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Ann Thorac Surg 2002;74:899-905
© 2002 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, University of Washington, Seattle, WA, USA
* Address reprint requests to Dr Mulligan, Division of Cardiothoracic Surgery, Box 356310, 1959 NE Pacific St, University of Washington, Seattle, WA 09195 USA
e-mail: msmdu{at}uwashington.edu
Presented at the Thirty-eighth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, January 28–30, 2002.
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Abstract |
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Methods. Alveola macrophages were rendered hypoxic (0.5%) for 2 hours and reoxygenated for 6 hours. The secreted chemokine content in the media was quantified by enzyme-linked immunosorbent assay, and nuclear protein was analyzed after electro-mobility shift assay. When employed, CSA was administered 30 minutes before hypoxia.
Results. Alveolar macrophages demonstrated a marked increase in the secretion of the chemokines, MIP-2, MIP-1
, CINC, and MCP-1, in response to hypoxia and reoxygenation. This increase was dependent on mRNA transcription and de novo protein synthesis. It was also blocked by a specific inhibitor of the nuclear translocation factor, NF-
B. Pretreatment with CSA (500 ng/mL) significantly reduced expression of chemokines and activation of NF-B.
Conclusions. Cyclosporine A attenuates the chemokine response of AMs in vitro to hypoxia and reoxygenation at the pretranscriptional level through modulation of NF-B. These findings suggest the potential mechanism of action of CSA’s protective effects in lung ischemia reperfusion injury.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) and the ß chemokines, MIP-1 and monocyte chemoattractant protein (MCP-1) (unpublished data). In this model, there is a biphasic increase in vascular permeability as well as a steady rise in neutrophil sequestration [3]. Although ultimately this injury is neutrophil dependent, significant neutrophil recruitment is not detected until 2 hours after reperfusion. In experimental LIRI, there is a burst of oxidants within 15 minutes of reperfusion [2]. The source of these oxidants must therefore be a resident cell rather than a recruited cell. The alveolar macrophage (AM) is a resident cell of the lung, which is the presumed source of cytokines and oxidants in a variety of models of acute inflammatory lung injury [4, 5]. Furthermore, AM depletion is protective against the development of nonischemic acute lung injury, and that protection is associated with reduced elaboration of soluble inflammatory mediators [6]. It is therefore likely that the AM is the key effector cell in the early response to LIRI.
Recently, interest has focused on the well-characterized immunosuppressive agent, cyclosporine A (CSA), and its ability to attenuate acute reperfusion injury. CSA is a hydrophobic undecapeptide that binds to the cytoplasmic protein cyclophilin, and this complex inhibits calcineurin. The inhibition of calcineurin prevents the activation of a number of nuclear transcription factors, including NF-AT, OCT-1, AT-1, and NF-B [7]. The transcription factor NF-B has a range of activities, including modulation of cell growth and death, regulation of immune function, and notable affects on acute reperfusion injury [8]. We recently demonstrated that pretreatment with CSA significantly reduced LIRI and NF-B activation in a rat model of in situ warm ischemic [9].
Therefore, knowing that chemokines play a key role in LIRI, that the initial source of these mediators may be the AM, and that administration of CSA in vivo reduces injury, we hypothesized that the protective effects of CSA may relate to a reduction in chemokine expression by the rat AM in response to hypoxia and reoxygenation.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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Alveolar macrophage (AM) harvest
Pathogen-free adult male Long-Evans rats (Simonsen Labs, Gilroy, CA) weighing 280 g to 320 g were used for all experiments. Animals were euthanized with 120 mg/kg of intraperitoneal pentobarbitol. A 14-gauge angiocatheter was inserted into the trachea through a midline neck incision and secured with a 4 to 0 braided silk suture. A median sternotomy was performed and the heart lung block rapidly excised. Intratracheal lavage of the lungs was performed 15 times with 6-mL to 9-mL volumes of cold phosphate-buffered saline (PBS) with at least 90% recovery of volume. The lavage fluid was incubated on ice during collection. The fluid was then centrifuged at 1,500 g for 8 minutes and the cell pellet resuspended in Dulbecco’s modified Eagle’s media (DMEM). Cells counts and viability were assessed by standard trypan blue exclusion methods. One million cells were plated in 24-well culture plates and the AMs allowed to adhere over 30 minutes in serum-free DMEM. Before stimulation, selected batches of cells were preincubated for 30 minutes then stimulated as described below with either actinomycin D (5 µg/mL), cycloheximide (25 µg/mL), Sn50 (Biommol; Plymouth Meeting, Pittsburg, PA)(1 to 25 µg/mL), CSA (500 ng/mL), or cremaphor (6,500 ng/mL).
