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1.  Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium 
Arthritis Research  2000;2(5):415-423.
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.
Introduction:
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.
Results:
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.
Discussion:
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.
PMCID: PMC17818  PMID: 11056676
chemokine receptor; cytokine; rheumatoid arthritis; T lymphocyte
2.  Role of the Chemokine Receptors CCR1, CCR2 and CCR4 in the Pathogenesis of Experimental Dengue Infection in Mice 
PLoS ONE  2010;5(12):e15680.
Dengue virus (DENV), a mosquito-borne flavivirus, is a public health problem in many tropical countries. Recent clinical data have shown an association between levels of different chemokines in plasma and severity of dengue. We evaluated the role of CC chemokine receptors CCR1, CCR2 and CCR4 in an experimental model of DENV-2 infection in mice. Infection of mice induced evident clinical disease and tissue damage, including thrombocytopenia, hemoconcentration, lymphopenia, increased levels of transaminases and pro-inflammatory cytokines, and lethality in WT mice. Importantly, infected WT mice presented increased levels of chemokines CCL2/JE, CCL3/MIP-1α and CCL5/RANTES in spleen and liver. CCR1-/- mice had a mild phenotype with disease presentation and lethality similar to those of WT mice. In CCR2-/- mice, lethality, liver damage, levels of IL-6 and IFN-γ, and leukocyte activation were attenuated. However, thrombocytopenia, hemoconcentration and systemic TNF-α levels were similar to infected WT mice. Infection enhanced levels of CCL17/TARC, a CCR4 ligand. In CCR4-/- mice, lethality, tissue injury and systemic inflammation were markedly decreased. Despite differences in disease presentation in CCR-deficient mice, there was no significant difference in viral load. In conclusion, activation of chemokine receptors has discrete roles in the pathogenesis of dengue infection. These studies suggest that the chemokine storm that follows severe primary dengue infection associates mostly to development of disease rather than protection.
doi:10.1371/journal.pone.0015680
PMCID: PMC3012079  PMID: 21206747
3.  ERK1/2 mitogen-activated protein kinase selectively mediates IL-13–induced lung inflammation and remodeling in vivo 
Journal of Clinical Investigation  2005;116(1):163-173.
IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13–induced inflammation and alveolar remodeling. There were associated decreases in IL-13–induced chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of α1-antitrypsin. IL-13–induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13–induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13–induced diseases and disorders.
doi:10.1172/JCI25711
PMCID: PMC1319220  PMID: 16374521
4.  CCL26-Targeted siRNA Treatment of Alveolar Type II Cells Decreases Expression of CCR3-Binding Chemokines and Reduces Eosinophil Migration: Implications in Asthma Therapy 
The underlying inflammation present in chronic airway diseases is orchestrated by increased expression of CC chemokines that selectively recruit leukocyte populations into the pulmonary system. Human CCL26 signals through CC chemokine receptor 3 (CCR3), is dramatically upregulated in challenged asthmatics, and stimulates recruitment of eosinophils (EOSs) and other leukocytes. CCL26 participates in regulation of its receptor CCR3 and modulates expression of a variety of chemokines in alveolar type II cells. Utilizing the A549 alveolar type II epithelial cell culture model, we carried out studies to test the hypothesis that CCL26-siRNA treatment of these cells would ameliorate Th2-driven release of the eotaxins and other CCR3 ligands that would, in turn, decrease recruitment and activation of EOSs. Results demonstrate that CCL26-siRNA treatments decreased interleukin-4-induced CCL26 and CCL24 expression by >70%. CCL26-directed small-interfering RNA (siRNA) treatments significantly decreased release of CCL5 (RANTES), CCL15 (MIP-1δ), CCL8 (MCP-2), and CCL13 (MCP-4). In bioactivity assays it was shown that EOS migration and activation were reduced up to 80% and 90%, respectively, when exposed to supernatants of CCL26-siRNA-treated cells. These results provide evidence that CCL26 may be an appropriate target for development of new therapeutic agents designed to alleviate the underlying inflammation associated with chronic diseases of the airways.
doi:10.1089/jir.2008.0051
PMCID: PMC2941665  PMID: 19203252
5.  Why CCR2 and CCR5 Blockade Failed and Why CCR1 Blockade Might Still Be Effective in the Treatment of Rheumatoid Arthritis 
PLoS ONE  2011;6(7):e21772.
