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• W2080232806 abstract "CCL21 expression by lymphatic endothelial cells (LECs) is essential for migration of CCR7+ immune cells from skin to regional lymph nodes (LNs). We investigated the importance of mitogen-activated protein kinase (MAPK) signaling in CCL21 expression by ECs in vitro and in vivo. Normal human dermal lymphatic microvascular ECs (HMVEC-dLy) stimulated in vitro with oncostatin M (OSM) expressed high amounts of CCL21 mRNA. CCL21 protein expression by HMVEC-dLy was also markedly increased by OSM compared with unstimulated cultures. Marked phosphorylation of MAPK 44/42 was detected in HMVEC-dLy stimulated by OSM. CCL21 expression by HMVEC-dLy was blocked by a JAK inhibitor 1, JAK3 inhibitor, and U0126 (a MAPK kinase inhibitor) in vitro, all of which blocked phosphorylation of MAPK 44/42. In addition, injection of U0126 into murine skin significantly decreased CCL21 mRNA and protein expression. Moreover, injection of U0126 before sensitization decreased migration of dendritic cells to draining LNs and decreased contact hypersensitivity responses. In summary, these results suggest that the MAPK pathway is important for CCL21 expression by LECs in vitro and in vivo. Blocking MAPK signaling within skin may offer a novel approach to treatment of inflammatory skin diseases. CCL21 expression by lymphatic endothelial cells (LECs) is essential for migration of CCR7+ immune cells from skin to regional lymph nodes (LNs). We investigated the importance of mitogen-activated protein kinase (MAPK) signaling in CCL21 expression by ECs in vitro and in vivo. Normal human dermal lymphatic microvascular ECs (HMVEC-dLy) stimulated in vitro with oncostatin M (OSM) expressed high amounts of CCL21 mRNA. CCL21 protein expression by HMVEC-dLy was also markedly increased by OSM compared with unstimulated cultures. Marked phosphorylation of MAPK 44/42 was detected in HMVEC-dLy stimulated by OSM. CCL21 expression by HMVEC-dLy was blocked by a JAK inhibitor 1, JAK3 inhibitor, and U0126 (a MAPK kinase inhibitor) in vitro, all of which blocked phosphorylation of MAPK 44/42. In addition, injection of U0126 into murine skin significantly decreased CCL21 mRNA and protein expression. Moreover, injection of U0126 before sensitization decreased migration of dendritic cells to draining LNs and decreased contact hypersensitivity responses. In summary, these results suggest that the MAPK pathway is important for CCL21 expression by LECs in vitro and in vivo. Blocking MAPK signaling within skin may offer a novel approach to treatment of inflammatory skin diseases. antibody antigen acutely transforming retrovirus AKT8 in rodent T cell lymphoma contact hypersensitivity dendritic cell endothelial cell glyceraldehyde-3-phosphate dehydrogenase normal human dermal lymphatic microvascular EC lymphatic EC lymph node lymphatic vascular EC-1 mitogen-activated protein kinase microvascular EC MAPK/extracellular signal-regulated kinase kinase oncostatin M phosphate-buffered saline signal transducers and activators of transcription vascular endothelial growth factor receptor 3 CCL21 and its receptor, CCR7, are critically involved in the migration of antigen (Ag)-presenting dendritic cells (DCs; Forster et al., 1999Forster R. Schubel A. Breitfeld D. et al.CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1814) Google Scholar; Gunn et al., 1999Gunn M.D. Kyuwa S. Tam C. et al.Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.J Exp Med. 1999; 189: 451-460Crossref PubMed Scopus (870) Google Scholar; Saeki et al., 1999Saeki H. Moore A.M. Brown M.J. et al.Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.J Immunol. 1999; 162: 2472-2475PubMed Google Scholar) from peripheral tissues to draining lymph nodes (LNs) via afferent lymphatic vessels. The expression of CCL21 is restricted to high endothelial venules, stromal cells in secondary lymphoid organs (Gunn et al., 1998Gunn M.D. Tangemann K. Tam C. et al.A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes.Proc Natl Acad Sci USA. 1998; 95: 258-263Crossref PubMed Scopus (833) Google Scholar), and lymphatic vessels in multiple organs including skin (Saeki et al., 1999Saeki H. Moore A.M. Brown M.J. et al.Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.J Immunol. 1999; 162: 2472-2475PubMed Google Scholar). DCs from CCR7-deficient mice are strikingly impaired in their ability to enter lymphatic vessels and migrate to secondary lymphoid organs (Forster et al., 1999Forster R. Schubel A. Breitfeld D. et al.CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1814) Google Scholar; Ohl et al., 2004Ohl L. Mohaupt M. Czeloth N. et al.CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions.Immunity. 2004; 21: 279-288Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar). Moreover, there is a marked reduction of naive T cells and disorganization of the DC network within LN of mice homozygous for the paucity of LN T-cell (plt) mutation, which is characterized by the lack of CCL21 expression in LN (Nakano et al., 1998Nakano H. Mori S. Yonekawa H. et al.A novel mutant gene involved in T-lymphocyte-specific homing into peripheral lymphoid organs on mouse chromosome 4.Blood. 1998; 91: 2886-2895Crossref PubMed Google Scholar; Gunn et al., 1999Gunn M.D. Kyuwa S. Tam C. et al.Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.J Exp Med. 1999; 189: 451-460Crossref PubMed Scopus (870) Google Scholar; Fagarasan et al., 2000Fagarasan S. Shinkura R. Kamata T. et al.Alymphoplasia (aly)-type nuclear factor κB-inducing kinase (NIK) causes defects in secondary lymphoid tissue chemokine receptor signaling and homing of peritoneal cells to the gut-associated lymphatic tissue system.J Exp Med. 2000; 191: 1477-1486Crossref PubMed Scopus (105) Google Scholar). Collectively, these data suggest that CCL21 and CCR7 are vital for normal migration and retention of specific T cells and DCs within secondary lymphoid organs. Oncostatin M (OSM) is a member of the IL-6 family of cytokines with pleiotropic biological effects (Tanaka and Miyajima, 2003Tanaka M. Miyajima A. Oncostatin M, a multifunctional cytokine.Rev Physiol Biochem Pharmacol. 2003; 149: 39-52Crossref PubMed Google Scholar). Produced by a number of inflammatory cells, including activated T cells, monocytes, and DCs (Suda et al., 2003Suda T. Chida K. Todate A. et al.Oncostatin M production by human dendritic cells in response to bacterial products.Cytokine. 2003; 17: 335-340Crossref Scopus (52) Google Scholar; Tanaka and Miyajima, 2003Tanaka M. Miyajima A. Oncostatin M, a multifunctional cytokine.Rev Physiol Biochem Pharmacol. 2003; 149: 39-52Crossref PubMed Google Scholar), OSM can exert both pro- and anti-inflammatory effects, depending on the model system examined (Pelletier and Martel-Pelletier, 2003Pelletier J.P. Martel-Pelletier J. Oncostatin M: foe or friend?.Arthritis Rheum. 2003; 48: 3301-3303Crossref PubMed Scopus (25) Google Scholar). Endothelial cells (ECs) express high numbers of OSM receptors, and OSM has been shown to upregulate expression of several cytokines and chemokines in human umbilical vein endothelial cell (Modur et al., 1997Modur V. Feldhaus M.J. Weyrich A.S. et al.Oncostatin M is a proinflammatory mediator: in vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules.J Clin Invest. 1997; 100: 158-168Crossref PubMed Scopus (158) Google Scholar). Furthermore, OSM is a potent growth factor for AIDS-associated Kaposi’s sarcoma (Miles et al., 1992Miles S.A. Martinez-Maza O. Rezai A. et al.Oncostatin M as a potent mitogen for AIDS-Kaposi’s sarcoma-derived cells.Science. 1992; 255: 1432-1434Crossref PubMed Scopus (231) Google Scholar; Nair et al., 1992Nair B.C. DeVico A.L. Nakamura S. et al.Identification of a major growth factor for AIDS-Kaposi’s sarcoma cells as oncostatin M.Science. 1992; 255: 1430-1432Crossref PubMed Scopus (204) Google Scholar; Murakami-Mori et al., 1995Murakami-Mori K. Taga T. Kishimoto T. et al.AIDS-associated Kaposi’s sarcoma (KS) cells express oncostatin M (OM)-specific receptor but not leukemia inhibitory factor/OM receptor or interleukin-6 receptor: complete block of OM-induced KS cell growth and OM binding by anti-gp130 antibodies.J Clin Invest. 