Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.

The homing of proinflammatory (M1) and the "alternatively activated" anti-inflammatory (M2) macrophages plays a different role in the process of inflammation. Chemokines are the major mediators of macrophage chemotaxis, but how they differentially regulate M1 and M2 macrophages remains largely unclear. In the present study, we attempted to screen chemokines that differentially induce chemotaxis of M1 and M2 macrophages and to explore the underlying mechanism. Among the 41 chemokines that specifically bind to 20 chemokine receptors, CCL19, CCL21, CCL24, CCL25, CXCL8, CXCL10, and XCL2 specifically induced M1 macrophage chemotaxis, whereas CCL7 induced chemotaxis of both M1 and M2 macrophages. Whereas the differential effects of these chemokines on M1/M2 macrophage chemotaxis could be attributable to the predominant expression of their cognate receptors on the macrophage subsets, CCR7, the receptor for CCL19/CCL21, appeared to be an exception. Immunoblot analysis indicated an equivalent level of CCR7 in the whole cell lysate of M1 and M2 macrophages, but CCL19 and CCL21 only induced M1 macrophage chemotaxis. Both immunoblot and confocal microscopy analyses demonstrated that CCR7 was predominantly expressed on the cell surface of M1 but in the cytosol of M2 macrophages before ligand stimulation. As a result, CCL19 or CCL21 induced activation of both MEK1-ERK1/2 and PI3K-AKT cascades in M1 but not in M2 macrophages. Intriguingly, CCL19/CCL21-mediated M1 macrophage chemotaxis was blocked by specific inhibition of PI3K rather than MEK1. Together, these findings suggest that recruitment of M1 and M2 macrophages is fine tuned by different chemokines with the involvement of specific signaling pathways.

Most T lymphocytes are generated within the thymus. It is unclear, however, how newly generated T cells relocate out of the thymus to the circulation. The present study shows that a CC chemokine CCL19 attracts mature T cells out of the fetal thymus organ culture. Another CC chemokine CCL21, which shares CCR7 with CCL19 but has a unique C-terminal extension containing positively charged amino acids, failed to show involvement in thymic emigration. Neonatal appearance of circulating T cells was defective in CCL19-neutralized mice as well as in CCR7-deficient mice but not in CCL21-neutralized mice. In the thymus, CCL19 is predominantly localized in the medulla including endothelial venules. These results indicate a CCL19- and CCR7-dependent pathway of thymic emigration, which represents a major pathway of neonatal T cell export.

It is unclear where within tissues subsets of effector and memory CD8 T cells persist during viral infection and whether their localization affects function and long-term survival. Following lymphocytic choriomeningitis virus infection, we found most killer cell lectin-like receptor G1 (KLRG1)(lo)IL-7R(hi) effector and memory cells, which are long-lived and high proliferative capacity, in the T cell zone of the spleen. In contrast, KLRG1(hi)IL-7R(lo) cells, which appear terminally differentiated and have shorter life spans, were exclusively localized to the red pulp. KLRG1(lo)IL-7R(hi) T cells homed to the T cell zone using pertussis toxin-sensitive chemokine receptors and appeared to contact gp38(+) stromal cells, which produce the chemokines CCL19 and CCL21 and the T cell survival cytokine IL-7. The transcription factors T-bet and B lymphocyte-induced maturation protein-1 controlled effector CD8 T cell splenic migration. Effector CD8 T cells overexpressing T-bet homed to the red pulp, whereas those lacking B lymphocyte-induced maturation protein-1 homed to the T cell zone. Upon memory formation, CD62L(+) memory T cells were predominantly found in the T cell zone, whereas CD62L(-) cells were found in the red pulp. Thus, effector and memory CD8 T cell subset localization within tissues is linked to their differentiation states, and this may identify anatomical niches that regulate their longevity and homeostasis.

Ectopic formation of secondary lymphoid tissue is initiated by the local attraction of naive T and B cells. In this study, we describe a novel type of organized lymphoid structure in the lung of human idiopathic pulmonary fibrosis, with key features of lymphoid neogenesis, including: 1) recently activated CD40 ligand (CD40L)+ T cells; 2) variable numbers of activated CD40+/CD40L+ B cells, sometimes organized in follicles; 3) fully mature dendritic cells (DC) expressing CD40, CD83, CD86, and DC-lysosome-associated membrane protein; 4) the expression of the chemokine CCL21; 5) the presence of vessels with characteristics of high endothelial venules; and 6) a dense network of follicular DC. Surprisingly, these structures are devoid of CCR7+ naive T cells, proliferating lymphocytes, and germinal centers, suggesting that newly recruited activated DC and Ag-experienced lymphocytes can drive lymphoid neogenesis and that factors present within the lymphoid aggregates, such as CD40L, are essential to induce DC maturation.

