Neutrophils in Cystic Fibrosis Display a Distinct Gene Expression Pattern Minou Adib-Conquy,1 Thierry Pedron,2,3 Anne-France Petit-Bertron,1 Olivier Tabary,4 Harriet Corvol,4,5 Jacky Jacquot,4 Annick Clément,4,5 and Jean-Marc Cavaillon1 1Unit Cytokines & Inflammation, Institut Pasteur, Paris, France; 2Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France; 3INSERM, U786, Paris, France; 4INSERM U 719 and Université Pierre & Marie Curie, Faculté de Médecine Saint-Antoine, Paris, France; 5AP-HP, Service de Pédiatrie-Pneumologie, Hôpital Armand Trousseau, Paris, France We compared gene expression in blood neutrophils (polymorphonuclear leukocytes, or PMNs) collected from healthy subjects with those of cystic fibrosis (CF) patients devoid of bacterial colonization. Macroarray analysis of 1050 genes revealed upregula- tion of 62 genes and downregulation expression of 27 genes in CF blood PMNs. Among upregulated genes were those coding for vitronectin, some chemokines (particularly CCL17 and CCL18), some interleukin (IL) receptors (IL-3, IL-8, IL-10, IL-12), all three colony- stimulating factors (G-, M-, GM-CSF), numerous genes coding for molecules involved in signal transduction, and a few genes under the control of γ-interferon. In contrast, none of the genes coding for adhesion molecules were modulated. The upregulation of six genes in CF PMNs (coding for thrombospondin-1, G-CSF, CXCL10, CCL17, IKKε, IL-10Ra) was further confirmed by qPCR. In addi- tion, the increased presence of G-CSF, CCL17, and CXCL10 was confirmed by ELISA in supernatants of neutrophils from CF patients. When comparison was performed between blood and airway PMNs of CF patients, there was a limited difference in terms of gene expression. Only the mRNA expression of amphiregulin and tumor necrosis factor (TNF) receptor p55 were significantly higher in air- way PMNs. The presence of amphiregulin was confirmed by ELISA in the sputum of CF patients, suggesting for the first time a role of amphiregulin in cystic fibrosis. Altogether, this study clearly demonstrates that blood PMNs from CF patients display a profound modification of gene expression profile associated with the disease, suggesting a state of activation of these cells. Online address: http://www.molmed.org doi: 10.2119/2007-00081.Adib-Conquy INTRODUCTION response to platelet-activating factor and the neutrophils of CF patients. We com- The properties of circulating neu- untreated or opsonized zymosan (8). In pared PMNs derived from blood and air- trophils from cystic fibrosis (CF) patients contrast, circulating PMNs display a de- way of CF patients to blood PMNs from differ from those of healthy subjects. creased chemotactic response to leuko- healthy individuals. We performed a These observations probably reflect that triene B4 (9) and decreased chlorination macroarray analysis to investigate the mutation or deletion of the CF trans- of phagocytosed bacteria (1). Other mod- mRNA expression of 1050 different genes membrane conductance regulator (CFTR) ifications of cell-surface markers have coding for cytokines, chemokines and may lead to disturbance of blood poly- been reported such as reduced expres- their receptors, apoptosis-related mole- morphonuclear leukocytes (PMNs), ei- sion of Fcγ RIII (CD16) (8) and reduced cules, cellular signaling molecules, and ther directly (1) or as a consequence of shedding of L-selectin (CD62L) upon different cellular metabolism actors. an ongoing inflammation and infection stimulation with either IL-8 or formyl- (2). Particularly, circulating PMNs from methionyl-leucyl-phenylalanin (FMLP) MATERIALS AND METHODS CF patients appear to be primed and can (10). Apoptosis of blood PMNs appears release increased levels of interleukin similar between CF patients and healthy Patient Characteristics (IL)-8 (3), elastase (4), lipoxygenase prod- subjects, but the interaction of CF airway Five CF patients (two boys, three girls) ucts (5), myeloperoxidase (6), and chlo- PMNs with CF respiratory epithelial cells with different genotypes (∆F508/∆F508; ramine (7) in response to proper activa- reduces their apoptosis (11,12). G542X/G542X; N1303K/347delCC; tors. Furthermore, CF blood PMNs In this study, for the first time, we un- ∆F508/G542X; ∆F508/NI), mean age display enhanced oxidative activity in dertook an analysis of gene expression in 10.7 ± 5.6 years, clinical score 80 ± 10, were included in this study. In all pa- tients, the diagnosis of CF was con- Address correspondence and reprint requests to Jean-Marc Cavaillon, Unit Cytokines & firmed by a sweat chloride concentration Inflammation, Institut Pasteur, 28 rue Dr Roux, 75015 Paris, France. Phone: 331 45 68 82 38; of >60 Meq/L and by CFTR gene muta- Fax: 331 40 61 35 92; E-mail: [email protected] tions (13). Results of physical examina- Submitted July 19, 2007; Accepted for publication November 1, 2007. tion, chest radiographs, Schwachman- 36 | ADIB-CONQUY ET AL. | MOL MED 14(1-2)36-44, JANUARY-FEBRUARY 2008 RESEARCH ARTICLE Kulczycki score (14), pulmonary