GENERAL THORACIC
Functional Roles of Tumor Necrosis Factor-Alpha and Interleukin 1-Beta in Hypoxia and Reoxygenation Heather E. Merry, MD, Patrick Phelan, MD, Matthew Doaks, BS, Minqing Zhao, MD, PhD, and Michael S. Mulligan, MD Division of Thoracic Surgery, Department of Surgery, University of Washington, Seattle, Washington
Background. Intercellular signaling plays an important role in the development of lung ischemia–reperfusion injury. However, the role of specific mediators remains poorly characterized. Alveolar macrophages (AM) produce soluble mediators early in reperfusion, which modulate the responses of endothelial and epithelial cells to oxidative stress. There is a burst of proinflammatory cytokine production in a variety of cells; however, interleukin 1-beta (IL-1b) and tumor necrosis factor-alpha (TNF-a) localize to the AM. We hypothesized that these cytokines account for the costimulatory effects that AM exert on endothelial and epithelial cells. Methods. Activated AM media was placed on cultured rat type 2 pneumocytes and pulmonary artery endothelial cells, which were then subjected to hypoxia and reoxygenation. To assess the contributions of IL-1b and TNFa, the cells were treated with control media or media that had been depleted of IL-1b or TNF-a. To deplete specific cytokines, activated media was passed through a column
with immobilized IL-1b or TNF-a antibodies. Nuclear translocation of transcription factors, mitogen-activated protein kinase activation, and cytokine and chemokine production were assessed. Results. Depletion of IL-1b or TNF-a effectively eliminated the ability of AM media to enhance the response of endothelial and epithelial cells to oxidative stress. There were significant reductions in monocyte chemotactic protein 1 and cytokine-induced neutrophil chemoattractant (CINC) production (p < 0.05) at 4 hours of reperfusion. Additionally there was decreased nuclear translocation of nuclear factor-kappa B, and extracellular signal-regulated kinase phosphorylation. Conclusions. Interleukin 1-beta and TNF-a are critical mediators in the intercellular communication pathways that allow the AM to enhance the response of surrounding cells to oxidative stress. (Ann Thorac Surg 2015;99:1200–5) Ó 2015 by The Society of Thoracic Surgeons
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with effective, but incomplete, protection. Furthermore, simultaneous blockade of TNF-a and IL1-ß was additive in providing a protective benefit [12]. This suggests that these cytokines are working through independent parallel pathways to drive the early phase of LIRI. Within 15 minutes of reperfusion there is a burst of TNF-a and IL-1b, which localizes to the alveolar macrophage (AM) [12]. Although clinically impractical, depletion of AM with gadolinium chloride or clondronate leads to significant protection in an experimental model of LIRI [13, 14]. This suggests that the AM plays a key coordinating role in this early phase of LIRI and that likely this is through production of TNF-a and IL-1b. This series of experiments is designed to characterize the role of the AM in amplifying the early response in LIRI. Previously we have shown that oxidative stress triggers mitogen-activated protein kinase activation leading to upregulation of proinflammatory transcription factors (such as nuclear factor-kappa B [NFkB], early growth response protein 1 [EGR-1], and activator protein 1 [AP-1]), which in turn causes elaboration of proinflammatory mediators in three primary lung cell types: pulmonary artery endothelial cells (PAEC), type 2 pneumocytes (T2P), and AM [15–18]. We hypothesize that in addition to the direct effect of oxidative stress, the AM plays a key role in priming surrounding cells to the
ung ischemia–reperfusion injury (LIRI) remains a significant complication after lung transplantation, leading to early graft dysfunction and the development of bronchiolitis obliterans [1, 2]. Previous work has demonstrated that the pathogenesis of LIRI is biphasic in nature [3]. The late phase is dependent on neutrophil recruitment and activation, and is characterized by increased vascular permeability and a heterogeneous chemokine and cytokine milieu, whereas the early phase is neutrophil independent and characterized by a predominance of tumor necrosis factor-alpha (TNF-a) and interleukin 1-beta (IL-1b). Both TNF-a and IL-1b are proinflammatory cytokines that lead to leukocyte chemoattraction, phagocyte stimulation, enhancement of downstream cytokine and chemokine production, and variable effects on cell growth and death. Previous work has demonstrated a functional role for these cytokines in multiple models of ischemia– reperfusion injury, including liver [4, 5], heart [6, 7], kidney [8], brain [9], gut [10], limb [11], and lung [12] ischemia and reperfusion. In an in vivo experimental model of LIRI, blockade of TNF-a or IL-1b was associated Accepted for publication Nov 17, 2014. Address correspondence to Dr Mulligan, University of Washington Medical Center, 1959 NE Pacific St, Box 356310, Seattle, WA 98195; e-mail: [email protected].
