The chemokines CCL19, CCL21 and CCL25, by signalling through the receptors CCR7 or CCR9, play critical roles in leukocyte homing. They also bind another heptahelical surface protein, CCX-CKR. CCX-CKR cannot couple to typical chemokine receptor signalling pathways or mediate chemotaxis, and its function remains unclear. We have proposed that it controls chemokine bioavailability. Here, using transfected HEK293 cells, we have shown that both CCX-CKR and CCR7 mediate rapid CCL19 internalisation upon initial chemokine exposure. However, internalised CCL19 was more efficiently retained and degraded after uptake via CCX-CKR. More importantly, CCR7 rapidly became refractory for CCL19 uptake, but the sequestration activity of CCX-CKR was enhanced. These properties endowed CCX-CKR with an impressive ability to mediate progressive sequestration and degradation of large quantities of CCL19, and conversely, prevented CCR7-expressing cells from extensively altering their chemokine environment. These differences may be linked to the routes of endocytosis used by these receptors. CCX-CKR, unlike CCR7, was not critically dependent on beta-arrestins or clathrin-coated pits. However, over-expression of caveolin-1, which stabilises caveolae, blocked CCL19 uptake by CCX-CKR while having no impact on other chemokine receptors, including CCR7. These data predict that CCX-CKR scavenges extracellular chemokines in vivo to modify responses through CCR7.

The development of secondary lymphoid organs, such as lymph nodes (LNs), in the embryo results from the reciprocal action between lymphoid tissue inducer (LTi) cells and stromal cells. However, the initial events inducing LN anlagen formation before the LTi stromal cells cross-talk interactions take place are not fully elucidated. In this study, we show that the inguinal LN anlagen in mouse embryos developed from mesenchymal cells surrounding the lymph sacs, spherical structures of endothelial cells that bud from veins. Using inguinal and mesenteric LNs (mLNs), we provide evidence supporting a two-step maturation model for stromal cells: first, ICAM-1(-)VCAM-1(-) mesenchymal precursor cells become ICAM-1(int)VCAM-1(int) cells, in a process independent of LTi cells and lymphotoxin beta receptor (LTbetaR) signaling. The second step involves the maturation of ICAM-1(int)VCAM-1(int) cells to ICAM-1(high)VCAM-1(high) mucosal addressin cell adhesion molecule-1(+) organizer cells and depends on both LTi cells and LTbetaR. Addition of alphaLTbetaR agonist to LN organ cultures was sufficient to induce ICAM-1(int)VCAM-1(int) cells to mature. In LtbetaR(-/-) embryos, both inguinal and mLN stromal cells showed a block at the ICAM-1(int)VCAM-1(int) stage, and, contrary to inguinal LNs, mLNs persist longer and contained LTi cells, which correlated with the sustained gene expression of Il-7, Cxcl13, and, to a lesser degree, Ccl21. Taken together, these results highlight the importance of the signals and cellular interactions that induce the maturation of stromal cells and ultimately lead to the formation of lymphoid tissues.

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.

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.

The chemokines (CKs) CXCL13, CCL21, and CXCL12 are known to play differential roles in the organization of the lymphoid tissues and the development of lymphoid malignancies. We investigated the expression of these CKs and their receptors in the salivary glands of Sjogren's syndrome patients with lymphoepithelial lesions (lymphoepithelial sialadenitis or LESA) and in MALT lymphoma to understand their involvement in salivary gland lymphomagenesis. We demonstrate that within salivary glands with LESA and MALT lymphoma the lymphoid CKs CXCL13 and CCL21 are selectively associated with areas of reactive lymphoid proliferation, whereas no significant expression of these molecules was detected in the malignant lymphoid aggregate. Conversely, CXCL12 was observed predominantly in infiltrated ducts and malignant B cells. Accordingly, CXCL13 and CCL21 transcript levels were significantly increased in LESA samples while CXCL12 levels were increased in MALT lymphoma and isolated tumor cells. Low levels of CK receptors were detected on lymphoma-extracted lymphocytes, suggesting down-regulation in the abundance of ligands. Our findings suggest that in salivary gland MALT lymphoma the lymphoid CKs CXCL13 and CCL21 are directly implicated in the organization of ectopic reactive lymphoid tissue, whereas CXCL12 is associated with the infiltrated epithelium and malignant B cell component and is possibly involved in the regulation of malignant B cell survival.