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0021772
PMCID: PMC3128605  PMID: 21747955
6.  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
7.  Effect of CCR5 receptor antagonists on endocytosis of the human CCR5 receptor in CHO-K1 cells 
British Journal of Pharmacology  2008;153(7):1513-1527.
Background and purpose:
The CCR5 chemokine receptor is a member of the G protein-coupled receptor (GPCR) family that is expressed by macrophages, memory T-lymphocytes and dendritic cells and is activated by chemotactic proteins (e.g. MIP-1α [CCL3], MIP-1β [CCL4] and RANTES [CCL5]). CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus-1 (HIV-1) and some chemokines can inhibit HIV-1 infection by stimulating CCR5 receptor endocytosis. The aim of this study was to evaluate the effect of CCR5 antagonists on CCR5 endocytosis.
Experimental approach:
The effects of CCR5 agonists and antagonists on receptor internalization in CHO cells, expressing a C-terminal green fluorescent protein-tagged human CCR5 receptor (CCR5-GFP), were quantified using a confocal imaging plate reader.
Key results:
MIP-1α [CCL3], MIP-1β [CCL4] and RANTES [CCL5] were all able to stimulate potently the internalization of CCR5-GFP. This effect was inhibited by the non-peptide antagonist TAK 779. The CCR5 peptide antagonist met-RANTES antagonized MIP-1α-mediated increases in intracellular free calcium but was also able to stimulate a substantial internalization of the human CCR5-GFP receptor. However, CHO cells exhibited an aminopeptidase activity that was able to metabolize sufficient met-RANTES into an agonist metabolite capable of stimulating calcium mobilization via CCR5 receptors in naïve cells.
Conclusions and implications:
These data suggest that there is an endogenous aminopeptidase activity on the surface of CHO cells, that produces a slow internalization of the receptor following a time-dependent conversion of receptor-bound met-RANTES from a CCR5 receptor antagonist into a CCR5 agonist molecule.
doi:10.1038/sj.bjp.0707691
PMCID: PMC2437898  PMID: 18223665
receptor internalization; CCR5 receptor; met-RANTES; G-protein-coupled receptors; antagonist stimulated; endosomes; aminopeptidase
8.  Photoluminescent Mesoporous Silicon Nanoparticles with siCCR2 Improve the Effects of Mesenchymal Stromal Cell Transplantation after Acute Myocardial Infarction 
Theranostics  2015;5(10):1068-1082.
Background: Despite the benefits of mesenchymal stromal cell (MSC) transplantation in cardiac tissue, detailed in vivo observations have shown that MSCs only survive for a brief period after transplantation due to harsh microenvironmental conditions, including ischemia, inflammation and anoikis, in the infarcted myocardium. Thus, new strategies are needed to enhance MSC survival and inhibit cardiac remodeling. Studies have now demonstrated that chemokine [C-C motif] ligand 2 (CCL2) and its cognate receptor C-C chemokine receptor 2 (CCR2) promote excessive Ly6Chigh inflammatory monocyte infiltration at the infarct in response to ischemic myocardial injury. Therefore, decreasing the activities of these monocytes immediately after acute myocardial infarction (AMI) could be beneficial for AMI patients. Objectives: This study tested the hypothesis that therapeutic siRNA-loaded photoluminescent mesoporous silicon nanoparticles (PMSNs) targeting CCR2 expression in Ly6Chigh inflammatory monocytes decrease the accumulation of these cells in the infarct, improve the efficacy of MSC transplantation and attenuate myocardial remodeling.
Methods: PMSNs carrying therapeutic siCCR2 were first synthesized without the inclusion of fluorescent materials or dyes. After AMI BALB/c mice were established, 105 5-ethynyl-2'- deoxyuridine (EdU)-labeled MSCs suspended in 100 µl of phosphate buffered saline (PBS) were injected into the border zone of the infarct of each mouse. PMSNs-siCCR2 (25 µg/g) were also intravenously injected via the tail vein immediately following AMI induction. Control mice were injected with an equal amount of PMSNs without siCCR2. PMSNs-siCCR2 were examined in vivo using near-infrared imaging technology. The therapeutic effects of PMSNs-siCCR2 for MSC transplantation were determined at the mRNA, protein and functional levels.
