Vitamin A plays an essential role in the maintenance of gut homeostasis but its interplay with chemokines has not been explored so far. Using an in vitro model system we studied the effects of human colonic epithelial cells (Caco2, HT-29, and HCT116) derived inflammatory stimuli on monocyte-derived dendritic cells and macrophages. Unstimulated Caco2 and HT-29 cells secreted CCL19, CCL21, and CCL22 chemokines, which could attract dendritic cells and macrophages and induced CCR7 receptor up-regulation by retinoic-acid resulting in dendritic cell migration. The chemokines Mk, CXCL16, and CXCL7 were secreted by all the 3 cell lines tested, and upon stimulation by IL-1β or TNF-α this effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1β. In the presence of ATRA the supernatants of these cells induced CD103 expression on monocyte-derived dendritic cells and when conditioned by ATRA and cocultured with CD4(+) T-lymphocytes they reduced the proportion of Th17 T-cells. However, in the macrophage-T-cell cocultures the number of these effector T-cells was increased. Thus cytokine-activated colonic epithelial cells trigger the secretion of distinct combinations of chemokines depending on the proinflammatory stimulus and are controlled by retinoic acid, which also governs dendritic cell and macrophage responses.

BACKGROUND

Circulation of leukocytes via blood, tissue and lymph is integral to adaptive immunity. Afferent lymphatics form CCL21 gradients to guide dendritic cells and T cells to lymphatics and then to draining lymph nodes (dLN). Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 (VEGFR-3) are the major lymphatic growth factor and receptor. We hypothesized these molecules also regulate chemokine gradients and lymphatic migration.

METHODS

CD4 T cells were injected into the foot pad or ear pinnae, and migration to afferent lymphatics and dLN quantified by flow cytometry or whole mount immunohistochemistry. Vascular endothelial growth factor receptor 3 or its signaling or downstream actions were modified with blocking monoclonal antibodies (mAbs) or other reagents.

RESULTS

Anti-VEGFR-3 prevented migration of CD4 T cells into lymphatic lumen and significantly decreased the number that migrated to dLN. Anti-VEGFR-3 abolished CCL21 gradients around lymphatics, although CCL21 production was not inhibited. Heparan sulfate (HS), critical to establish CCL21 gradients, was down-regulated around lymphatics by anti-VEGFR-3 and this was dependent on heparanase-mediated degradation. Moreover, a Phosphoinositide 3-kinase (PI3K)α inhibitor disrupted HS and CCL21 gradients, whereas a PI3K activator prevented the effects of anti-VEGFR-3. During contact hypersensitivity, VEGFR-3, CCL21, and HS expression were all attenuated, and anti-heparanase or PI3K activator reversed these effects.

CONCLUSIONS

Vascular endothelial growth factor C/VEGFR-3 signaling through PI3Kα regulates the activity of heparanase, which modifies HS and CCL21 gradients around lymphatics. The functional and physical linkages of these molecules regulate lymphatic migration from tissues to dLN. These represent new therapeutic targets to influence immunity and inflammation.

Dermal dendritic cells and epidermal Langerhans cells are APCs that migrate from skin to draining lymph nodes (LN) to drive peripheral tolerance and adaptive immunity. Their migration requires the chemokine receptor CCR7, which directs egress from the skin via dermal lymphatic vessels and extravasation into the LN parenchyma from lymph in the subcapsular sinus. CCR7 is activated by two chemokines: CCL19 and CCL21. CCL21 alone is sufficient for the migration of APCs from skin to LN. CCL19 and CCL21 also bind atypical chemokine receptor (ACKR) 4. ACKR4-mediated CCL21 scavenging by lymphatic endothelial cells lining the subcapsular sinus ceiling stabilizes interfollicular CCL21 gradients that direct lymph-borne CCR7(+)APCs into the parenchyma of mouse LN. In this study, we show that ACKR4 also aids APC egress from mouse skin under steady-state and inflammatory conditions. ACKR4 plays a particularly prominent role during cutaneous inflammation when it facilitates Langerhans cell egress from skin and enables the accumulation of dermal dendritic cells in skin-draining LN. Stromal cells in mouse skin, predominantly keratinocytes and a subset of dermal lymphatic endothelial cells, express ACKR4 and are capable of ACKR4-dependent chemokine scavenging in situ. ACKR4-mediated scavenging of dermal-derived CCL19, rather than CCL21, is critical during inflammation, because the aberrant trafficking of skin-derived APCs inAckr4-deficient mice is completely rescued by genetic deletion ofCcl19 Thus, ACKR4 on stromal cells aids the egress of APCs from mouse skin, and, during inflammation, facilitates CCR7-dependent cell trafficking by scavenging CCL19.

