Arthur M. Mandelin

TH-17 cells in rheumatoid arthritis

IntroductionThe aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-γ.MethodsPeripheral blood (PB) mononuclear cells from normal and RA donors and mononuclear cells from RA SF were examined either without stimulation or after pretreatment with IL-23 followed by stimulation with phorbol myristate acetate (PMA) plus ionomycin (P/I). The abundance of TH-17 cells in RA SF was determined by flow cytometry. IL-17 levels were quantified in synovial tissue from RA, OA and normal individuals by ELISA and IL-23 was identified in SFs by ELISA. RA SF and control in vitro differentiated macrophages were either untreated or treated with the toll-like receptor (TLR) 2 ligand peptidoglycan, and then IL-23, IL-27 and IFN-γ mRNA levels were quantified by real-time polymerase chain reaction (RT-PCR).ResultsTreatment with P/I alone or combined with IL-23 significantly increased the number of TH-17 cells in normal, RA PB and RA SF. With or without P/I plus IL-23, the percentage of TH-17 cells was higher in RA SF compared with normal and RA PB. IL-17 levels were comparable in OA and normal synovial tissues, and these values were significantly increased in RA synovial tissue. Although IL-17 was readily detected in RA SFs, IL-23 was rarely identified in RA SF. However, IL-23 mRNA was significantly increased in RA SF macrophages compared with control macrophages, with or without TLR2 ligation. IL-27 mRNA was also significantly higher in RA SF compared with control macrophages, but there was no difference in IL-27 levels between RA and control macrophages after TLR2 ligation. IFN-γ mRNA was also detectable in RA SF macrophages but not control macrophages and the increase of IFN-γ mRNA following TLR2 ligation was greater in RA SF macrophages compared with control macrophages.ConclusionThese observations support a role for TH-17 cells in RA. Our observations do not strongly support a role for IL-23 in the generation of TH-17 cells in the RA joint, however, they suggest strategies that enhance IL-27 or IFN-γ might modulate the presence of TH-17 cells in RA.

IL-17 contributes to angiogenesis in rheumatoid arthritis.

Angiogenesis is an early and a critical event in the pathogenesis of rheumatoid arthritis (RA). Neovascularization is dependent on endothelial cell activation, migration and proliferation, and inhibition of angiogenesis may provide a novel therapeutic approach in RA. In this study, we document a novel role of IL-17 in mediating angiogenesis. Local expression of IL-17 in mouse ankles increases vascularity. We further demonstrate that IL-17 is angiogenic by showing its ability to promote blood vessel growth in Matrigel plugs in vivo. Additionally, IL-17, in concentrations present in the RA joint, induces human lung microvascular endothelial cell (HMVEC) migration mediated through the PI3K/AKT1 pathway. Furthermore, suppression of the PI3K pathway markedly reduces IL-17–induced tube formation. We also show that both IL-17–induced HMVEC chemotaxis and tube formation are mediated primarily through IL-17 receptor C. Neutralization of either IL-17 in RA synovial fluids or IL-17 receptor C on HMVECs significantly reduces the induction of HMVEC migration by RA synovial fluid. Finally, RA synovial fluid immunoneutralized with anti–IL-17 and antivascular endothelial growth factor does not reduce HMVEC migration beyond the effect detected by immunodepleting each factor alone. These observations identify a novel function for IL-17 as an angiogenic mediator in RA, supporting IL-17 as a therapeutic target in RA.

Portability of an algorithm to identify rheumatoid arthritis in electronic health records.

OBJECTIVES Electronic health records (EHR) can allow for the generation of large cohorts of individuals with given diseases for clinical and genomic research. A rate-limiting step is the development of electronic phenotype selection algorithms to find such cohorts. This study evaluated the portability of a published phenotype algorithm to identify rheumatoid arthritis (RA) patients from EHR records at three institutions with different EHR systems. MATERIALS AND METHODS Physicians reviewed charts from three institutions to identify patients with RA. Each institution compiled attributes from various sources in the EHR, including codified data and clinical narratives, which were searched using one of two natural language processing (NLP) systems. The performance of the published model was compared with locally retrained models. RESULTS Applying the previously published model from Partners Healthcare to datasets from Northwestern and Vanderbilt Universities, the area under the receiver operating characteristic curve was found to be 92% for Northwestern and 95% for Vanderbilt, compared with 97% at Partners. Retraining the model improved the average sensitivity at a specificity of 97% to 72% from the original 65%. Both the original logistic regression models and locally retrained models were superior to simple billing code count thresholds. DISCUSSION These results show that a previously published algorithm for RA is portable to two external hospitals using different EHR systems, different NLP systems, and different target NLP vocabularies. Retraining the algorithm primarily increased the sensitivity at each site. CONCLUSION Electronic phenotype algorithms allow rapid identification of case populations in multiple sites with little retraining.

