The inflammation accompanies and exacerbates cerebral ischaemia. The infiltrated leucocytes are thought to contribute to tissue injury in stroke patients. GRO-alpha (CXCL1) is a potent neutrophil chemoattractant which may play an important role in pathophysiology of stroke. 23 ischaemic stroke patients and 15 controls have been studied. CSF and blood sampling together with cranial CT were performed within first 24 hours of stroke. CXCL1 levels were determined by ELISA, and volume of stroke-related brain CT hypodense areas was calculated. Stroke patients displayed significantly higher CSF CXCL1 level than controls (65.6+/-22.3 vs 43.8+/-2.3 pg/ml; p<0.01). Serum CXCL1 levels in stroke patients did not differ from controls. CSF CXCL1 level correlated positively with volume of brain CT hypodense areas (p<0.001). The results suggest that CXCL1 may be involved in inflammatory reaction during an early phase of ischaemic stroke.

BACKGROUND

Recent evidence has demonstrated that environment and genetic factors play pivotal roles in diabetes and its related complications. The significant contributory role of cytokines in pathogenesis of type 2 diabetes is also well documented. This study was aimed to examine and compare both <em>CXCL1</em> (GRO-alpha) and <em>CXCL1</em>0 (IP-10) circulating levels in type 2 diabetic patients and healthy controls.

METHODS

Peripheral blood samples were collected from 100 type 2 diabetic patients and 150 healthy controls. Circulating <em>CXCL1</em> and <em>CXCL1</em>0 levels were measured by ELISA.

RESULTS

Elevated serum levels of both <em>CXCL1</em> and <em>CXCL1</em>0 were found in type 2 diabetic patients in comparison to controls.

CONCLUSIONS

Elevated levels of <em>CXCL1</em> and <em>CXCL1</em>0 could possibly be used as a marker of inflammation and angiogenesis/angiostasis in type 2 diabetes.

During acute Vesicular Stomatitis Virus (VSV) infection of the mouse central nervous system, neutrophils, natural killer (NK) cells, macrophages, and CD4+ and CD8+ T cells are recruited from the circulation in response to chemokines and cytokines. This study elucidated the production of these factors and infiltration of these peripheral cells. Chemokines that were observed included CCL1, CXCL1CXCL1 (MIP-2), CCL2 (MCP-1), and CCL11 (eotaxin). Cytokines produced in response to the infection include IL-1 and interferon-gamma, but not type I interferons. Neutrophils are the first recruited cell type, appearing as early as 24 h after intranasal application of the virus. NK cells follow, but T cells are not detected until 6 days postinfection.

The PTH receptor (PTHR1) is expressed on osteoblasts and responds to PTH or PTHrP in an endocrine or autocrine/paracrine manner, respectively. A microarray study carried out on PTHR1-positive osteoblasts (Kusa 4b10 cells) identified the cysteine-X-cysteine (CXC) family chemokine ligand 1 (Cxcl1) as a novel immediate PTH/PTHrP-responsive gene. Cxcl1 is a potent neutrophil chemoattractant with recognized roles in angiogenesis and inflammation, but a role in bone biology has not been described. Cxcl1 mRNA levels were up-regulated 1 h after either PTH or PTHrP treatment of differentiated Kusa 4b10 osteoblasts (15-fold) and mouse calvarial osteoblasts (160-fold) and in rat metaphyseal bone (5-fold) 1 h after a single sc injection of PTH. Furthermore, PTH treatment stimulated a 10-fold increase in secreted Cxcl1 protein by both Kusa 4b10 cells and calvarial osteoblasts. Immunohistochemistry and PCR demonstrated that CXCR2, the receptor for Cxcl1, is highly expressed in osteoclast precursors (hemopoietic cells) but is predominantly undetectable in the osteoblast lineage, suggesting that osteoblast-derived Cxcl1 may act as a chemoattractant for osteoclast precursors. Confirming this hypothesis, recombinant Cxcl1 dose-dependently stimulated migration of osteoclast precursors in cell culture studies, as did conditioned media from Kusa 4b10 cells treated with PTH. These data indicate that local action through the PTHR1 could stimulate cells of the osteoblast lineage to release a chemokine capable of attracting osteoclast precursors to the bone environment.

