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CHEMOKINE (C-C MOTIF) LIGAND 21
LATEST LITERATURE
1: Immunology and cell biology, 2010 Aug 31, 20(9)
The lymphoid chemokine CCL21 triggers LFA-1 adhesive properties on human dendritic cells.
[Abstract]Dendritic cells (DCs) are the most potent APCs, involved in the induction of immunity and tolerance. Recently we showed that during differentiation of human DCs from monocyte precursors, Lymphocyte function-associated antigen-1 (LFA-1)-binding capacity is lost, although integrin expression levels were maintained constant, suggesting a different regulation mechanism of this integrin on different cell types. However, the exact role of LFA-1 in DC adhesion and migration remains obscure. Chemokines are potent regulators of integrin function, influencing migratory and adhesive properties of leukocytes. Here, we show that upon vaccination of cancer patients with human DCs, cells that have migrated in vivo into the lymph nodes upregulated the active form of LFA-1. We further show that exposure of human DCs to the lymphoid chemokine CCL21 specifically restores the high-affinity form of LFA-1 and induces binding to its ligand ICAM-1 under low shear stress. Our data indicate that on DCs LFA-1 may function as an inducible anchor during lymphatic transmigration or within the lymph nodes. A thorough understanding of the adhesive events during the DC life cycle will help to improve the outcome of DC-based antitumor clinical trials.Immunology and Cell Biology advance online publication, 31 August 2010; doi:10.1038/icb.2010.103.
2: International immunology, 2010 Aug 25, 20(9)
Inflammation-induced secretion of CCL21 in lymphatic endothelium is a key regulator of integrin-mediated dendritic cell transmigration.
[Abstract]Tissue inflammation induces rapid mobilization of antigen-charged dendritic cells (DCs), which migrate to draining lymph nodes via afferent lymphatics to elicit the immune response. This increase in DC trafficking has been shown to require integrin-dependent adhesion to ICAM-1 and VCAM-1, expressed on inflamed lymphatic endothelium. In addition, both constitutive- and inflammation-induced DC migration involves the chemokine CCL21, which most likely triggers integrin activation on DC via its receptor CCR7. Recently, however, conflicting evidence has suggested that DC entry occurs independently of integrins, implying that the role of CCL21 in lymphatics is purely chemotactic. Hence, while CCL21 is reported to be inducible during inflammation, the details of this induction and the role of CCL21 during initial DC trafficking are unclear. Here, we have characterized both the production of CCL21 and the mechanism of its action in DC transmigration using primary human dermal lymphatic endothelial cells (HDLECs) and a mouse model of skin contact hypersensitivity. We showed that CCL21 is constitutively expressed intracellularly but rapidly secreted after exposure to the inflammatory cytokine tumour necrosis factor (TNF) alpha following de novo RNA and protein synthesis. Furthermore, using in vitro transmigration assays, we showed that endogenous HDLEC-derived CCL21 stimulates DC translymphatic migration by a predominantly chemotactic mechanism in resting HDLEC and by a beta2 integrin-mediated mechanism in TNFalpha-stimulated HDLEC. These results imply a direct role for CCL21 in lymphatic transmigration that involves the selective use of integrin activation in inflammation.
3: Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver, 2010 Jul 5,
The CCL21/CCR7 pathway plays a key role in human colon cancer metastasis through regulation of matrix metalloproteinase-9.
[Abstract]PURPOSE: CC chemokine receptor 7 (CCR7) and matrix metalloproteinase-9 (MMP-9) have been associated with lymph node metastasis in human colon cancer. Studies have suggested a potential link between CCR7 and MMP-9 in cancer; however, the molecular mechanism by which C-C ligand 21/CCR7 promotes tumour dissemination in human colon cancer is not well understood. Thus, we aimed to determine whether MMP-9 is regulated by the C-C ligand 21/CCR7 in human colon cancer. METHOD: RNA interference technology was employed to detect effect of CCR7 deficiency on the expression of MMP-9 in SW480 human colon cancer cells. We also evaluated the ability of CCR7 short hairpin RNA to inhibit MMP-9 production and tumour invasion in a xenografted mouse model by using whole-body fluorescence imaging and gelatin zymography. RESULT: We found that CCR7 short hairpin RNA significantly inhibited C-C ligand 21/CCR7-induced up-regulation of MMP-9 in SW480 cells. Furthermore, knockdown of CCR7 significantly limited the production of MMP-9 and colon cancer metastasis in a xenografted mouse model. Mice that received SW480/control cells had progressively enlarging tumours and more lymphatic metastases, and these animals did not survive as long as mice that received SW480/CCR7(-) cells. CONCLUSION: MMP-9 and CCR7 may be useful targets for the treatment of lymphatic metastasis in colon cancer.
