CCL21 Protein Research Articles

Enhancing Th17 cells drainage through meningeal lymphatic vessels alleviate neuroinflammation after subarachnoid hemorrhage

BackgroundSubarachnoid hemorrhage (SAH) is a severe cerebrovascular disorder primarily caused by the rupture of aneurysm, which results in a high mortality rate and consequently imposes a significant burden on society. The occurrence of SAH initiates an immune response that further exacerbates brain damage. The acute inflammatory reaction subsequent to SAH plays a crucial role in determining the prognosis. Th17 cells, a subset of T cells, are related to the brain injury following SAH, and it is unclear how Th17 cells are cleared in the brain. Meningeal lymphatic vessels are a newly discovered intracranial fluid transport system that has been shown to drain large molecules and immune cells to deep cervical lymph nodes. There is limited understanding of the role of the meningeal lymphatic system in SAH. The objective of this research is to explore the impact and underlying mechanism of drainage Th17 cells by meningeal lymphatics on SAH.MethodsTreatments to manipulate meningeal lymphatic function and the CCR7-CCL21 pathway were administered, including laser ablation, injection of VEGF-C geneknockout, and protein injection. Mouse behavior was assessed using the balance beam experiment and the modified Garcia scoring system. Flow cytometry, enzyme-linked immunosorbent assays (ELISA), and immunofluorescence staining were used to study the impact of meningeal lymphatic on SAH drainage. Select patients with unruptured and ruptured aneurysms in our hospital as the control group and the SAH group, with 7 cases in each group. Peripheral blood and cerebrospinal fluid (CSF) samples were assessed by ELISA and flow cytometry.ResultsMice with SAH showed substantial behavioral abnormalities and brain damage in which immune cells accumulated in the brain. Laser ablation of the meningeal lymphatic system or knockout of the CCR7 gene leads to Th17 cell aggregation in the meninges, resulting in a decreased neurological function score and increased levels of inflammatory factors. Injection of VEGF-C or CCL21 protein promotes Th17 cell drainage to lymph nodes, an increased neurological function score, and decreased levels of inflammatory factors. Clinical blood and CSF results showed that inflammatory factors in SAH group were significantly increased. The number of Th17 cells in the SAH group was significantly higher than the control group. Clinical results confirmed Th17 cells aggravated the level of neuroinflammation after SAH.ConclusionThis study shows that improving the drainage of Th17 cells by meningeal lymphatics via the CCR7-CCL21 pathway can reduce brain damage and improve behavior in the SAH mouse model. This could lead to new treatment options for SAH.

Medullary stromal cells synergize their production and capture of CCL21 for T-cell emigration from neonatal mouse thymus.

The release of newly selected αβT cells from the thymus is key in establishing a functional adaptive immune system. Emigration of the first cohorts of αβT cells produced during the neonatal period is of particular importance, because it initiates formation of the peripheral αβT-cell pool and provides immune protection early in life. Despite this, the cellular and molecular mechanisms of thymus emigration are poorly understood. We examined the involvement of diverse stromal subsets and individual chemokine ligands in this process. First, we demonstrated functional dichotomy in the requirement for CCR7 ligands and identified CCL21, but not CCL19, as an important regulator of neonatal thymus emigration. To explain this ligand-specific requirement, we examined sites of CCL21 production and action and found Ccl21 gene expression and CCL21 protein distribution occurred within anatomically distinct thymic areas. Although Ccl21 transcription was limited to subsets of medullary epithelium, CCL21 protein was captured by mesenchymal stroma consisting of integrin α7+ pericytes and CD34+ adventitial cells at sites of thymic exit. This chemokine compartmentalization involved the heparan sulfate-dependent presentation of CCL21 via its C-terminal extension, explaining the absence of a requirement for CCL19, which lacks this domain and failed to be captured by thymic stroma. Collectively, we identified an important role for CCL21 in neonatal thymus emigration, revealing the importance of this chemokine in initial formation of the peripheral immune system. Moreover, we identified an intrathymic mechanism involving cell-specific production and presentation of CCL21, which demonstrated a functional synergy between thymic epithelial and mesenchymal cells for αβT-cell emigration.

