CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) IN HUMAN LEUKOCYTES

AIM: 1) to evaluate the cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and functional activity in monocytes; 2) to create immortalized cell lines from human B-lymphocyte cells characterized by different genotypes; 3) to evaluate CFTR protein expression in immortalized B cells. BACKGROUND: Cystic Fibrosis (CF), the most common autosomal severe disorder in Caucasians, is caused by mutations in the CFTR gene. Although CF is a multi-organ disease, the lung pathology is the main cause of morbidity and mortality of CF patients. It is characterized by chronic inflammation as a consequence of persistent bacterial infections by several opportunistic pathogens. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of CFTR in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes and display a remarkable variability in cell morphology although common molecular and cellular functions. Although expression of CFTR in alveolar macrophages has been described, its expression has not been reported in monocytes that are more accessible for expression studies and functional analysis tests than macrophages. Evaluation of expression and functional activity of CFTR in peripheral blood mononuclear cells (PBMC) is a pre-requisite to evaluate their role and their potential use in diagnostic and developing new drugs acting on the molecular defect of CF. METHODS: Purification of monocytes and lymphocyte B cells from whole blood; production of Epstein-Barr Virus (EBV); immortalization of Lymphocytes B cells by EBV; RNA isolation and CFTR mRNA analysis by reverse-transcription and polymerase chain reaction (PCR); quantitative real-time PCR (RT-qPCR); western Blotting; Flow cytometry assay; immunofluorescence; cell depolarization assay; Nasal Potential Differences (NPDs) assay; analysis of cell depolarization assay data. RESULTS: In this study western blotting using a polyclonal and two monoclonal anti-CFTR antibodies that recognize different epitopes detected all known forms of CFTR. Flow cytometry and confocal microscopy analysis confirmed expression of CFTR protein expression and its membrane localization. Increased fluorescence intensity, corresponding to membrane depolarization, was observed only when non-CF monocytes were stimulated with CFTR agonist, while CF monocytes did not show fluorescence variation. These results suggested a correlation between CFTR activity and membrane depolarization and data were confirmed using a specific CFTR inhibitor, CFTR (inh)-172. This approach was compared to NPD measurements performed in a subset of the same patients subjected to this analysis. Results obtained by NPD overlapped those obtained by the analysis of monocytes from non-CF donors and CF patients. B-lymphocytes were then immortalized by EBV and were tested as potential cell models for CFTR activity assays. The major glycosylated form of CFTR was detected in immortalized non-CF EVB-transformed B cell line by a monoclonal anti-CFTR antibody, but a band with minor molecular weight was also detected with this antibody and with a polyclonal anti-CFTR antibody. Flow cytometry and confocal assay allowed us to confirm CFTR expression and membrane location in these cell lines. Membrane depolarization test was applied in EBV-transformed B cells and the results confirmed a stimulus induced membrane depolarization in non- CF cells. CONCLUSION: We have demonstrated that CFTR proteins are expressed in human monocytes as a variant recognized by a specific antibody. Its molecular weight is consistent with a lower level of post-translational processing and its loss in patients carrying a homozygous non-sense mutation confirmed its presence in human monocytes. Flow cytometry could be also a useful method to evaluate CFTR expression. We demonstrated that it can distinguish between non-CF and HTZ subjects and CF patients analyzing stained CD14/Rb-AF488 monocytes. Single-cell membrane depolarization analysis confirmed that, upon stimulation with CFTR agonists, normal monocytes displayed a highly reproducible membrane depolarization activity consistent with the expression of functional CFTR. Single-cell depolarization assay could be performed within a few hours after blood collection. It is also easily repeatable with a minimal discomfort and risk for the patient and it could thus allow a time-course evaluation of effects of any particular therapy on CFTR expression or functional activity. A specific activity index was devised that appears capable to discriminate among CF and non-CF cells. Overlapping NPD data and functional activity data, we observed a perfect correspondence. Since NPD is a reference diagnostic test applied when a subject has borderline sweat test and at least one unidentified CFTR mutation, we might promote the evaluation of CFTR activity in monocytes by optical techniques as a useful tool to assess CFTR activity for basic and translational research, including drug development and diagnosis. As primary cells are available in limited amounts, we have taken advantage of the observation that CFTR-associated Cl- permeability has been demonstrated in lymphocytes. So, immortalized-B-cells could be useful as cellular model to study CFTR expression and activity. We observed a form of CFTR that likely represents a processed isoform possibly linked to specific calpain activity in lymphocyte cells as demonstrated in the literature. Furthermore, the index obtained by single-cell fluorescence imaging discriminated between non-CF and CF groups as shown in monocytes. All these results demonstrated that CFTR protein is expressed and is active in human lymphocytes and EBV-transformed B cells opening interesting perspectives in this field. Indeed, these cells can be exploited to evaluate the response of specific mutations to newly developed drugs acting directly or indirectly on the basic defect of CF.