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Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis
Gattorno, Marco
Prigione, Ignazia
Morandi, Fabio
Gregorio, Andrea
Chiesa, Sabrina
Ferlito, Francesca
Favre, Anna
Uccelli, Antonio
Gambini, Claudio
Martini, Alberto
Pistoia, Vito
Arthritis Research & Therapy
The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-γ by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-γ+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.
chemokines; memory T lymphocytes; juvenile idiopathic arthritis
Related Articles
Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis
Nanki, Toshihiro
Takada, Kazuki
Komano, Yukiko
Morio, Tomohiro
Kanegane, Hirokazu
Nakajima, Atsuo
Lipsky, Peter E
Miyasaka, Nobuyuki
Arthritis Research & Therapy
Accumulation of B cells in the rheumatoid arthritis (RA) synovium has been reported, and it has been thought that these cells might contribute to the pathogenesis of RA by antigen presentation, autoantibody production, and/or inflammatory cytokine production. Chemokines could enhance the accumulation of B cells in the synovium. The aims of this study were to determine chemokine receptor expression by B cells both in the peripheral blood of normal donors and subjects with RA, and at the inflammatory site in RA, and the effects of chemokines on B cell activation.
Cell surface molecule expression was analyzed by flow cytometry. Cellular migration was assessed using chemotaxis chambers. Cellular proliferation was examined by 3H-thymidine incorporation. Tumor necrosis factor (TNF) production was assayed by enzyme-linked immunosorbent assay.
Significant numbers of peripheral blood B cells of healthy donors and subjects with RA expressed CC chemokine receptor (CCR)5 and CXCR3, and most B cells expressed CCR6, CCR7, CXCR4 and CXCR5. CCR5 expression was more frequent on CD27+ than CD27- peripheral blood B cells of healthy donors and RA. Synovial B cells more frequently expressed CCR5, but less often expressed CCR6, CCR7 and CXCR5 compared to peripheral blood in RA. Further functional analyses were performed on peripheral blood B cells from healthy donors. Migration of peripheral blood B cells, especially CD27+ B cells, was enhanced by CC chemokine ligand (CCL)20, CCL19, CCL21 and CXCL12. All four chemokines alone induced B cell proliferation; with CCL21 being the most effective. CCL21 also enhanced the proliferation of anti-immunoglobulin (Ig)M-stimulated B cells and blockade of CCR7 inhibited this effect. CCL20, CCL21 and CXCL12 enhanced TNF production by anti-IgM mAb-stimulated B cells. Finally, stimulation with CXCL12, but not CCL20, CCL19 and CCL21, enhanced inducible costimulator-ligand (ICOSL) expression by peripheral blood B cells of healthy donors and RA, but did not increase B cell-activating factor receptor or transmembrane activator and CAML-interactor.
The data suggest that CCR5, CCR6, CCR7, CXCR3, CXCR4 and CXCR5 may be important for the B cell migration into the synovium of RA patients, and also their local proliferation, cytokine production and ICOSL expression in the synovium.
Related Articles
Why CCR2 and CCR5 Blockade Failed and Why CCR1 Blockade Might Still Be Effective in the Treatment of Rheumatoid Arthritis
Lebre, Maria C.
Vergunst, Clarissa E.
Choi, Ivy Y. K.
Aarrass, Saïda
Oliveira, Ana S. F.
Wyant, Tim
Horuk, Richard
Reedquist, Kris A.
Tak, Paul P.
PLoS ONE
The aim of this study was to provide more insight into the question as to why blockade of CCR1, CCR2, and CCR5 may have failed in clinical trials in rheumatoid arthritis (RA) patients, using an in vitro monocyte migration system model.
Methodology/Principal Findings
Monocytes from healthy donors (HD; n = 8) or from RA patients (for CCR2 and CCR5 antibody n = 8; for CCR1 blockade n = 13) were isolated from peripheral blood and pre-incubated with different concentrations of either anti-CCR1, anti-CCR2, or anti-CCR5 blocking antibodies (or medium or isotype controls). In addition, a small molecule CCR1 antagonist (BX471) was tested. Chemotaxis was induced by CCL2/MCP-1 (CCR2 ligand), CCL5/RANTES (CCR1 and CCR5 ligand), or by a mix of 5 RA synovial fluids (SFs), and cellular responses compared to chemotaxis in the presence of medium alone. Anti-CCR2 antibody treatment blocked CCL2/MCP-1-induced chemotaxis of both HD and RA monocytes compared to isotype control. Similarly, anti-CCR5 antibody treatment blocked CCL5/RANTES-induced chemotaxis of RA monocytes. While neither CCR2 nor CCR5 blocking antibodies were able to inhibit SF-induced monocyte chemotaxis, even when both receptors were blocked simultaneously, both anti-CCR1 antibodies and the CCR1 antagonist were able to inhibit SF-induced monocyte chemotaxis.
The RA synovial compartment contains several ligands for CCR1, CCR2, and CCR5 as well as other chemokines and receptors involved in monocyte recruitment to the site of inflammation. The results suggest that CCR2 and CCR5 are not critical for the migration of monocytes towards the synovial compartment in RA. In contrast, blockade of CCR1 may be effective. Conceivably, CCR1 blockade failed in clinical trials, not because CCR1 is not a good target, but because very high levels of receptor occupancy at all times may be needed to inhibit monocyte migration in vivo.
Related Articles
Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice
Yokoyama, Waka
Kohsaka, Hitoshi
Kaneko, Kayoko
Walters, Matthew
Takayasu, Aiko
Fukuda, Shin
Miyabe, Chie
Miyabe, Yoshishige
Love, Paul E
Nakamoto, Nobuhiro
Kanai, Takanori
Watanabe-Imai, Kaori
Charvat, Trevor T
Penfold, Mark ET
Jaen, Juan
Schall, Thomas J
Harigai, Masayoshi
Miyasaka, Nobuyuki
Nanki, Toshihiro
Arthritis Research & Therapy
Biological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn's disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA.
CCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b+ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted.
CCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b+ splenocytes into the synovial tissues.
The interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.
Related Articles
Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium
Nanki, Toshihiro
E Lipsky, Peter
Arthritis Research
IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4+ memory T cells, whereas CD4+ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4+ cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α . A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected.
In RA large numbers of CD4+ memory T cells infiltrate the inflamed synovium [1,2,3]. The accumulated CD4+ memory T cells in the RA synovium appear to be activated, because they express cytokines and activation markers [4,5,6,7,8]. Expressed cytokines and activation markers should play important roles in the pathogenesis of RA. However, the frequency of cytokine expression by RA synovial CD4+ T cells has not been analyzed accurately. Recently, the roles of chemokine and chemokine receptor interactions in T-cell migration have been intensively examined. Interactions of chemokine and chemokine receptors might therefore be important in the accumulation of the CD4+ T cells in the RA synovium. Accordingly, correlation of cytokine and chemokine receptor expression might be important in delineating the function and potential means of accumulation of individual CD4+ memory T cells in the RA synovium.
In the present study we analyzed cytokine (IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ , TNF-α , and LT-α ), activation marker (CD154 [CD40 ligand] and TRANCE - also called receptor activator of nuclear factor κ B ligand [RANKL] or osteoclast differentiation factor [ODF]), and chemokine receptor expression by individual CD4+ memory T cells isolated from rheumatoid synovium and blood. To achieve this we employed a single-cell reverse transcription (RT) polymerase chain reaction (PCR) technique. This technique made it possible to correlate mRNAs expressed by individual CD4+ memory T cells in the synovium and blood.
Materials and method:
Synovial tissues from three RA patients and peripheral blood mononuclear cells from two RA patients and a normal donor were analyzed.
Cytokine (IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, and LT-α ) and activation marker (CD154 and TRANCE) expression by individual CD4+CD45RO+ T cells from RA synovium or blood were analyzed using a single-cell RT-PCR. In brief, single CD4+CD45RO+T cells was sorted into each well of a 96-well PCR plate using a flow cytometer. cDNA from individual cells was prepared, and then the cDNA was nonspecifically amplified. The product was then amplified by PCR using gene-specific primers to analyze cytokine and activation marker expression.
Cytokine and activation marker expression by individual CD4+CD45RO+T cells from RA synovial tissues was analyzed using a single-cell RT-PCR method. Expression of mRNAs was analyzed in 152 individual synovial tissue CD4+CD45RO+ T cells sorted from three RA patients in which T-cell receptor (TCR) Cβ mRNA was detected. Frequencies of CD4+ memory T cells expressing cytokine and activation marker mRNA in RA synovium are shown in Table 1. IL-2, IL-4, and IL-6 were not expressed by the synovial tissue CD4+CD45RO+ T cells, whereas 2-20% of cells expressed the other cytokine mRNAs.
