| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Department of Surgery, Division of Cardiothoracic Surgery, University of Washington Medical Center, Seattle, Washington
b Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Washington Medical Center, Seattle, Washington
Accepted for publication October 16, 2007.
* Address correspondence to Dr Mulligan, Division of Cardiothoracic Surgery, University of Washington Medical Center, Box 356310, 1959 NE Pacific St, Seattle, WA 98195 (Email: msmmd{at}u.washington.edu).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and MethodsResultsCommentReferences |
|---|
Methods: Macrophages were exposed to hypoxia and reoxygenation and the media collected. Cultured endothelial cells were then exposed to macrophage media and maintained at normoxia or subjected to hypoxia and reoxygenation. To assess any reciprocal effects of endothelial cell products on macrophage activation, macrophages were likewise exposed to activated endothelial cell media.
Results: Exposure of endothelial cells to activated alveolar macrophage media enhanced chemokine secretion in response to hypoxia and reoxygenation. In the reciprocal experiment, activated endothelial cell media increased the production of macrophage inflammatory protein 1
from macrophages.
Conclusions: Alveolar macrophages drive the development of lung reperfusion injury, by enhancing the production of proinflammatory chemokines from endothelial cells, which impart a degree of positive feedback on alveolar macrophages.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
|---|
In vivo models have demonstrated the existence of a biphasic response to ischemia and reperfusion [4]. The early response is transient and involves a marked increase in vascular permeability together with a short oxidant release caused by the nuclear translocation of proinflammatory mediators that lead to the secretion of inflammatory cytokines and chemokines [5–7]. The early phase is neutrophil-independent and therefore coordinated by a resident cell such as the alveolar macrophage.
Alveolar macrophages generate a variety of cytokines, chemokines, growth factors, and arachidonic metabolites [8] in response to oxidative stress. These include tumor necrosis factor- and interleukin-1β, which lead to the upregulation of cell adhesion molecules and the recruitment of neutrophils. This pulmonary artery endothelial cell (PAEC) is integral in this process through secretion of chemoattractants and upregulation of adhesion molecules [9]. We believe that through the secretion of soluble mediators, alveolar macrophages prime PAECs, augmenting their response to an initial oxidative stimulus, causing the accumulation and migration of neutrophils into the lung interstitial space and the eventual development of reperfusion lung injury. Previous work with in vivo and in vitro models has demonstrated an important role for the chemokines, cytokine-induced neutrophil chemoattractant (CINC), and macrophage inflammatory protein 1- (MIP-1) in promoting the inflammatory injury with oxidative stress in the lung [10–14].
To determine how alveolar macrophages influence the response of PAECs to oxidative stress, studies were performed using a cell culture model of hypoxia and reoxygenation as a surrogate for LIRI. Primary cultures of alveolar macrophages and PAECs were subjected to 2 hours of hypoxia and either 15 minutes or 4 hours of reoxygenation to simulate the early and late responses to oxidative stress.
We propose that crosstalk between a coordinating cell, alveolar macrophages, and other constitutive cells, such as PAECs, is responsible for the cascade ultimately resulting in LIRI. In this study, we will examine the interaction between alveolar macrophages and PAECs.
|
Material and Methods |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
|---|
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 the Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the Institute of Health.
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. A median sternotomy was performed, and the heart lung block was rapidly excised. Intratracheal lavage of the lungs was performed 15 times with 3- to 10-mL volumes of cold phosphate-buffered saline (PBS) yielding a 90% recovery. Collected lavage fluid was centrifuged at 1500g for 10 minutes, and the cell pellet was resuspended in Roswell Park Memorial Institute (RPMI) serum-free standard growth media (Gibco BRL, Gaithersburg, MD).
Cells counts and viability were assessed by trypan blue exclusion methods, and RPMI was added until a density of 500,000 cells/mL was reached. One milliliter of this cellular media was loaded for each well of a 12-well culture plate (Fisher Scientific, Pittsburgh, PA), and alveolar macrophages were incubated at 37°C for 60 minutes to allow adherence to take place [12]. Media was then substituted with fresh RPMI containing 5% heat-inactivated fetal bovine serum (HI FBS).
Hypoxia and Reoxygenation
We generated three types of alveolar macrophage media for subsequent exposure to PAECs. Control media was generated by plating alveolar macrophages and leaving them unstimulated for 6 hours. Early alveolar macrophage media was collected from alveolar macrophages that underwent 2 hours of hypoxia and 15 minutes of reoxygenation. This media would be used to investigate the effects of alveolar macrophage products released during hypoxia or early in reoxygenation and would attempt to simulate the "early phase" of LIRI. Late alveolar macrophage media was generated by subjecting alveolar macrophages to 2 hours of hypoxia, followed by 4 hours of reoxygenation. This would be used to investigate the influence of alveolar macrophage products generated later in reperfusion.
