Laboratory of Ecotoxicology, UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre cedex, France.
Laboratory of Ecotoxicology, UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre cedex, France.
Aquat Toxicol. 2014 Aug;153:98-109. doi: 10.1016/j.aquatox.2013.11.012. Epub 2013 Nov 28.
In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK571) blockers, we show that MXR activity is only carried out by MRP-type transporters in M. edulis hemocytes. In addition, cell-type-gated flow cytometry and calculation of the MXR activity factor indicate that ABCC-efflux activity is higher and more inducible in eosinophilic granulocytes than in other hemocyte subtypes. We conclude that, in the hemocytes of M. edulis, MXR phenotype is mediated by an ABCC/MRP-type transporter activity principally supported by eosinophilic granulocytes. A role for ABC transporters in hemocyte migration is discussed.
在海洋和河口物种中，免疫毒性和/或免疫调节机制是异生物、微生物和环境理化变化相互作用的交叉点。在贻贝类动物中，免疫完全依赖于由细胞共同执行的先天反应，这些细胞被称为血细胞，存在于这些生物体的开放血淋巴循环系统中。然而，血细胞并不是一个同质的免疫细胞群体，因为可以通过细胞化学、流式细胞术或细胞迁移分析来区分贻贝血细胞的不同亚型。先前的研究还表明，这些细胞能够通过 ATP 结合盒（ABC）转运体的活性将异生物排出，从而赋予多异生物抗性（MXR）表型。Mytilidae 中存在与脊椎动物 B/P-糖蛋白（P-gp）和 C/多药耐药相关蛋白（MRP）相对应的 ABC 转运体。在此，我们研究了不同贻贝血细胞亚群中 ABCB 和 ABCC 介导的外排作用的相对贡献，这些贻贝是从诺曼底（法国）受化学污染物影响不同的地区收集的。RT-PCR 分析为血细胞中存在 ABCB 和 ABCC 转运体转录本提供了证据。用单克隆抗体 UIC2 对 ABCB/P-gp 的免疫检测在活血细胞中显示，表达仅限于铺展细胞的颗粒结构。通过将流式细胞术与精确的库尔特细胞尺寸测量相结合，以获得细胞体积归一化荧光浓度，用 calcein-AM 作为荧光探针测量外排转运体活性。在这些条件下，来自 Yport（对照地点）的血细胞的基础荧光水平高于来自勒阿弗尔港的细胞，后者的贻贝更容易接触持久性污染物。通过使用特定的 ABCB/P-gp（维拉帕米、PSC833、佐西喹达）和 ABCC/MRP（MK571）阻断剂，我们表明 MXR 活性仅由 M. edulis 血细胞中的 MRP 型转运体进行。此外，细胞类型门控流式细胞术和 MXR 活性因子的计算表明，嗜酸性粒细胞中的 ABCC 外排活性更高且更可诱导。我们得出结论，在 M. edulis 的血细胞中，MXR 表型是由一种 ABCB/MRP 型转运体活性介导的，主要由嗜酸性粒细胞支持。讨论了 ABC 转运体在血细胞迁移中的作用。