CCR7 is a chemokine receptor expressed on the surfaces of T cells, B cells, and mature dendritic cells that controls cell migration in response to the cognate ligands CCL19 and CCL21. CCR7 is critical for the generation of an adaptive T cell response. However, the roles of CCR7 in the host defense against pulmonary infection and innate immunity are not well understood. We investigated the role of CCR7 in the host defense against acute pulmonary infection with Pseudomonas aeruginosa. We intranasally infected C57BL/6 mice with P. aeruginosa and characterized the expression of CCR7 ligands and the surface expression of CCR7 on pulmonary leukocytes. In response to infection, expression of CCL19 and expression of CCL21 were oppositely regulated, and myeloid dendritic cells upregulated CCR7 expression. We further examined the effects of CCR7 deficiency on the inflammatory response to P. aeruginosa infection. We infected Ccr7(-/-) and wild-type mice with P. aeruginosa and characterized the accumulation of pulmonary leukocytes, production of proinflammatory mediators, neutrophil activation, and bacterial clearance. CCR7 deficiency led to an accumulation of myeloid dendritic cells and T cells in the lung in response to infection. CCR7 deficiency resulted in higher expression of CD80 and CD86 on dendritic cells; increased production of interleukin-12/23p40 (IL-12/23p40), gamma interferon (IFN-gamma), and IL-1 alpha; increased neutrophil respiratory burst; and, ultimately, increased clearance of acute P. aeruginosa infection. In conclusion, our results suggest that CCR7 deficiency results in a heightened proinflammatory environment in response to acute pulmonary P. aeruginosa infection and contributes to more efficient clearance.

Migration of dendritic cells (DCs) plays an important role in T-cell-mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll-like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS-induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild-type (WT), but not in TLR4(-/-) bone marrow-derived (BM) DCs indicating that TLR4 signaling is essential for LPS-induced swelling. As a consequence, LPS-treatment enhanced the migratory activity along a chemokine (CCL21)-gradient in WT, but not in TLR4-deficient BMDCs suggesting that the LPS/TLR4-induced swelling response facilitates DC migration. Moreover, the role of calcium-activated potassium channels (K(Ca) 3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS-challenged BMDCs. We found that the LPS-induced swelling of K(Ca) 3.1-deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS-induced increase in [Ca(2+)](i) detected in WT DCs was reduced in K(Ca) 3.1-deficient DCs. Finally, directed migration of LPS-challenged K(Ca) 3.1-deficient DCs was low compared to WT DCs indicating that activation of K(Ca) 3.1 is involved in LPS-induced DC migration. These findings suggest that both TLR4 and K(Ca) 3.1 contribute to the migration of LPS-activated DCs as an important feature of the adaptive immune response.

To investigate T-cell functional molecules and inflammatory cytokines and to assess T-cell apoptosis in malnourished infants, 64 infants from undernourished women and 28 healthy control infants were recruited to the study. Malnourished infants showed a significant decrease in the levels of circulating IL-2 and IL-7 and increases in the levels of IL-1β, IL-6, IL-10 and TNF-α, as measured by flow cytometry. There was a significant reduction in the number of CD3(+) T cells and an increase in apoptotic T cells, which was associated with an up-regulation of CD95 and PD-1 expression on CD3(+) T cells in malnourished compared to control infants. Significant reductions were also observed in the phosphorylation of AKT and STAT5 and in the expression of CCR7 and CXCR4 receptors in malnourished children, and these reductions were associated with a significant reduction in T-cell migratory capacity to their ligands CCL21 and CXCL12, respectively, as measured using an in vitro chemotaxis assay. Taken together, these data suggest that lymphocytes from malnourished infants are short-lived and dysfunctional.

Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co-cultured with USSC, as evidenced by the up-regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin-12 production. So, USSCs mature iDCs, thereby redirecting the antigen-uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.

OBJECTIVE

To investigate the effects of intravenous (IV) infusion of human mesenchymal stem/stromal cells (hMSCs) on activation and migration of CCR7(+) antigen presenting cells (APCs) in allogeneic corneal transplantation.

METHODS

We first analyzed the cellular and molecular profiles of draining lymph nodes (DLNs) in early and late phases after syngeneic or allogeneic corneal transplantation in mice, and then investigated the effects of hMSCs on APCs expressing CCR7, a key molecule implicated in APC migration to DLNs.

RESULTS

After early transplantation, the numbers of MHC class II(+)CD11b(+)CD11c(-), MHC class II(+)CD11b(-)CD11c(+), and MHC II(+)CD11b(+)CD11c(+) cells as well as the levels of APC-derived cytokines (IL-12a and IL-12b) driving the Th1 response were increased in both syngeneic and allogeneic transplants indicating activation of APCs. In late phase, the numbers of CD3(+)CD4(+)CD8(-) and CD3(+)CD4(-)CD8(+) cells and the levels of T cell-derived cytokines were increased in allogeneic transplants, but not in syngeneic transplants indicating immune rejection. The peri-transplant infusion of IV hMSCs significantly reduced the numbers of CCR7(+)CD11b(+) or CCR7(+)CD11c(+) cells in DLNs and the cornea in the early phase. Also, the expression of CCR7 and its ligands, CCL19, CCL21, and CXC3R as well as IL-12 were markedly decreased by hMSCs in the cornea and DLNs.

CONCLUSIONS

IV hMSCs reduced the activation and migration of CCR7(+) APCs in the cornea and DLNs in allogeneic corneal transplantation.

Dendritic cells (DCs), as potent antigen presenting cells, are increasingly used for immunotherapeutic approaches, predominantly in oncology. Low efficiency of injected Ag-pulsed DC homing to draining lymph nodes (DLNs) is one of the factors that affect the efficacy of therapy. As Langerhans cell emigration was enhanced after skin mast cell degranulation, we investigated the effect of local mast cell activation on exogenous bone marrow-derived DCs (BM-DCs) homing to DLNs. Product of activated MC/9 mast cells enhanced chemotaxis of BM-DCs to CCL21 in vitro. Intradermal injection of compound 48/80 (c48/80) induced local skin mast cell obvious degranulation and boosted exogenous BM-DC homing to DLNs. Both Ag-specific lymphocyte proliferation and TH1/TH2 cytokine production increased after HBsAg-pulsed BM-DC was injected into c48/80 pretreated mice. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy.

BACKGROUND

B-cell dysregulation has been implicated but not fully characterized in pediatric opsoclonus-myoclonus syndrome (OMS), a neuroblastoma-associated neuroinflammatory disorder.

OBJECTIVE

To assess the role of B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), two critical B cell-modulating cytokines, as potential biomarkers of disease activity and treatment biomarkers in OMS.

METHODS

Soluble BAFF and APRIL were measured in cerebrospinal fluid (CSF) and serum by ELISA in 433 children (296 OMS, 109 controls, 28 other inflammatory neurological disorders (OIND)). BAFF-R receptors on circulating CD19+ B cells were measured by flow cytometry. A blinded scorer rated motor severity on the OMS Evaluation Scale. Immunotherapies were evaluated cross-sectionally and longitudinally.

RESULTS

The mean CSF BAFF concentration, which was elevated in untreated OMS and OIND, correlated with OMS severity category (P = 0.006), and reduction by adrenocorticotropic hormone or corticotropin (ACTH) (-61%) or corticosteroids (-38%) was seen at each level of severity. In contrast, CSF APRIL was normal in OMS and OIND and unaffected by immunotherapy. When the entire OMS dataset was dichotomized into 'high' versus 'normal' CSF BAFF concentration, the phenotype of the high group included greater motor severity and number of CSF oligoclonal bands, and a higher concentration of inflammatory chemokines CXCL13 and CXCL10 in CSF and CXCL9 and CCL21 in serum. Serum APRIL was 6.7-fold higher in the intravenous immunoglobulins (IVIg) group, whereas serum BAFF was 2.6-fold higher in the rituximab group. The frequency of B cell BAFF-R expression was similar in untreated and treated OMS. Longitudinal studies of CSF BAFF revealed a significant decline in ACTH-treated patients (with or without rituximab) (P < 0.0001). Longitudinal studies of serum APRIL showed a 2.9-fold increase after 1 to 2 g/kg IVIg monotherapy (P = 0.0003).