Ó 2015 by The Society of Thoracic Surgeons Published by Elsevier
0003-4975/$36.00 http://dx.doi.org/10.1016/j.athoracsur.2014.11.042
MERRY ET AL CYTOKINES IN LUNG REPERFUSION INJURY
Abbreviations and Acronyms AM AP-1 EGR-1 EMSA FBS IL-1b LIRI MCP-1 NFkB PAEC PBS PBSt
= = = = = = = = = = = =
alveolar macrophages activator protein 1 early growth response protein 1 electromobility shift assay fetal bovine serum interleukin 1-beta lung ischemia–reperfusion injury monocyte chemotactic protein 1 nuclear factor-kappa B pulmonary artery endothelial cells phosphate-buffered saline phosphate-buffered saline with Tween TNF-a = tumor necrosis factor-alpha T2P = type 2 pneumocytes
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culture plates. Media was then substituted with fresh RPMI containing 5% heat-inactivated fetal bovine serum (FBS).
Pulmonary Artery Endothelial Cell Culture
Material and Methods
Heart-lung blocks were rapidly excised as described above. Endotracheal lavage of the lungs was performed 15 times with 6 to 9 mL of PBS containing 0.25 mmol/L EDTA to deplete alveolar macrophages. Strips of peripheral lungs (2 mm) were excised from all lung lobes. The peripheral tissue was minced, rinsed in RPMI, transferred to a dispase (10 mg/mL) solution, and incubated for 60 minutes at 37 C [15, 16]. The cell suspension was homogenized and incubated for an additional 5 minutes. Complete media with 10% FBS was added to terminate the reaction, and the cellular suspension was filtered through a 100-mm mesh. The filtrate was centrifuged at 800g for 8 minutes, and the cell pellet was resuspended in supplemented RPMI media and plated on gelatin-coated culture dishes. Media was changed every 48 hours until the cells were confluent. Once confluent, the cells were labeled for 8 hours with 4 mg/mL acetylated low-density lipoproteins, which bind selectively to endothelial cells. Cells were separated using flow cytometry (FAC STAR Plus, San Jose, CA). The pure cultures of endothelial cells were then maintained in RPMI with 10% FBS. All cells used in these experiments were from passages 4 through 10.
Reagents
Type 2 Pneumocyte Culture
All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise specified.
A rat type 2 pneumocyte cell line, RLE-6TN [19] (American Tissue Cell Company, Manassas, VA) was maintained in Ham’s F-12 culture media containing 10% heat-inactivated FBS. Cells were cultured in 12-well plates at a density of 105 cells/mL. Culture media was replenished every 48 hours until 95% confluence was reached. Cell counts and viability were assessed by standard trypan blue exclusion techniques [20].
effects of oxidative stress and amplifying their response. Furthermore, we hypothesize that the AM does this through two parallel pathways, one TNF-a–dependent and one IL-1b–dependent. To elaborate these pathways and determine the downstream effects of these cytokines, we used a series of media transfer experiments with specific cytokine depletion.
