Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRalphabeta NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Valpha14i-positive and approximately 50% of Valpha14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Valpha14i-negative than Valpha14i-positive NKT cells. In spleen only, subsets of Valpha14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-gamma/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4(-) Valpha14i-negative NKT cells that were L-selectin(high) and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Valpha14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1(+) Valpha14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Valpha14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4(+) or CD4(-)CD8(-) and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Valpha14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.     

The expression and role of CXC chemokines in colorectal cancer

Cancer is a life-threatening disease world-wide and colorectal cancer is the second common cause of cancer mortality. The interaction between tumor cells and stromal cells plays a crucial role in tumor initiation and progression and is partially mediated by chemokines. Chemokines predominantly participate in the chemoattraction of leukocytes to inflammatory sites. Nowadays, it is clear that CXC chemokines and their receptors (CXCR) may also modulate tumor behavior by several important mechanisms: regulation of angiogenesis, activation of a tumor-specific immune response by attracting leukocytes, stimulation of tumor cell proliferation and metastasis. Here, we review the expression and complex roles of CXC chemokines (CXCL1 to CXCL16) and their receptors (CXCR1 to CXCR6) in colorectal cancer. Overall, increased expression levels of CXC chemokines correlate with poor prognosis.     

The Chemokine CXCL16 and Its Receptor,CXCR6, as Markers and Promoters of Inflammation-Associated Cancers

Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes.     

CXCR6/CXCL16 functions as a regulator in metastasis and progression of cancer

Metastasis is considered the obvious mark for most aggressive cancers. However, little is known about the molecular mechanism of the regulation of cancer metastasis. Recent evidence increasingly suggests that the interaction between chemokines and chemokine receptors is pivotal in the process of metastasis. The chemokine receptor CXCR4 and its ligand CXCL12, for example, have been reported to play a vital role in cancer metastasis. Another chemokine and chemokine receptor pair, the CXCL16/CXCR6 axis, has been studied by several independent research groups. Here, we summarize recent advances in our knowledge of the function of CXC chemokine receptor CXCR6 and its ligand CXCL16 in regulating metastasis and invasion of cancer. CXCR6 and CXCL16 are up-regulated in multiple cancer tissue types and cancer cell lines relative to normal tissues and cell lines. In addition, both CXCR6 and CXCL16 levels increase as tumor malignancy increases. Trans-membranous CXCL16 chemokine reduces proliferation while soluble CXCL16 chemokine enhances proliferation and migration. TM-CXCL16 functions as an inducer for lymphocyte build-up around tumor sites. High trans-membranous CXCL16 expression correlates with a good prognosis. Moreover, the Akt/mTOR signal pathway is involved in activating the CXCR6/CXCL16 axis. These findings suggest multiple opportunities for blocking the CXCR6/CXCL16 axis and the Akt/mTOR signal pathway in novel cancer therapies.     

CXCR4/CXCL12 在肿瘤中的研究进展

研究表明趋化因子及其受体在胚胎发育、干细胞迁移以及各种免疫反应中发挥重要作用,是许多生理及病理过程中细胞运动的重要因素。趋化因子受体CXCR4是一个由352个氨基酸构成的、7次跨膜的G蛋白偶联受体。趋化因子CXCL12为其特异性受体。研究发现,CXCR4/CXCL12在多种肿瘤中都有表达,在肿瘤的生长、血管生成、转移等方面发挥着重要作用。与正常组织相比,肿瘤组织及转移灶CXCR4高表达。因此,对CXCR4/CXCL12轴在肿瘤病生理中的作用机制进行进一步研究,很可能为肿瘤的治疗及对肿瘤转移的预防提供一个新的思路。我们现在就对其在肿瘤病生理中的作用做一综述。     

The chemokine,CXCL16, and its receptor,CXCR6, are constitutively expressed in human annulus fibrosus and expression of CXCL16 is up-regulated by exposure to IL-1ß in vitro

Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization.     

Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection

Acute allograft rejection is a major complication postlung transplantation and is the main risk factor for the development of bronchiolitis obliterans syndrome. Acute rejection is characterized by intragraft infiltration of activated mononuclear cells. The ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11) are potent chemoattractants for mononuclear cells and act through their shared receptor, CXCR3. Elevated levels of these chemokines in bronchoalveolar lavage fluid have been associated with human acute lung allograft rejection. This led to the hypothesis that the expression of these chemokines during an allogeneic response promotes the recruitment of mononuclear cells, leading to acute lung allograft rejection. We performed studies in a rat orthotopic lung transplantation model of acute rejection, and demonstrated increased expression of CXCL9 and CXCL10 paralleling the recruitment of mononuclear cells and cells expressing CXCR3 to the allograft. However, CXCL9 levels were 15-fold greater than CXCL10 during maximal rejection. Inhibition of CXCL9 decreased intragraft recruitment of mononuclear cells and cellular expression of CXCR3, resulting in lower acute lung allograft rejection scores. Furthermore, the combination of low dose cyclosporin A with anti-CXCL9 therapy had more profound effects on intragraft leukocyte infiltration and in reducing acute allograft rejection scores. This supports the notion that CXCL9 interaction with cells expressing CXCR3 has an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of acute lung allograft rejection.     

MIP-2 recruits NKT cells to the spleen during tolerance induction

Peripheral tolerance occurs after intraocular administration of Ag and is dependent on an increase in splenic NKT cells. New data here show that macrophage inflammatory protein-2 (MIP-2) is selectively up-regulated in tolerance-conferring APCs and serves to recruit NKT cells to the splenic marginal zone, where they form clusters with APCs and T cells. In the absence of the high-affinity receptor for MIP-2 (as in CXCR2-deficient mice) or in the presence of a blocking Ab to MIP-2, peripheral tolerance is prevented, and Ag-specific T regulatory cells are not generated. Understanding the regulation of lymphocyte traffic during tolerance induction may lead to novel therapies for autoimmunity, graft acceptance, and tumor rejection.     

CXCR5 may be involved in the attraction of human metastatic neuroblastoma cells to the bone marrow

Introduction Up-regulation of some chemokine receptors on tumor cells is associated with increased metastatic potential. In this respect, limited information is available on chemokine receptor in human neuroblastoma (NB). Objects Purpose of the study was to identify chemokines/chemokine receptors involved in bone marrow (BM) localization of metastatic NB cells in view of the development of targeted therapeutic strategies. CD45⁻ metastatic NB cells were isolated from the BM of six patients by immunomagnetic bead manipulation. Some experiments were carried out using a panel of human neuroblastoma cell lines (GI-ME-N, GI-LI-N, LAN-5, HTLA-230, SH-SY-5Y and IMR-32). Immunophenotypic analyses were performed by flow cytometry. Cell migration assays were carried out using transwell systems. Calcium ion mobilization, chemokine receptor internalization and cell proliferation were investigated by flow cytometry. Results In all BM samples, CXCR5 was expressed by the majority of primary neuroblasts and mediated their chemotaxis in response to CXCL13. Primary metastatic NB cells from all BM samples expressed CXCR6, but were not attracted by soluble CXCL16. Studies performed with two CXCR6⁺ NB cell lines showed that the mechanism whereby neuroblasts did not migrate to CXCL16 was likely related to defective calcium ion mobilization. Conclusions CXCR5 is the first chemokine receptor so far identified able to attract in vitro primary metastatic NB cells. CXCR6 may be involved in retention of metastatic neuroblasts in the BM through interaction with CXCL16 expressing stromal cells in the absence of signal transduction.     

The Cytomegalovirus UL146 Gene Product vCXCL1 Targets Both CXCR1 and CXCR2 as an Agonist

