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Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons.

ABSTRACT: The soluble form of the chemokine fractalkine/CX(3)CL1 regulates microglia activation in the central nervous system (CNS), ultimately affecting neuronal survival. This study aims to determine whether CXCL12, another chemokine constitutively expressed in the CNS (known as stromal cell-derived factor 1; SDF-1), regulates cleavage of fractalkine from neurons. To this end, ELISA was used to measure protein levels of soluble fractalkine in the medium of rat neuronal cultures exposed to SDF-1. Gene arrays, quantitative RT-PCR, and Western blot were used to measure overall fractalkine expression in neurons. The data show that the rate of fractalkine shedding in healthy cultures positively correlates with in vitro differentiation and survival. In analogy to non-neuronal cells, metalloproteinases (ADAM10/17) are involved in cleavage of neuronal fractalkine as indicated by studies with pharmacologic inhibitors. Moreover, treatment of the neuronal cultures with SDF-1 stimulates expression of the inducible metalloproteinase ADAM17 and increases soluble fractalkine content in culture medium. The effect of SDF-1 is blocked by an inhibitor of both ADAM10 and -17, but only partially affected by a more specific inhibitor of ADAM10. In addition, SDF-1 also up-regulates expression of the fractalkine gene. Conversely, exposure of neurons to an excitotoxic stimulus (i.e. NMDA) inhibits alpha-secretase activity and markedly diminishes soluble fractalkine levels, leading to cell death. These results, along with previous findings on the neuroprotective role of both SDF-1 and fractalkine, suggest that this novel interaction between the two chemokines may contribute to in vivo regulation of neuronal survival by modulating microglial neurotoxic properties.

SUBMITTER: Cook A

PROVIDER: S-EPMC2856264 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons.

Cook Anna A Hippensteel Randi R Shimizu Saori S Nicolai Jaclyn J Fatatis Alessandro A Meucci Olimpia O

The Journal of biological chemistry 20100202 14

The soluble form of the chemokine fractalkine/CX(3)CL1 regulates microglia activation in the central nervous system (CNS), ultimately affecting neuronal survival. This study aims to determine whether CXCL12, another chemokine constitutively expressed in the CNS (known as stromal cell-derived factor 1; SDF-1), regulates cleavage of fractalkine from neurons. To this end, ELISA was used to measure protein levels of soluble fractalkine in the medium of rat neuronal cultures exposed to SDF-1. Gene ar ...[more]

PMID: 20124406

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Project description:BackgroundFractalkine (CX3CL1) is involved in the development of numerous inflammatory conditions including metabolic diseases. However, changes in the circulatory fractalkine levels in type-2 diabetes (T2D) and their relationship with inflammatory chemokines/cytokines remain unclear. The aim of the study was to determine the T2D-associated modulations in plasma fractalkine levels and investigate their relationship with circulatory chemokines/cytokines.MethodsA total of 47 plasma samples were collected from 23 T2D and 24 non-diabetic individuals selected over a wide range of body mass index (BMI). Clinical metabolic parameters were determined using standard commercial kits. Fractalkine and chemokines/cytokines were measured using Luminex X-MAP® technology. C-reactive protein (CRP) was measured by ELISA. The data were compared using unpaired t-test and the dependence between two variables was assessed by Pearson's correlation coefficient (r).ResultsPlasma fractalkine levels were significantly higher (P = 0.005) in T2D patients (166 ± 14.22 pg/ml) as compared with non-diabetics (118 ± 8.90 pg/ml). In T2D patients, plasma fractalkine levels correlated positively (P ≤ 0.05) with inflammatory chemokines/cytokines including CCL3 (r = 0.52), CCL4 (r = 0.85), CCL11 (r = 0.51), CXCL1 (r = 0.67), G-CSF (r = 0.91), IFN-α2 (r = 0.97), IL-17A (r = 0.79), IL-1β (r = 0.97), IL-12P70 (r = 0.90), TNF-α (r = 0.58), and IL-6 (r = 0.60). In non-diabetic individuals, fractalkine levels correlated (P ≤ 0.05) with those of CCL4 (r = 0.49), IL-1β (r = 0.73), IL-12P70 (r = 0.41), and TNF-α (r = 0.50). Notably, plasma fractalkine levels in T2D patients associated with systemic inflammation (CRP) (r = 0.65, P = 0.02).ConclusionsThe altered plasma fractalkine levels associate differentially with inflammatory chemokines/cytokines in T2D patients which may have implications for T2D immunopathogenesis.

Project description:ObjectiveWe have previously shown the existence of a muscle-pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated.MethodsGene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1-50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1.ResultsBoth CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in β-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFα on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes.ConclusionsWe demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

Dataset Information

Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons.

Publications

Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons.

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