Alteration in regulatory T cells and programmed cell death 1-expressing regulatory T cells in active generalized vitiligo and their clinical correlation.

Vitiligo is an autoimmune depigmentation disease, and defects in regulatory T cells (Tregs) have been proposed in the pathogenesis of generalized vitiligo (GV). However, the role of programmed cell death (PD)1(+) Tregs has not been studied. To investigate the status of Tregs, PD1(+) Tregs and associated parameters in active GV (aGV) during the first episode of disease attack and to establish the clinical correlation. The percentages of circulating Tregs, PD1(+) Tregs and CD3(+) CD4(+) PD1(+) T cells were evaluated in 50 patients with aGV and 51 controls. Expression levels of FOXP3, TGFB1, CTLA4 and genes for chemokine receptors (CCR4, CCR7) and their ligands (CCL21, CCL22) were quantified in peripheral blood and in lesional, perilesional, nonlesional and normal skin sections. The corresponding proteins were immunolocalized in tissue of aGV. The percentage of Tregs was decreased (P=0·001) and that of PD1(+) Tregs increased (P=0·001) in peripheral blood of patients with aGV compared with controls. The abundance of TGFB1 and CCL21 mRNA was significantly decreased in the peripheral blood of patients with aGV. Significant differences in forkhead box P3, transforming growth factor-β and CCL21 protein expression were found in skin sections. Deficiency in Treg frequency and decreased expression of Treg-associated parameters (TGFB and CCL21) suggested a possible defect in Tregs that may alter their suppression function and skin homing in aGV. The increased PD1(+) Tregs suggests that the PD1/PD ligand pathway may be involved in aGV and may have a role in Treg exhaustion. Further study is required to delineate the effect of PD1 in regulating Treg function in aGV.

The homeostatic chemokine CCL21 predicts mortality in aortic stenosis patients and modulates left ventricular remodeling.

BackgroundCCL21 acting through CCR7, is termed a homeostatic chemokine. Based on its role in concerting immunological responses and its proposed involvement in tissue remodeling, we hypothesized that this chemokine could play a role in myocardial remodeling during left ventricular (LV) pressure overload.Methods and ResultsOur main findings were: (i) Serum levels of CCL21 were markedly raised in patients with symptomatic aortic stenosis (AS, n = 136) as compared with healthy controls (n = 20). (ii) A CCL21 level in the highest tertile was independently associated with all-cause mortality in these patients. (iii) Immunostaining suggested the presence of CCR7 on macrophages, endothelial cells and fibroblasts within calcified human aortic valves. (iv). Mice exposed to LV pressure overload showed enhanced myocardial expression of CCL21 and CCR7 mRNA, and increased CCL21 protein levels. (v) CCR7−/− mice subjected to three weeks of LV pressure overload had similar heart weights compared to wild type mice, but increased LV dilatation and reduced wall thickness.ConclusionsOur studies, combining experiments in clinical and experimental LV pressure overload, suggest that CCL21/CCR7 interactions might be involved in the response to pressure overload secondary to AS.

CCR7 directs the recruitment of T cells into inflamed pancreatic islets of nonobese diabetic (NOD) mice

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreatic islets. The migration of T cells from blood vessels into pancreas is critical for the development of islet inflammation and β cell destruction in T1D. To define the roles of C-C chemokine receptor type 7 (CCR7) in recruitment of T cells into islets, we used laser capture microdissection to isolate tissue from inflamed islets of nonobese diabetic (NOD) mice and uninflamed islets of BALB/c and young NOD mice. RT-PCR analyses detected mRNAs for CCR7 and its chemokine ligands CCL19 (ELC; MIP-3β) and CCL21 (SLC) in captures from inflamed, but not from uninflamed, islets. Immunohistology studies revealed that high endothelial venules in inflamed islets co-express CCL21 protein and MAdCAM-1 (an adhesion molecule that recruits lymphocytes into islets). Desensitization of lymphocyte CCR7 blocked about 75 % of T cell migration from the bloodstream into inflamed islets, but had no effect on B cell migration into islets. These results indicate that CCR7 and its ligands are important in the recruitment of T cells into inflamed islets and thus in the pathogenesis of T1D.

