The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. This study was performed to determine the contribution of endogenous gp96 in TLR-mediated macrophage activation in RA.
RA synovial fluids (SFs) were employed to activate macrophages and HEK-TLR2 and HEK-TLR4 cells. Neutralizing antibodies to TLR2, TLR4 and gp96 were employed to inhibit activation. RA SF macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of TNFα or IL-8 mRNA. Arthritis was induced employing the K/BxN serum transfer model, gp96 expression determined by Immunoblot analysis, ELISA and immunohistochemistry. The arthritis was treated with neutralizing anit-gp96 or control serum.
RA SF induced the activation of macrophages and HEK-TLR2 and HEK-TLR4 cells. RA SF-induced macrophage and HEK-TLR2 activation was suppressed by neutralizing anti-gp96 antibody only when high (>800 ng/ml), but not low (<400 ng/ml), concentrations of gp96 were present. Neutralization of RA SF macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting its role in RA, joint tissue gp96 expression was induced in the K/BxN serum transfer model of RA, and neutralizing antibodies to gp96, ameliorated joint inflammation on clinical and histologic examination.
These observations support the role of gp96 as an endogenous TLR2 ligand in RA and identify the TLR2 pathway as a therapeutic target.
The aim of the study was to characterize the expression, regulation and pathogenic role of TLR7 and TLR8 in rheumatoid arthritis (RA).
Expression of TLR7 and TLR8 was demonstrated in RA, osteoarthritis (OA) and normal (NL) synovial tissues (ST) employing immunohistochemistry. We next examined the mechanism by which TLR7 and TLR8 ligation mediates proinflammatory response by Western blot analysis and ELISA. Expression of TLR7 and TLR8 in RA monocytes was correlated to disease activity score (DAS28) and TNF-α levels. Further the effect of TLR7 ligation in RA monocytes was determined on synovial fluid (SF) mediated TNF-α transcription.
TLR7/TLR8 are predominately expressed in RA ST lining and sublining macrophages. We show that NF-κB and/or PI3K pathways are essential for TLR7/TLR8 induction of proinflammatory factors in RA peripheral blood (PB) differentiated macrophages. Expression of TLR7 in RA monocytes shows a strong correlation with DAS28 and TNF-α levels. In contrast, expression of TLR8 in these cells does not correlate with DAS28, TLR7 or TNF-α levels. We further demonstrate that RNA from RA SF but not RA or NL plasma could modulate TNF-α transcription from RA monocytes that can be downregulated by antagonizing TLR7 ligation or degradation of single stand (ss) RNA. Thus, ssRNA present in RA SF may function as a potential endogenous ligand for TLR7.
These results suggest that expression of TLR7 but not TLR8 may be a predictor for RA disease activity and anti-TNF-α responsiveness, and targeting TLR7 may suppress chronic progression of RA.
RA synovial tissue; RA monocytes/macrophages; TLR7; TLR8 and ssRNA
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.
IL-17-induced arthritis; CXCL1; CXCL5; Angiogenesis
Objective and design
Antiphospholipid antibodies (APA) have been associated with clinical cardiovascular disease, but it remains unclear whether APA are associated with sub-clinical atherosclerosis. This study examined the relationship between APA and sub-clinical atherosclerosis, measured as coronary artery calcification (CAC), in participants from the prospective Coronary Artery Risk Development in Young Adults (CARDIA) Study.
Subjects and method
2,203 black and white participants with sera available from the CARDIA year 7 examination and CAC measured by computed tomography at years 15 or 20 were selected.
Anti-β2-glycoprotein I (anti-β2-GPI) immunoglobulin (Ig) M, IgG, and IgA were positive in 7.0, 1.4, and 1.8 % of participants, respectively; anti-cardiolipin (aCL) IgM and IgG were positive in 1.5 and 1.0 %, respectively. 9.5 % of participants had CAC score >0 at year 15. Anti-β2-GPI IgM, IgG, IgA, and aCL IgG positivity were associated with CAC >0 at year 15 after adjustment for traditional cardiovascular risk factors; [odds ratios (95 % confidence intervals) were 1.7 (1.0, 3.1), 6.4 (2.4, 16.8), 5.6 (2.3, 13.2), and 5.1 (1.4, 18.6), respectively]. Anti-β2-GPI IgG was associated with year 20 CAC >0, and anti-β2-GPI IgA and aCL IgG were marginally associated.
