 phenotype than their peripheral blood counterparts.

Conclusion

Activated immunoglobulin class–switched CD27− and CD27+ memory B cells, indicating a phenotype of APCs with expression of costimulatory molecules, accumulate in the joints of patients with JIA and might be involved in the amplification of pathogenic T cell activation. These findings provide evidence that B cells play an antibody‐independent immunopathologic role in human chronic inflammatory arthritis of childhood.

     

Expression of hypoxia‐inducible factor 1α by macrophages in the rheumatoid synovium: Implications for targeting of therapeutic genes to the inflamed joint

Objective

To determine if the rheumatoid synovium is a suitable target for hypoxia‐regulated gene therapy.

Methods

Sequential sections of wax‐embedded synovial membrane samples were obtained from 10 patients with rheumatoid arthritis (RA), 10 with primary osteoarthritis (OA), and from 6 healthy controls. Membrane sections from each patient were immunostained for hypoxia‐inducible factor 1α (HIF‐1α) and CD68 (a pan–macrophage marker).

Results

HIF‐1α was expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone. There was markedly lower expression of HIF‐1α in OA synovia, and it was absent from all of the healthy synovia.

Conclusion

These observations indicate that macrophages transduced with a therapeutic gene under the control of a hypoxia‐inducible promoter could be administered to RA patients systemically. Migration of these cells to synovial tissue would result in the transgene being switched on in diseased joints but not in healthy tissues.

     

MRL/Mp CD4+,CD25− T cells show reduced sensitivity to suppression by CD4+,CD25+ regulatory T cells in vitro: A novel defect of T cell regulation in systemic lupus erythematosus

Objective

To investigate the hypothesis that loss of suppression mediated by peripheral CD4+,CD25+ regulatory T cells is a hallmark of systemic lupus erythematosus (SLE).

Methods

Mice of the MRL/Mp strain were studied as a polygenic model of SLE. Following immunomagnetic selection, peripheral lymphoid CD25+ and CD25− CD4+ T cells were cultured independently or together in the presence of anti‐CD3/CD28 monoclonal antibody–coated beads. Proliferation was assessed by measuring the incorporation of tritiated thymidine.

Results

While MRL/Mp CD4+,CD25+ regulatory T cells showed only subtle abnormalities of regulatory function in vitro, syngeneic CD4+,CD25− T cells showed significantly reduced sensitivity to suppression, as determined by crossover experiments in which MRL/Mp CD4+,CD25− T cells were cultured with H‐2–matched CBA/Ca CD4+,CD25+ regulatory T cells in the presence of a polyclonal stimulus.

Conclusion

Our findings highlight a novel defect of peripheral tolerance in SLE. Identification of this defect could open new opportunities for therapeutic intervention.

     

Adoptive Transfer of Human Gingiva‐Derived Mesenchymal Stem Cells Ameliorates Collagen‐Induced Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of Regulatory T Cell Differentiation

Objective

Current approaches offer no cures for rheumatoid arthritis (RA). Accumulating evidence has revealed that manipulation of bone marrow–derived mesenchymal stem cells (BM‐MSCs) may have the potential to control or even prevent RA, but BM‐MSC–based therapy faces many challenges, such as limited cell availability and reduced clinical feasibility. This study in mice with established collagen‐induced arthritis (CIA) was undertaken to determine whether substitution of human gingiva‐derived mesenchymal stem cells (G‐MSCs) would significantly improve the therapeutic effects.

Methods

CIA was induced in DBA/1J mice by immunization with type II collagen and Freund's complete adjuvant. G‐MSCs were injected intravenously into the mice on day 14 after immunization. In some experiments, intraperitoneal injection of PC61 (anti‐CD25 antibody) was used to deplete Treg cells in arthritic mice.

Results

Infusion of G‐MSCs in DBA/1J mice with CIA significantly reduced the severity of arthritis, decreased the histopathology scores, and down‐regulated the production of inflammatory cytokines (interferon‐γ and interleukin‐17A). Infusion of G‐MSCs also resulted in increased levels of CD4+CD39+FoxP3+ cells in arthritic mice. These increases were noted early after infusion in the spleens and lymph nodes, and later after infusion in the synovial fluid. The FoxP3+ Treg cells that were increased in frequency mainly consisted of Helios‐negative cells. When Treg cells were depleted, infusion of G‐MSCs partially interfered with the progression of CIA. Pretreatment of G‐MSCs with a CD39 or CD73 inhibitor significantly reversed the protective effect of G‐MSCs on CIA.

Conclusion

The role of G‐MSCs in controlling the development and severity of CIA mostly depends on CD39/CD73 signals and partially depends on the induction of CD4+CD39+FoxP3+ Treg cells. G‐MSCs provide a promising approach for the treatment of autoimmune diseases.