Alveolar Macrophage Harvest Pathogen-free adult male Long-Evans rats (Simonsen Labs, Gilroy, CA) weighing 250 to 300 g were used for all experiments. The University of Washington Animal Care Committee approved all experimental protocols. Animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). Animals were euthanized with 120 mg/kg of intraperitoneal pentobarbital. A 14-gauge angiocatheter was inserted into the trachea through a midline neck incision and secured with a 4-0 braided silk suture. The heart-lung block was rapidly excised through a median sternotomy. Intratracheal lavage with 50 mL of cold phosphatebuffered saline (PBS) was performed as previously described [18]. The collected fluid was centrifuged at 1,500g for 10 minutes, and the cell pellet was resuspended in serum-free RPMI (Gibco BRL, Grand Island, NY). Cell counts and viability were assessed by standard trypan blue exclusion methods. Cells were then plated at a density of 500,000 cells per well in a 12-well culture plate (Fisher Scientific, Pittsburgh, PA). The AM were incubated at 37 C for 60 minutes to allow adherence to the
Hypoxia and Reoxygenation Plated AM were placed in a humidified hypoxic chamber (Coy Lab Products, Grass Lake, MI) with a partial percentage of oxygen of 0.5% for 2 hours. After exposure to hypoxia they were transferred to a normoxic incubator for 15 minutes, at the end of which the supernatant was collected and stored at 70 C until used as experimental media. Plated T2P and PAEC were placed in a humidified hypoxic chamber at a partial pressure of 0.5% for 2 hours with either control media (F-12 or RPMI, respectively, with 5% FBS), activated AM media, TNF-a–depleted media, or IL-1b–depleted media. At the end of the hypoxic period, the cells were transferred to a normoxic humidified incubator for up to 4 hours. Media achieves atmospheric PO2 within 5 minutes [16, 17]. Negative controls remained in the normoxic incubator for up to 6 hours. After 15 minutes of reoxygenation (or 2 hours 15 minutes of normoxia for negative controls), nuclear protein was harvested for electromobility shift assay analysis.
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Pulmonary artery endothelial cells were tested for NFkB and EGR-1. Type 2 pneumocytes were tested for NFkB and AP-1. This testing strategy results from previous work that has shown that EGR-1 and AP-1 are active in PAEC and T2P, respectively [15–18]. At 4 hours of reoxygenation (or 6 hours of normoxia for negative controls), cell supernatants were harvested for chemokine analysis. All samples were stored at 80 C until the time of analysis. All experiments and analyses were performed in triplicate, and the results described were obtained in at least three independent experiments.
Cytokine Depletion Specific cytokine depletion of activated AM media was achieved by passing activated media through a 4% beaded agarose column (Pierce, Rockford, IL) that had polyclonal TNF-a or IL-1b antibodies bound to it.
Nuclear Protein Extraction Cells were harvested in a 100-mL solution of low-salt buffer containing MgCl2 (1.5 mmol/L), KCl (10 mmol/L), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 10 mmol/L). This solution was spun at 1,200g for 1 minute, and the resultant pellet was resuspended in 40 mL of a low-salt buffer solution containing MgCl2 (1.5 mmol/L), KCl (10 mmol/L), HEPES (10 mmol/L), 0.06% Nonidet P-40, and 0.01 mg/mL of leupeptin. This solution was spun a second time at 1,200g for 10 minutes, and the resulting pellet was resuspended in a buffer containing NaCl (420 mmol/L), HEPES (20 mmol/L), EDTA (0.2 mmol/L), MgCl2 (1.5 mmol/L), phenylmethylsulfonyl fluoride (0.5 mmol/L), dithiothreitol (0.5 mmol/L), and 25% glycerol, and incubated at 4 C for 20 minutes. The suspension was centrifuged for a third time (1,200g) for 10 minutes, and the supernatant containing the nuclear protein was collected and frozen at 80 C. Quantification of nuclear protein yield was achieved by means of bicinchoninic acid assay (Pierce). Fig 1. Baseline chemokine content in experimental media. There was no significant difference in monocyte chemotactic protein 1 (MCP-1) or CINC content between the activated alveolar macrophage (AM) media, the tumor necrosis factor-alpha (TNF-a)–depleted and interleukin 1-beta (IL-1b)–depleted media.
Ann Thorac Surg 2015;99:1200–5
Electromobility Shift Assay Nuclear protein in aliquots of 10 mg was incubated in a binding reaction with a double-stranded biotin-labeled oligonucleotide containing the consensus NFkB binding sequence 50 -GCCATTGGGGATTTC-CTCTTTACTGG-30 (Promega, Madison, WI). The binding reaction was performed at room temperature for 60 minutes, and protein was resolved on a 6% nondenaturing polyacrylamide gel at 100 V for 1 to 2 hours. The gels were dried and subsequently autoradiographed. Duplicate samples for each condition were analyzed from each experiment. Densitometry was performed with Image J software (Version 1.2, Silver Spring, MD) to assess relative signal intensity. Results were verified by at least three independent experiments.