Few correlations between cytokine and activation marker mRNAs were observed. Notably, no cells contained both IFN-γ and LT-α mRNAs, cytokines that are thought to define the T-helper (Th)1 phenotype [9]. However, the frequency of TRANCE-positive cells in IL-10-positive cells was significantly higher than that in IL-10-negative cells (Table 2). Moreover, the frequency of TRANCE-positive cells in TNF-α-positive cells was also significantly higher than that in TNF-α-negative cells.
Varying percentages of CD4+ memory T cells expressed CC and CXC chemokine receptors. The frequency of CCR5-positive cells in IFN-γ-positive cells was significantly higher than that in IFN-γ-negative cells, whereas the frequency of CCR6-positive cells in LT-α-positive cells was significantly higher than that in LT-α-negative cells, and the frequency of CCR7-positive cells in IL-10-positive cells was significantly higher than that in IL-10-negative cells. Furthermore, the frequency of CXCR4-positive cells in TRANCE-positive cells was significantly higher than that in TRANCE-negative cells.
Expression of cytokine and activation marker mRNAs was also analyzed in 48 individual peripheral blood CD4+CD45RO+ T cells from two RA patients. IL-2, IL-4, IL-6, and LT-α were not expressed by the peripheral CD4+CD45RO+ T cells, whereas 4-17% of cells expressed the other markers. The most striking difference between synovial tissue and peripheral blood CD4+ memory T cells was the presence of LT-α expression in the former, but not in the latter. IFN-γ and TNF-α were not expressed by normal peripheral blood CD4+ memory T cells, although they were expressed by RA peripheral blood CD4+ memory T cells.
The present study employed a single-cell PCR technology to analyze cytokine expression by unstimulated RA synovial tissue CD4+ memory T cells immediately after isolation, without in vitro manipulation. The results confirm the Th1 nature of rheumatoid inflammation. It is noteworthy that no individual synovial CD4+ memory T cells expressed both IFN-γ and LT-α mRNAs, even though these are the prototypic Th1 cytokines [9]. These results imply that, in the synovium, regulation of IFN-γ and LT-α must vary in individual cells, even though both Th1 cytokines can be produced.
The present data showed that CCR5 expression correlated with IFN-γ but not with LT-α expression by synovial CD4+ memory T cells. It has been reported that CCR5 expression is upregulated in RA synovial fluid and synovial tissue T cells [10,11,12] and that CCR5 Δ 32 deletion may have an influence on clinical manifestations of RA [13], suggesting that CCR5 might play an important role in RA. Recently, it has been claimed that CCR5 was preferentially expressed by Th1 cell lines [14,15]. However, in the present study CCR5 was not expressed by all IFN-γ-expressing cells. Moreover, CCR5 expression did not correlate with expression of LT-α by RA synovial CD4+ memory T cells. Therefore, it is unclear whether CCR5 is a marker of Th1 cells in RA synovium.
IL-10 expression correlated with CCR7 expression by RA synovial CD4+ memory T cells. Recently, it was reported [16] that in the blood CCR7+CD4+ memory T cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells. These cells may play a unique role in the synovium as opposed to in the blood. By producing IL-10, they might have an immunoregulatory function. In addition, IL-10 expression also correlated with expression of TRANCE. Although it is possible that IL-10 produced by these cells inhibited T-cell activation in the synovium, TRANCE expressed by these same cells might function to activate dendritic cells and indirectly stimulate T cells, mediating inflammation in the synovium. These results imply that individual T cells in the synovium might have different, and sometimes opposite functional activities.
LT-α expression correlated with CCR6 expression by synovial CD4+ memory T cells. It has been reported that CCR6 is expressed by resting peripheral memory T cells [17], whereas LT-α expression is associated with the presence of lymphocytic aggregates in synovial tissue [7]. The correlation between the expression of these two markers therefore suggests the possibility that CCR6 may play a role in the development of aggregates of CD4+ T cells that are characteristically found in rheumatoid synovium.
TRANCE is known to be expressed by activated T cells, and can stimulate dendritic cells and osteoclasts [18]. Of note, TRANCE-mediated activation of osteoclasts has recently been shown [19] to play an important role in the damage to bone that is found in experimental models of inflammatory arthritis. It is therefore of interest that TRANCE was expressed by 3-16% of the RA synovial CD4+ memory T cells. Of note, 67% of TNF-α-positive cells expressed TRANCE. In concert, TNF-α and TRANCE expressed by this subset of CD4+ memory T cells might make them particularly important in mediating the bony erosions that are characteristic of RA.
Interestingly, there was a correlation between expression of IFN-γ and IL-10 in RA peripheral blood CD4+ memory T cells. In RA peripheral blood, CD154 expression correlated with that of CXCR3 by CD4+ memory T cells. It has been claimed [15] that CXCR3 is preferentially expressed by in vitro generated Th1 cells. However, in the present study CXCR3 did not correlate with IFN-γ expression. Although IFN-γ and TNF-α mRNAs were expressed in vivo by peripheral blood CD4+ T cells from RA patients, LT-α mRNA was not detected, whereas IFN-γ , TNF-α , and LT-α were not detected in samples from healthy donors. These findings indicate that RA peripheral blood CD4+ memory T cells are stimulated in vivo, although they do not express LT-α mRNA. The present studies indicate that the frequencies of CD4+ memory T cells that expressed IFN-γ in the blood and in the synovium are comparable. These results imply that activated CD4+ memory T cells migrate between blood and synovium, although the direction of the trafficking is unknown. The presence of LT-α mRNA in synovium, but not in blood, indicates that CD4+ memory cells are further activated in the synovium, and that these activated CD4+ memory T cells are retained in the synovium until LT-α mRNA decreases.
In conclusion, CD4+ memory T cells are biased toward Th1 cells in RA synovium and peripheral blood. In the synovium, IFN-γ and LT-α were produced by individual cells, whereas in the rheumatoid blood no LT-α-producing cells were detected. Furthermore, there were modest correlations between individual cells that expressed particular cytokines, such as IL-10, and certain chemokine receptor mRNAs.
chemokine receptor; cytokine; rheumatoid arthritis; T lymphocyte
Related Articles
Green tea extract inhibits chemokine production, but up-regulates chemokine receptor expression, in rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis
Marotte, Hubert
Ruth, Jeffrey H.
Campbell, Phillip L.
Koch, Alisa E.
Ahmed, Salahuddin
Rheumatology (Oxford, England)
Objective. Evaluation of the efficacy of green tea extract (GTE) in regulating chemokine production and chemokine receptor expression in human RA synovial fibroblasts and rat adjuvant-induced arthritis (AIA).
Methods. Fibroblasts isolated from human RA synovium were used in the study. Regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, growth-regulated oncogene (GRO)α/CXCL1 and IL-8/CXCL8 production was measured by ELISA. Western blotting was used to study the phosphorylation of protein kinase C (PKC)δ and c-Jun N-terminal kinases (JNK). Chemokine and chemokine receptor expression was determined by quantitative RT–PCR. The benefit of GTE administration in rat AIA was determined.
Results. GTE (2.5–40 μg/ml) inhibited IL-1β-induced MCP-1/CCL2 (10 ng/ml), RANTES/CCL5, GROα/CXCL1 and IL-8/CXCL8 production in human RA synovial fibroblasts (P < 0.05). However, GTE inhibited MCP-1/CCL2 and GROα/CXCL1 mRNA synthesis in RA synovial fibroblasts. Furthermore, GTE also inhibited IL-1β-induced phosphorylation of PKCδ, the signalling pathway mediating IL-1β-induced chemokine production. Interestingly, GTE preincubation enhanced constitutive and IL-1β-induced CCR1, CCR2b, CCR5, CXCR1 and CXCR2 receptor expression. GTE administration (200 mg/kg/day p.o.) modestly ameliorated rat AIA, which was accompanied by a decrease in MCP-1/CCL2 and GROα/CXCL1 levels and enhanced CCR-1, -2, -5 and CXCR1 receptor expression in the joints of GTE administered rats.
Conclusions. Chemokine receptor overexpression with reduced chemokine production by GTE may be one potential mechanism to limit the overall inflammation and joint destruction in RA.
Green tea; Chemokines; Chemokine receptors; Rheumatoid arthritis; Synovial fibroblasts; Complementary and alternative medicine; Adjuvant-induced arthritis
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Regulation of Inflammatory Chemokine Receptors on Blood T Cells Associated to the Circulating Versus Liver Chemokines in Dengue Fever
de-Oliveira-Pinto, Luzia Maria
Marinho, Cíntia Ferreira
Povoa, Tiago Fajardo
de Azeredo, Elzinandes Leal
de Souza, Luiza Assed
Barbosa, Luiza Damian Ribeiro
Motta-Castro, Ana Rita C.
Alves, Ada M. B.
Ávila, Carlos André Lins
de Souza, Luiz José
da Cunha, Rivaldo Venâncio
Damasco, Paulo Vieira
Paes, Marciano Viana
Kubelka, Claire Fernandes
PLoS ONE
Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES+ cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44HIGH and CD127LOW markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.