func- healthy subjects, because we found in a bridization scanning were also described tion tests with determination of forced previous study (3) that induced sputa (16). After recording of the signals for vital capacity (FVC) and forced expira- from healthy subjects does not contain each gene with ArrayVision and quality tory volume in 1 s (FEV1), oxygen satu- enough PMNs for isolation and culture. control of hybridizations using the lu- ration (SaO2), and sputum quantitative As in the procedure described above for ciferase signal intensity, data correspon- bacterial cultures were recorded at the the blood PMNs, contaminating mono- ding to all the membranes were trans- time of the study. For pulmonary func- cytes were eliminated after incubation formed in a log2 scale and normalized tion tests, the mean values expressed as with pan-antihuman HLA class II–coated by a method derived from the variance percentage of predicted values were magnetic beads. We followed a similar analysis (ANOVA) to give an equal me- FVC 65.5% ± 18% and FEV1 55% ± 16%. cell-counting procedure and assessment dian signal to all membranes. This statis- None of the CF children tested positive of viability with trypan blue dye exclu- tical method estimates the weight and for Pseudomonas aeruginosa. The control sion. The purity of the PMN suspension significance of variability sources on ex- group included three healthy young was >99% as assessed by May-Grünwald- perimental data. For each condition, 4 bi- adults from the medical staff, mean age Giemsa staining. ological replicates were performed and 30.8 ± 3.2 years, without history of lung hybridized onto macroarrays, where disease and with normal lung function. Cultures of Blood PMNs PCR products were spotted in duplicate; Written informed consent was obtained PMNs were cultured in RPMI 1640 eight signals for one gene were used for from all CF patients or their guardians. supplemented with L-glutamine, antibi- one experimental condition. Compara- The study was approved by the Ethic otics (100 IU/mL penicillin, 100 µg/mL tive analyses between baseline (control Committee of St. Louis University Hos- streptomycin; Life Technology), and 5% blood) and experiment (CF blood or air- pital (Paris, France) (reg. no. CCPPRB heat-inactivated normal human serum way) were done with the dChip software 2004/15). For ethical reasons, it was not (pool of sera from healthy volunteers) in (18), using an unpaired Welch t test with possible to obtain blood samples from absence of any stimuli. Aliquots (0.5 mL; a P value threshold of 0.05. This software healthy children. 5 × 105 cells) of PMN suspension were in- was also used for hierarchical clustering cubated in a 5% CO2 incubator in 24-well using Euclidian distance and average as Isolation of PMNs from Blood Samples multidish plates (Nunc; ATGC Biotech- a linkage method. Before clustering, the The PMNs from venous blood of CF nology, Marne La Vallée, France) for 18 h expression values for one gene across all patients were obtained as described (3). at 37°C. At the end of the culture, the su- samples were standardized to have a In brief, blood PMNs from CF patients pernatants were harvested, centrifuged mean of zero. Increased or decreased val- were isolated by employing glucose dex- for 10 min at 300g and 15°C, and kept at ues were then ranged compared with tran and Ficoll (Amersham Pharmacia –20°C before cytokine measurements. this mean. Macroarray hybridization and Biotech, France) method. To allow the analysis were performed on blood- elimination of contaminating monocytes, Measurement of Amphiregulin, derived PMNs from four patients and on blood PMNs were further purified after CCL17, CXCL10, and G-CSF by ELISA airway-derived PMNs from three CF pa- incubation with pan-antihuman HLA Amphiregulin, CXCL10, and G-CSF tients. They were compared with blood class II–coated magnetic beads (Dyn- were measured with a DuoSet from R&D PMNs from three healthy controls. abeads M450; Dynal, Oslo, Norway) System (Abingdon, UK) and CCL17 with (3,15). The purity and viability of blood a Quantikine kit (R&D Systems) as rec- Quantitative Real-Time PCR PMNs were assessed by blue trypan and ommended by the manufacturer. Total RNA of PMN was prepared May-Grünwald-Giemsa staining. using the RNeasy Mini Kit (Qiagen). Pu- Macroarray Hybridization and rified RNA was reverse-transcribed with Isolation of PMNs from Airway Analysis Superscript II RNase H (Invitrogen) and Samples The macroarray experiments were car- an oligodT 12-18 primer (Invitrogen) ac- Spontaneous sputa were collected in ried out using membranes that were cording to the manufacturer’s protocol. sterile cups and processed immediately. characterized and validated in two pub- The expression levels of genes of interest Airway PMNs in sputa were isolated in lished studies (16,17). Briefly, PCR prod- and GAPDH were determined by real- accordance with a previously adopted ucts of 1050 human genes were spotted time quantitative PCR, using a Brilliant procedure (3).