Role of LOX-1 in Ang II-induced oxidative functional damage in renal tubular epithelial cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), plays an important role in angiotension II (Ang II)-induced hypertensive renal injury associated with pro-inflammatory responses, tubular functional damage and cellular apoptosis. In this study, we report on the role of LOX-1 in Ang II-induced oxidative functional damage and underlying signaling in human renal proximal tubular epithelial cells (HRPTEpiCs). The exposure to Ang II enhanced the expression of the NADPH oxidases (the p22phox, p47phox and Nox4 subunits), LOX-1 and the adhesion molecule, ICAM-1. It also promoted monocytic U937 cell adherences to HRPTEpiCs, increased reactive oxygen species formation and stimulated apoptosis, which was concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). Furthermore, the Ang II treatment disturbed the balance of the Bcl-2 family proteins, destabilized mitochondrial membrane potential, and subsequently triggered the release of cytochrome c into the cytosol, causing the activation of caspase-3. The NADPH oxidase inhibitors and LOX-1 small interfering RNA markedly ameliorated these detrimental effects by reducing LOX-1 expression and MAPK activation. The p38 and p44/42MAPK inhibitors also inhibited the Ang II-induced functional damage without affecting LOX-1 expression in the HRPTEpiCs. These observations suggest that LOX-1 mediates Ang II-induced renal tubular epithelial dysfunction. In addition, MAPK pathway activation occurs downstream of the Ang II/reactive oxygen species/LOX-1 cascade.     

Chemokines and common variable immunodeficiency; possible contribution of the fractalkine system (CX3CL1/CX3CR1) to chronic inflammation

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation. The chemokine Fractalkine (CX3CL1) and its receptor CX3CR1 is suggested to play an important role in the pathogenesis of several inflammatory disorders. We hypothesized that enhanced CX3CL1/CX3CR1 interaction could be involved in the chronic inflammation characterising subgroups of CVID. CVID patients were characterized by raised plasma levels of CX3CLl and enhanced expression of its corresponding receptor CX3CR1 on CD4(+) and CD8(+) T cells, including both CD45RA(+) and CD45RA(-) subsets. CX3CR1 expression was particularly enhanced in patients characterized by chronic inflammation in vivo. The high expression of the receptor in CVID patients was accompanied by enhanced chemotactic, adhesive, and other inflammatory cell responses to stimulation with CX3CL1. Our findings suggest that increased CX3CL1/CX3CR1 interaction could contribute to the inflammatory phenotype seen in subgroups of CVID patients.     

Pigment epithelium-derived factor (PEDF) blocks angiotensin II-induced T cell proliferation by suppressing autocrine production of interleukin-2

Angiotensin II (Ang II) elicits numerous inflammatory-proliferative responses in vascular cells, thereby being involved in atherosclerosis. We have previously shown that pigment epithelium-derived factor (PEDF) blocks the Ang II-induced endothelial cell activation, thus suggesting that PEDF may play a role in atherosclerosis. However, effects of PEDF on T cell activation, another key steps of atherosclerosis, remain to be elucidated. In this study, we examined whether PEDF could inhibit the Ang II-induced MOLT-3 T cell proliferation in vitro and the way that it might achieve this effect. Ang II significantly stimulated DNA synthesis in MOLT-3 T cells, which was inhibited by PEDF, olmesartan, an Ang II type I receptor blocker, an anti-oxidant N-acetylcysteine (NAC), or antibodies directed against IL-2. PEDF or NAC suppressed gene expression of interleukin-2 (IL-2) in Ang II-exposed MOLT-3 T cells. Furthermore, PEDF blocked the Ang II-induced reactive oxygen species (ROS) generation and NADPH oxidase activity in MOLT-3 T cells. These results demonstrate that PEDF inhibits the Ang II-induced T cell proliferation by blocking autocrine production of IL-2 via suppression of NADPH oxidase-mediated ROS generation. Blockade by PEDF of T cell activation may become a novel therapeutic target for atherosclerosis.     

