Fractalkine Expression Induces Endothelial Progenitor Cell Lysis by Natural Killer Cells

BACKGROUND: Circulating CD34(+) cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN) could target progenitor cell injury by Natural Killer (NK) cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+)-derived Endothelial Colony Forming Cells (ECFC) can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+) progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+) cells expressing FKN was identified as an independent variable inversely correlated to CD34(+) progenitor cell count. We further showed that treatment of CD34(+) circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+) progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients

The neurotoxic effect of 13, 19-didesmethyl and 13-desmethyl spirolide C phycotoxins is mainly mediated by nicotinic rather than muscarinic acetylcholine receptors.

International audienceSpirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric a7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the a4b2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes

FKN expression is induced on CD34⁺ circulating progenitors by inflammatory cytokines and KTR sera.

<p>FKN expression was assessed by flow cytometry after gating of CD34⁺ cells within PBMC isolated from healthy donors after 20 h treatment with (<b>A</b>) sera from transplant patients with high % of CD34⁺FKN⁺ cells or with (<b>B</b>) TNF-α (20 ng/ml) and IFN-γ (50 ng/ml). Serum from healthy blood donors or control medium served as controls. (<b>C</b>) FKN expression was also induced on CD34⁺ cells gated within CD133-purified progenitors after a 20 h treatment with sera from transplant patients.</p

CD34⁺ progenitors expressing FKN are found at higher occurrence in transplant recipients analyzed in reference to gender and matched controls.

<p>(<b>A</b>) % of CD34⁺ progenitors expressing FKN were evaluated by flow cytometry, in 168 kidney transplant recipients (KTR), and a subgroup of 58 KTR was also analyzed in reference to a gender- and age- matched healthy control group (CTL). (<b>B</b>) The % of individuals exhibiting low CD34⁺ cell count is higher in the group of transplant recipients, when analyzed in reference to a group of gender and age-matched controls (n = 58). The 2800 cells/ml cut off value for CD34⁺ cells corresponding to the 25 percentile observed in controls was used as a threshold of low CD34⁺ cell count.</p

Membrane FKN targets CD34⁺-derived ECFC lysis by NK cells.

<p>(<b>A</b>) Cytotoxicity assay performed using PBMC effector cells isolated from 7 independent blood donors as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026663#s4" target="_blank">Methods</a>. Each independent assay was performed in triplicate (n = 7) and analysed using an effector: target ratio = 100∶1. (<b>B</b>) Representative illustration of ECFC lysis. CMFDA labeled target cells were analyzed by epi-fluorescence microscopy on an inverted microscope Nikon Eclipse TE 2000-U with a Plan Fluor 4x /0.13 objective, images were acquired using NIS elements AR software. (<b>C</b>) FKN enhances ECFC lysis by purified NK cells. CMFDA cytotoxicity assay performed using purified NK cells as effectors, effector: target ratio = 10∶1, (n = 3). (<b>D</b>) Addition of FKN neutralizing antibody reduces ECFC lysis. FKN-transfected ECFC were treated with 30 µg/ml of anti-human FKN antibody before addition of PBMC (n = 3). (<b>E</b>) Lysis of FKN expressing ECFC resulted in increased microparticles release after 24 h of incubation with PBMC.ECFC were labeled with the lipophilic DiD tracer and transfected with pCDNA3.1 vector coding for membrane FKN (grey bars) or with pCDNA 3.1 empty vector (white bars) and cultured for 24 h. PBMC were added at an effector: target ratio of 50∶1 and co-cultured with target cells for 4 h and 24 h, (n = 5). DiD positive microparticles released by target cells were enumerated by calculation of </p