Results: PMSNs-siCCR2 circulated freely in vivo and were cleared in a relatively short period of time (t½=37 min) with no evidence of toxicity. The therapeutic PMSNs-siCCR2 showed higher levels of cellular accumulation in Ly6Chigh monocytes in the spleen and more efficient degradation of CCR2 compared with the control (8.04%±2.17% vs. 20.02%±4.55%, p<0.001). Subsequently, the PMSNs-siCCR2 decreased the accumulation of CD11b-positive monocytes at the infarct (49.3%±17.34% vs. 61.32%±22.43%, p<0.001) on day 1. Increased survival of transplanted MSCs (13±3/mm2 vs. 4±1/mm2, p<0.001) and significantly decreased TdT-mediated dUTP nick end labeling (TUNEL)+ cardiac myocytes (17.44%±6.26% vs. 39.49%±13.28%, p<0.001) were then identified in the infarct zone three days after AMI induction in the PMSNs-siCCR2 group. Three weeks after MSC injection, significant increases were observed in the vascular density (235.5±39.6/mm2 vs. 147.4±20.3/mm2, p<0.001) and the cardiac myosin-positive area (21.7%±8.4% vs. 13.2%±4.4%, p<0.001) of the infarct border zone. In addition, significant amelioration of left ventricular (LV) remodeling (thickness of the LV posterior walls) (0.84±0.11 mm vs. 0.61±0.08 mm, p<0.001) was also observed at the same time compared with the control group.
Conclusions: PMSNs-siCCR2-mediated CCR2 gene silencing in Ly6Chigh monocytes improved the effectiveness of MSC transplantation and selectively ameliorated myocardial remodeling after AMI. These results suggest that PMSNs-siCCR2 could potentially be used to develop an anti-inflammatory therapy for post-AMI MSC transplantation.
doi:10.7150/thno.11517
PMCID: PMC4508497  PMID: 26199646
AMI; PMSNs; siCCR2; MSCs; Ly6Chigh monocytes
9.  CCR5 as a Treatment Target in Pulmonary Arterial Hypertension 
Circulation  2014;130(11):880-891.
Background
Pulmonary arterial hypertension (PH), whether idiopathic or related to underlying diseases such as HIV infection, results from complex vessel remodeling involving both pulmonary-artery smooth muscle cell (PA-SMC) proliferation and inflammation. CCR5, a co-receptor for cellular HIV-1 entry expressed on macrophages and vascular cells, may be involved in the pathogenesis of PH. Maraviroc is a new CCR5 antagonist designed to block HIV entry.
Methods and Results
Marked CCR5 expression was found in lungs from patients with idiopathic PH, in mice with hypoxia-induced PH and in SIV-infected macaques, in which it was chiefly localized in the PA-SMCs. To assess the role for CCR5 in experimental PH, we used both gene disruption and pharmacological CCR5 inactivation in mice. Because maraviroc does not bind to murine CCR5, we used human-CCR5ki mice for pharmacological and immunohistochemical studies. Compared to wild-type mice, CCR5−/− mice or human-CCR5ki mice treated with maraviroc exhibited decreased PA-SMC proliferation and recruitment of perivascular and alveolar macrophages during hypoxia exposure. CCR5−/− mice reconstituted with wild-type bone-marrow cells and wild-type mice reconstituted with CCR5−/− bone-marrow cells were protected against PH, suggesting CCR5-mediated effects on PASMCs and macrophage involvement. The CCR5 ligands CCL5 and the HIV-1 gp120 protein increased intracellular calcium and induced growth of human and h-CCR5ki mouse PASMCs; maraviroc inhibited both effects. Maraviroc also reduced the growth-promoting effects of conditioned media from CCL5-activated macrophages derived from human-CCR5ki mice on PA-SMCs from wild-type mice.
Conclusions
The CCL5-CCR5 pathway represents a new therapeutic target in PH associated with HIV or with other conditions.
doi:10.1161/CIRCULATIONAHA.114.010757
PMCID: PMC4160408  PMID: 24993099
hypertension pulmonary; inflammation; remodeling; CCR5; smooth muscle cells
10.  Inflammatory Cytokines Induce Expression of Chemokines by Human Retinal Cells: Role in Chemokine Receptor Mediated Age-related Macular Degeneration 
Aging and Disease  2015;6(6):444-455.
