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1.  Ligation of TLR7 by rheumatoid arthritis synovial fluid single strand RNA induces transcription of TNF-α in monocytes 
Annals of the rheumatic diseases  2012;72(3):418-426.
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.
PMCID: PMC4281889  PMID: 22730373
RA synovial tissue; RA monocytes/macrophages; TLR7; TLR8 and ssRNA
2.  Anti-CXCL5 therapy ameliorates IL-17-induced arthritis by decreasing joint vascularization 
Angiogenesis  2011;14(4):443-455.
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.
PMCID: PMC4281892  PMID: 21779896
IL-17-induced arthritis; CXCL1; CXCL5; Angiogenesis
3.  Angiogenesis in rheumatoid arthritis is directly fostered by TLR5 ligation and indirectly through IL-17 induction 
Arthritis and rheumatism  2013;65(8):2024-2036.
In this study we focus on examining the impact of TLR5 on rheumatoid arthritis (RA) endothelial cell function and collagen induced arthritis (CIA) vascularization.
Endothelial migration and tube formation were employed to demonstrate the direct role of TLR5 ligation in angiogenesis. CIA mice were treated with TLR5 agonist, flagellin to document the effect of TLR5 ligation in RA pathology. CIA vascularization was examined by histology and joint cytokine levels and spleen TH-17 cells were quantified by ELISA and FACS analysis. Development of TH-17 cells by TLR5 ligation was validated in RA peripheral blood mononuclear cells.
Ligation of TLR5 to endogenous ligands expressed in RA synovial fluid contributes to endothelial infiltration and tube formation. Further, post onset treatment with flagellin, exacerbates CIA joint inflammation while in the control mice, disease activity reaches the plateau phase. We show that TLR5 enhanced disease severity is due to TH-17 cell differentiation and CIA joint vascularization. When the underlying mechanism was examined in RA peripheral blood mononuclear cells, we found that ligation of myeloid TLR5 and their production of TH-17 promoting cytokines was necessary for TH-17 cell polarization. Additionally we demonstrate that blockade of IL-17 cascade can markedly reduce endothelial migration activated by flagellin condition media suggesting that TLR5 ligation can mediate RA angiogenesis either directly through attracting endothelial cells or indirectly by fostering TH-17 cell development.
Our data demonstrate a novel role for TLR5 in RA angiogenesis hence TLR5 may be a promising new target for RA treatment.
PMCID: PMC3729873  PMID: 23666857
RA; TLR5; angiogenesis; CIA; TH-17 cells
4.  The novel role of IL-7 ligation to IL-7R in myeloid cells of rheumatoid arthritis and collagen induced arthritis 
Although role of IL-7 and IL-7R has been implicated in the pathogenesis of Rheumatoid arthritis (RA), the majority of the studies have focused on the impact of IL-7/IL-7R in T cell development and function. Our novel data, however, document that RA patients with greater disease activity have higher levels of IL-7, IL-7R and TNF-α in RA monocytes suggesting a feedback regulation between IL-7/IL-7R and TNF-α cascades in myeloid cells which is linked to chronic disease progression. Investigations into the involved mechanism showed that IL-7 is a novel and potent chemoattractant which attracts IL-7R+ monocytes through activation of the PI3K/AKT1 and ERK pathways at similar concentrations of IL-7 detected in RA synovial fluid. To determine whether ligation of IL-7 to IL-7R is a potential target for RA treatment and to identify their mechanism of action, collagen induced arthritis (CIA) was therapeutically treated with anti-IL-7 antibody or IgG control. Anti-IL-7 antibody treatment significantly reduces CIA monocyte recruitment and osteoclast differentiation as well as potent joint monocyte chemoattractants and bone erosion markers suggesting that both direct and indirect pathways may contribute to the observed effect. We also demonstrate that reduction in joint MIP-2 levels is responsible for suppressed vascularization detected in anti-IL-7 antibody treated mice compared to the control group. In conclusion we show for the first time that expression of IL-7/IL-7R in myeloid cells is strongly correlated with RA disease activity and that ligation of IL-7 to IL-7R contributes to monocyte homing, differentiation of osteoclasts and vascularization in the CIA effector phase.
