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1.  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
2.  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
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.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  Chemokine Receptor CCR1 Regulates Inflammatory Cell Infiltration after Renal Ischemia-Reperfusion Injury 
Neutrophils and macrophages rapidly infiltrate the kidney after renal ischemia-reperfusion injury, however specific molecular recruitment mechanisms have not been fully delineated for these cell types. Here we provide genetic and pharmacologic evidence supporting a positive role for the chemokine receptor CCR1 in macrophage and neutrophil infiltration in a 7 day mouse model of renal ischemia-reperfusion injury. By day 7, injured kidneys from mice lacking CCR1 contained 35% fewer neutrophils and 45% fewer macrophages than injured kidneys from wild type control mice. Pretreatment of WT mice with the specific CCR1 antagonist BX471 also suppressed neutrophil and macrophage infiltration in the model. Injured kidneys from mice lacking CCR1 also had reduced content of the CCR1 ligands CCL3 (MIP-1α) and CCL5 (RANTES) compared to injured kidneys from wild type controls, suggesting a leukocyte source for these inflammatory chemokines and existence of a CCR1-dependent positive feedback loop for leukocyte infiltration in the model. Local leukocyte proliferation and apoptosis were detected after injury, but were not dependent on CCR1. Also, the extent of necrotic and fibrotic damage and decline in renal function in injured kidneys was similar in wild type and CCR1-deficient mice. Thus, CCR1 appears to regulate trafficking of macrophages and neutrophils to kidney in a mouse model of renal ischemia-reperfusion injury, however this activity does not appear to affect tissue injury.
PMCID: PMC2633769  PMID: 19050287
Molecules-chemokines; Cells-monocytes/macrophages; Processes-inflammation
19.  Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice 
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/s13075-014-0445-9
PMCID: PMC4201712  PMID: 25248373
20.  Effects of chemokines on proliferation and apoptosis of human mesangial cells 
BMC Nephrology  2004;5:8.
Background
Proliferation and apoptosis of mesangial cells (MC) are important mechanisms during nephrogenesis, for the maintenance of glomerular homeostasis as well as in renal disease and glomerular regeneration. Expression of chemokines and chemokine receptors by intrinsic renal cells, e.g. SLC/CCL21 on podocytes and CCR7 on MC is suggested to play a pivotal role during these processes. Therefore the effect of selected chemokines on MC proliferation and apoptosis was studied.
Methods
Proliferation assays, cell death assays including cell cycle analysis, hoechst stain and measurement of caspase-3 activity were performed.
Results
A dose-dependent, mesangioproliferative effect of the chemokine SLC/CCL21, which is constitutively expressed on human podocytes was seen via activation of the chemokine receptor CCR7, which is constitutively expressed on MC. In addition, in cultured MC SLC/CCL21 had a protective effect on cell survival in Fas-mediated apoptosis. The CXCR3 ligands IP-10/CXCL10 and Mig/CXCL9 revealed a proproliferative effect but did not influence apoptosis of MC. Both the CCR1 ligand RANTES/CCL5 and the amino-terminally modified RANTES analogue Met-RANTES which blocks CCR1 signalling had no effect on proliferation and apoptosis.
Conclusions
The different effects of chemokines and their respective receptors on proliferation and apoptosis of MC suggest highly regulated, novel biological functions of chemokine/chemokine receptor pairs in processes involved in renal inflammation, regeneration and glomerular homeostasis.
doi:10.1186/1471-2369-5-8
PMCID: PMC493268  PMID: 15265234
Mesangial cell; proliferation; apoptosis; chemokine; chemokine receptor
21.  Low CCR7-Mediated Migration of Human Monocyte Derived Dendritic Cells in Response to Human Respiratory Syncytial Virus and Human Metapneumovirus 
PLoS Pathogens  2011;7(6):e1002105.
Human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV) largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC) is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC) with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV-stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to lymphatic tissue contributes to reduced adaptive responses to these viruses.
