In the event of a deliberate or accidental radiological emergency, the skin would likely receive substantial ionizing radiation (IR) poisoning, which could negatively impact cellular proliferation, communication, and immune regulation within the cutaneous microenvironment. Indeed, as we have previously shown, local IR exposure to the murine ear causes a reduction of two types of cutaneous dendritic cells (cDC), including interstitial dendritic cells of the dermis and Langerhans cells of the epidermis, in a dose- and time-dependent manner. These APCs are critical regulators of skin homeostasis, immunosurveillance, and the induction of T and B cell-mediated immunity, as previously demonstrated using conditional cDC knockout mice. To mimic a radiological emergency, we developed a murine model of sublethal total body irradiation (TBI). Our data would suggest that TBI results in the reduction of cDC from the murine ear that was not due to a systemic response to IR, as a loss was not observed in shielded ears. We further determined that this reduction was due, in part, to the upregulation of the chemoattractant CCL21 on lymphatic vessels as well as CCR7 expressed on cDC. Migration as a potential mechanism was confirmed using CCR7(-/-) mice in which cDC were not depleted following TBI. Finally, we demonstrated that the loss of cDC following TBI results in an impaired contact hypersensitivity response to hapten by using a modified contact hypersensitivity protocol. Taken together, these data suggest that IR exposure may result in diminished immunosurveillance in the skin, which could render the host more susceptible to pathogens.

In this report, we identify an important function for CD30 signals in the effective segregation of B and T lymphocytes in the murine spleen, additional to the recognized requirement for lymphotoxin signals. We show that CD30 signals are not required for transcription or protein expression of homeostatic chemokines, but CD30-deficient mice display impaired B/T segregation. This defect correlates with defective expression as detected by Abs of the transmembrane mucin-type protein podoplanin on T zone stroma, although expression at other sites is normal. Defective segregation is not intrinsic to CD30-deficient lymphocytes which segregate normally following transfer into RAG-deficient mice and significantly up-regulate the expression of both CCL21 and podoplanin on T zone stroma of RAG-deficient mice. During development, induction of expression of the CD30 ligand by lymphoid tissue inducer cells and podoplanin by T zone stroma are temporally linked, and the spatial association of these cells suggests that lymphoid tissue inducer cells are capable of providing the CD30 signals. Finally, we show that the appearance of podoplanin on T zone stroma in development is associated with B/T segregation of splenic white pulp areas. Our studies indicate that homeostatic chemokine expression by itself is not sufficient for B/T segregation and our data point to a significant role for podoplanin or molecules associated with podoplanin expressing stroma in the effective segregation of lymphocytes.

Memory/effector T cells recirculate through extralymphoid tissues by entering from blood and egressing via afferent lymph. Although T cell entry into effector sites is key to inflammation, the relevance of T cell egress to this process is unknown. In this study, we found that Ag recognition at the effector site reduced the tissue egress of proinflammatory Th1 cells in a mouse model of delayed hypersensitivity. Transgenic expression of "tissue exit receptor" CCR7 enhanced lymphatic egress of Ag-sequestered Th1 cells from the inflamed site and alleviated inflammation. In contrast, lack of CCR7 on Th1 cells diminished their tissue egress while enhancing inflammation. Lymph-borne Th1 and Th17 cells draining the inflamed skin of sheep migrated toward the CCR7 ligand CCL21, suggesting the CCR7-CCL21 axis as a physiological target in regulating inflammation. In conclusion, exit receptors can be targeted to modulate T cell dwell time and inflammation at effector sites, revealing T cell tissue egress as a novel control point of inflammation.