Restoration of CCL21 Accelerates Lymph Node (LN) and Spleen Regeneration Post-Transplant and Improves Anti-Pathogen and Anti-Tumor Effector Responses

Abstract 212 Introduction:BMT conditioning causes substantial stromal cell injury, which may hinder lymphopoiesis and immune recovery post-transplant. The C-C chemokine CCL21, produced by LN fibroblastic reticular cells (FRCs), is critical for the trafficking of CCR7 expressing T cells into secondary lymphoid organs (i.e. spleen, LN). We first reported that radiation conditioning markedly reduces FRCs, CCL21 expression, and lymphoid tissue inducer cells that are critical for lymphoid organogenesis. As a consequence of these events, mice have substantial T cell lymphopenia and poor responses to pathogens and neoantigens (Kelly et al, Blood, 2010). We hypothesized that CCL21 deficiency would preclude the recruitment of recent thymic emigrants to secondary lymphoid organs where they would receive necessary signals for their survival. Moreover, within the secondary lymphoid organs, T cells would be unable to properly migrate to B cell zones. Thus, we tested whether restoring CCL21 protein could improve LN stromal cell architecture, T cell trafficking and consequently, T cell effector responses. [Display omitted] Methods/Results:C57BL/6 mice were lethally irradiated, rescued with congenic T cell depleted bone marrow, and given CCL21 cDNA containing or null adenovirus vector transduced dendritic cells (DCs) that were pulsed with lysates from either vesicular stomatitis or attenuated Listeria monocytogenes expressing the nominal antigen ovalbumin (VSV-OVA; Listeria-OVA). The DC vaccines were given intramuscularly beginning 21 days post-transplant and then weekly for three total doses. On day 42, mice were challenged with either VSV-OVA or Listeria-OVA and euthanized on day 43 or 50, respectively. Compared to DC/null, DC/CCL21 vaccine substantially improved spleen and LN architecture and markedly increased ER-TR7+ LN stromal cell density. These architectural changes were associated with significantly increased CCL21 expression (p<0.05 for both CCL21 density per B cell area and normalization to non-BMT controls determined by ELISA), resulting in a significant increase in total LN cells, CD8+ T cells and lymphoid tissue inducer cells (all p<0.05). Expression of the B cell chemoattractant, CXCL13, and B cell follicle number were significantly increased (p<0.05) in the spleen and LN of DC/CCL21 vaccinated mice and T cells were found juxtaposed to B cells within B cell zones. Compared to DC/null, DC/CCL21 LN CD8+ T cell number normalized vs non-BMT controls, and clearance of Listeria-OVA by the liver was significantly (p<0.05) improved and similar to non-BMT controls (3.5 vs 1.8 vs 1.2 × 105 colony-forming units, respectively). Similarly, VSV-OVA clearance was significantly improved in DC/CCL21 vs DC/null vaccines, both less than non-BMT controls (13.5 vs 26 vs 8 splenic plaque-forming units, respectively; all group-wise comparisons, p<0.05). In order to determine whether improved immune function could enhance the endogenous CD8+ T cell response to acute myeloid leukemia (AML) challenge post-BMT, congeneic BMT recipients were vaccinated with DC/CCL21 or DC/null pre-loaded with AML lysates. All mice received a lethal dose (106) of C57BL/6 AML cells expressing firefly luciferase (C1498) on d.42 post-BMT (see Figure). Compared to the uniform lethality in both DC/null vaccines and non-vaccinated, non-BMT controls, mice receiving DC/CCL21 had a significant increase (p=.0241 compared to DC/null) in survival with 50% of mice surviving long-term. Conclusions:Taken together, these data indicate that DC/CCL21 vaccines can potently restore LN and splenic architecture as well as improve endogenous T cell responses to pathogens and AML challenge. Because DC/CCL21 vaccines are in clinical trial for patients with lung cancer or melanoma, our studies may provide the foundation for future clinical trials of DC/CCL21 vaccination in patients receiving pre-transplant conditioning regimens. If our preclinical data prove translatable, DC/CCL21 vaccination could increase immune reconstitution in BMT recipients. This could lead to decreased relapse and increased pathogenic responses to life-threatening infections and decreasing morbidity and mortality post transplant. Disclosures:No relevant conflicts of interest to declare.

