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N-acetylcysteine promotes apoptotic cell clearance and lung inflammation through inhibition of RhoA

Changsuk Moon, Ye-Ji Lee, Hyun-Jeong Park, Young H. Chong, Jihee Lee Kang

Online Data Supplement

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METHODS Supplemental Materials and Methods

Reagents LPS (Escherichia coli lipopolysaccharide, 055:B5), NAC and zymosan A were purchased from the Sigma Chemical Company (St. Louis, MO). H2-dichlorofluorescein diacetate (H2DCFDA), dihydroethidium (DHE), and calcein-AM were purchased from Molecular Probes (Eugene, OR). The antibodies used in this study were: anti-IB- rabbit polyclonal, antiphospho-IB- (Serine 32) (New England Biolabs, Inc., Beverly, MA). Y-27632, a specific inhibitor of RhoA kinase, was obtained from CalbiochemNovabiochem Corp. (San Diego, CA) and the G-LISA RhoA Activation Assay was obtained from Cytoskeletal Inc. (Denver, CO). [-32P]-dATP was obtained from Amersham (Waltham, MA) and DNA polymerase klenow fragment and dNTPs from Intron Biotechnology (Seoul, Korea).

Animal Protocols Specific pathogenfree male BALB/C mice (Daehan Biolink) weighing 20~25 g were used in all experiments. The Animal Care Committee of the Ewha Medical Research

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Institute approved the experimental protocol. Mice were cared for and handled in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mouse pharyngeal aspiration was performed as described (E1). Animals were anesthetized with a mixture of ketamine and xylazine (45 and 8 mg/kg, i.p., respectively). Following anesthesia, animals were placed individually on a board in a near vertical position and tongues were withdrawn with a lined forceps. 30 l of the test solution containing LPS (4.5 mg/kg) was then placed posterior in the throat and aspirated into lungs. Control mice were administrated sterile saline (0.9 % NaCl). Mice revived unassisted after approximately 10-20 min. Animals were administered NAC (200 mg/kg), i.p. 2 h before LPS treatment and every 12 h (E2) and euthanized at 1, 3, or 6 d post-LPS. For RhoA/Rho kinase pathway inhibition experiments, a Rho kinase inhibitor, Y-27632 (10 or 1 mg/kg) was administered i.p. 30 min before LPS and once a day thereafter (E3). Animals were euthanized at 3 d post-LPS.

Isolation of Bronchoalveolar Lavage (BAL) cells, Lung Tissue, and Cell Counts BAL was performed through a tracheal cannula using 0.7 ml aliquots of ice-cold Ca2+/Mg2+-free phosphate-buffered medium (145 mM NaCl, 5 mM KCl, 1.9 mM

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NaH2PO4, 9.35 mM Na2HPO4, and 5.5 mM dextrose; at pH 7.4) to a total of 3.5 ml for each mouse. BAL samples so obtained were centrifuged at 500 x g for 5 min at 4C, and cell pellets were washed and resuspended in phosphate-buffered medium. Cell counts were determined using an electronic Coulter Counter fitted with a cell sizing analyzer (Coulter Model ZBI with a channelizer 256; Coulter Electronics, Bedfordshire, UK (E4). Neutrophils and alveolar macrophages were identified by their characteristic of cell diameter. BAL cells were isolated and cytospins made to assess phagocytic indices (E5) and the proportion of neutrophils that were apoptotic. After BAL, lungs were removed, immediately frozen in liquid nitrogen, and stored at -70C.

Primary Cell Isolation and Culture Suspended alveolar macrophages were over 95% viable, as determined by trypan blue dye exclusion. Alveolar macrophages were isolated by adhesion (60 min) and cultured in serum-free X-vivo medium (Biowhittaker, Wakersville, MA). Neutrophils were isolated and prepared from normal blood using Percoll gradient centrifugation, as previously described (E6).

Cell lines and Culture

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Murine J774 macrophages (American Type Culture Collection (ATCC) were cultured in DMED supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 g/ml streptomycin, and 100 U/ml penicillin in humidified 10% CO2 at 37C. Human leukemia Jurkat T cell line was obtained from ATCC and was cultured in RPMI 1640 containing 10% heat-inactivated FBS supplemented with penicillin-streptomycinglutamine, and incubated at 37C in 5% CO2.