CONCLUSIONS

Striking distinctions in BAFF/APRIL signaling were found. OMS displayed heterogeneity in CSF BAFF expression, which met many but not all criteria as a potential biomarker of disease activity. We speculate that CSF BAFF may have more utility in a biomarker panel than as a stand-alone biomarker, and that the selective upregulation of both serum APRIL by IVIg and BAFF by rituximab, as well as downregulation of CSF BAFF by ACTH/steroids, may have utility as treatment biomarkers.

Chemokines constitute a group of over 40 secreted peptides that are important for the control of leukocyte migration both during homeostasis and inflammation. Recent studies have implicated the ligands CCL19 and CCL21 and their receptor, CCR7, in the specific migration of naïve lymphocytes and mature dendritic cells to secondary lymphoid organs during immune homeostasis. However, the role that these molecules play during immune priming is not well understood. In this study, using CCL19((8-83)), a novel N-terminal truncation mutant, we have investigated the role of CCL19 in a primary allogeneic immune response, a response of particular relevance to transplant rejection. This antagonist specifically inhibited wild type CCL19-induced chemotaxis and intracellular calcium mobilization without affecting that of CCL21. The treatment of mice with CCL19((8-83)) did not globally inhibit the recruitment of cells into lymph nodes; however, it inhibited the generation of cytotoxic T lymphocytes toward allogeneic dendritic cells. This is the first evidence that CCL19 plays a role in immune priming.

In recent decades, the CXC chemokine receptor 7 (CCR7) [corrected] and its ligand CCL21 have been extensively reported to be associated with tumorigenesis. Meanwhile, Slug signaling induces the epithelial-mesenchymal transition (EMT) process in chondrosarcoma development. In the present study, we explored the functions of CCL21/CCR7 [corrected] in Slug-mediated EMT in the chondrosarcoma. We analyzed protein expression of CCR7 [corrected] and Slug in human chondrosarcoma samples. Effects of CCR7 [corrected] on chondrosarcoma cells were assessed by in vitro assays. Additionally, CCR7 [corrected] pathways were further investigated by pharmacological and genetic approaches. We found that the altered CCR7 [corrected] (81.7 %) and Slug (85.0 %) expression in human chondrosarcoma tissues were significantly associated with grade, recurrence, and 5-year overall survival. According to in vitro assays, CCL21 stimulation induced the expression of phosph-ERK, phosph-AKT, Slug and N-cadherin in SW1353 cells, while the expression of E-cadherin was down-regulated. Furthermore, Slug signaling modulated E- to N-cadherin switch, which was influenced by the kinase inhibitor PD98059 and LY294002. In addition, the genetic silencing of Slug inhibited the capacity of migration and invasion of SW1353 cells. In conclusion, CCL21/CCR7 [corrected] pathway activates ERK and PI3K/AKT signallings to up-regulate Slug pathway, leading to the occurrence of EMT process in human chondrosarcoma. This study lays a new foundation for molecule-targeted therapy of human chondrosarcoma.

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon's (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.

Lymphoid tissues are sites of soluble and cell-associated antigen sampling of peripheral tissues, and they are key compartments for the generation of cellular and humoral immune responses. Hilar lymph nodes (HiLNs), which drain the lungs, were examined to understand the effects of simian immunodeficiency virus (SIV) infection on this compartment of the immune system. Histologic and messenger RNA (mRNA) expression profiling approaches were used to determine the numbers, types, and distributions of SIV viral RNA cells and to identify differentially expressed genes in HiLNs during SIV infection. SIV RNA cells were found to be primarily CD68 and localized to paracortical and medullary regions early in infection, whereas they resided mainly in paracortex during AIDS. As SIV infection progressed, CXCL9, CXCL10, interferon-gamma, and Toll-like receptor 3 levels all increased. In contrast, CCL19 increased early in infection but decreased during AIDS, whereas CCL21 decreased progressively throughout infection. Finally, local levels of cellular activation were increased throughout infection. Taken together, these findings indicate that SIV infection leads to an inflammatory environment in lung-draining lymph nodes that is characterized by type 1 cytokines and chemokines and likely has an impact on the nature and strength of immune responses to pulmonary pathogens.