Large DNA viruses, such as herpesvirus and poxvirus, encode proteins that target and exploit the chemokine system of their host. UL146 and UL147 in the cytomegalovirus (CMV) genome encode the two CXC chemokines vCXCL1 and vCXCL2. In this study, vCXCL1 was probed against a panel of the 18 classified human chemokine receptors. In calcium mobilization assays vCXCL1 acted as an agonist on both CXCR1 and CXCR2 but did not activate or block any of the other 16 chemokine receptors. vCXCL1 was characterized and compared with CXCL1/GROα, CXCL2/GROβ, CXCL3/GROγ, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2 and CXCL8/IL-8 in competition binding, calcium mobilization, inositol triphosphate turnover, and chemotaxis assays using CXCR1- and CXCR2-expressing Chinese hamster ovary, 300.19, COS7, and L1.2 cells. The affinities of vCXCL1 for the CXCR1 and CXCR2 receptors were 44 and 5.6 nm, respectively, as determined in competition binding against radioactively labeled CXCL8. In calcium mobilization, phosphatidylinositol turnover, and chemotaxis assays, vCXCL1 acted as a highly efficacious activator of both receptors, with a rather low potency for the CXCR1 receptor but comparable with CXCL5 and CXCL7. It is suggested that CMV uses the UL146 gene product expressed in infected endothelial cells to attract neutrophils by activating their CXCR1 and CXCR2 receptors, whereby neutrophils can act as carriers of the virus to uninfected endothelial cells. In that way a lasting pool of CMV-infected endothelial cells could be maintained.     

Chemokines generally exhibit scavenger receptor activity through their receptor-binding domain

Chemokines are a family of cytokines that induce directed migration of various types of leukocytes through specific interactions with a group of seven transmembrane receptors. Scavenger receptors are a heterogenous family of transmembrane molecules that commonly bind and uptake oxidized low density lipoprotein and bacteria. Here, we show that not only CXC chemokine 16 (CXCL16)/SR-PSOX, a transmembrane chemokine with scavenger receptor activity, but also 12 out of 15 chemokines examined efficiently bound scavenger receptor ligands in competition with cells expressing their specific chemokine receptors. Furthermore both the chemotactic and scavenger receptor activities of SR-PSOX/CXCL16 were similarly impaired in a series of mutants altered in the chemokine domain, indicating that SR-PSOX/CXCL16 binds scavenger receptor ligands as well as CXCR6 using highly overlapping binding motifs. Taken together, chemokines generally have scavenger receptor-like activity through their receptor-binding domain, suggesting a close evolutionary relationship between chemokines and scavenger receptors.     

Prognostic value of the expression of chemokines and their receptors in regional lymph nodes of melanoma patients

Chemokines and their receptors have been reported to drive immune cells into tumours or to be directly involved in the promotion or inhibition of the development of tumours. However, their expression in regional lymph node (LN) tissues in melanoma patients remains unknown. The present study investigated the relationship between the expression of mRNA of chemokines and their receptors and clinicopathology of the regional LN tissues of skin cutaneous melanoma (SKCM) patients available in The Cancer Genome Atlas. The relationship between chemokines and their receptors and the composition of immune cells within the tumour was analysed. In SKCM regional LN tissues, the high expression of 32 types of chemokines and receptors, namely CCL2, 4-5, 7-8, 13, 22-25, CCR1-9, CXCL9-13, 16, CXCR3, 5, 6, XCL1-2 and XCR1 in LN was associated with favourable patient prognosis. Conversely, high expression of CXCL17 was an indicator of poor prognosis. The expression of mRNA for CXCL9-11, 13, CXCR3, 6, CCL2, 4, 5, 7, 8, 25, CCR1, 2, 5, and XCL1, 2 in regional LN tissues was positively correlated with the fraction of CD8-positive T cells and M1 macrophages, and was negatively correlated with M0 macrophages. CCR4, 6-9, CCL13, 22, 23 and XCR1 were positively correlated with the fraction of memory B cells and naive T cells, and negatively correlated with M0 macrophages and resting mast cells, suggesting that chemokines and their receptors may affect the prognosis of patients by guiding immune cells into the tumour microenvironment to eliminate tumour cells.     

Critical role for CXCR3 chemokine biology in the pathogenesis of bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival post-lung transplantation and is characterized by a persistent peribronchiolar inflammation that eventually gives way to airway fibrosis/obliteration. Acute rejection is the main risk factor for the development of BOS and is characterized by a perivascular/bronchiolar leukocyte infiltration. The specific mechanism(s) by which these leukocytes are recruited have not been elucidated. The CXC chemokines (monokine induced by IFN-gamma (MIG)/CXC chemokine ligand (CXCL)9, IP-10/CXCL10, and IFN-inducible T cell alpha chemoattractant (ITAC)/CXCL11) act through their shared receptor, CXCR3. Because they are potent leukocyte chemoattractants and are involved in other inflammation/fibroproliferative diseases, we hypothesized that the expression of these chemokines during an allogeneic response promotes the persistent recruitment of mononuclear cells, leading to chronic lung rejection. We found that elevated levels of MIG/CXCL9, IFN-inducible protein 10 (IP-10)/CXCL10, and ITAC/CXCL11 in human bronchoalveolar lavage fluid were associated with the continuum from acute to chronic rejection. Translational studies in a murine model demonstrated increased expression of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 paralleling the recruitment of CXCR3-expressing mononuclear cells. In vivo neutralization of CXCR3 or its ligands MIG/CXCL9 and IP-10/CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated BOS. This supports the notion that ligand/CXCR3 biology plays an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of BOS.     