Histone-bound DNA Fragmentation Assay of BAL cells DNA fragmentation assay was used to provide quantitative analysis of lung cell apoptosis. The BAL cells (105/well) were analyzed for histone-bound DNA fragments using an ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN). In brief, the cells were lysed with lysis buffer at room temperature and the cell lysate was mixed with an antibody solution at room temperature for 2 hours. The wells were washed, and the substrate was then added. After incubation for 10 min at 37C, optical density was measured using a microplate reader at a wavelength of 405 nm.

Flow Cytometry To measure cell membrane changes associated with apoptosis, BAL cells (1x106

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cells/ml) were incubated with Annexin V-FITC (Annexin V Apoptosis Detection Kit; PharMingen, Becton Dickinson, San Diego, CA) and propidium iodide for 15 min in the dark at room temperature. The cells were analyzed on a FACSCalibur system (BD Biosciences, San Jose, CA) with CellQuest software (BD Biosciences) and ModFitLT software (Verity Software House, http://www.vsh.com). The combination of conjugated Annexin V and propidium iodide allowed for the discrimination between early apoptotic cells (Annexin positive), late apoptosis and/or necrotic cells (Annexin V and propidium iodide positive) and viable cells (unstained).

Lactate Dehydrogenase Assays Lactate dehydrogenase (LDH) activity was measured in the first aliquot of the acellular BAL fluid. LDH is a cytoplasmic enzyme that is released when the cell membrane is damaged or lysed. LDH activity was determined by monitoring the LDHcatalyzed oxidation of pyruvate coupled with the reduction of NAD at 340 nm using a commercial kit (Roche diagnostic Systems, Montclair, NJ).

Viability Assays of Alveolar Macrophages Suspended alveolar macrophages were stained with trypan blue (0.08%, 1min) and

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examined with light microscopy. DNA fragmentation of apoptotic cells was detected in the BAL cells using the enzyme terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) kit (Promega, Madison), according to the manufactures protocols. Briefly, the cells (105 alveolar macrophages/ml) on glass coverslips were fixed in 45 paraformaldehyde at room temperature for 25 min and then permeabilized in 0.2% triton X-100 solution in PBS at room temperature for 5 min. Negative staining controls were stained without rTdT Enzyme. Positive staining controls were normal control alveolar macrophages treated with DNase I at 37C for 20 min to cause chromosomal DNA fragmentation. The samples were biotinylated at 37C for 1 h and the endogenous peroxidase were blocked in 0.3% H2O2. The samples were treated with Streptavidin-HRP at room temperature for 30 min and diaminobenzinine (DAB) and cells were counted with the light microscope. Positive staining is indicated by blackbrown.

ROS Production Intracellular ROS generation was measured with 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) using a modified method of Qin et al. (E7). The non-fluorescent compound, H2DCF-DA accumulates within cells upon de-acetylation and then reacts

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with ROS to form fluorescent DCF. Briefly, BAL cells (2x106cells/ml) were stained with 10 M H2DCF-DA in PBS for 30 min at 37C. DCF fluorescence intensity was measured at 488 nm excitation and at 525 nm emission using a fluorescence microplate reader (Molecular Devices, CA). Increases in intracellular ROS were divided by the saline control value and expressed as fold induction. For image analysis of intracellular ROS generation in alveolar macrophages, dihydroethidium (DHE) assays were performed according to a modified protocol from Sanlioglu et al. (E8). Briefly, alveolar macrophages (105 cells) were plated in a 24-well tissue culture plate on glass coverslips and incubated in DMEM without serum containing 10 M DHE for 30 min. The cells were fixed with 2% paraformaldehyde for 15 min. Nuclei were stained with Hoechst no. 33258 (5 g/ml, Calbiochem) for 10 min at room temperature, and slides were then mounted. Images were collected using an LSM Image Examiner on a confocal laser scanning microscope (LSM-5 Pascal Exciter, Carl Zeiss, Germany). In the presence of intracellular ROS, DHE is converted to ethidium and detected as bright red nuclear staining. The red DHE staining was quantified by creating masks and measuring fluorescent mean intensity of staining using LSM image examiner software (Carl Zeiss).

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ROS production in zymosan-stimulated alveolar macrophages was quantified by using DCF-DA assays. Zymosan-stimulated ROS production was determined by unopsonized zymosan A (Sigma, St. Louis, MO) at 2 mg/ml immediately before measurement. Unopsonized zymosan A has been shown to stimulate ROS production from alveolar macrophages but not neutrophils, which only respond to opsonized particles (E9, E10). Increases in zymosan-stimulated ROS production were divided by the saline control value and expressed as fold induction.