Chemokine and chemokine receptor signalling pairs play a crucial role in regulation of cell migration, morphogenesis, and cell activation. Expressed in mammals on activated T and NK cells, chemokine receptor CXCR3 binds interferon-γ inducible chemokines CXCL9-11 and CCL21. Here we sequenced the carp CXCR3 chemokine receptor and showed its relationship to CXCR3a receptors found in other teleosts. We found high expression of the CXCR3 gene in most of the organs and tissues of the immune system and in immune-related tissues such as gills and gut, corroborating a predominantly immune-related function. The very high expression in gill and gut moreover indicates a role for CXCR3 in cell recruitment during infection. High in vivo expression of CXCR3 at later stages of inflammation, as well as its in vitro sensitivity to IFN-γ2 stimulation indicate that in carp, CXCR3 is involved in macrophage-mediated responses. Moreover, as expression of the CXCR3 and CXCb genes coincides in the focus of inflammation and as both the CXCb chemokines and the CXCR3 receptor are significantly up-regulated upon IFN-γ stimulation it is hypothesized that CXCb chemokines may be putative ligands for CXCR3.

We reported previously that the production of IL-23 is impaired in DCs from mice that lack expression of the chemokines CCL19 and CCL21, which share the receptor CCR7, suggesting that these chemokines are required for IL-23 expression. However, the molecular mechanism of CCR7-mediated IL-23 production in DCs is unknown. We found that CCL19 and CCL21 stimulated DCs through CCR7 and induced transcription of IL-23p19 mRNA and IL-23 production in splenic and BMDC. Stimulation of DCs with CCR7 ligands induced phosphorylation of MAPK family members and of Akt, but only a specific PI3K inhibitor, LY294002, not inhibitors of ERK, JNK, or p38, decreased IL-23p19 transcription and IL-23 production. In DCs stimulated with CCL19 or CCL21, I κ B α was degraded, and NF-κ B was translocated into the nucleus. Prevention of NF-κ B activation blocked chemokine-mediated IL-23p19 transcription. A PI3K inhibitor abolished NF-κ B activation and IL-23 production. Based on these findings, we concluded that PI3K and NF-κ B signaling pathways play a critical role in CCR7-mediated IL-23 production in murine DCs. As IL-23 contributes to Th17 cell generation, and Th17 cells are pathogenic in autoimmune diseases, precise elucidation of these mechanisms would contribute to the development of strategies to control autoimmune diseases.

The present study has investigated the relationship between pancreatic lymphatics, infiltrating cells, and insulitic development after a single injection of complete Freund's adjuvant (CFA) given at an early age in the nonobese diabetic (NOD) mice. No CFA-treated NOD mice developed hyperglycemia, whereas most CFA-untreated mice died of diabetes at the age of 20-30 weeks. In untreated NOD mice, the increased infiltration of dendritic cells (DCs) and T-lymphocytes into the pancreatic islets appeared to be consistent with the increased expression of the secondary lymphoid chemokine (CCL21) and CD(31) by the endothelial cell lining of inter- and intralobular lymphatics. As the infiltration became severe, the reaction products of CCL21 and CD(31) were distributed in the nucleus and cytoplasm of lymphatic endothelial cells (LECs), through which DCs and T-lymphocytes migrated frequently. Administration of CFA reduced the number of infiltrating DCs and T-lymphocytes, but did not affect macrophage infiltration. The peri-insulitis occurred in numerous islets of CFA-treated NOD mice without the appearance of the intraislet infiltration and islet-associated lymphoid-like tissues. Furthermore, significant suppression of CCL21 and CD(31) was demonstrated on the infiltrating cells to the islets and islet-associated lymphatics. The abluminal endothelial cell lining of lymphatic vessels exhibited weaker immunoreactivity of CCL21 and CD(31) in comparison with the luminal surfaces. The reaction product of 5'-nucleotidase (5'-Nase) was evenly deposited on LECs, which were the absence of open junctions, cytoplasmic protrusions, and vesicles. CFA treatment influenced the migratory processes of the infiltrating cell, which were closely related with structural changes of pancreatic lymphatics and inhibited insulitic development. These findings suggest that in CFA-treated NOD mice, the suppression of insulitis and prevention of diabetes are secondary to the functional modulation of pancreatic lymphatics and infiltrating cells.