Prepared alveolar macrophages were incubated in a humidified hypoxic chamber (Coy Lab Products, Grass Lake, MI) with 0.5% oxygen for 2 hours at 37°C. Reoxygenation was achieved by removing the plate from the hypoxic chamber and placing it into a normoxic humidified incubator for either 15 minutes or 4 hours. At the end of the experiment, media rich in proinflammatory products was aspirated and stored at –80°C. Samples were either analyzed for baseline chemokine content by enzyme linked immunosorbent assay (ELISA) or used to stimulate PAECs. This baseline amount produced by alveolar macrophages would be subtracted from total amounts in media collected at the end of the coculture experiments. The correction would allow specific assessments of the chemokine response of the PAECs.
Pulmonary Artery Endothelial Cell Culture
Pathogen-free, adult male, 21-day-old Long-Evans rats (Simonsen Labs, Gilroy, CA) were used in all experiments. 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. A median sternotomy was performed and the heart lung block rapidly excised. Endotracheal lavage of the lungs was performed 15 times with 6 to 9 mL phosphate buffered saline (PBS) containing 0.25 mM ethylenediaminetetraacetic acid (EDTA) to deplete alveolar macrophages [15].
Two-millimeter strips of peripheral lungs 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. The cell suspension was homogenized and incubated for an additional 5 minutes at 37°C. A total of 10 mL complete media containing 10% FBS was added to terminate the reaction, and the cellular suspension was then filtered through a 100-µm mesh. The filtrate was spun at 800g for 8 minutes, resuspended in supplemented RPMI media, and plated on gelatin-coated culture dishes.
The media was changed every 48 hours during the incubation period until confluent [16]. Cells were labeled for 8 hours with 4 µg/mL acetylated low-density lipoproteins, which bind selectively to endothelial cells. Cells were separated using flow cytometry (FAC STAR Plus, San Jose, CA). This results in a pure culture of endothelial cells. All cells used in these experiments were from passages 3 to 6.
Treatment of Pulmonary Artery Endothelial Cell Culture With Alveolar Macrophage Media
Before placing PAECs in the hypoxic chamber, plated cell media was exchanged with control, early, or late alveolar macrophage media. Negative control PAECs were incubated under normoxic conditions at 37°C, whereas PAECs subjected to hypoxia and reoxygenation were incubated in 0.5% oxygen for 2 hours at 37°C. Reoxygenation was achieved by removing the plate from the hypoxic chamber and placing it in a normoxic humidified incubator for either 15 minutes or 4 hours. At the end of the experiment, media was collected and stored at –80°C for ELISA analysis.
Reciprocal Experiment Treatment of Alveolar Macrophages
The PAECs maintained in RPMI were placed into the same groups used to pretreat alveolar macrophages. Media from the first group (control) was not subjected to hypoxia and reoxygenation, the second group underwent 2 hours hypoxia and 15 minutes reoxygenation, generating early PAEC products, and the third group was subjected to 2 hours of hypoxia, followed by 4 hours of reoxygenation secreting late PAEC products.
The PAEC media was replenished with RPMI containing 5% HI FBS, and cells were then incubated in a humidified hypoxic chamber for 2 hours at 37°C. As previously described, reoxygenation was achieved by removing the plate from the hypoxic chamber and placing it into a normoxic humidified incubator for either 15 minutes or 4 hours.
As end time points were reached, PAEC media was collected and stored at –80°C. The samples were similarly analyzed for baseline chemokine content by ELISA, or combined with harvested alveolar macrophages.
To begin the experiment, the alveolar macrophage media was exchanged with media generated from one of the three pretreated PAEC groups. Alveolar macrophages were incubated in the hypoxic chamber and reoxygenated as previously described. At the end of the experiment, the media was collected for ELISA analysis.
Enzyme Linked Immunoassay of Cytokine or Chemokine Content
Sandwich ELISAs for CINC, MIP-1, and MCP-1 have been developed in our laboratory (Peprotech, Rocky Hills, NJ) as previously described [1].