The addition of polysialic acid to N- and/or O-linked glycans, referred to as polysialylation, is a rare posttranslational modification that is mainly known to control the developmental plasticity of the nervous system. Here we show that CCR7, the central chemokine receptor controlling immune cell trafficking to secondary lymphatic organs, carries polysialic acid. This modification is essential for the recognition of the CCR7 ligand CCL21. As a consequence, dendritic cell trafficking is abrogated in polysialyltransferase-deficient mice, manifesting as disturbed lymph node homeostasis and unresponsiveness to inflammatory stimuli. Structure-function analysis of chemokine-receptor interactions reveals that CCL21 adopts an autoinhibited conformation, which is released upon interaction with polysialic acid. Thus, we describe a glycosylation-mediated immune cell trafficking disorder and its mechanistic basis.

Dendritic cells (DCs) are potent antigen-presenting cells that likely play multiple roles in human immunodeficiency virus type 1 (HIV-1) pathogenesis. We used the simian immunodeficiency virus (SIV)/macaque model to study the effects of infection on homeostatic chemokine expression and DC localization directly in secondary lymphoid tissues. SIV infection altered the expression of chemokines (CCL19/MIP-3beta, CCL21/ 6Ckine, and CCL20/MIP-3alpha) and of chemokine receptors (CCR7 and CCR6) that drive DC trafficking. CCL19/MIP-3beta, CCL20/MIP-3alpha, CCR6, and CCR7 expression increased in lymph nodes during the early systemic burst of viral replication (acute infection), whereas CCL21/6Ckine expression progressively decreased throughout disease to AIDS. Parallel with the SIV-induced perturbations in chemokine expression were changes in the expression of the DC-associated markers, DC-SIGN, DC-LAMP, and DECTIN-1. During AIDS, DC-LAMP mRNA expression levels were significantly reduced in lymph nodes and spleen, and DC-SIGN levels were significantly reduced in spleen. These findings suggest that the disruption of homeostatic chemokine expression is responsible, in part, for alterations in the networks of antigen-presenting cells in lymphoid tissues, ultimately contributing to systemic immunodeficiency.

HIV-1 infection is associated with B-cell abnormalities, such as hypergammaglobulinemia, poor immunization responses, and loss of serologic memory. To determine whether altered expression of chemokine receptors and their ligands may play a role in B-cell dysfunctions during HIV-1 infection, the expression of CXC chemokine receptor 4 (CXCR4), CXCR5, and CC chemokine receptor 7 (CCR7) and their respective ligands on CD19(+) B cells were examined in HIV-1-infected patients and controls. We report a decreased CXCR5 expression on B cells from patients (P < .05), a phenomenon associated with a low CD4 T-cell count (< 350 cells/microL). Interestingly, an increased expression of CXC chemokine ligand 13 (CXCL13), the ligand for CXCR5, was found in peripheral B cells from HIV-1-infected patients. Moreover, on B-cell activation in vitro, CXCL13 was secreted in culture. CXCL13(+) B cells were also found in the lymph nodes of HIV-1-infected patients, but not in control tissue. B-cell migration toward CXCL13, CXCL12, and CC chemokine ligand 21 (CCL21), ligands for CXCR5, CXCR4, and CCR7 was also evaluated. In patients with a low CD4 T-cell count, migration toward all ligands was increased. Our findings indicate that altered expression of the chemokine receptor-ligand pair, CXCR5/CXCL13, may participate in the establishment of B-cell dysfunctions during HIV-1 infection.

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.