Enzyme-Linked Immunosorbent Assay of Cytokine or Chemokine Content Sandwich enzyme-linked immunosorbent assays for CINC, macrophage inflammatory protein 1a, and monocyte chemotactic protein 1 (MCP-1) were performed per the manufacturer’s instructions. Fifty microliters of a 10-mg/mL anti-chemokine antibody was added (Peprotech, Rocky Hills, NJ) to a carbonatecoating buffer solution (pH ¼ 9.6). The solution was plated in a 96-well immunoassay plate (Dynex Technologies, Chantilly, VA) and incubated overnight at room temperature. The plate was washed with PBS containing 0.05% Tween (PBSt) before blocking nonspecific binding sites with a 1% bovine serum albumin PBS solution for 1 hour at room temperature. Samples and standards were diluted 1:1 in PBS, and 100 mL was added to each well and incubated at room temperature for 2 hours. The wells were washed in PBSt, and between 0.5 and 2 mg/mL of secondary biotinylated antibody (Peprotech) was added to each well and incubated for 2 hours at room temperature. Wells were washed in PBSt for a third time, and incubated for 20 minutes with a streptavidin–horseradish-peroxidase
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Regulation of Transcription Factors by Interleukin 1Beta and Tumor Necrosis Factor-Alpha During 2 Hours of Hypoxia and 15 Minutes of Reoxygenation in Type 2 Pneumocytes and Pulmonary Artery Endothelial Cells
Fig 2. Electromobility shift assay for nuclear factor-kappa B (NFkB) nuclear translocation in pulmonary artery endothelial cells. There was a marked increase in NFkB translocation relative to the control (Ctrl) media with activated alveolar macrophage (AM) media at 15 minutes of reoxygenation (p < 0.001). However, with tumor necrosis factor-alpha (TNF-a) depletion there was suppression of NFkB below the level of control media (p < 0.005). (HR ¼ hypoxia–reperfusion; IL-1b ¼ interleukin 1-beta; OD ¼ optical density.)
conjugate (Pierce). The assay was developed by adding o-phenylenediamine dihydrochloride substrate and analyzed on a spectrophotometer. The linear sensitivity range of the assays has been determined, and the assays show no cross-reactivity with one other. Samples and standards were run in triplicate, and well-to-well variation did not exceed 5%.
Statistical Analysis All data are presented as mean values ( standard deviation) unless otherwise designated. Comparisons between groups were made using analysis of variance, and statistical significance was defined for all tests as a probability value of less than 0.05.
Previously we have shown that the proinflammatory transcription factors NFkB, AP-1, and EGR-1 participate in the early development of lung ischemia–reperfusion injury [15–18]. Treatment with activated AM media resulted in enhanced nuclear translocation of NFkB at 15 minutes of reoxygenation in both PAEC (Fig 2) and T2P (Fig 3). Depletion of IL-1b reduced NFkB nuclear translocation in the PAEC and T2P (50% and 57%, respectively) when compared with activated AM media alone. Depletion of IL-1b resulted in NFkB nuclear translocation levels that were 25% higher than control media in PAEC, but equivalent to control media in T2P. Similarly, treatment with activated AM media resulted in augmentation of nuclear translocation of EGR-1 in PAEC and AP-1 in T2P at 15 minutes of reoxygenation compared with control media (Fig 4). Treatment with IL1b–depleted media resulted in 85% and 89% reductions in the translocation of EGR-1 and AP-1, respectively, when compared with activated AM media alone. However with IL-1b depletion there was a 70% reduction of activity compared to control media for both EGR-1 and AP-1. Depletion of TNF-a resulted in a reduction in nuclear translocation of EGR-1 and AP-1 when compared with the activated AM media (50% and 65%, respectively). No protection was observed when TNF-a–depleted media was compared with control media (25% increase and equivalent, respectively).
Effect of Tumor Necrosis Factor-Alpha and Interleukin 1-Beta on Secretory Response of Type 2 Pneumocytes and Pulmonary Artery Endothelial Cells Subjected to Hypoxia and Reoxygenation Previous work from our laboratory has demonstrated that T2P and PAEC secrete CINC and MCP-1 in response to hypoxia and reoxygenation [15–18]. This response is further augmented by soluble mediators produced by AM.