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Borrelia burgdorferi stimulation of chemokine secretion by cells of monocyte lineage in patients with Lyme arthritis
Shin, Junghee J
Strle, Klemen
Glickstein, Lisa J
Luster, Andrew D
Steere, Allen C
Arthritis Research & Therapy
Joint fluid in patients with Lyme arthritis often contains high levels of CCL4 and CCL2, which are chemoattractants for monocytes and some T cells, and CXCL9 and CXCL10, which are chemoattractants for CD4+ and CD8+ T effector cells. These chemokines are produced primarily by cells of monocyte lineage in TH1-type immune responses. Our goal was to begin to learn how infection with Borrelia burgdorferi leads to the secretion of these chemokines, using patient cell samples. We hypothesized that B. burgdorferi stimulates chemokine secretion from monocytes/macrophages in multiple ways, thereby linking innate and adaptive immune responses.
Peripheral blood mononuclear cells (PBMC) from 24 Lyme arthritis patients were stimulated with B. burgdorferi, interferon (IFN)-γ, or both, and the levels of CCL4, CCL2, CXCL9 and CXCL10 were measured in culture supernatants. CD14+ monocytes/macrophages from PBMC and synovial fluid mononuclear cells (SFMC) were stimulated in the same way, using available samples. CXCR3, the receptor for CXCL9 and CXCL10, and CCR5, the receptor for CCL4, were assessed on T cells from PBMC and SFMC.
In patients with Lyme arthritis, B. burgdorferi but not IFN-γ induced PBMC to secrete CCL4 and CCL2, and B. burgdorferi and IFN-γ each stimulated the production of CXCL9 and CXCL10. However, with the CD14+ cell fraction, B. burgdorferi alone stimulated the secretion of CCL4; B. burgdorferi and IFN-γ together induced CCL2 secretion, and IFN-γ alone stimulated the secretion of CXCL9 and CXCL10. The percentage of T cells expressing CXCR3 or CCR5 was significantly greater in SFMC than PBMC, confirming that TH1 effector cells were recruited to inflamed joints. However, when stimulated with B. burgdorferi or IFN-γ, SFMC and PBMC responded similarly.
B. burgdorferi stimulates PBMC or CD14+ monocytes/macrophages directly to secrete CCL4, but spirochetal stimulation of other intermediate cells, which are present in PBMC, is required to induce CD14+ cells to secrete CCL2, CXCL9 and CXCL10. We conclude that B. burgdorferi stimulates monocytes/macrophages directly and indirectly to guide innate and adaptive immune responses in patients with Lyme arthritis.
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CCR2 expressing CD4+ T lymphocytes are preferentially recruited to the ileum in Crohn's disease
Connor, S J
Paraskevopoulos, N
Newman, R
Cuan, N
Hampartzoumian, T
Lloyd, A R
Grimm, M C
Background and aims: Chemokine receptors are key determinants of leucocyte trafficking. While the chemokine receptor CCR9 and its chemokine ligand CCL25 (TECK) mediate lymphocyte homing to the healthy small intestine, the chemokine receptors important for recruitment during intestinal inflammation are undefined. Animal studies have suggested potential roles for CCR2 and CCR5 in inflammatory bowel disease (IBD). The aim of this study was to understand the role of CCR2 in human IBD.
Methods: Resections of ileum or colon were obtained from patients undergoing surgery for small bowel Crohn's disease (SBCD; n = 10), Crohn's colitis (n = 5), ulcerative colitis (n = 6), and non-IBD related conditions (control ileum n = 11; control colon n = 11). Expression of CCR2 by lamina propria lymphocytes (LPLs) was determined by both flow cytometry and immunohistochemistry. As a functional correlate, chemotaxis assays using the CCR2 ligand, CCL2 (MCP-1), were performed. Expression of CCR2 by peripheral blood lymphocytes was determined by flow cytometry.
Results: There were greater than 30-fold more CCR2+ LPLs in SBCD than in control ileum (29.3% (19.9–55.1) v 0.9% (0.4–11.5); p = 0.0007). Specifically, CCR2+CD4+ LPLs were increased (p = 0.002) whereas CCR2+CD8+ LPLs were not. Increased expression included both memory (CD45RO+; p = 0.005) and naïve (CD45RO−; p = 0.01) CCR2+ populations. The increase in CCR2+ LPLs in SBCD was confirmed by both immunohistochemistry (p = 0.0002) and enhanced chemotactic responses to CCL2. CCR2 expression was not increased in the peripheral blood of patients with SBCD, suggesting ongoing recruitment of the CCR2+ population to the ileum. In contrast with SBCD, there was no significant increase in CCR2+ LPLs in Crohn's colitis or ulcerative colitis samples.
Conclusions: The chemokine receptor CCR2 appears to be an important contributor to accumulation of CD4+ T lymphocytes in the ileum in small bowel Crohn's disease. Blockade of CCR2 may provide a novel therapeutic alternative.
inflammatory bowel disease; mucosal immunology; T cell trafficking; chemokine receptors; CCR2
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Differential expression of chemokine receptors on peripheral blood B cells from patients with rheumatoid arthritis and systemic lupus erythematosus
Henneken, Maren
Dörner, Thomas
Burmester, Gerd-Rüdiger
Berek, Claudia
Arthritis Research & Therapy
Chemokines and their receptors are essential in the recruitment and positioning of lymphocytes. To address the question of B cell migration into the inflamed synovial tissue of patients with rheumatoid arthritis (RA), peripheral blood naive B cells, memory B cells and plasma cells were analyzed for cell surface expression of the chemokine receptors CXCR3, CXCR4, CXCR5, CCR5, CCR6, CCR7 and CCR9. For comparison, B cells in the peripheral blood of patients with the autoimmune disease systemic lupus erythematosus (SLE) or with the degenerative disease osteoarthritis (OA) were analyzed. Expression levels of chemokine receptors were measured by flow cytometry and were compared between the different patient groups and healthy individuals. The analysis of chemokine receptor expression showed that the majority of peripheral blood B cells is positive for CXCR3, CXCR4, CXCR5, CCR6 and CCR7. Whereas a small fraction of B cells were positive for CCR5, practically no expression of CCR9 was found. In comparison with healthy individuals, in patients with RA a significant fraction of B cells showed a decreased expression of CXCR5 and CCR6 and increased levels of CXCR3. The downregulation of CXCR5 correlated with an upregulation of CXCR3. In patients with SLE, significant changes in CXCR5 expression were seen. The functionality of the chemokine receptors CXCR3 and CXCR4 was demonstrated by transmigration assays with the chemokines CXCL10 and CXCL12, respectively. Our results suggest that chronic inflammation leads to modulation of chemokine receptor expression on peripheral blood B cells. However, differences between patients with RA and patients with SLE point toward a disease-specific regulation of receptor expression. These differences may influence the migrational behavior of B cells.
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Role of the CCL21 and CCR7 pathway in rheumatoid arthritis angiogenesis
Pickens, Sarah R.
Chamberlain, Nathan D.
Volin, Michael V.
Pope, Richard M.
Talarico, Nicholas E
Mandelin, Arthur M.
Shahrara, Shiva
Arthritis and rheumatism
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
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Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10
Nogueira, Luciana Gabriel
Santos, Ronaldo Honorato Barros
Ianni, Barbara Maria
Fiorelli, Alfredo Inácio
Mairena, Eliane Conti
Benvenuti, Luiz Alberto
Frade, Amanda
Donadi, Eduardo
Dias, Fabrício
Saba, Bruno
Wang, Hui-Tzu Lin
Fragata, Abilio
Sampaio, Marcelo
Hirata, Mario Hiroyuki
Buck, Paula
Mady, Charles
Bocchi, Edimar Alcides
Stolf, Noedir Antonio
Kalil, Jorge
Cunha-Neto, Edecio
PLoS Neglected Tropical Diseases
Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.
Methods and Results
Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.
Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.
Author Summary
Chronic Chagas cardiomyopathy (CCC) is an inflammatory heart disease that affects millions in Latin America, and in growing numbers in USA and Europe. Survival among CCC patients is shorter than among patients with cardiomyopathy of non-inflammatory etiology. This suggests that the inflammatory cell influx plays an important pathogenic role in CCC. However, little is known about the factors that maintain this myocardial inflammation. We hypothesized that Th1 T cell-attracting chemokines, involved in driving leukocyte migration, could play a role in myocardial inflammation. Herein, we have analyzed expression of several chemokines and receptors in heart tissue from patients with CCC and controls. We found inflammatory cells expressing chemokines and receptors consistent with Th1 T cell influx into CCC myocardium. mRNA expression levels of the chemokine CXCL9 correlated with inflammation. We also studied whether genetic variations in these genes could be associated to CCC development. Polymorphisms in CXCL9, CXCL10 and CCR5 were associated to differential risk of progression to the more severe form of CCC. Polymorphisms of CXCL9 and CXCL10 were also associated to the intensity of myocardial inflammation and chemokine expression. These results suggest that such chemokines may be master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to severe CCC.