These findings indicate that APA positivity during young adulthood is a risk factor for subsequent sub-clinical atherosclerosis and might play a role in the pathogenesis of atherosclerosis.
Antiphospholipid antibodies; Cardiovascular disease; Coronary atherosclerosis; Coronary artery calcification; EBCT; MDCT
The 96-kDa glycoprotein (gp96) is an endoplasmic reticulum (ER) resident molecular chaperone. Under physiologic conditions, gp96 facilitates the transport of toll-like receptors (TLRs) to cell or endosomal membranes. Under pathologic circumstances such as rheumatoid arthritis, gp96 translocates to the cell surface and extracellular space, serving as an endogenous danger signal promoting TLR signaling. Macrophages play a central role in regulating innate and adaptive immunity, and are the major source of proinflammatory cytokines and chemokines in rheumatoid arthritis (RA). Macrophage numbers in the sublining of RA synovial tissue correlate with clinical response. This review focuses on the recent findings that implicate gp96 induced macrophage activation mediated through TLR signaling in the pathogenesis of RA and provides insights concerning the targeting gp96 and the TLR signaling pathway as therapeutic approaches for patients with RA and possibly other chronic inflammatory conditions.
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 toll like receptor (TLR) 2 and 4 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 to peripheral blood monocytes from RA or healthy controls. The transcription of TLR2, TNFα 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.
gp96; grp94; macrophages; TLR2; TLR4; Rheumatoid Arthritis
Monocytes recruited into tissues from peripheral blood differentiate into macrophages, which are critical in the pathogenesis of many diseases. There is limited data concerning the global changes in the expression of genes during monocyte to macrophage differentiation, and how the patterns of change identify the mechanism contributing to macrophage differentiation or function. Employing microarray technology, we examined the transcriptional profile of in vitro adherence-induced differentiation of primary human monocytes into macrophages. We found the significant up regulation of genes contributing to the functions of macrophages, including those regulating to immunity and defense; lipid, fatty acid and steroid metabolism; cell adhesion, carbohydrate metabolism; amino acid metabolism and endocytosis. In contrast, the vast majority of transcription factors affected were down regulated during monocyte to macrophage differentiation, suggesting that transcriptional repression may be important for the transition from monocytes to macrophages. However, a limited number of transcription factors were up regulated, among these was C/EBPα, which may contribute to differentiation by regulating down stream genes, which are a characteristic of differentiated macrophages. These observations suggest that examination of the transcriptional profile in monocytes and macrophages in patients may identify relevant therapeutic targets in diseases mediated by macrophages.
mononuclear phagocytes; gene regulation; transcription factors
CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal–modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1α (MIP-1α; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat.
Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA.
Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1α and MIP-1β at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor α, interleukin-1β, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint.
The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.
These studies were performed to determine the role of CCL21 and its corresponding receptor CCR7 in the pathogenesis of Rheumatoid Arthritis (RA).
Histological studies were performed to compare the expression of CCR7 and CCL21 in RA synovial tissues. Next the role of CCL21 and/or CCR7 in angiogenesis was examined employing 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, endothelial chemotaxis and tube formation.
In this study, we document that CCR7 and CCL21 colocolize in VWF+ cells where their expression is elevated in RA synovial tissue. Hence the ability to induce angiogenesis was examined for CCR7 ligands, CCL19 and CCL21. CCL21, but not CCL19, at concentrations present in the RA joint, induces human microvascular endothelial cell (HMVEC) migration that is mediated through CCR7 ligation. Further, suppression of the PI3K pathway markedly reduces CCL21-induced HMVEC chemotaxis and tube formation, however suppression of ERK and JNK pathways has no effect on these processes. Neutralization of either CCL21 in RA synovial fluids or CCR7 on HMVECs significantly reduces the induction of HMVEC migration and/or tube formation by RA synovial fluid. We further demonstrate that CCL21 is angiogenic, by showing its ability to promote blood vessel growth in matrigel plugs in vivo at concentrations present in RA joint.