Results Baseline Media Analysis To ensure that there was no dilution of the media with the specific cytokine depletion, the media was analyzed for baseline content of CINC and MCP-1 (Fig 1). Additionally to assess the efficacy of the cytokine depletion, baseline levels of TNF-a and IL-1b were measured. There was no significant difference in MCP-1 or CINC content between the media. Depletion of TNF-a or IL-1b was successful to levels below the detection limit of the enzyme-linked immunosorbent assay (
Functional Roles of Tumor Necrosis Factor-Alpha and Interleukin 1-Beta in Hypoxia and Reoxygenation Heather E. Merry, MD, Patrick Phelan, MD, Matthew Doaks, BS, Minqing Zhao, MD, PhD, and Michael S. Mulligan, MD Division of Thoracic Surgery, Department of Surgery, University of Washington, Seattle, Washington
Background. Intercellular signaling plays an important role in the development of lung ischemia–reperfusion injury. However, the role of specific mediators remains poorly characterized. Alveolar macrophages (AM) produce soluble mediators early in reperfusion, which modulate the responses of endothelial and epithelial cells to oxidative stress. There is a burst of proinflammatory cytokine production in a variety of cells; however, interleukin 1-beta (IL-1b) and tumor necrosis factor-alpha (TNF-a) localize to the AM. We hypothesized that these cytokines account for the costimulatory effects that AM exert on endothelial and epithelial cells. Methods. Activated AM media was placed on cultured rat type 2 pneumocytes and pulmonary artery endothelial cells, which were then subjected to hypoxia and reoxygenation. To assess the contributions of IL-1b and TNFa, the cells were treated with control media or media that had been depleted of IL-1b or TNF-a. To deplete specific cytokines, activated media was passed through a column
with immobilized IL-1b or TNF-a antibodies. Nuclear translocation of transcription factors, mitogen-activated protein kinase activation, and cytokine and chemokine production were assessed. Results. Depletion of IL-1b or TNF-a effectively eliminated the ability of AM media to enhance the response of endothelial and epithelial cells to oxidative stress. There were significant reductions in monocyte chemotactic protein 1 and cytokine-induced neutrophil chemoattractant (CINC) production (p < 0.05) at 4 hours of reperfusion. Additionally there was decreased nuclear translocation of nuclear factor-kappa B, and extracellular signal-regulated kinase phosphorylation. Conclusions. Interleukin 1-beta and TNF-a are critical mediators in the intercellular communication pathways that allow the AM to enhance the response of surrounding cells to oxidative stress. (Ann Thorac Surg 2015;99:1200–5) Ó 2015 by The Society of Thoracic Surgeons
L
with effective, but incomplete, protection. Furthermore, simultaneous blockade of TNF-a and IL1-ß was additive in providing a protective benefit [12]. This suggests that these cytokines are working through independent parallel pathways to drive the early phase of LIRI. Within 15 minutes of reperfusion there is a burst of TNF-a and IL-1b, which localizes to the alveolar macrophage (AM) [12]. Although clinically impractical, depletion of AM with gadolinium chloride or clondronate leads to significant protection in an experimental model of LIRI [13, 14]. This suggests that the AM plays a key coordinating role in this early phase of LIRI and that likely this is through production of TNF-a and IL-1b. This series of experiments is designed to characterize the role of the AM in amplifying the early response in LIRI. Previously we have shown that oxidative stress triggers mitogen-activated protein kinase activation leading to upregulation of proinflammatory transcription factors (such as nuclear factor-kappa B [NFkB], early growth response protein 1 [EGR-1], and activator protein 1 [AP-1]), which in turn causes elaboration of proinflammatory mediators in three primary lung cell types: pulmonary artery endothelial cells (PAEC), type 2 pneumocytes (T2P), and AM [15–18]. We hypothesize that in addition to the direct effect of oxidative stress, the AM plays a key role in priming surrounding cells to the
ung ischemia–reperfusion injury (LIRI) remains a significant complication after lung transplantation, leading to early graft dysfunction and the development of bronchiolitis obliterans [1, 2]. Previous work has demonstrated that the pathogenesis of LIRI is biphasic in nature [3]. The late phase is dependent on neutrophil recruitment and activation, and is characterized by increased vascular permeability and a heterogeneous chemokine and cytokine milieu, whereas the early phase is neutrophil independent and characterized by a predominance of tumor necrosis factor-alpha (TNF-a) and interleukin 1-beta (IL-1b). Both TNF-a and IL-1b are proinflammatory cytokines that lead to leukocyte chemoattraction, phagocyte stimulation, enhancement of downstream cytokine and chemokine production, and variable effects on cell growth and death. Previous work has demonstrated a functional role for these cytokines in multiple models of ischemia– reperfusion injury, including liver [4, 5], heart [6, 7], kidney [8], brain [9], gut [10], limb [11], and lung [12] ischemia and reperfusion. In an in vivo experimental model of LIRI, blockade of TNF-a or IL-1b was associated Accepted for publication Nov 17, 2014. Address correspondence to Dr Mulligan, University of Washington Medical Center, 1959 NE Pacific St, Box 356310, Seattle, WA 98195; e-mail: [email protected].