Induction of Apoptosis Human neutrophils were stained with 2 M calcein-AM (Molecular Probes, Inc. Eugene, OR) for 20 min in PBS (Golpon HA, FASEB J, 2004). This method allowed us to distinguish them from the endogenous apoptotic neutrophils when the PI was determined. After the dye was removed by washing in medium, neutrophils were cultured at 2.5x106/ml in RPMI with 10% FBS at 5% CO2 at 37C for 24 h before use. Jurkat T cells were exposed to UV irradiation at 254 nm for 10 min and cultured in RPMI 1640 (Media Tech Inc.) for 2.5 h at 5% CO2 at 37C before use. All cells were approximately 70% apoptotic by evaluation of nuclear morphology at the light microscopic level (E11).

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In Vitro Phagocytosis Assays Alveolar macrophages (105) from mice treated with saline, LPS, LPS+NAC, or saline+NAC were plated in each well of a 4-chamber slide glass plate at 37C for 2 h. Primary alveolar macrophages from nave mice and J774 macrophages (105 AM/24-well plates) were treated with NAC (0.5 mM) or Y-27632 (10 M) for 30 min before LPS (10 g/ml) or H2O2 (10 M) stimulation for 20 min (E12, E13). The cells were washed three times before adding the following targets: media controls, viable, apoptotic Jurkat T cells or apoptotic human neutrophils labeled fluorescently were added to macrophage cultures in a ratio 10:1 in 1ml media and incubated for 90 min. Samples were washed prior to Wrights Giemsa staining to remove uningested apoptotic cells. Phagocytosis was quantified using the phagocytic index ((number of apoptotic bodies)/200 total macrophages) x 100) previously described (E14). Each condition was tested in duplicate and the reader was blinded to the sample identification during analysis.

RhoA Activity Assays RhoA activity was measured in the lysates collected from alveolar macrophages (106/well) using an ELISA-based RhoA Activation Assay biochem Kit (G-LISA,

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Cytoskeleton, Denver, CO) according to the manufacturers instructions. Briefly, the cell lysates were added to the wells of the RhoA-GTP affinity plate that coated with the Rhotekin binding domain of RhoA for 30 min. The active GTP-bound form of RhoA was measured using indirect immunodetection followed by a colorimetric reaction at 490 nm on a microplate spectrophotometer. Rho activation pull-down assay was performed in lung tissue homogenate according to the manufacturers indications (Millipore, Temeccula, CA). Active Rho in lung tissue homogenate samples was isolated using Sepharose-bound Rhotekin. Samples were incubated for 1 h, washed, boiled, and run on a 12% SDS-PAGE gel. To ensure equal loading, 50 l of whole tissue lysate were run on the gel for each condition and total Rho levels were evaluated.

ELISAs First BAL fluid samples were assayed using TNF- and MIP-2 (BioSource International, Camarillo, CA), and TGF-1 enzyme-linked immunosorbent assay (ELISA) kits (R &D Systems, Minneapolis, MN) according to the manufacturers instructions. Alveolar macrophages (105/well) from the saline, LPS, LPS+NAC group were plated in a 24 well tissue culture plate and allowed to adhere for 60 min at 37C.

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The wells washed three times to remove nonadherent cells. Cells were cultured with media, viable or apoptotic Jurkat cells (106/well) for an additional 18 h and after centrifugation the acellular supernatants were harvested and TNF- and TGF-1 were measured by ELISA. Myeloperoxidase (MPO) is an enzyme that is found predominantly in the azurophilic granules of neutrophils. Tissue MPO activity correlates significantly with the number of neutrophils determined histochemically in inflamed tissues (E15), and thus, it is frequently utilized to estimate tissue nutrophil infiltration. Lung tissue samples were homogenized in 50 mM potassium phosphate buffer (PB, pH 6.0), and suspended in 50 mM PB containing 0.5% hexadecyltrimethylammonium bromide (HEATAB) (Sigma Chemical Co.). After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 12,000 rpm for 10 min at 4 C. Aliquots were added to the reaction mixture containing 50 mM PB, 20 mM H2O2 solution, and 0-dianisidine (0.167 mg/ml) for 15 min. The reaction was stopped by the addition of 100 l of 0.1% sodium azide, and the optical density at 570 nm was measured. MPO activity was expressed as U/100 mg tissue.