OBJECTIVE

To discover methylated-differentially expressed genes (MDEGs) in hepatocellular carcinoma (HCC) and to explore relevant hub genes and potential pathways.

METHODS

The data of expression profiling GSE25097 and methylation profiling GSE57956 were gained from GEO Datasets. We analyzed the differentially methylated genes and differentially expressed genes online using GEO2R. Functional and enrichment analyses of MDEGs were conducted using the DAVID database. A protein-protein interaction (PPI) network was performed by STRING and then visualized in Cytoscape. Hub genes were ranked by cytoHubba, and a module analysis of the PPI network was conducted by MCODE in Cytoscape software.

RESULTS

In total, we categorized 266 genes as hypermethylated, lowly expressed genes (Hyper-LGs) referring to endogenous and hormone stimulus, cell surface receptor linked signal transduction and behavior. In addition, 161 genes were labelled as hypomethylated, highly expressed genes (Hypo-HGs) referring to DNA replication and metabolic process, cell cycle and division. Pathway analysis illustrated that Hyper-LGs were enriched in cancer, Wnt, and chemokine signalling pathways, while Hypo-HGs were related to cell cycle and steroid hormone biosynthesis pathways. Based on PPI networks, PTGS2, PIK3CD, CXCL1, ESR1, and MMP2 were identified as hub genes for Hyper-LGs, and CDC45, DTL, AURKB, CDKN3, MCM2, and MCM10 were hub genes for Hypo-HGs by combining six ranked methods of cytoHubba.

CONCLUSIONS

In the study, we disclose numerous novel genetic and epigenetic regulations and offer a vital molecular groundwork to understand the pathogenesis of HCC. Hub genes, including PTGS2, PIK3CD, CXCL1, ESR1, MMP2, CDC45, DTL, AURKB, CDKN3, MCM2, and MCM10, can be used as biomarkers based on aberrant methylation for the accurate diagnosis and treatment of HCC.

Obesity is a chronic inflammation with increased serum levels of insulin, insulin-like growth factor 1 (IGF1), and interleukin-17 (IL-17). The objective of this study was to test a hypothesis that insulin and IGF1 enhance IL-17-induced expression of inflammatory chemokines/cytokines through a glycogen synthase kinase 3β (GSK3B)-dependent mechanism, which can be inhibited by melatonin. We found that insulin/IGF1 and lithium chloride enhanced IL-17-induced expression of C-X-C motif ligand 1 (Cxcl1) and C-C motif ligand 20 (Ccl20) in the Gsk3b(+/+) , but not in Gsk3b(-/-) mouse embryonic fibroblast (MEF) cells. IL-17 induced higher levels of Cxcl1 and Ccl20 in the Gsk3b(-/-) MEF cells, compared with the Gsk3b(+/+) MEF cells. Insulin and IGF1 activated Akt to phosphorylate GSK3B at serine 9, thus inhibiting GSK3B activity. Melatonin inhibited Akt activation, thus decreasing P-GSK3B at serine 9 (i.e., increasing GSK3B activity) and subsequently inhibiting expression of Cxcl1 and Ccl20 that was induced either by IL-17 alone or by a combination of insulin and IL-17. Melatonin's inhibitory effects were only observed in the Gsk3b(+/+) , but in not Gsk3b(-/-) MEF cells. Melatonin also inhibited expression of Cxcl1, Ccl20, and Il-6 that was induced by a combination of insulin and IL-17 in the mouse prostatic tissues. Further, nighttime human blood, which contained high physiologic levels of melatonin, decreased expression of Cxcl1, Ccl20, and Il-6 in the PC3 human prostate cancer xenograft tumors. Our data support our hypothesis and suggest that melatonin may be used to dampen IL-17-mediated inflammation that is enhanced by the increased levels of insulin and IGF1 in obesity.

OBJECTIVE

To further characterize, in a rat model, the effects of atherosclerosis-induced chronic bladder ischemia on bladder function and associated changes in oxidative stress markers and proinflammatory cytokines.