OBJECTIVE

The chemokine CC-ligand 21/secondary lymphoid tissue chemokine (CCL21/SLC) regulates the homing of naïve T cells and dendritic cells that express CC-chemokine receptor 7 (CCR7) from distant sites to lymphoid tissue such as lymph nodes. We hypothesized that CCL21/SLC regulates the migration of CCR7-bearing melanoma cells from a primary lesion to regional tumor-draining lymph nodes.

METHODS

Quantitative real-time reverse transcriptase-PCR (qRT) assay and immunohistochemistry (IHC) were used to assess the level of CCR7 expression in melanoma cell lines and in primary and metastatic melanoma tumors. Cell migration assay using melanoma cell lines was performed under the induction of CCL21/SLC. The CCL21/SLC expression level in tumor-draining sentinel lymph nodes (SLNs) was assessed by both qRT assay and IHC.

RESULTS

Melanoma cell lines and tumors demonstrated heterogeneous expression of CCR7 mRNA by qRT assay. There was strong functional correlation between CCR7 mRNA expression and cell migration induced by CCL21/SLC. IHC evidence of CCR7 expression in primary melanomas significantly (P = 0.02) correlated with Breslow thickness. Assessment of SLN from 55 melanoma patients by qRT assay demonstrated that CCL21/SLC mRNA expression level was significantly (P = 0.008) higher in pathologically melanoma-negative SLNs than in melanoma-positive SLNs.

CONCLUSIONS

This report demonstrates a potential mechanism for recruitment and homing of CCR7(+) metastatic melanoma cells to tumor-draining lymph nodes, which express CCL21/SLC. The study also suggests that lymph nodes bearing metastasis may suppress CCL21/SLC production.

OBJECTIVE

The chemokines CCL19 and CCL21 bind CCR7, which is involved in the organization of secondary lymphoid tissue and is expressed during chronic tissue inflammation. We investigated the expression of CCL21 and CCR7 in chronic hepatitis C. The effects of CCL21 on hepatic stellate cells (HSCs) were also studied.

METHODS

Expression of CCL21 was assessed by in situ hybridization and immunohistochemistry. CCR7 on T cells was analyzed by flow cytometry. Cultured human HSCs were studied in their activated phenotype.

RESULTS

In patients with chronic hepatitis C, expression of CCL21 and CCR7 was up-regulated. CCL21 was detected in the portal tracts and around inflammatory lymphoid follicles, in proximity to T lymphocytes and dendritic cells, which contributed to expression of this chemokine. Expression of CCR7 was also increased in patients with primary biliary cirrhosis. Intrahepatic CD8(+) T lymphocytes isolated from patients with chronic hepatitis C had a significantly higher percentage of positivity for CCR7 than those from healthy controls, and the expression of CCR7 was associated with that of CXCR3. Cultured HSCs expressed functional CCR7, the activation of which stimulated cell migration and accelerated wound healing in an in vitro model. Exposure of HSCs to CCL21 triggered several signaling pathways, including extracellular signal-regulated kinase, Akt, and nuclear factor kappaB, resulting in induction of proinflammatory genes.

CONCLUSIONS

Expression of CCL21 during chronic hepatitis C is implicated in the recruitment of T lymphocytes and the organization of inflammatory lymphoid tissue and may promote fibrogenesis in the inflamed areas via activation of CCR7 on HSCs.

Migration of autoaggressive T cells across the blood-brain barrier (BBB) is critically involved in the initiation of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The direct involvement of chemokines in this process was suggested by our recent observation that G-protein-mediated signaling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo. To search for chemokines present at the BBB, we performed in situ hybridizations and immunohistochemistry and found expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in venules surrounded by inflammatory cells. Their expression was paralleled by the presence of their common receptor CCR7 in inflammatory cells in brain and spinal cord sections of mice afflicted with EAE. Encephalitogenic T cells showed surface expression of CCR7 and the alternative receptor for CCL21, CXCR3. They specifically chemotaxed towards both CCL19 or CCL21 in a concentration dependent and pertussis toxin-sensitive manner comparable to naive lymphocytes in vitro. Binding assays on frozen sections of EAE brains demonstrated a functional involvement of CCL19 and CCL21 in adhesion strengthening of encephalitogenic T lymphocytes to inflamed venules in the brain. Taken together our data suggest that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue are involved in T lymphocyte migration into the immunoprivileged central nervous system during immunosurveillance and chronic inflammation.