Blocking MAPK Signaling Downregulates CCL21 in Lymphatic Endothelial Cells and Impairs Contact Hypersensitivity Responses

CCL21 expression by lymphatic endothelial cells (LECs) is essential for migration of CCR7+ immune cells from skin to regional lymph nodes (LNs). We investigated the importance of mitogen-activated protein kinase (MAPK) signaling in CCL21 expression by ECs in vitro and in vivo. Normal human dermal lymphatic microvascular ECs (HMVEC-dLy) stimulated in vitro with oncostatin M (OSM) expressed high amounts of CCL21 mRNA. CCL21 protein expression by HMVEC-dLy was also markedly increased by OSM compared with unstimulated cultures. Marked phosphorylation of MAPK 44/42 was detected in HMVEC-dLy stimulated by OSM. CCL21 expression by HMVEC-dLy was blocked by a JAK inhibitor 1, JAK3 inhibitor, and U0126 (a MAPK kinase inhibitor) in vitro, all of which blocked phosphorylation of MAPK 44/42. In addition, injection of U0126 into murine skin significantly decreased CCL21 mRNA and protein expression. Moreover, injection of U0126 before sensitization decreased migration of dendritic cells to draining LNs and decreased contact hypersensitivity responses. In summary, these results suggest that the MAPK pathway is important for CCL21 expression by LECs in vitro and in vivo. Blocking MAPK signaling within skin may offer a novel approach to treatment of inflammatory skin diseases.

Tissue inflammation modulates gene expression of lymphatic endothelial cells and dendritic cell migration in a stimulus-dependent manner

AbstractChemokines and adhesion molecules up-regulated in lymphatic endothelial cells (LECs) during tissue inflammation are thought to enhance dendritic cell (DC) migration to draining lymph nodes, but the in vivo control of this process is not well understood. We performed a transcriptional profiling analysis of LECs isolated from murine skin and found that inflammation induced by a contact hypersensitivity (CHS) response up-regulated the adhesion molecules ICAM-1 and VCAM-1 and inflammatory chemokines. Importantly, the lymphatic markers Prox-1, VEGFR3, and LYVE-1 were significantly down-regulated during CHS. By contrast, skin inflammation induced by complete Freund adjuvant induced a different pattern of chemokine and lymphatic marker gene expression and almost no ICAM-1 up-regulation in LECs. Fluorescein isothiocyanate painting experiments revealed that DC migration to draining lymph nodes was more strongly increased in complete Freund adjuvant-induced than in CHS-induced inflammation. Surprisingly, DC migration did not correlate with the induction of CCL21 and ICAM-1 protein in LECs. Although the requirement for CCR7 signaling became further pronounced during inflammation, CCR7-independent signals had an additional, albeit moderate, impact on enhancing DC migration. Collectively, these findings indicate that DC migration in response to inflammation is stimulus-specific, mainly CCR7-dependent, and overall only moderately enhanced by LEC-induced genes other than CCL21.