Ó 2015 by The Society of Thoracic Surgeons Published by Elsevier
0003-4975/$36.00 http://dx.doi.org/10.1016/j.athoracsur.2014.11.042
MERRY ET AL CYTOKINES IN LUNG REPERFUSION INJURY
Abbreviations and Acronyms AM AP-1 EGR-1 EMSA FBS IL-1b LIRI MCP-1 NFkB PAEC PBS PBSt
= = = = = = = = = = = =
alveolar macrophages activator protein 1 early growth response protein 1 electromobility shift assay fetal bovine serum interleukin 1-beta lung ischemia–reperfusion injury monocyte chemotactic protein 1 nuclear factor-kappa B pulmonary artery endothelial cells phosphate-buffered saline phosphate-buffered saline with Tween TNF-a = tumor necrosis factor-alpha T2P = type 2 pneumocytes
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culture plates. Media was then substituted with fresh RPMI containing 5% heat-inactivated fetal bovine serum (FBS).
Pulmonary Artery Endothelial Cell Culture
Material and Methods
Heart-lung blocks were rapidly excised as described above. Endotracheal lavage of the lungs was performed 15 times with 6 to 9 mL of PBS containing 0.25 mmol/L EDTA to deplete alveolar macrophages. Strips of peripheral lungs (2 mm) were excised from all lung lobes. The peripheral tissue was minced, rinsed in RPMI, transferred to a dispase (10 mg/mL) solution, and incubated for 60 minutes at 37 C [15, 16]. The cell suspension was homogenized and incubated for an additional 5 minutes. Complete media with 10% FBS was added to terminate the reaction, and the cellular suspension was filtered through a 100-mm mesh. The filtrate was centrifuged at 800g for 8 minutes, and the cell pellet was resuspended in supplemented RPMI media and plated on gelatin-coated culture dishes. Media was changed every 48 hours until the cells were confluent. Once confluent, the cells were labeled for 8 hours with 4 mg/mL acetylated low-density lipoproteins, which bind selectively to endothelial cells. Cells were separated using flow cytometry (FAC STAR Plus, San Jose, CA). The pure cultures of endothelial cells were then maintained in RPMI with 10% FBS. All cells used in these experiments were from passages 4 through 10.
Reagents
Type 2 Pneumocyte Culture
All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise specified.
A rat type 2 pneumocyte cell line, RLE-6TN [19] (American Tissue Cell Company, Manassas, VA) was maintained in Ham’s F-12 culture media containing 10% heat-inactivated FBS. Cells were cultured in 12-well plates at a density of 105 cells/mL. Culture media was replenished every 48 hours until 95% confluence was reached. Cell counts and viability were assessed by standard trypan blue exclusion techniques [20].
effects of oxidative stress and amplifying their response. Furthermore, we hypothesize that the AM does this through two parallel pathways, one TNF-a–dependent and one IL-1b–dependent. To elaborate these pathways and determine the downstream effects of these cytokines, we used a series of media transfer experiments with specific cytokine depletion.
Alveolar Macrophage Harvest Pathogen-free adult male Long-Evans rats (Simonsen Labs, Gilroy, CA) weighing 250 to 300 g were used for all experiments. The University of Washington Animal Care Committee approved all experimental protocols. Animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). Animals were euthanized with 120 mg/kg of intraperitoneal pentobarbital. A 14-gauge angiocatheter was inserted into the trachea through a midline neck incision and secured with a 4-0 braided silk suture. The heart-lung block was rapidly excised through a median sternotomy. Intratracheal lavage with 50 mL of cold phosphatebuffered saline (PBS) was performed as previously described [18]. The collected fluid was centrifuged at 1,500g for 10 minutes, and the cell pellet was resuspended in serum-free RPMI (Gibco BRL, Grand Island, NY). Cell counts and viability were assessed by standard trypan blue exclusion methods. Cells were then plated at a density of 500,000 cells per well in a 12-well culture plate (Fisher Scientific, Pittsburgh, PA). The AM were incubated at 37 C for 60 minutes to allow adherence to the
Hypoxia and Reoxygenation Plated AM were placed in a humidified hypoxic chamber (Coy Lab Products, Grass Lake, MI) with a partial percentage of oxygen of 0.5% for 2 hours. After exposure to hypoxia they were transferred to a normoxic incubator for 15 minutes, at the end of which the supernatant was collected and stored at 70 C until used as experimental media. Plated T2P and PAEC were placed in a humidified hypoxic chamber at a partial pressure of 0.5% for 2 hours with either control media (F-12 or RPMI, respectively, with 5% FBS), activated AM media, TNF-a–depleted media, or IL-1b–depleted media. At the end of the hypoxic period, the cells were transferred to a normoxic humidified incubator for up to 4 hours. Media achieves atmospheric PO2 within 5 minutes [16, 17]. Negative controls remained in the normoxic incubator for up to 6 hours. After 15 minutes of reoxygenation (or 2 hours 15 minutes of normoxia for negative controls), nuclear protein was harvested for electromobility shift assay analysis.