4: Immunity, 2010 May 28, 32(5)
Double Duty for CCL21 in Dendritic Cell Trafficking.
[Abstract]Mechanisms controlling leukocyte adhesion, propulsion and directional migration have not been fully integrated. In this issue of Immunity, Schumann et al. (2010) propose that DCs swarm to T cell zones using immobilized CCL21 for adhesive random migration and soluble CCL21 for steering.
5: Glycobiology, 2010 May 20, 130(6)
Polysialylated neuropilin-2 enhances human dendritic cell migration through the basic C-terminal region of CCL21.
[Abstract]Dendritic cell (DC) migration to secondary lymphoid organs is a critical step to properly exert its role in immunity; and predominantly depends on the interaction of the chemokine receptor CCR7 with its ligands CCL21 and CCL19. Polysialic acid (PSA) has been recently reported to control CCL21-directed migration of mature DCs. Here; we first demonstrate that PSA present on human mature monocyte-derived dendritic cells did not enhance chemotactic responses to CCL19. We have also explored the molecular mechanisms underlying the selective enhancing effect of PSA on CCL21-driven chemotaxis of DCs. In this regard; we found out that prevention of DC polysialylation decreased CCL21 activation of JNK and Akt signaling pathways; both associated with CCR7-mediated chemotaxis. We also report that the enhanced PSA-mediated effect on DC migration towards CCL21 relied on the highly basic C-terminal region of this chemokine; and depended on the PSA acceptor molecule neuropilin-2 (NRP2) and on the polysialyltransferase ST8SiaIV. Altogether; our data indicate that the CCR7/CCL21/NRP2/ST8SiaIV functional axis constitutes an important guidance clue for DC targeting to lymphoid organs.
6: Experimental hematology, 2010 May 17, 130(6)
Analysis Of Migratory And Prosurvival Pathways Induced By The Homeostatic Chemokines Ccl19 And Ccl21 In B-Chronic Lymphocytic Leukemia.
[Abstract]OBJECTIVE: The CCR7 chemokine receptor has been reported to promote homing of B-chronic lymphocytic leukemia (CLL) cells into lymph nodes (LNs) and support their survival, but the mechanisms mediating these effects are largely unknown. We investigated the role of different signaling pathways triggered by CCR7 engagement by its ligands, the chemokines CCL19 and CCL21, in the control of CLL migration and survival. METHODS: Chemotaxis and apoptosis assays were performed in the presence of pharmacologic inhibitors and genetic mutants of the PI3K-, Rho GTPase- and MAPK- signaling cascades to assess the role of these pathways on primary CLL migration and survival in response to CCR7 activation. Kinase activation was determined by immunoblotting and pull-down experiments. RESULTS: CLL chemotactic activity induced by CCL19 or CCL21 was markedly reduced by inhibitors of PI3K and the Rho effector molecule ROCK, and also by the expression of dominant negative forms of PI3K and RhoA, whereas constitutively activated PI3K and RhoA mutants strongly promoted CLL migration. In contrast, MAPKs were not significantly involved in CLL migration to CCL19/CCL21. Conversely, ERK and JNK, along with PI3K, had a role in CCR7-mediated CLL cell survival. Biochemical experiments confirmed that CCL19/21 induced PI3K-dependent phosphorylation of Akt/PKB, activation of the Rho/ROCK/MLC pathway and MAPKs phosphorylation. CONCLUSIONS: The role of PI3K, Rho GTPases and MAPKs in CCR7-mediated CLL cells migration and survival suggests that these signal transduction pathways could represent promising targets for CLL therapy.
7: Arthritis care & research, 2010 May, 62(5)
Association of a rheumatoid arthritis susceptibility variant at the CCL21 locus with premature mortality in inflammatory polyarthritis patients.