An adaptive immune response is initiated in the T cell area of secondary lymphoid organs, where antigen-presenting dendritic cells may induce proliferation and differentiation in co-localized T cells after T cell receptor engagement. The chemokines CCL19 and CCL21 and their receptor CCR7 are essential in establishing dendritic cell and T cell recruitment and co-localization within this unique microenvironment. It is shown that systemic application of a fusion protein that consists of CCL19 fused to the Fc part of human IgG1 induces effects similar to the phenotype of CCR7-/- animals, like disturbed accumulation of T cells and dendritic cells in secondary lymphoid organs. CCL19-IgG further inhibited their co-localization, which resulted in a marked inhibition of antigen-specific T cell proliferation. The immunosuppressive potency of CCL19-IgG was tested in vivo using murine models for TH1-mediated immune responses (delayed-type hypersensitivity) and for transplantation of different solid organs. In allogeneic kidney transplantation as well as heterotopic allogeneic heart transplantation in different strain combinations, allograft rejection was reduced and organ survival was significantly prolonged by treatment with CCL19-IgG compared with controls. This shows that in contrast to only limited prolongation of graft survival in CCR7 knockout models, the therapeutic application of a CCR7 ligand in a wild-type environment provides a benefit in terms of immunosuppression.

Mesenchymal stromal cells (MSCs) are highly interesting candidates for clinical applications in regenerative medicine. Due to their low occurrence in human tissues, extensive in vitro expansion is necessary to obtain sufficient cell numbers applicable as a clinical dose in the context of cellular therapy. Current cell culture media formulations for the isolation and expansion of MSCs include fetal calf serum (FCS), human AB serum (ABS), or human platelet lysate (PL) as a supplement. However, these established supplements are inherently ill-defined formulations that contain a variety of bioactive molecules in varying batch-to-batch compositions and the risk of transmitting pathogens that escape routine screening procedures. In this study, we have comparatively characterized the capacity of commonly used basal media, such as the Minimum Essential Medium alpha (αMEM), Dulbecco's modified Eagle's medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), and RPMI 1640 as well as human- and animal-derived supplements, that is, PL, ABS, and FCS to stimulate cell proliferation. MSC proliferation was observed to be optimal in the PL-supplemented αMEM. Using a combinatorial approach, we then assessed a library of soluble factors, including mitogens (TGF-β1, Activin A, bFGF, EGF, IGF-I, PDGF-BB, and VEGF), chemokines (CCL21, CCL25, CXCL12, and RANTES), proteins (human serum albumin), lipids (e.g., oleic acid, linoleic acid, and arachidonic acid), and hormones (dexamethasone, insulin, and TSH), to create a defined medium as well as coating of cell culture surfaces to promote robust MSC proliferation in vitro. A combination of recombinant human factors partially met the nutritional requirements of bone marrow-derived MSCs, and was able to promote cell proliferation comparable to about 5% PL if supplemented with auxiliary 0.6%-1.2% PL. Maximal MSC proliferation was achieved by combining 5% PL with a cocktail of recombinant factors and did not depend on coating of cell culture surfaces.

Cytokines are a protein family of regulatory factors derived from tumors and their environmental components that contribute to the growth, invasion and metastasis of breast cancer. However, the way in which tumor progression and cytokines regulate each other is not well understood. In this study, we used an oligoDNA microarray to assess the kinetic expression profile of cytokine genes in tumor tissues and lymph nodes during the progression of tumor growth in mice that had been subcutaneously challenged with breast adenocarcinoma SB5b cells. Our results demonstrated that IL-15, IL-17, IL-18 and IL-18 binding protein (IL-18bp), which are associated with inflammation, were increased in tumor tissues. Conversely, chemokines and their receptors, including CXCR4/CXCL12, CCR7/CCL21, CCL9, CXCL9 and CCL12, were overexpressed in lymph nodes during tumor growth. Furthermore, RT-PCR and Western blot analysis revealed that IL-18, a pro-angiogenic factor in tumors, was up-regulated in tumor tissues. Interestingly, CCR3, IL-1R2, SOCS and IL-20 were up-regulated in tumor tissues, but down-regulated in lymph nodes during tumor growth. This result suggests that the expression of cytokines and cytokine-related genes was differentially regulated, which resulted in a beneficial effect for tumor progression.