Gastric cancer (GC), which is mainly induced by Helicobacter pylori (H. pylori) infection, is one of the leading causes of cancer-related death in the developing world. Active inflammation initiated by H. pylori infection and maintained by inherent immune disorders promotes carcinogenesis and postoperative recurrence. However, the presence with H. pylori in tumors has been linked to a better prognosis, possibly due to the induction of antitumor immunity. Tumor infiltrations of tumor-associated macrophages, myeloid-derived suppressor cells, neutrophils, Foxp3(+) regulatory T cells are correlated with poor prognosis. Tumor infiltrating CD8(+) cytotoxic T lymphocytes, dendritic cells, and CD45RO T cells are generally associated with good prognosis of GC, although some subsets of these immune cells have inverse prognosis prediction values. High ratios of Foxp3(+)/CD4(+) and Foxp3(+)/CD8(+) in tumors are associated with a poor prognosis; whereas high Th1/Th2 ratio in tumors predicts a good prognosis. High levels of interleukin (IL)-6, IL-10, IL-32, and chemokine C-C motif ligands (CCL)7 and CCL21 in circulation, high expression of CXC chemokine receptor 4, chemokine C-C motif receptor (CCR)3, CCR4, CCR5, CCR7, hypoxia-inducible factor-1α, signal transducer activator of transcription-3, cyclooxygenase-2, and orphan nuclear receptor 4A2 in tumors are associated with an unfavorable prognosis. Increased serum levels of matrix metalloproteinases (MMP)-3, MMP-7, and MMP-11 and increased levels of MMP-9, MMP-12, and MMP-21 in tumors are consistently associated with poor survival of GC. Further emphasis should be put on the integration of these biomarkers and validation in large cohorts for personalized prediction of GC postoperative prognosis.

OBJECTIVE

Activation of T cells by dendritic cells (DC) is thought to play a pivotal role in induction and maintenance of Crohn's disease. Detailed analyses however concerning the phenotype and maturation of DC as well as the mechanisms underlying their recruitment are still lacking for Crohn's disease.

METHODS

Different myeloid and plasmacytoid DC subsets were characterised by immunohistochemistry. Expression of the so-called "lymphoid" chemokines CCL19, CCL20, and CCL21 was determined by real time reverse transcription-polymerase chain reaction in Crohn's disease and normal controls. Furthermore, expression of CCL19, CCL20, and CCL21 as well as their receptors CCR6 (for CCL20) and CCR7 (for CCL19 and CCL21) was characterised by immunohistochemistry and, in addition, their cellular localisation was determined by double immunofluorescence investigations.

RESULTS

Colonic tissue affected by Crohn's disease was characterised by an increased number of mature myeloid DC forming clusters with proliferating T cells. In keeping with their advanced maturation, DC possess the chemokine receptor CCR7. Increased expression of the CCR7 ligands CCL19 by DC themselves as well as CCL21 by reticular cells and lymphatic vessels was observed in Crohn's disease, thereby causing the matured DC to be trapped at the site of inflammation.

CONCLUSIONS

Our results demonstrate that autocrine and paracrine actions of lymphoid chemokines in Crohn's disease may lead to increased numbers of mature DC away from their usual migration to lymphoid organs and result in the development of a tertiary lymphatic tissue within the bowel wall maintaining the autoimmune inflammation in Crohn's disease.

Whenever neurons in the CNS are injured, microglia become activated. In addition to local activation, microglia remote from the primary lesion site are stimulated. Because this so-called secondary activation of microglia is instrumental for long-term changes after neuronal injury, it is important to understand how microglia activity is controlled. The remote activation of microglia implies that the activating signals are transported along neuronal projections. However, the identity of these signals has not yet been identified. It is shown here that glutamate-treated neurons rapidly express and release the chemokine CCL21. We also provide evidence that neuronal CCL21 is packed in vesicles and transported throughout neuronal processes to reach presynaptic structures. Chemotaxis assays show that functional CCL21 is released from endangered neurons and activate microglia via the chemokine receptor CXCR3. Based on these findings, we suggest that neuronal CCL21 is important in directed neuron-microglia signaling and that this communication could account for the remote activation of microglia, far distant from a primary lesion.