Hypoxia and reoxygenation (H&R)
Plated cells 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. Hypoxic DMEM supplemented with 5% fetal bovine serum was used in these experiments (media was allowed to equilibrate overnight in the chamber to assure consistent hypoxia). Reperfusion was achieved by transferring the plate to a normoxic humidified incubator for up to 6 hours. For nuclear protein harvest, experiments were performed in a 10-mL petri dish with 10 million cells and 5 mL of media. Viability was demonstrated using trypan blue exclusion and MTT (3,4,5, dimethylthiazol-2yl 2,5, diphenyltetrazolium bromide) metabolism.
Nuclear protein extraction
The cells were harvested in 1 mL of low-salt buffer (MgCl2 1.5 mmol/L, KCl 10 mmol/L, and Hepes 10 mmol/L). This solution was centrifuged at 1,200 g for 1 minute and the supernatant removed. The resultant pellet was resuspended in 40 µL of a low-salt buffer (containing Noniodet P-40 and 0.01 µg/mL leupeptin). The supernatant was then centrifuged again (1,200 g) for 10 minutes. The resultant pellet was suspended in buffer containing 420 mmol/L NaCl, 20 mmol/L Hepes, 0.2 mmol/L EDTA, 1.5 mmol/L MgCl2, 0.5 mmol/L PMS, 0.5 mmol/L DDT, and 25% glycerol, and incubated at 4°C for 20 minutes. This solution was then centrifuged (1,200 g) for 10 minutes, the pellet discarded, and the supernatant containing the nuclear protein stored at -70° C. Quantification of nuclear protein was performed using the bichinoic acid assay (BCA).
EMSA (electro-mobility shift assay)
Nuclear protein samples (10-µg aliquots) were incubated in a binding reaction with a double-stranded 32P end-labeled oligonucleotide containing the human consensus NF-B binding site sequence 5'-GCCATTGGGGATTTCCTCTTTACTGG-3 [10]. This sequence is homologous to the rat consensus NF-B site. The NF-B binding reaction was carried out at room temperature for 60 minutes, and the bound proteins were resolved on a 6% nondenaturing polyacrylamide gel at 100 V for 1 to 2 hours. The gels were dried and autoradiographed. Duplicate samples for each condition were analyzed. Densitometry was performed with Image J software (Version 1.2) to assess relative signal intensity.
Protein analysis
At the end of the hypoxia and reoxygenation experiment, the media were isolated and spun at 1,000 g for 2 minutes. The resultant supernatant was snap-frozen after the addition of a protease inhibitor cocktail (1 µg/mL leupeptin, 1 µg/mL aprotinin, 5 µg/mL trypsin inhibitor, and 1 µg/mpepstatin A). Protein content of media was assessed with enzyme-linked immunosorbent assay (ELISA) techniques.