NADPH oxidase-dependent formation of reactive oxygen species contributes to angiotensin II-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells

The objective of the present study was to investigate the role of NADPH oxidase-dependent formation of reactive oxygen species (ROS) in the angiotensin II (Ang II)-induced epithelial-mesenchymal transition (EMT) and in the accumulation of extracellular matrix (ECM) in rat peritoneal mesothelial cells (RPMCs). Primary cultured RPMCs were incubated with serum-free media for 24 h in order to arrest and synchronize cell growth. The cells were treated with Ang II (10?? M) up to 48 h. Cells were pretreated with an Ang II type I receptor antagonist (losartan, 10?? M), or an inhibitor of NADPH, oxidase diphenyleneiodonium (DPI) (10?? M), for 1 h before addition of Ang II. The dichlorofluorescein (DCF)-sensitive cellular ROS were measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression for the NADPH oxidase subunit p47phox, plasminogen activator inhibitor-1 (PAI-1), α-smooth muscle actin (α-SMA) and E-cadherin. PAI-1, α-SMA and p47phox protein expression were examined by Western blot analysis. Ang II significantly induced the production of intracellular ROS. DPI and losartan inhibited Ang II-induced ROS generation by 86.8% and 77.4% (p<0.05), respectively. Ang II significantly stimulated NADPH oxidase subunit p47phox mRNA and protein expression in RPMCs. Both losartan and DPI inhibited Ang II-induced up-regulation of p47phox mRNA by 37.3% and 67.8% (p<0.05), respectively. Ang II also stimulated α-SMA mRNA and protein expression and down-regulated E-cadherin mRNA expression in RPMCs. This effect was suppressed by both losartan and DPI. Ang II significantly up-regulated PAI-1 mRNA and protein expression and these were significantly suppressed by both losartan and DPI. In conclusion, NADPH oxidase-dependent formation of ROS mediates Ang II dependent EMT and accumulation of ECM in rat peritoneal mesothelial cells. NADPH oxidase may represent a potential therapeutic target in the management of peritoneal fibrosis.     

Hypertrophy of the ligament flavum in degenerative lumbar stenosis associated with the increased expression of fractalkine (CX3CL1)/CX3CR1 chemokine

Abstract

Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in chronic inflammatory disease, but its role in spinal diseases is unknown. The purpose of this study is to investigate the role of CX3CL1/CX3CR1 chemokine on hypertrophy of the ligamentum flavum (LF) in degenerative lumbar stenosis (DLS) compared with that of non-degenerative spinal condition (NDS) of the lumbar spine and correlation between expression of CX3CL1/CX3CR1 chemokine and thickness of LF. The mRNA concentrations of CX3CL1/CX3CR1 chemokine were analyzed in the surgically obtained LF specimens from DLS (n?=?10) and NDS (n?=?11) by real-time PCR. The localization of CX3CL1/CX3CR1 chemokine within the LF was determined using immunohistochemical study. Plasma levels of soluble FKN (sFKN) were measured by enzyme-linked immunosorbent assay, respectively. The thickness of the LF was measured with axial T1-weighted MRI. The cells that express CX3CL1/CX3CR1 chemokine ratio in the LF observed in DLS group were substantially higher than in NDS group. In ELISA, the plasma levels of sFKN was significantly increased in DLS compared with patients in the other groups (p?=?0.006). There was greater CX3CL1/CX3CR1 expression in DLS as quantified by RT-PCR (p?=?0.004, 0.010). Thickness of LF in patients was significantly correlated with serum CX3CL1 level (R^2?=?0.824, p?=?0.003) and with mRNA expression of CX3CL1/CX3CR1 (R^2?=?0.671, p?=?0.000) (R^2?=?0.514, p?=?0.001). This study identified for the first time that increases in CX3CL1 and CX3CR1-expressing cells are significantly related to LF hypertrophy.     