[Abstract]OBJECTIVE: To investigate whether recently identified rheumatoid arthritis (RA) susceptibility loci are also associated with disease severity, specifically all-cause and cardiovascular disease (CVD) mortality, in patients with inflammatory polyarthritis (IP). METHODS: Subjects with recent-onset IP were recruited from the Norfolk Arthritis Register. Seventeen RA susceptibility single-nucleotide polymorphisms (SNPs) were tested using Sequenom MassArray iPLEX chemistry. Vital status was ascertained from central records. The association of SNP allele carriage with mortality risk was assessed using Cox proportional hazards models after adjusting by sex. The mortality risks of those SNP alleles found to be associated were then stratified by baseline anti-citrullinated peptide (anti-CCP) antibody and shared epitope (SE) status. RESULTS: All SNPs were successfully genotyped in 2,324 IP subjects. The presence of 2 copies of the risk allele rs2812378 mapping to the CCL21 gene predicted all-cause mortality (hazard ratio [HR] 1.40, 95% confidence interval [95% CI] 1.04-1.87), whereas risk allele carriage also predicted increased CVD mortality (HR 1.33, 95% CI 1.01-1.75). The highest mortality risks were seen in anti-CCP antibody-positive subjects with 2 copies of the CCL21 risk alleles and 2 copies of the SE (all-cause HR 3.20, 95% CI 1.52-6.72; CVD HR 3.73, 95% CI 1.30-10.72). CONCLUSION: In this large study, we found that carriage of CCL21 risk alleles was associated with premature mortality in IP independently of anti-CCP antibody and SE status. Interestingly, CCL21 expression has been reported in atherosclerotic plaques supporting the thesis that the increased CVD mortality in IP patients may be mediated by shared inflammatory mechanisms.
8: Science (New York, N.Y.), 2010 Mar 25,
Induction of Lymphoidlike Stroma and Immune Escape by Tumors That Express the Chemokine CCL21.
[Abstract]Tumor manipulation of host immunity is important for tumor survival and invasion. Many cancers secrete CCL21, a chemoattractant for various leukocytes and lymphoid tissue inducer cells, which drive lymphoid neogenesis. CCL21 expression by melanoma tumors in mice was associated with an immunotolerant microenvironment, which included the induction of lymphoidlike reticular stromal networks, an altered cytokine milieu, and the recruitment of regulatory leukocyte populations. In contrast, CCL21-deficient tumors induced antigen-specific immunity. CCL21-mediated immune tolerance was dependent on host rather than tumor expression of the CCL21 receptor, CCR7, and could protect distant, coimplanted CCL21-deficient tumors and nonsyngeneic allografts from rejection. We suggest that by altering the tumor microenvironment, CCL21-secreting tumors shift the host immune response from immunogenic to tolerogenic, thereby facilitating tumor progression.
9: Circulation, 2010 Mar 15,
Targeting Lymphatic Vessel Activation and CCL21 Production by Vascular Endothelial Growth Factor Receptor-3 Inhibition Has Novel Immunomodulatory and Antiarteriosclerotic Effects in Cardiac Allografts.
[Abstract]BACKGROUND: -Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts. Methods and Results-Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C(+) inflammatory cell and hyaluronan receptor-1 (LYVE-1)(+) lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3(+), contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein-positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8(+) effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62(+) dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4(+) T cells in chronically rejecting mouse cardiac allografts. Conclusions-These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel-targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.
10: The Journal of investigative dermatology, 2010 Feb 25,
Induction of Targeted Cell Migration by Cutaneous Administration of a DNA Vector Encoding a Biologically Active Chemokine CCL21.
[Abstract]Skin inflammation can induce local expression of CCL21, which is subsequently drained to lymph nodes (LNs) influencing their cellular composition. To determine whether the same can be achieved by dermal administration of a plasmid DNA (pDNA) encoding CCL21, we generated a pDNA-based gene construct allowing high-level expression of CCL21. Expression and secretion of biologically active CCL21 were confirmed in vitro by immunohistochemistry, western blot analysis, ELISA, and transwell chemotactic assays. In vivo experiments showed cellular expression of transgenic CCL21 after particle-mediated gene gun delivery of pDNA into skin. CCL21 was expressed in the epidermis, consequently secreted into the upper dermis, and transported into the draining LNs, which resulted in increased CCL21 concentration, total cell number, and frequencies of CD11c(+) DCs and CD4(+)/CD62L(+) na?ve, CD4(+)/CD62L(-), and CD8(+)/CD62L(-) effector memory T-cells (expressing CCL21 receptors CCR7 or CXCR3), as well as retention of adoptively transferred T-lymphocytes, in the draining LNs of plt/plt mice (lacking endogenous expression of CCL21). Our studies show that biologically active CCL21 can be overexpressed by genetic means in vitro and in vivo. This strategy allows reconstitution of a genetic defect and colocalization of different cell types in the secondary lymphoid organs, an important prerequisite for targeted cell migration.Journal of Investigative Dermatology advance online publication, 25 February 2010; doi:10.1038/jid.2010.31.