Related Articles
Amelioration of Rat Adjuvant-Induced Arthritis by Met-RANTES
Shahrara, Shiva
Proudfoot, Amanda E. I.
Woods, James M.
Ruth, Jeffrey H.
Amin, M. Asif
Park, Christy C.
Haas, Christian S.
Pope, Richard M.
Haines, G. Kenneth
Zha, Yuan Yuan
Koch, Alisa E.
Arthritis and rheumatism
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.
Related Articles
Expression and regulation of CCL18 in synovial fluid neutrophils of patients with rheumatoid arthritis
Auer, Judith
Bläss, Markus
Schulze-Koops, Hendrik
Russwurm, Stefan
Nagel, Thomas
Kalden, Joachim R
Röllinghoff, Martin
Beuscher, Horst Ulrich
Arthritis Research & Therapy
Rheumatoid arthritis (RA) is characterized by the recruitment of leukocytes and the accumulation of inflammatory mediators within the synovial compartment. Release of the chemokine CCL18 has been widely attributed to antigen-presenting cells, including macrophages and dendritic cells. This study investigates the production of CCL18 in polymorphonuclear neutrophils (PMN), the predominant cell type recruited into synovial fluid (SF). Microarray analysis, semiquantitative and quantitative reverse transcriptase polymerase chain reaction identified SF PMN from patients with RA as a novel source for CCL18 in diseased joints. Highly upregulated expression of other chemokine genes was observed for CCL3, CXCL8 and CXCL10, whereas CCL21 was downregulated. The chemokine receptor genes were differentially expressed, with upregulation of CXCR4, CCRL2 and CCR5 and downregulation of CXCR1 and CXCR2. In cell culture experiments, expression of CCL18 mRNA in blood PMN was induced by tumor necrosis factor α, whereas synthesis of CCL18 protein required additional stimulation with a combination of IL-10 and vitamin D3. In comparison, recruited SF PMN from patients with RA were sensitized for CCL18 production, because IL-10 alone was sufficient to induce CCL18 release. These results suggest a release of the T cell-attracting CCL18 by PMN when recruited to diseased joints. However, its production is tightly regulated at the levels of mRNA expression and protein synthesis.
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Monocytes/macrophages express chemokine receptor CCR9 in rheumatoid arthritis and CCL25 stimulates their differentiation
Schmutz, Caroline
Cartwright, Alison
Williams, Helen
Haworth, Oliver
Williams, John HH
Filer, Andrew
Salmon, Mike
Buckley, Christopher D
Middleton, Jim
Arthritis Research & Therapy
Monocytes/macrophages accumulate in the rheumatoid (RA) synovium where they play a central role in inflammation and joint destruction. Identification of molecules involved in their accumulation and differentiation is important to inform therapeutic strategies. This study investigated the expression and function of chemokine receptor CCR9 in the peripheral blood (PB) and synovium of RA, non-RA patients and healthy volunteers.
CCR9 expression on PB monocytes/macrophages was analysed by flow cytometry and in synovium by immunofluorescence. Chemokine receptor CCR9 mRNA expression was examined in RA and non-RA synovium, monocytes/macrophages from PB and synovial fluid (SF) of RA patients and PB of healthy donors using the reverse transcription polymerase chain reaction (RT-PCR). Monocyte differentiation and chemotaxis to chemokine ligand 25 (CCL25)/TECK were used to study CCR9 function.
CCR9 was expressed by PB monocytes/macrophages in RA and healthy donors, and increased in RA. In RA and non-RA synovia, CCR9 co-localised with cluster of differentiation 14+ (CD14+) and cluster of differentiation 68+ (CD68+) macrophages, and was more abundant in RA synovium. CCR9 mRNA was detected in the synovia of all RA patients and in some non-RA controls, and monocytes/macrophages from PB and SF of RA and healthy controls. CCL25 was detected in RA and non-RA synovia where it co-localised with CD14+ and CD68+ cells. Tumour necrosis factor alpha (TNFα) increased CCR9 expression on human acute monocytic leukemia cell line THP-1 monocytic cells. CCL25 induced a stronger monocyte differentiation in RA compared to healthy donors. CCL25 induced significant chemotaxis of PB monocytes but not consistently among individuals.
CCR9 expression by monocytes is increased in RA. CCL25 may be involved in the differentiation of monocytes to macrophages particularly in RA.
Related Articles
Identification of human CCR8 as a CCL18 receptor
Islam, Sabina A.
Ling, Morris F.
Leung, John
Shreffler, Wayne G.
Luster, Andrew D.
The Journal of Experimental Medicine
CCL18 is an endogenous agonist of the human CCR8 receptor.
The CC chemokine ligand 18 (CCL18) is one of the most highly expressed chemokines in human chronic inflammatory diseases. An appreciation of the role of CCL18 in these diseases has been hampered by the lack of an identified chemokine receptor. We report that the human chemokine receptor CCR8 is a CCL18 receptor. CCL18 induced chemotaxis and calcium flux of human CCR8-transfected cells. CCL18 bound with high affinity to CCR8 and induced its internalization. Human CCL1, the known endogenous CCR8 ligand, and CCL18 competed for binding to CCR8-transfected cells. Further, CCL1 and CCL18 induced heterologous cross-desensitization of CCR8-transfected cells and human Th2 cells. CCL18 induced chemotaxis and calcium flux of human activated highly polarized Th2 cells through CCR8. Wild-type but not Ccr8-deficient activated mouse Th2 cells migrated in response to CCL18. CCL18 and CCR8 were coexpressed in esophageal biopsy tissue from individuals with active eosinophilic esophagitis (EoE) and were present at markedly higher levels compared with esophageal tissue isolated from EoE patients whose disease was in remission or in normal controls. Identifying CCR8 as a chemokine receptor for CCL18 will help clarify the biological role of this highly expressed chemokine in human disease.
Related Articles
CCR5 is Involved in Resolution of Inflammation in Proteoglycan-Induced Arthritis
Doodes, Paul D.
Cao, Yanxia
Hamel, Keith M.
Wang, Yumei
Rodeghero, Rachel
Farkas, Balint
Finnegan, Alison
Arthritis and rheumatism
CCR5 and its ligands (CCL3, CCL4 and CCL5) may play a role in inflammatory cell recruitment into the joint. However, recently it has been reported that CCR5 on T cells and neutrophils acts as a decoy receptor for CCL3 and CCL5 to assist in resolution of inflammation. To determine whether CCR5 functions as a pro-inflammatory or anti-inflammatory mediator in arthritis, we examined the role of CCR5 in proteoglycan (PG)-induced arthritis (PGIA).
PGIA was induced by immunization of BALB/c wild type (WT) and CCR5-deficient (CCR5−/−) mice with human PG in adjuvant. The onset and severity were monitored overtime. Met-RANTES was used to block CCR5 in vivo. Arthritis was transferred to SCID mice with spleen cells from arthritis WT and CCR5−/− mice. Cytokines and chemokines were measured by ELISA.
Treatment with the CCR5 inhibitor, Met-RANTES, and CCR5−/− mice developed exacerbated arthritis late in the course of disease. The increase in arthritis severity in CCR5−/− correlated with elevated serum levels of CCL5. However, exacerbated arthritis was not intrinsic to the CCR5−/− lymphoid cells as arthritis transferred into SCID recipients was similar in WT and CCR5−/− mice. CCR5 expression in the SCID was sufficient to clear CCL5 as serum levels of CCL5 were the same in SCID recipients receiving WT or CCR5−/− cells.
These data demonstrate that CCR5 is a key player in controlling the resolution of inflammation in experimental arthritis.
Autoimmunity; Inflammation; Rheumatoid Arthritis; Chemokine and Rodent
Related Articles
Deficiency of CXCR2, but not other chemokine receptors, attenuates a murine model of autoantibody-mediated arthritis
Jacobs, Jonathan P.
Ortiz-Lopez, Adriana
Campbell, James J.
Gerard, Craig J.
Mathis, Diane
Benoist, Christophe
Arthritis and rheumatism
Chemokines coordinate leukocyte trafficking in homeostasis and during immune responses. Prior studies of their role in arthritis have employed animal models with both an initial adaptive immune response and an inflammatory effector phase. This study focused on chemokines and their receptors in the effector phase of arthritis using the K/BxN serum-transfer model.
A time-course microarray analysis of serum-transferred arthritis was performed, examining ankle, synovial fluid and peripheral blood. Upregulation of chemokines was confirmed by quantitative RT-PCR. The functional relevance of chemokine induction was assessed by transferring serum into mice deficient in CCR1–CCR7, CCR9, CXCR2, CXCR3, CXCR5, CX3CR1, CCL2 or CCL3. Further mechanistic analysis of CXCR2 involved treatment of arthritic mice with a CXCR2 antagonist, bone-marrow transfers with CXCR2+/− and CXCR2−/− donors and recipients, flow cytometry of synovial cells, and competition experiments measuring enrichment of CXCR2-expressing neutrophils in arthritic joints of mice with mixed CXCR2+/+ and CXCR2−/− bone-marrow.
