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1.  The Role of Glycoprotein 96 in the Persistent Inflammation of Rheumatoid Arthritis 
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.
doi:10.1016/j.abb.2012.12.008
PMCID: PMC3558611  PMID: 23257071
2.  Gp96 Perpetuates the Persistent Inflammation of Rheumatoid Arthritis 
Arthritis and rheumatism  2012;64(11):3638-3648.
Objective
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.
Methods
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.
Results
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.
Conclusions
These observations support the role of gp96 as an endogenous TLR2 ligand in RA and identify the TLR2 pathway as a therapeutic target.
doi:10.1002/art.34610
PMCID: PMC3473144  PMID: 22777994
3.  Role of the CCL21 and CCR7 pathway in rheumatoid arthritis angiogenesis 
Arthritis and rheumatism  2012;64(8):2471-2481.
Objective
These studies were performed to determine the role of CCL21 and its corresponding receptor CCR7 in the pathogenesis of Rheumatoid Arthritis (RA).
Methods
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.
Results
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.
Conclusion
These observations identify a novel function for CCL21 as an angiogenic mediator in RA, supporting CCL21/CCR7 as a therapeutic target in RA.
doi:10.1002/art.34452
PMCID: PMC3409328  PMID: 22392503
CCL21; CCR7; RA synovial fluid; angiogenesis and migration
4.  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.
doi:10.4049/jimmunol.1102977
PMCID: PMC3614068  PMID: 22661088
RA monocytes; TLR5; TNF-α; DAS28; RA fibroblasts and RA differentiated macrophages
5.  Prostate secretions from men with CP/CPPS inhibit proinflammatory mediators 
The Journal of urology  2010;184(4):1536-1542.
Purpose
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.
Results
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.
Conclusions
These results may have implications for the design of specific diagnostics and therapeutics targeted at complete resolution of prostate inflammatory insults.
doi:10.1016/j.juro.2010.05.086
PMCID: PMC3660015  PMID: 20723928
Chronic pelvic pain syndrome; prostatitis; chemokines; inflammation; pelvic pain
6.  Characterization of IL-7 and IL-7R in the pathogenesis of Rheumatoid Arthritis 
Arthritis and rheumatism  2011;63(10):2884-2893.
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1002/art.30493
PMCID: PMC3614067  PMID: 21647866
IL-7; IL-7R; RA synovial tissue fibroblast; macrophages and proangiogenic factors
7.  Myeloid Cell-Specific Fas Expression is Required to Prevent Systemic Autoimmunity 
Arthritis and Rheumatism  2012;64(3):808-820.
OBJECTIVE
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.
METHODS
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.
RESULTS
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.
CONCLUSION
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.
doi:10.1002/art.34317
PMCID: PMC3290732  PMID: 22143975
8.  Transcriptional diversity during monocyte to macrophage differentiation 
Immunology letters  2008;117(1):70-80.
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.
doi:10.1016/j.imlet.2007.12.012
PMCID: PMC2838727  PMID: 18276018
mononuclear phagocytes; gene regulation; transcription factors
9.  Characterization of CCL19 and CCL21 in Rheumatoid Arthritis 
Arthritis and rheumatism  2011;63(4):914-922.
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1002/art.30232
PMCID: PMC3079365  PMID: 21225692
CCL19; CCL21; RA synovial tissue fibroblast; macrophages and proangiogenic factors
10.  More than just B-cell inhibition 
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.
doi:10.1186/ar3439
PMCID: PMC3239370  PMID: 21878134
11.  Activated TLR Signaling in Atherosclerosis among Women with Lower Framingham Risk Score: The Multi-Ethnic Study of Atherosclerosis 
PLoS ONE  2011;6(6):e21067.
Background
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.
Results
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.
Conclusion
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.
doi:10.1371/journal.pone.0021067
PMCID: PMC3116882  PMID: 21698167
12.  Innate Immunity and Rheumatoid Arthritis 
doi:10.1016/j.rdc.2010.03.004
PMCID: PMC2937347  PMID: 20510234
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.
doi:10.4049/jimmunol.0901942
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.
doi:10.4049/jimmunol.0903271
PMCID: PMC2857761  PMID: 20173024
IL-17; angiogenesis; HMVECs; migration; rheumatoid arthritis
15.  ROLE OF TOLL LIKE RECEPTORS IN RHEUMATOID ARTHRITIS 
Current rheumatology reports  2009;11(5):357-364.
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.
PMCID: PMC2913446  PMID: 19772831
16.  The synovial lining micromass system: Towards RA in a dish? 
Arthritis and rheumatism  2010;62(3):643-646.
doi:10.1002/art.27297
PMCID: PMC2878733  PMID: 20187157
17.  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 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.
doi:10.4049/jimmunol.0801563
PMCID: PMC2814438  PMID: 19342676
gp96; grp94; macrophages; TLR2; TLR4; Rheumatoid Arthritis
18.  Bim-BH3 mimetic therapy is effective at suppressing inflammatory arthritis through the activation of myeloid cell apoptosis 
Arthritis and rheumatism  2010;62(2):441-451.
Objective
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.
Methods
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.
Results
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.
Conclusion
These data demonstrate that BH3 mimetic therapy may have significant potential for RA treatment.
doi:10.1002/art.27198
PMCID: PMC2848986  PMID: 20112357
Bim; arthritis; macrophages; apoptosis
19.  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.
doi:10.4049/jimmunol.0802246
PMCID: PMC2811490  PMID: 19265168
IL-17; monocyte; migration; rheumatoid arthritis
20.  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.