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Pulmonary artery endothelial cells were tested for NFkB and EGR-1. Type 2 pneumocytes were tested for NFkB and AP-1. This testing strategy results from previous work that has shown that EGR-1 and AP-1 are active in PAEC and T2P, respectively [15–18]. At 4 hours of reoxygenation (or 6 hours of normoxia for negative controls), cell supernatants were harvested for chemokine analysis. All samples were stored at 80 C until the time of analysis. All experiments and analyses were performed in triplicate, and the results described were obtained in at least three independent experiments.
Cytokine Depletion Specific cytokine depletion of activated AM media was achieved by passing activated media through a 4% beaded agarose column (Pierce, Rockford, IL) that had polyclonal TNF-a or IL-1b antibodies bound to it.
Nuclear Protein Extraction Cells were harvested in a 100-mL solution of low-salt buffer containing MgCl2 (1.5 mmol/L), KCl (10 mmol/L), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 10 mmol/L). This solution was spun at 1,200g for 1 minute, and the resultant pellet was resuspended in 40 mL of a low-salt buffer solution containing MgCl2 (1.5 mmol/L), KCl (10 mmol/L), HEPES (10 mmol/L), 0.06% Nonidet P-40, and 0.01 mg/mL of leupeptin. This solution was spun a second time at 1,200g for 10 minutes, and the resulting pellet was resuspended in a buffer containing NaCl (420 mmol/L), HEPES (20 mmol/L), EDTA (0.2 mmol/L), MgCl2 (1.5 mmol/L), phenylmethylsulfonyl fluoride (0.5 mmol/L), dithiothreitol (0.5 mmol/L), and 25% glycerol, and incubated at 4 C for 20 minutes. The suspension was centrifuged for a third time (1,200g) for 10 minutes, and the supernatant containing the nuclear protein was collected and frozen at 80 C. Quantification of nuclear protein yield was achieved by means of bicinchoninic acid assay (Pierce). Fig 1. Baseline chemokine content in experimental media. There was no significant difference in monocyte chemotactic protein 1 (MCP-1) or CINC content between the activated alveolar macrophage (AM) media, the tumor necrosis factor-alpha (TNF-a)–depleted and interleukin 1-beta (IL-1b)–depleted media.
Ann Thorac Surg 2015;99:1200–5
Electromobility Shift Assay Nuclear protein in aliquots of 10 mg was incubated in a binding reaction with a double-stranded biotin-labeled oligonucleotide containing the consensus NFkB binding sequence 50 -GCCATTGGGGATTTC-CTCTTTACTGG-30 (Promega, Madison, WI). The binding reaction was performed at room temperature for 60 minutes, and protein was resolved on a 6% nondenaturing polyacrylamide gel at 100 V for 1 to 2 hours. The gels were dried and subsequently autoradiographed. Duplicate samples for each condition were analyzed from each experiment. Densitometry was performed with Image J software (Version 1.2, Silver Spring, MD) to assess relative signal intensity. Results were verified by at least three independent experiments.