These observations identify a novel function for CCL21 as an angiogenic mediator in RA, supporting CCL21/CCR7 as a therapeutic target in RA.
CCL21; CCR7; RA synovial fluid; angiogenesis and migration
The innate immune system plays an important role in rheumatoid arthritis (RA) pathogenesis. Previous studies support the role of TLR2 and 4 in RA and experimental arthritis models however the regulation and pathogenic effect of TLR5 is undefined in RA. In this study we show that TLR5 is elevated in RA and osteoarthritis (OA) synovial tissue lining and sublining macrophages and endothelial cells compared to normal individuals. Further, expression of TLR5 is elevated in RA synovial fluid macrophages and RA peripheral blood (PB) monocytes compared to RA and normal PB in vitro differentiated macrophages. We also found that TLR5 on RA monocytes is an important modulator of TNF-α in RA synovial fluid and that TLR5 expression on these cells strongly correlates with RA disease activity and TNF-α levels. Interestingly, TNF-α has a feed back regulation with TLR5 expression in RA monocytes, while expression of this receptor is regulated by IL-17 and IL-8 in RA macrophages and fibroblasts. We show that RA monocytes and macrophages are more responsive to TLR5 ligation compared to fibroblasts despite the proinflammatory response being mediated through the same signaling pathways in macrophages and fibroblasts. In conclusion we document the potential role of TLR5 ligation in modulating transcription of TNF-α from RA synovial fluid and the strong correlation of TLR5 and TNF-α with each other and with disease activity score in RA monocytes. Our results suggest that expression of TLR5 may be a predictor for RA disease progression and that targeting TLR5 may suppress RA.
RA monocytes; TLR5; TNF-α; DAS28; RA fibroblasts and RA differentiated macrophages
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.
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.
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.
Electronic phenotype algorithms allow rapid identification of case populations in multiple sites with little retraining.
Automated learning; biomedical informatics; discovery and text and data mining methods; electronic health record; genetic; improving the education and skills training of health professionals; infection control; knowledge representations; linking the genotype and phenotype; medical informatics; natural language processing; other methods of information extraction; phenotype algorithms DNA databank machine learning; phenotype identification; phenotyping; rheumatoid arthritis; rheumatology; translational research – application of biological knowledge to clinical care
In the past, numerous chemokines have been shown to be present in the expressed prostatic secretions (EPS) of patients with CP/CPPS. This study examined the functional effects of chemokines in the EPS of patients with CPPS.
Materials and Methods
The functional effects of EPS on human monocytes were studied by examining monocyte chemotaxis in response to MCP-1, a major chemoattractant previously identified in CP/CPPS. Effects on cellular signaling were determined by quantifying intracellular calcium elevation in monocytes and activation of NF-κB in benign prostate epithelial cells.
Our results show that the MCP-1 present in EPS is non-functional, with an inability to mediate chemotaxis of human monocytes or mediate signaling in either monocytes or prostate epithelial cells. Moreover, this absence of functionality could be extended to other proinflammatory cytokines such as IL-1β and TNFα, when incubated with the EPS from CPPS patients. The mechanism underlying this apparent ability to modulate pro-inflammatory cytokines involves heat labile extracellular proteases that mediate inhibition of both immune and prostate epithelial cell function.
These results may have implications for the design of specific diagnostics and therapeutics targeted at complete resolution of prostate inflammatory insults.
Chronic pelvic pain syndrome; prostatitis; chemokines; inflammation; pelvic pain
The aim of the study was to characterize the expression of IL-7 and IL-7R in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages, endothelial cells and RA synovial tissue fibroblasts.
Expression of IL-7 and IL-7R was demonstrated in RA and normal synovial tissues employing immunohistochemistry. Expression and regulation of IL-7 and IL-7R was determined in RA peripheral blood in vitro differentiated macrophages, RA synovial tissue fibroblasts and human microvascular endothelial cells (HMVECs) by real-time RT-PCR and/or flow cytometry. Next, IL-7 activated macrophages, RA fibroblasts and endothelial cells were examined for production of proangiogenic factors employing ELISA.