The simultaneous presence of dendritic, T- and B-cells in the central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, suggests that interactions among these cell types might be instrumental in the local induction and maintenance of autoimmune reactions. In this study, we explored the possibility that such aberrant leukocyte recruitment in the CNS could be sustained by "lymphoid" chemokines which orchestrate dendritic cell and lymphocyte homing to lymphoid organs. Transcripts for CCL19 and CCL21 and their common receptor CCR7 were induced in the CNS of mice undergoing relapsing-remitting and chronic-relapsing EAE. While CCL21 immunoreactivity was confined to the endothelium of some inflamed blood vessels, CCL19 was expressed by many infiltrating leukocytes and some astrocytes and microglia in the CNS parenchyma. CCR7+ cells accumulated in inflammatory lesions during EAE progression, when abundant infiltration of the CNS by mature dendritic cells, B-cells and cells expressing naive T-cell markers also occurred. These findings suggest that CCL19 and CCL21 produced in the EAE-affected CNS may be critical for the homing of antigen presenting cells and lymphocytes, resulting in continuous local antigenic stimulation and maintenance of chronic neuroinflammation.

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-gamma-inducing cytokines, IFN-alpha beta and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-alpha beta. Indeed, IFN-alpha beta neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4(+), and CD8(+) cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-alpha beta-induced expression of CXCL10, neutralization of IFN-alpha beta significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-alpha beta may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.

Lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or lymphotoxin-alpha but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles in the thyroid.

Chemokines play critical roles in leukocyte recruitment into sites of inflammation such as rheumatoid arthritis (RA). While chemokines immobilized on endothelium (solid-phase), but not soluble chemokines, direct rolling leukocytes to firmly adhere to endothelium, soluble and solid-phase chemokine gradients may play important roles in leukocyte extravasation into the tissue. In this study, we have sought to determine (1) if chemokines can be immobilized on structures in the extravascular space, (2) the mechanisms by which chemokines may be immobilized, and (3) if different chemokines have similar potentials to form solid-phase gradients. While secreted alkaline phosphatase (SEAP)-tagged chemokines SLC (CCL21), TARC (CCL17), and RANTES (CCL5) bound to mast cells and the extracellular matrix (ECM) in RA synovium under physiologic salt conditions, MCP1 (CCL2), MIP1 alpha (CCL3), MIP1 beta (CCL4), and fractalkine (FKN, CX3CL1) fusion proteins did not detectably bind. Chemokine binding to ECM and mast cells in situ and to immobilized heparin was inhibited by high salt and glycosaminoglycans (GAGs) heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, but not by dextran or hyaluronan, indicating that the chemokines bind to highly sulfated GAGs. Chemokine binding to synovial structures correlated strongly with avidity of chemokine binding to heparin (SLC>> TARC>> RANTES>> MIP1 beta>> MCP1>> MIP1 alpha>> FKN). A RANTES mutant with decreased avidity for heparin was not able to bind to ECM or mast cells. Thus, these data indicate that chemokines can bind to ECM and mast cell granule constituents in situ via interactions with GAGs. Further, only a subset of chemokines were able to bind efficiently to structures in the extravascular space, indicating that chemokines may form different types of gradients based on their GAG binding ability and that chemotactic gradients in tissues may be quite complex.

The COX-2 product prostaglandin E(2) (PGE(2)) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE(2) production or PGE(2)-mediated signaling through the PGE(2) receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE(2), but very little is known about the mechanism by which PGE(2) affects NK effector activity. We now report the direct effects of PGE(2) on the NK cell. Endogenous murine splenic NK cells express all four PGE(2) receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE(2), the EP4 agonist PGE(1)-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE(2), the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE(1)-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE(2) production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.

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.

BACKGROUND

Migration of metastatic tumor cells from the bloodstream into lymph nodes is thought to be facilitated by expression of the chemokine receptors CCR7, CXCR4 and, for B cell-derived tumors, CXCR5. Expression of their respective chemokine ligands (CCL19, CCL21, CXCL12 and CXCL13) by endothelial cells inside the lymph nodes facilitates the trans-endothelial migration (TEM) of these cells through high endothelial venules into the lymph node parenchyma. It is known that CXCR7, a second CXCL12 receptor, regulates TEM of CXCR4+CXCR7+ tumor cells towards a CXCL12 source. In this study, we set out to assess the potential stimulation by CXCL12 of tumor cell TEM towards other chemokines and whether CXCR7 might be able to regulate such effects.