Quinacrine (QC) is an anti-inflammatory drug that has been used for the treatment of malaria and rheumatoid diseases. The mechanism(s) underlying the anti-inflammatory activity of QC remains poorly understood. We recently reported the QC-mediated inhibition of the NF-kappaB pathway using an in vitro model. To test this potential mechanism in vivo, we used the contact hypersensitivity response (CHS) to chemical allergen sensitization and challenge in mice as a model of skin inflammation. The results indicated that QC treatment inhibited NF-kappaB activation in the skin during allergen sensitization. This inhibition was reflected by decreased mRNA expression and protein production of the NF-kappaB-dependent cytokines TNF-alpha and IL-1beta and the chemokine CCL21 in the skin. The decreases in these cytokines resulted in reduced migration of allergen-presenting dendritic cells from the skin into skin-draining lymph nodes and markedly decreased activation of effector CD8+ T cells for the CHS response to allergen challenge (inhibitory concentration 50% or IC50 was 55 mg/kg). These findings reveal a previously unrecognized mechanism of QC-mediated inhibition of inflammation.

Infection by human immunodeficiency virus type 1 (HIV-1) is associated with decreases in peripheral CD4(+) T cells and development of lymphadenopathy. The precise mechanisms by which HIV-1 induces these changes have not been elucidated. T-cell trafficking through lymphoid tissues is facilitated by CCL21-mediated entry and sphingosine-1-phosphate (S1P)-mediated egress. Having previously determined that HIV-1 envelop glycoprotein, gp120, directly alters T-cell migration, we investigated whether gp120 without HIV-1 infection could influence the responses of CD4(+) T cells to the signals involved in T-cell trafficking through lymph tissue. Incubation of normal human T cells with gp120 for 1 h resulted in reprogramming of CD4 T-cell migratory responses by increasing sensitivity to CCL20 and CCL21 and complete inhibition of migration to S1P. Incubation of human T cells with gp120 prior to injection into NOD.CB17-Prkdc(scid)/J mice resulted in increases in lymph node accumulation of CD4(+) T cells, with reciprocal decreases in blood and spleen compared to T cells not exposed to gp120. The effects of gp120 required CD4 signaling mediated through p56(lck). These findings suggest that gp120 alone can alter CD4(+) influx and efflux from lymph nodes in a fashion consistent with the development of lymphopenia and lymphadenopathy.

Dendritic cells (DCs) play a key role in the host immune response to infections. Mycobacterium tuberculosis (MTB) can inhibit the maturation of DCs and impair their ability to stimulate T cell proliferation. Here, we assessed in vitro migratory behavior of human monocyte-derived DCs (MoDC) when infected with various MTB strains (H37Rv and prevalent clinical strains S7 and S10 from South India). The migration of Rv and S7 infected MoDC towards secondary lymphoid chemokine (CCL21) was 50% lower after 1 day of infection compared to LPS stimulation. This reduced cell migration may be due to a block in the chemokine receptor switch from CCR5 to CCR7 expression on MoDC. Only clinical strain S10 infected MoDC showed an up-regulation of CCR7 and down-regulation of CCR5 expression, similar to LPS stimulated MoDC. While Rv and S7 infected MoDC did not display any alteration in expression of these receptors. Similarly, Rv and S7 infected MoDC did not induce IL-8, IP-10 and MCP-1 chemokine production. This reduction in chemokine levels was reflected in the reduced chemoattraction of CD4(+) T cells also. These findings suggest that there is variation in the stimulation of MoDC with different clinical strains of MTB and this variation may be dependent upon the virulence of the strain.