1995; 96: 1319-1327Crossref PubMed Scopus (48) Google Scholar), a tumor comprised morphologically of cells with the characteristics of lymphatic ECs (LECs; Beckstead et al., 1985Beckstead J.H. Wood G.S. Fletcher V. Evidence for the origin of Kaposi’s sarcoma from lymphatic endothelium.Am J Pathol. 1985; 119: 294-300PubMed Google Scholar). In a previous study, we revealed that OSM enhanced CCL21 expression by microvascular EC (MEC) and increased the efficiency of DC trafficking to LNs (Sugaya et al., 2006Sugaya M. Fang L. Cardones A.R. et al.Oncostatin M enhances CCL21 expression by microvascular endothelial cells and increases the efficiency of dendritic cell trafficking to lymph nodes.J Immunol. 2006; 177: 7665-7672Crossref PubMed Scopus (35) Google Scholar). It was somewhat surprising that MEC could be stimulated to produce CCL21 by OSM because CCL21 expression is associated with lymphatic vessels in vivo (Gunn et al., 1998Gunn M.D. Tangemann K. Tam C. et al.A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes.Proc Natl Acad Sci USA. 1998; 95: 258-263Crossref PubMed Scopus (833) Google Scholar). LEC differ from blood vascular EC by expressing a characteristic set of proteins, including lymphatic vascular EC-1 (LYVE)-1, podoplanin, vascular endothelial growth factor receptor 3 (VEGFR3), and Prox-1 (Jackson, 2003Jackson D.G. The lymphatics revisited: new perspectives from the hyaluronan receptor LYVE-1.Trends Cardiovasc Med. 2003; 13: 1-7Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Interestingly, human dermal MEC isolated and cultured from neonatal (Cardones et al., 2006Cardones A.R. Leitner W.W. Murakami T. et al.Genetic immunization with LYVE-1 cDNA yields function-blocking antibodies against native protein.Microvas Res. 2006; 71: 32-39Crossref PubMed Scopus (3) Google Scholar) and adult skin (Kriehuber et al., 2001Kriehuber E. Breiteneder-Geleff S. Groeger M. et al.Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.J Exp Med. 2001; 194: 797-808Crossref PubMed Scopus (422) Google Scholar; Podgrabinska et al., 2002Podgrabinska S. Braun P. Velasco P. et al.Molecular characterization of lymphatic endothelial cells.Proc Natl Acad Sci USA. 2002; 99: 16069-16074Crossref PubMed Scopus (364) Google Scholar) are heterogeneous, with a significant population of cells expressing lymphatic-associated proteins, including podoplanin and LYVE-1. In this current report, we revealed that OSM treatment of primary human dermal MEC led to an enhanced expression of a large number of LEC-specific genes including CCL21. We also showed that purified LEC stimulated with OSM in vitro expressed high amounts of CCL21 mRNA and protein via activation of the mitogen-activated protein kinase (MAPK) 44/42 pathway. MAPK/extracellular signal-regulated kinase kinase (MEK) inhibition within skin before hapten sensitization decreased migration of DCs to draining LNs and decreased contact hypersensitivity (CHS) responses. As CCL21 expression was previously believed to be specific for LECs (when compared with blood ECs), we asked the question as to whether OSM could stimulate expression of other genes preferentially expressed in LECs. Using complementary DNA microarray analyses, we found that primary human dermal MEC stimulated with 100ngml−1 of OSM for 2 days upregulated expression of CCL21 along with four other genes classically associated with LECs (Table 1). Specifically, mRNA levels for VEGFR3, LYVE-1, podoplanin, and Prox-1 were all markedly upregulated in human dermal MEC exposed to OSM (Table 1). Many other less well-known genes (82 in all) that are preferentially expressed in lymphatic endothelium when compared with blood vessel endothelium (Hong et al., 2004Hong Y.K. Foreman K. Shin J.W. et al.Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus.Nat Genet. 