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4 4 4 4 4 4 Volatile organic compounds enhance allergic airway inflammation
in an experimental mouse model
Ulrike Bnisch, Alexander Bhme, Tibor Kohajda, Iljana Mgel, Nicole Schtze, Martin von Bergen, Jan C. Simon, Irina Lehmann, Tobias Polte
SUPPORTING INFORMATION
METHODS
Sampling and measurements of VOCs
For the determination of VOCs emitted from PVC flooring, stainless steel sampling tubes filled with Tenax TA were used and preconditioned at 280C under a steady flow of nitrogen for 40 min. Air samples (15 l) in mice cages with installed PVC flooring on the top covers were adsorbed to these tubes. Analysis was carried out with Markes TD-100 Thermal Desorber (Markes International Ltd., USA) coupled to an Agilent 7890A gas chromatographic system with an Agilent 5975C quadrupole mass spectrometer (Agilent Technologies Inc., USA). For separation an Rxi-1ms capillary column (Restek, 60 m, 0.32 mm i.d., 1 m d f ) w i t h h e l i u m a s c a r r i e r g a s w a s u s e d . F o r M S d e t e c t i o n e l e c t r o n i o n i z a t i o n ( E I ) w i t h 7 0 e V w a s a p p l i e d a n d m a s s f r a g m e n t s w e r e d e t e c t e d b e t w e e n 3 3 a n d 3 6 0 m / z i n t h e t o t a l i o n c u r r e n t m o d e ( T I C ) . A r e f e r e n c e s t a n d a r d l i b r a r y ( N I S T M S S e a r c h 2 . 0 , N a t i o nal Institute of Standards and Technology) as well as reference substances were used to identify the VOCs. For the quantification of the VOCs calibration curves were created with the use of reference substances.
Samples of PVC flooring (3cm) were cut into small pieces and placed in a 10 ml head space vial to determine VOC pattern. The samples were equilibrated at a defined temperature for 30min and a 250l aliquot of the head space air was injected to the GC-MS.
To determine concentrations of TXIB and NMP inside the inhalation chamber air sample were collected in gas sampling bulbs (137 mL) during exposure almost daily (except on weekends). Afterwards, sampling bulbs filled with TXIB containing air were flushed with nitrogen 5.0 (= purity > 99.9999 %) at 17 mL/min for at least one hour and escaping TXIB was trapped on three succeeding sorbent tubes filled with Tenax (Chrompack, Germany). Required flow rate and time had been determined in preliminary tests employing gas sampling bulbs with known TXIB concentrations. Sampling bulbs filled with NMP were equipped with a twister (polydimethylsiloxane coated stirring bar from Gerstel, Germany), and NMP was collected from the air phase for 24 h. Subsequent analysis of the adsorbed compound was performed on a 6890N gas chromatograph equipped with a 5973N mass selective detector (both from Agilent, USA), a HP-5 MS capillary column (length: 30 m, i.d.: 0.25 mm), and a thermodesorption unit (Gerstel, Germany). Received peak areas were converted to concentrations in terms of g/m employing external calibrations, which had been prepared by charging sorbent tubes with different amounts of TXIB diluted in toluene and NMP diluted in iso-propanol, respectively, followed by GC analysis as outlined above. Here, concentration ranges from 2.90 to 146 g/m for both, TXIB and NMP.