House dust mite (HDM), the most common allergen, activate both the IgE-associated and innate immune responses. To clarify the process of sensitization, we investigated the role of the CCL21, CCL19, and CCR7 axis in a mouse model of HDM-induced allergic asthma. HDM inhalation without systemic immunization resulted in a HDM-specific IgE response. CCR7-knockout (CCR7KO) mice exhibited greater airway inflammation and IgE responses compared to wild-type mice. We examined FoxP3 expression in these mice to clarify the contribution of regulatory cells to the responses. FoxP3 expression was higher in the lungs but not in the lymph nodes of CCR7KO mice compared to wild-type mice. In CCR7KO mice, FoxP3-positive cells were found in lung, but we observed higher release of IL-13, IL-5, TGF-β, IL-17, and HMGB1 in bronchoalveolar lavage fluid. We demonstrate here that immuno-regulation through CCR7 expression in T cells plays a role in HDM-specific sensitization in the airway.

Chemokine CCL21 can effectively attract CCR7(+) dendritic cells (DCs), however its role in this event is poorly understood. In this report, we investigated the effect of exogenous CCL21 expressed in breast cancer MCF-7 on human monocyte-derived DCs. CCL21-transfected MCF-7 stimulation prompted DC functions: migration, antigen-uptake and presentation. The stimulated DCs facilitated Th1 type cytokines production, perforin-forming CD8(+) T cell transformation and final T cell-associated clearance of MCF-7. Moreover, the MCF-7-resourced CCL21 protected DCs from apoptosis significantly, involving up-regulations of Bcl-2 expression and NF-kappaB activity, and reduction of caspase-3. This study provides evidence that tumor-derived CCL21 increases the presentation and apoptosis resistance of DCs, suggesting such a mechanism may be useful for the improvement of tumor cell immunogenicity and anti-tumor response.

BACKGROUND

In this study, we report the influence of CCL21 and its receptor, CCR7, on the progression of pancreatic cancer and illuminates the correlation between the CCL21/CCR7 axis and the angiogenesis and lymphangiogenesis of pancreatic adenocarcinoma (PAC).

METHODS

A total of 30 patients with pancreatic cancer was involved in the current study. The expression of CCL21 and CCR7 in cancerous tissues, paracancerous tissues and normal pancreas were investigated using real-time PCR, Western blot and immunohistochemistry, respectively. In addition, we assessed microvessel density (MVD) and microlymphatic vessel density (MLVD) in tumor tissues using immunohistochemistry.

RESULTS

Compared to paracancerous tissues and normal pancreas, CCL21 expression in cancerous tissues was detected at a significantly low level. In contrast, the CCR7 expression was considerably higher in cancerous tissues than in normal pancreas and paracancerous tissues. Additionally, a significant correlation between the expression pattern of the CCL21/CCR7 axis and clinicopathological features, such as lymph node metastasis, was identified. Furthermore, we found that CCL21 expression was significantly associated with MVD but not significantly associated with MLVD, while CCR7 expression was significantly associated with MLVD but not significantly associated with MVD.

CONCLUSIONS

The chemotactic interaction between CCR7 and its ligand, CCL21, may be a critical event during progression in pancreatic cancer, and its underlying mechanism may be induction of angiogenesis and lymphangiogenesis regulated by this chemotactic interaction.

BACKGROUND

We investigated the expression and function of CC chemokine receptors (CCR) on highly-purified kidney and blood dendritic cells isolated from mice in which dendritic cells were mobilized with fms-like tyrosine 3 kinase ligand (Flt3L).

METHODS

CCR and CC chemokine expression were determined by RNase protection assay or flow cytometry, and dendritic cell migratory responses assayed using Transwell chambers. Chemokine production in renal tissue was detected by immunofluorescence staining. Trafficking of fluorochrome-labeled dendritic cells was monitored in vivo.