Statistical Analysis
All data are presented as mean values (± standard deviation) unless otherwise designated. Initial comparisons between groups were made using a two-tailed Student t test, and statistical significance was defined for all tests as p < 0.05. Given the multiple experimental conditions, linear regression models were used to assess the impact of treatment of the cells (cell condition) and treatment of the media (media condition). Linear regression models were used to avoid issues related to multiple comparisons. The models were formulated as follows: Y = β0 + β1 (cell condition) + β2 (media condition), where Y equals CINC, MIP-1, and MCP-1 in separate models. A total of six models were created. Cell condition and media condition were modeled with dummy variables. Standardized residuals were plotted to assess assumptions of linear models. Because of the concern of heteroscedasticity (a critical assumption when performing linear regression or analysis of variance), Huber/White-corrected standard errors were used [17]. We assessed the data for influential points and outliers. Interaction terms between the media conditions and cell conditions were assessed in the models using the Wald statistic. Analyses were performed with Stata 8.0 software (StataCorp, College Station, TX). Cell culture wells were run in triplicate and experiments were repeated three times.
|
Results |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
|---|
increased under the influence of hypoxia and reoxygenation but did not increase significantly in PAECs.
Effect of Alveolar Macrophage Products on Rat Pulmonary Artery Endothelial Cell Response
When PAECs were exposed to hypoxia and reoxygenation in the presence of preconditioned media, both early and late conditioned alveolar macrophage media provoked a dramatic increase in the production of MCP-1 (p < 0.001) and CINC (p < 0.001) from PAECs subjected to 2 hours of hypoxia and 15 minutes of reoxygenation. This increase was several times greater than that noted with either cell type alone (Table 1). No further enhancement could be demonstrated after 4 hours of reoxygenation. Secretion of MIP-1 by PAECs was not augmented until these cells were stimulated by media generated late after alveolar macrophage media (p < 0.001).
|
in response to reoxygenation (p < 0.001). This augmentation was most effective under the stimulation of late PAEC products.
|
|
Comment |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
|---|
by the PAECs. Secretion of CINC and MCP-1 secretions were enhanced by products released from the alveolar macrophage early in the reoxygenation period as effectively as the response seen late in reoxygenation. Secretion of MIP-1 was enhanced by alveolar macrophage secretions that were produced after 4 hours of reoxygenation.
In the reciprocal experiment, late PAEC products were capable of enhancing the alveolar macrophage production of MIP-1 in response to hypoxia and reoxygenation. Late PAEC products were capable of stimulating a MCP-1 response by alveolar macrophages but did not augment their response to reoxygenation. Similarly, PAEC products were capable of stimulating a CINC response by alveolar macrophages but did not augment their response to hypoxia and reoxygenation.
Early alveolar macrophage products augment both CINC and MCP-1 responses of PAEC to reoxygenation and therefore provide the key physiologic stimulus. Late PAEC augment MIP-1 in the alveolar macrophage and therefore exert a positive feedback relationship on MIP-1 production.
Experimental models have aided our understanding of the physiopathology of acute lung injury. Early in the posthypoxic period, activation of the alveolar macrophage stimulates the secretion of proinflammatory cytokines and chemokines. These soluble messengers are responsible for amplifying the response in other cell types, including the PAEC. Although there remain critical steps in this hypothesis that are unsolved, this experiment provides important information about the early role of alveolar macrophages in coordinating the response of PAECs in response to oxidative stress.
The paradigm for the transmigration of leukocytes is open to debate, but we believe that the key effector cell in the development of injury is the alveolar macrophage. In response to oxidative stress, alveolar macrophages secrete proinflammatory mediators [12] that prime surrounding PAECs to secrete a quantity of chemokine far greater than what they would secrete independently [7].
These studies have directly implicated alveolar macrophage secretory products as capable of enhancing the response of constitutive lung cells to oxidative stress, thus supporting that the alveolar macrophage is the key coordinating cell in the inflammatory response to oxidative stress in the lung. In further studies, we aim to identify which mediators are coordinating the costimulatory responses.
|
References |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
|---|
B: ten years later Cell 1996;87:13-20.[Medline]Related Article
This article has been cited by other articles:
|
|
 |
|
  A. S. McCourtie, A. S. Farivar, S. M. Woolley, H. E. Merry, P. S. Wolf, B. Mackinnon-Patterson, J. C. Keech, E. FitzSullivan, and M. S. Mulligan Alveolar Macrophage Secretory Products Effect Type 2 Pneumocytes Undergoing Hypoxia-Reoxygenation Ann. Thorac. Surg., December 1, 2008; 86(6): 1774 - 1779. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. P. van der Kaaij and A. J.J.C. Bogers Invited Commentary Ann. Thorac. Surg., December 1, 2008; 86(6): 1779 - 1780. [Full Text] [PDF]
|
|
|
|
|
|
J. S. Allan Invited Commentary Ann. Thorac. Surg., March 1, 2008; 85(3): 1060 - 1060. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
