Increased group IV cytosolic phospholipase A2 activity in lungs of sheep after smoke inhalation injury

Taeko Fukuda, Dae Kyong Kim, Mi-Reyoung Chin, Charles A. Hales, and Joseph V. Bonventre

Medical Services, Massachusetts General Hospital, Charlestown 02129; and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Increased phospholipase A2 (PLA2) activity was measured in cytosolic fractions of lungs from sheep exposed to smoke from burning cotton or to synthetic smoke consisting of carbon and acrolein, a cotton smoke toxin. Three peaks of PLA2 activity were identified by heparin-Sepharose chromatography. The heparin-nonbinding PLA2 activity was twofold higher in the extracts from lungs exposed to smoke than in normal lungs. This activity was identified as the group IV 85-kDa cytosolic PLA2 (cPLA2). The activities of the forms of PLA2 that bound to heparin did not change after smoke exposure. Those activities showed a pH optimum of 9.0, required a millimolar Ca2+ concentration for full activity, and were inhibited by 5 mM dithiothreitol. One activity eluted at an NaCl concentration typical for group Ib and V PLA2 and had the expected substrate specificity. The other form of lung PLA2 that bound heparin was a group II PLA2. Lung myeloperoxidase activity increased progressively with increased exposure to smoke. cPLA2 was identified in sheep neutrophils. With 30 breaths of smoke exposure, there was an increase in cPLA2 activity without a difference in immunoreactivity on Western blot, indicating that the increased activity was not due to increased amounts of protein. In conclusion, smoke induces increases in resident lung cell cPLA2 activity that is likely responsible for eicosanoid production, leading to lung inflammation and bronchoconstriction.

eicosanoids; polymorphonuclear leukocytes; acrolein; cotton smoke


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LUNG INJURY FROM SMOKE INHALATION is a major cause of mortality (34). Smoke inhalation initiates an inflammatory cascade, with leukocyte infiltration, cytokine release, and generation of mediators such as reactive oxygen species, proteases, and products of arachidonic acid (AA) metabolism (27), with a subsequent increase in vascular permeability (28).

Heat, except when inhaled as steam, is not the primary agent causing lung parenchymal injury. Rather, chemical toxins carried in either the gas or particle phase confer most of the damage (19). Acrolein, a potent aldehyde that is a major component of smoke from burning wood, cotton, cigarettes, and smog, has a severe irritant effect on the airways (25) and is a known cause of high-permeability pulmonary edema (10).

Phospholipases A2 (EC. 3.1.1.4) play key roles in the production of intracellular and extracellular chemical mediators of inflammation, such as eicosanoids. Phospholipases A2 hydrolyze fatty acids bound at the sn-2 position of glycerophospholipids (37, 38). A major product of phospholipase A2 (PLA2) action on membrane lipids is AA, the eicosanoid metabolites of which include prostaglandins and leukotrienes that modulate membrane channel activity and signal transduction, are vasoactive and chemotactic, and are implicated in many pathophysiological mechanisms of inflammation and tissue injury (3, 4). Eicosanoids have been identified in bronchoalveolar lavage fluid (BALF) and lung lymph after inhalation of smoke from burning cotton or synthetic smoke consisting of acrolein adherent to cotton particles (12, 28). Blockade of the cyclooxygenase and lipoxygenase cascades with BW-755C substantially ameliorates the appearance of pulmonary edema after cotton smoke or acrolein smoke inhalation, demonstrating a causative role for eicosanoids in smoke-induced injury (11).

The superfamily of mammalian phospholipases A2 is composed of a number of members that vary in size, tissue distribution, Ca2+ dependency, substrate preference, and sensitivity to dithiothreitol (DTT) (7). Four related genes encode four Ca2+-dependent secretory phospholipases A2 (groups I, IIa, IIc, and V), with approximate molecular masses of 14 kDa and requirements for 10-4 to 10-3 M Ca2+ concentration for effective hydrolysis of substrates. Group I (29) and II phospholipases A2 (26) have been found in the lung. Inflammatory cytokines, such as tumor necrosis factor, interleukin-1, and interleukin-6 induce transcription of the group II PLA2 gene, resulting in increased eicosanoid generation in a variety of target cells (31).