Enzyme-Linked Immunosorbent Assay of Cytokine or Chemokine Content Sandwich enzyme-linked immunosorbent assays for CINC, macrophage inflammatory protein 1a, and monocyte chemotactic protein 1 (MCP-1) were performed per the manufacturer’s instructions. Fifty microliters of a 10-mg/mL anti-chemokine antibody was added (Peprotech, Rocky Hills, NJ) to a carbonatecoating buffer solution (pH ¼ 9.6). The solution was plated in a 96-well immunoassay plate (Dynex Technologies, Chantilly, VA) and incubated overnight at room temperature. The plate was washed with PBS containing 0.05% Tween (PBSt) before blocking nonspecific binding sites with a 1% bovine serum albumin PBS solution for 1 hour at room temperature. Samples and standards were diluted 1:1 in PBS, and 100 mL was added to each well and incubated at room temperature for 2 hours. The wells were washed in PBSt, and between 0.5 and 2 mg/mL of secondary biotinylated antibody (Peprotech) was added to each well and incubated for 2 hours at room temperature. Wells were washed in PBSt for a third time, and incubated for 20 minutes with a streptavidin–horseradish-peroxidase
MERRY ET AL CYTOKINES IN LUNG REPERFUSION INJURY
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Regulation of Transcription Factors by Interleukin 1Beta and Tumor Necrosis Factor-Alpha During 2 Hours of Hypoxia and 15 Minutes of Reoxygenation in Type 2 Pneumocytes and Pulmonary Artery Endothelial Cells
Fig 2. Electromobility shift assay for nuclear factor-kappa B (NFkB) nuclear translocation in pulmonary artery endothelial cells. There was a marked increase in NFkB translocation relative to the control (Ctrl) media with activated alveolar macrophage (AM) media at 15 minutes of reoxygenation (p < 0.001). However, with tumor necrosis factor-alpha (TNF-a) depletion there was suppression of NFkB below the level of control media (p < 0.005). (HR ¼ hypoxia–reperfusion; IL-1b ¼ interleukin 1-beta; OD ¼ optical density.)
conjugate (Pierce). The assay was developed by adding o-phenylenediamine dihydrochloride substrate and analyzed on a spectrophotometer. The linear sensitivity range of the assays has been determined, and the assays show no cross-reactivity with one other. Samples and standards were run in triplicate, and well-to-well variation did not exceed 5%.
Statistical Analysis All data are presented as mean values ( standard deviation) unless otherwise designated. Comparisons between groups were made using analysis of variance, and statistical significance was defined for all tests as a probability value of less than 0.05.
Previously we have shown that the proinflammatory transcription factors NFkB, AP-1, and EGR-1 participate in the early development of lung ischemia–reperfusion injury [15–18]. Treatment with activated AM media resulted in enhanced nuclear translocation of NFkB at 15 minutes of reoxygenation in both PAEC (Fig 2) and T2P (Fig 3). Depletion of IL-1b reduced NFkB nuclear translocation in the PAEC and T2P (50% and 57%, respectively) when compared with activated AM media alone. Depletion of IL-1b resulted in NFkB nuclear translocation levels that were 25% higher than control media in PAEC, but equivalent to control media in T2P. Similarly, treatment with activated AM media resulted in augmentation of nuclear translocation of EGR-1 in PAEC and AP-1 in T2P at 15 minutes of reoxygenation compared with control media (Fig 4). Treatment with IL1b–depleted media resulted in 85% and 89% reductions in the translocation of EGR-1 and AP-1, respectively, when compared with activated AM media alone. However with IL-1b depletion there was a 70% reduction of activity compared to control media for both EGR-1 and AP-1. Depletion of TNF-a resulted in a reduction in nuclear translocation of EGR-1 and AP-1 when compared with the activated AM media (50% and 65%, respectively). No protection was observed when TNF-a–depleted media was compared with control media (25% increase and equivalent, respectively).
Effect of Tumor Necrosis Factor-Alpha and Interleukin 1-Beta on Secretory Response of Type 2 Pneumocytes and Pulmonary Artery Endothelial Cells Subjected to Hypoxia and Reoxygenation Previous work from our laboratory has demonstrated that T2P and PAEC secrete CINC and MCP-1 in response to hypoxia and reoxygenation [15–18]. This response is further augmented by soluble mediators produced by AM.
Results Baseline Media Analysis To ensure that there was no dilution of the media with the specific cytokine depletion, the media was analyzed for baseline content of CINC and MCP-1 (Fig 1). Additionally to assess the efficacy of the cytokine depletion, baseline levels of TNF-a and IL-1b were measured. There was no significant difference in MCP-1 or CINC content between the media. Depletion of TNF-a or IL-1b was successful to levels below the detection limit of the enzyme-linked immunosorbent assay (