ELISA
Sandwich ELISAs for MIP-2, CINC, MIP-1, MCP-1, and RANTES have been developed in our laboratory. Fifty microliters of a 5-µg/mL protein A-purified specific antichemokine antibody (Peprotech, Rocky Hills, NJ) in a carbonate-coating buffer solution (pH 9.6) was added to a 96-well (Dynex) immunoassay plate. The plate is incubated overnight at 4°C and washed with PBS (0.05% Tween.) Nonspecific binding was blocked with bovine serum albumin (2%) in DMEM (30 minutes, 37°C). Sample or standards were loaded at a volume of 50 µL and incubated for 1 hour at 37°C. A primary affinity-purified biotinylated antibody (Peprotech) specific to the epitope being studied (0.5 to 2 µg/mL) was added to each well and incubated for 1 hour at 37°C. After a 20-minute incubation with a streptavidin-horseradish-peroxidase conjugate(HRP) (Pierce, Rockford, IL), the assay was developed by adding o-phenylenediamine dihydrochloride substrate. The reaction was stopped by the addition of 50 µL of 3 mol/L H2SO4. The linear sensitivity range of the assays has been determined and the assays show no cross-reactivity with each other. Samples and standards were run in triplicate, and well-to-well variation did not exceed 5%.
Statistical analysis
All data were presented as mean values ± the standard error of the mean unless otherwise designated. Comparisons between two groups were made using a two-tailed Student’s t test and between multiple groups were done with analysis of variance. Statistical significance was defined for all tests as a p less than 0.05.
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Results |
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secretion increased by 3.69-fold (5.15 ± 0.75 to 24.13 ± 5.84 ng/mL, p < 0.03), MIP-2 by 2.90-fold (17.85 ± 3.38 to 69.95 ± 14.4 ng/mL, p < 0.04), CINC by 82% (1.02 ± 0.11 to 1.85 ± 0.19 ng/mL, p < 0.004), and MCP-1 by 52% (10.93 ±0.1 to 16.63 ±1.21 ng/mL, p < 0.005) (Fig 1).
The secretion of the chemokine RANTES did not significantly increase (data not shown).
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(76.4%), and MCP-1 (97.9%) (Table 2). This, therefore, demonstrates the dependence on synthesis of de novo protein synthesis in both basal and stimulated chemokine secretion.
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and MCP-1 by stimulated AMs were all significantly reduced, in fact, below even basal secretory levels as seen in the negative control. Therefore, this demonstrates that chemokine secretion is dependent on de novo transcription (Table 2).
Activation of NF-B by hypoxia and reoxygenation is required for chemokine secretion
To further investigate the transcriptional regulation of these chemokines, we studied the nuclear translocation and activation of NF-B: a potent proinflammatory transcription factor known to bind to the 5I-flanking region of several chemokine genes. Nuclear translocation of the active NF-B was detected by EMSA (Fig 2).
Densitometry demonstrates that NF-B is activated in AMs exposed to hypoxia. This signal disappears after 2 hours of reperfusion. Cold competition with nonradiolabeled oligonucleotide of NF-B consensus results in a diminution of the band in the hypoxia-treated group, thus demonstrating that this band is specific for NF-B.
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B on chemokine expressionB activation to transcriptional upregulation of the chemokines was assessed with Sn50. This synthetic peptide contains the nuclear localization sequence of NF-B and thus blocks nuclear translocation of NF-B. AMs were treated 1 hour before stimulation with either 1, 5, or 25 µg/mL of Sn50. Media was analyzed for protein content by ELISA. There is a dose-dependant inhibition of chemokine secretion in response to inhibition of NF-B translocation (Fig 3).
Therefore, NF-B is an important event in the transcriptional upregulation of chemokine expression by AMs in response to hypoxia.
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B activation, as demonstrated by a diminished band in nuclear protein isolated from AMs pretreated with CSA and then exposed to hypoxia compared with positive controls (Fig 2).
