CXCR6 is expressed on T cells in both T helper type 1 (Th1) inflammation and allergen-induced Th2 lung inflammation but is only a weak mediator of chemotaxis

Numerous chemokine receptors are increased in number on T cells in inflamed tissues. Our objective was to examine CXCR6 expression on lymphocytes during immune and inflammatory reactions and its potential for mediating T-cell recruitment. The cDNA for rat CXCR6 was cloned and monoclonal antibodies (mAbs) to CXCR6 were developed. CXCR6 was present on 4-6% of CD4 and CD8 T cells in blood, normal lymph nodes (LNs) and the spleen, primarily on memory T cells. In vitro antigen re-stimulation of LN T cells from animals with autoimmune arthritis and experimental autoimmune encephalomyelitis (EAE) increased the proportion of CXCR6(+) T cells to 35-50% and anti-T-cell receptor (TCR) activation to 60-80%. In vivo, after antigen challenge of LNs there was only a small increase in CXCR6(+) T cells on the lymphoblasts in the LNs, and a much higher percentage of T cells were CXCR6(+) in virus-induced peritoneal exudates (approximately 47%) and in allergen-induced lung inflammation (33%). Chemotaxis of CXCR6-expressing inflammatory T cells to CXCL16 was poor, but that to CXCL10 was robust. We conclude that few T cells in normal and antigen-challenged LNs are CXCR6(+), whereas a high proportion of in vitro activated T cells and T cells from inflammatory sites are CXCR6(+), but these cells migrate poorly to CXCL16. This suggests that CXCR6 may contribute to T-cell positioning and activation, rather than recruitment. CXCR6 is also expressed on T cells not only in T helper type 1 (Th1) inflammation (arthritis and EAE) but also, as shown here, in Th2 inflammation, where it is increased after allergen challenge.     

Critical involvement of atypical chemokine receptor CXCR7 in allergic airway inflammation

Trafficking and recruitment of immune cells to the site of inflammation with spatial and temporal synchronization is crucial for the development of allergic airway inflammation. Particularly, chemokines are known to be key players in these processes. Previous studies revealed that the CXCL12/CXCR4 axis plays an important role in regulating allergic airway inflammation. However, the role of CXCR7, a recently discovered second receptor for CXCL12, in regulating airway inflammation has not been explored. Initially, CXCR7 was considered as a decoy receptor; however, numerous subsequent studies revealed that engagement of CXCR7 triggered its own signalling or modulated CXCR4‐mediated signalling. In the present study, we detected the expression of CXCR7 in airway epithelial cells. Use of a lentiviral delivery system to knock down the expression of CXCR7 in the lung of sensitized mice abrogated the cardinal features of asthma, indicating that CXCR7 plays a role in regulating allergic airway inflammation. The activation of mitogen‐activated protein kinase and Akt signalling in response to CXCL12 in the mouse epithelial cell line MLE‐12 was reduced when CXCR7 expression was knocked down. However, either knockdown or overexpression of CXCR7 in MLE‐12 did not affect CXCL12‐mediated calcium influx, indicating that CXCR7 does not modulate CXCR4‐mediated signalling, and that it functions as a signalling receptor rather than a decoy receptor. Finally, we found that the expression of chemokine CCL2 is regulated by CXCR7/CXCL12‐mediated signalling through β‐arrestin in airway epithelial cells. Hence, regulating the expression of CCL2 in airway epithelial cells may be one mechanism by which CXCR7 participates in regulating allergic airway inflammation.     

CXCR1/CXCR2拮抗剂G31P抗中性粒细胞介导的炎症作用研究

目的 建立肺炎动物模型,使用人CXCR1/CXCR2受体拈抗剂G31P,治疗与中性粒细胞相关的炎性疾病.方法 检测G31P能否阻断人IL-8对中性粒细胞趋化以及阻断支气管上皮细胞A549释放IL-8;建立pcDNA3.0-CXCR1、2、4转染的肾细胞HEK293,检测G31P阻断IL-8对细胞株的趋化作用;建立肺炎动物模型,计数肺泡灌洗液(BALF)中性粒细胞数,进行肺组织病理学观察.结果 实验证实G31P可以阻断中性粒细胞趋化和IL-8介导的CXCR1、CXCR2转染的HEK293细胞株的趋化作用,抑制A549释放炎性介质;G31P治疗组中性粒细胞比例下降,病理检测有明显差异.结论 G31P可以阻断ELR+CXC趋化因子对中性粒细胞的趋化作用,阻断ELR+CXC趋化因子与中性粒细胞表面受体CXCR2的结合,阻断肺泡上皮细胞和血管内皮细胞表面的CXCR2,从而进一步阻止中性粒细胞介导的炎症反应.     

CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice

Polymorphonuclear neutrophil (PMN) extravasation/sequestration in the lung and a dysregulated inflammatory response characterize the pathogenesis of acute lung injury (ALI). Previously, we have shown that hemorrhage (Hem) serves to prime PMN such that subsequent septic challenge [cecal ligation and puncture (CLP)] produces a pathological, inflammatory response and consequent lung injury in mice. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) are murine CXC chemokines found elevated in the lungs and plasma following Hem/CLP and have been reported by others to share a common receptor (CXCR2). Based on these data, we hypothesize that blockade of CXCR2 immediately following Hem would suppress KC and MIP-2 priming of PMN, thereby reducing the inflammatory injury observed following CLP. To assess this, Hem mice (90 min at 35+/-5 mmHg) were randomized to receive 0, 0.4, or 1 mg antileukinate (a hexapeptide inhibitor of CXCRs) in 100 microl phosphate-bufferd saline (PBS)/mouse subcutaneously, immediately following resuscitation (Ringer's lactate-4x drawn blood volume). Twenty-four hours post-Hem, mice were subjected to CLP and killed 24 h later. The results show that blockade of CXCR2 significantly (P<0.05, Tukey's test) reduced PMN influx, lung protein leak, and lung-tissue content of interleukin (IL)-6, KC, and MIP-2 and increased tissue IL-10 levels. Plasma IL-6 was significantly decreased, and IL-10 levels increased in a dose-dependent manner compared with PBS-treated mice. A differential effect was observed in plasma levels of KC and MIP-2. KC showed a significant reduction at the 0.4 mg antileukinate dose. In contrast, plasma MIP-2 was significantly elevated at both doses compared with the PBS-treated controls. Together, these data demonstrate that blockade of CXCR2 signaling attenuates shock-induced priming and ALI observed following Hem and subsequent septic challenge in mice.     

CXCR2在妊娠早期小鼠子宫内膜的表达

目的研究CXCR2在妊娠早期小鼠子宫内膜的表达情况.方法应用免疫组织化学和图像分析技术,观察CXCR2在妊娠1d、4d、5d、6d小鼠子宫内膜的表达部位及蛋白水平的变化规律;HE染色后光镜下观察子宫内膜的形态学变化.结果免疫组化染色结果显示:在子宫内膜腔上皮,CXCR2于妊娠4d表达最强,妊娠5d开始逐渐下降,妊娠6d降至妊娠1d水平;在子宫内膜腺上皮和基质,CXCR2的表达水平随妊娠天数的增加而逐渐升高.HE染色可见妊娠1d子宫内膜腔上皮和基质中有大量中性粒细胞浸润;妊娠4d腔上皮中未见中性粒细胞,基质中则有大量中性粒细胞浸润;妊娠5d和妊娠6d腔上皮中未见中性粒细胞,基质中中性粒细胞数量甚少.结论CXCR2可能通过与其配体白细胞介素-8结合而参与小鼠胚泡着床过程.     