METHODS

Adult Sprague-Dawley male rats were divided into three groups (arterial endothelial injury: AI, sham, naïve). The AI group (n = 14) underwent endothelial injury of the iliac arteries and received a 2% cholesterol diet. The sham group (n = 12) underwent sham operation and received a 2% cholesterol diet. The naïve group (n = 12) received a regular diet. After 8 weeks, cystometrograms (CMG) without anesthesia or restraint were performed. In bladders from each group, oxidative stress markers (8-hydroxy-2'-deoxyguanosine: 8-OHdG; malondialdehyde: MDA) and proinflammatory cytokines (IL-8 like cytokine CXCL1/CINC-1, TNF-α, IL-6) were quantified. Histological examination of the iliac arteries was also performed.

RESULTS

At 8 weeks, the body and bladder wet weights were not significant different among the three groups. The micturition interval in the AI group decreased significantly compared with those in the other two groups, but maximum pressure during micturition did not change. The iliac arteries in the AI group revealed thickening of intima as well as diffuse media fibrosis at the sites of balloon injury. The levels of oxidative stress markers and proinflammatory cytokines were significantly higher in the AI than in the other groups.

CONCLUSIONS

Oxidative stress and inflammation may be key factors in the development of bladder overactivity in atherosclerosis-induced chronic bladder ischemia.

BACKGROUND

CXC chemokines are induced by inflammatory stimuli in epithelial cells and some, like MIG/CXCL9, IP-10/<em>CXCL1</em>0 and I-TAC/<em>CXCL1</em>1, are antibacterial for Streptococcus pyogenes.

RESULTS

SpeB from S. pyogenes degrades a wide range of chemokines (i.e. IP10/<em>CXCL1</em>0, I-TAC/<em>CXCL1</em>1, PF4/CXCL4, GROalpha/<em>CXCL1</em>, GRObeta/CXCL2, GROgamma/CXCL3, ENA78/CXCL5, GCP-2/CXCL6, NAP-2/CXCL7, SDF-1/<em>CXCL1</em>2, BCA-1/<em>CXCL1</em>3, BRAK/<em>CXCL1</em>4, SRPSOX/<em>CXCL1</em>6, MIP-3alpha/CCL20, Lymphotactin/XCL1, and Fractalkine/CX3CL1), has no activity on IL-8/CXCL8 and RANTES/CCL5, partly degrades SRPSOX/<em>CXCL1</em>6 and MIP-3alpha/CCL20, and releases a 6 kDa CXCL9 fragment. <em>CXCL1</em>0 and <em>CXCL1</em>1 loose receptor activating and antibacterial activities, while the CXCL9 fragment does not activate the receptor CXCR3 but retains its antibacterial activity.

CONCLUSIONS

SpeB destroys most of the signaling and antibacterial properties of chemokines expressed by an inflamed epithelium. The exception is CXCL9 that preserves its antibacterial activity after hydrolysis, emphasizing its role as a major antimicrobial on inflamed epithelium.

Immune recognition of tumor targets by specific cytotoxic lymphocytes is essential for the effective rejection of tumors. A phase I clinical trial of ipilimumab (an antibody that blocks CTLA-4 function) in combination with bevacizumab (an antibody that inhibits angiogenesis) in patients with metastatic melanoma found favorable clinical outcomes were associated with increased tumor endothelial activation and lymphocyte infiltration. To better understand the underlying mechanisms, we sought features and factors that changed as a function of treatment in patients. Ipilimumab plus bevacizumab (Ipi-Bev) increased tumor vascular expression of ICAM1 and VCAM1. Treatment also altered concentrations of many circulating cytokines and chemokines, including increases of <em>CXCL1</em>0, IL1α, TNFα, <em>CXCL1</em>, IFNα2, and IL8, with decreases in VEGF-A in most patients. IL1α and TNFα induced expression of E-selectin, <em>CXCL1</em>, and VCAM1 on melanoma tumor-associated endothelial cells (TEC) in vitro and promoted adhesion of activated T cells onto TEC. VEGFA inhibited TNFα-induced expression of ICAM1 and VCAM1 and T-cell adhesion, which was blocked by bevacizumab. <em>CXCL1</em>0 promoted T-cell migration across TEC in vitro, was frequently expressed by melanoma cells, and was upregulated in a subset of tumors in treated patients. Robust upregulation of <em>CXCL1</em>0 in tumors was accompanied by increased T-cell infiltration. Ipi-Bev also augmented humoral immune responses recognizing targets in melanoma, tumor endothelial, and tumor mesenchymal stem cells. Our findings suggest that Ipi-Bev therapy augments immune recognition in the tumor microenvironment through enhancing lymphocyte infiltration and antibody responses. IL1α, TNFα, and <em>CXCL1</em>0, together with VEGF neutralization, contribute to Ipi-Bev-induced melanoma immune recognition. Cancer Immunol Res; 4(10); 858-68. ©2016 AACR.