PMCID: PMC3686279  PMID: 23606539
monocyte migration; collagen induced arthritis; disease correlation; IL-7; IL-7R and TNF-α
5.  Tocilizumab in the treatment of rheumatoid arthritis and beyond 
Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by joint pain, swelling, stiffness, and progressive destruction of the small joints of the hands and feet. Treatment of RA has improved over the past decade. With multiple cytokines well-known now to play a role in the pathogenesis of RA, including tumor necrosis factor alpha, interleukin (IL)-1β, and IL-6, many targeted biological treatments against these cytokines have emerged, changing the treatment of this disease. Tocilizumab (TCZ) is a recombinant humanized monoclonal antibody against the IL-6 receptor and has been approved in many countries, including the United States, for the treatment of moderate to severe RA in patients who have not adequately responded to one or more disease-modifying antirheumatic drugs (DMARDs) or cannot tolerate other approved drug classes for RA. The aim of this review is to discuss the role of IL-6 in RA, and to provide an overview of the mode of action, pharmacokinetics, and safety of TCZ. Furthermore, efficacy studies of TCZ as both monotherapy and combination therapy will be evaluated. There have been several important clinical trials evaluating the efficacy and safety of TCZ in RA patients; this review summarizes this data from 14 key trials with emphasis on Phase III trials. Review of these trials provides strong evidence that its use, both as monotherapy and in combination with methotrexate or other DMARDs, is an effective treatment in reducing the signs and symptoms of RA. TCZ showed tolerable safety but care is required for its use since there are some important safety concerns including elevated liver enzymes, elevated low-density lipoprotein, infections, and gastrointestinal perforations. Additionally, given the efficacy of TCZ in the treatment of RA, this review discusses how TCZ may be beneficial in the treatment of other autoimmune diseases, spinal disease, cardiovascular disease, organ transplantation, and malignancies where elevated levels of IL-6 may play a role in the pathogenesis of these diseases.
PMCID: PMC3974690  PMID: 24729685
tocilizumab; IL-6; rheumatoid arthritis; biologics
6.  Role of the CCL21 and CCR7 pathway in rheumatoid arthritis angiogenesis 
Arthritis and rheumatism  2012;64(8):2471-2481.
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.
PMCID: PMC3409328  PMID: 22392503
CCL21; CCR7; RA synovial fluid; angiogenesis and migration
7.  TLR5; a novel and unidentified inflammatory mediator in Rheumatoid Arthritis that correlates with disease activity score and joint TNF-α levels 
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.
PMCID: PMC3614068  PMID: 22661088
RA monocytes; TLR5; TNF-α; DAS28; RA fibroblasts and RA differentiated macrophages
8.  Prostate secretions from men with CP/CPPS inhibit proinflammatory mediators 
The Journal of urology  2010;184(4):1536-1542.
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.
PMCID: PMC3660015  PMID: 20723928
Chronic pelvic pain syndrome; prostatitis; chemokines; inflammation; pelvic pain
9.  Characterization of IL-7 and IL-7R in the pathogenesis of Rheumatoid Arthritis 
Arthritis and rheumatism  2011;63(10):2884-2893.
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.
PMCID: PMC3614067  PMID: 21647866
IL-7; IL-7R; RA synovial tissue fibroblast; macrophages and proangiogenic factors
10.  Local expression of IL-27 ameliorates collagen induced arthritis 
Arthritis and rheumatism  2011;63(8):2289-2298.
To determine the mechanism of action of IL-27 against rheumatoid arthritis (RA).
Adenovirus containing IL-27 transcript was constructed and was locally delivered into the ankles of collagen-induced arthritis (CIA) mice. Progression of arthritis was assessed in treated versus untreated mice by measuring ankle circumference and through histological analysis. IL-17 and its downstream targets as well as cytokines promoting TH-17 cell differentiation were quantified by ELISA in CIA ankles locally expressing adenoviral IL-27 versus controls. Ankles from both treatment groups were immunostained for neutrophil and monocyte migration. Last, vascularization was quantified by histology and by determining ankle hemoglobin levels.