Pretreatment in vivo reduces the AM chemokine response in vitro
To determine whether the in vitro effects of CSA reflected a potential mechanism of protection in vivo, the following studies were undertaken. CSA (5 mg/kg) or cremaphor (65 mg/kg) (the carrier of CSA) was administered to animals 3 hours before lavage of lungs. The cells were then stimulated with either hypoxia and reoxygenation or LPS 200 pg/mL (a standard agent to test macrophage responsiveness) for 8 hours. MIP-2 was selected as a representative chemokine for these studies. CSA pretreatment in vivo reduced basal secretion of MIP-2 (Fig 4)
from 25.3 ± 2.6 to 2.5 ± 0.4 ng/mL. Stimulated secretion was also reduced from 45.8 ± 8.3 to 6.3 ± 2 ng/mL. In contrast to the response to hypoxia and reoxygenation, CSA did not affect the secretion of MIP-2 by AMs in response to LPS.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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, CINC, and MCP-1. The expression of these proteins is dependent on de novo protein and RNA synthesis, which in turn is regulated by the transcription factor NF-B. This is demonstrated not only by the nuclear translocation of NF-B in stimulated cells but also by the dose-dependant inhibition of chemokine secretion by Sn50 (which inhibits nuclear translocation of NF-B). Pretreatment of AMs before hypoxia and reoxygenation with CSA attenuated the expression of these chemokines and reduced the activation of NF-B. Therefore, it is highly likely that CSA exerts its inhibitory effect on chemokine secretion in response to oxidant stress through a reduction in activation of NF-B.
Hypoxia alone has been shown to activate NF-B in both cell lines and freshly harvested AMs [11, 12]. In these same studies, acute hypoxia also increased mRNA for TNF and MIP-1. In a rat AM cell line, in vitro models of oxidant stress using hydrogen peroxide demonstrate an upregulation of mRNA for MIP-2 and MIP 1 [13, 14].We have demonstrated in this study that NF-B upregulation secondary to hypoxia likely mediates these increases in chemokine mRNA and ultimately results in translational release of protein.
Cellular adherence itself may result in activation of NF-B. Debate exists as to whether this stimulation is sufficient or not to produce chemokines [15, 16]. In our study, we observed relatively high basal levels of chemokines in response to plating. This did not confound the response to hypoxia and reoxygenation but may have enhanced it. In vivo AMs are loosely adherent to endothelium and epithelium. On activation, they strongly attach and spread; such intercellular interactions are difficult to produce in vitro [16]. In our experiment, plating may have acted as a permissive costimulatory factor that is not inconsistent with requirements for optimum secretion in vivo.
Cyclosporine A reduces the secretion of cytokines, TNF, and IL-1ß and the chemokine IL-8 in both basal- and in lipopolysacchride (LPS)-stimulated human AMs [17, 18]. Macrophages from transplant patients receiving CSA show reduced expression of IL-1 in response to LPS [19, 20], thus demonstrating a profound relevant affect of CSA on macrophage immune function. In our studies, we have demonstrated that AMs harvested from CSA-treated animals secrete less chemokine in response to hypoxia and reoxygenation but not LPS stimulation.
In vitro studies on macrophages have delineated the mechanism by which CSA may inhibit NF-B activation (Fig 5).
These studies suggest that inhibition of calcineurin blocks the degradation of IB (the cytoplasmic protein that binds NF-B in its inactive form) [21]. In addition, processing of proform precursors of NF-B to its functional heterodimeric form may also be inhibited. The net result is diminished concentration of active NF-B in the nucleus, which leads to decreased production of mRNA for proinflammatory cytokines, neutrophil adhesion molecules, procoagulant proteins, and vasoconstrictive substances [8].
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B activation. Mitogen-activated protein (MAP) kinase pathways are highly conserved signaling cascades that participate in a diverse array of biological processes. CSA inhibits these pathways at multiple levels [7]. It has been suggested that MAP kinases may also help regulate NF-B.