Culture and treatment of bone marrow-derived dendritic cells (BMDC)
Bone marrow cells were collected from nave Balb/c mice, depleted of red blood cells using ammonium chloride, and grown in RPMI medium containing 10% FCS and 20 ng/ml rmGM-CSF for 7 days. Cells were characterized by staining with an anti-CD11c Phycoerythrin (PE) mAb and 2 x 106 cells/ml were given to 24-well plates and exposed to NMP or TXIB (both 5-100 (g/m3) in the presence of LPS (1 (g/ml) for 18 hrs. To adjust experimental conditions of the in vivo mouse scenario to an in vitro approach employing DCs as test organisms partitioning behaviour of NMP or TXIB between air and mouse, and aqueous medium and cells had to be considered: First, accumulation of NMP/TXIB into mouse was quantified using compounds concentration in the air and the logarithmic octanol/air partition coefficient (log Koa). The latter forms a model parameter to describe the partitioning behaviour of NMP/TXIB between an organic phase (= mouse) and air, and was calculated using ChemProp-software ADDIN EN.CITE Schrmann19972501[1]2501250117Schrmann, G., Khne, R., Kleint, F., Ebert, R.-U., Rothenbacher, C. and Herth, P. A software system for automatic chemical property estimation from molecular structure.Quantitative structure-activity relationships in environmental science - VII Chen, F. and Schrmann, G., Eds., Pensacola (FL), USA.Quantitative structure-activity relationships in environmental science - VIIChen, F. and Schrmann, G., Eds., Pensacola (FL), USA.Quantitative structure-activity relationships in environmental science - VIIChen, F. and Schrmann, G., Eds., Pensacola (FL), USA.1997[ HYPERLINK \l "_ENREF_1" \o "Schrmann, 1997 #2501" 1]. Secondly, to maintain the designated concentrations the required amount, which had to be added to the cell testing scenario was calculated considering NMP or TXIBs partitioning behaviour between water and an organic phase given by the logarithmic octanol/water partition coefficient (log Kow) ADDIN EN.CITE Schrmann19972501[1]2501250117Schrmann, G., Khne, R., Kleint, F., Ebert, R.-U., Rothenbacher, C. and Herth, P. A software system for automatic chemical property estimation from molecular structure.Quantitative structure-activity relationships in environmental science - VII Chen, F. and Schrmann, G., Eds., Pensacola (FL), USA.Quantitative structure-activity relationships in environmental science - VIIChen, F. and Schrmann, G., Eds., Pensacola (FL), USA.Quantitative structure-activity relationships in environmental science - VIIChen, F. and Schrmann, G., Eds., Pensacola (FL), USA.1997[ HYPERLINK \l "_ENREF_1" \o "Schrmann, 1997 #2501" 1]. The supernatant was collected for cytokine measurement.
Co-culture of OVA-pulsed DCs and CD4+ T cell
Bone marrow-derived DCs (Balb/c) were pulsed for 24 h with OVA (200 g/ml), some cells in the presence of NMP or TXIB (100 (g/m3, 3 h). 5 x 105 OVA-pulsed DCs were co-cultured with 5 x 105 separated CD4+ T cells (Milteny Biotek, Bergisch-Gladbach, Germany, > 95%) from DO11.10 mice which are transgenic for T cell receptor recognizing OVA specific peptide ADDIN EN.CITE Murphy19902103[2]2103210317Murphy, K. M.Heimberger, A. B.Loh, D. Y.Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivoScienceScience1720-32504988Amino Acid SequenceAnimalsAntigens, CD4/*immunologyAntigens, CD8Antigens, Differentiation, T-Lymphocyte/*immunologyMiceMice, TransgenicMicroscopy, ElectronMolecular Sequence DataOvalbumin/immunologyPhagocytosisPhenotypeReceptors, Antigen, T-Cell/genetics/*immunologyT-Lymphocytes/cytology/*immunology/ultrastructureThymus Gland/cytology/*immunology1990Dec 212125367http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2125367 [ HYPERLINK \l "_ENREF_2" \o "Murphy, 1990 #2103" 2] and cultured for 72 h to measure cytokine production in the supernatant ADDIN EN.CITE Peterson19981186[3]1186118617Peterson, J. D.Herzenberg, L. A.Vasquez, K.Waltenbaugh, C.Department of Microbiology-Immunology, Northwestern University, Medical School, Chicago, IL 60611-3072, USA.Glutathione levels in antigen-presenting cells modulate Th1 versus Th2 response patternsProc Natl Acad Sci U S AProc Natl Acad Sci U S A3071-6956AnimalsAntibodies/bloodAntigen-Presenting Cells/*immunologyCyclophosphamide/pharmacologyCytokines/*biosynthesisDietEthanol/pharmacologyFemaleGlutathione/*deficiencyInterferon-gamma/biosynthesisInterleukin-12/biosynthesisInterleukin-4/biosynthesisMaleates/pharmacologyMiceMice, Inbred C57BLMice, TransgenicReceptors, Antigen, T-Cell/immunologyTh1 Cells/*immunologyTh2 Cells/*immunologyVaccination1998Mar 179501217http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9501217 [ HYPERLINK \l "_ENREF_3" \o "Peterson, 1998 #1186" 3].
Transfer of OVA-pulsed DCs
Immunization by intratracheal injection of OVA-pulsed DCs was done as described Lambrecht et al. ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_4" \o "Lambrecht, 2000 #2131" 4, HYPERLINK \l "_ENREF_5" \o "Kuipers, 2009 #2132" 5]. Briefly, bone marrow-derived DCs were pulsed for 24 h with OVA (200 g/ml), some cells in the presence of NMP or TXIB (100 (g/m3). 2 x 106 cells in 60 (l PBS were adoptively transferred into the trachea in anesthetized nave Balb/c mice. From day 10 onward, mice were challenged i.n. with OVA for 3 consecutive days. AHR was measured on day 13 and mice were sacrificed the following day.
