10.00 PBS OVA OVA+isotype antibody 8.00 OVA+anti-HMGB1. PBS Methatroline (mg/ml)

Transcription

1 RESEARCH ARTICLE Penh (100% of PBS) 1 PBS anti-hmgb p=0.054 Cellular & Molecular Immunology advance online publication, PBS Methatroline (mg/ml) Neutrophil isolation and culture Neutrophil were isolated using a standard protocol from the blood of by Percoll (GE Healthcare) density gradient centrifugation. Briefly, K 2 EDTA tubes were used to collect the blood. Percoll gradients with a density of were prepared by mixing 9.5 ml Percoll with 1.5 ml PBS (103) and4mlh 2 O. After removal of erythrocytes by dextran sedimentation, the leukocyte-rich supernatant was carefully layered on Percoll gradient, and centrifuged at 400g for 30 min at 20 uc. Then the cell pellet was resuspended in 2 ml PBS, and remaining RBCs were lysed by adding 50 ml RBC-lysis buffer (Boster, China) for 5 min at room temperature. Then, tubes were cen- ; doi: /cmi Supplementary Information accompanies the paper on trifuged at 300g for 5 min at 20 uc, supernatant was carefully Supplemental Figure 1 Effect of anti-hmgb1 on -induced AHR. Data are mean6s.e.m., n58/group. As expected, -induced asthmatic Cellular & Molecular Immunology s website ( pellet was resuspended in 4 ml RPMI-1640 cell culture medium removed, and cells were washed twice with PBS. Finally, cell mice had a significant increase in AHR, anti-hmgb1 antibody administration to -induced mice resulted in reduction tendency in AHR detected by non-invasive methods, but there were no significant difference (P50.054). Supplementary Figure 1 without FBS and cells/ml were plated onto 24 wells for Supplementary Figure 2 resting 1 h in an incubator at 37 uc and cells were treated with Supplementary Figure 3 HMGB1 and HMGB1/IL-1b complex for 30 min. Nontreated Supplementary Figure 4 cells were used as controls. Supplementary Figure 5 Alveolar macrophages cell isolation MATERIALS AND METHODS AM were isolated by BALF. Normal subjects attended the Primary culture of human bronchial epithelial cells (PBECs) bronchoscopy were pretreated with atropine. Oxygen was The normal human bronchial epithelial cells were obtained administered via nasal prongs. Three 60 ml aliquots of warmed from bronchi removed from three patients with lung cancer normal saline were instilled in the right middle lobe via a undergoing lobectomy in the first affiliated hospital of Guangxi wedged fibreoptic bronchoscope (Olympus P240), aspirated Medical University and the study had approved by the Ethics BALF was immediately cooled to 4 uc and filtered through a Committee of Guangxi Medical University. In all cases, specimens were taken from the bronchi distant from the cancer area. at 500g for 10 min. BAL cells were washed twice with sterile sterile gauze to remove particulate debris before centrifugation Bronchi were longitudinally opened and subsequently washed HBSS. Cells were.85% viable as assessed by trypan blue exclusion. Macrophages were purified by adhesion as described.[16] in ice-cold PBS balanced salt solution including penicillin and streptomycin. Dissect bronchi by removing all excess connective tissue and cutting into small tissue segments sized approxiing 5% fetal calf serum, 2 mm L-glutamine and 1% antibiotic The cells were suspended (10 6 cells/ml) in RPMI 1640 containmately 3 6 mm 2. Then explant bronchial tissue are plated onto antimycotic solution and incubated in 24-well plates at 37 uc in collagen-coated (Sigma) plastic 24-well tissue culture plates a humidified atmosphere of 5% CO 2 and 95% air. After 12 h, (Coring). and cultured for days in a mixture of Airway more than 98% of adherent cells were macrophages and layered Epithelial Cell Growth Medium (PromoCell) and at 37 uc ina onto additional Percoll gradients to reach a more higher purity humidified incubator (5% CO 2 ). Then epithelial cells trypsinized and were seeded in 12-well plates ( /well), before HMGB1 and HMGB1/IL-1b complex for 24 h. Non-treated ranging between 95% and 99%. Cells were treated with being challenged with HMGB1 or HMGB1-IL-1b complexes cells were used as controls. for 24 h. AHR measurement with non-invasive method (previous experiment) Twenty-four hours after the final aerosol challenge, AHR to methacholine (Sigma) was determined in unrestrained conscious mice by body plethysmography (Buxco Electronics). Each mouse was exposed to aerosolized PBS, which was followed by increasing concentrations of aerosolized methacholine solutions (3.12, 6.25, 12.5 and 25 mg/ml; Sigma) for 3 min each, airway resistance (enhanced pause (Penh)) values were measured for 5 min. Data were expressed as the percentage increase in the Penh after challenge with each concentration of methacholine, where the baseline Penh (after PBS challenge) was expressed as 100%.