慢性乙肝患者外周血单个核细胞中CXCR1、CXCR2及IL-8 mRNA表达与干扰素治疗关系

目的:了解乙型肝炎病毒(HBV)慢性感染患者外周血单个核细胞(PBMC)中CXCR1、CXCR2及IL-8 mRNA表达水平及与α干扰素(IFN-α)治疗的关系。方法:采用实时定量PCR法动态观察30例慢性乙型肝炎患者接受IFN-α治疗前、治疗3个月、6个月后其外周血单个核细胞CXCR1、CXCR2及IL-8 mRNA表达水平。结果:治疗前慢性乙肝患者CXCR1、CXCR2及IL-8 mRNA表达水平分别为(0.44740.0386)、(0.4720 0.0458)、(1.1897 0.1028),均高于正常对照组(n=36),其中CXCR1及IL-8 mRNA水平升高显著,差异有统计学意义(P<0.01)。治疗过程中CXCR1、CXCR2及IL-8表达水平均显著下降。IFN-α治疗6个月后CXCR1、CXCR2及IL-8 mRNA表达水平分别为(0.41290.0395)、(0.4461 0.0477)、(0.8660 0.1307),与治疗前相比,差异有显著性(P<0.01或P<0.05)。治疗前的CX-CR1、CXCR2及IL-8的表达水平在HBV高复制组(HBV-DNA>106,n=22)明显高于HBV低复制组(HBV-DNA<106,n=8),差异有统计学意义(P<0.05)。结论:慢性乙肝患者外周血单个核细胞中CXCR1和IL-8表达水平显著升高,在干扰素治疗后,其表达水平下调,证明其可能与慢性乙肝炎症的发生机制相关。     

Systemic inflammation in acute intermittent porphyria: a case–control study

This study aimed to examine whether acute intermittent porphyria (AIP) is associated with systemic inflammation and whether the inflammation correlates with disease activity. A case–control study with 50 AIP cases and age‐, sex‐ and place of residence‐matched controls was performed. Plasma cytokines, insulin and C‐peptide were analysed after an overnight fast using multiplex assay. Long pentraxin‐3 (PTX3) and complement activation products (C3bc and TCC) were analysed using enzyme‐linked immunosorbent assay (ELISA). Urine porphobilinogen ratio (U‐PBG, µmol/mmol creatinine), haematological and biochemical tests were performed using routine methods. Questionnaires were used to register AIP symptoms, medication and other diseases. All 27 cytokines, chemokines and growth factors investigated were increased significantly in symptomatic AIP cases compared with controls (P < 0·0004). Hierarchical cluster analyses revealed a cluster with high visfatin levels and several highly expressed cytokines including interleukin (IL)‐17, suggesting a T helper type 17 (Th17) inflammatory response in a group of AIP cases. C3bc (P = 0·002) and serum immunoglobulin (Ig)G levels (P = 0·03) were increased significantly in cases with AIP. The U‐PBG ratio correlated positively with PTX3 (r = 0·38, P = 0·006), and with terminal complement complex (TCC) levels (r = 0·33, P = 0·02). PTX3 was a significant predictor of the biochemical disease activity marker U‐PBG in AIP cases after adjustment for potential confounders in multiple linear regression analyses (P = 0·032). Prealbumin, C‐peptide, insulin and kidney function were all decreased in the symptomatic AIP cases, but not in the asymptomatic cases. These results indicate that AIP is associated with systemic inflammation. Decreased C‐peptide levels in symptomatic AIP cases indicate that reduced insulin release is associated with enhanced disease activity and reduced kidney function.     

Sequence and expression analysis of rainbow trout CXCR2, CXCR3a and CXCR3b aids interpretation of lineage-specific conversion,loss and expansion of these receptors during vertebrate evolution

The chemokine receptors CXCR1–3 bind to 11 chemokines (CXCL1–11) that are clustered on the same chromosome in mammals but are largely missing in ray-finned fish. A second CXCR1/2, and a CXCR3a and CXCR3b gene have been cloned in rainbow trout. Analysis of CXCR1–R3 genes in lobe-finned fish, ray-finned fish and tetrapod genomes revealed that the teleostomian ancestor likely possessed loci containing both CXCR1 and CXCR2, and CXCR3a and CXCR3b. Based on this synteny analysis the first trout CXCR1/2 gene was renamed CXCR1, and the new gene CXCR2. The CXCR1/R2 locus was shown to have further expanded in ray-finned fish. In relation to CXCR3, mammals appear to have lost CXCR3b and birds both CXCR3a and CXCR3b during evolution. Trout CXCR1–R3 have distinct tissue expression patterns and are differentially modulated by PAMPs, proinflammatory cytokines and infections. They are highly expressed in macrophages and neutrophils, with CXCR1 and CXCR2 also expressed in B-cells.     

Quantitative evaluation of CXCL8 and its receptors (CXCR1 and CXCR2) gene expression in Iranian patients with multiple sclerosis

CXCL8 and its receptors (CXCR1 and CXCR2) play important roles in CNS development, neuronal survival, modulation of excitability, and neuroimmune response. The aim of this study is to evaluate gene expression of CXCL8 and CXCR1/CXCR2 in peripheral blood cells (PBCs) of Iranian patients with relapsing remitting (RR) form of Multiple sclerosis (MS). We explored the mRNA expression of CXCL8 and its receptors in PBCs of 49 RR-MS patients in remitting status and 60 healthy controls by quantitative Real-Time PCR. Median expression of CXCL8 mRNA in peripheral blood of MS patients decreased more than 3-fold compared to control group (p?<?0.001), while there were not significant differences in CXCR1 and CXCR2 gene expression between MS patients and healthy subjects (p?=?0.159 and p?=?0.248, respectively). There was a significant negative correlation of CXCR2 expression with EDSS (rs?=??0.432, p?=?0.004). It appears that decreased expression of CXCL8 may lead to a raised risk of MS.     

A synthetic, non-peptide CXCR2 antagonist blocks MIP-2-induced neutrophil migration in mice

Non-peptide antagonists of chemokine receptors are considered an intriguing alternative for the treatment of acute and chronic diseases. Particularly the recruitment of neutrophils to inflammatory sites often causes harmful side effects and is mediated by chemokine ligands of the CXC chemokine receptor 2 (CXCR2). Hence, this receptor has been proposed as an important target for novel drugs. This study investigates the potential of the non-peptide CXCR2 antagonist SB 455821 to block neutrophil migration in mice. By using bone marrow derived neutrophils we established a migration assay which revealed SB 455821 as a potent inhibitor of macrophage inflammatory protein 2 (MIP-2)-induced neutrophil migration in vitro (IC50-20 nM). In vivo, injection of MIP-2 into the peritoneal cavities of mice markedly increased neutrophil numbers in peritoneal lavages which were reduced to control levels by co-administration of SB 455821 indicating that the compound effectively binds to the receptor under physiological conditions and exhibits biological activity in vivo. Nevertheless, using intraperitoneal injection of zymosan as a complex inflammatory stimulus, SB 455821 was unable to block neutrophil recruitment to the peritoneal cavity of mice possibly due to other chemotactic mediators overruling signals derived from CXCR2 ligands. Our data show that SB 455821 blocks MIP-2-induced neutrophil migration in vitro and after injection in mice suggesting that selective CXCR2 antagonists may be useful drugs in diseases where neutrophil accumulation plays a major role and leads to exacerbation of acute or chronic inflammations.     

CXCR4 inhibitor attenuates allergen-induced lung inflammation by down-regulating MMP-9 and ERK1/2

Chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor chemokine receptor 4 (CXCR4) have been recognized to play a crucial role in the pathogenesis of bronchial asthma, but the underlying molecular mechanisms are yet to be fully addressed. In the present report we demonstrated that CXCL12/CXCR4 signaling mediates allergic airway inflammation through induction of matrix metalloproteinase 9 (MMP-9) in a murine asthmatic model. We noted that administration of AMD3100, a specific CXCR4 antagonist, significantly attenuated OVA-induced asthmatic responses along with reduced epithelial MMP-9 expression. Our studies in a bronchial epithelial cell line, 16HBE cells, further revealed that CXCL12/CXCR4 signaling synergizes with IL-13 to enhance epithelial MMP-9 expression. Our mechanistic studies demonstrated that CXCL12/CXCR4 enhances epithelial MMP-9 expression by inducing ERK1/2 expression and activation. Together, these studies would bring novel insight into the understanding for the role of CXCL12/CXCR4 signaling in asthmatic responses during the course of bronchial asthma development.     

