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Ann Thorac Surg 2003;76:253-259
© 2003 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, University of Washington Medical Center, Seattle, Washington, USA
Accepted for publication February 12, 2003.
* Address reprint requests to Dr Mulligan, Division of Cardiothoracic Surgery, University of Washington Medical Center, 1959 NE Pacific St., Seattle, WA 98195, USA
e-mail: msmmd{at}u.washington.edu
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Abstract |
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METHODS: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received either recombinant IL-4 or recombinant IL-10, or antibodies to IL-4 or IL-10 immediately before reperfusion. Lung injury was quantitated by permeability indices, lung parenchymal neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) effluent. Expression of IL-4 and IL-10 was determined by immunoblotting, and mRNA expression for early response cytokines was evaluated by ribonuclease protection assays.
RESULTS: IL-4 and IL-10 protein expression was significant after 2 hours of reperfusion. Animals receiving anti-IL-4 (p = 0.05) and anti-IL-10 (p = 0.01) antibodies demonstrated increased permeabilities compared with positive controls. Lung tissue neutrophil accumulation (p < 0.004) and BAL leukocyte content (p < 0.04) were also significantly increased in animals receiving anti-IL-10 antibodies. Conversely, animals receiving recombinant IL-4 and recombinant IL-10 demonstrated reduced permeabilities and lung MPO content. Both anti-IL-4 and anti-IL-10 treatment increased mRNA expression for a number of early response cytokines, including TNF-
and IL-1ß.
CONCLUSIONS: IL-4 and IL-10 are expressed in response to LIRI and function to decrease injury severity. These effects are partly due to modulated expression of early proinflammatory cytokines.
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Introduction |
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(TNF-) and interleukin-1ß (IL-1ß), is clinically relevant. Although several regulatory cytokines, including interleukin-4 (IL-4) and interleukin-10 (IL-10), are being investigated in clinical trials, a definition of their molecular effects remains incomplete.
Interleukin-4 and IL-10 are Th-2 cytokines that modulate acute inflammatory processes. IL-10 is involved in lymphoid and myeloid cell development and activation [4], and limits the secretion of proinflammatory cytokines TNF- and IL-1ß by macrophages [5]. Though IL-4 also exerts antiinflammatory effects through decreased macrophage production of TNF- and IL-1ß, the modulation appears less dramatic compared with IL-10 [5, 6].
Recombinant IL-10 (rIL-10) attenuates experimental reperfusion injury of the heart, intestine, liver, and hindlimb [7–10], and ameliorates acute lung injury secondary to liver or hindlimb ischemia [10, 11]. Though exogenous IL-10 has been illustrated to reduce direct lung ischemia reperfusion injury (LIRI), the molecular mechanism of this protection was not investigated [12]. Furthermore, the physiologic relevance of these effects was unknown because the role of endogenous IL-10 was not studied. Endogenous IL-10 has previously been demosntrated to be protective as treatment with antibody IL-10, which enhanced the development of acute immune complex alveolitis in rats [13]. Regarding IL-4, recent reports suggest provision of exogenous IL-4 is protective in LPS-induced inflammation [14] and hepatic reperfusion injury [15]. However, although administration of rIL-4 reduced immune complex-induced lung injury, blockade of endogenous IL-4 was without effect [13].
Given the complexity and redundancy of the lung’s response to inflammation, simultaneous modulation of multiple proinflammatory cytokines is hypothetically attractive. If IL-10 and IL-4 are expressed early, subsequent to oxidative stress, they could potentially retard the development of reperfusion injury. Conversely, antagonism of these mediators would allow injury to progress unchecked to a more severe degree. In the present study we hypothesized that administering antibodies to IL-10 and IL-4 would be injurious in an in vivo model of direct lung ischemia reperfusion, whereas recombinant formulations would afford a degree of protection. These effects would likely occur through modulated early proinflammatory cytokine expression, namely TNF- and IL-1ß.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Animal model
Long-Evans rats (Simonsen Laboratories, Gilroy, CA), weighing 280 to 320 g, were used for all experiments. The University of Washington Animal Care Committee approved all experimental protocols. Animals were anesthetized with 35 mg of intraperitoneal pentobarbital, and were subsequently shaved and prepared. A 14-gauge angiocatheter was inserted into the trachea through a midline neck incision. Animals were placed on a Harvard Rodent Ventilator (Harvard Apparatus Inc., Holliston, MA) with a standardized inspired oxygen content of 60%, a rate of 90 breaths per minute, and 2 cm H2O of positive end-expiratory pressure. Maximal peak pressures were maintained below 10 cm H20. All animals received 0.2 mg of atropine intramuscularly and were anesthetized with inhaled halothane. A left anterolateral thoracotomy in the fifth intercostal space was performed, the left lung was mobilized atraumatically, and the inferior pulmonary ligament was divided sharply. At this time all animals received 50 U of intravenous heparin in saline (total volume 500 µL). Five minutes later, the left pulmonary artery, vein, and main stem bronchus were occluded with a noncrushing microvascular clamp, maintaining the lung in a partially inflated state. Lungs were kept moist with periodic applications of warm saline, and the incision was covered to minimize evaporative losses. The ischemic period was held constant at 90 minutes, after which the clamp was removed and the lung ventilated and reperfused for periods up to 4 hours. At the conclusion of reperfusion, a midline incision from the neck to the pubis was created to access the chest and abdominal cavities. Blood samples were obtained from the inferior vena cava just before sacrifice. The heart-lung block was excised and the pulmonary circulation was flushed through the main pulmonary artery with 20 mL of saline. The lungs were then separated from mediastinal tissues.
Lung permeability index
To quantitate reperfusion-induced vascular injury, permeability indices were measured. 125I-radiolabeled bovine serum albumin (BSA) was obtained from NEN Life Sciences (Boston, MA). Before use of the 125I-BSA in vivo, serial dilutions were performed to obtain an activity of approximately 800,000 counts per minute (cpm). This dilution of 125I-BSA was then brought to a final volume of 500 µL in a 1% BSA/PBS solution. Five minutes before removal of the hilar clamp, or at an equivalent time in sham animals, the 125I-BSA mix was intravenously injected through a penile vein. Immediately before sacrificing the animals, 1 mL of blood was drawn from the inferior vena cava. Subsequently, the heart-lung block was excised and flushed as previously described. The radioactivity counts were quantitated in the left and right lungs, and in the inferior vena cava blood sample using a gamma counter. The permeability index was expressed as the ratio of the cpm in the left lung to 1.0-mL inferior vena caval blood, and is represented as follows:
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Negative controls did not undergo any surgical manipulation. Sham/thoracotomy only animals were placed on the ventilator, had a left thoracotomy, and were ventilated for up to 5.5 hours. An ischemia only group underwent thoracotomy, occlusion of the left hilum for 90 minutes, and was sacrificed before reperfusion. Positive controls underwent 90 minutes of ischemia followed by 4 hours of reperfusion.
To determine the effects of IL-4 and IL-10 on reperfusion injury, treated animals received either rIL-4 or rIL-10 (10 µg), or a polyclonal rabbit antirat IL-4 or IL-10 antibody. For the majority of studies, anti-IL-4 and anti-IL-10 antibodies were administered at a dose of 250 µg, because this dose had previously been utilized in a rat model of immune complex alveolitis with excellent results [13]. A dose response for anti-IL-10 was also performed as subsequent experiments tested the effects of 500 µg and 100 µg on permeability indices, myeloperoxidase assays, and alveolar leukocyte content.
Antibodies and recombinant cytokines were dissolved in 500 µL of sterile PBS immediately before administration, and were injected through a penile vein 5-minutes before the removal of the vascular clamp from the left hilum. Nonspecific rabbit IgG was administered similarly as a control. All groups contained at least 4 animals to achieve statistical significance.
Myeloperoxidase assay
Tissue MPO content quantitated lung parenchymal neutrophil sequestration as described previously [16]. Lungs were homogenized in a phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB). Samples were assayed for the ability to decompose H202 in the presence of O-dianisidine dihydrochloride by the change in absorption at 460 nm over 1 minute. MPO activity was measured in negative controls in animals subjected to 90 minutes of ischemia and up to 4 hours of reperfusion, in the IL-4 (250 µg) and IL-10 antibody treated (250 µg and 100 µg) groups, and in both the rIL-10 and rIL-4 treated groups. All groups contained at least 4 animals.
Bronchoalveolar lavage
Additional animals underwent BAL at the time of sacrifice. Through the tracheostomy, lungs were lavaged individually by clamping the contralateral hilum and instilling 3 mL of saline that was flushed and aspirated three times. This fluid was centrifuged (1500g x 8 minutes at 4°C) to pellet the cells, and the supernatant was snap frozen for subsequent cytokine analysis after the addition of a protease cocktail inhibitor (leupeptin 1 µg/mL, aprotinin 1 µg/mL, trypsin inhibitor 5 µg/mL, and pepstatin A 1 µg/mL). The red blood cells were lysed and the pellet was resuspended in normal saline. Total nucleated cells were counted using a hemacytometer (Hausser Scientific, Reading, PA). All groups contained at least 4 animals.