Heat Shock Protein 96 Is Elevated in Rheumatoid Arthritis and Activates Macrophages Primarily via TLR2 Signaling

Macrophages are important mediators of chronic inflammation and are prominent in the synovial lining and sublining of patients with rheumatoid arthritis (RA). Recently, we demonstrated increased TLR2 and TLR4 expression and increased response to microbial TLR2 and TLR4 ligands in macrophages from the joints of RA. The current study characterized the expression of the 96-kDa heat shock glycoprotein (gp96) in the joints of RA and its role as an endogenous TLR ligand to promote innate immunity in RA. gp96 was increased in RA compared with osteoarthritis and arthritis-free control synovial tissues. The expression of gp96 strongly correlated with inflammation and synovial lining thickness. gp96 was increased in synovial fluid from the joints of RA compared with disease controls. Recombinant gp96 was a potent activator of macrophages and the activation was mediated primarily through TLR2 signaling. The cellular response to gp96 was significantly stronger with RA synovial macrophages compared with peripheral blood monocytes from RA or healthy controls. The transcription of TLR2, TNF-α, and IL-8, but not TLR4, was significantly induced by gp96, and the induction was significantly greater in purified RA synovial macrophages. The expression of TLR2, but not TLR4, on synovial fluid macrophages strongly correlated with the level of gp96 in the synovial fluid. The present study documents the potential role of gp96 as an endogenous TLR2 ligand in RA and provides insight into the mechanism by which gp96 promotes the chronic inflammation of RA, identifying gp96 as a potential new therapeutic target.

IL-17–Mediated Monocyte Migration Occurs Partially through CC Chemokine Ligand 2/Monocyte Chemoattractant Protein-1 Induction

Rheumatoid arthritis (RA) is a chronic inflammatory disease that is mediated, in part, by proinflammatory factors produced by RA synovial tissue (ST) fibroblasts and macrophages, resulting in monocyte migration from the blood to the ST. To characterize the potential role of IL-17 in monocyte migration, RA synovial fibroblasts and macrophages were activated with IL-17 and examined for the expression of monocyte chemokines. The two potentially important monocyte chemoattractants identified were CCL20/MIP-3α and CCL2/MCP-1, which were significantly induced in RA synovial fibroblasts and macrophages. However, in vivo, only CCL2/MCP-1 was detectable following adenovirus IL-17 injection. We found that IL-17 induction of CCL2/MCP-1 was mediated by the PI3K, ERK, and JNK pathways in RA ST fibroblasts and by the PI3K and ERK pathways in macrophages. Further, we show that neutralization of CCL2/MCP-1 significantly reduced IL-17–mediated monocyte recruitment into the peritoneal cavity. We demonstrate that local expression of IL-17 in ankle joints was associated with significantly increased monocyte migration and CCL2/MCP-1 levels. Interestingly, we show that RA synovial fluids immunoneutralized for IL-17 and CCL2/MCP-1 have similar monocyte chemotaxis activity as those immunoneutralized for each factor alone. In short, CCL2/MCP-1 produced from cell types present in the RA joint, as well as in experimental arthritis, may be responsible, in part, for IL-17–induced monocyte migration; hence, these results suggest that CCL2/MCP-1 is a downstream target of IL-17 that may be important in RA.

