Ten milliliters of sterile saline was instilled into a lung subsegment (either the right middle lobe or lingula) followed by instillation of LPS (in 10 ml saline) from Escherichia coli (United States Pharmacopeial Convention, Lot G, Bureau of Biologics, United States Food and Drug Administration, Rockville, MD) into the contralateral lung, using a flexible video bronchoscope. The subjects were randomized to left or right lungs for saline or LPS instillation. The subjects received LPS at a dose of 4 ng/kg body weight. A bilateral bronchoalveolar lavage (BAL) was performed 6 hours post-challenge; seven successive 20 ml aliquots of pre-warmed 0.9% saline were instilled in the saline-challenged subsegment of the lung and each aspirated immediately with low suction. This procedure was repeated in the LPS-challenged subsegment of the contralateral lung.
Extracted molecule
total RNA
Extraction protocol
BAL fluid (BALF) was immediately centrifuged for 10 min at 1200 rpm at 4°C. BALF cells were passed over a 40 µm nylon filter (BD Falcon, Bedford, MA) and resuspended in ice-cold sterile automated magnetic cell sorting and separation (autoMACS) buffer (PBS, 0.5% bovine serum albumin, 2 mM EDTA; pH = 7.4). Subsequently, cells were incubated for 15 min with CD71 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C. Cells were washed again in autoMACS buffer and purified by autoMACS (Miltenyi Biotec). Total and viable cell counts were determined before and after the isolation procedure using a Burker-Turk haemocytometer and trypan blue (Emergo, Landsmeer, the Netherlands). Additionally, cytospins were prepared before and after autoMACS and stained with Giemsa. Total and differential cell counts revealed a recovery of 35-40% macrophages and a purity of isolated macrophages of >95% in all groups. After isolation, alveolar macrophages were dissolved in RNAeasy lysis Buffer (buffer RLT, QIAGEN, Hilden, Germany) and stored at -80°C until used for RNA isolation with RNeasy Mini kit (QIAGEN, Hilden, Germany). Hence, in each subject alveolar macrophages were harvested from a lung segment instilled with LPS and from a contralateral lung segment instilled with sterile saline.
Label
biotin
Label protocol
cDNA was synthesized from 50 ng of total RNA using the WT-OvationTM System (NuGEN, San Carlos, CA, USA) powered by Ribo-SPIATM technology. Fragmented cDNA was end labeled with a biotin-conjugated nucleotide analog (DLR-1a; Affymetrix, Santa Clara, CA, USA) using terminal transferase (Roche Diagnostics, Mannheim, Germany).
Hybridization protocol
Fragmented and labeled cDNA was hybridized for 18 h at 50°C in a hybridization solution containing 7% DMSO. Hybridization was performed using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix). After washing, chips were stained with streptavidin-phycoerythrin according to Affymetrix EukGE-WS2v4 protocol using the Fluidic FS450 station.
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner 3000. Affymetrix GeneChip Operating Software version 1.4 (GCOS) was used to manage Affymetrix GeneChip array data and to automate the control of GeneChip fluidics stations and scanners.
Data processing
Expression data were generated using the Robust Multi-array Average (RMA) method implemented in the Affy package of the Bioconductor microarray analysis environment. Before the analysis of differentially expressed genes, a filter was applied to minimize the technical noise. In both groups, the genes with an expression level under a threshold value corresponding to the overall median value from all microarrays were removed. Linear model for microarray, as implemented in the LIMMA package available on the Bioconductor environment, was selected to identify differentially expressed genes