11: PloS one, 2010, 5(1)
TWEAK activates the non-canonical NFkappaB pathway in murine renal tubular cells: modulation of CCL21.
[Abstract]TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFkappaB activation suggestive of engagement of the non-canonical NFkappaB pathway. We now explore TWEAK-induced activation of NFkappaB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFalpha activated different DNA-binding NFkappaB complexes. TWEAK-induced sustained NFkappaB activation was associated with NFkappaB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFalpha used as control), induced a delayed increase in CCL21a mRNA (3.5+/-1.22-fold over control) and CCL21 protein (2.5+/-0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFkappaB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFalpha. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFkappaB2 and RelB translocation and CCL21a mRNA (1.5+/-0.3-fold over control) and CCL21 protein (1.6+/-0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2+/-0.9 vs 1.3+/-0.6-fold over healthy control) or deficiency of TWEAK (2+/-0.9 vs 0.8+/-0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1+/-1.4 vs 1.8+/-1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFkappaB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.
12: Annals of neurology, 2009 Mar 18, 66(4)
CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia.
[Abstract]OBJECTIVE: Myasthenia gravis (MG), a neuromuscular disease mediated by anti-acetylcholine receptor (AChR) autoantibodies, is associated with thymic hyperplasia characterized by ectopic germinal centers that contain pathogenic antibody-producing B cells. Our thymic transcriptome study demonstrated increased expression of CCL21, a recruiter of immune cells. Accordingly, we are investigating its implication in MG pathogenesis. METHODS: The expression of CCL21 and its CCR7 receptor was analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting, respectively. Chemotaxis of T and B cells to CCL21 was measured by transwell assay. The nature of the thymic cells overexpressing CCL21 was investigated by immunochemistry and laser-capture microdissection combined with real-time PCR. RESULTS: We demonstrate that CCL21 is overexpressed specifically in hyperplastic MG thymuses, whereas there is no variation in CCR7 levels on blood cells. We show that although CCL21 attracts both human T and B cells, it acts more strongly on naive B cells. CCL21 overexpression is normalized in corticoid-treated MG patients, suggesting that targeting this chemokine could represent a new selective treatment, decreasing the abnormal peripheral lymphocyte recruitment. Moreover, we locate protein and messenger RNA overexpression of CCL21 to specific endothelial vessels. Investigation of the nature of these vessels demonstrated different angiogenic processes in MG thymuses: high endothelial venule angiogenesis and lymphangiogenesis. Unexpectedly, CCL21 overexpression originates from afferent lymphatic endothelial vessels. INTERPRETATION: We postulate that thymic overexpression of CCL21 on specialized lymphatic vessels results in abnormal peripheral lymphocyte recruitment, bringing naive B cells in contact with the inflammatory environment characteristic of MG thymuses, where they can be sensitized against AChR. Ann Neurol 2009;66:521-531.
13: PloS one, 2009, 4(9)
Interaction of polysialic acid with CCL21 regulates the migratory capacity of human dendritic cells.
[Abstract]Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). Immature DCs (iDCs) are situated in the periphery where they capture pathogen. Subsequently, they migrate as mature DCs (mDCs) to draining lymph nodes to activate T cells. CCR7 and CCL21 contribute to the migratory capacity of the DC, but it is not completely understood what molecular requirements are involved. Here we demonstrate that monocyte-derived DCs dramatically change ST8Sia IV expression during maturation, leading to the generation of polysialic acid (polySia). PolySia expression is highly upregulated after 2 days Toll-like receptor-4 (TLR4) triggering. Surprisingly, only immunogenic and not tolerogenic mDCs upregulated polySia expression. Furthermore, we show that polySia expression on DCs is required for CCL21-directed migration, whereby polySia directly captures CCL21. Corresponding to polySia, the expression level of CCR7 is maximal two days after TLR4 triggering. In contrast, although TLR agonists other than LPS induce upregulation of CCR7, they achieve only a moderate polySia expression. In situ we could detect polySia-expressing APCs in the T cell zone of the lymph node and in the deep dermis. Together our results indicate that prolonged TLR4 engagement is required for the generation of polySia-expressing DCs that facilitate CCL21 capture and subsequent CCL21-directed migration.