RESULTS

Freshly-isolated renal dendritic cells expressed mRNA for CCR1, 2, 5, and 7 and CCR1 and 5 protein. They did not migrate to inducible chemokines--CCL3 [macrophage inflammatory protein (MIP)-1alpha], CCL5 [regulated upon activation, normal T cell expressed and secreted (RANTES)], or CCL20 (MIP-3alpha). Following lipopolysaccharide (LPS) stimulation, the dendritic cells down-regulated CCR1, 2, and 5 expression, up-regulated or sustained signals for CCR7, and migrated to the constitutively expressed ligands CCL19 (MIP-3beta) and CCL21 (secondary lymphoid tissue chemokine). Normal kidneys expressed weak message for CCL2, 3, and 4, with stronger signals for CCL5 and 19. Intrarenal CCL5 production was enhanced by Flt3L administration, in association with marked increases in interstitial CD45+ mononuclear cells. Mobilized blood dendritic cells migrated to CCR2 and CCR5 ligands and trafficked to renal intertubular sites following adoptive (intravenous) transfer. Their migration to the CCR5 ligand MIP-1beta (CCL4) and homing to kidneys of Flt3L-treated recipients were inhibited by CCR5 antagonism.

CONCLUSIONS

These data implicate specific CCR and their ligands in regulation of the dendritic cell constituency of the kidney. CCR5 antagonism inhibits their directed migration and intrarenal accumulation.

Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin- and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (168)RSKSKKFRR(176) sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.

Lymphocyte homing to peripheral lymph nodes (PLNs) is mediated by multistep interactions between lymphocytes and high endothelial venules (HEVs). Heparan sulfate (HS) has been implicated in the presentation of chemokines on the surface of HEVs during this process. However, it remains unclear whether this cell surface presentation is a prerequisite for lymphocyte homing. In this study, we generated conditional knockout (cKO) mice lacking Ext1, which encodes a glycosyltransferase essential for HS synthesis, by crossing Ext1(flox/flox) mice with GlcNAc6ST-2-Cre transgenic mice expressing Cre recombinase in HEVs. Immunohistochemical studies indicated that HS expression was specifically eliminated in PLN HEVs but retained in other blood vessels in the cKO mice. The accumulation of a major secondary lymphoid tissue chemokine, CCL21, on HEVs was also abrogated without affecting CCL21 mRNA levels, indicating that HS presents CCL21 on HEVs in vivo. Notably, a short-term lymphocyte homing assay indicated that lymphocyte homing to PLNs was diminished in the cKO mice by 30-40%. Consistent with this result, contact hypersensitivity responses were also diminished in the cKO mice. The residual lymphocyte homing to PLNs in the cKO mice was dependent on pertussis toxin-sensitive Gi protein signaling, in which lysophosphatidic acid-mediated signaling was partly involved. These results suggest that chemokine presentation by HS on the surface of HEVs facilitates but is not absolutely required for lymphocyte homing.

Myeloid-derived suppressor cells (MDSCs) expand in tumor-bearing host. They suppress anti-tumor immune response and promote tumor growth. Chemokines play a vital role in recruiting MDSCs into tumor tissue. They can also induce the generation of MDSCs in the bone marrow, maintain their suppressive activity, and promote their proliferation and differentiation. Here, we review CCL2/CCL12-CCR2, CCL3/4/5-CCR5, CCL15-CCR1, CX3CL1/CCL26-CX3CR1, CXCL5/2/1-CXCR2, CXCL8-CXCR1/2, CCL21-CCR7, CXCL13-CXCR5 signaling pathways, their role in MDSCs recruitment to tumor tissue, and their correlation with tumor development, metastasis and prognosis. Targeting chemokines and their receptors may serve as a promising strategy in immunotherapy, especially combined with other strategies such as chemotherapy, cyclin-dependent kinase or immune checkpoints inhibitors.