The intracellular high-molecular-mass (85-kDa) group IV PLA2 [cytosolic PLA2 (cPLA2)] has been implicated in Ca2+- and receptor-mediated liberation of arachidonic acid (AA) (4, 9, 15, 31). cPLA2 has been purified from various tissues including bovine lung, bovine kidney, pig spleen (13), human monocytic U937 cells (6, 32), rabbit platelets (16), and the rat macrophage cell line RAW 264.7 (22). cPLA2 is important for methacholine-induced bronchial reactivity (35). cPLA2 activity is also activated by reactive oxygen species (30), which are important mediators of smoke-induced lung injury.

To evaluate whether increased levels of PLA2 activity might contribute to eicosanoid production and inflammation after short exposures to smoke, we identified and characterized PLA2 activity in sheep lungs after exposure to smoke from burning cotton or to a synthetic smoke containing only carbon and acrolein, which is likely the major toxin in cotton smoke. In the acrolein-carbon- or cotton smoke-exposed lungs, there was an increase in PLA2 activity that was identified biochemically and immunochemically as cPLA2. With longer periods of smoke exposure, myeloperoxidase (MPO) activity in the lung was increased, which suggests that neutrophil infiltration may be responsible for some but not all of the increased lung cPLA2 activity. This increased cPLA2 activity, with its substrate specificity for AA-containing phospholipids, may account for the generation of inflammatory mediators that lead to pulmonary edema and respiratory distress.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. 1-Acyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoethanolamine (2-[1-14C]AA-GPE; 55.1 mCi/mmol) and 1-stearoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphocholine (2-[1-14C]AA-GPC; 56.7 mCi/mmol) were purchased from Amersham. Heparin-Sepharose HiTrap, Mono Q, and Superose 12 columns were obtained from Pharmacia LKB Biotechnology. All other chemicals were of the highest purity available from commercial sources.

Preparation of animals and harvesting of lungs. Domesticated sheep weighing from 25 to 27 kg were anesthetized with intravenous pentobarbital sodium (25 mg/kg induction; 150- to 200-mg maintenance dose given intermittently to maintain deep anesthesia), intubated with a cuffed endotracheal tube (10 mm ID, 33 cm long), and mechanically ventilated with 35% oxygen with a volume ventilator (Harvard Apparatus, Millis, MA) set initially at a tidal volume of 15 ml/kg at 15 breaths/min and 2 Torr positive end-expiratory pressure. The respiratory rate was adjusted to achieve an arterial PCO2 of 33-42 Torr.

Blood gases and pH were measured at 38°C with an Instrumentation Laboratory 1306 blood gas analyzer (Watertown, MA). An oral tube was passed into the stomach to evacuate the contents. A catheter was inserted into a femoral vein to permit infusion of lactated Ringer solution at a rate sufficient to maintain a pulmonary capillary wedge pressure of 5 mmHg. A right thoracotomy was performed, and a lymph fistula was established in the caudal mediastinal lymph node with the use of a modification of the technique of Staub et al. (33). The distal node coming from the abdomen was ligated with a double suture to decrease contamination. As the thorax was closed, a tube was placed and connected to a sealed collection system (Pleur-Evac, Deknatel, Floral Park, NY) with -20 cmH2O applied. A Swan-Ganz pulmonary arterial catheter (model 93A-13H-7F, American Edwards Laboratories, Santa Ana, CA) was inserted via an internal jugular vein and positioned in the pulmonary artery by continuously following the wave forms on the monitor. Pulmonary arterial pressure, intermittent pulmonary capillary wedge pressure, and tracheal pressure were monitored throughout the experiments with transducers (model P23 XL, Spectromed, Oxnard, CA) mounted at the midthoracic level. Data were continuously recorded on a Gould (Cleveland, OH) model 3400 chart recorder. Cardiac output was determined in duplicate by using thermal dilution and a Cardiac OutPUT computer (COM-1, American Edwards Laboratories).