2004; 36: 683-685Crossref PubMed Scopus (302) Google Scholar; Podgrabinska et al., 2002) were also upregulated (Supplementary Table S1 online). The two complementary DNA microarray experiments revealed strikingly similar results, confirming the reproducibility of our findings. To assess the effects of OSM, IL-6, or IL-31 on differentiation of human dermal MEC, we also performed quantitative RT-PCR using mRNA from MEC stimulated with OSM, IL-6, or IL-31. Consistent with the microarray experiments and our previous report, OSM treatment strongly increased CCL21 mRNA expression in MEC (Supplementary Figure S1 online). Moreover, OSM or IL-6 treatment increased LYVE-1 mRNA and decreased VEGFR1 mRNA expression (Supplementary Figure S1 online). These results suggested that OSM and IL-6 could cause preferential differentiation from MEC into LEC. To clarify the effects of OSM, IL-6, or IL-31 on terminally differentiated LEC, which was more relevant to in vivo situation, we decided to use normal human dermal lymphatic MECs (HMVEC-dLy), purified LEC expressing podoplanin (>95%), for the following experiments.Table 1OSM increases expression of classic LEC-specific genes in dermal MECGenePrimer pairExperiment 1Experiment 2LYVE-1219059_s_at6.94.7220037_s_at9.815.6VEGFR3210316_at3.34.0Podoplanin204879_at2.55.9208233_at2.22.5221898_at13.930.1Prox-1207401_at6.65.9228656_at10.18.8242119_at1.82.2CCL21204606_at10.45.3Abbreviations: LEC, lymphatic endothelial cell; LYVE-1, lymphatic vascular EC-1; MEC, microvascular EC; OSM, oncostatin M; VEGFR3, vascular endothelial growth factor receptor 3. Open table in a new tab Download .pdf (.11 MB) Help with pdf files Supplementary Information Abbreviations: LEC, lymphatic endothelial cell; LYVE-1, lymphatic vascular EC-1; MEC, microvascular EC; OSM, oncostatin M; VEGFR3, vascular endothelial growth factor receptor 3. To determine the effects of IL-6 family cytokines including on LEC in vitro, we treated HMVEC-dLy with OSM, IL-6, or IL-31 and measured mRNA levels of CCL21 by quantitative real-time RT-PCR (Figure 1a). Under serum-free conditions, upregulation of CCL21 mRNA was detected in HMVEC-dLy at 48hours as well as 72hours after addition of OSM (10 and 100ngml−1), but not after addition of other cytokines (IL-6 and IL-31; Figure 1a). We next treated HMVEC-dLy with OSM (100ngml−1) and measured protein levels of CCL21 (Figure 1b). Under serum-free conditions, supernatant CCL21 levels of HMVEC-dLy were increased by stimulation with OSM at 72hours (Figure 1b). Thus, OSM treatment increased transcriptional and translational expression of CCL21 in human LEC. IL-6 family cytokines including OSM have previously been shown to stimulate several signaling pathways, such as MAPK, signal transducers and activators of transcription 3 (STAT3), and phosphoinositide-3 kinase/Akt (acutely transforming retrovirus AKT8 in rodent T cell lymphoma; El Mabrouk et al., 2007El Mabrouk M. Sylvester J. Zafarullah M. Signaling pathways implicated in oncostatin M-induced aggrecanase-1 and matrix metalloproteinase-13 expression in human articular chondrocytes.Biochem Biophys Acta. 2007; 1773: 309-320Crossref PubMed Scopus (39) Google Scholar; Lee et al., 2007Lee Y.J. Heo J.S. Suh H.N. et al.Interleukin-6 stimulates alpha-MG uptake in renal proximal tubule cells: involvement of STAT3, PI3K/Akt, MAPKs, and NF-kappaB.Am J Physiol Renal Physiol. 2007; 293: 1036-1046Crossref Scopus (30) Google Scholar; Zhang et al., 2008Zhang Q. Putheti P. Zhou Q. et al.Structures and biological functions of IL-31 and IL-31 receptors.Cytokine Growth Factor Rev. 2008; 19: 347-356Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). To determine whether these pathways were involved in CCL21 expression in OSM-stimulated LEC, we examined phosphorylation of MAPK 44/42, STAT3, and Akt. Using HMVEC-dLy, we found only slightly detectable MAPK 44/42 phosphorylation and no detectable STAT3 and Akt phosphorylation in unstimulated cells (Figure 2a and data not shown). By contrast, incubation with 100ngml−1 OSM, but not IL-6 or IL-31, for 15minutes as well as 30minutes markedly induced MAPK 44/42 phosphorylation in these cells (Figure 2a). Incubation with 