Cyclosporine A may also act further upstream, influencing the oxidative pathway at the mitochodrial level. Peroxynitrite, a potent nitrogen-centered free radical, is produced by the reaction of superoxide and nitric oxide early in reperfusion [3]. It has a verse range of biological effects, including directly increasing mitochondrial calcium efflux through nonspecific pores, which in turn results in mitochondrial dysfunction, a hallmark of oxidative injury [22]. In an in vivo cardiac model of ischemia and reperfusion, CSA (0.1 µM) is protective. However, this protection does not relate to reduced opening of the mitochondrial pores [23]. Therefore, the effects of CSA on mitochondrial calcium efflux are likely not to becally important. Any effects on peroxynitrite production may relate to inhibition of inducible nitric oxide synthase (iNOS) [24]. By reducing nitric oxide (NO) production, CSA may indirectly blunt the production of peroxynitrite. We are currently evaluating the individual contributions of these mechanisms to protection conferred by CSA pretreatment in vivo. Studies to date that examined the effects of CSA in NO metabolism, however, have been inconsistent. Therefore, the most consistent findings suggest that CSA’s effect on AM response to hypoxia and reoxygenation likely relates to the transcriptional regulation through NF-B
The AM is likely the key effector cell that initiates LIRI. This is supported by in situ mRNA localization of IL-1ß, MCP-1, and MIP-2 in this model to the AM [25]. We have also demonstrated by immunohistochemistry studies that TNF and IL-1ß get expressed rapidly within the early reperfusion period and can be localized to the macrophage (unpublished data). Furthermore, in the isolated perfused lung model, exposure to gadolinium chloride (GdCl3) a drug that inhibits macrophages, results in improved oxygenation and lower pulmonary vascular resistance 30 minutes into reperfusion [26]. We have recently demonstrated that AM depletion with clodrinote-encapsulated liposomes in a rat model of warm ischemic results in a reduction of tissue neutrophil sequestration and vascular permeability to I125-BSA [27]. These findings further reinforce the central role of the AM in LIRI.
In summary, we have presented a highly plausible mechanism of action for CSA reducing LIRI. This study is of course is limited because the effects of CSA on other resident cell types (eg, epithelium and endothelium) were not studied. Despite this, the model is validated by the fact that in vivo administration of CSA to an animal, subsequent harvest of AMs, and stimulation in vitro with hypoxia and reoxygenation blunts chemokine response of the AM. The concentration of CSA employed in this study (500 ng/mL) is comparable with blood concentrations that are clinically relevant. Therefore, using clinically recommended doses may be likely to affect AMs and potentially reduce reperfusion injury in surgical practice. The therapeutic implications of selectively inhibiting the key effector cell in LIRI offers great promise.
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Discussion |
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B and what altering NF-B may do in the system to your cells. As you know, NF-B regulates the expression of many genes, including, importantly, those for cellular response to injury. It is the major transcriptional regulator of bcl-x1 and actually plays a role for bcl-2 as well, the two most important antiapoptotic proteins that exist in many cells.
My first question is, did you examine the viability of the macrophages after you inhibited the expression of NF-B? Is it possible that the macrophages are either sick or dying and that is why the cytokine expression is low?
Secondly, if you plan to use this clinically, how would you go about using it? I would suggest that downregulating NF-B in a whole-body situation by any agent with greater effect on NF-B expression than cyclosporine may actually render the lung more susceptible to injury because of downregulating antiapoptotic protein expression. Thank you very much.
DR NAIDU: Thank you very much for that question. With regard to the first question, macrophage viability was confirmed with tryphan blue exclusion and MTT metabolism. We also assessed macrophage function in response to LPS. Cells responded vigorously to LPS and this response was not affected by cyclosporine. With regard to the second question, the clinical use of cyclosporine might involve pretreatment of donors or recipients. It could be added to the donor lung reperfusion solution. Some combinations of strategies might be more effective. It might be objectionable (to the abdominal surgeons) to give it systemically. Interestingly, in our experience with 100 recipients who have received it preoperatively, none has demonstrated any untoward effects. NF-B has numerous proinflammatory effects beyond those on bcl-2 and bcl-x. I think it is difficult to predict how the balance of effects in this model will be felt. Cyclosporine inhibition of NF-B may ultimately not effect bcl-x or bcl-2. That should be measured directly.
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B: ten years later. Cell 1996;87:13-20.[Medline]
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= negative controls; 
= positive controls.)
) (65 mg/kg) and then subjected to either hypoxia and reoxygenation or lipopolysaccharide (LPS) (200 pg/ml) stimulation. Data are expressed as mean ± standard error of the mean (n = 4). (MIP = macrophage inflammatory protein.)