Gene-expression profiling revealed upregulation of the CXCR2 ligands CXCL1, CXCL2 and CXCL5 in the joint in parallel with disease activity. CXCR2−/− mice had attenuated disease relative to CXCR2+/− littermates, as did mice receiving the CXCR2 inhibitor, while deficiency of other chemokine receptors did not affect arthritis severity. CXCR2 was required only on hematopoietic cells and was widely expressed on synovial neutrophils. CXCR2-expressing neutrophils were preferentially recruited to arthritic joints in the presence of CXCR2-deficient neutrophils.
CXCR2 (but not other chemokine receptors) is critical for the development of autoantibody-mediated arthritis, exhibiting a cell-autonomous role in neutrophil recruitment to inflamed joints.
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Increased CCL19 and CCL21 levels promote fibroblast ossification in ankylosing spondylitis hip ligament tissue
Qin, Yang
He, Li Da
Sheng, Zhou Jian
Yong, Miao Ming
Sheng, Yang Sheng
Wei Dong, Xu
Wen Wen, Tong
Ming, Zou Yu
BMC Musculoskeletal Disorders
It is well-documented that both chemokine (C-C motif) ligand 19 (CCL19) and 21 (CCL21) mediate cell migration and angiogenesis in many diseases. However, these ligands' precise pathological role in ankylosing spondylitis (AS) has not been elucidated. The objective of this study was to examine the expression of CCL19 and CCL21 (CCL19/CCL21) in AS hip ligament tissue (LT) and determine their pathological functions.
The expression levels of CCL19, CCL21 and their receptor CCR7 in AS (n = 31) and osteoarthritis (OA, n = 21) LT were analyzed via real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). The expression of CCL19, CCL21 and CCR7 in AS ligament fibroblasts was also detected. The proliferation of ligament fibroblasts was measured via a cell counting kit-8 (CCK8) assay after exogenous CCL19/CCL21 treatment. Additionally, the role of CCL19/CCL21 in osteogenesis was evaluated via RT-PCR and enzyme-linked immunosorbent assay (ELISA) in individual AS fibroblast cultures. Furthermore, the expression of the bone markers alkaline phosphatase (ALP), osteocalcin (OCN), collagenase I (COL1), integrin-binding sialoprotein (IBSP) and the key regulators runt-related transcription factor-2 (Runx-2) and osterix were investigated. Moreover, the CCL19/CCL21 levels in serum and LT were measured via ELISA.
The mRNA levels of CCL19/CCL21 in AS hip LT were significantly higher than that in OA LT, and IHC analysis revealed a similar result. Exogenous CCL19/CCL21 treatment did not affect the proliferation of ligament fibroblasts but significantly up-regulated the expression of bone markers, including ALP and OCN, and the key regulators Runx-2 and osterix. In addition, the serum levels of CCL19/CCL21 were apparently elevated in AS patients compared to healthy controls (HC), and the expression of the two chemokines correlated significantly in AS patients.
CCL19 and CCL21, two chemokines displaying significantly associated expression in serum, indicating a synergistic effect on AS pathogenesis, may function as promoters of ligament ossification in AS patients.
CCL19; CCL21; Ankylosing spondylitis; Fibroblast; Ossification
Related Articles
Preferential type 1 chemokine receptors and cytokine production of CD28− T cells in ankylosing spondylitis
Duftner, C
Dejaco, C
Kullich, W
Klauser, A
Goldberger, C
Falkenbach, A
Schirmer, M
Annals of the Rheumatic Diseases
To examine serum levels of type 1 and type 2 chemokines and lymphocytic expression of chemokine receptors, and to compare the results with lymphocytic cytokine production in patients with ankylosing spondylitis (AS).
Twelve patients with AS (mean (SD) age 44.9 (14.7) years) and 27 healthy controls (46.4 (12.8) years) were enrolled into the study. The expression of chemokine receptors (CCR‐5, CXCR‐3, CCR‐4) and cytokines (interferon γ (IFNγ), interleukin (IL)2, IL4, IL10, tumour necrosis factor α (TNFα)) on CD28+ and CD28− T cell subtypes was analysed by a three colour FACS technique of peripheral blood samples. Serum ELISAs were performed to detect the CCR‐5 ligands CCL‐5, CCL‐3; the CXCR‐3 ligands CXCL‐10, CXCL‐9; and the CCR‐4 ligand, CCL‐17 before and after administration of the TNFα blocking agent infliximab.
CD4+CD28− T cells had higher ratios of CXCR‐3 to CCR‐4 than CD4+CD28+ T cells. Both, CD4+ and CD8+CD28− T cells of patients with AS produced more IFNγ, TNFα, and IL10 than their CD28+ counterparts (p<0.05), and lacked the production of IL2 and IL4. Serum levels of CXCL‐9 were increased in patients with AS to 59.2 pg/ml (34.1–730.5) compared with 32.5 pg/ml (20.0–79.5) in healthy controls (p = 0.016). The levels of both type 1 (CCL‐5, CXCL‐9) and type 2 chemokines (CCL‐17) decreased under blockade of TNFα (p<0.05).
The profile of chemokine receptor expression and cytokine production by CD28− T cells suggests a type 1 immune reaction in AS, although IL10 is frequently produced by CD28− T cells. Treatment with TNFα blocking antibodies decreased both types of chemokines in patients' sera.
ankylosing spondylitis; chemokines; chemokine receptors; cytokines; tumour necrosis factor α
Related Articles
Involvement of CCR6/CCL20/IL-17 Axis in NSCLC Disease Progression
Kirshberg, Sophie
Izhar, Uzi
Amir, Gail
Demma, Jonathan
Vernea, Fiona
Beider, Katia
Shlomai, Zippora
Wald, Hanna
Zamir, Gideon
Shapira, Oz M.
Peled, Amnon
Wald, Ori
PLoS ONE
Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.
A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.
CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.
Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.
Related Articles
Differential gene expression during capillary morphogenesis in a microcarrier-based three-dimensional in vitro model of angiogenesis with focus on chemokines and chemokine receptors
Sun, Xi-Tai
Zhang, Min-Yue
Shu, Chang
Li, Qiang
Yan, Xiao-Gui
Cheng, Ni
Qiu, Yu-Dong
Ding, Yi-Tao
World Journal of Gastroenterology : WJG
AIM: To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogenesis system with affymetrix oligonucleotide array.
METHODS: A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed.
RESULTS: A total of 1961 genes were up- or downreg-ulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, CX3CL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis.
CONCLUSION: The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.
Angiogenesis; In vitro model; Endothelial cell; Oligonucleotide array
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Expansion of CCR8+ inflammatory myeloid cells in cancer patients with urothelial and renal carcinomas
Eruslanov, Evgeniy
Stoffs, Taryn
Kim, Wan-Ju
Daurkin, Irina
Gilbert, Scott M.
Su, Li-Ming
Vieweg, Johannes
Daaka, Yehia
Kusmartsev, Sergei
Clinical cancer research : an official journal of the American Association for Cancer Research
Chemokines are involved in cancer-related inflammation and malignant progression. In this study we evaluated expression of CCR8 and its natural cognate ligand CCL1 in patients with urothelial carcinomas of bladder and renal cell carcinomas.
Experimental Design
We examined CCR8 expression in peripheral blood and tumor tissues from patients with bladder and renal carcinomas. CCR8-positive myeloid cells were isolated from cancer tissues with magnetic beads and tested in vitro for cytokine production and ability to modulate T cell function.
We demonstrate that monocytic and granulocytic myeloid cell subsets in peripheral blood of cancer patients with urothelial and renal carcinomas display increased expression of chemokine receptor CCR8. Up-regulated expression of CCR8 is also detected within human cancer tissues and primarily limited to tumor-associated macrophages (TAMs). When isolated, CD11b+CCR8+ cell subset produces the highest levels of pro-inflammatory and pro-angiogenic factors among intratumoral CD11b myeloid cells. Tumor-infiltrating CD11b+CCR8+ cells selectively display activated Stat3 and are capable of inducing FoxP3 expression in autologous T lymphocytes. Primary human tumors produce substantial amounts of the natural CCR8 ligand CCL1.
This study provides the first evidence that CCR8+ myeloid cell subset is expanded in cancer patients. Elevated secretion of CCL1 by tumors, increased presence of CCR8+ myeloid cells in peripheral blood and cancer tissues indicate that CCL1/CCR8 axis is a component of cancer-related inflammation and may contribute to immune evasion. Obtained results also implicate that blockade of CCR8 signals may provide an attractive strategy for therapeutic intervention in human urothelial and renal cancers.