14: Journal of immunology (Baltimore, Md. : 1950), 2009 Oct 1, 183(7)
Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment.
[Abstract]In the paracortex of the lymph node (LN), T zone fibroblastic reticular cells (TRCs) orchestrate an immune response by guiding lymphocyte migration both physically, by creating three-dimensional (3D) cell networks, and chemically, by secreting the chemokines CCL19 and CCL21 that direct interactions between CCR7-expressing cells, including mature dendritic cells and naive T cells. TRCs also enwrap matrix-based conduits that transport fluid from the subcapsular sinus to high endothelial venules, and fluid flow through the draining LN rapidly increases upon tissue injury or inflammation. To determine whether fluid flow affects TRC organization or function within a 3D network, we regenerated the 3D LN T zone stromal network by culturing murine TRC clones within a macroporous polyurethane scaffold containing type I collagen and Matrigel and applying slow interstitial flow (1-23 microm/min). We show that the 3D environment and slow interstitial flow are important regulators of TRC morphology, organization, and CCL21 secretion. Without flow, CCL21 expression could not be detected. Furthermore, when flow through the LN was blocked in mice in vivo, CCL21 gene expression was down-regulated within 2 h. These results highlight the importance of lymph flow as a homeostatic regulator of constitutive TRC activity and introduce the concept that increased lymph flow may act as an early inflammatory cue to enhance CCL21 expression by TRCs, thereby ensuring efficient immune cell trafficking, lymph sampling, and immune response induction.
15: Ai zheng = Aizheng = Chinese journal of cancer, 2009 Jul, 28(7)
[Role of CCL21/CCR7 in invasion of colorectal carcinoma cell line SW480.]
[Abstract]Background and Objective: Chemokine receptor CCR7 is up-regulated in gastrointestinal carcinomas and is significantly associated with lymphatic invasion and lymph node metastasis. This study was to investigate the role and mechanism of CCL21/CCR7 in invasion of colorectal carcinoma cell line SW480. Methods: The invasive capacity of SW480 cells was examined using Wound healing assay and Transwell assay. Expression of matrix metalloproteinase-9 (MMP-9) was measured by Western blot. SW480 cells were pre-incubated with CCL21 for 2 h before exposure to VP-16 (20 ng/mL). Cell proliferation was measured using MTT assay. Cell apoptosis was analyzed by flow cytometry and Hoechst33258 staining. Results: Compared to the control group, more cells in the CCL21 treatment group migrated into the gap at same time points; the count of SW480 cells penetrating through the membrane after the treatment of 100ng/mL CCL21 increased significantly [(113+/-7) vs. (48+/-4)] (P<0.05); and the relative expression of MMP-9 in the CCL21 treatment group was enhanced evidently [(0.83+/-0.02) vs. (0.38+/-0.01)] (P<0.05). Although CCL21 alone did not promote proliferation of SW480 cells, pre-incubation of cells with 100ng/mL CCL21 attenuated the inhibitory effect of VP-16 on proliferation of SW480 from 68.3% to 47.4%, and reduced the apoptotic rate from (65.2+/-5.2)% to (48.7+/-3.1)%. Conclusion: CCL21 enhances the invasive ability of SW480 cells, induces MMP-9 expression, and promotes the survival of SW480 cells under the suboptimal circumstance in vitro.
16: Blood, 2009 Jul 16, 114(3)
CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.
[Abstract]CD4(+) T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)-transgenic (tg) CD4(+) T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3Kdelta(D910A/D910A) or PI3Kgamma-deficient TCR-tg CD4(+) T cells showed similar responsiveness to CCL21 costimulation as control CD4(+) T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4(+) T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca(2+) signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.
17: Cancer microenvironment : official journal of the International Cancer Microenvironment Society, 2009 May 6, 182(11)
Regulated Expression of CCL21 in the Prostate Tumor Microenvironment Inhibits Tumor Growth and Metastasis in an Orthotopic Model of Prostate Cancer.