Chemokine reeptor-3 (CCR-3) was shown to be associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). AMD is a vision threatening retinal disease that affects the aging population world-wide. Retinal pigment epithelium and choroid in the posterior part of the retina are the key tissues targeted in the pathogenesis of CNV in AMD. We used human retinal pigment epithelial (HRPE) and choroidal fibroblast (HCHF) cells, prepared from aged adult human donor eyes, to evaluate the expression of major CCR-3 ligands, CCL-5, CCL -7, CCL-11,CCL-24 and CCL-26. Microarray analysis of gene expression in HRPE cells treated with inflammatory cytokine mix (ICM= IFN-γ+TNF-α+IL-1β) revealed 75 and 23-fold increase in CCL-5 and CCL-7 respectively, but not CCL-11, CCL-24 and CCL-26. Chemokine secretion studies of the production of CCL5 and CCL7 by HRPE corroborated with the gene expression analysis data. When the HRPE cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent manner. Similar to the gene expression data, the ICM did not enhance HRPE production of CCL-11, CCL-24 and CCL-26. CCL-11 and CCL-26 were increased with IL-4 treatment and this HRPE production was augmented in the presence of TNF-α and IL1β. When HCHF cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent fashion. IL-4 induced low levels of CCL-11 and CCL-26 in HCHF and this production was significantly enhanced by TNF-α. Under these conditions, neither HRPE nor HCHF were demonstrated to produce CCL-24. These data demonstrate that chronic inflammation triggers CCL-5 and CCL-7 release by HRPE and HCHF and the subsequent interactions with CCR3 may participate in pathologic processes in AMD.
doi:10.14336/AD.2015.0323
PMCID: PMC4657816  PMID: 26618046
Age-related macular degeneration; Retinal pigment epithelium; Retina; Inflammation; Choroidal neovascularization; Chemokines; CCR3
11.  Expression of CCL20 and Its Corresponding Receptor CCR6 Is Enhanced in Active Inflammatory Bowel Disease, and TLR3 Mediates CCL20 Expression in Colonic Epithelial Cells 
PLoS ONE  2015;10(11):e0141710.
Background
The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production.
Methods
A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn’s disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using in situ hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay.
Results
CCL20 and CCR6 mRNA abundances were increased during active inflammation (CCL20 5.4-fold in ulcerative colitis and 4.2-fold in Crohn’s disease; CCR6 1.8 and 2.0, respectively). CCL20 and CCR6 mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and TLR3 silencing reduced CCL20 mRNA and protein levels.
Conclusions
The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn’s disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3.
doi:10.1371/journal.pone.0141710
PMCID: PMC4633243  PMID: 26536229
12.  CCR5 is Involved in Resolution of Inflammation in Proteoglycan-Induced Arthritis 
Arthritis and rheumatism  2009;60(10):2945-2953.
Objective
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).
Methods
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.
Result
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.
Conclusion
These data demonstrate that CCR5 is a key player in controlling the resolution of inflammation in experimental arthritis.
doi:10.1002/art.24842
PMCID: PMC2826270  PMID: 19790057
Autoimmunity; Inflammation; Rheumatoid Arthritis; Chemokine and Rodent
13.  Post-transcriptional silencing of CCR3 downregulates IL-4 stimulated release of eotaxin-3 (CCL26) and other CCR3 ligands in alveolar type II cells 
Cytokine  2008;44(3):342-351.
Trafficking and inflammation in airway diseases are, in part, modulated by members of the CC chemokine family, eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26), which transduce signals through their CCR3 receptor. In this context, we hypothesized that transfecting alveolar type II epithelial cells with CCR3-targeted siRNA or antisense (AS-ODN) sequences will downregulate cellular synthesis and release of the primary CCR3 ligands CCL26 and CCL24 and will modulate other CCR3 ligands. The human A549 alveolar type II epithelium-like cell culture model was used for transfection and subsequent effects on CCR3 agonists. siRNAs were particularly effective. PCR showed a 60-80% decrease in mRNA and immunoblots showed up to 75-84% reduction of CCR3 in siRNA treated cells. CCR3-siRNA treatments reduced IL-4 stimulated CCL26 release and constitutive CCL24 release by 65% and 80%, respectively. Release of four additional CCR3 agonists RANTES, MCP-2, MCP-3 and MCP-4 was also significantly reduced by CCR3-siRNA treatments of the alveolar type II cells. Activation of eosinophils, assessed as superoxide anion generation, was reduced when eosinophils were treated with supernatants of A549 cells pretreated with CCR3-targeted siRNAs or AS-ODNs. Collectively, the data suggest that post-transcriptional regulation of CCR3 receptors may be a potential therapeutic approach for interrupting proinflammatory signaling.
doi:10.1016/j.cyto.2008.09.006
PMCID: PMC2661111  PMID: 19038554
alveolar type II cells; CCR3 receptor; eotaxins; siRNA; antisense oligonucleotides
14.  Extramedullary Myelopoiesis in Malaria Depends on Mobilization of Myeloid-Restricted Progenitors by IFN-γ Induced Chemokines 
PLoS Pathogens  2013;9(6):e1003406.