Zymographic Analysis of Matrix Metalloproteinase-9 (MMP-9)

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The gelatinolytic activities of BAL fluid samples were determined by zymography using gelatin copolymerized with acrylamide in gel, as described previously (E16). Aliquots, normalized for equal amounts of protein (5 g) were electrophoresed in 10% SDS-PAGE gels using 0.1% gelatin as substrate without boiling under non-reducing conditions. After removing SDS with 2.5% Triton X-100 for 2 h, gels were incubated for 20 h at 37C in 50 mM Tris-Cl (pH 7.4) containing 10 mM CaCl2 and 0.02% NaN3. The gels were then stained for 1 h in 7.5% acetic acid/10% propanol-2 containing 0.5% Coomassie Brilliant Blue G250 and destained in the same solution without dye. Positions of gelatinolytic activity were unstained on a heavily stained background, and clear zymogram bands were photographed using negative Polaroid 665 film and MMP9 activities were quantified by densitometry using an UltroScan XL laser densitometer (LKB, Model 2222-020). To confirm MMP-9 activity, aliquots of BAL fluid were analyzed by Western blotting using antihuman MMP-9 monoclonal antibody (data not shown).

Measurement of Total Potein BAL sample protein concentration was used as an indicator of blood-pulmonary epithelial cell barrier integrity. Total protein was measured according to the method of

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Hartree (E17), using bovine serum albumin as a standard.

Lung Histology Lungs were fixed with 4% formalin in 0.1 M phosphate buffer, pH 7.2 at room temperature for 24 h, and embedded in optimal cutting temperature mold and frozen at 20C for 1 h. Sections (5 m) were stained with hematoxylin and eosin (H&E). Light microscopic analysis of lungs was performed by blinded observation.

Western Blot Analysis Lung tissue homogenate samples and total cell lysates (50 g protein/lane) were separated on 10% SDS-polyacrylamide gels. Separated proteins were electrophoretically transferred onto nitrocellulose paper and blocked for 1 h at room temperature with Tris-buffered SAL containing 3% BSA. The membranes were then incubated at room temperature for 1 hour with an anti-rabbit phospho-myosin phosphatase target subunit (MYPT-1) at threonine 853, or an anti-rabbit phospho-IB- (Ser32)/IB- and visualized by chemiluminescence (ECL).

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Nuclear Extracts Nuclear extracts were prepared using a modification of the method described by Lee et al. (E18). Nuclear extracts were prepared from lung tissue samples. Aliquots of frozen tissue were placed in a Precellys tissue homogenizer (Bertin Technol., Cedex, France), containing 0.5 ml of buffer A (100 mM HEPES, pH 7.9, 10 mM KCl, 0.1 M EDTA, 0.5 mM DTT, 0.6% Nonidet P-40, and 0.5 mM PMSF) to lyse cells. Supernatants containing intact nuclei, were incubated on ice for 5 min, and centrifuged for 10 min at 5000 rpm. Nuclear pellets were resuspended in buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.42 M NaCl, 1 mM EDTA, and 0.5 mM PMSF) for 30 min on ice. The supernatants, which contained nuclear proteins, were collected by centrifuging at 10,000 rpm for 2 min and stored at 70C.

Electrophoretic Mobility Shift Assay (EMSA) Binding reaction mixtures (10 l), containing 5 g (4 l) of nuclear extract protein, 2 g poly (dI-dC)poly (dI-dC) (Sigma Co., St. Louis. MO), and 40,000 cpm 32P-labeled probe in binding buffer (4 mM HEPES, pH 7.9, 1 mM MgCl2, 0.5 mM DTT, 2% glycerol, and 20 mM NaCl) were incubated for 30 min at room temperature. ProteinDNA complexes were separated on 5% non-denaturing polyacrylamide gels in 1 TBE

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buffer, and autoradiographed. Autoradiograph signals of activated NF-B were quantitated by densitometry using an UltroScan XL laser densitometer (LKB, Model 2222-020, Bromma, Sweden) to determine band intensities. The oligonucleotide used as a probe for EMSA was a double-stranded DNA fragment, containing the NF-B consensus sequence (5'-CCTGTGCTCCGGGAATTTCCCTGGC C-3'), and labeled with [-32P]-dATP (Amersham, Buckinghamshire, UK) using DNA polymerase Klenow fragment (Life Technologies, Gaithersburg, MD). Cold competition was performed by adding 100 ng unlabeled double-stranded probe to reaction mixtures.

Statistical Analysis Values are expressed as mean SEM. Analysis of variance (ANOVA) was applied for multiple comparisons, and Tukeys post hoc test was applied where appropriate. Students t test was used for comparisons of two sample means. of <0.05 was considered statistically significant. A P value

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