A transient ischemic attack (TIA) can cause reversible and delayed impairment of cognition, but the specific mechanisms are still unclear. Annexin a1 (ANXA1) is a phospholipid-binding protein. Here, we confirmed that cognition and hippocampal synapses were impaired in TIA-treated mice, and this could be rescued by multiple mild stimulations (MMS). TIA promoted the interaction of ANXA1 and CX3CR1, increased the membrane distribution of CX3CR1 in microglia, and thus enhanced the CX3CR1 and CX3CL1 interaction. These phenomena induced by TIA could be reversed by MMS. Meanwhile, the CX3CR1 membrane distribution and CX3CR1-CX3CL1 interaction were upregulated in primary cultured microglia overexpressing ANXA1, and the spine density was significantly reduced in co-cultured microglia overexpressing ANXA1 and neurons. Moreover, ANXA1 overexpression in microglia abolished the protection of MMS after TIA. Collectively, our study provides a potential strategy for treating the delayed synaptic injury caused by TIA.
Animals ; Annexin A1/metabolism* ; CX3C Chemokine Receptor 1/metabolism* ; Chemokine CX3CL1 ; Cognition ; Dendritic Spines/metabolism* ; Ischemic Attack, Transient ; Mice ; Microglia/metabolism*

OBJECTIVE: To explore the mechanism by which fractalkine (CX3CL1; FKN) inhibits lipopolysaccharide (LPS)-induced immunological response in RAW264.7 cells. METHODS: A RAW264.7 cell model overexpressing FKN was established by transfection with the lentiviral vector CX3CL1. The effects of LPS, ICG-001 (a Wnt/β-catenin signaling pathway inhibitor), either alone or in combination, on M1 polarization of na?ve and FKN-overexpressing RAW264.7 cells were evaluated by detecting of intereukin-6 (IL-6) and tumor necrosis factor-α (TNF- RESULTS: The RAW264.7 cell model of FKN overexpression was successfully established. In na?ve RAW264.7 cells, treatment with both ICG-001 and LPS, as compared with LPS alone, significant promoted TNF- CONCLUSIONS FKN overexpression suppresses LPS-induced M1 type polarization of RAW264.7 cells by activating Wnt/β-catenin signaling pathway.
Animals ; Chemokine CX3CL1 ; Lipopolysaccharides/pharmacology* ; Macrophages ; Mice ; RAW 264.7 Cells ; Tumor Necrosis Factor-alpha ; Wnt Signaling Pathway

Patients with diabetic peripheral neuropathy experience debilitating pain that significantly affects their quality of life (Abbott et al., 2011), by causing sleeping disorders, anxiety, and depression (Dermanovic Dobrota et al., 2014). The primary clinical manifestation of painful diabetic neuropathy (PDN) is mechanical hypersensitivity, also known as mechanical allodynia (MA) (Callaghan et al., 2012). MA's underlying mechanism remains poorly understood, and so far, based on symptomatic treatment, it has no effective therapy (Moore et al., 2014).
Animals ; CX3C Chemokine Receptor 1/physiology* ; Chemokine CX3CL1/physiology* ; Diabetes Mellitus, Experimental/complications* ; Diabetes Mellitus, Type 1/complications* ; Diabetic Neuropathies/etiology* ; Hyperalgesia/etiology* ; Mice ; Mice, Inbred C57BL ; Spinal Cord/physiology* ; Streptozocin/pharmacology*

Signal transducer and activator of transcription 3 (STAT3) and Chemokine CX3C ligand 1 (Fractalkine/CX3CL1) play important roles in vascular inflammation and injury. To study if STAT3 promotes vascular endothelial cell proliferation and migration through fractalkine, we overexpressed or knocked down STAT3 in vascular endothelial cells, and used quantitative real-time PCR and Western blotting to determine the effect of STAT3 on fractalkine expression. The wild type and STAT3 binding site mutant fractalkine promoter luciferase reporter plasmids were constructed, and luciferase activity assays were used to explore the effect of STAT3 on the transcriptional activity of the fractalkine promoter. MTT assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on the proliferation rate of vascular endothelial cells. Scratch assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on vascular endothelial cell migration. There results showed that overexpression of STAT3 could promote fractalkine expression, and knockdown of STAT3 could down-regulate fractalkine expression. STAT3 could directly bind to the promoter of fractalkine to promote its transcriptional activity via binding the GAS site of the fractalkine promoter. Knockdown of STAT3 could inhibit the migration of vascular endothelial cell, and overexpression of fractalkine antagonized this inhibition. Our data concluded that STAT3 promotes the proliferation and migration of vascular endothelial cell by binding the GAS site of the fractalkine promoter to promote fractalkine transcriptional activity and expression.
Cell Proliferation ; Chemokine CX3CL1 ; Endothelial Cells ; Promoter Regions, Genetic ; STAT3 Transcription Factor