Lung lymph was continuously collected from below the level of the lymph fistula and measured every 30 min. The samples were placed immediately on ice at the time of collection and were later centrifuged for 10 min at 2,300 rpm at 4°C. Protein content was determined on the supernatant with a protometer (National Instrument, Baltimore, MD). Sytemic and pulmonary arterial pressures and cardiac output were monitored.

Exposure to smoke. The animals were allowed to stabilize on the anesthetic for 1-1.5 h, during which time baseline measurement of the hemodynamic parameters and lymph flow were measured every 30 min. Smoke generation and administration were done in one of two ways, involving either a smoke generator on which Hales et al. (10) have previously published or real smoke from burning cotton (11).

The synthetic smoke system allowed the delivery of smoke containing only one toxin at a time. Each breath of room air from the ventilator was passed through the smoke generator, which divided flow into two streams according to the settings of the two needle valves. The resultant flows were measured on flow meters (Lab Crest, Waltham, MA). One stream was fed into an ultrasonic nebulizer (model US-1, Puritan-Bennett, Los Angeles, CA) that was filled with distilled water and the toxin acrolein. The other stream was fed through a jet into a cup filled with carbon particles (D-Dacro G-60, Fisher Scientific, Fair Lawn, NJ) with a mean geometric diameter of 3.9 ± 2 (SD) µm as determined by a particle impactor (Anderson Sampler, Atlanta, GA). The carbon particles were then elutriated into the exit tube. The airflows from the nebulizer and carbon chimney were mixed and fed through a thermostat-controlled heating system (Hot-Watt 6 Fo10, Danvers, MA, and a Fenwall controller 551, Ashland, MA) into a condensing section, allowing hot gases and carbon to cool to body temperature. The vaporization-condensation process produces an aerosol that has characteristics independent of the ultrasonic nebulizer. In addition, the carbon provides nucleation sites, thus being incorporated into the droplets. The smoke was then provided to the animal through an endotracheal tube. Immediately after exposure to smoke in room air, the inspired fraction of O2 was increased to 0.35.

The toxin used in the present study was acrolein (100 µl, density 0.841 g/ml; Kodak, Rochester, NY) that was mixed with 100 ml of distilled water and placed in the nebulizer that was set to deliver 5 ml of distilled water and toxin over the 10-min exposure. Carbon particles were weighed before being placed in the chimney. The temperature of the heater was set at 120°C, and the condensing section was cooled by airflow to lower the effluent gas temperature to 40°C. Liquid remaining in the nebulizer and carbon remaining in the chimney at the end of exposure were weighed. The concentrations of inhaled acrolein in the gas mixture at the first, middle, and last minutes of acrolein smoke exposure were 50-100 parts/million (10).

Real smoke was generated with a modified Bee smoker (The Bee Keeper, Woburn, MA) as originally described by Walker et al. (39) and subsequently modified by Kimaura et al. (17). Ten pure cotton pledgits (14 g) were packed in the chamber and ignited instantly with a blowtorch. The smoker was attached to the sheep via the endotracheal tube while 16 breaths of smoke were delivered. The sheep was then returned to the ventilator while the smoker was recharged and refired. A total of 15-130 breaths was delivered to each sheep. The tidal volume was 640 ± 35 ml as determined by measuring exhaled minute ventilation from the sheep and dividing by the respiratory rate. We have grouped together sheep receiving 40-50 breaths of cotton smoke and those receiving 120-130 breaths of cotton smoke, referring to them as 40- or 120-breath groups, respectively. In all cases, the sheep exposed to acrolein-carbon received 120 breaths.