CX3CL1 up-regulation is associated with recruitment of CX3CR1+ mononuclear phagocytes and T lymphocytes in the lungs during cigarette smoke-induced emphysema

CX3CR1 is expressed on monocytes, dendritic cells, macrophages, subsets of T lymphocytes, and natural killer cells and functions in diverse capacities such as leukocyte adhesion, migration, and cell survival on ligand binding. Expression of the CX3CL1 gene, whose expression product is the sole ligand for CX3CR1, is up-regulated in human lungs with chronic cigarette smoke-induced obstructive lung disease. At present, it is unknown whether CX3CL1 up-regulation is associated with the recruitment and accumulation of immune cells that express CX3CR1. We show that mice chronically exposed to cigarette smoke up-regulate CX3CL1 gene expression, which is associated with an influx of CX3CR1⁺ cells in the lungs. The increase in CX3CR1⁺ cells is primarily comprised of macrophages and T lymphocytes and is associated with the development of emphysema. In alveolar macrophages, cigarette smoke exposure increased the expression of both CX3CR1 and CX3CL1 genes. The inducibility of CX3CR1 expression was not solely dependent on a chronic stimulus because lipopolysaccharide up-regulated CX3CR1 in RAW264.7 cells in vitro and in mononuclear phagocytes in vivo. Our findings suggest a mechanism by which macrophages amplify and promote CX3CR1⁺ cell accumulation within the lungs during both acute and chronic inflammatory stress. We suggest that one function of the CX3CR1-CX3CL1 pathway is to recruit and sustain divergent immune cell populations implicated in the pathogenesis of cigarette smoke-induced emphysema.     

The Chemokine CX3CL1 (Fractalkine) and its Receptor CX3CR1: Occurrence and Potential Role in Osteoarthritis

Chemokines are molecules able to induce chemotaxis of monocytes, neutrophils, eosinophils, lymphocytes and fibroblasts. The complex chemokine acts in many physiological and pathological phenomena, including those occurring in the articular cartilage. To date, chemokine CX3CL1 (fractalkine) is the only member of the CX3C class of chemokines with well-documented roles in endothelial cells. CX3CL1 is a unique chemokine that combines properties of chemoattractant and adhesion molecule. The main roles of CX3CL1 include promotion of leukocyte binding and adhesion as well as activation of the target cells. The soluble chemokine domain of CX3CL1 is chemotactic for T cells and monocytes. CX3CL1 acts via its receptor, CX3CR1, which belongs to a family of G protein-coupled receptors. Stimulation of CX3CR1 activates both CX3CL1-dependent and integrin-dependent migrations of cells with synergistically augmented adhesion. Genetic polymorphisms of CX3CR1 may significantly modify the biological roles of CX3CL1, especially in pathologic conditions. Osteoarthritis (OA) is the most common joint disease, affecting approximately 7–8 % of the general population. Development of OA is largely driven by low-grade local background inflammation involving chemokines. The importance of CX3CL1/CX3CR1 signalling in the pathophysiology of OA is still under investigation. This paper, based on a review of the literature, updates and summarises the current knowledge about CX3CL1/CX3CR1 in OA and indicates possible interactions with a potential for therapeutic targeting.     

CX3CL1 expression in the conjunctiva is involved in immune cell trafficking during toxic ocular surface inflammation

Inappropriate expression of the chemokine CX3CL1 is reportedly known to act on inflammatory conditions in extraocular immune diseases. We studied the expression and effects of CX3CL1 in human patients, cultured human conjunctival cells, and transgenic mice exposed to benzalkonium chloride (BAC), a commonly used preservative in ophthalmic medications despite its proinflammatory properties, to determine whether CX3CL1 is involved in conjunctival inflammation. We report that CX3CL1 expression is increased in the conjunctiva of patients receiving BAC-containing medication, and correlates with clinical inflammation. BAC enhances the production of CX3CL1 in a conjunctival epithelial cell line, through the tumor-necrosis factor-α pathway, which attracts specific leukocyte subsets. In vivo, BAC-induced macrophage infiltration and subsequent inflammation of the conjunctiva is decreased in CX3CR1-deficient mice as compared with CX3CR1^+/+ controls. This translational study opens new avenue to investigate ocular surface disorders by focusing on chemokine-related inflammation and immune cell trafficking in the ocular conjunctival mucosa.     