The thymic stromal niche normally directs the production and export of a self-tolerant T cell repertoire. Many models of spontaneous autoimmunity, however, develop thymic architectural abnormalities before disease onset. Although this is suspected to affect central tolerance induction, creating an autoimmune predisposition, in-depth analysis of the microenvironment within these thymi is lacking, such that the mechanisms and likely direct effects on the T cell repertoire are unknown or speculative. Here we show that NZB mice, the first described model for systemic autoimmunity, demonstrate a complex thymic phenotype, including a lack of the autoimmune regulator (Aire), early defects in thymic epithelial cell (TEC) expansion, and evidence for altered NF-kappaB2 signaling. Analysis of medullary TEC revealed a numerical loss of the Aire-expressing MHC class II(high) (mTEC-high) subset as well reduced Aire protein and mRNA per cell. RelB expression was also reduced, while chemokines CCL19 and CCL21 were increased. Unexpectedly, the proportion of cortex and medulla in the NZB mice was normal from 36 wk, despite worsening architectural abnormalities. These data show that the NZB defect is more complex than previously appreciated, segregating into early numerical TEC deficiencies that correct with age, late degeneration of the niche architecture that does not affect TEC number, and a persistent reduction in Aire and RelB expression per cell acquired upon mTEC-high differentiation.

Visceral leishmaniasis is associated with atrophy and histological disorganization of splenic compartments. In this paper, we compared organized and disorganized splenic lymphoid tissue from dogs naturally infected with Leishmania infantum assessing the size of the white pulp compartments, the distribution of T, B and S100+ dendritic cells, using immunohistochemistry and morphometry and the expression of CCR7 and the cytokines, CXCL13, lymphotoxin (LT)-α, LT-β, CCL19, CCL21, TNF-α, IL-10, IFN-γ and TGF-β, using by real time RT-PCR. The lymphoid follicles and marginal zones were smaller (3.2 and 1.9 times, respectively; Mann-Whitney, P<0.02) in animals with disorganized splenic tissue in comparison to those with organized splenic lymphoid tissue. In spleens with disorganized lymphoid tissue, the numbers of T cells and S100+ dendritic cells were decreased in the follicles, and the numbers of B cells were reduced in both the follicles and marginal zones. CXCL13 mRNA expression was lower in animals with disorganized lymphoid tissue (0.5±0.4) compared to those with organized lymphoid tissue (2.7±2.9, both relative to 18S expression, P = 0.01). These changes in the spleen were associated with higher frequency of severe disease (7/12) in the animals with disorganized than in animals with organized (2/13, Chi-square, P = 0.01) splenic lymphoid tissue. The data presented herein suggest that natural infection with Leishmania infantum is associated with the impairment of follicular dendritic cells, CXCL13 expression, B cell migration and germinal center formation and associates these changes with severe clinical forms of visceral leishmaniasis. Furthermore the fact that this work uses dogs naturally infected with Leishmania infantum emphasizes the relevance of the data presented herein for the knowledge on the canine and human visceral leishmaniasis.

Understanding the mechanism of lymph node metastasis, a poor prognostic sign for prostate cancer, and the further dissemination of the disease is important to develop novel treatment strategies. Recent studies have reported that C-C chemokine receptor 7 (CCR7), whose ligand is CCL21, is abundantly expressed in lymph node metastasis and promotes cancer progression. Tumor necrosis factor-α (TNF-α) is chronically produced at low levels within the tumor microenvironment. The aim of this study was to determine whether TNF-α promotes prostate cancer dissemination from metastatic lymph nodes through activation of the CCL21/CCR7 axis. First, human prostate cancer cells were determined to express both TNF-α and CCR7. Second, low concentrations of TNF-α were confirmed to induce CCR7 in prostate cancer cells through phosphorylation of ERK. Finally, CCL21 was found to promote the migration of prostate cancer cells through phosphorylation of the protein kinase p38. Our results suggest that TNF-α leads to the induction of CCR7 expression and that the CCL21/CCR7 axis might increase the metastatic potential of prostate cancer cells in lymph node metastasis.