All hemodynamic measurements were continued for 4 h after smoke exposure. At the end of this 4-h period, the sheep were killed with intravenous thiopental and potassium chloride. The lungs were removed and kept at -80°C. For gravimetric analysis, the lungs were trimmed of extra parenchymal airway and blood vessels, blotted dry, weighed, and then placed in an oven at 80°C for 48 h. The lung was weighed and dried for another 24 h at 80°C. If the weight fell between the two readings, the lung was dried for another 24 h.

PLA2 assay. PLA2 activity was assayed with ethanol-solubilized 2-[1-14C]AA-GPE (55.1 mCi/mol; Amersham). The standard PLA2 assay buffer (100 µl) contained 100 mM Tris · HCl, 5 mM CaCl2, and 0.5 nmol of radioactive phospholipids (~65,000 counts/min) at pH 9.0. The reaction was carried out at 37°C for 30 min and was stopped by adding 0.56 ml of Dole's reagent (78% propan-2-ol, 20% n-heptane, and 2% of 2 N H2SO4 in water) (8). Water (0.11 ml) was added, and the sample was vortex mixed and centrifuged at 10,000 g for 2 min, then 0.15 ml of the upper phase was transferred to a new Eppendorf tube to which 50 mg of silica gel and 0.8 ml of n-heptane were added, vortex mixed, and centrifuged again at 10,000 g for 2 min. Finally, 0.8 ml of supernatant, which contained released fatty acid, was counted for radioactivity in a beta liquid scintillation counter. Protein concentration was determined with the Bradford protein assay (Bio-Rad Laboratories, Melville, NY), with bovine serum albumin (BSA) as the standard.

Heparin-Sepharose HiTrap column chromatography. Sheep lungs, frozen at -80°C, were thawed at 37°C, minced, and homogenized in six volumes (vol/wt) of homogenizing buffer (50 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 0.15 M NaCl) with a polytron (Brinkmann homogenizer). The homogenates were centrifuged at 1,000 g for 20 min. All subsequent manipulations of lung tissue for characterization were performed at 4°C in a cold room or on ice unless otherwise indicated. The supernatants were centrifuged at 100,000 g for 60 min at 4°C. The resulting supernatants were filtered with MILEX-HA (0.45-µm-pore size; Millipore, Bedford, MA), diluted in the same volume of buffer A (50 mM Tris · HCl, pH 7.4, and 1 mM EDTA), and applied at a flow rate of 2 ml/min to a heparin-Sepharose column (HiTrap 5 ml, Pharmacia LKB Biotechnology) preequilibrated with buffer A. The column was washed with 40 ml of buffer A. Bound fractions were eluted at a flow rate of 2 ml/min, with a 40-ml linear gradient of 0-2.0 M NaCl. Fractions (2 ml) were collected and assayed for PLA2 activity.

Ion-exchange chromatography on Mono Q fast-performance liquid column. The pooled heparin-nonbinding fractions of sheep lung were loaded onto a Mono Q fast-performance liquid chromatography (FPLC) column (0.5 × 5 cm; Pharmacia LKB Biotechnology) preequilibrated with buffer A. Proteins were eluted at a flow rate of 1.0 ml, with a 20-ml linear gradient of 0-1.0 M NaCl. Fractions (1.0 ml) were collected and assayed for PLA2 activity.

Gel-filtration chromatography on Superose 12 FPLC column. The active pool (fractions 9-11; 3 ml) from the Mono Q column was concentrated to ~200 µl with a Centricon 10 (Amicon, Danvers, MA). The concentrated sample was injected onto a Superose 12 FPLC column preequilibrated with 50 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 0.5 M NaCl. Proteins were eluted at a flow rate of 0.5 ml/min. Fractions (1.0 ml) were collected and assayed for PLA2 activity. For molecular-mass determination, a mixture of molecular-mass markers, RNase A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), BSA (67 kDa), and blue dextran 2000 (2,000 kDa), was injected onto the column under the same conditions.