Our results demonstrate that ectopic expression of IL-27 in CIA ameliorates inflammation, lining hypertrophy, and bone erosion compared to the control group. Serum and joint IL-17 levels were significantly reduced in the IL-27 treatment group compared to controls. Two of the main cytokines which induce TH-17 cell differentiation and IL-17 downstream target molecules were greatly downregulated in CIA ankles receiving forced expression of IL-27. The control mice had higher levels of vascularization and monocyte trafficking compared to those mice ectopically expressing IL-27.
Our results suggest that increased IL-27 levels relieve arthritis in CIA ankles. This amelioration of arthritis involves a reduction in CIA serum and joint IL-17 levels and results in decreased IL-17-mediated monocyte recruitment and angiogenesis. Hence, IL-27 may be a therapeutic target in RA.
PMCID: PMC3115512  PMID: 21384333
IL-27; collagen induced arthritis; IL-17; IL-1 and IL-6
11.  Characterization of CCL19 and CCL21 in Rheumatoid Arthritis 
Arthritis and rheumatism  2011;63(4):914-922.
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.
PMCID: PMC3079365  PMID: 21225692
CCL19; CCL21; RA synovial tissue fibroblast; macrophages and proangiogenic factors
13.  IL-17-mediated monocyte migration occurs partially through CCL2/MCP-1 induction1 
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.
PMCID: PMC2858914  PMID: 20228199
IL-17; CCL2/MCP-1; macrophages; synovial tissue fibroblasts; monocytes; rheumatoid arthritis
14.  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 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.
PMCID: PMC2857761  PMID: 20173024
IL-17; angiogenesis; HMVECs; migration; rheumatoid arthritis
15.  Interleukin-18 as an in vivo mediator of monocyte recruitment in rodent models of rheumatoid arthritis 
Arthritis Research & Therapy  2010;12(3):R118.
The function of interleukin-18 (IL-18) was investigated in pertinent animal models of rodent rheumatoid arthritis (RA) to determine its proinflammatory and monocyte recruitment properties.
We used a modified Boyden chemotaxis system to examine monocyte recruitment to recombinant human (rhu) IL-18 in vitro. Monocyte recruitment to rhuIL-18 was then tested in vivo by using an RA synovial tissue (ST) severe combined immunodeficient (SCID) mouse chimera. We defined monocyte-specific signal-transduction pathways induced by rhuIL-18 with Western blotting analysis and linked this to in vitro monocyte chemotactic activity. Finally, the ability of IL-18 to induce a cytokine cascade during acute joint inflammatory responses was examined by inducing wild-type (Wt) and IL-18 gene-knockout mice with zymosan-induced arthritis (ZIA).
We found that intragraft injected rhuIL-18 was a robust monocyte recruitment factor to both human ST and regional (inguinal) murine lymph node (LN) tissue. IL-18 gene-knockout mice also showed pronounced reductions in joint inflammation during ZIA compared with Wt mice. Many proinflammatory cytokines were reduced in IL-18 gene-knockout mouse joint homogenates during ZIA, including macrophage inflammatory protein-3α (MIP-3α/CCL20), vascular endothelial cell growth factor (VEGF), and IL-17. Signal-transduction experiments revealed that IL-18 signals through p38 and ERK½ in monocytes, and that IL-18-mediated in vitro monocyte chemotaxis can be significantly inhibited by disruption of this pathway.
Our data suggest that IL-18 may be produced in acute inflammatory responses and support the notion that IL-18 may serve a hierarchic position for initiating joint inflammatory responses.
PMCID: PMC2911912  PMID: 20565717
16.  IL-17 induces monocyte migration in rheumatoid arthritis 
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.
PMCID: PMC2811490  PMID: 19265168
IL-17; monocyte; migration; rheumatoid arthritis
17.  Monocyte chemoattractant protein-1 (MCP) and macrophage inflammatory protein-1α (MIP) as possible biomarkers for the chronic pelvic pain syndrome 
The Journal of urology  2008;179(5):1857-1862.
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).
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.
PMCID: PMC2786778  PMID: 18353390
chronic pelvic pain syndrome; prostatitis; cytokines; monocyte chemoattractant protein; macrophage inflammatory protein
18.  TH-17 cells in rheumatoid arthritis 
The 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.
Peripheral 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).
Treatment 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.
These 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.
