Materials and Methods
Patient Selection
Patients with ARDS that were enrolled in the Acute Lung Injury Specialized Center of Clinically Oriented Research randomized trial of granulocyte-macrophage colony stimulating factor conducted at the University of Michigan between July 2004 and October 2007 were studied. Samples from eighteen patients were included in the microarray analysis, whereas samples from a larger group of patients were used for measurement of S100A12 and IL-1R2 quantitation. Criteria for ARDS were as follows: Acute onset of illness with: 1) PaO2/FiO2 ≤ 300; 2) bilateral infiltrates consistent with pulmonary edema on frontal chest radiograph; 3) requirement for positive pressure ventilation via an endotracheal tube; 4) no clinical evidence of left atrial hypertension; 5) if measured, pulmonary arterial wedge pressure ≤ 18 mm Hg; and 6) the aforementioned criteria occurring together within a 24-hour interval. These subjects underwent serial bronchoscopy with BAL and peripheral blood collection at various time points post onset of ARDS. For the microarray analysis, a subset of 18 patients were studied. In this subset, sample collection was performed between days 0–4 from the time patients fulfilled the diagnostic criteria for ARDS and only samples collected from patients randomized to the placebo control arm of the study or the observational study were utilized. Of the 18 patients enrolled in the microarray sub-study, 17 patients had blood samples drawn for buffy coat analysis, and 7 patients underwent bronchoscopy for the collection of BAL for AM analysis. Serial blood and BAL samples were also collected from an additional 70 ARDS patients for a total of 88 samples analyzed for cytokine levels by ELISA. As with microarray samples, only patients from the observational or placebo arms were analyzed. A single blood and BAL sample were collected from 5 healthy subjects following informed s and under protocols approved by the University of Michigan Institutional Review Board, which served as controls for both the microarray and ELISA analyses. All control subjects were less than 55 years of age, on no medications, and were life-long non-smokers. Demographic and clinical data of the microarray study population are summarized in Table 1. Demographic and clinical data of the larger population are summarized in Table 2.
Sample Preparation
Peripheral whole blood samples were collected from ARDS patients and healthy controls into heparin-containing Vacutainer® tubes (Becton-Dickinson, Franklin, NJ, USA) and centrifuged (1500 rpm for 10 minutes at room temperature). The buffy coat cell layer was carefully aspirated and buffy coat cells were suspended in Trizol® reagent (Invitrogen; Grand Island, NY). Samples were stored at −20°C. The remaining plasma was stored at −80°C.
Bronchoalveolar lavage fluid was obtained from control subjects and ARDS patients by bronchoscopy using a standard technique as previously described. The BAL was centrifuged (1500 rpm at 4°C for 10 minutes). Cell free supernantant was removed and stored (−70°C). Cells were resuspended and plated in plastic culture dishes containing media (RPMI, manufacturer, state, USA). After a one hour incubation (5% CO2, 37°C), non-adherent cells were removed by washing, and adherent macrophages were resuspended in Trizol® reagent and stored (−20°C). Cell differentials post adherence revealed >90% macrophages by morphology, and highly expressed both CD163 and CD14. The mRNA expression of CD163 and CD14 by AM isolated from ARDS patients was not different than that expressed by AM collected from control patients (data not shown).
Microarray Analysis
Frozen buffy coat and AM samples were shipped to Centocor (Radnor, PA).
The quantity and purity of RNA were measured using a Thermo Scientific NanoDrop 1000 Spectrophotometer (NanoDrop technologies, Berlin, Germany) while the quality of RNA was determined by running aliquots on the 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Samples with a RNA integrity number <6.5 or a concentration <50 ng/μl were excluded from the study while samples with a RNA integrity number ≥6.5 and a concentration ≥50 ng/μl were labelled. TotalRNA was reverse transcribed and product cDNA amplified, fragmented, and labelled using the Ovation RNA Amplification and cDNA Biotin System (NuGen). Labelled cDNA was hybridized to Affymetrix GeneChip HT HG-U133+ PM Array plates. After washing and staining cell files were produced from scanned images (High Throughput Array Plate Scanner, Affymetrix) using Affymetrix Control Console software.
ELISA
Cell-free BAL fluid and plasma from healthy controls and ARDS patients were assayed for human IL-1R2 using a Duo-Set ELISA (R&D Systems, Minneapolis, MN) and S100A12 using a CircuLex EN-RAGE ELISA Kit (MBL International, Woburn, MA). Assays were performed in accordance with the manufacturers' instructions.
Statistical Analysis
Statistical analyses of microarrays were performed using ArrayStudio Version 5.0 (Omicsoft, NC). Data were normalized using robust multichip average method and log2-transformed and assessed by probset intensity, principle component analysis, hierarchical cluster analysis and sample correlation. General linear model with proper contrast tests were used. Differential expressed genes were identified using following criteria: a fold change of at least 2, raw p-value < 0.01 and estimated least squares mean intensity greater than or equal to 4 for at least one group in the comparison. Corrected p values using the Benjamini-Hochberg method were generated through the use of IPA (Ingenuity® Systems, www.ingenuity.com) to control for false discovery. Pathway and network analyses were also generated through the use of IPA.
Plasma and BAL IL-1R2 and S100A12 ELISA data were Log10 transformed and/or range scaled, as required, to achieve normal distribution for the performance of parametric statistical tests. The mean normalized concentrations of IL-1R2 and S100A12 in healthy controls were compared to the respective mean concentrations of these cytokines in ARDS patients using a one-way analysis of variance followed by a Dunnett's post-test for multiple comparisons. The strength of associations between ARDS patient plasma and BAL cytokine concentrations and clinical phenotype data (e.g., APACHE III scores) were determined by logistic regression analysis. Statistical analyses were performed using GraphPad Prism 6.0 software for Windows (GraphPad Software, San Diego, CA).