Characterization of CCL19 and CCL21 in rheumatoid arthritis

OBJECTIVE To characterize the expression of CCL19 and CCL21 in rheumatoid arthritis (RA) synovial tissue (ST) and to examine their regulation and pathogenetic role in macrophages and RA ST fibroblasts. METHODS Expression of CCL19 and CCL21 in RA and normal ST was demonstrated by immunohistochemistry analysis. CCL19 and CCL21 levels in synovial fluid (SF) from patients with osteoarthritis (OA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), and RA were quantified by enzyme-linked immunosorbent assay (ELISA). Regulation of CCL19 and CCL21 expression in in vitro-differentiated RA peripheral blood macrophages as well as RA ST fibroblasts was determined by real-time reverse transcription-polymerase chain reaction. Proangiogenic factor production in CCL19- and CCL21-activated in vitro-differentiated peripheral blood macrophages and RA ST fibroblasts was examined by ELISA. RESULTS CCL19 and CCL21 were elevated in RA ST compared to tissue from normal controls. Levels of CCL19 and CCL21 were greatly increased in RA and PsA SF versus OA SF. In RA macrophages and fibroblasts, expression of CCL19 was increased by stimulation with lipopolysaccharide, tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β). However, CCL21 expression was modulated only by IL-1β in RA fibroblasts, and by TNFα and RA SF in RA macrophages. CCL19 and CCL21 activation induced vascular endothelial growth factor and angiotensin I (Ang I) production in RA ST fibroblasts and secretion of IL-8 and Ang I from macrophages. CONCLUSION The findings of the present study identify, for the first time, regulators of CCL19 and CCL21 in RA fibroblasts and in vitro-differentiated RA peripheral blood macrophages and demonstrate a novel role of CCL19/CCL21 in angiogenesis in RA.

Local expression of interleukin-27 ameliorates collagen-induced arthritis

OBJECTIVE To determine the mechanism of action of interleukin-27 (IL-27) against rheumatoid arthritis (RA). METHODS Adenovirus containing IL-27 transcript was constructed and was locally delivered into the ankles of mice with collagen-induced arthritis (CIA). Progression of arthritis was determined in treated and untreated mice by measuring ankle circumference and through histologic analysis. IL-17 and its downstream targets as well as cytokines promoting Th17 cell differentiation were quantified by enzyme-linked immunosorbent assay in CIA mouse ankles locally expressing adenoviral IL-27 as well as in control-treated mouse ankles. Ankles from both treatment groups were immunostained for neutrophil and monocyte migration (macrophages in the tissue). Finally, vascularization was quantified by histology and by determining ankle hemoglobin levels. RESULTS Ectopic expression of IL-27 in CIA mice ameliorated inflammation, lining hypertrophy, and bone erosion as compared with control-treated CIA mice. Serum and joint levels of IL-17 were significantly reduced in the IL-27-treated group compared with the control-treated group. Two of the main cytokines that induce Th17 cell differentiation and IL-17 downstream target molecules were greatly down-regulated in CIA mouse ankles receiving forced expression of IL-27. The control mice had higher levels of vascularization and monocyte trafficking than did mice ectopically expressing IL-27. CONCLUSION Our results suggest that increased levels of IL-27 relieve arthritis in CIA mouse ankles. This amelioration of arthritis involves a reduction in CIA mouse serum and joint levels of IL-17 and results in decreased IL-17-mediated monocyte recruitment and angiogenesis. Hence, the use of IL-27 may be a strategy for treatment of patients with RA.

Characterization of interleukin‐7 and interleukin‐7 receptor in the pathogenesis of rheumatoid arthritis

