American Journal of Pathology, Vol. 140, No. 4, April 1992 Copyright C American Association of Pathologists
Role of Platelet-activating Factor in Pancreatitis-associated Acute Lung In jury in the Rat Weiguo Zhou,* Mark 0. McCollum,t Barry A. Levine,t and Merle S. Olson* From the Audie L. Murphy Veterans Memorial Hospital, and the Departments of Biochemistry and Surgery, t The University of Texas Health Science Center, San Antonio, Texas
Acute necrotizing pancreatitis induced by infusion of bile salt into the pancreatic duct in rats is consistently associated with acute lung injury similar to the adult respiratory distress syndrome. The role of platelet-activating factor (PAF) in this pancreatitisassociated remote organ failure (lung injury) was investigated. Pulmonary tissue levels of PAF were increased gradually and reached a level of 1345 ± 455 pg/g (6 times the control level) at 12 hours after induction of pancreatitis, whereas pancreatic PAF levels were undetectable and blood PAF remained unchanged This local pulmonary PAF accumulation occurred at approximately the same time as the progression of lung injury. Pulmonary responses detected (i.e., eicosanoid production; leukocytic infiltration, Evan's blue extravasation, P-glucuronidase release) were attenuated to varying degrees by treatment of rats in which pancreatitis was initiated with the PAF receptor antagonists (WEB2170 and BN52021). Rat lung lavages were examined after a 12-hour course ofpancreatitis and no changes in PAF concentration, surfactant content, and phospholipase A2 (PLA2) activity were noted Intravenous administration of PLA2 promoted pulmonary PAF production in experimental rats with pancreatitis but not in normal rats. This observation indicates that PLA2, which was determined to be elevated in plasma during pancreatitis, may be responsible for the accumulation of PAF in the lung. In conclusion, pancreatitis-associated lung injury appears to result from an endogenous inflammatory response in which PAF may play an important role. (Am J Pathol 1992,
Sixty percent of deaths from acute pancreatitis occur within the first 7 days of illness and are almost always associated with acute respiratory failure. This course of events is indistinguishable both clinically and pathologi-
cally from the adult respiratory distress syndrome.1 The events that link these two entities, acute pancreatitis and pulmonary failure, are not fully understood. However, the release of pancreatic-derived proteolytic or hydrolytic enzymes into the systemic circulation coupled with pulmonary surfactant deficiency probably determines the development of lung injury.3 A number of animal models of acute pancreatitis are currently available. The model of acute necrotizing pancreatitis induced by infusion of bile salt into the pancreatic duct is particularly appealing and unique in that increased vascular permeability and diffuse lung injury develop fully and rapidly.3'4 This study ascertained whether the pulmonary insult associated with pancreatitis involves an endogenous inflammatory reaction in the lung in which complex interactions occur between multiple cell types and mediators. Of particular interest was the role of platelet-activating factor (PAF) in remote organ failure (i.e., lung injury) since PAF is a potent endogenous inflammatory signal5 and a well-established mediator of lung injury.6'7
Materials and Methods Materials 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine was purchased from BACHEM Bioscience Inc. (Bubendorf, Switzerland) and polar lipid standards were purchased from Avanti Polar Lipids Inc. (Birmingham, AL). WEB2170 was a gift from Boehringer-lngelheim (Ridgefield, CT). [3H]serotonin (20.4 Ci/mmol) was purchased from E. 1. du Pont de Nemours & Co. (Boston, MA). L-3phosphatidylcholine, 1,2-di [1 -14C] palmitoyl (113 mCi/ mmol) was purchased from Amersham Corp. (Arlington Heights, IL). [3H]eicosanoid RIA kits were purchased from Advanced Magnetics Inc. (Cambridge, MA). All Supported by grants from the NIH (AM 19473) (MSO), the Robert A. Welch Foundation (AQ728) (MSO), and the Veterans Administration Medical Research Services (BAL). Accepted for publication November 18, 1991. Address reprint requests to Dr. Merle S. Olson, Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760.
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other chemicals used were purchased from Sigma (St. Louis, MO).
EGTA. The culture medium was pooled for measurement of spontaneous PAF release from alveolar macrophages.