100ngml−1 OSM and IL-6, but not IL-31, for 15 or 30minutes markedly induced STAT3 phosphorylation in these cells (Figure 2a). Incubation with 100ngml−1 OSM, but not IL-6 or IL-31, for 15 as well as 30minutes only slightly induced Akt phosphorylation in these cells (data not shown). Thus, the MAPK 44/42 pathway seems most important for CCL21 expression in OSM-treated human LEC. To investigate signaling pathways important for CCL21 expression in OSM-stimulated LEC, we used JAK inhibitor 1, an inhibitor of JAK family kinases, JAK3 inhibitor, and U0126, a MEK inhibitor. MAPK 44/42 phosphorylation in HMVEC-dLy was blocked highly by U0126, moderately by JAK inhibitor 1, and slightly by JAK3 inhibitor (Figure 2b). JAK inhibitor 1, but not JAK3 inhibitor or U0126, blocked phosphorylation of STAT3 in HMVEC-dLy (Figure 2b). CCL21 protein expression by OSM-treated HMVEC-dLy was blocked highly by U0126, moderately by JAK inhibitor 1, and slightly by JAK3 inhibitor (Figure 2c). The suppression of CCL21 protein expression correlated with degree of blockade of MAPK 44/42 phosphorylation, but not STAT3 phosphorylation, suggesting that CCL21 expression by HMVEC-dLy is mainly dependent on MAPK signaling. To assess the importance of MAPK signaling in CCL21 expression in vivo, we injected the ears of C57BL/6 mice with DMSO or 100μM U0126, a MEK inhibitor, and extracted total skin RNA 24hours later. Quantitative real-time RT-PCR was performed to measure CCL21, CCR7, and LYVE-1 mRNA expression relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). CCL21 mRNA expression was decreased by U0126 injection, whereas CCR7 and LYVE-1 mRNA expression remained unchanged (Figure 3a and data not shown). We also lysed the ears using homogenizer 24hours later, and western blotting was performed to detect CCL21 protein expression. CCL21 protein expression was decreased by U0126 injection (Figure 3b). Therefore, U0126 treatment decreased transcriptional and translational expression of CCL21 in mouse skin. Based on the data above, we predicted that intradermal injection of skin with U0126 would result in decreased DC migration from peripheral tissues (i.e., skin) to draining LN in vivo, presumably by decreasing expression of CCL21 by LEC. To test this hypothesis, we painted FITC onto murine ears, followed by injecting U0126 or DMSO, and after 24hours, draining LNs were collected for flow cytometry. In this experiment, only migratory DCs were stained strongly positive for major histocompatibility complex class II, whereas resident DCs were lost in major histocompatibility complex class II-negative population together with B cells (Figure 3c). The total cell numbers in LNs were not statistically different between the two groups. After treatment with U0126, the frequency of migratory DCs and Ag-bearing migratory DCs in draining LNs was decreased as expected (Figure 3d). To determine whether blocking MAPK signaling within skin can decrease CHS responses, we injected the ears of C57BL/6 mice with DMSO or 100μM U0126 1 day before sensitization or elicitation. Injection with U0126 1 day before sensitization significantly decreased CHS responses compared with DMSO treatment (Figure 4a). In contrast, when U0126 was injected 1 day before elicitation, CHS responses were not impaired (Figure 4b). Mice without sensitization did not show ear swelling as expected (data not shown). We also evaluated CHS responses histopathologically. The ear swelling and cellular infiltration 24hours after the challenge were less prominent in mice injected with U0126 1 day before sensitization compared with mice injected with DMSO (Figure 4c). Therefore, blocking MAPK signaling within skin during the sensitization phase can decrease CHS responses. To assess specific CCL21 effect, we next injected the ears of C57BL/6 mice with phosphate-buffered saline (PBS), 20μgml−1 anti-mouse CCL21 antibody (Ab), or 25μgml−1 recombinant mouse CCL21 1 day before sensitization or elicitation. As expected, injection with anti-mouse CCL21 Ab 1 day before sensitization significantly