Dendritic cells (DCs) are key regulators of protective immune responses and tolerance to (self-)Ags. Therefore, the scientific rationale for the use of tolerogenic DC therapy in the fields of allergies, autoimmunity, and transplantation medicine is strong. In this study, we analyzed the tolerogenic capacity of IL-10-modulated DC (IL-10DC) subpopulations to identify a DC subset that combines potent immunosuppressive activities with valuable immune properties for clinical implementation. IL-10DCs consist of two phenotypically distinct subpopulations: CD83highCCR7+ IL-10DCs and CD83lowCCR7- IL-10DCs. Suppressor assays with activated effector T cells revealed that CD4+ regulatory T cells generated by CD83high IL-10DCs (iTreg+) exhibited a significantly higher suppressive capacity compared with CD4+ regulatory T cells generated by CD83low IL-10DCs (iTreg-). In this context, iTreg+ displayed a more activated phenotype (proliferation, cytokine production) compared with iTreg- In contrast to CD83low IL-10DCs, CD83high IL-10DCs exerted a strong migratory capacity toward the secondary lymphoid organ chemokine CCL21 and retained a functionally stable phenotype under inflammatory conditions. In addition, CD83high IL-10DCs expressed significantly higher levels of surface and soluble CD25. Functional analysis demonstrated that IL-10DC-related soluble CD25 efficiently inhibited the proliferation of activated T cells and that blockade of CD25 function abolished the induction of regulatory T cells by IL-10DCs, indicating a critical role for IL-10DC-related CD25 in shifting the immune response toward an iTreg- controlled tolerance reaction. In conclusion, the selective use of the CD83high IL-10DC subset may result in a higher efficacy of tolerance induction in vivo and may support the development of novel DC vaccination strategies for transplantations, as well as for allergic and autoimmune diseases.

Cancer and dendritic cells recognize and migrate toward chemokines secreted from lymphatics and use this mechanism to invade the lymphatic system, and cancer cells metastasize through it. The lymphatic-secreted chemokine ligand CCL21 has been identified as a key regulatory molecule in the switch to a metastatic phenotype in melanoma and breast cancer cells. However, it is not known whether CCL21 inhibition is a potential therapeutic strategy for inhibition of metastasis. Here, we describe an engineered CCL21-soluble inhibitor, Chemotrap-1, which inhibits migration of metastatic melanoma cells in vivo. Two-hybrid, pull-down, and coimmunoprecipitation assays allowed us to identify a naturally occurring human zinc finger protein with CCL21 chemokine-binding properties. Further analyses revealed a short peptide (∼70 amino acids), with a predicted coiled-coil structure, which is sufficient for association with CCL21. This CCL21 chemokine-binding peptide was then fused to the Fc region of human IgG1 to generate Chemotrap-1, a human chemokine-binding Fc fusion protein. Surface plasmon resonance and chemotaxis assays showed that Chemotrap-1 binds CCL21 and inhibits CCL21-induced migration of melanoma cells in vitro with subnanomolar affinity. In addition, Chemotrap-1 blocked migration of melanoma cells toward lymphatic endothelial cells in vitro and in vivo. Finally, Chemotrap-1 strongly reduced lymphatic invasion, tracking, and metastasis of CCR7-expressing melanoma cells in vivo. Together, these results show that CCL21 chemokine inhibition by Chemotrap-1 is a potential therapeutic strategy for metastasis and provide further support for the hypothesis that lymphatic-mediated metastasis is a chemokine-dependent process.