Determination of PAF
Male Sprague-Dawley rats weighing 250-300 g, fed with standard laboratory chow ad libitum, were anesthetized lightly with ether to undergo laparotomy. Acute necrotizing pancreatitis was induced by retrograde infusion of 5% sodium taurocholate (1.2 ml/kg) into the pancreatic duct via catheter and syringe pump at a constant infusion rate (0.3 ml/min). Control rats received a saline infusion intraductally. The abdominal wounds were closed and the rats were returned to their cages with free access to water and food after surgery. Groups of rats (n > 5) were sacrificed and sampled at various timed intervals. In a second series of experiments, six rats were both pre- and post-treated with a PAF receptor antagonist, WEB2170. WEB2170 (dissolved in saline with the aid of 0.1 N HCI) was administered subcutaneously 1 hour before induction of pancreatitis, and every 4 hours thereafter at a dose of 5 mg/kg. Another six pancreatitic rats were treated with BN52021 (dissolved in saline containing 2% ethanol, 5 mg/kg IV via the tail vein every 4 hours). Control rats were injected with saline alone. Rats were killed and analyzed at 12 hours after induction of pancreatitis. In a third series of experiments, groups of six rats each were injected with 500 units of PLA2 from porcine pancreas as a bolus via the femoral vein. Control rats received saline. All animals were killed for pulmonary PAF measurement at 30 minutes after PLA2 challenge.
Lung Lavage and Alveolar Macrophage Preparation Rats underwent whole-lung lavage via tracheal cannula with 10 ml of Hanks buffer containing 0.25% bovine serum albumin (BSA). The aspirated lavage fluid was then placed into a ice-chilled tube. This procedure was repeated two times, recovering an average of 18 ml of fluid out of the initial 20 ml instilled. An alveolar macrophage pellet was obtained by centrifugation of lung lavage fluid at 600 x g for 10 minutes. The cell pellet was resuspended in 2 ml of RPMI 1640 medium (Gibco) containing 0.25% BSA. The cell suspension was then placed in 35-mm plastic Petri dishes and incubated at 370C for 1 hour. Nonadherent cells were removed from adherent macrophages by washing with RPMI 1640 medium. Purified macrophages were incubated in RPMI 1640 medium for 2 hours. Cell counting was performed by resuspending the cells into 2 ml with
As previously described, samples of pancreatic and pulmonary tissues were freeze-clamped between aluminum tongs cooled in liquid nitrogen and stored at - 700C until analysis.8 Blood samples were withdrawn from the right ventricle at the time of sacrifice and placed immediately into ice-chilled tubes containing heparin. After centrifugation at 40C, 0.2 ml of plasma was removed into 3.8 ml of chloroform/methanol/water (1/2/0.8). Samples of tissue, plasma, cells, lung lavage, or culture medium were employed in the lipid extraction, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses for PAF. PAF activity was assayed using a [3H] serotonin secretion bioassay in washed rabbit platelets.8 Characterization of PAF was performed as described previously.8 -
Determination of Eicosanoids Samples of freeze-clamped lungs were powdered, weighed (0.5 g), and homogenized in 2.5 ml of 0.05 M Tris-HCI buffer (pH 7.4) using a Yamato LSC homogenizer for 2 min. Homogenate (1 ml) was added to 3 ml of absolute ethanol and allowed to stand for 5 minutes. Water (16 ml) was added, vortexed, and centrifuged at 3000 x g for 10 minutes. The supernatant was pooled and acidified to pH 3.0 with 1 M HCI. The acidified sample was then passed through "activated" Sep-Pak C18 Cartridges (Minipore Corp., Milford, MA), washed with 15% ethanol (20 ml) and petroleum ether (10 ml) and eluted with methyl formate (10 ml). Finally the eluted fraction was collected and stored at - 800C until assay. The RIAs for eicosanoids were performed according to the procedure outlined in the instruction manuals for the Advanced Magnetics Inc. RIA kits.
Phospholipid Assay Lyso-phosphatidylcholine (lyso-PC) and phosphatidylcholine (PC), both of which are essential components of pulmonary surfactant present in lung lavage, also were assessed. The phospholipids were separated by TLC8 and each individual phospholipid was quantitated by phosphorus assay.9
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Enzyme Assays Amylase release into the serum after induction of pancreatitis was determined by the quantitative kinetic method using a Sigma Diagnostics amylase reagent. For PLA2 activity, the substrate was L-3phosphatidylcholine, 1,2-di [1 _14C] palmitoyl. The reaction was buffered with 50 ,ul of 0.1 M Tris-HCI (pH 7.4) containing 20 nM of labelled substrate, 2 mM CaC02, and 0.35% cholate; 50 ,u of lung lavage or plasma was added and the mixture was incubated at 37°C for 20 minutes.10 PLA2 activity was quantitated radiometrically by the formation of lyso-PC isolated by TLC.8 Myeloperoxidase (MPO) was extracted from homogenized lung tissue by suspending the material in 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer (PH 6.0) before sonication in an ice bath for 10 seconds. The specimens were subjected to freezing and thawing three times, after which sonication was repeated. Suspensions were then centrifuged at 40,000 x g for 10 minutes and the resultant supernatant containing MPO was assayed spectrophotometrically at 250C by following the increase in absorbance at 470 nm resulting from the oxidation of guaiacol.11 An enzyme unit is defined as the amount of enzyme that produces an increase of 1 absorbance unit per minute. ,B-glucuronidase (a lysosomal enzyme) activity in lung lavage was used as another parameter of general cytotoxicity and was measured spectrophotometrically using a Sigma Diagnostics b-glucuronidase kit (catalog no. 325-A). Lung lavage fluid was obtained by lavaging whole lungs with 2 ml of saline. A Sigma unit of P-glucuronidase activity is defined as 1 ,ug of phenolphthalein liberated from phenolphthalein glucuronic acid per hour at 560C.