The atypical chemokine receptor CCX-CKR scavenges homeostatic chemokines in circulation and tissues and suppresses Th17 responses

AbstractOur previous in vitro studies led to proposals that the atypical chemokine receptor CCX-CKR is a scavenger of CCR7 ligand homeostatic chemokines. In the present study, we generated CCX-CKR−/− mice and confirm this scavenger function in vivo. Compared with wild-type mice, CCX-CKR−/− have a 5-fold increase in the level of CCL21 protein in blood, and 2- to 3-fold increases in CCL19 and CCL21 in peripheral lymph nodes. The effect of these protein increases on immunity was investigated after immunization with MOG35-55 peptide emulsified in complete Freund adjuvant (CFA). The subsequent characteristic paralysis develops with enhanced kinetics and severity in CCX-CKR−/− versus wild-type mice. Despite this effect, antigen-specific immune responses in the draining lymph nodes are diminished in CCX-CKR−/− mice. Instead, the earlier onset of disease is associated with enhanced T-cell priming in the CCX-CKR−/− spleen and a skewing of CD4+ T-cell responses toward Th17 rather than Th1. This observation correlates with increased expression of IL-23 in the CCX-CKR−/− spleen and increased CCL21 levels in the central nervous system postimmunization. The early onset of disease in CCX-CKR−/− mice is reversed by systemic administration of neutralizing anti-CCL21 antibodies. Thus, by regulating homeostatic chemokine bioavailability, CCX-CKR influences the localization, kinetics, and nature of adaptive immune responses in vivo.

CD93/AA4.1: A Novel Regulator of Inflammation in Murine Focal Cerebral Ischemia

The stem-cell marker CD93 (AA4.1/C1qRp) has been described as a potential complement C1q-receptor. Its exact molecular function, however, remains unknown. By using global expression profiling we showed that CD93-mRNA is highly induced after transient focal cerebral ischemia. CD93 protein is upregulated in endothelial cells, but also in selected macrophages and microglia. To elucidate the potential functional role of CD93 in postischemic brain damage, we used mice with a targeted deletion of the CD93 gene. After 30 min of occlusion of the middle cerebral artery and 3 d of reperfusion these mice displayed increased leukocyte infiltration into the brain, increased edema, and significantly larger infarct volumes (60.8 +/- 52.2 versus 23.9 +/- 16.6 mm(3)) when compared with wild-type (WT) mice. When the MCA was occluded for 60 min, after 2 d of reperfusion the CD93 knockout mice still showed more leukocytes in the brain, but the infarct volumes were not different from those seen in WT animals. To further explore CD93-dependent signaling pathways, we determined global transcription profiles and compared CD93-deficient and WT mice at various time points after induction of focal cerebral ischemia. We found a highly significant upregulation of the chemokine CCL21/Exodus-2 in untreated and treated CD93-deficient mice at all time points. Induction of CCL21 mRNA and protein was confirmed by PCR and immunohistochemistry. CCL21, which was formerly shown to be released by damaged neurons and to activate microglia, contributes to neurodegeneration. Thus, we speculate that CD93-neuroprotection is mediated via suppression of the neuroinflammatory response through downregulation of CCL21.

TWEAK Activates the Non-Canonical NFκB Pathway in Murine Renal Tubular Cells: Modulation of CCL21

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 NFκB activation suggestive of engagement of the non-canonical NFκB pathway. We now explore TWEAK-induced activation of NFκB2 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 TNFα activated different DNA-binding NFκB complexes. TWEAK-induced sustained NFκB activation was associated with NFκB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFα 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 NFκB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFα. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFκB2 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 NFκB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.