CX3CR1 defines functionally distinct intestinal mononuclear phagocyte subsets which maintain their respective functions during homeostatic and inflammatory conditions

Intestinal mononuclear phagocytes (iMNP) are critically involved in mucosal immunity and tissue homeostasis. Two major non-overlapping populations of iMNP have been identified in mice. CD103(+) iMNP represent a migratory population capable of inducing tolerogenic responses, whereas CX3CR1(+) iMNP are resident cells with disease-promoting potential. CX3CR1(+) iMNP can further be subdivided based on differential expression of CX3CR1. Using CX3CR1(GFP/+) ×RAG2(-/-) mice, we demonstrate that CX3CR1(hi) and CX3CR1(lo) iMNP clearly differ with respect to their morphological and functional properties. Compared with CX3CR1(hi) iMNP, CX3CR1(lo) iMNP are polarised towards pro-inflammatory responses already under homeostatic conditions. During a CD4(+) T-cell-induced colitis, CX3CR1(lo) cells accumulate in the inflamed mucosa and upregulate the expression of pro-inflammatory cytokines and triggering receptor expressed on myeloid cells-1 (TREM-1). In contrast, CX3CR1(hi) iMNP retain their non-inflammatory profile even during intestinal inflammation. These findings identify two functionally distinct iMNP subsets based on differential expression of CX3CR1 and indicate an unanticipated stability of iMNP.     

Fractalkine/CX3CL1 enhances GABA synaptic activity at serotonin neurons in the rat dorsal raphe nucleus

Serotonin (5-hydroxytryptamine; 5-HT) has an important role in mood regulation, and its dysfunction in the central nervous system (CNS) is associated with depression. Reports of mood and immune disorder co-morbidities indicate that immune-5-HT interactions may mediate depression present in immune compromised disease states including HIV/AIDS, multiple sclerosis, and Parkinson's disease. Chemokines, immune proteins that induce chemotaxis and cellular adhesion, and their G-protein coupled receptors distribute throughout the CNS, regulate neuronal patterning, and mediate neuropathology. The purpose of this study is to investigate the neuroanatomical and neurophysiological relationship between the chemokine fractalkine/CX3CL1 and its receptor CX3CR1 with 5-HT neurons in the rat midbrain raphe nuclei (RN). Immunohistochemistry was used to examine the colocalization of CX3CL1 or CX3CR1 with 5-HT in the RN, and whole-cell patch-clamp recordings in rat brain slices were used to determine the functional impact of CX3CL1 on 5-HT dorsal raphe nucleus (DRN) neurons. Greater than 70% of 5-HT neurons colocalize with CX3CL1 and CX3CR1 in the RN. CX3CL1 localizes as discrete puncta throughout the cytoplasm, whereas CX3CR1 concentrates to the perinuclear region of 5-HT neurons and exhibits microglial expression. CX3CL1 and CX3CR1 also colocalize with one another on individual RN cells. Electrophysiology studies indicate a CX3CL1-mediated enhancement of spontaneous inhibitory postsynaptic current (sIPSC) amplitude and dose-dependent increase of evoked IPSC (eIPSC) amplitude without affecting eIPSC paired–pulse ratio, a finding observed selectively in 5-HT neurons. CX3CL1's effect on eIPSC amplitude is blocked by pretreatment with an anti-CX3CL1 neutralizing antibody. Thus, CX3CL1 enhances postsynaptic GABA receptor number or sensitivity on 5-HT DRN neurons under conditions of both spontaneous and synaptically-evoked GABA release. CX3CL1 may indirectly inhibit 5-HT neurotransmission by increasing the sensitivity of 5-HT DRN neurons to GABA inputs. Therapies targeting CX3CL1 may treat serotonin related mood disorders, including depression experienced by patients with compromised immune systems.     