Resolution of a variety of acute bacterial and parasitic infections critically relies on the stimulation of myelopoiesis leading in cases to extramedullary hematopoiesis. Here, we report the isolation of the earliest myeloid-restricted progenitors in acute infection with the rodent malaria parasite, Plasmodium chabaudi. The rapid disappearance of these infection-induced myeloid progenitors from the bone marrow (BM) equated with contraction of the functional myeloid potential in that organ. The loss of BM myelopoiesis was not affected by the complete genetic inactivation of toll-like receptor signaling. De-activation of IFN-γ signaling completely abrogated the contraction of BM myeloid progenitors. Radiation chimeras of Ifngr1-null and control BM revealed that IFN-γ signaling in an irradiation-resistant stromal compartment was crucial for the loss of early myeloid progenitors. Systemic IFN-γ triggered the secretion of C-C motif ligand chemokines CCL2 and CCL7 leading to the egress of early, myeloid-committed progenitors from the bone marrow mediated by their common receptor CCR2. The mobilization of myeloid progenitors initiated extramedullary myelopoiesis in the spleen in a CCR2-dependent manner resulting in augmented myelopoiesis during acute malaria. Consistent with the lack of splenic myelopoiesis in the absence of CCR2 we observed a significant persistence of parasitemia in malaria infected CCR2-deficient hosts. Our findings reveal how the activated immune system mobilizes early myeloid progenitors out of the BM thereby transiently establishing myelopoiesis in the spleen in order to contain and resolve the infection locally.
Author Summary
Malaria in man and in most animal models is accompanied by splenomegaly. At the same time, the spleen is the main organ for the control resolution of the parasitemia. This process initially depends mostly on the innate immune system and requires increased production of myeloid cells. We investigated the number of bone marrow (BM) LIN− cells which includes hematopoietic stem cells and progenitors during infection of mice with Plasmodium chabaudi and observed a significant reduction. Using a refined definition for early myeloid-restricted progenitors we could show that the loss of these cells in malaria equated with contraction of BM myelopoiesis. Since absence of IFN-γ receptor on stromal cells was sufficient to block this contraction we investigated the effect of IFN-γ on chemokine secretion. We observed a huge upregulation of CCL2/CCL7 serum levels and an increase in Ccl2/Ccl7 transcription in the BM at peak parasitemia. Egress from the BM of early myeloid progenitors was critically dependent on the chemokine receptor CCR2. Their mobilization resulted in extramedullary myelopopiesis in the spleen which contributed to the clearance of parasite-infected erythrocytes. Our study defined the molecular signals and interaction of various cell types leading to the establishment of splenic myelopoiesis in a mouse model of malaria.
doi:10.1371/journal.ppat.1003406
PMCID: PMC3675198  PMID: 23762028
15.  Upregulation of CCL20 and Recruitment of CCR6+ Gastric Infiltrating Lymphocytes in Helicobacter pylori Gastritis▿  
Infection and Immunity  2007;75(9):4357-4363.
Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. There is increased T-cell infiltration at the site of infection with H. pylori. CCR6, a specific β-chemokine receptor for CCL20 (MIP-3α/LARC/exodus), has recently been reported to mediate lymphocyte homeostasis and immune responses in mucosal tissue, and it may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation. In this study, we investigated the role of CCR6 and its ligand, CCL20, in inducing an inflammatory response in the gastric mucosa during H. pylori infection. Gastric infiltrating T lymphocytes were isolated from endoscopic biopsy specimens of H. pylori gastritis patients and analyzed for the expression of the CCR6 chemokine receptor. Our results demonstrated that there was significantly increased CCR6 expression in CD3+ T cells infiltrating the gastric mucosa, and the CCR6 ligand, the CCL20 chemokine, was selectively expressed in inflamed gastric tissues. The production of CCL20 was upregulated in response to H. pylori in gastric epithelial cells when there was stimulation by the proinflammatory cytokines interleukin-1β and tumor necrosis factor alpha. Furthermore, recombinant CCL20 induced lymphocyte chemotaxis migration in fresh gastric T cells ex vivo, indicating that the gastric T cells could migrate toward inflammatory sites via CCR6/CCL20 interaction. Our results suggest that the interaction between CCL20 and CCR6 may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation in Helicobacter infection.
doi:10.1128/IAI.01660-06
PMCID: PMC1951156  PMID: 17562763
16.  Chemokine Receptor CCR6-Dependent Accumulation of γδ T Cells in Injured Liver Restricts Hepatic Inflammation and Fibrosis 
Hepatology (Baltimore, Md.)  2013;59(2):630-642.