decreased CHS responses compared with PBS treatment (Figure 4d). On the contrary, injection with recombinant mouse CCL21 did not increase CHS response, suggesting that CCL21 expression in the ear was sufficient (Figure 4d). Surprisingly, injection with anti-mouse CCL21 Ab 1 day before elicitation also significantly decreased CHS responses compared with PBS treatment (Figure 4e). Therefore, blocking CCL21 during either sensitization or elicitation phase suppressed CHS, but adding recombinant CCL21 did not affect CHS. In this study, we found that OSM enhanced CCL21 mRNA and protein expression by HMVEC-dLy, purified LEC (Figure 1). We then demonstrated that OSM induced high levels of phosphorylation of MAPK 44/42 and STAT3 in HMVEC-dLy (Figure 2a), which is consistent with previous studies using a variety of cells (Thoma et al., 1994Thoma B. Bird T.A. Friend D.J. et al.Oncostatin M and leukemia inhibitory factor trigger overlapping and different signals through partially shared receptor complexes.J Biol Chem. 1994; 269: 6215-6222Abstract Full Text PDF PubMed Google Scholar; Stancato et al., 1997Stancato L.F. Sakatsume M. David M. et al.Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway.Mol Cell Biol. 1997; 17: 3833-3840Crossref PubMed Scopus (95) Google Scholar). We also showed that IL-6 induced enhanced phosphorylation of STAT3, but not MAPK44/42, and that IL-31 did not induce phosphorylation of STAT3 or MAPK44/42 in HMVEC-dLy (Figure 2a). OSM, IL-6, and IL-31 are members of the IL-6 family of cytokines, whose receptors are composed of different subunits. OSM receptor is a complex of OSMR and gp130, IL-6 receptor is a complex of IL-6R and gp130 (Tanaka and Miyajima, 2003Tanaka M. Miyajima A. Oncostatin M, a multifunctional cytokine.Rev Physiol Biochem Pharmacol. 2003; 149: 39-52Crossref PubMed Google Scholar), and IL-31 receptor is a complex of the OSMR and IL-31RA (Zhang et al., 2008Zhang Q. Putheti P. Zhou Q. et al.Structures and biological functions of IL-31 and IL-31 receptors.Cytokine Growth Factor Rev. 2008; 19: 347-356Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The differences in receptors may explain the different signaling pathways activated by these cytokines. We next showed that treatment of a MEK inhibitor downregulated CCL21 expression and decreased migration of DCs and Ag-bearing DCs to LNs in vivo (Figure 3). CCL21 and CCR7 interaction has been reported to be critically involved in the migration of Ag-presenting DCs (Forster et al., 1999Forster R. Schubel A. Breitfeld D. et al.CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1814) Google Scholar; Gunn et al., 1999Gunn M.D. Kyuwa S. Tam C. et al.Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.J Exp Med. 1999; 189: 451-460Crossref PubMed Scopus (870) Google Scholar; Saeki et al., 1999Saeki H. Moore A.M. Brown M.J. et al.Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.J Immunol. 1999; 162: 2472-2475PubMed Google Scholar). Importantly, our results elucidate the underlying molecular regulation involved in CCL21-mediated DC migration. We have also shown that blocking MAPK signaling or neutralization of CCL21 within skin before sensitization can decrease CHS (Figures 4a, c and d), which are consistent with previous reports (Engeman et al., 2000Engeman T.M. Gorbachev A.V. Gladue R.P. et al.Inhibition of functional T cell priming and contact hypersensitivity responses by treatment with anti-secondary lymphoid chemokine antibody during hapten sensitization.J Immunol. 2000; 164: 5207-5214Crossref PubMed Scopus (60) Google Scholar). In both CCR7-deficient and plt mice, where CCL21–CCR7 interactions were abrogated, T cells and CHS responses were increased (Schneider et al., 2007Schneider M.A. Meingassner J.G. Lipp M. et al.CCR7 is required for the in vivo function of CD4+ CD25+ regulatory T cells.J Exp Med. 2007; 204: 735-745Crossref PubMed Scopus (242) Google Scholar; Nakano et al., 2009Nakano H. Lin K.L. Yanagita M. et al.Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses.Nat Immunol. 