Western blot analysis for IL-4 and IL-10
Lungs were homogenized in a 10-mL solution containing 10 mmol/L HEPES (pH = 7.9), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5 mol/L PMSF, 0.6% NP-40, and a protease inhibitor cocktail. The homogenate was incubated on ice for 5 minutes and 1-mL aliquots were placed into microfuge tubes for analysis. Samples were centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant protein concentration was determined using the bicinchoninic assay (Pierce Co., Rockford, IL). The protein (40 µg) was loaded on PAGE-SDS gels (12%) and run at 100 V for 1 hour. Following transfer to a PVDF membrane, a Coomasie blue stain was performed to confirm equal protein transfer. The membranes were incubated with either an anti-IL-4 or anti-IL-10 polyclonal antibody (Peprotech) at a 1:1000 dilution overnight. An HRP-conjugated secondary antibody was applied for 1 hour, and the proteins were visualized using Pierce Supersignal reagents (Amersham, IL) and autoradiography. In order to define the patterns of IL-4 and IL-10 expression in injured lungs, IL-4 and IL-10 protein expressions were analyzed in negative controls and in animals undergoing ischemia and up to 4 hours of reperfusion.
Ribonuclease protection assay
Ribonuclease protection assays (RPA) were performed on lung extracts from multiple experimental groups (negative controls, animals that underwent 90 minutes of ischemia and 4 hours of reperfusion, and animals treated with either 250 µg of anti-IL-4 or 250 µg of anti-IL-10 antibody just before reperfusion). Lung RNA was isolated in guanidine thiocyanate, with two rounds of acid phenol/chloroform extraction and alcohol precipitation. RNA integrity was confirmed by agarose gel electrophoresis, and quantitated by optical density measurements (260 nm). RNA from each rat was evaluated using the Riboquant system (PharMingen, San Diego, CA). Rat template rCK1 was used for detection of cytokine mRNA. In vitro transcription was carried out in transcription buffer supplemented with (-32P) UTP (3000 Ci/mmol [Pierce supersignal reagents]) and T7 RNA polymerase. After DNase I treatment, the riboprobe was isolated by phenol/chloroform extraction and ammonium acetate/ethanol precipitation. Labeling efficiency was determined by measuring Chernokov activity in a scintillation counter. Each riboprobe was diluted to the optimal activity defined by the manufacturer, added to 20 µg of kidney RNA, heated to 90°C, allowed to cool to 56°C, and annealed overnight. After RNase and proteinase K treatment, protected RNA hybrids were purified by phenol/chloroform extraction and ammonium acetate/ethanol precipitation, and separated by electrophoresis on 5% polyacrylamide per 8 mol/L urea gels. Gels were dried and underwent autoradiography using Kodak Biomax MS2 Film (Eastman-Kodak, Rochester, NY). Samples for each condition were done in triplicate. Densitometric analysis of relative mRNA expression was then done with Image J software (Version 1.2) (Image Pro Plus Image J Software, Carlsbad, CA).
Statistical analysis
All data are presented as mean values (± the standard error of the mean) unless otherwise designated. Statistical analysis was done by analysis of variance (ANOVA). Results were corrected for multiple comparisons using Bonferroni’s method and statistical significance was defined for all tests as p less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Animals receiving 250 µg of antibody to IL-10 demonstrated a statistically significant (p < 0.01) increase in vascular permeability (1.51 ± 0.07) compared with positive controls. All animals that received 500 µg of anti IL-10 antibody died within 1 hour of reperfusion. The lungs were grossly hemorrhagic and the lung injury appeared severe. Further studies planned at this dose, including MPO and BAL leukocyte counts, were not performed. Administration of 100 µg of anti-IL-10 increased the permeability index (1.36 ± 0.01) significantly (p = 0.02) when compared with positive controls, but was less dramatic than that seen with 250 µg of anti-IL-10 antibody. This general trend of 100 µg being less dramatic than 250 µg persisted throughout our experiments.
Animals receiving 250 µg of antibody to IL-4 had a significant increase in permeability at 4 hours of reperfusion (1.12 ± 0.06) compared with positive controls, representing a 57% increase in injury (p < 0.05). This augmentation was less than that seen with 250 µg of anti-IL-10, so a 100-µg dose was not investigated.
Administration of rIL-4 and rIL- 10 markedly reduced vascular permeability in injured lungs by 49% (p < 0.001) and 66% (p < 0.001), respectively, compared with positive controls (Table 1).
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Animals receiving antibody to IL-4 had a slight, but not statistically significant (p = 0.24), increase in MPO activity at 4 hours of reperfusion (0.43 ± 0.06) when compared with positive controls (0.41 ± 0.04).
Treatment with 250 µg of anti-IL-10 antibody was associated with an increase in MPO activity to 0.53 ± 0.04 (p < 0.004), representing a 30% increase in neutrophil accumulation in lung parenchyma; 100 µg of anti-IL-10 did not alter lung MPO significantly (0.44 ± 0.07) relative to positive controls.