Chemokine (C-X3-C motif) ligand 1 (CX₃CL1, also known as fractalkine) and its receptor chemokine (C-X3-C motif) receptor 1 (CX₃CR1) are widely expressed in immune cells and non-immune cells throughout organisms. However, their expression is mostly cell type-specific in each tissue. CX₃CR1 expression can be found in monocytes, macrophages, dendritic cells, T cells, and natural killer (NK) cells. Interaction between CX3CL1 and CX₃CL1 can mediate chemotaxis of immune cells according to concentration gradient of ligands. CX₃CL1 expressing immune cells have a main role in either pro-inflammatory or anti-inflammatory response depending on environmental condition. In a given tissue such as bone marrow, brain, lung, liver, gut, and cancer, CX₃CL1 expressing cells can maintain tissue homeostasis. Under pathologic conditions, however, CX₃CL1 expressing cells can play a critical role in disease pathogenesis. Here, we discuss recent progresses of CX3CL1/CX₃CL1 in major tissues and their relationships with human diseases.
Bone Marrow ; Brain ; Chemokine CX3CL1 ; Chemotaxis ; Dendritic Cells ; Homeostasis ; Humans ; Ligands ; Liver ; Lung ; Macrophages ; Monocytes ; Organ Specificity ; T-Lymphocytes

BACKGROUND: Fractalkine (CX3CL1) is a chemokine with a unique CX3C motif and is produced by endothelial cells stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interferon-γ. There have been several reports that the caspase/calpain system is activated in endotoxemia, which leads to cellular apoptosis and acute inflammatory processes. We aimed to determine the role of the caspase/calpain system in cell viability and regulation of fractalkine production in LPS-treated endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with 0.01–100 μg/mL of LPS to determine cell viability. The changes of CX3CL1 expression were compared in control, LPS (1 μg/mL)-, IL-1α (1 μg/mL)-, and IL-1β (1 μg/mL)-treated HUVECs. Cell viability and CX3CL1 production were compared with 50 μM of inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. RESULTS: Cell viability was significantly decreased from 1 to 100 μg/mL of LPS. Cell viability was significantly restored with inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. The expression of CX3CL1 was highest in IL-1β-treated HUVECs. CX3CL1 production was highly inhibited with a calpain inhibitor and significantly decreased with the individual inhibitors of caspase-1, caspase-3, and caspase-9. CONCLUSION: The caspase/calpain system is an important modulator of cell viability and CX3CL1 production in LPS-treated endothelial cells.
Apoptosis ; Calpain* ; Caspase 3 ; Caspase 9 ; Cell Survival ; Chemokine CX3CL1* ; Endothelial Cells* ; Endotoxemia ; Human Umbilical Vein Endothelial Cells ; Interleukins ; Lipopolysaccharides ; Tumor Necrosis Factor-alpha

BACKGROUND/AIMS: Abnormal immune regulation and increased intestinal permeability augmenting the passage of bacterial molecules that can activate immune cells, such as monocytes/macrophages, have been reported in irritable bowel syndrome (IBS). The aim was to compare the maturation phenotype of monocytes/macrophages (CD14+) from IBS patients and controls in the presence or absence of Escherichia coli lipopolysaccharides (LPS), in vitro. METHODS: Mononuclear cells were isolated from peripheral blood of 20 Rome II-IBS patients and 19 controls and cultured with or without LPS for 72 hours. The maturation phenotype was examined by flow cytometry as follows: M1-Early (CD11c⁺CD206⁻), M2-Advanced (CD11⁻CD206⁺CX3CR1⁺); expression of membrane markers was reported as mean fluorescence intensity (MFI). The Mann-Whitney test was used and significance was set at P < 0.05. RESULTS: In CD14+ cells, CD11c expression decreased with vs without LPS both in IBS (MFI: 8766.0 ± 730.2 vs 12 920.0 ± 949.2, P < 0.001) and controls (8233.0 ± 613.9 vs 13 750.0 ± 743.3, P < 0.001). M1-Early cells without LPS, showed lower CD11c expression in IBS than controls (MFI: 11 540.0 ± 537.5 vs 13 860.0 ± 893.7, P = 0.040), while both groups showed less CD11c in response to LPS (P < 0.01). Furthermore, the percentage of “Intermediate” (CD11c⁺CD206⁺CX3CR1⁺) cells without LPS, was higher in IBS than controls (IBS = 9.5 ± 1.5% vs C = 4.9 ± 1.4%, P < 0.001). Finally, fractalkine receptor (CX3CR1) expression on M2-Advanced cells was increased when treated with LPS in controls but not in IBS (P < 0.001). CONCLUSIONS: The initial phase of monocyte/macrophage maturation appears to be more advanced in IBS compared to controls. However, the decreased CX3CR1 in patients with IBS, compared to controls, when stimulated with LPS suggests a state of immune activation in IBS.
Chemokine CX3CL1 ; Escherichia coli ; Flow Cytometry ; Fluorescence ; Humans ; In Vitro Techniques ; Irritable Bowel Syndrome* ; Lipopolysaccharides* ; Membranes ; Monocytes ; Permeability ; Phenotype*

OBJECTIVEA study was conducted to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of regulated upon activation normal T-cell expressed and secreted (RANTES) and fractalkine in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were incubated with different concentrations of Pg-LPS (200, 500, and 1000 ng x mL(-1)) for 1, 6, 12, and 24 h, respectively. Then real time quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent method (ELISA) were adopted to detect the protein levels and mRNA levels of RANTES and fractalkine.