2009; 10: 394-402Crossref PubMed Scopus (251) Google Scholar). In these mice, however, CCL19–CCR7 interactions were also abrogated. Moreover, defect in CCL21–CCR7 interactions from birth may have caused immunological changes in these mice. Interestingly, neutralization of CCL21 within skin before elicitation decreased CHS (Figure 4e), suggesting that CCL21 expression was also important in the elicitation phase. On the contrary, blocking MAPK signaling within skin before elicitation did not affect CHS responses. The effects of U0126 injected into the skin cannot be expected to be limited to CCL21 expression by LECs. U0126 might have abrogated regulatory mechanisms for CHS responses. In addition, we injected DMSO as a negative control in experiments using U0126, which was dissolved in DMSO. Injection of DMSO into the elicitation site may have induced MPAK-independent ear-swelling responses, increasing ear thickness of baseline (day 0), which may account for no significant differences between DMSO and U0126. Our data and those of others have suggested that CCL21 expression in dermal LECs is associated with DC migration and CHS responses (Engeman et al., 2000Engeman T.M. Gorbachev A.V. Gladue R.P. et al.Inhibition of functional T cell priming and contact hypersensitivity responses by treatment with anti-secondary lymphoid chemokine antibody during hapten sensitization.J Immunol. 2000; 164: 5207-5214Crossref PubMed Scopus (60) Google Scholar). Migration of dermal DCs into the draining LNs also has a central role in various types of skin inflammation. CCL21 expression is upregulated in the skin lesions of psoriasis and lichen planus, as well as allergic and irritant contact dermatitis (Eberhard et al., 2004Eberhard Y. Ortiz S. Ruiz Lascano A. et al.Up-regulation of the chemokine CCL21 in the skin of subjects exposed to irritants.BMC Immunol. 2004; 5: 7Crossref PubMed Scopus (33) Google Scholar; Serra et al., 2004Serra H.M. Eberhard Y. Martín A.P. et al.Secondary lymphoid tissue chemokine (CCL21) is upregulated in allergic contact dermatitis.Int Arch Allergy Immunol. 2004; 133: 64-71Crossref PubMed Scopus (20) Google Scholar; Manzo et al., 2007Manzo A. Bugatti S. Caporali R. et al.CCL21 expression pattern of human secondary lymphoid organ stroma is conserved in inflammatory lesions with lymphoid neogenesis.Am J Pathol. 2007; 171: 1549-1562Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Blocking MAPK signaling or neutralization of CCL21 can be a, to our knowledge, previously unreported approach to the treatment of these diseases. The MAPK signaling pathway has been a major clinical focus in oncology research, and many inhibitors targeting this pathway have been pursued (Wang et al., 2007Wang J.Y. Wilcoxen K.M. Nomoto K. et al.Recent advances of MEK inhibitors and their clinical progress.Curr Top Med Chem. 2007; 7: 1364-1378Crossref PubMed Scopus (63) Google Scholar; Wong, 2009Wong K.K. Recent developments in anti-cancer agents targeting the Ras/Raf/ MEK/ERK pathway.Recent Pat Anticancer Drug Discov. 2009; 4: 28-35Crossref PubMed Scopus (135) Google Scholar). Several oral MEK inhibitors, such as PD0325901 and AZD6244, have been evaluated in clinical trials (Adjei et al., 2008Adjei A.A. Cohen R.B. Franklin W. et al.Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers.J Clin Oncol. 2008; 26: 2139-2146Crossref PubMed Scopus (489) Google Scholar; Haura et al., 2010Haura E.B. Ricart A.D. Larson T.G. et al.A phase II study of PD-0325901, an oral MEK inhibitor, in previously treated patients with advanced non-small cell lung cancer.Clin Cancer Res. 2010; 16: 2450-2457Crossref PubMed Scopus (188) Google Scholar; LoRusso et al., 2010LoRusso P.M. Krishnamurthi S.S." @default.
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• W2080232806 date "2011-09-01" @default.
• W2080232806 modified "2023-10-01" @default.
• W2080232806 title "Blocking MAPK Signaling Downregulates CCL21 in Lymphatic Endothelial Cells and Impairs Contact Hypersensitivity Responses" @default.
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