[Abstract]Currently there are no curative therapies available for patients with metastatic prostate cancer. Thus, novel therapies are needed to treat this patient population. Immunotherapy represents one promising approach for the elimination of occult metastatic tumors. However, the prostate tumor microenvironment (TME) represents a hostile environment capable of suppressing anti-tumor immunity and effector cell function. In view of this immunosuppressive activity, we engineered murine prostate cancer cells with regulated expression (tet-on) of CCL21. Prostate tumor cells implanted orthotopically produced primary prostate tumors with predictable metastatic disease in draining lymph nodes and distant organs. Expression of CCL21 in the prostate TME enhanced survival, inhibited tumor growth and decreased the frequency of local (draining lymph node) and distant metastasis. Therefore, these studies provide a strong rationale for further evaluation of CCL21 in tumor immunity and its use in cancer immunotherapy.
18: Brain, behavior, and immunity, 2009 May 3, 182(11)
CCL21-induced calcium transients and proliferation in primary mouse astrocytes: CXCR3-dependent and independent responses.
[Abstract]CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in damaged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10(-9)M and 10(-8)M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high concentrations of CCL21 (10(-7)M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 promoted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (chemokine-) receptor, which is activated at high CCL21 concentrations.
19: Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences, 2009 Mar, 38(2)
[Construction of murine CCL21 eukaryotic expression plasmid and chemotactic function of its products]
[Abstract]OBJECTIVE: To construct the murine CCL21 eukaryotic expression plasmid, and to investigate the chemotactic function of its products. METHODS: Murine CCL21 cDNA was amplified by RT-PCR from murine total RNA, and was inserted into eukaryotic expression plasmid pcDNA3.1 after confirmation of sequencing. The recombinant CCL21 plasmid was transferred into mouse forestomach carcinoma (MFC) cells and the chemotactic function of expressed products was detected by chemotaxis assay. RESULT: Gene sequencing, gel electrophoresis of PCR products and restrictive digestion proved the successful construction of CCL21, and its expression was confirmed by Western Blot. The transfected tumor cells had a significant chemotactic function to DC. Conclusion: The recombinant murine CCL21 eukaryotic expression plasmid has been successfully constructed, and its expression products in tumor cells have a marked chemotactic function to DC.
20: Journal of immunology (Baltimore, Md. : 1950), 2009 Apr 15, 182(8)
CD30 is required for CCL21 expression and CD4 T cell recruitment in the absence of lymphotoxin signals.
[Abstract]Lymphoid tissue inducer cells express a diverse array of tumor necrosis family ligands, including those that bind CD30 and the lymphotoxin beta receptor. Both of these signaling pathways have been linked with B/T segregation in the spleen. In this study, we have dissected a lymphotoxin-independent CD30-dependent signal for the induction of expression of the T zone chemokine, CCL21. Reduced expression of CCL21 due to CD30 deficiency was functionally significant: mice deficient in both lymphotoxin and CD30 (dKO) signals had significantly smaller accumulations of lymphocytes in their splenic white pulp areas, with no evidence of focal aggregation of T cells. Furthermore, recruitment of wild-type CD4 T cells was poor in dKO mice compared with both wild-type or lymphotoxin-deficient mice. Phylogeny suggests that CD30 signals predated those through the lymphotoxin beta receptor. We suggest that CD30 signals from lymphoid tissue inducer cells were a primitive mechanism to recruit and prime CD4 T cells. This would have been a stepping stone in the evolution of the highly organized lymphotoxin dependent B and T white pulp areas within which CD4-dependent memory Ab responses now develop.
21: Oncology reports, 2009 Jan, 21(1)
Decreased expression of the chemokine CCL21 in human colorectal adenocarcinomas.
[Abstract]Recent studies have revealed participation of chemokines in cancer by regulating leukocyte movement to modify local immunoresponse. The chemokine CCL21 has been identified to play a pivotal role in homing and localization of immune cells to lymphoid tissue and into organ of non-lymphoid origin. In the cancer biology CCL21 seems to have multifaceted roles. CCL21 attracts CCR7 bearing cells especially T and dendritic cells but also various cancer cells. Besides the antitumour role as leukocyte recruiting, CCL21 has been shown to facilitate dendritic cell functions and to exert an angiostatic effect. To gain insight into the possible influence of CCL21 on colorectal cancer (CRC) we determined whether the CCL21 is altered in CRC tissue. Collectively, by using ELISA we noted a significant lower CCL21 level in cancer tissue compared with paired normal tissue. Patients with a tumour localized in the rectum revealed significantly lower level of CCL21 than patients with a tumour localized in the colon both compared with paired normal tissue. We used immunohistochemistry and found heterogeneous immunoreactivity predominantly within areas of stromal cells mainly in macrophages. We also used a TaqMan system to investigate two single-nucleotide polymorphisms rs 11574915 and rs 2812377 with supposed effect on CRC. No significant difference was observed between CRC and control subjects regarding genotype and allelic distributions or associations to clinical characteristics or CCL21 tissue levels. Our study implied that lower level of CCL21 in CRC tissue supports the idea that cancer is related to immunodeficiency probably depending on regulatory factors produced by tumour cells and that the different levels of CCL21 in rectum and colon may reflect divergent mechanisms in colorectal carcinogenesis. Further studies are needed to clarify whether the CCL21 level has an impact on CRC progression and survival rate.