RESULTSThe RANTES protein levels and mRNA levels, as well as fractalkine mRNA levels, were significantly higher in all experimental groups of 1, 6, and 12 h than in the control group (P<0.05), except the expression of RANTES mRNA in 200 ng x mL(-1) group of 12 h and RANTES protein in 200 ng x mL(-1) group of 1 h. The expression levels of RANTES mRNA and fractalkine mRNA were highest in 1000 ng x mL(-1) group of 6 h and were 4.88- and 6.20-fold higher, respectively, than those in the control group. The expression levels of RANTES protein, mRNA, and fractalkine mRNA decreased 6 h after stimulation, and were significantly higher than those in the control group (P<0.05) in the RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis 500 ng x mL(-1) group of 24 h. There was a significant difference between the expression of fractalkine mRNA in 1000 ng x mL(-1) group of 6 and 12 h than in the control group (P<0.05).

CONCLUSIONPg-LPS infection might up-regulate the expression of RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis.

Atherosclerosis ; Cells, Cultured ; Chemokine CCL5 ; genetics ; metabolism ; Chemokine CX3CL1 ; analysis ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Porphyromonas gingivalis ; immunology ; isolation & purification ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

Subcutaneous injection of BmK I could be adopted to well establish a novel pain model. Moreover, 5-hydroxytryptamine (serotonin, 5-HT) receptor is involved in regulating animal pain-related behaviors. However, the underlying mechanism of 5-HT3R on BmK I-induced pain remains unclear. Animal behavioral testing, RT-PCR and Western blotting were used to yield the following results: first, intraplantar (i.pl.) injection of BmK I (10 μg) induced elevated mRNA and protein levels of 5-HT3AR in bilateral L4-L5 spinal cord; Second, intrathecal (i.t.) injection of ondansetron (a specific antagonist of 5-HT3AR) reduced spontaneous pain responses, attenuated unilateral thermal and bilateral mechanical hypersensitivity elicited by BmK I; Microglia could be activated by BmK I (i.pl.) in both sides of L4-L5 spinal cord, and this effect was reversed by intrathecal pre-treatment with 5-HT3AR antagonist. Meanwhile, the 5-HT3AR in L4-L5 spinal cord was almost co-localized with NeuN (a marker of nerve cell), but not co-expressed with Iba-1 (a marker of microglia). Finally, the expression level of CX3CL1 and CX3CR1 was reduced by intrathecal pre-treatment with ondansetron. Our results indicate that both 5-HT3AR signaling pathway and microglia are activated in the process of induction and maintenance of BmK I-induced pain nociception. Meanwhile, our results suggest that the neuronal 5-HT3AR may communicate with microglia indirectly via CX3CL1 which is involved in regulating the BmK I-induced hyperalgesia and sensitization.
Animals ; Behavior, Animal ; Chemokine CX3CL1 ; metabolism ; Hyperalgesia ; chemically induced ; Inflammation ; physiopathology ; Injections, Spinal ; Microglia ; drug effects ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin, 5-HT3 ; metabolism ; Scorpion Venoms ; adverse effects ; Spinal Cord ; metabolism ; physiopathology

OBJECTIVE: To investigate the distribution of mast cells in nasal polyps. METHOD: Biopsy specimens from patients with nasal polyps (n = 20) and control patients (n = 8) were obtained and included in this study. The distribution of mast cells in nasal polyps and the expression of chemokines (CCL5, CCL11, CX3CL1, IL-8, IL-6) in the epithelial cells of normal nasal mucosa and nasal polyps was determined by immunohistochemistry. RESULT: Mast cells migrate to intraepithelial in nasal polyps and the expression of chemokines (CCL5, CCL11, CX3CL1, IL-8) was up regulated in the epithelial cells of nasal polyps compare to normal nasal mucosa. CONCLUSION Our findings showed that mast cells migrate to intraepithelial in nasal polyps and the over expression of chemotaxins (CCL5, CCL11, CX3CL1, IL-8) may be response for mast cells' migration in nasal polyps. Mast cells might be associated with the development of nasal polyps.
Chemokine CCL11 ; metabolism ; Chemokine CCL5 ; metabolism ; Chemokine CX3CL1 ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Immunohistochemistry ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Mast Cells ; metabolism ; pathology ; Nasal Mucosa ; cytology ; metabolism ; Nasal Polyps ; metabolism ; pathology ; Up-Regulation