VOC exposure of epithelial cells
Human alveolar epithelial cells (A549, ATCC No: CCL-185; LGC Promochem, Wesel, Germany) were cultured and exposed to VOC using an air-liquid cell culture model as described previously ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_6" \o "Lehmann, 2008 #2508" 6, HYPERLINK \ l " _ E N R E F _ 7 " \ o " F e l t e n s , 2 0 1 0 # 2 3 1 2 " 7 ] . B r i e f l y , t i s s u e c u l t u r e i n s e r t s ( t r a n s w e l l s ) w i t h 0 . 4 m p o l y e s t e r m e m b r a n e s ( C o r n i n g , N Y , U S A ) w e r e p l a c e d i n t o 6 - w e l l p l a t e s a n d 2 x 1 0 5 A 5 4 9 c e l l s w e r e s e e d e d i n t o e a c h c u l t u r e i n s e r t a n d g r o w n f o r 2 d a y s . After replacement of culture medium with exposure medium consisting of equal volumes RPMI 1640 and CO2 independent medium (Invitrogen, Karlsruhe, Germany) transwells-containing dishes were transferred into pre-warmed flasks, exposed to the selected VOC (diluted in methylcyclopentane) by addition of 10 l d i r e c t l y i n t o t h e g l a s s f l a s k . F o r c o n t r o l s , 1 0 l m e t h y l c y c l o p e n t a n e w e r e a d d e d a n d c e l l s w e r e i n c u b a t e d f o r 2 4 h a t 3 7 C .
W e s t e r n B l o t
L u n g t i s s u e s f r o m m i c e a f t e r s h o r t - t e r m e x p o s u r e t o d i f f e r e n t c o n c e n t r a t i o n s o f N M P ( 1 9 a n d 5 1 ( g / m 3 ) o r T X I B ( 9 a n d 32 (g/m3) on two consecutive days for 6 hours were homogenized 18 hours later in 500 l lysis buffer (2% SDS, 20% glycerol, 0.5 mM EDTA, 250 mM Tris/HCl pH 8.4 including 0.5% Complete Proteinase Inhibitor Cocktail). Human alveolar epithelial cells were washed with cold PBS and lysed by applying 200 l lysis buffer. Protein concentrations were measured using DC Protein Assay according to the manufacturer's instructions (Bio-Rad Laboratories, Munich, Germany). Proteins were separated under denaturing conditions on 4-12% NuPAGE Bis-Tris gradient gels (Invitrogen, Karlsruhe, Germany) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). Blotting efficiency was controlled by staining the gels with Ponceau S (Merck, Darmstadt, Germany). Membranes were blocked using ECL blocking reagent (GE Healthcare, Freiburg, Germany) and proteins were detected using GSTP-1- (Kamiya Biomedical Company) and - a c t i n - ( S i g m a - A l d r i c h ) s p e c i f i c a n t i b o d i e s a s w e l l a s h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d s e c o n d a r y a n t i - m o u s e I g G - s p e c i f i c a n t i b o d i e s ( B i o - R a d , M u n i c h , G e r m a n y ) . B a n d s c o r r e s p o n d i n g t o t h e p r o t e i n s o f i n t e r e s t w e r e v i s u a l i s e d u s i n g t h e E C L A d v a n c e W e s t e r n Blot Detection Kit (GE Healthcare) and a FluorChem 8900 imager (Alpha Innotech, San Leandro, USA) and quantified using the free software ImageJ (NIH, USA; http://rsbweb.nih.gov/ij).
Measurement of 8-isoprostane
In a short-term protocol mice were exposed 6 hours/day to NMP (19 and 51 (g/m3) and TXIB (9 and 32 (g/m3) on two consecutive days. 18 hours after the last VOC exposure (independently from the protocol) lung tissues were homogenized in buffer (0.1 M phosphate, 1 mM EDTA, 10 M indomethacin and 0.05% butylated hydroxytoluene, BHT at pH 7.4) using lysing matrix A (FastPrep24 homogenizer, MP Biomedicals, LLC, Eschwege, Germany). Aliquots of the obtained supernatants were hydrolyzed (15% KOH) and deproteinized (ethanol containing 0.01% BHT). 8-isoprostane concentration was determined by specific immunoassay according to manufacturers instructions (Cayman Chemical Company, Ann Arbor, MI, USA). Protein concentration of 5 l homogenates were measured at 700 nm using DC Protein Assay (Bio-Rad, Mnchen, Ger m a n y ) c o m p a r i n g i t t o d i l u t e d s e r i e s o f b o v i n e s e r u m a l b u m i n a s s t a n d a r d .
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