METHODS

The human Burkitt's lymphoma cell line NC-37, which expresses CXCR4, CXCR5, CXCR7 and CCR7, was selected as a model system. TEM of these cells through a human HUVEC endothelial cell monolayer was used as the main model system for these studies. Regulation of their TEM behavior by various concentrations of the various cognate chemokines for the above-mentioned receptors, placed in either the source or target wells of modified Boyden chamber migration plates, was assessed by quantifying the number of cells migrated under each experimental condition.

RESULTS

Exposure of CXCR4⁺CXCR7⁺ cancer cells to CXCL12 greatly potentiated their TEM towards the chemokines CCL19 and CXCL13. This CXCL12-potentiated TEM was inhibited by the second CXCR7 chemokine ligand, CXCL11, as well as CXCR7-specific small molecule antagonists and antibodies. In contrast, the CXCR4 antagonist AMD3100 was less effective at inhibiting CXCL12-potentiated TEM. Thus, CXCR7 antagonists may be effective therapeutic agents for blocking CXCL12-mediated migration of CXCR4⁺CXCR7⁺ tumor cells into lymph nodes, regardless of whether the cancer cells follow a CXCL12 gradient or whether serum CXCL12 stimulates their migration towards CCR7 and CXCR5 chemokines in the lymph nodes.

Recent studies demonstrate that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis. However, the mechanism by which COX-2 increases the invasion of cancer cells to lymph node is unclear. CCR7 is a chemokine receptor that plays important roles in the mediation of migration of leukocytes and dendritic cells toward lymphatic endothelial cells (LECs) that express receptor ligand CCL21. We found that treatment of prostaglandin E(2) or ectopic expression of COX-2 in MCF-7 cells up-regulated CCR7 expression. On the contrary, knockdown of COX-2 by small hairpin RNA reduced CCR7 in COX-2-overexpressing MDA-MB-231 cells. Interaction of CCR7 and CCL21 was important for the migration of breast cancer cells toward LECs because antibodies against these two molecules inhibited the migration. We also found that COX-2 increased CCR7 expression via the EP2 and EP4 receptor in breast cancer cells. EP2 and EP4 agonists stimulated CCR7 in MCF-7 cells, whereas antagonists or small hairpin RNA of EP2 and EP4 attenuated CCR7 in MDA-MB-231 cells. Protein kinase A and AKT kinase were involved in COX-2-induced CCR7. Pathological analysis demonstrated that COX-2 overexpression was associated with CCR7, EP2, and EP4 expressions in breast tumor tissues. In addition, CCR7 expression in COX-2-overexpressing tumors was significantly correlated with lymph node metastasis. Collectively, we suggest that CCR7 is a down-stream target for COX-2 to enhance the migration of breast cancer cells toward LECs and to promote lymphatic invasion.

OBJECTIVE

To examine whether dendritic cells (DCs) are constituents of muscle inflammation in juvenile dermatomyositis (DM).

METHODS

The types, numbers, and activation state of DC subsets in inflamed muscle tissue from patients with juvenile DM and in noninflamed muscle tissue from control subjects were examined by multicolor immunofluorescence. Chemokine expression of the muscle-infiltrating cells was examined by laser capture microdissection and quantitative polymerase chain reaction.

RESULTS

Plasmacytoid DCs were the predominant component of the inflamed muscle tissue from patients with juvenile DM. These cells were identified by coexpression of CD4 and CD123, but not CD11c, and also expressed CD83, indicating maturity of the cells. In contrast, in noninflamed muscle, plasmacytoid DCs were scarce and did not express CD83. Mononuclear cells surrounding the blood vessels of inflamed muscle contained abundant transcripts of CCL19 and CCL21, but very little CCL18 transcripts. In contrast, cells from noninflamed muscle contained negligible amounts of CCL19 and CCL21, but had high amounts of CCL18. Both the inflamed and noninflamed muscle tissue had equivalent levels of CXCL12 transcripts, but inflamed muscle contained more transcripts of the CXCL12 receptor CXCR4.