PMCID: PMC2575607  PMID: 18710567
19.  CXCL16-Mediated Cell Recruitment to Rheumatoid Arthritis Synovial Tissue and Murine Lymph Nodes Is Dependent Upon the MAPK Pathway 
Arthritis and rheumatism  2006;54(3):765-778.
Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor α (TNFα), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process.
Using a human RA ST–SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo.
CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFα. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFα in vitro, suggesting that CXCL16 protein may function in large part independently of TNFα.
Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.
PMCID: PMC1472704  PMID: 16508941
20.  Amelioration of Rat Adjuvant-Induced Arthritis by Met-RANTES 
Arthritis and rheumatism  2005;52(6):1907-1919.
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.
PMCID: PMC1282452  PMID: 15934086
21.  Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues 
The focal adhesion kinase (FAK) family kinases, including FAK and proline-rich kinase 2 (Pyk)2, are the predominant mediators of integrin αvβ3 signaling events that play an important role in cell adhesion, osteoclast pathology, and angiogenesis, all processes important in rheumatoid arthritis (RA). Using immunohistochemical and western blot analysis, we studied the distribution of phospho (p)FAK, pPyk2, pSrc, pPaxillin and pPLCγ in the synovial tissue (ST) from patients with RA, osteoarthritis (OA) and normal donors (NDs) as well as in RA ST fibroblasts and peripheral blood differentiated macrophages (PB MΦs) treated with tumor necrosis factor-α (TNFα) or interleukin-1β (IL1β). RA and OA STs showed a greater percentage of pFAK on lining cells and MΦs compared with ND ST. RA ST fibroblasts expressed pFAK at baseline, which increased with TNFα or IL1β stimulation. Pyk2 and Src were phosphorylated more on RA versus OA and ND lining cells and MΦs. pPyk2 was expressed on RA ST fibrobasts but not in MΦs at baseline, however it was upregulated upon TNFα or IL1β activation in both cell types. pSrc was expressed in RA ST fibroblasts and MΦs at baseline and was further increased by TNFα or IL1β stimulation. pPaxillin and pPLCγ were upregulated in RA versus OA and ND lining cells and sublining MΦs. Activation of the FAK family signaling cascade on RA and OA lining cells may be responsible for cell adhesion and migration into the diseased STs. Therapies targeting this novel signaling pathway may be beneficial in RA.
PMCID: PMC2212559  PMID: 17963503
22.  Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue 
Arthritis Research  2002;4(3):201-208.
Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. We have compared the distribution of the receptor tyrosine kinase (Tie) and the Ang ligands in synovial tissues from normal subjects and those with rheumatoid arthritis (RA) and osteoarthritis (OA).
Immunohistochemical analysis was used to determine the expression of Ang-1, Ang-2, Tie1 and Tie2 in synovial tissue of normal subjects and those with RA and OA. Ang-1, Ang-2, Tie1 and Tie2 mRNA and protein expression were quantified in synovial tissues and RA synovial tissue fibroblasts with real-time reverse transcription polymerase chain reaction and western blot analysis.
In RA, Ang-1 positive immunostaining on lining cells, macrophages and endothelial cells was significantly higher than in OA and normal synovial tissue. The expression pattern of Ang-2 in synovial tissue was similar in RA and OA, whereas the Ang-2 expression was low in normal tissue. Synovial tissue from subjects with RA and OA showed a significant upregulation of Tie1 on lining cells, macrophages and endothelial cells compared to that from normal subjects. Tie2 was significantly upregulated in the RA and OA synovial tissue lining cells, macrophages and smooth muscle cells compared to normal synovial tissue. Generally Ang-1, Ang-2, Tie1 and Tie2 mRNA levels were higher in RA synovial tissue compared to normal and OA synovial tissues, and RA synovial tissue fibroblasts. Western blot analysis also demonstrated greater Tie1 and Tie2 protein expression in RA and OA synovial tissue compared to RA synovial tissue fibroblasts. In conclusion, the dominance of Ang-1 mRNA and protein expression over Ang-2 is in agreement with an active neovascularization in RA synovial tissue.
PMCID: PMC111023  PMID: 12010571
angiopoietin; mRNA; rheumatoid arthritis; Tie receptor expression

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