OBJECTIVE

Lung macrophages are critically involved in respiratory diseases. This study assessed the effects of the PDE4 inhibitor roflumilast and its active metabolite, roflumilast N-oxide on the release of a range of chemokines (CCL2, 3, 4, CXCL1, 8, 10) and of TNF-α, from human lung macrophages, stimulated with bacterial lipopolysaccharide LPS.

METHODS

Lung macrophages isolated from resected human lungs were incubated with roflumilast, roflumilast N-oxide, PGE(2), the COX inhibitor indomethacin, the COX-2 inhibitor NS-398 or vehicle and stimulated with LPS (24 h). Chemokines, TNF-α, PGE(2) and 6-keto PGF(1α) were measured in culture supernatants by immunoassay. COX-2 mRNA expression was assessed with RT-qPCR. PDE activities were determined in macrophage homogenates.

RESULTS

Expression of PDE4 in lung macrophages was increased after incubation with LPS. Roflumilast and roflumilast N-oxide concentration-dependently reduced the LPS-stimulated release of CCL2, CCL3, CCL4, CXCL1CXCL1 or CXCL8 was not altered. This reduction by the PDE4 inhibitors was further accentuated by exogenous PGE(2) (10 nM) but abolished in the presence of indomethacin or NS-398. Conversely, addition of PGE(2) (10 nM), in the presence of indomethacin restored inhibition by roflumilast. LPS also increased PGE(2) and 6-keto PGF(1α) release from lung macrophages which was associated with an up-regulation of COX-2 mRNA.

CONCLUSIONS

Roflumilast and roflumilast N-oxide reduced LPS-induced release of CCL2, 3, 4, <em>CXCL1</em>0 and TNF-α in human lung macrophages.

Neutrophil chemokine receptor expression can be altered by exposure to Toll-like receptor (TLR) agonists, a process that is thought to have the potential to localize neutrophils to sites of infection. In order to investigate this process in more detail, we examined the regulation of highly pure neutrophil CXCR1 and CXCR2 expression and function by selective agonists of TLR2 (Pam(3)CSK(4)) and TLR4 (lipopolysaccharide, LPS). CXCR1 and CXCR2 were down-regulated by TLR engagement. CXCR2 loss was more rapid and showed a dependence upon soluble helper molecules (LPS binding protein and CD14) that was not evident for CXCR1, suggesting differential coupling of LPS signalling to CXCR1 and CXCR2 loss. However, TLR engagement in highly pure neutrophils did not result in complete loss of chemokine receptors, and LPS-treated neutrophils remained able to mount a respiratory burst to CXCL8 and CXCL1, and were able to migrate towards CXCL8 in assays of under-agarose chemotaxis. Thus, although treatment of purified human neutrophils with TLR2 and TLR4 agonists modifies chemokine receptor expression, remaining receptors remain functionally competent.

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone-forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF-β2, GDF15, FGF3, FGF19, CXCL1, galectins, and β2-microglobulin, which represent both novel and previously published secreted proteins. RT-PCR using human versus mouse-specific primers showed that TGFβ2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFβ2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.