CONCLUSIONS

These findings are consistent with the idea that plasmacytoid DCs are mediators of muscle inflammation in juvenile DM. The abundance of CD83+ plasmacytoid DCs in perivascular areas and the overexpression of CCL19 and CCL21 in perivascular cellular foci suggest that in situ activation and maturation of resident plasmacytoid DCs are central to the initiation and perpetuation of muscle inflammation in juvenile DM.

Chemokine receptors are essential mediators of the metastatic spread in various cancer types; however their precise function in the development of secondary tumors remains poorly understood. We report here a novel property of the chemokine receptors CXCR4 and CCR7 in inhibiting detachment-induced cell death--anoikis, which is believed to be one of the major blocks in the metastatic spread of various neoplasms. Activation of these chemokine receptors by their respective ligands, CXCL12 and CCL21 specifically reduced the sensitivity of metastatic breast cancer cells to anoikis by a distinct mechanism of selective regulation of pro-apoptotic Bmf and anti-apoptotic Bcl-xL proteins. Consequently, functional CXCR4 and CCR7 increased cell survival in the absence of correct ECM attachment both in vitro and in vivo. We also demonstrated that preventing chemokine-induced reduction in Bmf levels significantly attenuated breast cancer metastasis in an experimental mouse model. These results provide evidence for a previously unknown axis in malignant tumors, which connects chemokine receptors with deregulated apoptosis in the absence of the appropriate cell--ECM interaction and may offer novel targets for therapeutic intervention for the treatment of metastatic breast and potentially other tumors.

BACKGROUND

Ectopic lymphoid neogenesis (LN) occurs in rheumatoid synovium, where it is thought to drive local antigen-dependent B cell development and autoantibody production. This process involves the expression of specific homing chemokines and the development of high endothelial venules (HEV).

OBJECTIVE

To investigate whether these mechanisms occur in psoriatic arthritis (PsA) synovium, where autoantibodies have not been described and the organisation and function of B cells is not clear, and to analyse their clinical correlates.

METHODS

Arthroscopic synovial biopsy specimens from patients with PsA before and after tumour necrosis factor alpha blockade were characterised by immunohistochemical analysis for T/B cell segregation, peripheral lymph node addressin (PNAd)-positive HEV, and the expression of CXCL13, CCL21 and CXCL12 chemokines in relation to the size of lymphoid aggregates.

RESULTS

Lymphoid aggregates of variable sizes were observed in 25 of 27 PsA synovial tissues. T/B cell segregation was often observed, and was correlated with the size of lymphoid aggregates. A close relationship between the presence of large and highly organised aggregates, the development of PNAd+ HEV, and the expression of CXCL13 and CCL21 was found. Large organised aggregates with all LN features were found in 13 of 27 tissues. LN in PsA synovitis was not related to the duration, pattern or severity of the disease. The synovial LN pattern remained stable over time in persistent synovitis, but a complete response to treatment was associated with a regression of the LN features.

CONCLUSIONS

LN occurs frequently in inflamed PsA synovial tissues. Highly organised follicles display the characteristic features of PNAd+ HEV and CXCL13 and CCL21 expression, demonstrating that the microanatomical bases for germinal centre formation are present in PsA. The regression of LN on effective treatment indicates that the pathogenic and clinical relevance of these structures in PsA merits further investigation.

Homeostatic chemokines such as CCL19, CCL21, and CXCL13 are known to elicit chemotaxis from naive T and B cells and play a critical role in lymphocyte homing to appropriate zones within secondary lymphoid organs (SLO). Here we tested whether CCL21 and CXCL13 modulate murine lymphocyte motility in the absence of concentration gradients, using videomicroscopy to directly observe the migration of single cells. CCL21 treatment of T cells induced rapid polarization and sustained random migration with average speeds of 5.16 +/- 2.08 microm/min; B cell migration (average velocity 4.10 +/- 1.58 microm/min) was similarly induced by CXCL13. Migration required the presence of both chemokine and adhesion ligands and was sustained for >24 h. Furthermore, in in vitro assays modeling the relative infrequency of Ag-specific T cell-dendritic cell (DC) encounters during primary immune responses, we found that CCL21 addition to T-DC cocultures accelerated the kinetics of CD69 up-regulation and enhanced by 2-fold the proliferation of Ag-specific T cells in a manner dependent on G-protein-coupled receptor signaling in T cells. These results suggest that homeostatic chemokines could substantially impact the dynamics and priming of lymphocytes within SLO even in the absence of significant concentration gradients.