Immunoprecipitation of PLA2 activity. Packed protein A-Sepharose beads saturated with rabbit anti-porcine spleen 100-kDa PLA2 antiserum or rabbit normal serum were prepared as previously described (13). Rabbit antiserum made against porcine spleen 100-kDa cPLA2 or rabbit preimmune serum was mixed with packed protein A-Sepharose beads preequilibrated with 20 mM Tris · HCl buffer (pH 7.5) containing 5 mg/ml of BSA (2:1 vol/vol) and incubated for 24 h at 4°C with constant shaking. The beads were washed six times with 20 mM Tris · HCl (pH 7.5) containing 1 mM EDTA and 5 mg/ml of BSA. Fifty microliters of protein A-Sepharose beads were incubated for 4, 8, and 16 h at 4°C with 450 µl of the active pool of PLA2 activity collected from Mono Q FPLC with constant shaking. The beads were then pelleted by centrifuging at 10,000 g for 5 min, and the supernatants were assayed for PLA2 activity.

Immunoblotting. Proteins were separated by SDS-PAGE (10% gels) (18) and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell) in 25 mM Tris · HCl (pH 8.3)-190 mM glycine-20% methanol. Nonspecific binding of antisera to nitrocellulose was prevented by preincubation of the nitrocellulose in 3% BSA in Tris-buffered saline (24.8 mM Tris · HCl, pH 8.0, 2.7 mM KCl, and 137 mM NaCl) for 2 h at room temperature. The blocked nitrocellulose membrane was incubated with 1:5,000 diluted anti-cPLA2 antiserum overnight at room temperature with constant shaking. Unbound antibodies were removed with three washes of Tris-buffered saline containing 0.1% Tween 20, and the sites of antibody binding were developed with an enhanced chemiluminescence system (Amersham).

MPO activity. MPO activity, used as an indicator of leukocyte, primarily neutrophil, infiltration, was measured in lung tissue after 2 h of smoke exposure or on non-smoke-exposed lung tissue. An aliquot (0.4 ml) of the 40,000-g supernatants of lung homogenates, prepared as described by Bradley et al. (5), was added to 2.6 ml of reaction mixture containing 50 mM potassium phosphate buffer (pH 6.0), 0.2 mg o-dianisidine dihydrochloride/ml, and 0.0006% H2O2. Absorbance was measured at 460 nm. Assay linearity was confirmed. MPO activity, normalized to protein content of the supernatant, is expressed as the percentage of the levels in normal lung.

Statistics. Data are expressed as means ± SE. Differences among mean values of PLA2 specific activities and among groups were evaluated by analysis of variance. Student's t-test was used for comparisons between two groups, and differences were considered significant if P was <0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic and functional effects of smoke inhalation. Pulmonary arterial pressure did not rise significantly after 15 breaths of cotton smoke (Table 1), although by 4 h after smoke, lung lymphatic clearance of protein had risen. This suggested an increase in lung microvascular permeability because the lymph-to-plasma protein ratio was constant (Table 2). However, the wet-to-dry weight ratio did not rise (Fig. 1), presumably because the lymph drainage was adequate to prevent pulmonary edema. In two sheep that received 30 breaths of smoke and five that received 40-50 breaths, there was a modest rise in pulmonary arterial pressure of 3-4 mmHg, which was partly, but not entirely, due to a rise in cardiac output because pulmonary vascular resistance rose (P < 0.05; Table 1). Pulmonary vascular resistance was calculated as (mean pulmonary artery pressure - mean pulmonary capillary wedge pressure)/cardiac output. Lung lymph flow and lymph protein clearance rose significantly (Table 2) but the lymph-to-plasma protein ratio fell. Thus lung microvascular fluid movement into the extravascular space was likely due to a combination of pressure rise and an increase in microvascular permeability. Again, the increase in lymph flow prevented a significant increase in pulmonary edema as the lung wet-to-dry weight ratio did not rise significantly in this small group, although there was a small trend up in this ratio with an increased number of breaths of smoke (Fig. 1). Both 120-130 breaths of cotton smoke and synthetic smoke with acrolein-carbon caused mild pulmonary edema (Fig. 1) as well as an increase in lymph flow and lymph protein clearance (Table 2). Pulmonary arterial pressure rose with acrolein-carbon exposure and probably contributed to increased lymph flow, although the lymph-to-plasma protein ratio did not fall, showing that there was also a change in microvascular permeability. High-dose smoke, unlike lower doses, did not increase pulmonary arterial pressure or pulmonary vascular resistance (Table 1). Thus the increased lymph flow and wet-to-dry weight ratio of the lung were likely due to increases in microvascular permeability.