CRTH2 is a prominent effector in contact hypersensitivity-induced neutrophil inflammation

Chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes, CRTH2, is a cognate receptor for prostaglandin (PG) D(2) and, in humans, is suggested to play a functional role in Th2-dependent allergic inflammation. While peripheral blood leukocytes expressing high levels of surface CRTH2 have been detected in disease, little is known of the functional significance of CRTH2 in disease etiology. We have utilized a Th2-dependent murine model of FITC-induced contact hypersensitivity to assess the role, if any, CRTH2-PGD(2) may play in the elicitation or maintenance of such pathobiology. Expression of both PGD(2) and CRTH2 in lesional skin was paralleled by the release of the chemoattractants LTB(4) and the chemokine KC, as well as a profuse dermal neutrophilic and eosinophilic infiltrate, closely paralleling the acute inflammatory pathology observed in human atopic dermatitis. A small molecule CRTH2 antagonist, but not a selective PGD(2)R (DP) receptor antagonist, was able to completely abrogate these responses. Inflammatory cascades mediated by CRTH2 ligation may therefore represent an important early step in the elicitation and maintenance of Th2-dependent skin inflammation.     

Involvement of exosomes in lung inflammation associated with experimental acute pancreatitis

A frequent complication of acute pancreatitis is the lung damage associated with the systemic inflammatory response. Although various pro‐inflammatory mediators generated at both local and systemic levels have been identified, the pathogenic mechanisms of the disease are still poorly understood. In recent years, exosomes have emerged as a new intercellular communication system able to transfer encapsulated proteins and small RNAs and protect them from degradation. Using an experimental model of taurocholate‐induced acute pancreatitis in rats, we aimed to evaluate the role of exosomes in the extent of the systemic inflammatory response. Induction of pancreatitis increased the concentration of circulating exosomes, which showed a different proteomic profile to those obtained from control animals. A series of tracking experiments using PKH26‐stained exosomes revealed that circulating exosomes effectively reached the alveolar compartment and were internalized by macrophages. In vitro experiments revealed that exosomes obtained under inflammatory conditions activate and polarize these alveolar macrophages towards a pro‐inflammatory phenotype. Interestingly, the proteomic analysis of circulating exosomes during acute pancreatitis suggested a multi‐organ origin with a relevant role for the liver as a source of these vesicles. Tracking experiments also revealed that the liver retains the majority of exosomes from the peritoneal cavity. We conclude that exosomes are involved in the lung damage associated with experimental acute pancreatitis and could be relevant mediators in the systemic effects of pancreatitis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression of the CXCR6 on polymorphonuclear neutrophils in pancreatic carcinoma and in acute, localized bacterial infections

The chemokine receptor CXCR6 has been described on lymphoid cells and is thought to participate in the homing of activated T-cells to non-lymphoid tissue. We now provide evidence that the chemokine receptor CXCR6 is also expressed by activated polymorphonuclear neutrophils (PMN) in vivo: Examination of biopsies derived from patients with pancreatic carcinoma by confocal laser scan microscopy revealed a massive infiltration of PMN that expressed CXCR6, while PMN of the peripheral blood of these patients did not. To answer the question whether CXCR6 expression is a property of infiltrated and activated PMN, leucocytes were collected from patients with localized soft tissue infections in the course of the wound debridement. By cytofluorometry, the majority of these cells were identified as PMN. Up to 50% of these PMN were also positive for CXCR6. Again, PMN from the peripheral blood of these patients were nearly negative for CXCR6, as were PMN of healthy donors. In a series of in vitro experiments, up-regulation of CXCR6 on PMN of healthy donors by a variety of cytokines was tested. So far, a minor, although reproducible, effect of tumour necrosis factor (TNFalpha) was seen: brief exposure with low-dose TNFalpha induced expression of CXCR6 on the surface of PMN. Furthermore, we could show an increased migration of PMN induced by the axis CXCL16 and CXCR6. In summary, our data provide evidence that CXCR6 is not constitutively expressed on PMN, but is up-regulated under inflammatory conditions and mediates migration of CXCR6-positive PMN.     