The chemokines CCL21 and CCL19, and cell bound TNF family ligand lymphotoxin beta (LTbeta), have been associated with numerous chronic inflammatory diseases. A general role in chronic inflammatory diseases cannot be assumed however; in the case of allergic inflammatory disease, CCL21/CCL19 and LTbeta have not been associated with the induction, recruitment, or effector function of Th2 cells nor dendritic cells to the lung. We have examined the induction of allergic inflammatory lung disease in mice deficient in CCL21/CCL19 or LTbeta and found that both kinds of mice can develop allergic lung inflammation. To control for effects of priming differences in knockout mice, adoptive transfers of Th2 cells were also performed, and they showed that such effector cells had equivalent effects on airway hyper-responsiveness in both knockout background recipients. Moreover, class II positive antigen presenting cells (B cells and CD11c+ dendritic cells) showed normal recruitment to the peribronchial spaces along with CD4 T cells. Thus, the induction of allergic responses and recruitment of both effector Th2 cells and antigen presenting cells to lung peribronchial spaces can develop independently of CCL21/CCL19 and LTbeta.

Lymphoid chemokines CCL19 and CCL21 are crucial for the recruitment of circulating naive T cells into lymph nodes. However, it is not completely known how they contribute to the development of allergic diseases. To determine whether the lack of CCL19 and CCL21 affects allergic airway inflammation, CCL19- and CCL21-deficient [paucity of lymph node T cells (plt/plt)] and wild-type (WT) mice were immunized intra-peritoneally and then challenged intra-nasally with chicken ovalbumin (OVA). Plt/plt mice developed more severe allergic airway inflammation characterized by increased eosinophils and lymphocytes in bronchoalveolar lavage (BAL) and profound inflammation in peribronchiolar and perivascular regions than did WT mice. CD4+ alpha4 integrin+ and CD4+ beta7 integrin+ T cells were significantly increased in the BAL of OVA-immunized and OVA-challenged (OVA/OVA) plt/plt mice compared with OVA/OVA WT mice. Moreover, there were higher levels of IL-4 and IL-13 mRNAs and lower levels of IL-2 and IFN-gamma mRNAs in inflamed lungs of OVA/OVA plt/plt mice compared with OVA/OVA WT mice. Plt/plt mice produced higher levels of total and OVA-specific IgE antibody. Thus, our results suggest that lack of lymphoid chemokines CCL19 and CCL21 enhances allergic airway inflammation by modulating the recruitment of CD4+ T cells into the lung, the balance between Th1 and Th2 cytokines and the IgE production.

OBJECTIVE

The lymphatic chemokine CCL21 is required for dendritic cell (DC) migration from tissues to lymph nodes, which helps establish tolerance to foreign yet harmless antigens. We demonstrate that CCL21 is almost completely absent from SAMP1/YitFc (SAMP) mice, which spontaneously develop chronic ileitis that resembles Crohn's disease, and that DC migration is severely impaired in these mice compared with AKR mice (controls). Toll-like receptor agonists like the Toll-like receptor 7 agonist R848 induce DC maturation and mobilization.

METHODS

We collected intestinal and other tissues and mesenteric lymph nodes (MLN) from SAMP mice. Expression of CCL21 was measured by quantitative reverse transcription polymerase chain reaction and immunofluorescence analyses; spontaneous and induced migration of DCs were assessed by flow cytometry. We analyzed production of retinoic acid by DCs and their ability to induce development of regulatory T cells. Mice were fed R848 to determine its effects on migration of DCs and development of ileitis in SAMP mice.

RESULTS

SAMP mice expressed almost no CCL21 in any tissue tested. Their CD11b(+)CD103(+) DCs were defective in migration from the ileal lamina propria to the MLN. DCs from SAMP mice also had a greatly reduced ability to produce retinoic acid and induce development of regulatory T cells compared with control mice. Young SAMP mice had reduced CCL21 expression and decreased DC migration before developing ileitis. Administration of R848 to adult SAMP mice increased migration of DC to the MLN and development of regulatory T cells there, and reduced the severity of ileitis.