OBJECTIVE To characterize the expression of interleukin-7 (IL-7) and IL-7 receptor (IL-7R) in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages, endothelial cells, and synovial tissue fibroblasts in RA. METHODS Expression of IL-7 and IL-7R in RA and normal synovial tissue was demonstrated by immunohistochemistry. Expression and regulation of IL-7 and IL-7R in RA peripheral blood in vitro-differentiated macrophages, RA synovial tissue fibroblasts, and human microvascular endothelial cells (HMVECs) were determined by real-time reverse transcription-polymerase chain reaction and/or flow cytometry. Enzyme-linked immunosorbent assay was used to examine production of proangiogenic factors by IL-7-activated macrophages, RA fibroblasts, and endothelial cells. RESULTS IL-7 and IL-7R were coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells. Expression of IL-7 and its receptor was significantly elevated in RA synovial fluid and peripheral blood macrophages as well as RA fibroblasts, compared to normal cells. Toll-like receptor 4 ligation (with lipopolysaccharide) and tumor necrosis factor α (TNFα) stimulation modulated expression of IL-7 and IL-7R on RA macrophages and HMVECs. However, in RA fibroblasts, lipopolysaccharide and TNFα activation increased expression of IL-7R only. IL-7 also mediated RA pathogenesis by inducing production of potent proangiogenic factors from macrophages and endothelial cells. CONCLUSION We have identified, for the first time, regulators of IL-7 and IL-7R expression in RA fibroblasts, RA peripheral blood in vitro-differentiated macrophages, and endothelial cells. Our results document a novel role of IL-7 in RA angiogenesis.

Role of the CCL21 and CCR7 Pathways in rheumatoid arthritis angiogenesis

OBJECTIVE To determine the role of CCL21 and its receptor CCR7 in the pathogenesis of rheumatoid arthritis (RA). METHODS Histologic studies were performed to compare the expression of CCR7 and CCL21 in RA synovial tissue. Next, the role of CCL21 and/or CCR7 in angiogenesis was examined using in vitro chemotaxis, tube formation, and in vivo Matrigel plug assays. Finally, the mechanism by which CCL21 mediates angiogenesis was determined by Western blot analysis and endothelial cell chemotaxis and tube formation assays. RESULTS CCL21, but not CCL19, at concentrations present in the RA joint, induced human microvascular endothelial cell (HMVEC) migration that was mediated through CCR7 ligation. Suppression of the phosphatidylinositol 3-kinase pathway markedly reduced CCL21-induced HMVEC chemotaxis and tube formation; however, suppression of the ERK and JNK pathways had no effect on these processes. Neutralization of either CCL21 in RA synovial fluid or CCR7 in HMVECs significantly reduced the induction of HMVEC migration and/or tube formation by RA synovial fluid. We further demonstrated that CCL21 is angiogenic, by showing its ability to promote blood vessel growth in Matrigel plugs in vivo at concentrations that are present in RA joints. CONCLUSION Angiogenesis is dependent on endothelial cell activation, migration, and proliferation, and inhibition of angiogenesis may provide a novel therapeutic approach in RA. This study identified a novel function of CCL21 as a mediator of RA angiogenesis, supporting CCL21/CCR7 as a therapeutic target in RA.

Anti-CXCL5 therapy ameliorates IL-17-induced arthritis by decreasing joint vascularization

IL-17-induced joint inflammation is associated with increased angiogenesis. However, the mechanism by which IL-17 mediates angiogenesis is undefined. Therefore, the pathologic role of CXCL1 and CXCL5 was investigated in arthritis mediated by local expression of IL-17, employing a neutralizing antibody to each chemokine. Next, endothelial chemotaxis was utilized to examine whether endothelial migration was differentially mediated by CXCL1 and CXCL5. Our results demonstrate that IL-17-mediated disease activity was not affected by anti-CXCL1 treatment alone. In contrast, mice receiving anti-CXCL5 demonstrated significantly reduced clinical signs of arthritis, compared to the mice treated with IgG control. Consistently, while inflammation, synovial lining thickness, bone erosion and vascularization were markedly reduced in both the anti-CXCL5 and combination anti-CXCL1 and 5 treatment groups, mice receiving anti-CXCL1 antibody had clinical scores similar to the control group. In contrast to joint FGF2 and VEGF levels, TNF-α was significantly reduced in mice receiving anti-CXCL5 or combination of anti-CXCL1 and 5 therapies compared to the control group. We found that, like IL-17, CXCL1-induced endothelial migration is mediated through activation of PI3K. In contrast, activation of NF-κB pathway was essential for endothelial chemotaxis induced by CXCL5. Although CXCL1 and CXCL5 can differentially mediate endothelial trafficking, blockade of CXCR2 can inhibit endothelial chemotaxis mediated by either of these chemokines. These results suggest that blockade of CXCL5 can modulate IL-17-induced inflammation in part by reducing joint blood vessel formation through a non-overlapping IL-17 mechanism.