Underlying chronic inflammation alters the profile and mechanisms of acute neutrophil recruitment

Chronically inflamed tissues show altered characteristics that include persistent populations of inflammatory leukocytes and remodelling of the vascular network. As the majority of studies on leukocyte recruitment have been carried out in normal healthy tissues, the impact of underlying chronic inflammation on ongoing leukocyte recruitment is largely unknown. Here, we investigate the profile and mechanisms of acute inflammatory responses in chronically inflamed and angiogenic tissues, and consider the implications for chronic inflammatory disorders. We have developed a novel model of chronic ischaemia of the mouse cremaster muscle that is characterized by a persistent population of monocyte‐derived cells (MDCs), and capillary angiogenesis. These tissues also show elevated acute neutrophil recruitment in response to locally administered inflammatory stimuli. We determined that Gr1^low MDCs, which are widely considered to have anti‐inflammatory and reparative functions, amplified acute inflammatory reactions via the generation of additional proinflammatory signals, changing both the profile and magnitude of the tissue response. Similar vascular and inflammatory responses, including activation of MDCs by transient ischaemia–reperfusion, were observed in mouse hindlimbs subjected to chronic ischaemia. This response demonstrates the relevance of the findings to peripheral arterial disease, in which patients experience transient exercise‐induced ischaemia known as claudication.These findings demonstrate that chronically inflamed tissues show an altered profile and altered mechanisms of acute inflammatory responses, and identify tissue‐resident MDCs as potential therapeutic targets. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Stat2 loss disrupts damage signalling and is protective in acute pancreatitis

The severity of sterile inflammation, as seen in acute pancreatitis, is determined by damage-sensing receptors, signalling cascades and cytokine production. Stat2 is a type I interferon signalling mediator that also has interferon-independent roles in murine lipopolysaccharide-induced NF-κB-mediated sepsis. However, its role in sterile inflammation is unknown. We hypothesised that Stat2 determines the severity of non-infective inflammation in the pancreas. Wild type (WT) and Stat2^−/− mice were injected i.p. with caerulein or l -arginine. Specific cytokine-blocking antibodies were used in some experiments. Pancreata and blood were harvested 1 and 24 h after the final dose of caerulein and up to 96 h post l -arginine. Whole-tissue phosphoproteomic changes were assessed using label-free mass spectrometry. Tissue-specific Stat2 effects were studied in WT/Stat2^−/− bone marrow chimera and using Cre-lox recombination to delete Stat2 in pancreatic and duodenal homeobox 1 (Pdx1)-expressing cells. Stat2^−/− mice were protected from caerulein- and l -arginine-induced pancreatitis. Protection was independent of type I interferon signalling. Stat2^−/− mice had lower cytokine levels, including TNF-α and IL-10, and reduced NF-κB nuclear localisation in pancreatic tissue compared with WT. Inhibition of TNF-α improved (inhibition of IL-10 worsened) caerulein-induced pancreatitis in WT but not Stat2^−/− mice. Phosphoproteomics showed downregulation of MAPK mediators but accumulation of Ser412-phosphorylated Tak1. Stat2 deletion in Pdx1-expressing acinar cells (Stat2^{flox/Pdx1-cre}) reduced pancreatic TNF-α expression, but not histological injury or serum amylase. WT/Stat2^−/− bone marrow chimera mice were protected from pancreatitis irrespective of host or recipient genotype. Stat2 loss results in disrupted signalling in pancreatitis, upstream of NF-κB in non-acinar and/or bone marrow-derived cells. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Monitoring the allergic inflammation

Individual symptoms of allergy such as asthma, dermatitis, rhinitis have many different underlying mechanisms. The detailed characterization of the inflammatory mechanisms underlying symptom development in the individual patient is important in order to optimally control treatment. Measurement of eosinophil cationic protein (ECP) in sputum or blood and eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) in urine may be used to read the involvement of the eosinophil granulocyte in the process. An important information as eosinophil dominated processes seem to be particularly sensitive to corticosteroid treatment. The possibilities to measure the involvement of other inflammatory cells exist today, but are only used to a small extent. The dream would be that every patient with an inflammatory disease is characterized with respect to the profile of involving cells and mediators. Such information would provide us with a unique understanding of the underlying mechanisms of the development of disease symptoms and the possibilities of treating these.