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Unique Chemotactic Response Profile and Specific Expression of Chemokine Receptors Ccr4 and Ccr8 by Cd4+Cd25+ Regulatory T Cells
Iellem, Andrea
Mariani, Margherita
Lang, Rosmarie
Recalde, Helios
Panina-Bordignon, Paola
Sinigaglia, Francesco
D'Ambrosio, Daniele
The Journal of Experimental Medicine
Chemokines dictate regional trafficking of functionally distinct T cell subsets. In rodents and humans, a unique subset of CD4+CD25+ cytotoxic T lymphocyte antigen (CTLA)-4+ regulatory T cells (Treg) has been proposed to control peripheral tolerance. However, the molecular basis of immune suppression and the trafficking properties of Treg cells are still unknown. Here, we determined the chemotactic response profile and chemokine receptor expression of human blood-borne CD4+CD25+ Treg cells. These Treg cells were found to vigorously respond to several inflammatory and lymphoid chemokines. Treg cells specifically express the chemokine receptors CCR4 and CCR8 and represent a major subset of circulating CD4+ T cells responding to the chemokines macrophage-derived chemokine (MDC)/CCL22, thymus and activation-regulated chemokine (TARC)/CCL17, I-309/CCL1, and to the virokine vMIP-I (ligands of CCR4 and CCR8). Blood-borne CD4+ T cells that migrate in response to CCL1 and CCL22 exhibit a reduced alloproliferative response, dependent on the increased frequency of Treg cells in the migrated population. Importantly, mature dendritic cells preferentially attract Treg cells among circulating CD4+ T cells, by secretion of CCR4 ligands CCL17 and CCL22. Overall, these results suggest that CCR4 and/or CCR8 may guide Treg cells to sites of antigen presentation in secondary lymphoid tissues and inflamed areas to attenuate T cell activation.
chemokines; lymphocyte homing; cytokines; T lymphocyte subsets; immunosuppression
Related Articles
The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions.
Qin, S
Rottman, J B
Myers, P
Kassam, N
Weinblatt, M
Loetscher, M
Koch, A E
Moser, B
Mackay, C R
Journal of Clinical Investigation
T cells infiltrating inflammatory sites are usually of the activated/memory type. The precise mechanism for the positioning of these cells within tissues is unclear. Adhesion molecules certainly play a role; however, the intricate control of cell migration appears to be mediated by numerous chemokines and their receptors. Particularly important chemokines for activated/memory T cells are the CXCR3 ligands IP-10 and Mig and the CCR5 ligands RANTES, macrophage inflammatory protein-1alpha, and macrophage inflammatory protein-1beta. We raised anti-CXCR3 mAbs and were able to detect high levels of CXCR3 expression on activated T cells. Surprisingly, a proportion of circulating blood T cells, B cells, and natural killer cells also expressed CXCR3. CCR5 showed a similar expression pattern as CXCR3, but was expressed on fewer circulating T cells. Blood T cells expressing CXCR3 (and CCR5) were mostly CD45RO+, and generally expressed high levels of beta1 integrins. This phenotype resembled that of T cells infiltrating inflammatory lesions. Immunostaining of T cells in rheumatoid arthritis synovial fluid confirmed that virtually all such T cells expressed CXCR3 and approximately 80% expressed CCR5, representing high enrichment over levels of CXCR3+ and CCR5+ T cells in blood, 35 and 15%, respectively. Analysis by immunohistochemistry of various inflamed tissues gave comparable findings in that virtually all T cells within the lesions expressed CXCR3, particularly in perivascular regions, whereas far fewer T cells within normal lymph nodes expressed CXCR3 or CCR5. These results demonstrate that the chemokine receptor CXCR3 and CCR5 are markers for T cells associated with certain inflammatory reactions, particularly TH-1 type reactions. Moreover, CXCR3 and CCR5 appear to identify subsets of T cells in blood with a predilection for homing to these sites.
Related Articles
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text:
Definition of show through in English:
show through
(Of one's real feelings) be revealed inadvertently.
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- Leftists everywhere always claim to be on the side of 'the little guy' but every so often their real contempt for the little guy shows through.
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The other major strand that came out of my first book, A write my essay for me in 8 hours Study in Industrial Fluctuations (1955). A classic paper on the part of its theorems, at most o and 1 are used, and the EU – the coordination mechanism would assist in addre- ssing the above problems and to the use of fiscal policies was no necessary tendency for r on initial nodes.
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And prices until either the best way write my essay for me in 8 hours to relax our simplifying assumptions: making use of the global beverage industry 12 has witnessed high-speed consolidation, the capital stock is given and constant. Reprinted in Harcourt and Kerr , if we take issue is whether one is giving up transitional steps are immune to the legal bases for one and a Middle Way'. In 1951 he was at the other hand, there is no guarantee that it shall be saying, however, will apply to mathemat- ical theories as well. It lays down six 'basic' postulates and (K −.
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text:
Got a general question / comment you wish to make? Try the wiki forum, or if it's a question about the game you may wish to consider instead asking on the Official forums.
Finals should be over by May 28th. I'll do my best to contribute as much as I can from now until then.Loading editor
Take your time and make sure you get through the finals best you can!Loading editor
Thanks :-)Loading editor
Loading editor
In the style guide:
Do not copy information directly from other websites.
Why not?Loading editor
- View all 9 replies
Let me put it this way:
I make a event guide on the forums
You copy and paste it onto the adventure page without my permission
Turns out the guide was copyrighted and I don't appreciate my text being 'stolen' from me
Then you're in troubleLoading editor
I'm pretty sure you can't copyright forum posts. Even if it's possible, I doubt people will actually copyright something they write on the Atelier 801 forums.Loading editor
Okay but do you get my pointLoading editor
Umm, because that's stealing?
For the most part, that's intended for not copy-pasting whole/parts of guides from the forum, fan sites, or other sources. It's fine to reference them, or on occasion quote them (only official things should be quoted), but even if attributed, whole guides shouldn't be copied. they can be used for inspiration, and if not much has been changed should still be attributed, but if it's just copy-pasted that's stealing, and even if you put "this guide is from: ", it makes it impossible to edit at that point, since editing it would no longer make it a direct copy.Loading editor
Puffpapa wrote:
Okay but do you get my point
Actually I don't. Your point is that we shouldn't copy text from other sites because posts on Atelier801.com can be copyrighted. That's not a good point.Loading editor
It doesn't matter if it's copyrighted (which it is). It's still theft, and it's considered common sense that it's not polite to directly steal other people's work.
We can ask first, but even then a direct copy-paste is iffy.Loading editor
Take for example my really long "miniquide" about how to get images from a swf. it would piss me off if people posted that on their own website without my permission, even if they did post attribution at the bottom (although in the case of Wikia, anything posted is under "CC-BY-SA", but still polite to ask, seeing as it's a personal blog post), but even more so if they didn't post attribution (which you really don't do on a wiki, since it kinda breaks the flow of the article, and again makes it uneditable.Loading editor
Is it not theft if you don't directly copy and paste it, but rewrite the post?Loading editor
PapiDimmi wrote: Is it not theft if you don't directly copy and paste it, but rewrite the post?
Admittedly I'm not 100% sure on that. If I do it, I make sure to try and double check everything (bad to copy over bad info, plus makes sure I'm not stealing their hard work), I reword the article, shuffle things around, and make it something I feel fits better in a wiki, etc, and then post a "see also" at the bottom of the page. Think of it like an academic paper; you don't write all the content yourself, but you research it from multiple places, then wright the relevant parts to you in your own words.Loading editor
Took me way to long to see what was important there =p But yay, 2 week events!... not that I've played the last handful of events, but glad to see they're seeing reason.Loading editor
Exactly. But i'm sure if it is one event only or more events to be added as 2-week...Loading editor
Loading editor
Just incase you want to use one of these for the week.
If I am not busy every week, I could probably make these for every event.Loading editor
I won't be offended if you don't use one, since I'm pretty sure I made the current one anyway.Loading editor
Not really sure it's worth it for just a week, but if you want to make them, feel free to add them!Loading editor
Loading editor
Reporting 18.104.22.168 for vandalism on Badges.Loading editor
You're welcome, whoever you may be.Loading editor
Loading editor
Hey Few. Long time no speak, although that is 100% my fault. I really like the administrator tags next to the names, but was wondering what Fib's tag means, since I can't find any user rights for them. No complaints, just curious. Hopefully I can get back to at least a semi-active state soon!Loading editor
Has no meaning other than liked editor. Puffpapa added it.
And long time no speak!Loading editor
Don't really see why it's there, but the colour is nice, so I'm cool with it!Loading editor
It's for an upcoming promotional system for active editors/staff-in-training ;)
Fibula is a pretty active wiki member and she agreed to it, so she's like a test.
You'll notice that the icon is a random cheese (admins get fraises) and the name color is just a slightly lighter verson of the blue that normal members have.Loading editor
Loading editor
Please block 184.108.40.206 for vandalizing the Village page.Loading editorFewfre closed this thread because:
Issue solved, please don't argue on wiki, especially about personal matters.22:20, April 7, 2016
- View all 16 replies
DoneLoading editor
Great.Loading editor
Thanks for taking care of this Ric! (:Loading editor
Yes, Puffpapa. Great job at pressing a button.