Fibrocyte: New participant in the pathogenesis of renal fibrosis

Progressive fibrosis is a common pathological finding in various organs, resulting in organ failure. Renal fibrosis is a progressive disease caused by diverse clinical entities. Irrespective and independent of the primary lesion, it is the severity of renal fibrosis that correlates best with the loss of renal function and the risk for progression to renal failure. A circulating population of cells (termed fibrocytes) that display leukocyte markers such as CD45 and CD34 as well as mesenchymal markers including type I collagen has been described. Fibrocytes have been reported to rapidly enter sites of tissue injury and contribute to the pathogenesis of fibrotic conditions. Fibrocytes express CCR7 and migrate in response to secondary lymphoid tissue chemokine (SLC/CCL21). We hypothesized that CCR7-positive fibrocytes contribute to renal fibrosis. To investigate this idea, renal fibrosis was induced by unilateral ureteral obstruction in mice. CD45- and type I collagen-dual positive fibrocytes infiltrated in the fibrotic kidneys and the number of infiltrated fibrocytes increased with the progression of the extent of fibrosis in an experimental murine renal fibrosis model. Most infiltrated fibrocytes in the kidneys were positive for CCR7. In addition, CCL21 protein co-localized with high endothelial venulelike vessels in fibrotic kidneys. The blockade of CCL21/CCR7 signaling reduced the number of infiltrating fibrocytes as well as the extent of renal fibrosis, which was confirmed by the reduction of the amount of hydroxyproline and the levels of renal transcripts of pro α1 chain of type I collagen and transforming growth factor-β1. These results suggest that CCR7-positive fibrocytes infiltrate into the kidney via CCL21-positive vessels, thereby, contributing to the pathogenesis of renal fibrosis. Thus, regulating the recruitment and activation of fibrocytes by modulation of CCL21/CCR7 signaling may provide a novel therapeutic approach for combatting organ fibrosis.

Decreased Enteral Stimulation Alters Mucosal Immune Chemokines

Migration of lymphocytes into and through the mucosal immune system depends upon adhesion molecules to attract circulating cells and chemokines to stimulate diapedesis into tissues. Decreased enteral stimulation significantly reduces mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) levels, an adhesion molecule critical for homing of T and B cells to Peyer's patches (PP), which reduces PP and intestinal T and B cells. We studied the effect of type and route of nutrition on tissue specific chemokines in PP (CXCL-12, -13 and CCL-19, -20 and -21), small intestine (SI; CCL-20, -25 and -28) and lung (CXCL-12, CCL-28). Intravenously cannulated male Institute of Cancer Research (ICR) mice were randomized to chow or parenteral nutrition (PN) for 5 days. PP, SI, and lung chemokine mRNA levels were measured using real-time qRT-polymerase chain reaction, and analyzed semiquantitatively by the DeltaDeltaCt method. Protein levels were quantified using enzyme-linked immunosorbent assay (ELISA) techniques, and groups compared using Student's t-test. PP CXCL13 protein significantly decreased, whereas CCL21 protein increased significantly in the parenterally fed group. Parenteral feeding significantly decreased SI CCL20 and CCL 25 protein levels. CCL28 decreased significantly in the SI and lung of intravenously fed animals. mRNA levels changed in the opposite direction (compared with protein) for all chemokines except CCL28. Decreased enteral stimulation significantly alters key mucosal immune chemokine protein levels at multiple sites. In general, PN (and concomitant lack of enteral stimulation) results in decreased levels of chemokines that control lymphocyte migration within the mucosal immune system.

The Chemokine Binding Protein M3 Prevents Diabetes Induced by Multiple Low Doses of Streptozotocin

Multiple injections of low-dose streptozotocin (MLDS) induce lymphocytic insulitis and diabetes in rodents. To test whether the influx of inflammatory cells was associated with changes in the expression of chemokines, we measured the expression of all known chemokine ligands by real-time quantitative PCR in isolated islets. With the exception of CCL20 and CCL19, chemokines were not significantly expressed in islets from wild-type mice before MLDS treatment. Ten days after treatment, the expression of several chemokines, including CXCL9, CCL1, CXCL10, and CCL21, was dramatically up-regulated. The expression of CCL1, CXCL9, and CCL21 protein was confirmed by immunohistochemistry and was mostly associated with the infiltrating cells. The mouse herpesvirus 68-encoded chemokine decoy receptor M3 can broadly engage these chemokines with high affinity. To test whether a blockade of chemokine function would alter the onset or magnitude of insulitis and diabetes, we used transgenic mice expressing M3 in beta cells (rat insulin promoter (RIP)-M3 mice). RIP-M3 mice were normoglycemic and responded normally to glucose challenge but were remarkably resistant to diabetes induced by MLDS. Islets from MLDS-treated RIP-M3 mice had fewer inflammatory cells and expressed lower levels of chemokines than those from MLDS-treated controls. The role of M3 in chemokine blockade during insulitis was further supported by in vitro experiments demonstrating that multiple chemokines up-regulated during islet inflammation are high-affinity M3 ligands that can be simultaneously sequestered. These results implicate chemokines as key mediators of insulitis and suggest that their blockade may represent a novel strategy to prevent insulitis and islet destruction.