IL-7 and IL-7R were coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells. Consistently, expression of IL-7 and its receptor were significantly elevated in RA synovial fluid and peripheral blood macrophages as well as RA fibroblasts compared to normal cells. TLR4 ligation and stimulation with TNF-α modulated expression of IL-7 and IL-7R on RA macrophages and HMVECs. However, in RA fibroblasts only expression of IL-7R was increased by LPS and TNF-α activation. IL-7 also mediated RA pathogenesis by inducing production of potent proangiogenic factors from macrophages and endothelial cells.
We identify, 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 and we document a novel role of IL-7 in RA angiogenesis.
IL-7; IL-7R; RA synovial tissue fibroblast; macrophages and proangiogenic factors
The death receptor Fas is a critical mediator of the extrinsic apoptotic pathway. While the role that Fas plays in mediating lymphoproliferation has been extensively investigated, the impact of myeloid cell-specific loss of Fas has yet to be examined.
Mice with Fas flanked by loxP sites (Fasflox/flox) were crossed with mice expressing Cre under control of the murine lysozyme M gene promoter (CreLysM), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage. The genotype for CreLysMFasflox/flox mice was verified by real-time PCR and flow cytometric analysis. Flow cytometric analysis was also employed to characterize myeloid, dendritic, and lymphoid cell distribution and activation in bone marrow, blood and spleen. Luminex-based assays and ELISAs were used to measure serum cytokine/chemokine and immunglobulin levels. Immunohistochemical and immunofluorescent analyses were utilized to examine renal damage or dysfunction.
CreLysMFasflox/flox mice exhibited an SLE-like disease including leukocytosis, splenomegaly, hypergammaglobulinemia, anti-nuclear autoantibody and proinflammatory cytokine production, and glomerulonephritis. Loss of Fas in myeloid cells increased levels of both Gr-1low and Gr-1intermediate blood monocytes and splenic macrophages, and in a paracrine manner, incited activation of conventional dendritic cells and lymphocytes in CreLysMFasflox/flox mice.
Taken together, these results suggest that loss of Fas in myeloid cells is sufficient to induce inflammatory phenotypes in mice reminiscent of an SLE-like disease. Thus, Fas in myeloid cells may be considered a suppressor systemic autoimmunity.
The aim was to characterize the expression of CCL19 and CCL21 in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages and RA synovial tissue fibroblasts.
Expression of CCL19 and CCL21 was demonstrated in RA and normal (NL) synovial tissues employing immunohistochemistry. CCL19 and CCL21 levels were quantified in fluids from osteoarthritis (OA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA) and RA using ELISA. Regulation of CCL19 and CCL21 expression was determined in RA peripheral blood in vitro differentiated macrophages as well as RA synovial tissue fibroblasts by real-time RT-PCR. CCL19 and CCL21 activated peripheral blood in vitro differentiated macrophages and RA synovial tissue fibroblasts were examined for proangiogenic factor production employing ELISA.
CCL19 and CCL21 were elevated in RA synovial tissue compared to NL controls. Levels of CCL19 and CCL21 were greatly increased in RA and PsA synovial fluid versus OA synovial fluid. In RA macrophages and fibroblasts, expression of CCL19 was increased by LPS, TNF-α and IL-1β stimulation. However, CCL21 expression was modulated by IL-1β in RA fibroblasts as well as TNF-α and RA synovial fluid in RA macrophages. CCL19 and CCL21 activation induced VEGF and Ang-1 production from RA synovial tissue fibroblasts and secretion of IL-8 and Ang-1 from macrophages.
We identify, for the first time, regulators of CCL19 and CCL21 in RA fibroblasts and RA peripheral blood in vitro differentiated macrophages and we document a novel role of CCL19/21 in RA angiogenesis.
CCL19; CCL21; RA synovial tissue fibroblast; macrophages and proangiogenic factors
Despite tremendous advances in the therapy of rheumatoid arthritis (RA), there remains interest in oral agents that may offer benefits that are similar to, or better than, those of biologic therapies. In their paper, Chang and colleagues demonstrate the effectiveness of a Bruton tyrosine kinase (Btk) inhibitor in two models of RA. Btk inhibition impacts several pathways affecting both B-cell and macrophage activation, making it a promising target in RA. However, other kinase inhibitors have failed to transition from animal models to human therapy, so it remains to be seen whether a Btk inhibitor will have a role in the RA treatment armamentarium.