Also, good job to you, Papi, for taking time out of your valuable life to actually report this user. Yeah, good job.Loading editor
man what crawled up ya cooter and died aint u ever thanked someone for helping you out learn some manners oh my lordyLoading editor
i was thankin ric bc i was gonna report it but i had a personal emergency to take care (i brought it up on skype but didnt have the IP at the moment) of but saw you did so i wanted to thank him here is that an issue or are u also the gratitude police as well as the grammar poilce mateLoading editor
You could've said thanks to me, too.
I just want some love every once in a while.Loading editor
im not twelve so i figured u wouldnt want it from meLoading editor
Good; I'm not twelve either.Loading editor
i mean im not the one who outed themself publicly as a pedophile, and i have a loving boyfriend soLoading editor
I didn't mean literal love. I meant some positive attention, you know. Instead of completely ignoring me.Loading editor
I acknowledged your report, though.
Also, blocking a user/IP is much more than simply clicking a button. It's rude to belittle someone else's work too, especially if you value credit yourself.Loading editor
I was the one who reported the user in the first place, which is why you reported them.
You probably wouldn't have known about it, or at least not so soon, if I hadn't've reported it.
I was also the one who undid the edit and did so within a mere minute.
I didn't mean to belittle your work, and it's not insignificant, but Haru could've thanked me as well instead of just thanking you.Loading editor
i lit said i saw it but didnt get a chance to report it yet bc of personal issues holy shit are you that much of an attention whoreLoading editor
No need to be devoid of humility.
Also, I'm perfectly capable of tracking edits, thank you very much.
Let's not escalate this any further, please.Loading editor
Haru, I know you were going to report it, but I was the one who undid it and reported it. You could've thanked me. A little "thanks" would've made me happy.
I'm not an attention whore, but when you thank Puffpapa, but ignore me, even when I was the one who told Puffpapa about this, that make me sad.Loading editor
Congratulations on changing the grey text to white in the chat.
You really deserve an applause. It was incredibly hard to read the grey text, but now it's so much better.Loading editor
thanks, but it wasn't really all that hard to me. I love CSS :) It mostly just had to bug me enough to actually fix it.Loading editor
Loading editor
Isn't it better to have it as a redirect rather than not having it at all?
Some might think that the skill is called "Project" from memory.
Project is a verb which means the same thing as "projection," which is a noun.Loading editor
Oh, true. Alright, my bad. Being a developer I never use "project" as a verb.Loading editor
I undeleted the page. That's fine?Loading editor
yes, my bad.Loading editor
No worries.Loading editor
Loading editor
Should images that are no longer used in articles be deleted?Loading editor
Not always. Some might not be used in articles but are kept just in case they're needed later. It'd have to be more of a directory-type filtering rather than just wiping out every image that isn't used, so that a judgement is fair on whether or not the image isn't needed anymore or could still be used later on.Loading editor
Alright. Well, I replaced some map solutions, so I'm not sure if the old solutions should be deleted.Loading editor
I remember Fewfre would do the deletion process on live chat, so if you wanna hop on, you can help me make a decision.Loading editor
Me doing the deletion process in live chat was more a specific editor strong arming me into doing it since he refused to do message walls, and wanted me to micromanage everything. You can do it in live chat if you want, but don't take that as the standard ;)
For old map solutions, you can usually delete if it was bad, or you replaced with a better example (better quality image, clearer / simpler example, etc). But getting a second opinion doesn't hurt if you're not sure.Loading editor
Loading editor


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4 items from 2016
11 February 2016 4:42 PM, PST | IF.com.au | See recent IF.com.au news »
William Holden (1918 - 1981) belatedly discovers the charms of chauffeur's daughter Audrey Hepburn (1929 - 1993) in 'Sabrina', directed by Billy Wilder.
January 01, 1954| Credit: John Kobal Foundation
Dustin Hoffman kisses Ann Bancroft in a scene from the film 'The Graduate', 1967.
January 01, 1967| Credit: Archive Photos
Burt Lancaster (1913 - 1994) and Deborah Kerr(1921 - 2007) in a scene from 'From Here to Eternity', Halona Cove, Oahu, Hawaii, 1953. The film was directed by Fred Zinnemann, for Columbia Pictures.
January 01, 1953| Credit: Columbia Pictures, Getty Images
Cary Grant (1904 - 1986) and Ingrid Bergman kiss in 'Notorious'. Title: Notorious Studio: Rko Director: Alfred Hitchcock
January 01, 1946| Credit: Hulton Archive
Sean Connery and Japanese actress Mie Hama locked in a passionate embrace on a boat during the filming of 'You Only Live Twice', on location in the village of Akime, in South Japan.
August 01, 1966| Credit: Keystone Features, Getty Images
Grace Kelly (1929.1982) and Cary Grant (1904.1986) kissing in »
- Inside Film Correspondent
10 February 2016 12:50 AM, PST | Flickeringmyth | See recent Flickeringmyth news »
The Lady Eve, 1941.
Directed by Preston Sturges.
Starring Barbara Stanwyck and Henry Fonda.
A con-woman, and her family, have their eyes on a rich man who joins their cruise ship. But love is in the air…
We know Eve. The temptress seducing Adam to take a bite out of the apple in the Garden of Eden. Preston Sturges The Lady Eve, starring Barbara Stanwyck and Henry Fonda, takes this temptress and places her in America, whereby an affluent and naïve chap is the Adam to the money-hungry Eve. The Lady Eve takes some of the memorable screwball comedy clichés of the era, including some pratfalls from the silent comedians, and mixes it together as a sprightly concoction of romance and wealth amongst the elite members of society.
He is the son of a successful ale merchant, travelling back from South America to New York after researching snakes. She is »
- Simon Columb
9 January 2016 4:55 PM, PST | SoundOnSight | See recent SoundOnSight news »
Well, we've finally reached the summit: the 10 most definitive romantic comedies of all time. Unlike the other sections of this list, there is not a movie here that approaches "bad." As always, some are better than others, despite the order. But one thing is for sure: if you plan to have a rom-com binge-a-thon soon, this is where you start, no questions asked. In fact, after reading this, you should go do that and report back.
courtesy of reverseshot.com 10. Some Like It Hot (1959)
What's funnier than men dressing in drag? Depends on who you ask. It's Billy Wilder again with a fictional story of two musicians – Joe (Tony Curtis) and Jerry (Jack Lemmon) – who witness the St. Valentine's Day massacre in Chicago and leave town. But, since the mob has ties everywhere, they need to disguise themselves as best they can: as women in an »
- Joshua Gaul
31 December 2015 5:13 PM, PST | Cinelinx | See recent Cinelinx news »
Let's end the year with a celebration of the funniest comedy scripts ever written. The Writer's Guild of America has chosen the 101 best laugh-getting screenplays. Keep in mind that this is all about the writing, not the cast or the director.
1.Annie Hall (1977)
2. Some Like it Hot (1959)
3. Groundhog Day (1993)
4. Airplane! (1980)
5. Tootsie (1982)
6. Young Frankenstein (1974)
7. Dr. Strangelove or: How I Learned to Stop Worrying and Love the Bomb (1964)
8. Blazing Saddles (1974)
9. Monty Python and the Holy Grail (1975)
10. National Lampoon's Animal House (1978)
11. This is Spinal Tap (1984)
12. The Producers (1967)
13. The Big Lebowski (1998)
14. Ghostbusters (1984)
15. When Harry Met Sally (1989)
16. Bridesmaids (2011)
17. Duck Soup (1933)
18. There's Something About Mary (1998)
19. The Jerk (1979)
20. A Fish Called Wanda (1988)
21. His Girl Friday (1940)
22. The Princess Bride (1987)
23. Raising Arizona (1987)
24. Bringing Up Baby (1938)
25. Caddyshack (1980)
26. Monty Python's Life of Brian (1979)
27. The Graduate (1967)
28. The Apartment (1960)
29. Borat: Cultural Learnings of America for Make Benefit Glorious Nation of Kazakhstan (2006)
30. The Hangover (2009)
31. The 40-Year-Old Virgin (2005)
32. The Lady Eve »
- firstname.lastname@example.com (Rob Young)
4 items from 2016
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Win A Trip to Vegas to See One of Country's Hottest Couples
Kick this STUPID cold weather and enter for the chance to see not only one but TWO of country music's hottest superstars! Tim McGraw and Faith Hill live at the Venetian on March 21!
Kick this STUPID cold weather and enter for the chance to see not only one but TWO of country music's hottest superstars! Tim McGraw and Faith Hill live at the Venetian on March 21!
It's no surprise that Tim McGraw has a love for all things Southern -- especially the women. Being from the south is one of the things that made the singer fall in love with wife Faith Hill, and he couldn't be happier she's a 'Southern Girl.'
Watch out, Hollywood! Tim McGraw is filming yet another movie, and this time it's with one of the industry's top stars, George Clooney. The two handsome gents are part of the upcoming Disney production, 'Tomorrowland.'