Malignant lymphocyte migration into lymph nodes is an important aspect of chronic lymphocytic leukemia (CLL), yet little is known about the processes involved. Here we demonstrate that CLL cells migrate across vascular endothelium in response to at least 3 chemokines, namely, CCL21, CCL19, and CXCL12. Moreover, transendothelial cell migration (TEM) in response to CCL21 and CCL19 was significantly higher for the malignant B cells of patients who had clinical lymph node involvement as compared with those of patients lacking such organomegaly. Furthermore, the expression of CCR7, the receptor for both CCL21 and CCL19, correlated with clinical lymphadenopathy, and blocking of CCR7 inhibited CLL cell TEM. By using immunohistochemistry we demonstrated that CCL21 and CCL19, but not CXCL12, are located in high endothelial venules and are, therefore, in an appropriate location to induce TEM. Regarding the adhesion receptors involved in TEM, alpha4 (most likely in association with beta1) and alphaLbeta2 were shown to be important in CLL cell TEM in vitro, but only the level of alpha4 expression correlated with the presence of clinical lymphadenopathy. The present studies are the first to shed light on the factors determining CLL cell entry into nodes and define the phenotype of circulating malignant cells likely to determine the pattern of lymph node enlargement in the disease.

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node-like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin(+) T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell-rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter-CXCL13-transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell-rich zone.

The chemokine receptor CCR7 represents an important determinant for circulating lymphocytes to enter lymph nodes (LN) via high endothelial venules. High endothelial venules also represent the major site of entry for plasmacytoid dendritic cells (pDC). In the steady-state, murine pDC have been suggested to home to LN engaging the chemokine receptors CXCR3, CXCR4, and CCR5, whereas responsiveness to CCR7 ligands is thought to be acquired only upon activation. In this study, we show that already resting pDC express minute amounts of CCR7 that suffice to trigger migration to CCL19/CCL21 in vitro. Upon activation with TLR ligands, CCR7 levels on pDC are strongly increased. Notably, CCR7-deficient mice display substantially reduced pDC counts in LN but not in bone marrow and spleen. Adoptive cell transfer experiments revealed that under both steady-state as well as inflammatory conditions, the homing of CCR7-deficient pDC is severely impaired, indicating that the reduced cell counts of naive pDC observed in CCR7(-/-) mice reflect an intrinsic homing defect of pDC. Together, these observations provide strong evidence that similar to naive lymphocytes, nonstimulated pDC exploit CCR7 to gain entry into LN. This adds to the repertoire of chemokine receptors permitting them to enter diverse tissues.

Tissue inflammation induces rapid mobilization of antigen-charged dendritic cells (DCs), which migrate to draining lymph nodes via afferent lymphatics to elicit the immune response. This increase in DC trafficking has been shown to require integrin-dependent adhesion to ICAM-1 and VCAM-1, expressed on inflamed lymphatic endothelium. In addition, both constitutive- and inflammation-induced DC migration involves the chemokine CCL21, which most likely triggers integrin activation on DC via its receptor CCR7. Recently, however, conflicting evidence has suggested that DC entry occurs independently of integrins, implying that the role of CCL21 in lymphatics is purely chemotactic. Hence, while CCL21 is reported to be inducible during inflammation, the details of this induction and the role of CCL21 during initial DC trafficking are unclear. Here, we have characterized both the production of CCL21 and the mechanism of its action in DC transmigration using primary human dermal lymphatic endothelial cells (HDLECs) and a mouse model of skin contact hypersensitivity. We showed that CCL21 is constitutively expressed intracellularly but rapidly secreted after exposure to the inflammatory cytokine tumour necrosis factor (TNF) α following de novo RNA and protein synthesis. Furthermore, using in vitro transmigration assays, we showed that endogenous HDLEC-derived CCL21 stimulates DC translymphatic migration by a predominantly chemotactic mechanism in resting HDLEC and by a β2 integrin-mediated mechanism in TNFα-stimulated HDLEC. These results imply a direct role for CCL21 in lymphatic transmigration that involves the selective use of integrin activation in inflammation.