Chronic liver injury promotes hepatic inflammation, representing a prerequisite for organ fibrosis. We hypothesized a contribution of chemokine receptor CCR6 and its ligand, CCL20, which may regulate migration of T-helper (Th)17, regulatory, and gamma-delta (γδ) T cells. CCR6 and CCL20 expression was intrahepatically up-regulated in patients with chronic liver diseases (n = 50), compared to control liver (n = 5). Immunohistochemistry revealed the periportal accumulation of CCR6+ mononuclear cells and CCL20 induction by hepatic parenchymal cells in liver disease patients. Similarly, in murine livers, CCR6 was expressed by macrophages, CD4 and γδ T-cells, and up-regulated in fibrosis, whereas primary hepatocytes induced CCL20 upon experimental injury. In two murine models of chronic liver injury (CCl4 and methionine-choline-deficient diet), Ccr6−/− mice developed more severe fibrosis with strongly enhanced hepatic immune cell infiltration, compared to wild-type (WT) mice. Although CCR6 did not affect hepatic Th-cell subtype composition, CCR6 was explicitly required by the subset of interleukin (IL)-17- and IL-22-expressing γδ T cells for accumulation in injured liver. The adoptive transfer of WT γδ, but not CD4 T cells, into Ccr6−/− mice reduced hepatic inflammation and fibrosis in chronic injury to WT level. The anti-inflammatory function of hepatic γδ T cells was independent of IL-17, as evidenced by transfer of Il-17−/− cells. Instead, hepatic γδ T cells colocalized with hepatic stellate cells (HSCs) in vivo and promoted apoptosis of primary murine HSCs in a cell-cell contact-dependent manner, involving Fas-ligand (CD95L). Consistent with γδ T-cell-induced HSC apoptosis, activated myofibroblasts were more frequent in fibrotic livers of Ccr6−/− than in WT mice. Conclusion: γδ T cells are recruited to the liver by CCR6 upon chronic injury and protect the liver from excessive inflammation and fibrosis by inhibiting HSCs.
doi:10.1002/hep.26697
PMCID: PMC4139146  PMID: 23959575
17.  CC-chemokine receptor 7 and its ligand CCL19 promote mitral valve interstitial cell migration and repair 
Journal of Biomedical Research  2015;29(6):456-464.
Abstract
The effect of CC-chemokine receptor 7 (CCR7) and CC-chemokine ligand 19 (CCL19) on rheumatic mitral stenosis is unknown. This study aimed to explore the roles of CCR7 and CCL19 in rheumatic mitral stenosis by measuring the expression of CCR7 and CCL19 in human mitral valves from rheumatic mitral stenosis patients. Additionally, we examined their effects on human mitral valve interstitial cells (hMVICs) proliferation, apoptosis and wound repair. CCR7 and CCL19 expression was measured in the mitral valves from rheumatic mitral stenosis patients (n = 10) and compared to normal mitral valves (n = 5). CCR7 was measured in cultured hMVICs from rheumatic mitral stenosis patients and normal donors by RT-PCR and immunofluorescence. The cells were also treated with exogenous CCL19, and the effects on wound healing, proliferation and apoptosis were assayed. In the rheumatic mitral valves, valve interstitial cells expressed CCR7, while mononuclear cells and the endothelium expressed CCL19. Healthy mitral valves did not stain positive for CCR7 or CCL19. CCR7 was also detected in cultured rheumatic hMVICs or in normal hMVICs treated with CCL19. In a wound healing experiment, wound closure rates of both rheumatic and normal hMVICs were significantly accelerated by CCL19. These effects were abrogated by a CCR7 neutralizing antibody. The CCR7/CCL19 axis did not influence the proliferation or apoptosis of hMVICs, indicating that wound healing was due to increased migration rates rather than increased proliferation. In conclusion, CCR7 and CCL19 were expressed in rheumatic mitral valves. The CCR7/CCL19 axis may regulate remodeling of rheumatic valve injury through promoting migratory ability of hMVICs.
doi:10.7555/JBR.29.20150031
PMCID: PMC4662207  PMID: 26668580
CC-chemokine receptor 7; CC-chemokine ligand 19; rheumatic mitral stenosis; migration; wound repair
18.  Hepatic Macrophage Migration and Differentiation Critical for Liver Fibrosis Is Mediated by the Chemokine Receptor C-C Motif Chemokine Receptor 8 in Mice 
Hepatology (Baltimore, Md.)  2012;55(3):898-909.