CONCLUSIONS

Loss of CCL21 signaling and DC migration is required for development of ileitis in SAMP mice. Reagents such as R848, which activate DC migration to the MLN, may be developed as treatments for patients with Crohn's disease.

Dendritic cells (DCs) are one of the promising tools for enhancing antigen-specific immune responses in clinical settings. Many studies have been performed thus far to verify the efficacy of the DC vaccine in cancer patients; however, the responses have not always been satisfactory, partly because of DC incompetence. To obtain DCs potentially applicable for vaccination of cancer patients, our group sought to establish the strategy of DC generation mainly by modulating culture periods and maturation stimuli. Novel mature DCs that can be generated from monocytes within 3 days by using a combination of OK432 (Streptococcus pyogenes preparation), low-dose prostaglandin E2 (PGE2), and interferon-alpha (OPA-DCs) were developed. They strongly express CD83, CD86, and CCR7 and have potent ability to migrate to CCL21. In addition, they were able to activate natural killer and T helper 1 (TH1) cells and to induce peptide-antigen-specific cytotoxic T lymphocytes more significantly than monocyte-derived DCs stimulated with a conventional cytokine cocktail of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and PGE2 (monocyte-conditioned medium [MCM]-mimic DCs). The profound ability of OPA-DCs to stimulate these effectors is attributable to their higher expression of IL-12p70, IL-23, and IL-27 than MCM-mimic DCs, which was supported by the findings that the neutralization of IL-12p70 and IL-23 reduced the TH1 priming ability of OPA-DCs. Even when from advanced gastric or colonic cancer patients, OPA-DCs displayed abilities of migration and TH1 induction comparable to those from healthy subjects. Therefore, OPA-DCs may serve as a feasible vaccine with the potential to enhance TH1-dominant and cytolytic immune responses against cancers.

C-C chemokine receptor 7 (CCR7) and its chemoattractant agonist CCL21 promote cell migration and expression of pro-inflammatory proteins in an atherogenic environment. Since A(2A) adenosine receptor activation reduces migration and inflammatory effects, we examined its effect on CCR7 expression and migration. CCR7 protein expression decreased by about a third in macrophages treated with A(2A) receptor agonist CGS 21680 (p = 0.028, n = 7) and was reversed with antagonist, although mRNA levels increased twofold (p = 0.001, n = 3). Furthermore, macrophages treated with CGS 21680 showed a significant decrease in migration (p = 0.0311, n = 7). These results suggest that A(2A) adenosine receptor activation not only modulates CCR7 expression in both normal and inflammatory environments but also regulates macrophage migration to CCR7-specific chemoattractants.

C-type lectin receptors play important roles in immune cell interactions with the environment. We described CD302 as the simplest, single domain, type I C-type lectin receptor and showed it was expressed mainly on the myeloid phagocytes in human blood. CD302 colocalized with podosomes and lamellopodia structures, so we hypothesized that it played a role in cell adhesion or migration. In this study, we used mouse models to obtain further insights into CD302 expression and its potential immunological function. Mouse CD302 transcripts were, as in humans, highest in the liver, followed by lungs, lymph nodes (LN), spleen, and bone marrow. In liver, CD302 was expressed by hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. A detailed analysis of CD302 transcription in mouse immune cells revealed highest expression by myeloid cells, particularly macrophages, granulocytes, and myeloid dendritic cells (mDC). Interestingly, 2.5-fold more CD302 was found in migratory compared with resident mDC populations and higher CD302 expression in mouse M1 versus M2 macrophages was also noteworthy. CD302 knockout (CD302KO) mice were generated. Studies on the relevant immune cell populations revealed a decrease in the frequency and numbers of migratory mDC within CD302KO LN compared with wild-type LN. In vitro studies showed CD302KO and wild-type DC had an equivalent capacity to undergo maturation, prime T cells, uptake Ags, and migrate toward the CCL19/CCL21 chemokines. Nevertheless, CD302KO migratory DC exhibited reduced in vivo migration into LN, confirming a functional role for CD302 in mDC migration.