胰岛素通过活性氧的产生促进VEGF表达及血管平滑肌细胞迁移和增殖

 目的:探讨活性氧（reactive oxygen species, ROS）在胰岛素促进的血管平滑肌细胞迁移和增殖中的作用及分子机制。方法:采用原代培养的大鼠主动脉血管平滑肌细胞,应用DCF-DA荧光探针检测细胞内ROS的生成;应用实时定量PCR、Western blotting和ELISA法检测mRNA和蛋白的表达;应用转染报告基因的方法检测基因的转录活性;划痕法测定细胞迁移;CCK-8法测定细胞增殖。结果:胰岛素处理后血管平滑肌细胞内ROS产生明显增加。过氧化氢酶和NADPH氧化酶抑制剂二亚苯基碘鎓（DPI）明显抑制胰岛素促进的ROS生成及p-Akt、p-p70S6K1和p-ERK1/2蛋白的表达。过氧化氢酶和DPI明显降低胰岛素促进的血管内皮生长因子（vascular endothelial growth factor, VEGF）的mRNA和蛋白表达及转录激活。抑制ROS产生明显抑制胰岛素刺激的血管平滑肌细胞迁移和增殖。结论: 胰岛素通过NADPH氧化酶途径促进血管平滑肌细胞ROS产生。ROS介导了胰岛素促进的Akt/p70S6K1和ERK信号通路的激活、VEGF表达及血管平滑肌细胞的迁移和增殖。     

PP60c-Src在血管紧张素II诱导的大鼠血管平滑肌细胞信息转导中的作用(英)

目的：本研究通过观察c-Src在AngII对大鼠血管平滑肌细胞(VSMC)丝裂原激活的蛋白激酶(MAPK)的激活和c-Fos蛋白表达中的影响，以进一步阐明AngII促VSMC增殖的细胞内信息转导机制。方法： 原代和传代培养SD大鼠主动脉VSMC，以脂质体包裹反义c-Src寡脱氧核苷酸(oligodeoxynucleotides, ODNs)转染培养的VSMC以抑制c-Src蛋白表达和激酶活性。以未转染的VSMC为对照，观察10-7mol/L AngII刺激对转染的VSMC的MAPK活性和c-Fos蛋白表达的影响。蛋白免疫沉淀和酶自身磷酸化率法测定c-Src激酶活性；髓鞘碱性蛋白(MBP)底物磷酸化率测定MAPK激酶活性；Western blotting免疫印迹法测定c-Src和c-Fos蛋白表达情况。结果： 转染不同浓度反义c-Src ODNs的VSMC，c-Src蛋白含量呈浓度依赖性降低，c-Src激酶活性也显著 抑制，以Ang II刺激经转染反义c-Src ODNs的VSMC，c-Src激酶活性增幅仅为对照组的8.7%；MAPK活性仅为对照的1.6%；c-Fos蛋白表达的增幅为对照组的30.0%。结论： AngII可诱导VSMC c-Src激活和细胞内信息转导，且AngII引起的MAPK和c-fos的激活依赖于c-Src的激活，提示c-Src是AngII促血管平滑肌细胞增殖的重要信息分子。     

CX3CR1 RNAi inhibits hypoxia‐induced microglia activation via p38MAPK/PKC pathway

There is accumulating evidence which demonstrates that chronic cerebral ischaemia can induce white matter lesions (WMLs), and microglia‐activation‐mediated cytokines and proteases releasing during the ischaemia might play a vital role in pathogenesis. In addition, hypoxia‐induced upregulated expression of fractalkine promotes the activation of microglia and their migration to the lesions through interaction with its receptor CX3CR1. However, the specific mechanisms involved in fractalkine/CX3CR1‐mediated microglial activation have not been fully identified. In the present study, we constructed lentivirus encoding shRNA against CX3CR1 and transduced into microglial cells in under hypoxic conditions. Moreover, we analysed the proliferation, cytokine secretion and signal‐pathway activation of the microglia. We found that CX3CR1 RNAi‐mediated gene downregulation could attenuate hypoxic‐induced microglial proliferation, cytokine secretion [including tumuor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β)] and matrix metalloproteinase‐2 (MMP‐2) synthesis. These effects were shown to be nediated through p38MAPK/PKC activation. Therefore, our results reveal a novel mechanism of fractalkine/CX3CR1 involvement in activation of microglia. Thus CX3CR1 RNAi might provide a therapeutic strategy which could be useful in chronic cerebral ischaemia.     