Infection of mice with murine gammaherpesvirus 68 (MHV-68) is a well-characterized small animal model for the study of gammaherpesvirus infection. MHV-68 belongs to the same herpesvirus family as herpesvirus saimiri (HVS) of New World squirrel monkeys and human herpesvirus 8 (HHV-8) (also referred to as Kaposi's sarcoma-associated herpesvirus [KSHV]). The open reading frame ORF74 of HVS, KSHV, and MHV-68 encodes a protein with homology to G protein-coupled receptors and chemokine receptors in particular. ORF74 of KSHV (human ORF74 [hORF74]) is highly constitutively active and has been implicated in the pathogenesis of Kaposi's sarcoma. MHV-68-encoded ORF74 (mORF74) is oncogenic and has been implicated in viral replication and reactivation from latency. Here, we show that mORF74 is a functional chemokine receptor. Chemokines with an N-terminal glutamic acid-leucine-arginine (ELR) motif (e.g., KC and macrophage inflammatory protein 2) act as agonists on mORF74, activating phospholipase C, NF-kappaB, p44/p42 mitogen-activated protein kinase, and Akt signaling pathways and inhibiting formation of cyclic AMP. Using (125)I-labeled <em>CXCL1</em>/growth-related oncogene alpha as a tracer, we show that murine <em>CXCL1</em>0/gamma interferon-inducible protein 10 binds mORF74, and functional assays show that it behaves as an antagonist for this virally encoded G protein-coupled receptor. Profound differences in the upstream activation of signal transduction pathways between mORF74 and hORF74 were found. Moreover, in contrast to hORF74, no constitutive activity of mORF74 could be detected.

Structural cell migration plays a central role in the pathophysiology of several diseases, including asthma. Previously, we established that IL-17-induced (CXCL1, CXCL2, and CXCL3) production promoted airway smooth muscle cell (ASMC) migration, and consequently we sought to investigate the molecular mechanism of CXC-induced ASMC migration. Recombinant human CXCL1, CXCL2, and CXCL3 were used to assess migration of human primary ASMCs from normal and asthmatic subjects using a modified Boyden chamber. Neutralizing Abs or small interfering RNA (siRNA) knockdown and pharmacological inhibitors of PI3K, ERK1/2, and p38 MAPK pathways were used to investigate the receptors and the signaling pathways involved in CXC-induced ASMC migration, respectively. We established the ability of CXCL2 and CXCL3, but not CXCL1, to induce ASMC migration at the tested concentrations using normal ASMCs. We found CXCL2-induced ASMC migration to be dependent on p38 MAPK and CXCR2, whereas CXCL3-induced migration was dependent on p38 and ERK1/2 MAPK pathways via CXCR1 and CXCR2. While investigating the effect of CXCL2 and CXCL3 on asthmatic ASMC migration, we found that they induced greater migration of asthmatic ASMCs compared with normal ones. Interestingly, unlike normal ASMCs, CXCL2- and CXCL3-induced asthmatic ASMC migration was mainly mediated by the PI3K pathway through CXCR1. In conclusion, our results establish a new role of CXCR1 in ASMC migration and demonstrate the diverse mechanisms by which CXCL2 and CXCL3 mediate normal and asthmatic ASMC migration, suggesting that they may play a role in the pathogenesis of airway remodeling in asthma.

We sought to identify cells and cytokines in bronchoalveolar lavage (BAL) fluids that distinguish asthma from healthy control subjects and those that distinguish controlled asthma from uncontrolled asthma. Following informed consent, 36 human subjects were recruited for this study. These included 11 healthy control subjects, 15 subjects with controlled asthma with FEV1≥80% predicted and 10 subjects with uncontrolled asthma with FEV1 <80% predicted. BAL fluid was obtained from all subjects. The numbers of different cell types and the levels of 48 cytokines were measured in these fluids. Compared to healthy control subjects, patients with asthma had significantly more percentages of eosinophils and neutrophils, IL-1RA, IL-1α, IL-1β, IL-2Rα, IL-5, IL-6, IL-7, IL-8, G-CSF, GROα (CXCL1), MIP-1β (CCL4), MIG (CXCL9), RANTES (CCL5) and TRAIL in their BAL fluids. The only inflammatory markers that distinguished controlled asthma from uncontrolled asthma were neutrophil percentage and IL-8 levels, and both were inversely correlated with FEV1. We examined whether grouping asthma subjects on the basis of BAL eosinophil % or neutrophil % could identify specific cytokine profiles. The only differences between neutrophil-normal asthma (neutrophil≤2.4%) and neutrophil-high asthma (neutrophils%>2.4%) were a higher BAL fluid IL-8 levels, and a lower FEV1 in the latter group. By contrast, compared to eosinophil-normal asthma (eosinophils≤0.3%), eosinophil-high asthma (eosinophils>0.3%) had higher levels of IL-5, IL-13, IL-16, and PDGF-bb, but same neutrophil percentage, IL-8, and FEV1. Our results identify neutrophils and IL-8 are the only inflammatory components in BAL fluids that distinguish controlled asthma from uncontrolled asthma, and both correlate inversely with FEV1.