PURPOSE
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.
RESULTS
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.
CONCLUSIONS
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.
doi:10.1016/j.juro.2008.01.028
PMCID: PMC2786778  PMID: 18353390
chronic pelvic pain syndrome; prostatitis; cytokines; monocyte chemoattractant protein; macrophage inflammatory protein
21.  APIGENIN, A DIETARY FLAVONOID, SENSITIZES HUMAN T CELLS FOR ACTIVATION-INDUCED CELL DEATH BY INHIBITING PKB/Akt AND NF-κB ACTIVATION PATHWAY 
Immunology letters  2008;121(1):74-83.
Resistance of T cells to activation induced cell death (AICD) is associated with autoimmunity and lymphoproliferation. We found that apigenin (4′,5,7-Trihydroxyflavone), a nonmutagenic dietary flavonoid, augmented both extrinsic and intrinsic pathways of apoptosis in recurrently activated, but not in primarily stimulated, human blood CD4+ T cells. Apigenin potentiated AICD by inhibiting NF-κB activation and suppressing NF-κB-regulated anti-apoptotic molecules, cFLIP, Bcl-xL, Mcl-1, XIAP and IAP, but not Bcl-2. Apigenin suppressed NF-κB translocation to nucleus and inhibited I-κBα phosphorylation and degradation in response to TCR stimulation in re-activated peripheral blood CD4 T cells, as well as in leukemic Jurkat T cell lines. Among the pathways that lead to NF-κB activation upon TCR stimulation, apigenin selectively inhibited PI3K-PKB/Akt, but not PKC-θ activation in the human T cells, and synergized with a PI3K inhibitor to markedly augment AICD. Apigenin also suppressed expression of anti-apoptotic cyclooxygenase 2 (COX-2) protein in activated human T cells, but it did not affect activation of Erk MAPKinase. Thus, in chronically activated human T cells, relatively non-toxic apigenin can suppress anti-apoptotic pathways involving NF-κB activation, and especially cFLIP and COX-2 expression that are important for functioning and maintenance of immune cells in inflammation, autoimmunity and lymphoproliferation.
doi:10.1016/j.imlet.2008.08.004
PMCID: PMC2610846  PMID: 18812189
Apoptosis; T cells; Human; Signal Transduction; Autoimmunity
22.  The cyclin dependent kinase domain of p21 is a suppressor of IL-1β-mediated inflammation in activated macrophages 
European journal of immunology  2009;39(3):820-825.
Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here we demonstrate that p21(WAF1/CIP1), an established suppressor of cell cycle progression, is an inhibitor of IL-1β synthesis in macrophages. Mice deficient for p21 (p21−/−) display increased susceptibility to endotoxic shock which is associated with increased serum levels of IL-1β. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21−/− mice. Analysis of isolated macrophages, which are one of the central producers of IL-1β, reveals that deficiency for p21 led to more IL-1β mRNA and pro-protein synthesis following toll-like receptor (TLR) ligation. The increase in IL-1β pro-protein is associated with elevated secretion of active IL-1β by p21−/− macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1β secretion as well. A peptide mapping strategy shows that the cyclin dependent kinase (CDK) binding domain of p21 is sufficient to reduce the secretion of IL-1β by p21−/− macrophages. These data suggest a novel role for p21 and specifically for the CDK binding domain of p21(WAF1/CIP1) in inhibiting inflammation.
doi:10.1002/eji.200838683
PMCID: PMC2734089  PMID: 19189309
p21; macrophages; IL-1β; endotoxic shock; inflammation
23.  TNFα-induced macrophage death via caspase-dependent and independent pathways 
Macrophages are the principal source of TNFα, yet they are highly resistant to TNFα-mediated cell death. Previously, employing in vitro differentiated human macrophages, we showed that following the inhibition of NF-κB, TNFα-induced caspase-8 activation contributes to DNA fragmentation but is not necessary for the loss of the inner mitochondrial transmembrane potential (ΔΨm) or cell death. We here extend these observations to demonstrate that, when NF-κB is inhibited in macrophages, TNFα alters lysosomal membrane permeability (LMP). This results in the release of cathepsin B with subsequent loss of ΔΨm and caspase-8 independent cell death. Interestingly, the cytoprotective, NF-κB-dependent protein A20 was rapidly induced in macrophages treated with TNFα. Ectopic expression of A20 in macrophages preserves LMP following treatment with TNFα, and as a result, mitochondrial integrity is safeguarded and macrophages are protected from cell death. These observations demonstrate that TNFα triggers both caspase 8-dependent and -independent cell death pathways in macrophages and identify a novel mechanism by which A20 protects these cells against both pathways.
doi:10.1007/s10495-009-0311-4
PMCID: PMC2692057  PMID: 19152111
apoptosis; caspase-8; cathepsin B; A20
24.  TH-17 cells in rheumatoid arthritis 
Introduction
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.
Methods
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).
Results
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.
Conclusion
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.
doi:10.1186/ar2477
PMCID: PMC2575607  PMID: 18710567
25.  Amelioration of Rat Adjuvant-Induced Arthritis by Met-RANTES 
Arthritis and rheumatism  2005;52(6):1907-1919.
Objective
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.
Methods
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.
Results
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.
Conclusion
The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.
doi:10.1002/art.21033
PMCID: PMC1282452  PMID: 15934086

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