Recruitment of CD16+ monocytes into synovial tissues is mediated by fractalkine and CX3CR1 in rheumatoid arthritis patients

CD16+ monocytes, identified as a minor population of monocytes in human peripheral blood, have been implicated in several inflammatory diseases, including rheumatoid arthritis (RA). Fractalkine (FKN, CX3CL1), a member of the CX3 C subfamily, is induced by pro-inflammatory cytokines, while a receptor for FKN, CX3CR1, is capable of mediating both leukocyte migration and firm adhesion. Here, we investigated the role of FKN and CX3CR1 in activation of CD16+ monocytes and their recruitment into synovial tissues in RA patients. High levels of soluble FKN were detected in the synovial fluid and sera of RA patients. Circulating CD16+ monocytes showed a higher level of CX3CR1 expression than CD16- monocytes in both RA patients and healthy subjects. High level expression of CX3CR1 was also seen in CD16+ monocytes localized to the lining layer in RA synovial tissue. In the in vitro culture experiments, IL-10 induced CX3CR1 expression on the surface of monocytes, and TNFalpha induced membrane-bound FKN as well as soluble FKN expression in synovial fibroblasts. Moreover, soluble FKN was capable of inducing IL-1beta and IL-6 by activated monocytes. These results suggest that FKN might preferentially mediate migration and recruitment of CD16+ monocytes, and might contribute to synovial tissue inflammation.     

CX3CL1/fractalkine is a novel regulator of normal and malignant human B cell function

CX(3)CL1, or fractalkine, the unique member of the CX(3)C chemokine family, exists as a transmembrane glycoprotein, as well as in soluble form, each mediating different biological activities, and is constitutively expressed in many hematopoietic and nonhematopoietic tissues. CX(3)CR1, the CX(3)CL1 exclusive receptor, is a classical GPCR, expressed on NK cells, CD14(+) monocytes, and some subpopulation of T cells, B cells, and mast cells. A recent paper by our group has demonstrated for the first time that highly purified human B cells from tonsil and peripheral blood expressed CX(3)CR1 at mRNA and protein levels. In particular, tonsil na?ve, GC, and memory B cells expressed CX(3)CR1, but only GC centrocytes were attracted by soluble CX(3)CL1, which with its receptor, are also involved in the pathogenesis of several inflammatory disorders, as well as of cancer. Previous studies have shown that CX(3)CR1 is up-regulated in different types of B cell lymphoma, as well as in B-CLL. Recently, we have demonstrated that the CX(3)CL1/CX(3)CR1 axis is involved in the interaction of B-CLL cells with their microenvironment. Taken together, our data delineate a novel role for the CX(3)CL1/CX(3)CR1 complex in the biology of normal B cells and B-CLL cells. These topics are the subject of this review article.     

Angiotensin II-Derived Reactive Oxygen Species Promote Angiogenesis in Human Late Endothelial Progenitor Cells Through Heme Oxygenase-1 via ERK1/2 and AKT/PI3K Pathways

Angiotensin II (Ang II), the main component of renin-angiotensin system, could mediate pathogenic angiogenesis in cardiovascular disorders. Late endothelial progenitor cells (EPCs) possess potent self-renewal and angiogenic potency superior to early EPCs, but few study focused on the cross-talk between Ang II and late EPCs. We observed that Ang II could increase reactive oxygen species (ROS) and promote capillary formation in late EPCs. Ang II-derived ROS could also upregulate heme oxygenase-1 (HO-1) expression, and treating late EPCs with HO-1 small interfering RNA or heme oxygenase inhibitor (HO inhibitor) could inhibit Ang II-induced tube formation and increase ROS level and apoptosis rate. In addition, PD98059 and LY294002 pretreatment attenuated Ang II-induced HO-1 expression. Accordingly, Ang II-derived ROS could promote angiogenesis in late EPCs by inducing HO-1 expression via ERK1/2 and AKT/PI3K pathways, and we believe HO-1 might be a promising intervention target in EPCs due to its potent proangiogenic, antioxidant, and antiapoptosis potentials.     