Interferon consensus sequence-binding protein (ICSBP) is a transcription factor belonging to the interferon regulatory factor (IRF) family, recently shown to play a critical role in dendritic cell (DC) differentiation. Here, we analyzed the role of ICSBP in the development and trafficking of epidermal Langerhans cells (LCs) and dermal DCs and the implications for initiation of a competent immune response. ICSBP-/- mice exhibited a reduced frequency of LCs and a delayed mobility of DCs from skin that reflected a slower turnover rate in lymph nodes during steady-state conditions. Even under inflammatory changes, ICSBP-/- DCs displayed reduced mobility from skin to lymph nodes and, as a consequence, failed to induce a contact hypersensitivity (CHS) response, suggesting that these DCs were unable to initiate a competent antigen (Ag)-specific T-cell-mediated immunity. Moreover, bone marrow (BM)-derived DCs from ICSBP-/- mice exhibited an immature phenotype and a severe reduction of interleukin 12 (IL-12) expression. These BM DCs also showed a marked defect in their migratory response to macrophage inflammatory protein 3 alpha (MIP-3 alpha), MIP-3 beta, and the CC chemokine CCL21/6Ckine, which was paralleled by an impaired expression of the CC chemokine receptors, CCR6 and CCR7. Together, these results indicate that ICSBP is critically required for the development and trafficking of skin DCs, thus playing a critical role in the DC-mediated initiation of T-cell immunity.

Homeostasis of T cell numbers in the periphery implies an ability of lymphocytes to sense cell numbers. Although the mechanisms are unknown, we find that the chemokine CCL21 (also known as TCA4, SLC, 6Ckine), a ligand for the chemokine receptor CCR7, can regulate homeostasis of CD4 (but not CD8) T cells. In the absence of CCR7 ligands, transferred CD4 T cells failed to expand in lymphopenic hosts, whereas in the presence of CCL21 overexpression, homeostatic CD4 T cell proliferation occurred even in nonlymphopenic recipients. Ag-specific CD4 T cells transferred into Ag-expressing mice proliferated and induced autoimmunity only in lymphopenic recipients. Pancreatic expression of CCL21 was sufficient to replace the requirement for lymphopenia in the progression of autoimmune disease. These results suggest that CD4 T cells use local concentrations of CCR7 ligands as an index of T cell steady state numbers and that homeostatic expansion of the T cell population may be a contributing factor in the development of autoimmune disease.

We previously demonstrated that mesenchymal stem cells (MSCs) ameliorated experimental autoimmune uveoretinitis (EAU) in rats. Recently, MSC-derived exosomes (MSC-Exo) were thought to carry functions of MSCs. In this study, we tested the effect of local administration of human MSC-Exo on established EAU in the same species. Rats with EAU induced by immunization with interphotoreceptor retinol-binding protein 1177-1191 peptide were treated by periocular injections of increasing doses of MSC-Exo starting at the disease onset for 7 consecutive days. The in vitro effects of MSC-Exo on immune cell migration and responder T cell proliferation were examined by chemotactic assays and lymphocyte proliferation assays, respectively. We found that MSC-Exo greatly reduced the intensity of ongoing EAU as their parent cells by reducing the infiltration of T cell subsets, and other inflammatory cells, in the eyes. Furthermore, the chemoattractive effects of CCL2 and CCL21 on inflammatory cells were inhibited by MSC-Exo. However, no inhibitory effect of MSC-Exo on IRBP-specific T cell proliferation was observed. These results suggest that MSC-Exo effectively ameliorate EAU by inhibiting the migration of inflammatory cells, indicating a potential novel therapy of MSC-Exo for uveitis.

To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21-but not the absence of lymph flow-completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient.