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Ann Thorac Surg 2006;82:472-478
© 2006 The Society of Thoracic Surgeons
Division of Thoracic Surgery, University of Washington Medical Center, Seattle, Washington
Accepted for publication March 14, 2006.
* Address correspondence to Dr Mulligan, University of Washington Medical Center, 1959 NE Pacific St, Box 356310, Seattle, WA 98195. (Email: msmmd{at}u.washington.edu).
Presented at the Poster Session of the Forty-second Annual Meeting of The Society of Thoracic Surgeons, Chicago, IL, Jan 30–Feb 1, 2006.
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Abstract |
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METHODS: We administered recombinant interleukin-6 to rat lungs through the intratracheal route before inducing left lung ischemia and reperfusion. Multiple in-vivo indicators of left lung injury were studied, as were transactivation patterns for nuclear factor kappa B and signal transduction and activators of transcription-3. Downstream effects on the elaboration of proinflammatory chemokines and cytokines were also studied.
RESULTS: Recombinant interleukin-6 reduced endothelial disruption and neutrophil sequestration in left lung and alveolar spaces, resulting in improved oxygenation after ischemia and 4 hours of reperfusion. This protection was associated with decreased nuclear factor kappa B and signal transduction and activators of transcription-3 nuclear translocation early in reperfusion, and diminished proinflammatory mediator secretion late in reperfusion.
CONCLUSIONS: Further studies focusing on the effects of recombinant interleukin-6 in large animal models are warranted, as this may be a novel strategy to improve outcomes after lung transplantation. Intratracheal administration may focus its efficacy on the lung while reducing effects on other organ systems during organ procurement.
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Introduction |
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Interleukin-6 (IL-6) is a 21-kDa pleiotropic cytokine that modulates processes as diverse as proliferation and differentiation, immunologic responses, and inflammation. It is produced by a number of cells including macrophages, endothelial cells, neutrophils, and lymphocytes, all of which have been shown experimentally to contribute to lung reperfusion injury [2, 4, 5]. The function of IL-6 appears to be organ specific and stimulus dependent, and exerts its effects through multiple complex signaling pathways [6]. Even within a particular organ system, such as the lung or liver, IL-6 can induce proinflammatory mediator expression or exert cytoprotective effects depending on the inciting stimulus [7]. This phenomenon, known as the signal orchestration model, attempts to explain how IL-6 can elicit proinflammatory or anti-inflammatory effects depending on the in-vivo environmental circumstances [16].
The orchestration of signal transduction in response to IL-6 is complex and involves at least two parallel pathways. It has also been suggested that these pathways regulate one another, although this is not well described. The first pathway involves the Janus-associated kinase–signal transducer and activators of transcription (STAT) family, while the second travels through the mitogen-activated protein kinases and nuclear factor kappa B (NF
B) [15]. Although our laboratory has demonstrated requirements for the NFB pathway in the development of in-vivo lung reperfusion injury [4, 16], the involvement of STAT proteins has not been explored in warm, physiologic lung reperfusion injury models. However, their importance to lung injury has been suggested in a number of publications. In rodent models of immune-complex alveolitis and lipopolysaccharide administration, there is an early and persistent activation of STAT-3 protein [17, 18]. Furthermore, activation of STAT-3 is induced in the lung by reactive oxygen and nitrogen species [17–19], which we have previously shown to contribute to tissue injury after lung reperfusion injury [20]. Lastly, in other organ systems, target genes for the STAT family are similar to NFB, including cytokines/chemokines, adhesion molecules, and inflammatory mediators; and STAT knock-out animals demonstrate decreased expression of mediators such as tumor necrosis factor-
(TNF-) and inducible nitric oxide synthase [21].
The role of IL-6 in lung transplantation has yet to be clearly defined, although there are publications in humans with contrasting results. Genetic polymorphisms of the IL-6 gene are associated with an earlier onset of bronchiolitis obliterans [8], while high levels of IL-6 in bronchoalveolar lavage fluid have been reported in refractory cases of acute rejection [9]. Although increased levels of IL-6 are found in injured airways and lung tissue after an inflammatory stimulus, the functional significance of that finding is unknown at present [9, 10].
More specifically, in ischemia-reperfusion (IR) models, there also have been contrasting reports regarding IL-6, although most support a protective function. When IL-6 was administered to rat donors in an intestinal IR model, graft survival was improved and neutrophil extravasation was reduced in the allograft as well as the lung [11]. This protection was associated with reduced secretion of proinflammatory mediators, including TNF-. Administration of exogenous IL-6 also reduced injury in IR models of the liver and retina [12, 13], while appearing to have an injurious effect in an in vitro coculture model involving cardiomyocytes and neutrophils [14]. Interleukin-6 has not been studied in a physiologic lung reperfusion injury model to date.
In these studies, we describe the function of IL-6 in a well-established rodent model of lung reperfusion injury [4, 5, 20, 24, 25], hypothesizing that its administration would confer protection against several indicators of in-vivo lung injury. We further sought to characterize STAT-3 protein expression after lung reperfusion injury, and whether the nuclear translocation of STAT-3 or NFB proteins was modulated by IL-6. Given the disparate effects that IL-6 has on other vascular beds after IR, we administered recombinant IL-6 through the intratracheal route, concentrating its effects to the lung in an attempt to reduce undesired effects on other organs.
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Material and Methods |
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Animal Model
Pathogen-free Long-Evans rats (Simonsen Labs, Gilroy, CA), weighing between 280 g and 320 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 Institute of Health (NIH Publication No. 86-23, revised 1996).
A warm, in-situ, ischemia-reperfusion hilar occlusion model, which has been previously described [4, 20, 24, 25], was used. In brief, animals were anesthetized with 35 mg intraperitoneal pentobarbital. A 14G angiocatheter was inserted into the trachea through a midline neck incision and secured with a 4-0 braided suture. Animals were then placed on a Harvard Rodent Ventilator (Harvard Apparatus, Boston, Massachusetts) with a standardized inspired oxygen content of 60% and 2 cm H2O of positive end-expiratory pressure. Maximal peak pressures did not exceed 10 cm H20. The animals were placed on their right side, and a left anterolateral thoracotomy in the fifth intercostal space was performed. The left lung was mobilized atraumatically, and the inferior pulmonary ligament divided sharply. All animals received 50 units of heparin dissolved in saline (total volume 500 µL) through a penile vein. Five minutes after heparin administration, the pulmonary hilum was occluded, during inspiration, with a noncrushing microvascular clamp, making sure to include the left main bronchus, artery, and vein. The period of ischemia was held constant at 90 minutes, after which the clamp was removed and the lung reperfused for up to 4 hours. Animals were administered 0.5 mL warm subcutaneous saline per hour to maintain hydration. At the end of reperfusion, a midline incision from the neck to the pubis was created for access to the chest and abdominal cavities. Blood samples were obtained from the inferior vena cava just before sacrifice, the heart-lung block was rapidly excised, and the pulmonary circulation was flushed through the main pulmonary artery with 10 mL normal saline. The lungs were then separated from mediastinal tissues.
To determine the effects of IL-6 on lung reperfusion injury, treated animals received 150 ng of recombinant rat IL-6 intratracheally 30 minutes before ischemia. This dose was chosen as it was the lowest efficacious dose used in another rodent model of IR injury, and in preliminary studies in our own model of lung reperfusion injury was also found to be effective [13]. Most publications utilizing IL-6 bolus or infusion administered doses in the microgram range [11, 22, 23]. A 150 µL solution, followed by 300 µL of air (to clear drug from dead space), was given through the tracheostomy tube with the animal in a left lateral decubitus position. We have previously shown, using radioactivity and dye studies, that this method of administration delivers more than 70% of agent to the left lung selectively [24]. Vehicle-treated animals were administered phosphate-buffered saline at equivalent volumes using the same timing regimen.
Three groups of animals were studied in in-vivo left lung injury studies. Negative controls did not undergo any surgical manipulation. Positive controls underwent the full experimental protocol, including 90 minutes of ischemia followed by 4 hours of reperfusion, and received intratracheal vehicle as just described. The intratracheal IL-6-treated animals received recombinant protein 30 minutes before ischemia, and were then subjected to 90 minutes of ischemia and as much as 4 hours of reperfusion (similar to positive controls). Lung injury was quantitated according to the following measurements.
Lung Permeability Index
To quantitate lung vascular injury, permeability indices were measured. The 125I radiolabeled bovine serum albumin was obtained from NEN Life Sciences (Boston, Massachusetts). Before using 125I-bovine serum albumin in vivo, serial dilutions were performed to obtain an activity of approximately 800,000 counts per minute (cpm). A 1% bovine serum albumin/phosphate-buffered saline solution was added to make a final volume of 500 µL. Five minutes before removal of the hilar clamp, the 125I-bovine serum albumin was intravenously injected. Immediately before animal sacrifice, 1 mL blood was withdrawn from the inferior vena cava. The heart-lung block was then excised and flushed as described previously. The counts were quantitated for both the left and right lung, as well as from the inferior vena cava blood sample using a gamma counter. The permeability index is expressed as the ratio of the cpm in the left lung to the cpm in 1 mL of inferior vena cava blood:
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Myeloperoxidase Assay
The myeloperoxidase assay was used to quantitate lung tissue neutrophil accumulation as previously described [20, 24, 25]. Change in absorbance at 460 nm over 1 minute was recorded.
Bronchoalveolar Lavage Inflammatory Cell Counts
Using the 14G angiocatheter placed for ventilation, the lungs were lavaged individually with 3 mL sterile saline. To facilitate individual lung bronchoalveolar lavage analysis, the contralateral hilum was occluded. At least 80% of the instilled fluid was recovered from each lung bronchoalveolar lavage. This fluid was centrifuged (1,500 rpm x 8 minutes at 4°C) to pellet the cells. The pellet was resuspended in 10 mL sterile water to lyse red blood cells, and this fluid was again centrifuged (1,500 rpm x 8 minutes at 4°C). The supernatant was discarded, and the cells were counted using a hemacytometer (Hausser Scientific, Horsham, PA).
Arterial pO2 Levels
At the end of the experimental protocol, the right hilum of rats were ligated for 10 minutes, and subsequently 0.2 mL blood was drawn up from the aorta and run through a blood gas analyzer (Chiron Diagnostics, Emeryville, California). Two groups were studied (the vehicle-treated positive controls and the intratracheal IL-6 animals at 4 hours of reperfusion), with at least 4 animals per group.
Electrophoretic Mobility Shift Assay
At the end of the experimental protocols, lungs were snap frozen in liquid nitrogen after flushing the pulmonary circulation with 20 mL saline. The frozen tissue was ground to a fine powder and suspended in 4 mL buffer containing 0.06% Nonidet P-40 (Sigma Chemical, St. Louis, MO), 150 mM NaCl, 10 mM HEPES, 1 mM EDTA, and 0.5 mM PMSF. The solution was homogenized and centrifuged for 15 s (12,000g). The pellet was discarded, and the supernatant was centrifuged again for 15 s (12,000g). The resultant pellet was suspended in 40 µL buffer containing 40 mM NaCl, 20 mM HEPES, 0.2 mM EDTA, 1.2 mM MgCl2, 0.5 mM PMSF, 0.5 mM DDT, 25% glycerol, 5 µg/mL aprotinin, and 5 µg/mL leupeptin at 4°C for 20 minutes. This solution was centrifuged for 5 minutes, the pellet discarded, and the supernatant containing the nuclear protein stored at -80°C. Quantification of nuclear protein was performed using the bicinchoninic acid assay.
Nuclear protein (10 µg) was incubated in a binding reaction with double stranded 32P end-labeled oligonucleotide containing the NFB (Promega, Madison, Wisconsin) or STAT-3 (Santa Cruz Biotechnology, Santa Cruz, California) binding-consensus sequence. Running unlabeled oligonucleotide probe in a cold competition binding reaction assessed the specificity of each probe. The binding reaction was carried out at room temperature for 60 minutes, and the proteins were resolved on a 6% nondenaturing polyacrylamide gel at 100v for 1 to 2 hours. The gels were dried and autoradiographed. Numerous samples for each timepoint were analyzed, and each electrophoretic mobility shift assay was performed three times to verify results. Densitometry was performed to assess relative differences in activation between groups using the image analysis software, Image Pro Plus (Media Cybernetics, Silver Spring, Maryland).
Enzyme-Linked Immunosorbent Assay
Cytokine-induced neutrophil chemoattractant and monocyte chemoattractant protein-1 (MCP-1) were quantified using sandwich enzyme-linked immunosorbent assays (Peprotech, Rocky Hills, New Jersey) developed in our laboratory and described previously [4, 5, 17]. The TNF- enzyme-linked immunosorbent assay was performed per the manufacturer's protocol (R&D Systems, Minneapolis, Minnesota). 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 ± SEM unless otherwise designated. Comparisons between multiple groups were made using one-way analysis of variance with a post-hoc Bonferroni modification for multiple comparisons. Statistical differences between groups were assessed using a two-tailed Student's t test. Statistical significance was defined for all tests as a p less than 0.05.
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Results |
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Myeloperoxidase Content
Negative control, unmanipulated lungs demonstrated a baseline change in myeloperoxidase content of 0.07 ± 0.04, which increased to 0.43 ± 0.07 at 4 hours of reperfusion after intratracheal vehicle administration. That was a significant increase (p < 0.001). Intratracheal IL-6 reduced myeloperoxidase content in 4-hour reperfused lungs to 0.26 ± 0.05, a statistically significant decrease (p = 0.01) that was 47% protective against lung parenchymal neutrophil accumulation.
Bronchoalveolar Lavage Cell Counts
When compared with unmanipulated negative controls (8.4 x 105 cells), there was a significant increase in bronchoalveolar lavage cell counts (165 x 105 ± 40 cells) in vehicle-treated, positive controls after 4 hours of reperfusion (p < 0.005). The predominant cell type in positive control animals was the neutrophil (> 98%), whereas the earlier periods demonstrated almost exclusively alveolar macrophages. Intratracheal IL-6 markedly reduced alveolar cell counts by 52% to 85 x 105 ± 14 cells (p = 0.005).
Arterial pO2 Levels
The average arterial pO2 level of the vehicle-treated positive control animals was 243 ± 11 mm Hg, down from 542 ± 11 mm Hg in negative controls. This change in oxygenation was statistically significant when comparing these two control groups (p < 0.001). Intratracheal IL-6 increased arterial oxygenation to 493 ± 13 mm Hg after ischemia and 4 hours of reperfusion. The increase was statistically significant when compared with positive control animals (p < 0.001).
Proinflammatory Transcription Factor Nuclear Translocation
Since we previously demonstrated in numerous publications that transcriptional activation of proinflammatory genes occurs early in reperfusion, we primarily studied the timepoint of ischemia followed by 15 minutes of reperfusion to assess whether recombinant IL-6 protein modulated transcription factor transactivation. Later timepoints (ischemia followed by 4 hours of reperfusion) were also studied to determine whether any effects of IL-6 treatment persisted later in IR. As seen on the representative electrophoretic mobility shift assay for NFB nuclear translocation (Fig 1), at all time points studied (early or late in reperfusion), the global transactivation seen in whole lung homogenates for NFB activation was abrogated by intratracheal IL-6 administration.
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, MCP-1, and cytokine-induced neutrophil chemoattractant protein levels in left lung bronchoalveolar lavage fluid after ischemia and 4 hours of reperfusion in vehicle-treated positive controls. Administering IL-6 through the intratracheal route reduced each of the aforementioned mediators to a significant degree relative to the vehicle-treated animals. The p values and the percent reduction in chemokine secretion after IL-6 administration are also shown in Table 2.