Acute lung injury (ALI) is a severe inflammatory condition whose pathogenesis is irrevocably linked to neutrophil emigration to the lung. Activation and recruitment of neutrophils to the lung is mostly attributable to local production of the chemokines. However, much of our understanding of neutrophil recruitment to the lung is based on studies focusing on early time points after initiation of injury. In this study, we sought to evaluate the extended temporal relationship between neutrophil chemotactic factor expression and influx of neutrophils into the lung after intratracheal administration of either LPS or bleomycin. In both models, results demonstrated two phases of neutrophil chemotactic factor expression; first, an early phase characterized by high levels of CXCL1/keratinocyte-derived chemokine, CXCL2/monocyte-inhibitory protein-2, and CXCL5/LPS-induced chemokine expression, and second, a late phase distinguished by increases in extracellular ATP. Furthermore, we show that strategies aimed at either enhancing ATP catabolism (ip ecto-5'-nucleotidase administration) or inhibiting glycolytic ATP production (ip 2-deoxy-d-glucose treatment) reduce extracellular ATP accumulation, limit vascular leakage, and effectively block the late, but not the early, stages of neutrophil recruitment to the lung after LPS instillation. In conclusion, this study illustrates that neutrophil recruitment to the lung is mediated by the time-dependent expression of chemotactic factors and suggests that novel strategies, which reduce extracellular ATP accumulation, may attenuate late neutrophil recruitment and limit lung injury during ALI.

Cordycepin (3' deoxyadenosine) has long been used in the study of in vitro assembled polyadenylation complexes, because it terminates the poly(A) tail and arrests the cleavage complex. It is derived from caterpillar fungi, which are highly prized in Chinese traditional medicine. Here we show that cordycepin specifically inhibits the induction of inflammatory mRNAs by cytokines in human airway smooth muscle cells without affecting the expression of control mRNAs. Cordycepin treatment results in shorter poly(A) tails, and a reduction in the efficiency of mRNA cleavage and transcription termination is observed, indicating that the effects of cordycepin on 3' processing in cells are similar to those described in in vitro reactions. For the CCL2 and CXCL1 mRNAs, the effects of cordycepin are post-transcriptional, with the mRNA disappearing during or immediately after nuclear export. In contrast, although the recruitment of RNA polymerase II to the IL8 promoter is also unaffected, the levels of nascent transcript are reduced, indicating a defect in transcription elongation. We show that a reporter construct with 3' sequences from a histone gene is unaffected by cordycepin, while CXCL1 sequences confer cordycepin sensitivity to the reporter, demonstrating that polyadenylation is indeed required for the effect of cordycepin on gene expression. In addition, treatment with another polyadenyation inhibitor and knockdown of poly(A) polymerase α also specifically reduced the induction of inflammatory mRNAs. These data demonstrate that there are differences in the 3' processing of inflammatory and housekeeping genes and identify polyadenylation as a novel target for anti-inflammatory drugs.