Inducible bronchus-associated lymphoid tissue (iBALT) is an organized tertiary lymphoid structure that is not pre-programmed but develops in response to infection or under chronic inflammatory conditions. Emerging research has shown that iBALT provides a niche for T-cell priming and B-cell education to assist in the clearance of infectious agents, highlighting the prospect that iBALT may be engineered and harnessed to enhance protective immunity against respiratory pathogens. Although iBALT formation is associated with several canonical factors of secondary lymphoid organogenesis such as lymphotoxin-α and the homeostatic chemokines, CXCL13, CCL19, and CCL21, these cytokines are not mandatory for its formation, even though they influence its organization and function. Similarly, lymphoid tissue-inducer cells are not a requisite of iBALT formation. In contrast, dendritic cells are emerging as pivotal players required to form and sustain the presence of iBALT. Regulatory T cells appear to be able to attenuate the development of iBALT, although the underlying mechanisms remain ill-defined. In this review, we discuss facets unique to iBALT induction, the cellular subsets, and molecular cues that govern this process, and the contribution of this ectopic structure toward the generation of immune responses in the pulmonary compartment.

Efficient immune defenses are facilitated by the organized microarchitecture of lymphoid organs, and this organization is regulated by the compartmentalized expression of lymphoid tissue chemokines. Mouse cytomegalovirus (MCMV) infection induces significant remodeling of splenic microarchitecture, including loss of marginal zone macrophage populations and dissolution of T and B cell compartmentalization. MCMV preferentially infected the splenic stroma, targeting endothelial cells (EC) as revealed using MCMV-expressing green fluorescent protein. MCMV infection caused a specific, but transient transcriptional suppression of secondary lymphoid chemokine (CCL21). The loss of CCL21 was associated with the failure of T lymphocytes to locate within the T cell zone, although trafficking to the spleen was unaltered. Expression of CCL21 in lymphotoxin (LT)-alpha-deficient mice is dramatically reduced, however MCMV infection further reduced CCL21 levels, suggesting that viral modulation of CCL21 was independent of LTalpha signaling. Activation of LTbeta-receptor signaling with an agonistic antibody partially restored CCL21 mRNA expression and redirected transferred T cells to the splenic T cell zone in MCMV-infected mice. These results indicate that virus-induced alterations in lymphoid tissues can occur through an LT-independent modulation of chemokine transcription, and targeting of the LT cytokine system can counteract lymphoid tissue remodeling by MCMV.

BACKGROUND

Unstable atherosclerotic plaque typically contains an infiltrate of activated macrophages and activated T cells. This study established a functional profile of plaque-residing dendritic cells (DC) to examine whether they can function as Ag-presenting cells to facilitate in situ T-cell activation.

METHODS

Carotid artery plaque tissues were collected from 19 asymptomatic and 38 symptomatic patients undergoing endarterectomy. Matched samples of normal coronary artery wall, stable nonruptured plaque, and eroded unstable plaque were harvested from patients with fatal myocardial infarction. Quantitative PCR and immunohistochemistry were used to analyze the tissues for markers of DC activation (CD83, CD86, CCL19,CCL21) and correlate them with T-cell activation (IFN-gamma,TNF-alpha).

RESULTS

Carotid artery plaques from patients with ischemic symptoms compared to asymptomatic patients were characterized by the presence of high amount of T-cells (P<0.01) and tissue production of high levels of the T-cell cytokines IFN-gamma (P=0.001) and TNF-alpha (P=0.006). Plaque tissues from patients with ischemic complications contained elevated levels of CD83 (P<0.001), a marker of DC activation, and the DC chemokines CCL19 (P=0.001) and CCL21 (P<0.02). Unstable coronary artery plaques were similarly correlated compared to carotid plaques from symptomatic patients with the accumulation of T cells (P=0.001) and the production of T cell chemokines IFN-gamma (P=0.001) and TNF-alpha (P=0.002). Immunohistochemistry confirmed the presence of CD83(+) DC in the shoulder region of unstable plaques, where they produced the T cell-attracting chemokines CCL19 and CCL21. Mapping of activated DC demonstrated close contact between mature DC and T cells expressing the activation marker CD40 ligand (CD40L).

CONCLUSIONS

Activated and fully mature DC are represented in the inflammatory infiltrate characteristic for unstable carotid and coronary atheroma. Such DC produce chemokines, and thus can regulate the cell traffic into the lesion. Through the expression of the costimulatory ligand CD86, plaque-residing DC can augment T-cell stimulation and provide optimal stimulation conditions for T lymphocytes, resembling the microenvironment in organized lymphoid tissues.