Chemokines critically control the infiltration of immune cells upon liver injury, thereby promoting hepatic inflammation and fibrosis. The chemokine receptor CCR8 can affect trafficking of monocytes/macrophages, monocyte-derived dendritic cells (DCs) and T-helper cell (Th) subsets, but its role in liver diseases is currently unknown. To investigate the functional role of CCR8 in liver diseases, ccr8−/− and wild-type (WT) mice were subjected to chronic experimental injury models of carbon tetrachloride (CCl4) administration and surgical bile duct ligation (BDL). CCR8 was strongly up-regulated in the injured liver. Ccr8−/− mice displayed attenuated liver damage (e.g., ALT, histology, and TUNEL) compared to WT mice and were also protected from liver fibrosis in two independent injury models. Flow cytometry revealed reduced infiltrates of liver macrophages, neutrophils and natural killer cells, whereas hepatic CD4+ T cells increased. The main CCR8-expressing cells in the liver were hepatic macrophages, and CCR8 was functionally necessary for CCL1-directed migration of inflammatory but not for nonclassical monocytes into the liver. Moreover, the phenotype of liver macrophages from injured ccr8−/− animals was altered with increased expression of DC markers and enhanced expression of T-cell-attracting chemokine macrophage inflammatory protein 1-alpha (MIP-1α/CCL3). Correspondingly, hepatic CD4+ T cells showed increased Th1 polarization and reduced Th2 cells in CCR8-deficient animals. Liver fibrosis progression, but also subsequent T-cell alterations, could be restored by adoptively transferring CCR8-expressing monocytes/macrophages into ccr8−/− mice during experimental injury.
Conclusions
CCR8 critically mediates hepatic macrophage recruitment upon injury, which subsequently shapes the inflammatory response in the injured liver, affecting macrophage/DC and Th differentiation. CCR8 deficiency protects the liver against injury, ameliorating initial inflammatory responses and hepatic fibrogenesis. Inhibition of CCR8 or its ligand, CCL1, might represent a successful therapeutic target to limit liver inflammation and fibrosis progression.
doi:10.1002/hep.24764
PMCID: PMC4533854  PMID: 22031018
19.  Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis 
Arthritis Research & Therapy  2009;11(5):R149.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/ar2823
PMCID: PMC2787286  PMID: 19804625
20.  Endocytosis and Recycling of the HIV Coreceptor Ccr5 
The Journal of Cell Biology  2000;151(6):1281-1294.
The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1α, and MIP-1β. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH2-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect.
Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6–9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES–induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein–coupled receptor trafficking through the recycling endosome compartment.
PMCID: PMC2190598  PMID: 11121442
chemokine receptor; endocytosis; CCR5; recycling endosome; HIV
21.  CC Chemokine Receptor 4 (CCR4) in human allergen-induced late nasal responses 
Allergy  2010;65(9):1126-1133.
Background
CC Chemokine receptor 4 (CCR4) is preferentially expressed on Th2 lymphocytes. CCR4-mediated inflammation may be important in the pathology of allergic rhinitis. Disruption of CCR4 – ligand interaction may abrogate allergen-induced inflammation.
Methods
Sixteen allergic rhinitics and six nonatopic individuals underwent both allergen and control (diluent) nasal challenges. Symptom scores and peak nasal inspiratory flow were recorded. Nasal biopsies were taken at 8 h post challenge. Sections were immunostained and examined by light or dual immunofluorescence microscopy for eosinophils, T-lymphocytes, CCR4+CD3+ and CXCR3+CD3+ cells and examined by in situ hybridization for CCR4, IL-4 and IFN-γ mRNA+ cells. Peripheral blood mononuclear cells were obtained from peripheral blood of nine normal donors and the CCR4+CD4+ cells assessed for actin polymerization in response to the CCR4 ligand macrophage-derived chemokine (MDC/CCL22) and the influence of a CCR4 antagonist tested.