Administration of rIL-4 and rIL- 10 reduced MPO content in injured lungs by 50% (p < 0.001) and 66% (p < 0.001), respectively, when compared with positive controls (Table 2).
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In animals treated with anti-IL-4 (164x106 cells) antibody there was a slight, but not statistically significant, increase in cell count compared with positive controls. However, animals treated with 250 µg of anti-IL-10 antibody had counts of 198x106 ± 14 cells, a statistically significant (p < 0.04) increase from positive controls. This represented a 32% increase in BAL leukocyte content. Anti-IL-10 antibody of 100 µg was also associated with an increase in cell counts to 176x106 cells, once again revealing effects not as dramatic as the higher dose (250 µg).
Immunoblotting for IL-4 and IL-10
Interleukin-10 protein was detected at 2, 3, and 4 hours of reperfusion. There was minimal expression in the negative controls, ischemia only, or animals reperfused for 1 hour or less (Fig 1). Similarly, IL-4 protein was also detected at 2, 3, and 4 hours of reperfusion, with no expression noted in negative controls (Fig 2).
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, and IFN-
. In anti-IL-4 treated lungs that underwent ischemia and 4 hours of reperfusion, there was increased expression of all inflammatory and regulatory cytokines compared with the positive control lungs. This effect was even more pronounced in animals receiving antibody to IL-10. Densitometric analysis for TNF-, IL-1ß and IFN- is illustrated in Figure 3.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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In the present study IL-4 and IL-10 expression increased after 2 hours of reperfusion. Antibody blockade of endogenous IL-4 and IL-10 augmented the inflammatory response, whereas administration of exogenous rIL-4 and rIL-10 ameliorated injury in animals subjected to direct lung ischemia and reperfusion. Therefore, these regulatory cytokines are functional in controlling reperfusion injury. Because IL-4 and IL-10 are expressed early after reperfusion, they are temporally positioned to help decrease the intensity of the inflammatory injury.
Prior studies have suggested that the activity of both IL-4 and IL-10 are partially mediated through effects on neutrophil accumulation. Though recombinant IL-10 has previously been reported to decrease neutrophil accumulation in reperfused lungs [19, 20], these experiments involved administration of pharmacologic doses of IL-10 at the onset of injury. Therefore, the physiologic relevance of these studies is questionable. Endogenous IL-4 and IL-10 appear to be released early in reperfusion, and likely mediate their effects primarily through modulating proinflammatory cytokine production. Neutrophil accumulation begins 2 hours after reperfusion and is maximal by 4 hours. Our data indicates that IL-10 and IL-4 protein are detectable at 2 hours of reperfusion. Therefore, the impact of endogenous IL-10 and IL-4 on the process of neutrophil recruitment that is already underway would be expected to be relatively modest. Nonetheless, removing the inhibitory effects of endogenous IL-4 and IL-10 did increase injury severity. More importantly, the impact of antibody to IL-4 and IL-10 on proinflammatory cytokine release (TNF-, IL-1ß) would be expected to have immediate effects on injury factors, such as vascular permeability, and secondary effects on neutrophil recruitment. This is demonstrated by the more pronounced protective effects of rIL-4 and rIL-10 when delivered before reperfusion. In those experiments the reductions in vascular permeability and MPO content were consistently dramatic.
There have been two studies recently that have, in part, confirmed our findings relating to the antiinflammatory role of IL-10. Itano and colleagues [21] found that intravenous adenovirus mediated human IL-10 gene transfer into lung isografts ameliorated subsequent reperfusion injury, improving gas exchange and decreasing lung parenchymal neutrophil sequestration. Tagawa and coworkers [22] used a similar rat lung transplantation model, but introduced the gene for human IL-10 to the donor by an endobronchial route 24-hours before injury. Similar factors of injury were noted to be improved. We have further implicated the antiinflammatory role of endogenous IL-10 in LIRI by demonstrating its increase in expression temporally during early reperfusion, as well as documenting its functional role in modulating lung vascular permeability and alveolar leukocyte sequestration.
The functional role of IL-4 in the development of LIRI has not been studied as extensively as IL-10. It appears to be expressed in response to reperfusion and it functions similarly to IL-10. Indeed, the activity of IL-4 as it pertains to reduced vascular permeability, lung MPO content, and alveolar leukocyte sequestration subsequent to LIRI is indeed novel. However, the protective effects of IL-4 appear to be less pronounced when compared with IL-10.
The antibody studies have therefore helped define the physiologic roles of endogenous IL-4 and IL-10 in this model, and the protective effects of the recombinant formulations of these cytokines suggest potential for development of related therapeutic strategies.
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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B activation, and lung injury induced by hepatic ischemia reperfusion. Am J Physiol 1999;277:919-923.
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), interleukin-1ß (
), and interferon-
) in animals treated with antibody to interleukin-4 and interleukin-10. (IL = interleukin.)