Crosstalk between lymphatic endothelial cells (LECs) and tumor cells in the tumor microenvironment plays a crucial role in tumor metastasis. Our previous study indicated chemokine (C-X-C motif) ligand 1 (CXCL1) from LECs stimulates the metastasis of gastric cancer. However, the mechanism is still unclear. Here, we successfully isolated tumor-associated LECs (T-LECs) and normal LECs (N-LECs) from clinical samples by magnetic-activated cell sorting system (MACS) and proved that CXCL1 expression was elevated in T-LECs compared with N-LECs in situ and vitro. Besides, we demonstrated that CXCL1 secreted by T-LECs promoted the migration, invasion, and adhesion of gastric cancer cells by upregulating integrin β1, MMP2, and MMP9. Furthermore, CXCL1 induced MMP2/9 expression by activating integrin β1-FAK-AKT signaling. In the animal model, CXCL1 overexpressed in LECs increased the lymph node metastasis of gastric cancer. In conclusion, CXCL1 expression in T-LECs was upregulated, and CXCL1 secreted by T-LECs promoted the lymph node metastasis of gastric cancer through integrin β1/FAK/AKT signaling, leading to MMP2 and MMP9 expression. Therefore, CXCL1 produced in T-LECs represents a potentially promising target for treating gastric cancer.

BACKGROUND

The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. Transforming growth factor-βeta (TGF-β) and Chemokine (C-X-C Motif) Ligand-1 (CXCL1) are cytokines involved in the colonization of distant sites by CTCs in several pre-clinical animal models. However, their role is poorly-investigated in patients with metastatic cancer. Here, we investigated whether circulating levels of TGF-β and CXCL1 are predictors of CTC seeding in preferential distant sites in patients with metastatic breast cancer.

METHODS

CTCs were isolated from the peripheral blood of 61 patients with metastatic breast cancer by immunomagnetic separation. Plasma samples were collected from the same patients and assayed for TGF-β and CXCL1 by enzyme-linked immunoassay.

RESULTS

Patients were grouped in CK1+/- (N<10), CK2+ (N ≥ 10<50) and CK3+ (N ≥ 50), according to the number (N) of cytokeratin 7/8-positive CTCs: the highest number of CK7/8-positive CTCs was detected in patients with negative Human epidermal growth factor receptor-2 (HER-2/NEU) status (p<0.0001) antigen, identified by the monoclonal antibody Ki-67 (Ki-67) ≥ 15% (p=0.003), Carcinoma antigen 15-3 (CA-15.3) ≥ 40 U/ml (p=0.004) and those with lung metastases (p=0.01). We found that elevated plasma concentrations of TGF-β and CXCL1 are predictive for the detection of CTCs. In particular, patients with CK3+ CTCs and plasma concentrations of TGF-β and CXCL1 higher than the median value had a poor prognosis in comparison to patients with CK1+/- CTCs and TGF-β and CXCL1 concentrations below the median value.

CONCLUSIONS

Our study shows that elevated circulating levels of TGF-β and CXCL1 are associated with a poor prognosis, and higher detection of CTCs and propensity of these cells to seed lung metastases in patients with breast cancer.

BACKGROUND

The up-regulation of CCAAT/enhancer binding protein delta (CEBPD) has frequently been observed in macrophages in age-associated disorders, including rheumatoid arthritis (RA). However, the role of macrophage CEBPD in the pathogenesis of RA is unclear.

RESULTS

We found that the collagen-induced arthritis (CIA) score and the number of affected paws in Cebpd(-/-) mice were significantly decreased compared with the wild-type (WT) mice. The histological analysis revealed an attenuated CIA in Cebpd(-/-) mice, as shown by reduced pannus formation and greater integrity of joint architecture in affected paws of Cebpd(-/-) mice compared with WT mice. In addition, immunohistochemistry analysis revealed decreased pannus proliferation and angiogenesis in Cebpd(-/-) mice compared with WT mice. CEBPD activated in macrophages played a functional role in promoting the tube formation of endothelial cells and the migration and proliferation of synoviocytes. In vivo DNA binding assays and reporter assays showed that CEBPD up-regulated CCL20, CXCL1, IL23A and TNFAIP6 transcripts through direct binding to their promoter regions. CCL20, IL23A, CXCL1 and TNFAIP6 contributed to the migration and proliferation of synoviocytes, and the latter two proteins were involved in tube formation of endothelial cells. Finally, two anti-inflammatory chemicals, inotilone and rosmanol, reduced the expression of CEBPD and its downstream targets and mitigated the above phenomena.

CONCLUSIONS

Collectively, our findings suggest that CEBPD and its downstream effectors could be biomarkers for the diagnosis of RA and potentially serve as therapeutic targets for RA therapy.