Little is known about mechanisms determining the homeostasis of lymphocytes within lymphoid organs. Applying different mouse models, including conditionally proficient Ccr7 gene-targeted mice, we now show that semimature steady state dendritic cells (sDCs) constitutively trafficking into lymph nodes (LNs) were essential contributors to T cell homeostasis in these organs. sDCs provided vascular endothelial growth factor known to support high endothelial venule formation, thus facilitating enhanced homing of T cells to LNs. The presence of sDCs led to increased CCL21 production in T-zone fibroblastic reticular cells. CCL21 is a ligand for CCR7 known to regulate homing as well as retention of T cells in LNs. In addition, we provide evidence that CCL21 binds to the surface of DCs via its heparin-binding domain, further explaining why T cells leave LNs more rapidly in the absence of sDCs. Together, these data reveal multiple roles for sDCs in regulating T cell homeostasis in LNs.

Dendritic cells (DCs) respond to chemotactic signals to migrate from sites of infection to secondary lymphoid organs where they initiate the adaptive immune response. The key chemokines directing their migration are CCL19, CCL21, and CXCL12, but how signals from these chemokines are integrated by migrating cells is poorly understood. Using a microfluidic device, we presented single and competing chemokine gradients to murine bone-marrow derived DCs in a controlled, time-invariant microenvironment. Experiments performed with counter-gradients revealed that CCL19 is 10-100-fold more potent than CCL21 or CXCL12. Interestingly, when the chemoattractive potencies of opposing gradients are matched, cells home to a central region in which the signals from multiple chemokines are balanced; in this region, cells are motile but display no net displacement. Actin and myosin inhibitors affected the speed of crawling but not directed motion, whereas pertussis toxin inhibited directed motion but not speed. These results provide fundamental insight into the processes that DCs use to migrate toward and position themselves within secondary lymphoid organs.

OBJECTIVE

To characterize the expression of CCL19 and CCL21 in rheumatoid arthritis (RA) synovial tissue (ST) and to examine their regulation and pathogenetic role in macrophages and RA ST fibroblasts.

METHODS

Expression of CCL19 and CCL21 in RA and normal ST was demonstrated by immunohistochemistry analysis. CCL19 and CCL21 levels in synovial fluid (SF) from patients with osteoarthritis (OA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), and RA were quantified by enzyme-linked immunosorbent assay (ELISA). Regulation of CCL19 and CCL21 expression in in vitro-differentiated RA peripheral blood macrophages as well as RA ST fibroblasts was determined by real-time reverse transcription-polymerase chain reaction. Proangiogenic factor production in CCL19- and CCL21-activated in vitro-differentiated peripheral blood macrophages and RA ST fibroblasts was examined by ELISA.

RESULTS

CCL19 and CCL21 were elevated in RA ST compared to tissue from normal controls. Levels of CCL19 and CCL21 were greatly increased in RA and PsA SF versus OA SF. In RA macrophages and fibroblasts, expression of CCL19 was increased by stimulation with lipopolysaccharide, tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β). However, CCL21 expression was modulated only by IL-1β in RA fibroblasts, and by TNFα and RA SF in RA macrophages. CCL19 and CCL21 activation induced vascular endothelial growth factor and angiotensin I (Ang I) production in RA ST fibroblasts and secretion of IL-8 and Ang I from macrophages.

CONCLUSIONS

The findings of the present study identify, for the first time, regulators of CCL19 and CCL21 in RA fibroblasts and in vitro-differentiated RA peripheral blood macrophages and demonstrate a novel role of CCL19/CCL21 in angiogenesis in RA.

Crohn's disease (CD) and ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), are chronic, relapsing, and tissue destructive lesions that are accompanied by the uncontrolled activation of effector immune cells in the mucosa. Recent estimates indicate that there are 1.3 million annual cases of IBD in the United States, 50% of which consists of CD and 50% of UC. Chemokines and cytokines play a pivotal role in the regulation of mucosal inflammation by promoting leukocyte migration to sites of inflammation ultimately leading to tissue damage and destruction. In recent years, experimental studies in rodents have led to a better understanding of the role played by these inflammatory mediators in the development and progression of colitis. However, the clinical literature on IBD remains limited. Therefore, the aim of this study was to evaluate systemic concentrations of key chemokines and cytokines in forty-two IBD patients with a range of disease activity compared to levels found in ten healthy donors. We found a significant increase in an array of chemokines including macrophage migration factor (MIF), CCL25, CCL23, CXCL5, CXCL13, CXCL10, CXCL11, MCP1, and CCL21 in IBD patients as compared to normal healthy donors (P<0.05). Further, we also report increases in the inflammatory cytokines IL-16, IFN-γ, IL-1β and TNF-α in IBD patients when compared to healthy donors (P<0.05). These data clearly indicate an increase in circulating levels of specific chemokines and cytokines that are known to modulate systemic level through immune cells results in affecting local intestinal inflammation and tissue damage in IBD patients. Blockade of these inflammatory mediators should be explored as a mechanism to alleviate or even reverse symptoms of IBD.