Evaluation of Vascular Permeability In a preliminary study, no change in lung weight was observed during the 12-hour course of pancreatitis. The Evan's blue dye extravasation method was used to quantitate the increase in vascular permeability. Rats were injected intravenously with the dye at a dose of 20 mg/kg. After 15 minutes, the animals were killed, the lungs were removed, and the wet weight was determined. The dye was then extracted from the tissue by incubation with 4 ml of formamide for 24 hours at 370C. The quantity of dye extracted was determined spectrophotometrically at 620 nm and calculated from a standard curve established with known amounts of Evan's blue dye. Results are expressed as milligrams of dye per gram of wet tissue.
Histologic Methods Portions of the lung and pancreas were removed and fixed immediately in 10% buffered formalin. Sections were stained with hematoxylin and eosin and examined by light microscopy.
Statistical Analysis All values in the tables and figures represent means ± SEM. One factor ANOVA with multiple comparisons was used, and significance was assigned to a P value < 0.01, determined using the Scheffe F test.
Histology and Serum Enzymology All animals showed enzymatic and microscopic evidence of acute pancreatitis after injection of bile salt into the pancreatic duct. A fatal necrotizing pancreatitis evolved over hours with widespread hemorrhage, little leukocytic infiltration, massive necrosis, ascites, and 100% mortality within 30 hours. Control rats receiving saline intraductally appeared healthy. The pancreas was edematous initially but resolved within 6 hours. Hyperamylasemia and enhanced PLA2 activity in the plasma were observed in pancreatitic rats (Figure 2), but not in control rats. All animals with experimental pancreatitis developed acute lung injury as early as 3 hours postductal injection. This lung injury was characterized by a mild acute inflammatory response. However, the lungs of these animals displayed progressive but patchy interstitial edema, intraalveolar hemorrhage and inflammatory cell infiltration (primarily neutrophils and macrophages) (Figure 1). The kinetics of pulmonary neutrophil infiltration after infusion of bile salt were determined by measuring pulmonary MPO content. MPO levels were maximal after 3 hours and gradually decreased over the next 9 hours; elevated levels of MPO persisted at 12 hours postinfusion of bile salt (Figure 3). Also, bronchoalveolar macrophage accumulation was increased (Table 1).
Measurement of PAF Pulmonary tissue PAF levels were determined in samples of lungs obtained 3, 6, 12 hours after infusion of bile salt into the pancreatic duct. The tissue levels of PAF in the
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K. _ a.
Figure 1. Light micrographs of the rat tlung (H & E, X-300). Above: Control lung. Below: Lung at 12 br after induction of acute pancreatitis (intraalveolar hemorrhage and leukocitic infiltration).
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rats without PLA2 challenge (222.5 ± 50.2 vs. 210 ± 37.2 pg per g) (Figure 5).
Effects of PAF Antagonists
(1) UO ct r--4
Time (hr) Figure 2. Amylase and PLA2 levels in plasma during acute pancreatitis. PLA2 activity is expressed as nmol phosphatidy1choline hydrolyzed per min per ml ofplasma. All values are the means SEM from five to seven rats
normal, untreated rats were 212.5 89 pg/g wet tissue and rose with increasing duration of the disease process, reaching a level of 1296.7 pg/g at 12 hours. In contrast, PAF levels in blood and lung lavage samples remained low and unchanged, and PAF in the injured pancreas was undetectable during the 12-hour kinetic study (Figure 4). Also, no difference was noted in spontaneous PAF release by alveolar macrophages from normal and pancreatitic rats (data not shown). Pulmonary tissue PAF levels were assessed at 30 minutes after PLA2 challenge (500 units/rat, IV). PLA2challenged rats in which pancreatitis had been ongoing for 6 hours showed a marked increase in pulmonary tissue PAF levels when compared with PAF levels in lungs from pancreatitic rats treated with intravenous saline instead of PLA2 (controls) (984.7 76.6 vs. 442.7 70.2 pg per g). However, normal rats after PLA2 challenge had pulmonary PAF levels similar to those seen in normal ±
Discussion 1.0 0.0' 0
Time (hr) Figure 3. Kinetics ofpulmonary MPO content during acute pancreatitis. Results are expressed as the means SEMfrom five to six rats.
Biochemical Analysis of Lung Lavage Lung lavage collected at 12 hours after bile salt infusion was analyzed. All of the animals, including both control rats and experimental rats, had essentially the same values of PLA2 activity, PAF, PC, and lyso-PC in their respective lavage samples. Data are shown in Table 2.
The effects of WEB2170 and BN52021 administration on the tissue levels of eicosanoids, MPO and Evan's blue extravasation, and on macrophage accumulation in and ,-glucuronidase release into lung lavage fluid were evaluated. As shown in Table 1, the tissue levels of TXB2 (the stable metabolite of TXA2), 6-keto PGFia (the end product of PGI2 metabolism), MPO and Evan's blue extravasation at 12 hours after bile salt infusion were: 27.2 ± 4.6 ng/g of wet weight of tissue (4 times normal levels), 37.6 ± 6.7 ng/g (3 times normal levels), 1.93 ± 0.35 U/g (2.5 times normal levels), and 136 ± 11 ng/g (2 times normal levels), respectively. The bronchoalveolar macrophage accumulation and 3-glucuronidase release were: 8.61 ± 1.91 x 106 cells/ml (5 times normal levels) and 24.9 ± 6.2 Sigma U/ml (6 times normal levels), respectively. When treated with PAF antagonists, TXB2 was reduced by 64% with WEB2170 treatment and 63% with BN52021 treatment, 6-keto PGF1i was reduced by 60% with WEB2170 treatment and 67% with BN52021 treatment, MPO was reduced by 26% with WEB2170 treatment and 35% with BN52021 treatment, Evan's blue extravasation was reduced by 15% with WEB2170 treatment and 40% with BN52021 treatment. Bronchoalveolar macrophage accumulation was reduced by 25% with WEB2170 treatment and 54% with BN52021 treatment, and ,B-glucuronidase release was reduced by 31% with WEB2170 treatment and 51% with BN52021 treatment.
The role of PAF in the respiratory system in both experimental models7 and clinical medicine,12 and in both physiology13 and pathophysiology7 has been a subject of great interest. PAF appears to be involved in many types of inflammatory processes,5 and has recently been implicated in specific disease states including bacterial meningitis,14 organ graft rejection,15 and cancer.16 The data presented in this communication demonstrate that:
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Table 1. Effects of PAF Antagonists on Pulmonary Responses
Pancreatitis + BN52021
10 6.7 t 0.6 11.6 t 3.3 0.79 t 0.14 65 t 4 1.72 t 0.41 24.9 t 6.2
7 27.2 t 4.6 37.6 t 6.7 1.93 t 0.35 136 ± 11 8.61 t 1.91 142.3 t 23.1
6 9.5 t 0.9* 14.9 t 2.3* 1.41 t 0.17* 112 t 15* 6.41 t 0.78* 98.2 t 15.3*
6 10.3 t 1.0* 12.2 t 2.7* 1.23 t 0.22* 89 ± 9* 3.13 t 0.82* 70.2 t 11.2*
No.ofrats TXB2 (ng/g) 6-keto PGFi,a (ng/g) MPO (U/g) Evans blue (,g/g) AM (x106 cells/ml) BG (Sigma U/ml)
Significant differences from the pancreatitis group (P < 0.05, Scheffe F test). Results are expressed as the means ± SEM. Rats were sacrificed at 12 hr after induction of acute pancreatitis with or without PAF antagonist treatment. WEB2170 was administered subcutaneously and BN52021 intravenously. AM = alveolar macrophages. BG = p-glucuronidase in lung lavage. *
1) PAF is produced in the lung after induction of acute pancreatitis, and 2) treatment of rats with a PAF receptor antagonist attenuated lung inflammation and injury. Therefore, PAF, as a common inflammatory signal, may contribute to the pathology of the acute lung injury in this model of acute pancreatitis. These findings are consistent with previous studies showing that pancreatitisinduced acute lung injury results from activation of the endogenous inflammatory response in the lung.4 Our studies describe a model of acute lung inflammation and injury associated with pancreatitis in which remote organ (pulmonary) failure is produced, at least in part, in conjuction with the elevation of PAF in lung tissue. It has been demonstrated that alveolar macrophages,17 lung mast cells,18 lung epithelial cells,19 and lung endothelial cells2O are all capable of synthesizing PAF. The present study did not demonstrate an increase in the levels of PAF in lung lavage fluid after induction of acute pancreatitis. Also, similar amounts of PAF were released by alveolar macrophages isolated from rats with and without pancreatitis. Therefore, we would conclude that alveolar macrophages likely were not the cellular 1600 O=* S