囊性纤维化传导跨膜调节基因编码CFTR离子通道,负责氯化物和碳酸氢盐穿过质膜的运输。基因突变导致离子运输受损,随后导致所有外分泌腺的分泌紊乱,因此,多器官疾病囊性纤维化(CF)。近年来,一些研究报道了免疫细胞中CFTR的表达,如免疫荧光所证明的,流式细胞术,和免疫印迹。然而,这些数据主要限于单细胞群体,并且根据所使用的方法显示出显著差异.这里,我们使用标准化方案在一组全面的免疫细胞中研究了CFTR转录和蛋白质表达.方法:我们使用高特异性单克隆CFTR抗体的组合应用了高分辨率Westernblot方案,这些抗体已经过优化,用于检测上皮细胞和通过流式细胞术分选的健康初级免疫细胞亚群中的CFTR,并使用永生化细胞系作为对照。通过肽竞争和酶促肽-N-糖苷酶-F(PNGase)消化控制CFTR蛋白检测的特异性。使用定量实时PCR分析CFTR转录物,并标准化为上皮T84细胞的水平作为参考。结果:原代CD4+T细胞可显示CFTRmRNA表达,NK细胞,以及分化的THP-1和JurkatT细胞。相比之下,我们未能检测到CD14+单核细胞和未分化THP-1细胞的CFTR转录本,以及B细胞和CD8+T细胞。通过用靶向CFTR蛋白的不同表位的四种CFTR抗体的组合进行免疫印迹可检测到突出的免疫反应性条带。然而,在非上皮来源的生物样本中,这些CFTR样蛋白带可以通过肽竞争或PNGase消化被揭露为假阳性,这意味着观察到的mRNA转录本不一定被翻译成CFTR蛋白,可以通过免疫印迹检测到。我们的结果证实,免疫细胞中的mRNA表达比上皮起源的细胞低很多倍。免疫细胞中的免疫反应性信号被证明是假阳性,并且可能是由具有相似表位的高亲和力蛋白质的存在引起的。非特异性结合(例如,Fab与糖基分支的相互作用)也可能导致假阳性信号。我们的发现强调了精确控制的必要性,如CFTR阴性细胞,以及肽竞争和糖酵解消化,以便通过免疫印迹鉴定真正的CFTR蛋白。我们的数据表明,此外,CFTR蛋白表达数据来自组织学等技术,对于不存在分子量或其他独立对照的情况,可以防止假阳性免疫反应性信号的掩盖,也必须仔细解释。
The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the
CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on
CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of
CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4+ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14+ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8+ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the
CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine
CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.