Author Summary
The respiratory viruses human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus virus (HMPV) and human parainfluenza virus (HPIV3), can re-infect humans throughout life without significant antigenic change, suggesting that immunity to these viruses is incomplete. In contrast, re-infection by influenza A virus (IAV) depends on antigenic change, suggestive of more complete immunity. Dendritic cells (DC) take up virus antigen at the site of infection, undergo maturation, and migrate to the lymphatic tissue to present antigen to T lymphocytes, orchestrating the immune response. In response to antigen uptake, DC switch chemokine receptors on their surface, decreasing expression of receptors for inflammatory chemokines in the infected tissue, and increasing expression of CCR7, the sole chemokine receptor that directs DC to migrate to lymphatic tissue. By stimulating human DC in vitro, we found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently induce the switching of these surface receptors. In cell migration assays, we showed that, compared with IAV-treated DC, HRSV- or HMPV-treated DC migrated less efficiently to CCL19, a chemokine that directs T cell migration to lymphatic tissue. Thus, during infection with HRSV and HMPV, inefficient migration of DC to the lymphatics could reduce the adaptive immune response to these viruses.
doi:10.1371/journal.ppat.1002105
PMCID: PMC3121884  PMID: 21731495
22.  Expression and Functional Significance of SOCS-1 and SOCS-3 in Astrocytes 
Astrocytes play a number of important physiological roles in Central Nervous System (CNS) homeostasis. Inflammation stimulates astrocytes to secrete cytokines and chemokines that guide macrophages/microglia and T-cells to sites of injury/inflammation, and herein we describe how these processes are controlled by the Suppressor Of Cytokine Signaling (SOCS) proteins, a family of proteins that negatively regulate adaptive and innate immune responses. In this study, we describe that the immunomodulatory cytokine IFN-β induces SOCS-1 and SOCS-3 expression in primary astrocytes at the transcriptional level. SOCS-1 and SOCS-3 transcriptional activity is induced by IFN-β through GAS elements within their promoters. Studies in STAT-1α deficient astrocytes indicate that STAT-1α is required for IFN-β-induced SOCS-1 expression, while STAT-3 siRNA studies demonstrate that IFN-β-induced SOCS-3 expression relies on STAT-3 activation. Specific siRNA inhibition of IFN-β-inducible SOCS-1 and SOCS-3 in astrocytes enhances their pro-inflammatory responses to IFN-β stimulation, such as heightened expression of the chemokines CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES) and CXCL10 (IP-10), and promoting chemotaxis of macrophages and CD4+ T-cells. These results indicate that IFN-β induces SOCS-1 and SOCS-3 in primary astrocytes in order to attenuate its own chemokine-related inflammation in the CNS.
PMCID: PMC2836124  PMID: 18713987
23.  Murine lung eosinophil activation and chemokine production in allergic airway inflammation 
Cellular & molecular immunology  2010;7(5):361-374.
Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.
doi:10.1038/cmi.2010.31
PMCID: PMC3045045  PMID: 20622891
allergy; chemokines; eosinophils; lung; mouse
24.  Murine lung eosinophil activation and chemokine production in allergic airway inflammation 
Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.
doi:10.1038/cmi.2010.31
PMCID: PMC3045045  PMID: 20622891
allergy; chemokines; eosinophils; lung; mouse
25.  Transforming growth factor-β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor-κB in airway smooth muscle cells 
Summary
Background
Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-β) may be involved in the process of airway remodelling.
Objective
We analysed the effects of TGF-β on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms.
Methods
HASM cells were cultured and treated with TGF-β and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids.
Results
IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-β alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-β. Activation by IL-4 or IL-4 plus TGF-β was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-β was inhibited by mutation of the binding site for nuclear factor-κB (NF-κB) in the promoter. Pretreatment with an inhibitor of NF-κB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-β, indicating the importance of NF-κB in the cooperative activation of CCL11 transcription by TGF-β and IL-4.
Conclusion
These results indicate that Th2 cytokines and TGF-β may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-κB may play pivotal roles in this process.
doi:10.1111/j.1365-2222.2010.03474.x
PMCID: PMC3712283  PMID: 20214667
airway smooth muscle cells; asthma; CCL11; eotaxin; NF-κB; TGF-β

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