2 AHR measurement (supplementary experiments) volume (10 ml/kg), frequency (120/min) and positive endexpiratory pressure (3 cmh 2 O). Baseline lung resistance was 1 PBS Twenty-four hours after the aerosol challenge, AHR to methacholine (Sigma) was determined in 1 unrestrained+anti-hmgb1 conscious recorded for 3 min and after the challenge with aerosolized p<0.05 mice by body plethysmography (Buxco Electronics). Each methacholine for 20 s at a serial twofold increment from 3.12 mouse was exposed to aerosolized PBS, 8.00which was followed to 50 mg/ml; data were continuously collected from 10 s to by increasing concentrations of aerosolized methacholine solutions (3.12, 6.25, 12.5, 25 and 50 mg/ml; 6.00 Sigma) for 3 min each, express the changes in the airway function of the mice. 2 min and maximum values of lung resistance were taken to airway resistance (enhanced pause (Penh)) values were measured for 5 min. Data were expressed as4.00 the percentage increase RESULTS in the Penh after challenge with each concentration of methacholine, where the baseline Penh (after PBS challenge) was 1. Effects of anti-hmgb1 on -induced AHR detected with non-invasive method (previous experiment). expressed as 100%. To confirm and assess the findings from 2. Effects of anti-hmgb1 on -induced AHR detected noninvasive body plethysmography, respiratory mechanics with non-invasive method and invasive method (additional was determined with invasive method during mechanical PBS 3.12vent- ilation. Mice were anesthetized with 1% pentobarbital sodium Methatroline (mg/ml) experiment). (75 mg/kg) and cannulated with a blunted needle. Mice were Supplemental Figure 2 Effect of anti-hmgb1 -induced AHR by non-invasive method. Data are mean6s.e.m., n58/group. Treatment with placed in a body chamber (Buxco Electronics) and mechanically ventilated. The following ventilator settings were used: anti-hmgb1 antibody resulted in a decrease in airway resistance compared with the group. tidal Penh (100% of PBS)

3 Lung resistance (cmh2o.ml-1.s-1) PBS +anti-hmgb1 p<0.05 PBS Methatroline (mg/ml) Supplemental Figure 3 Effect of anti-hmgb1 on -induced AHR by invasive method. Data are mean6s.e.m., n58/group. Treatment with anti-hmgb1 antibody resulted in a decrease in airway resistance compared with the group.

4 40 TGF b1 secretion from eosionphils (pg/ml) Contro group HMGB1-IL-1b complex # IL-1b 2ng/ml HMGB1 1000ng/ml Supplemental Figure 4 HMGB1/IL-1b complex enhanced TGF-b1 level in supernatants from eosinophils. After isolation from peripheral blood as described in the section on Materials and methods. EOS was cultured in RPMI-1640 medium and 50 mg/ml gentamicin (Hiclone) cells/ml were plated onto 24 wells and were treated with HMGB1 (250 ng/ml and 1000 ng/ml), IL-1b (0.5 ng/ml and 2 ng/ml) and HMGB1/IL-1b complex (HMGB1 250 ng/ml1il-1b 0.5 ng/ml, HMGB ng/ml1il-1b, 2 ng/ml) for 24 h. The cell culture supernatants were collected and then the TGF-b1 level in supernatant was assessed by ELISA ( # P,0.01 versus control group; data are mean6s.e.m.; n54).

5 BALF from mouse 88 kda PBS +isotype +antiantibody HMGB1 antibody Neutrophil 16-HBE PBEC 88 kda A549 HMGB1 (ng/ml) IL-1b (ng/ml) Supplemental Figure 5 Gelatinase production in BALF of mouse and supernatant from 16-HBE, PBECs, A549 and neutrophils analyzed by gelatin zymography /well cells (16-HBE, PBECs, A549) and cells/ml neutrophils were seeded into 24-well plates and then were treated with HMGB1 (1000 ng/ml), IL-1b (2 ng/ml) and HMGB1/IL-1b complex (HMGB ng/ml1il-1b 2 ng/ml) for 24 h (for epithelial cells) or 30 min (for neutrophil). Non-treated cells were used as controls. Then the cell culture supernatants were collected. At last equal volume of samples (30 ml; except 10 ml for neutrophil) were subjected to gelatin zymography as described in the section on Materials and methods.