Function of CD4+CD3− cells in relation to B- and T-zone stroma in spleen

AbstractLymphocytes from lymphotoxin (LT) α–deficient mice, which lack segregation of their B- and T-cell areas, acquire normal organization following adoptive transfer into RAG-deficient recipients, identifying a non-B non-T cell in the segregation process. Here we show that a CD4+CD3− accessory cell is tightly associated with discrete VCAM-1–expressing stromal cells in B- and T-cell areas of the mouse spleen. CD4+CD3− cells express high levels of LTα, LTβ, and tumor necrosis factor (TNF) α, which are the ligands for the LTβ receptor and TNFR1 expressed by stromal cells. The expression of these ligands is functional, as transferring CD4+CD3− cells derived from either embryonic or adult tissues into LTα-deficient mice organizes B/T segregation and up-regulates CCL21 protein expression in areas where T cells are segregated from B cells. We propose that the function of CD4+CD3− cells is to form a link between primed CD4 T cells and the underlying stromal elements, creating distinct microenvironments in which they enable effector responses.

Construction of eukaryotic expression vector for human CCL21 and characterization of its chemotactic activity

ObjectiveTo obtain recombinant human CCL21 with biological activity from eukaryotic expression system for further use in cancer gene therapy.MethodsA fragment of human CCL21 gene was obtained from pSK-hCCL21 plasmid digested by Xho I and BamH I, inserted into the responding sites of eukaryotic expression vector pVAX1, and then transfected into COS-7 cells by electroporation method. The expression of hCCL21 protein was detected by western blotting analysis. The in vitro chemotaxis assay was used to test the chemotactic function of the expression product to lymphocytes.ResultsHuman CCL21 protein was expressed by transfected COS-7 cells with recombinant plasmid containing hCCL21 gene, and was verified by western blotting. The in vitro chemotaxis assay demonstrated that human CCL21 protein had a potent chemotactic function to lymphocytes.ConclusionHuman CCL21 was successfully and transiently expressed in eukaryotic cells, which lays some foundation for the study of CCL21 gene therapy in murine tumor models.

Oncostatin M Enhances CCL21 Expression by Microvascular Endothelial Cells and Increases the Efficiency of Dendritic Cell Trafficking to Lymph Nodes

CCL21, a lymphatic endothelial cell (LEC)-derived chemokine, and its receptor CCR7 regulate dendritic cell (DC) trafficking to lymph nodes (LN), but it is unclear how CCL21 expression is regulated. Oncostatin M (OSM) is an IL-6-like cytokine synthesized by activated DC and other leukocytes. In vitro, OSM (but not TNF-alpha) stimulated CCL21 mRNA and protein expression by human dermal microvascular EC (DMEC) in an ERK1/2-dependent fashion. Conditioned medium from OSM-treated DMEC stimulated CCL21-dependent chemotaxis of mouse bone marrow-derived DC (BMDC). Cultured BMDC expressed OSM, which was increased with the addition of LPS. Topical application of the contact-sensitizing hapten, trinitrochlorobenzene, resulted in enhanced OSM expression in the skin, whereas cutaneous injection of TNF-alpha did not. Injection of OSM into the footpad increased CCL21 mRNA expression in the draining LN by approximately 10-fold and in mouse skin by approximately 4-fold without increasing CCR7 mRNA. In vitro, OSM increased the permeability of DMEC and lung microvascular EC monolayers to FITC-dextran beads, and, in vivo, it enhanced accumulation of Evans blue dye in draining LN by approximately 3-fold (p = 0.0291). Of note, OSM increased trafficking of BMDC injected in footpads to draining LN by 2-fold (p = 0.016). In summary, OSM up-regulates CCL21 expression in skin and draining regional LN. We propose that OSM is a regulator of CCL21 expression and endothelial permeability in skin, contributing to efficient migration of DC to regional LN.