How do chemokines navigate neutrophils to the target site: Dissecting the structural mechanisms and signaling pathways

Chemokines play crucial roles in combating microbial infection and initiating tissue repair by recruiting neutrophils in a timely and coordinated manner. In humans, no less than seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and two receptors (CXCR1 and CXCR2) mediate neutrophil functions but in a context dependent manner. Neutrophil-activating chemokines reversibly exist as monomers and dimers, and their receptor binding triggers conformational changes that are coupled to G-protein and β-arrestin signaling pathways. G-protein signaling activates a variety of effectors including Ca²⁺ channels and phospholipase C. β-arrestin serves as a multifunctional adaptor and is coupled to several signaling hubs including MAP kinase and tyrosine kinase pathways. Both G-protein and β-arrestin signaling pathways play important non-overlapping roles in neutrophil trafficking and activation. Functional studies have established many similarities but distinct differences for a given chemokine and between chemokines at the level of monomer vs. dimer, CXCR1 vs. CXCR2 activation, and G-protein vs. β-arrestin pathways. We propose that two forms of the ligand binding two receptors and activating two signaling pathways enables fine-tuned neutrophil function compared to a single form, a single receptor, or a single pathway. We summarize the current knowledge on the molecular mechanisms by which chemokine monomers/dimers activate CXCR1/CXCR2 and how these interactions trigger G-protein/β-arrestin-coupled signaling pathways. We also discuss current challenges and knowledge gaps, and likely advances in the near future that will lead to a better understanding of the relationship between the chemokine-CXCR1/CXCR2-G-protein/β-arrestin axis and neutrophil function.     

Mesenchymal stem cells exhibit firm adhesion, crawling, spreading and transmigration across aortic endothelial cells: effects of chemokines and shear

Mesenchymal stem cells (MSCs) have anti-inflammatory and immunosuppressive properties and may be useful in the therapy of diseases such as arteriosclerosis. MSCs have some ability to traffic into inflamed tissues, however to exploit this therapeutically their migratory mechanisms need to be elucidated. This study examines the interaction of murine MSCs (mMSCs) with, and their migration across, murine aortic endothelial cells (MAECs), and the effects of chemokines and shear stress. The interaction of mMSCs with MAECs was examined under physiological flow conditions. mMSCs showed lack of interaction with MAECs under continuous flow. However, when the flow was stopped (for 10 min) and then started, mMSCs adhered and crawled on the endothelial surface, extending fine microvillous processes (filopodia). They then spread extending pseudopodia in multiple directions. CXCL9 significantly enhanced the percentage of mMSCs adhering, crawling and spreading and shear forces markedly stimulated crawling and spreading. CXCL9, CXCL16, CCL20 and CCL25 significantly enhanced transendothelial migration across MAECs. The transmigrated mMSCs had down-regulated receptors CXCR3, CXCR6, CCR6 and CCR9. This study furthers the knowledge of MSC transendothelial migration and the effects of chemokines and shear stress which is of relevance to inflammatory diseases such as arteriosclerosis.     

Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6⁺ cells in liver, were found to crawl within hepatic sinusoids at 10–20 μm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.     

Intravascular Immune Surveillance by CXCR6+ NKT Cells Patrolling Liver Sinusoids

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6⁺ cells in liver, were found to crawl within hepatic sinusoids at 10–20 μm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.     

Pharmacological Inhibition of the Chemokine CXCL16 Diminishes Liver Macrophage Infiltration and Steatohepatitis in Chronic Hepatic Injury

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl₄)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.