BACKGROUND

Uterine leiomyomas are extremely common and a major cause of pelvic pain, bleeding, infertility, and the leading indication for hysterectomy. Familial and epidemiological studies provide compelling evidence that genetic alterations play an important role in leiomyoma development.

METHODS

Using Affymetrix U133A GeneChip we analysed expression profiles of 22,283 genes in paired samples of leiomyoma and adjacent normal myometrium. We compared our results with previously published data on gene expression in uterine leiomyoma and identified the overlapping gene alterations.

RESULTS

We detected 80 genes with average differences of>> or = 2-fold and false discovery rates of < 5% (14 overexpressed and 66 underexpressed). A comparative analysis including eight previous gene expression studies revealed eight prominent genes (ADH1, ATF3, CRABP2, CYR61, DPT, GRIA2, IGF2, MEST) identified by at least five different studies, eleven genes (ALDH1, CD24, CTGF, DCX, DUSP1, FOS, GAGEC1, IGFBP6, PTGDS, PTGER3, TYMS) reported by four studies, twelve genes (ABCA, ANXA1, APM2, CCL21, CDKN1A, CRMP1, EMP1, ESR1, FY, MAP3K5, TGFBR2, TIMP3) identified by three studies, and 40 genes reported by two different studies.

CONCLUSIONS

Review of gene expression data revealed concordant changes in genes regulating retinoid synthesis, IGF metabolism, TGF-beta signaling and extracellular matrix formation. Gene expression studies provide clues to the relevant pathways of leiomyoma development.

BACKGROUND

C-C chemokine receptor type 7 (CCR7) plays an important role in chemotactic and metastatic responses in various cancers, including breast cancer. In the present study, the authors demonstrated that microRNA (miRNA) let-7a downregulates CCR7 expression and directly influences the migration and invasion of breast cancer cells.

METHODS

The expression of CCR7, its ligand CCL21, and let-7a was detected in breast cancer cell lines and in breast cancer patient tissues. Synthetic let-7a and an inhibitor of let-7a were transfected into MDA-MB-231 and MCF-7 breast cancer cells, respectively, and cell proliferation, cell migration, and invasion assays were performed. To confirm the fact that 3'UTR of CCR7 is a direct target of let-7a, a luciferase assay for the reporter gene expressing the let-7a binding sites of CCR7 3'UTR was used. An in vivo invasion animal model system using transparent zebrafish embryos was also established to determine the let-7a effect on breast cancer cell invasion.

RESULTS

First, a higher expression of both CCR7 and CCL21 in malignant tissues than in their normal counterparts from breast cancer patients was observed. In addition, a reverse correlation in the expression of CCR7 and let-7a in breast cancer cell lines and breast cancer patient tissues was detected. Synthetic let-7a decreased breast cancer cell proliferation, migration, and invasion, as well as CCR7 protein expression in MDA-MB-231 cells. The let-7a inhibitor reversed the let-7a effects on the MCF-7 cells. The 3'UTR of CCR7 was confirmed as a direct target of let-7a by using the luciferase assay for the reporter gene expressing let-7a CCR7 3'UTR binding sites. Notably, when analyzing in vivo invasion, MDA-MB 231 cells after synthetic let-7a transfection were unable to invade the vessels in zebrafish embryos.

CONCLUSIONS

The results from the present study suggest that targeting of CCL21-CCR7 signaling is a valid approach for breast cancer therapy and that let-7a directly binds to the 3'UTR of CCR7 and blocks its protein expression, thereby suppressing migration and invasion of human breast cancer cells. Furthermore, the present study underscores the therapeutic potential of let-7a as an antitumor and antimetastatic manager in breast cancer patients.