Results
Allergic rhinitics had increased early and late phase symptoms after allergen challenge compared to diluent; nonatopics did not respond to either challenge. Eosinophils, but not total numbers of CD3+ T cells, were increased in rhinitics following allergen challenge. In rhinitics, there was an increase in CCR4+CD3+ protein-positive cells relative to CXCR3+CD3+ cells; CCR4 mRNA+ cells were increased and IL-4 increased to a greater extent than IFN-γ. CCR4+CD4+ T cells responded to MDC in vitro, and this response was inhibited by the selective CCR4 antagonist.
Conclusion
Lymphocyte CCR4 expression is closely associated with induction of human allergen-induced late nasal responses. Blocking CCR4-ligand interaction may provide a novel therapeutic approach in allergic disease.
doi:10.1111/j.1398-9995.2010.02327.x
PMCID: PMC3380530  PMID: 20148806
allergic rhinitis; CCR4; nasal mucosa; Th2-mediated inflammation
22.  Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1β 
British Journal of Pharmacology  2002;137(5):663-675.
C-C chemokine receptor-1 (CCR1) has been implicated in mediating a variety of inflammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP-1α/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines.We used radioligand binding and [35S]-GTPγS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3-hCCR1) to characterize a panel of chemokines (HCC-1, MIP-1α, MIP-1β, MIP-1δ, MPIF-1, MCP-2, MCP-3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced 125I-MIP-1α with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [35S]-GTPγS exchange.We then assessed the utility of HL-60 cells cultured with known differentiating agents (PMA, DMSO, dibutyryl-cAMP or retinoic acid) for investigating CCR1 pharmacology. In [35S]-GTPγS exchange assays, membranes from cells cultured with retinoic acid (4–6 days) were the most responsive to activation by MIP-1α and MPIF-1. FACS analysis and comparative pharmacology confirmed that these activities were mediated by CCR1.Using [35S]-GTPγS exchange assays, intracellular calcium flux and/or whole cell chemotaxis assays in HL-60(Rx) cells, we validated that MIP-1α was the most potent CCR1 ligand (MIP-1α>MPIF-1>RANTES⩾MIP-1β) although the ligands differed in their efficacy as agonists. MPIF-1 was the more efficacious (MPIF-1>RANTES=MIP-1α>>MIP-1β). 125I-MIP-1β binding in Ba/F3-hCCR1 and HL-60(Rx) membranes was competitively displaced by MIP-1α, MPIF-1 and MIP-1β. The binding Ki for these chemokines with 125I-MIP-1β were essentially identical in the two membrane systems.Lastly, MIP-1β antagonized [35S]-GTPγS exchange, Ca2+ flux and chemotaxis in HL-60(Rx) cells in response to robust agonists such as MIP-1α, RANTES and MPIF-1. Based on our results, we propose that MIP-1β could function as an endogenous inhibitor of CCR1 function.
doi:10.1038/sj.bjp.0704907
PMCID: PMC1573530  PMID: 12381680
Chemokine receptor CCR1; MIP-1β; HL-60; [35S]-GTPγS; chemotaxis
23.  Antigen stimulation induces HIV envelope gp120-specific CD4+ T cells to secrete CCR5 ligands and suppress HIV infection 
Virology  2007;369(1):214-225.
CD4+ T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4+ T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4+ T cells elicited production of high amounts of CCR5 chemokines MIP-1α (CCL3), MIP-1β (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-γ or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4+ T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1β and IFN-γ rapidly declined. Thus, vaccines that trigger HIV-specific CD4+ T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4+ T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.
doi:10.1016/j.virol.2007.07.031
PMCID: PMC2443714  PMID: 17765942
24.  Bone marrow CD34+ cells and megakaryoblasts secrete β-chemokines that block infection of hematopoietic cells by M-tropic R5 HIV 
Journal of Clinical Investigation  1999;104(12):1739-1749.
CD34+ cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34+ cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34+ cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34+ cells and CD34+KIT+ cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1α, and MIP-1β and that IFN-γ stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34+KIT+ cells, even by RT-PCR. Conditioned media from CD34+ cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34+ cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34+ cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.
J. Clin. Invest. 104:1739–1749 (1999).
PMCID: PMC409882  PMID: 10606628
25.  Regulation of Inflammatory Chemokine Receptors on Blood T Cells Associated to the Circulating Versus Liver Chemokines in Dengue Fever 
PLoS ONE  2012;7(7):e38527.
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.
doi:10.1371/journal.pone.0038527
PMCID: PMC3398008  PMID: 22815692

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