Endothelin-1 Activation of the Endothelin B Receptor Modulates Pulmonary Endothelial CX3CL1 and Contributes to Pulmonary Angiogenesis in Experimental Hepatopulmonary Syndrome

Hepatic production and release of endothelin-1 (ET-1) binding to endothelin B (ET_B) receptors, overexpressed in the lung microvasculature, is associated with accumulation of pro-angiogenic monocytes and vascular remodeling in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). We have recently found that lung vascular monocyte adhesion and angiogenesis in HPS involve interaction of endothelial C-X3-C motif ligand 1 (CX3CL1) with monocyte CX3C chemokine receptor 1 (CX3CR1), although whether ET-1/ET_B receptor activation influences these events is unknown. Our aim was to define if ET-1/ET_B receptor activation modulates CX3CL1/CX3CR1 signaling and lung angiogenesis in experimental HPS. A selective ET_B receptor antagonist, BQ788, was given for 2 weeks to 1-week CBDL rats. ET-1 (±BQ788) was given to cultured rat pulmonary microvascular endothelial cells overexpressing ET_B receptors. BQ788 treatment significantly decreased lung angiogenesis, monocyte accumulation, and CX3CL1 levels after CBDL. ET-1 treatment significantly induced CX3CL1 production in lung microvascular endothelial cells, which was blocked by inhibitors of Ca²⁺ and mitogen-activated protein kinase (MEK)/ERK pathways. ET-1–induced ERK activation was Ca²⁺ independent. ET-1 administration also increased endothelial tube formation in vitro, which was inhibited by BQ788 or by blocking Ca²⁺ and MEK/ERK activation. CX3CR1 neutralizing antibody partially inhibited ET-1 effects on tube formation. These findings identify a novel mechanistic interaction between the ET-1/ET_B receptor axis and CX3CL1/CX3CR1 in mediating pulmonary angiogenesis and vascular monocyte accumulation in experimental HPS.Cirrhosis and portal hypertension result in vascular alterations in several organ systems, which impair the function of these organs.^1,2 The hepatopulmonary syndrome (HPS) is one such complication, in which intrapulmonary vascular abnormalities cause abnormal arterial gas exchange in 5% to 30% of cirrhotic patients.^3–5 The presence of HPS significantly decreases quality of life and increases mortality in those affected.⁶ Currently, the pathogenesis of vascular alterations in HPS is incompletely understood, limiting the development of effective medical therapies.Chronic common bile duct ligation (CBDL) in the rat is an established experimental model of HPS that reproduces the physiological abnormalities of human disease.^7–16 These animals develop pulmonary microvascular dilations and also develop vascular remodeling, which is linked to the accumulation of intravascular monocytes and vascular endothelial growth factor-A (VEGF-A) production.^11,12,17,18 Pulmonary vasodilation results, in large part, from Ca²⁺-dependent and protein kinase B/Akt–independent activation of pulmonary vascular endothelial nitric oxide synthase (eNOS)/NO, by hepatic production and release of endothelin-1 (ET-1) and stimulation of lung vascular endothelin B (ET_B) receptors.^9,19 ET-1/ET_B receptor activation also enhances pulmonary vascular monocyte accumulation,¹⁴ thereby promoting angiogenesis through monocyte VEGF-A production and activation of Akt and ERK. However, how ET-1/ET_B receptor activation modulates monocyte accumulation and whether ET_B receptor activation has direct effects on angiogenesis are unknown.We have recently found that the chemokine ligand/receptor pair C-X3-C motif ligand 1 (CX3CL1)/CX3C chemokine receptor 1 (CX3CR1) is up-regulated in the pulmonary microvasculature after CBDL and contributes to monocyte accumulation and to angiogenesis after CBDL.²⁰ Interaction between the membrane-bound form of CX3CL1 with CX3CR1 is unique in the ability to cause firm adhesion between cell types (monocyte-endothelium).^21–24 In addition, the secreted soluble form of CX3CL1, through interaction with the CX3CR1 on endothelial cells, can drive angiogenesis through Akt and ERK activation.²⁵ However, whether ET-1/ET_B receptor activation after CBDL influences monocyte accumulation or angiogenesis by affecting lung CX3CL1/CX3CR1 expression has not been tested. Therefore, the aims of the present study were to define if the ET-1/ET_B receptor activation modulates the pulmonary CX3CL1/CX3CR1 axis and to determine how it contributes to vascular remodeling in experimental HPS. To do this, we assessed the effects of selective ET receptor inhibition on pulmonary angiogenesis and CX3CL1/CX3CR1 expression in vivo and evaluated the effects of exogenous ET-1 on pulmonary microvascular endothelial cell CX3CL1 expression and angiogenesis in vitro.     