Atherosclerosis is the leading cause of cardiovascular disease (CVD). Traditional risk factors can be used to identify individuals at high risk for developing CVD and are generally associated with the extent of atherosclerosis; however, substantial numbers of individuals at low or intermediate risk still develop atherosclerosis.
A case-control study was performed using microarray gene expression profiling of peripheral blood from 119 healthy women in the Multi-Ethnic Study of Atherosclerosis cohort aged 50 or above. All participants had low (<10%) to intermediate (10% to 20%) predicted Framingham risk; cases (N = 48) had coronary artery calcium (CAC) score >100 and carotid intima-media thickness (IMT) >1.0 mm, whereas controls (N = 71) had CAC<10 and IMT <0.65 mm. We identified two major expression profiles significantly associated with significant atherosclerosis (odds ratio 4.85; P<0.001); among those with Framingham risk score <10%, the odds ratio was 5.30 (P<0.001). Ontology analysis of the gene signature reveals activation of a major innate immune pathway, toll-like receptors and IL-1R signaling, in individuals with significant atherosclerosis.
Gene expression profiles of peripheral blood may be a useful tool to identify individuals with significant burden of atherosclerosis, even among those with low predicted risk by clinical factors. Furthermore, our data suggest an intimate connection between atherosclerosis and the innate immune system and inflammation via TLR signaling in lower risk individuals.
Rheumatoid arthritis (RA) is a chronic inflammatory disease which is in part mediated by proinflammatory factors produced by RA synovial tissue fibroblasts and macrophages, resulting in monocyte migration from the blood to the synovial tissue. In order 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 (Ad)-IL-17 injection. We found that IL-17 induction of CCL2/MCP-1 was mediated by PI3K, ERK, and JNK pathways in RA synovial tissue fibroblasts and 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 both 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 in part responsible 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.
IL-17; CCL2/MCP-1; macrophages; synovial tissue fibroblasts; monocytes; 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 microvascular endothelial cell (HMVEC) migration mediated through the PI3K/AKT1 pathway. Further, 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 (R) C. Neutralization of either IL-17 in RA synovial fluids or IL-17RC on HMVECs significantly reduces the induction of HMVEC migration by RA synovial fluid. Finally, RA synovial fluid immunoneutralized with IL-17 and VEGF does not reduce HMVEC migration beyond the effect detected with 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.
IL-17; angiogenesis; HMVECs; migration; rheumatoid arthritis
An increasing body of data supports the role of the innate immune system in the pathogenesis of rheumatoid arthritis (RA). Toll-like receptors (TLRs) are expressed by cells within the RA joint and a variety of endogenous TLR ligands are present within the inflamed joints of patients with RA. Further, a variety of animal models suggest that TLR signaling is important in the pathogenesis of disease. Overall, the data suggest that activation by endogenous TLR ligands may contribute to the persistent expression of pro-inflammatory cytokines by macrophages and the joint damage to cartilage and bone that occurs in RA. The data supports a potential role for suppression of TLR signaling as a novel therapeutic approach in patients with RA.
Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies which activate the apoptotic cascade may have potential as a future therapy for RA, however few therapeutics fit this category. Recently, therapies that mimic the action of Bcl-2 homology 3 (BH3) domain-only proteins such as Bim have shown success in preclinical studies of cancer but their potential in autoimmune disease is unknown.
Synovial tissue from RA and osteoarthritis (OA) patients were analyzed for expression of Bim and CD68 using immunohistochemistry. Macrophages from mice lacking (Bim−/−) were examined for response to lipopolysaccharide (LPS) using flow cytometry, real time PCR, ELISA, and immunoblot analysis. Bim−/− mice were stimulated with thioglycollate or LPS and examined for macrophage activation and cytokine production. Experimental arthritis was induced using the K/BxN serum-transfer model. A mimetic peptide corresponding to the BH3 domain of Bim (TAT-BH3) was administered as a prophylactic and as a therapeutic. Edema of the ankles and histopathogical analysis of ankle sections were used to determine severity of arthritis, cellular composition, and apoptosis.