22: AIDS (London, England), 2009 Jan 2, 23(1)
Enhanced levels of the CCR7 ligands CCL19 and CCL21 in HIV infection: correlation with viral load, disease progression and response to highly active antiretroviral therapy.
[Abstract]The CCR7 ligands, CCL19 and CCL21, coordinate lymph node homing of naive and central memory T cells. In untreated HIV-infected patients, serum levels of CCL19 and CCL21 showed a biphasic pattern during progression; a marked increase was followed by a decline in patients with advanced immunodeficiency. During highly active antiretroviral therapy, a decrease in CCL19/CCL21 levels was restricted to virologic responders. We suggest that dysregulation of CCR7 ligands may play an important role in progression of HIV infection.
23: Journal of immunology (Baltimore, Md. : 1950), 2008 Oct 1, 181(7)
Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21.
[Abstract]Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.
24: Blood, 2008 Dec 15, 112(13)
Anti-inflammatory effects of an inflammatory chemokine: CCL2 inhibits lymphocyte homing by modulation of CCL21-triggered integrin-mediated adhesions.
[Abstract]Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1- and LFA-1-mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.
25: Cancer research, 2008 Sep 1, 68(17)
CXCL13 is highly produced by S¨¦zary cells and enhances their migratory ability via a synergistic mechanism involving CCL19 and CCL21 chemokines.
[Abstract]Chemokine and chemokine receptors expressed by normal and neoplastic lymphocytes play a key role in cell recruitment into skin and lymph nodes. The aim of this study was to get further insights into the role of chemokines in pathogenesis and progression of cutaneous T-cell lymphoma (CTCL) with particular regard to S¨¦zary Syndrome (SS), a CTCL variant with blood involvement. Here, we show that functional CXCL13 homeostatic chemokine is strongly up-regulated in SS cells, well-detectable in skin lesions and lymph nodes, and measurable at high concentration in plasma of SS patients, at different levels during disease progression. Furthermore, we show that the addition of CXCL13 to CCL19 or to CCL21, the selective CCR7 agonists responsible for lymph node homing, strongly enhances the migration of CCR7+ SS cells. We also show that neutralization of the CCR7 receptor strongly impairs CCL19/21-induced chemotaxis of SS cells both in the absence or presence of CXCL13. Additional experiments performed to investigate the survival, adhesion, and metalloproteases secretion indicate that CXCL13 combined with CCL19 and CCL21 mainly affects the chemotaxis of SS cells. Our findings suggest that this newly described CXCL13 expression in SS represents a new pathogenetic mechanism of diagnostic significance.
26: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2008 Dec, 22(12)
Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles.
[Abstract]Neurons are highly polarized cells, and neuron-neuron communication is based on directed transport and release of neurotransmitters, neuropeptides, and neurotrophins. Directed communication may also be attributed to neuron-microglia signaling, since neuronal damage can induce a microglia reaction at specific sites only. However, the mechanism underlying this site-specific microglia reaction is not yet understood. Neuronal CCL21 is a microglia-activating chemokine, which in brain is solely found in endangered neurons and is therefore a candidate for neuron-microglia signaling. Here we present that neuronal CCL21 is sorted into large dense-core vesicles, the secretory granules of the regulated release pathway of neurons. Live-cell imaging studies show preferential sorting of CCL21-containing vesicles into axons, indicating its directed transport. Thus, mouse neurons express and transport a microglia activating factor very similar to signaling molecules used in neuron-neuron communication. These data show for the first time the directed transport of a microglia activating factor in neurons and corroborate the function of neuronal CCL21 in directed neuron-microglia communication.