CCR7 and its ligands, CCL19 and CCL21, are responsible for directing the migration of T cells and dendritic cells into lymph nodes, where these cells play an important role in the initiation of the immune response. Recently, we have shown that systemic application of CCL19-IgG is able to inhibit the colocalization of T cells and dendritic cells within secondary lymphoid organs, resulting in pronounced immunosuppression with reduced allograft rejection after organ transplantation. In this study, we demonstrate that the application of sustained high concentrations of either soluble or immobilized CCL19 and CCL21 elicits an inhibitory program in T cells. We show that these ligands specifically interfere with cell proliferation and IL-2 secretion of CCR7(+) cells. This could be demonstrated for human and murine T cells and was valid for both CD4(+) and CD8(+) T cells. In contrast, CCL19 had no inhibitory effect on T cells from CCR7 knockout mice, but CCR7(-/-) T cells showed a proliferative response upon TCR-stimulation similar to that of CCL19-treated wild-type cells. Furthermore, the inhibition of proliferation is associated with delayed degradation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) and the down-regulation of CDK1. This shows that CCR7 signaling is linked to cell cycle control and that sustained engagement of CCR7, either by high concentrations of soluble ligands or by high density of immobilized ligands, is capable of inducing cell cycle arrest in TCR-stimulated cells. Thus, CCR7, a chemokine receptor that has been demonstrated to play an essential role during activation of the immune response, is also competent to directly inhibit T cell proliferation.

OBJECTIVE

Patients with inflammatory rheumatic diseases (IRDs) have a higher morbidity and mortality from accelerated atherosclerosis than the general population. We hypothesized that patients with the combination of IRD and coronary artery disease (CAD) would have a certain inflammatory phenotype compared with CAD patients without this comorbidity.

METHODS

Four groups of patients were included: patients with IRD, referred to coronary artery bypass grafting (CABG) (CAD-IRD, n = 67), patients without IRD, referred to CABG (CAD, n = 52), patients with IRD without CAD (IRD, n = 32) and healthy controls (n = 30). Plasma levels of several inflammatory markers were analysed by enzyme immunoassays.

RESULTS

(i) Plasma levels of markers of endothelial cell activation [i.e. vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand factor] and osteoprotegerin (OPG) were significantly increased and plasma levels of CCL21 significantly decreased in CAD-IRD patients as compared with CAD patients without IRD. (ii) Within the CAD-IRD group, acute coronary syndrome was a significant predictor of OPG, suggesting an enhanced inflammatory response during plaque destabilization in CAD-IRD patients. (iii) Plasma levels of VCAM-1, OPG and CCL21, but not lipid parameters, IRD characteristics and several other inflammatory markers (e.g. CRP), were significant predictors of CAD-IRD as opposed to CAD in two logistic regression models.

CONCLUSIONS

Our findings further support a role for inflammation in the accelerated form of atherosclerosis in IRD patients, and suggest that certain inflammatory pathways, such as the enhanced endothelial cell activation and the RANK ligand/RANK/OPG system, may be of particular importance.

Most of the genetic risk for rheumatoid arthritis (RA) is conferred by 'shared epitope' (SE), encoding alleles of HLA-DRB1. Specific North American Native (NAN) populations have RA prevalence rates of 2-5%, representing some of the highest rates estimated worldwide. As many NAN populations also demonstrate a high background frequency of SE, we sought to determine whether other genetic factors contribute to disease risk in this predisposed population. RA patients (n=333) and controls (n=490) from the Cree/Ojibway NAN population in Central Canada were HLA-DRB1 typed and tested for 21 single-nucleotide polymorphisms (SNPs) that have previously been associated with RA, including PTPN22, TRAF1-C5, CTLA4, PADI4, STAT4, FCRL3, CCL21, MMEL1-TNFRSF14, CDK6, PRKCQ, KIF5A-PIP4K2C, IL2RB, TNFAIP3, IL10-1082G/A and REL. Our findings indicate that SE is prevalent and represents a major genetic risk factor for RA in this population (82% cases versus 68% controls, odds ratio=2.2, 95% confidence interval 1.6-3.1, P<0.001). We also demonstrate that in the presence of SE, the minor allele of MMEL1-TNFRSF14 significantly reduces RA risk in a dominant manner, whereas TRAF1-C5 increases the risk. These findings point to the importance of non-HLA genes in determining RA risk in a population with a high frequency of disease predisposing HLA-DRB1 alleles.