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Comment |
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We and others have shown that lung reperfusion injury in this model depends on transcriptional activation of proinflammatory chemokines and cytokines, such as TNF-, cytokine-induced neutrophil chemoattractant, and MCP-1 [4, 5, 16, 20]. Tumor necrosis factor- is a potent cytokine chemoattractive for leukocytes in general to areas of inflammation, and has been localized in this model to alveolar macrophages early in the reperfusion period (15 minutes) after ischemia, and to macrophages, endothelial, and epithelial cells later in reperfusion [25]. Early secretion of TNF- from macrophages primes surrounding lung cell types to amplify their proinflammatory response (unpublished data). Furthermore, the ability to antagonize TNF function confers the greatest degree of protection against lung reperfusion injury when compared with singular blockade of any cytokine or chemokine, and is even equivalent to broad spectrum chemokine inhibition [25]. Therefore, any reductions in TNF secretion secondary to IL-6 administration would hypothetically have dramatic effects on reducing reperfusion injury and preserving lung function. Additionally, IL-6 is shown in this work to modulate cytokine-induced neutrophil chemoattractant secretion, an alpha chemokine known to be a potent chemoattractant for neutrophils, as well as MCP-1, a beta chemokine chemoattractive for macrophages and lymphocytes. All three cell types are known to contribute to the development of acute lung injury to varying degrees after ischemia and reperfusion [2, 4].
Given the reductions in secretion of TNF, cytokine-induced neutrophil chemoattractant, and MCP-1, it is not surprising that the activation of one of their transcriptional regulators, NFB, was reduced by IL-6 treatment. Previously in both in-vivo lung reperfusion injury models and in-vitro hypoxia and reoxygenation models involving alveolar macrophages, antagonization of NFB significantly limited the expression of the aforementioned mediators [4, 16]. Although it is widely published that NFB up-regulates IL-6 expression in the lung, the phenomenon of exogenous IL-6 in turn limiting NFB nuclear translocation has been studied in only one prior work. In a rodent model of hemorrhagic shock, exogenous IL-6 dramatically decreased inflammation and tissue injury in both the lung and liver. That was associated with a reduction in neutrophil sequestration in these organs, and the anti-inflammatory effects were found to be mediated in part through attenuated NFB transactivation [26]. We demonstrate similar findings in the lungs after direct ischemia and reperfusion, as neutrophil accumulation was limited in vivo after IL-6 administration through a mechanism that likely involved, at least in part, abrogated NFB activity. Although NFB is known to up-regulate IL-6 expression, it may be that high levels of IL-6 protein in turn limit NFB activity in a negative feedback loop. That may be part of the reason that IL-6 is at times proinflammatory, while at other times anti-inflammatory in IR models.
We have described the activation pattern of STAT 3 after experimental ischemia and reperfusion of the lung. Our findings were similar to what had been described in a model of immune-complex alveolitis with regard to STAT 3 activation, as there was early STAT 3 nuclear translocation evident, which persisted at above-baseline levels for hours after the inciting event. Interestingly, we found that IL-6, classically described as an inducer of STAT 3, reduced STAT 3 activation both early and late after reperfusion in this model. The discrepancy may be for several reasons. We utilized in this work a very low dose of IL-6, and just as hypothesized with NFB, so too may STAT 3 activation patterns be affected by the relative levels of circulating IL-6 protein. Secondly, in inflammatory models administering exogenous IL-6 and demonstrating protection against tissue injury, study endpoints are typically physiologic and not mechanistic, and as such STAT translocation patterns have not been characterized. It may be that similar findings would be noted in those publications. Third, the signal orchestration model describing IL-6 signaling pathways describes divergent pathways activated by IL-6, and suggests that the pathways may have negative regulatory effects on the other. As such, a relative anti-inflammatory balance after IL-6 administration through one pathway (ie, NFB or STAT) may negatively regulate the other as well. There are several known negative regulators of STAT signaling, such as the suppressors of cytokine-signaling protein 3, and there may be an as of yet undescribed up-regulation of one of these proteins by IL-6 after lung reperfusion injury, resulting in decreased activation of STAT 3.
In conclusion, IL-6 protects rodent lungs from injury after ischemia and reperfusion of the lung, and is associated with reduced transactivation of both NFB and STAT 3, and decreased secretion of proinflammatory mediators. Intratracheal administration was an effective route, focusing efficacy on the lung, and it may reduce possible untoward effects on other organ systems. Further work in a large animal orthotopic model of lung transplantation is under way in the hope of characterizing these mechanistic findings further and providing additional insights into the complicated signaling pathways regulated by IL-6.
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This article has been cited by other articles:
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  T. K. Varghese Jr Invited commentary Ann. Thorac. Surg., August 1, 2006; 82(2): 478 - 479. [Full Text] [PDF]
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