The expression of Bim was reduced in RA synovial tissue as compared to controls, particularly in macrophages. Bim−/− macrophages displayed elevated expression of markers of inflammation and secreted more IL-1β following stimulation with LPS or thioglycollate. TAT-BH3 ameliorated arthritis development, reduced the number of myeloid cells in the joint, and enhanced apoptosis without inducing cytotoxicity.
These data demonstrate that BH3 mimetic therapy may have significant potential for RA treatment.
Bim; arthritis; macrophages; apoptosis
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease which is in part mediated by the migration of monocytes from blood to RA synovial tissue, where they differentiate into macrophages and secrete inflammatory cytokines and chemokines. The T cell cytokine IL-17 is expressed in the RA synovial tissue and synovial fluid. In order to better understand the mechanism by which IL-17 might promote inflammation, its role in monocyte trafficking was examined. In vivo, IL-17 mediates monocyte migration into sponges implanted into severe combined immunodeficient (SCID) mice. In vitro, IL-17 was chemotactic, not chemokinetic, for monocytes at the concentrations detected in the RA synovial fluid. Further, IL-17-induced monocyte migration was mediated by ligation to IL-17 receptor (R) A and C expressed on monocytes and was mediated through p38MAPK signaling. Finally, neutralization of IL-17 in RA synovial fluid or its receptors on monocytes significantly reduced monocyte migration mediated by RA synovial fluid. These observations suggest that IL-17 may be important in recruiting monocytes into the joints of patients with RA, supporting IL-17 as a therapeutic target in RA.
IL-17; monocyte; migration; rheumatoid arthritis
The chronic pelvic pain syndrome (CPPS) is characterized by pelvic pain, voiding symptoms and varying degrees of inflammation within expressed prostatic secretions (EPS). We evaluated the chemokines MCP-1 (CCL2) and MIP-1α (CCL3) in EPS to identify marker elevations associated with both inflammatory (IIIA) and non-inflammatory (IIIB) CPPS. In addition, chemokine levels were correlated with clinical pain as determined by the NIH chronic prostatitis symptom index (CPSI).
MATERIALS AND METHODS
EPS were collected by digital rectal examination and evaluated by ELISA for MCP-1 and MIP-1α in 154 patients; controls (n = 13), BPH (n = 54), CPPS IIIA (n = 37), CPPS IIIB (n = 50). MCP-1 and MIP-1α levels were compared between IIIA, IIIB, and the control subgroups and correlated against the CPSI and pain sub-score using a Spearman test.
Mean levels of MCP-1 in the control, inflammatory BPH, non-inflammatory BPH, inflammatory CPPS, and non-inflammatory CPPS were 599.4, 886.0, 1636.5, 3261.2, and 2272.7 pg/ml, respectively. Mean levels of MIP-1α in the control, inflammatory BPH, non-inflammatory BPH, IIIA CPPS, and IIIB CPPS were 140.1, 299.4, 238.7, 1057.8, and 978.4 pg/ml, respectively. For each cytokine, both CPPS subtypes had statistically higher levels than the control group and BPH patients (p=0.0002). Receiver operating curves utilizing MCP-1 levels greater than 704 pg/ml and MIP-1α greater than 146 pg/ml identified patients with CPPS with an accuracy of 90% from control patients. MIP-1α levels (p=0.0007) correlated with the pain sub-score of the CPSI while MCP-1 (p=0.71) did not.
MCP-1 and MIP-1α within the prostatic fluid in both CPPS subtypes provide candidate future biomarkers for CPPS. In addition, MIP-1α elevation in EPS provides a new marker for clinical pain in CPPS patients. Given these findings, prostatic dysfunction likely plays a role in the pathophysiology of some patients with this syndrome. These chemokines may serve as effective diagnostic markers and modulators against the chemokines could provide an attractive treatment strategy in individuals with CPPS.
chronic pelvic pain syndrome; prostatitis; cytokines; monocyte chemoattractant protein; macrophage inflammatory protein