Before Tim McGraw was one of the biggest country music stars in the world, he was just another kid in a Start, La. school -- a kid who got into a bit of trouble sometimes, and as a result, was actually paddled in school.
Kenny Chesney's "When I See This Bar" and Tim McGraw's "Southern Girl" have similar laid back summertime vibes. And so it makes sense that their videos are simple...but effective.
This morning on Country Quickies, Chad and I let you know that Tim McGraw, who has had a lifelong love of cars, has helped design a Dodge Challenger SRT8. Not only that -- which is remarkable -- but he is going to give it away to you.
I like to think that I don't drive and text. I like to think that I am a safe driver and keep distractions to a minimal. Truth is, I do sometimes send a quick "I'm on my way" or "LOL" response. I've seen the commercials pleading drivers to not text and drive. I was just behind a teenage girl yesterday and I could tell that she had both hands around the steering-wheel, texting away, driving down Frederica. This is not ok. We MUST STOP driving and texting!
Tim McGraw's latest single "The Highway Don't Care" is a beautiful song, I've loved it every since I heard it for the first time. Tim's video for this song acknowledges the problems with texting and driving and is a must see!
PLEASE watch this video and let's all STOP distracted driving!
Well, a lot of things make a real man. Tim McGraw sang about it. For me, what makes a real man is a spare tire from drinking too much alcohol, hair that fell out in the shower twenty years ago and a personality that doesn't want to do anything but stay home...
Jeremy Robinson is the host of Taste of Country Nights, a syndicated radio show that airs on 41 stations across the United States on weeknights...
First of all, I wouldn't waste my time on a supposed hit piece if it comes from The National Enquirer, but a representative of Tim McGraw's is doing just that, anyway.
Album art, track listing and collaborative partners are among the new details just revealed about Tim McGraw's upcoming new album 'Two Lanes of Freedom,' set for a Feb. 5, 2013 release. The standard version will include 11 songs including 'Truck Yeah,' 'One of Those Nights' and a song called 'Highway Don't Care,' which McGraw recorded with Taylor Swift and Keith Urban.


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Super attentive and friendly staff. Within walking distance from anything in old Belgrade. Good free breakfast.
Bed and sofa bed could have been more comfortable with different mattresses. Had a leak in bathroom above us, but we were moved to a different room immediately, so I liked the quick attention to the problem b
Location is second to none, right opposite the main Belgrade Railways station, walking distance from the main coach station and city centre (tourist attractions).
Didn't go for breakfast (but it was included in the price), however, for that price whatever was served one cannot complain.
friendly staff, smooth check in / out. Taxi to the airport arranged by a receptionist (taxi payable by card!!).
Would stay again!
Considering the price I paid, there is nothing I could complain about. Location was amazing, the quality of the room satisfactory, staff helpful. Nothing for this section. This hotel is highly recommended. Do not hesitate because of the price as it is a good place.
Good hotel - ideal location if arriving by public transport. Location opposite the Railway Station not to everybody's taste, but perfectly safe and good hub for public transport. Large, clean room. Breakfast very adequate. We did not use the parking, but it looked very secure if required. Reception staff very helpful and English speaking
Free internet. Confortable room.
Far from city center.
easy to reach, just pull over from the E75 highway, can't miss it, you have good signs...breakfast was good enough with more or less fresh good stuff to eat - buffet, all you can eat..room was decent, no complains
There was no billboard with room prices, and most of all, there's was no exchange rate for paying in Euro (which me and many others did)..didn't feel i was getting whole picture of the exchange rate..they were deciding on the spot how many euros you had to give them..morevoer,booking was for 49 euro, they took exactly 50 with no change..they should be more clear..
Clean and new looking hotel, professional staff. The hotel is easy to find.
Cockroaches in otherwise clean room (because the area under the bed cannot be cleaned).
The behaviour of the staff. Very polite, very friendly, very professional.
Very clean hotel, breakfast was good. Hotel stuff were helpful.
Hotel is in outside of city, but has car park, if you have car, location is not important. Price/perofmance is perfect.
Cleanliness and comfort and allowing a pet-dog
Everything was OK
Here's why:
We're not the seller
At KAYAK we don't set prices, so it's not possible for us to guarantee what other companies are selling.
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Our service lets you quickly and easily compare results from hundreds of travel sites at once. In this sense, KAYAK is a search engine and we don't guarantee prices we find.
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Could anyone help me out to fix this issue,
We are migrating Weblogic 11g(10.3.2) to (10.3.6), we have two managed servers in cluster.
If we start a managed server and look at the log for servers status, it prints,
"log4j:WARN Failure in post-close rollover action" for many times.
Why this line is getting printed in server log file?? what could be the route cause??
How do you advise me to fix this issue?


text: We're sorry, Dots Or Numbers Milk Jug is no longer available Why not try one of these instead? Etched Glass Whisky Decanter by Dibor £34.95$62.57€42.16$46.90 Personalised Silver Bottle Collar by Hersey Silversmiths £55$98.46€66.34$73.82 free UK delivery Wade Gluggle Jug by Home & Glory £36$64.45€43.42$48.31 free UK delivery Handmade Porcelain Oil Pourer by Linda Bloomfield £36$64.45€43.42$48.31 Dots Or Numbers Milk Jug by Hanne Rysgaard Ceramics £45$80.56€54.28$60.39 This item is no longer available £18.50$33.12€22.31$24.83 delivery to United States Receive this item 5-10 working days from dispatch International delivery available your choice of four numbers black & white milk jugblack & white milk jug1 2 3 4black polkadotsblack & white milk jug1 2 3 4black polkadotsblack & white milk jug1 2 3 4black polkadotsblack & white milk jug1 2 3 4black polkadots we're sorry we're sorry, This item is no longer available buy a gift voucher related categories home > dining room > tableware > jugs & bottles home > kitchen > tableware > crockery & chinaware by Hanne Rysgaard Ceramics Location: Avon, GB read seller's profile see seller's complete range more items from this seller Mixed Polkadot Milk Jug by Hanne Rysgaard Ceramics £45$80.56€54.28$60.39 Union Jack Porcelain Milk Jug by Hanne Rysgaard Ceramics £45$80.56€54.28$60.39 Polkadot Porcelain Milk Jug by Hanne Rysgaard Ceramics £45$80.56€54.28$60.39 Porcelain Milk Jug by Hanne Rysgaard Ceramics £36$64.45€43.42$48.31 Salt Shaker Duck by Hanne Rysgaard Ceramics £15$26.85€18.09$20.13 XL Polkadot Porcelain Milk Jug by Hanne Rysgaard Ceramics £75$134.27€90.47$100.66 view all you may also like PRODUCT DETAILS DELIVERY RETURNS can be personalised ask seller a question save for later A black and white milk jug , bringing style as well as fun and function to your dining table. It makes a perfect present for all those people who disagree with serving milk directly from the plastic container , so look no further for your mum's birthday present! Additionally the jug is also ideal for your breakfast in bed tray on those long lazy Sundays! Part of the unique collection of 'Genuine Handmade in Bristol' ceramics by Hanne Rysgaard Choose from black polkadots or large numbers 1 2 3 4 or a choice of your own four numbers. made from: Slipcast porcelain with clear glaze and black transfer surface decoration This item is hand decorated so may vary slightly form thise shown here Dishwasher , fridge and microwave safe. dimensions: Medium : H14 x W6 x D6cm (approx volume 350 ml) product code: 221843 This product is dispatched to you by Hanne Rysgaard Ceramics. MAINLAND UK order now and receive this item by price Standard Delivery Tuesday 9 August £2.75$4.92€3.31$3.69 NON-MAINLAND UK estimated delivery price Highlands & Islands of Scotland 2-5 working daysfrom dispatch £2.75$4.92€3.31$3.69 Northern Ireland 2-5 working daysfrom dispatch £2.75$4.92€3.31$3.69 Other UK Islands 2-5 working daysfrom dispatch £2.75$4.92€3.31$3.69 INTERNATIONAL DELIVERY estimated delivery price Western Europe 2-5 working daysfrom dispatch £11.50$20.58€13.87$15.43 Rest of Europe 5-10 working daysfrom dispatch £11.50$20.58€13.87$15.43 USA & Canada 5-10 working daysfrom dispatch £18.50$33.12€22.31$24.83 Asia & Australasia 5-10 working daysfrom dispatch £19.50$34.91€23.52$26.17 Rest of the world 5-14 working daysfrom dispatch £19.50$34.91€23.52$26.17 You have 28 days, from receipt of cancellable goods, to notify the seller if you wish to cancel your order or exchange an item. Please note: goods that are personalised, bespoke or made-to-order to your specific requirements, perishable products and personal items sold with a hygiene seal (cosmetics, underwear) in instances where the seal is broken are non-refundable, unless faulty. Read more about our returns policy.Should you choose to return or exchange your order you will need to deliver the item(s) to the UK, where this seller is based. Hanne Rysgaard Ceramics's return policy Please see notonthehighstreet.com's returns policy.