The ethnic review board of Ruijin hospital affiliated to Shanghai Jiaotong University school of medicine approved the study, and informed consent was obtained from all subjects. According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) and the Chinese guideline on Chronic Obstructive lung disease, 51 severe stable COPD patients who were in stage III and IV with FEV1 less than 50% predicted value were investigated in this study. Patients who had an exacerbation in the previous 6 weeks or who were hospitalized in the previous 3 months were excluded from the study. Patients on oral steroids or who concomitantly suffered from bronchiactasis, chronic fungus infections and asthma were also excluded.
Induced sputum processing
Spirometry was recorded 15 min after inhalation of 200ug salbutamol via a metered-dose inhaler. Subjects inhaled 3% saline at room temperature, nebulized via an ultrasonic nebulizer (ShuangYu® Ultrasonic nebulizer, China) at maximum output for 20 minutes. Subjects were encouraged to cough deeply. Sputum was coughed into polypropylene pots. Saliva was discarded. If bothersome symptoms occur, the nebulization should be stopped and the subject treated with salbutamol. After the sputum induction, spirometry was repeated. If the FEV1 falls by more than 20%, the subject was required to wait until it had returned to baseline value.
The sputum samples were kept at 4°C for not more than 2 h prior to further processing. The portion of the sample for cell counting was diluted with 8 ml 1% dithiothreitol (DTT) (shanghai ZiYi reagent, China) and gently vortexed. The resultant cell suspension was then filtered through 400 μm sieves. After centrifuge with 1500 rpm for 10 minutes, cell pellet were washed twice with PBS containing 2% FCS, then resuspended in PBS. Total cell counting was carried out on a hemacytometer and cell viability was calculated by using the trypan blue. Cells were adjusted to a concentration of 1 × 105/ml and use 0.5 ml cell suspension for flow cytometry staining. The remaining cells were centrifuged and resuspended to be used in later RT-PCR testing.
For extracellular staining, 10 ul mouse anti-human CD14 FITC monoclonal antibody and 10 ul of mouse anti-human TLR2 PE monoclonal antibody or relative IgG antibody were added, incubated for 1 hour. For intracellular staining, 10 ul mouse anti-human CD14 FITC monoclonal antibody was added to the cell suspension and incubated for 1 hour in room temperature first. The sample was then fixed with 4% paraformaldehyde for 20 minutes. The cells were resuspended with 200 ul 0.1% saponin for 10 minutes. Mouse anti-human TLR2 PE monoclonal antibody 10 ul or relative IgG antibody were added to the sample and incubated for 1 hour. (Antibodies all from eBioscience, USA) Cells were then resuspend in PBS and acquired on a flow-cytometry. The expression of extra and intracellular TLR2 from macrophages was identified by both positive staining for TLR2 and CD14 staining. (BECTON-DICKINSCN FACS Calibur, BD Biosciences) Cellquest software was used for the analysis (eBioscience, USA).
We examined the expression of TLR2mRNA and TNFαmRNA expression in the induced sputum from severe COPD subjects of long term ICS therapy compared to subjects on no long term therapy. Among all the sputum samples, 12 in ICS naïve group and 10 in ICS group have the remaining cells for RT-PCR testing after flowcytometry testing. Target gene expression was analysed using quantitative real-time PCR. Briefly, RNA was extracted and reverse-transcribed to cDNA. The oligonucletoide primers for TLR2 were: upper: 5′ GGG TCT TGG GGG TCA TCA T 3′; lower: 5′ CAG AGC CTG GAG GTT CAC A 3′; Primes for TNFαwas: upper:5′ AGA GGG AGA GAA GCA ACT ACA G 3′; lower: 5′ CCG TGG GTC AGT ATG TGA G 3′. Reactions were characterized by comparing the threshold cycle (CT) values. Taqman qPCR probes for TLR2 and TNFαmRNA were purchased in kit form, combined with the reference gene eukaryotic 18S ribosomal RNA in dupliate real-time PCRs chains (ABI prism7900 Real Time PCR Machine, Applied Biosystems, USA) and results were calculated using 2-ΔCt relative to the housekeeping gene (18S) and an internal calibrator. (2-ΔCt = the difference in threshold cycles for the test gene and beta actin).
Expression levels are expressed as means +/− SD. Comparisons between the two groups were performed by SPSS 11.0 version software of independent-samples T test. Use Pearson correlation test for determine the association between mRNA level and TLR2 expression. A p value < 0.05 was considered significant.