CX3CR1 polymorphisms are associated with atopy but not asthma in German children

Chemokines and their receptors are involved in many aspects of immunity. Chemokine CX3CL1, acting via its receptor CX3CR1, regulates monocyte migration and macrophage differentiation as well as T cell-dependent inflammation. Two common, nonsynonymous polymorphisms in CX3CR1 have previously been shown to alter the function of the CX3CL1/CX3CR1 pathway and were suggested to modify the risk for asthma. Using matrix-assisted laser desorption/ionization time-of-flight technology, we genotyped polymorphisms Val249Ile and Thr280Met in a cross-sectional population of German children from Munich (n = 1,159) and Dresden (n = 1,940). For 249Ile an odds ratio of 0.77 (95% confidence interval 0.63-0.96; p = 0.017) and for 280Met an odds ratio of 0.71 (95% confidence interval 0.56-0.89; p = 0.004) were found with atopy in Dresden but not in Munich. Neither polymorphism was associated with asthma. Thus, amino acid changes in CX3CR1 may influence the development of atopy but not asthma in German children. Potentially, other factors such as environmental effects may modify the role of CX3CR1 polymorphisms.     

Cytokine Regulation in Immune Diseases

Objective

Inflammation and hypoxia activate the fractalkine (CX3CL1) receptor (CX3CR1)-related signaling pathway. Tumor necrosis factor alpha (TNFα) induces CX3CL1, influencing a mechanism of CX3CL1 autoregulation by CX3CR1 expression. We compared spontaneous and lipopolysaccharide (LPS)-induced CX3CL1 and TNFα production by human placenta under normoxic vs. hypoxic conditions, with respect to CX3CR1 expression and its functional status.

Methods

Placental lobules of term placentae (N = 24) were perfused extracorporeally. CX3CL1 and TNFα concentrations were measured in the perfusion fluid by ELISA. LPS, anti-CX3CR1 antibodies and pirfenidone were used in respective subgroups. After perfusion, CX3CR1 expression was estimated in placental tissue using quantitative immunohistochemistry, and the final results were adjusted for the mean microvascular density.

Results

The highest increase in CX3CL1 concentration in response to LPS was observed in hypoxia (p < 0.05). Unlike in normoxia, anti-CX3CR1 administration in hypoxia significantly reduced the LPS-evoked response. CX3CR1 expression was augmented by hypoxia and reached 260.9 ± 41 (% ±SEM) of the reference value in normoxia. Positive immunostaining for CX3CR1 corresponded to the vascular endothelium. Pirfenidone inhibited hypoxia + LPS-related increase in TNFα production and prevented the up-regulation of CX3CR1.

Conclusion

The modulatory influence of TNFα on CX3CR1 expression in hypoxia and CX3CL1/CX3CR1 interaction may serve as a compensatory mechanism to preserve or augment the pro-inflammatory course of intercellular interactions in placental endothelium.