Microparticles (MPs) are small membrane-vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro-inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co-cultured with increasing numbers of MPs. The effects of supernatants from co-cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans-well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro-angiogenic ELR⁺ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co-cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR⁺ chemokines. Consistent with the specific induction of ELR⁺ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio-chamber assay, supernatants from RASFs co-cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro-angiogenic ELR⁺ chemokines. These pro-angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis.

BACKGROUND

Bone marrow-derived microglia that originates in part from hematopoietic cells, and more particularly from monocytes preferentially attach to amyloid deposition in brains of Alzheimer's disease (AD). However, the mechanism of monocytes recruited into the amyloid plaques with an accelerated process in AD is unclear.

RESULTS

Here we reported that monocytes from AD patients express significantly higher chemokine (C-X-C motif) ligand 1 (CXCL1) compared to age-matched controls. AD patient's monocytes or CXCL1-overexpressing THP-1 cells had enhanced ability of β-amyloid (Aβ)-induced transendothelial migration and Aβ-induced transendothelial migration for AD patient's monocytes or CXCL1-overexpressing THP-1 cells was almost abrogated by anti-CXCL1 antibody. Furthermore, monocytes derived from a transgenic mouse model of AD also expressed significantly higher CXCL1. CD11b⁺CD45(hi) population of cells that were recruited from the peripheral blood were markedly bolcked in APP mouse brain by anti-CXCL1 antibody. Accordingly, in response to Aβ, human brain microvascular endothelial cells (HBMEC) significantly up-regulated CXC chemokine receptor 2 (CXCR2) expression, which was the only identified receptor for CXCL1. In addition, a high level expression of CXCR2 in HBMEC significantly promoted the CXCL1-overexpressing THP-1 cells transendothelial migration, which could be was abrogated by anti-CXCR2 antibody. Further examination of possible mechanisms found that CXCL1-overexpressing THP-1 cells induced transendothelial electrical resistance decrease, horseradish peroxidase flux increase, ZO-1 discontinuous and occludin re-distribution from insoluble to soluble fraction through interacting with CXCR2. ROCK inhibitor, Y27632, could block CXCL1-overexpressing THP-1 cells transendothelial migration, whereas other inhibitors had no effects.

CONCLUSIONS

The present data indicate that monocytes derived from AD patients overexpressing CXCL1, which is a determinant for Aβ-induced transendothelial migration. CXCL1 expressed by monocytes and CXCR2 on HBMEC is involved in monocytes migrating from blood to brain in AD patients.

Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL1CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-α), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals.

As we showed previously, the extracellular matrix (ECM) derived from rat bladder carcinoma cells (804G-ECM) has positive effects on rat primary beta-cell function and survival in vitro. The aim of this study was to define beta-cell genes induced by this ECM with a specific focus on cytokines. Analysis of differential gene expression by oligonucleotide microarrays, RT-PCR, and in situ hybridization was performed to identify cytokine mRNA induced by this matrix. Four cytokines were overexpressed on 804G-ECM compared with poly-L-lysine: C-X-C motif ligand 1 (CXCL1), CXCL2, interferon-inducible protein-10, and IL-1beta. A time-course experiment indicated that maximal induction by 804G-ECM of CXCL1/2 and interferon-inducible protein-10 occurred at 4 h. Stimulation of CXCL1 release by beta-cells on 804G-ECM was confirmed at the protein level. Moreover, secreted CXCL1 was shown to be functionally active by attracting rat granulocytes. Preventing the interaction of beta1 integrins and laminin-5 (a major component of 804G-ECM) with specific antibodies resulted in a 40-50% inhibition of CXCL1 expression. Using the nuclear factor-kappaB pathway inhibitor Bay 11-7082 it is demonstrated that CXCL1 expression and secretion are dependent on nuclear factor-kappaB activation. IL-1 secreted by beta-cells plated on 804G-ECM was found to be a key soluble mediator because treatment of cells with the IL-1 receptor antagonist significantly reduced both CXCL1 gene expression and secretion. It is concluded that ECM induces expression of cytokines including CXCL1 with amplification by IL-1 acting via a positive autocrine feedback loop.