Analysis of Antitumor Activity Elicited by Vaccination with Combinations of Interleukin-12 DNA with gp100 DNA or the Chemokine CCL21In Vivo

The antitumor efficacy of human melanoma-associated antigen (hgp100) and chemokine CCL21 in combination with interleukin-12 (IL-12) was evaluated in a syngeneic melanoma mouse model. The rationale for this approach was based on previous studies showing that the efficacy of IL-12 therapy in melanoma patients correlated with the presence of antibodies against tumor-associated antigens. We have previously shown that application of xenogeneic human gp100 DNA (hgp100 DNA) is protective against mouse B16 melanoma. Furthermore, the chemokine CCL21 has the ability to chemoattract both dendritic cells (DCs) and T lymphocytes. We show here that intratumoral injection of IL-12-encoding DNA (IL-12 DNA) in combination with hgp100- encoding DNA (hgp100 DNA) into tumor-bearing mice led to a strong antitumor effect. Coapplication of IL- 12 DNA with CCL21-encoding DNA (CCL21 DNA) or recombinant CCL21 (recCCL21) protein also showed some efficacy. Triple therapy with IL-12 DNA, hgp100 DNA, and CCL21 DNA, however, showed less effect on tumor growth than double therapy with IL-12 DNA and hgp100 DNA. These findings open a new route of investigation of IL-12 and gp100 or other tumor-associated antigens in the immunotherapy of a variety of tumors.

Expression of CCL21 and 5???-Nase on Pancreatic Lymphatics in Nonobese Diabetic Mice

To obtain better insight into the modulation of lymphatic endothelial cells during the autoimmune process, alterations of the structures and histochemical features in the pancreatic lymphatics were studied in nonobese diabetic (NOD) mice. The expression of secondary lymphoid tissue chemokine (SLC/CCL21) and 5'-nucleotidase (5'-Nase) on pancreatic lymphatics was examined by histochemistry and immunoblot in NOD mice. As insulitis developed, the increased expression of CCL21 and podoplanin on pancreatic lymphatics was consistent with the increased number of cytoplasmic protrusions and vesicles, whereas 5'-Nase activity of lymphatics seemed to become decreased. The expression of CCL21 protein also showed an age-dependent increase in NOD pancreas, even though it was undetectable in normal controls. During the period of severe infiltration, reaction products of CCL21 and podoplanin were detected in the nucleus and cytoplasm of lymphatic endothelial cells. Dendritic cells and T lymphocytes frequently penetrated through the slender walls of lymphatics and adhered to the lymphatic luminal surfaces, precipitating with few 5'-Nase particles. In contrast to wild-type NOD mice, complete Freund adjuvant administration reduced CCL21 expression in NOD pancreas, suppressing the entry of activated dendritic cells into lymphatics. These findings suggest that CCL21 and 5'-Nase may be involved in the interaction between infiltrating cells and lymphatic vessels to induce the functional changes of lymphatic endothelial cells during insulitic and diabetic development.

Disparate lymphoid chemokine expression in mice and men: no evidence of CCL21 synthesis by human high endothelial venules

AbstractT-cell homing to secondary lymphoid tissues generally depends on chemokine-induced firm adhesion in high endothelial venules (HEVs) and is primarily mediated through the CC chemokine receptor 7 (CCR7) on lymphocytes. The CCR7 ligand designated CCL21 is considered the most important trigger because it appears constitutively expressed by murine HEVs. Surprisingly, when we analyzed human tissues, no CCL21 mRNA could be detected in HEVs. In fact, CCL21 mRNA was only expressed in extravascular T-zone cells and lymphatics, whereas immunostaining revealed CCL21 protein within HEVs. This suggests that T-cell recruitment to human lymphoid tissues depends on the transcytosis of lymphoid chemokines through HEV cells because there is at present no evidence of alternative chemokine production in these cells that could explain the attraction of naive T lymphocytes.