                              
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Table 1.   Hemodynamics before and after smoke exposure


                              
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Table 2.   Lymphatic parameters before and after smoke



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Fig. 1.   Blood corrected wet-to-dry (Wet/Dry) weight ratios in smoke-exposed lungs. No. of animals/group are the same as in Table 1. * P < 0.05 compared with control group.

PLA2 activity of 100,000-g supernatants of sheep lungs. PLA2 activities of the 100,000-g supernatants from sheep lungs after 10 min of exposure to acrolein-carbon smoke or burning cotton smoke or no treatment were assayed with 2-[1-14C]AA-GPE as the substrate (Fig. 2). PLA2 activities in both types of smoke-exposed lungs were significantly higher than those in normal lungs. There was no significant difference between the PLA2 activities of lungs exposed to acrolein-carbon or burning cotton smoke. As few as 15 breaths of cotton smoke resulted in a significant increase in lung PLA2 activity (see Correlation between PLA2 activity and MPO activity).



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Fig. 2.   Comparison of phospholipase A2 (PLA2) activity of 100,000-g supernatant from normal sheep lungs or lungs exposed to 120 breaths of smoke derived from acrolein or burning cotton. A: PLA2 activity after exposure to acrolein or cotton smoke. Each aliquot of 100,000-g supernatant was assayed for PLA2 activity with 1-stearyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoethanolamine (2-[1-14C]AA-GPE) for 30 min at 37°C. Results are means ± SE; n = 9 animals/group. B: assay measurement of PLA2 activity with varying amounts of 100,000-g supernatant protein added to assay. cpm, Counts/min.

Characterization of sheep lung PLA2 activities. The PLA2 activity of sheep lungs was resolved into three peaks by heparin-Sepharose chromatography (Fig. 3). Two peaks of PLA2 activities were present in the heparin-binding fractions and one in the heparin-nonbinding fractions. In the fractions that bound to heparin, the PLA2 activities in the two peaks eluted from the column were equivalent in normal and smoke-exposed lungs. In contrast, the activity in the heparin-nonbinding fractions from the smoke-exposed lungs was higher by more than threefold than that in the fractions from normal lungs.


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Fig. 3.   Heparin-Sepharose column chromatography of normal and acrolein- and burning cotton smoke-exposed sheep lungs. Each aliquot of filtered 100,000-g supernatant was diluted by the same volume of 50 mM Tris · HCl (pH 7.4) and 1 mM EDTA and applied to a heparin-Sepharose HiTrap (5-ml) column preequilibrated with the same buffer. Bound fractions were eluted at a flow rate of 2 ml/min, with a 40-ml linear gradient of 0-2.0 M NaCl. Fractions (2 ml) were collected, and a 40-µl aliquot from each fraction was assayed for PLA2 activity with 2-[1-14C]AA-GPE.

The heparin-binding activity was further characterized and compared with group Ib and IIa PLA2 activities derived from bovine pancreas and rat platelets, respectively. Supernatants (100,000 g) of rat platelets alone or a mixture of purified pancreatic PLA2 with BSA (Sigma, St. Louis, MO) and 100,000-g supernatants of rat platelets were applied to a heparin-5PW FPLC column (0.75 × 7.5 cm; TosoHaas, Montgomeryville, PA). The first peak of heparin-binding lung PLA2 activity eluted in the same fractions as those of porcine pancreatic group I PLA2. The second peak of heparin-binding PLA2 activity eluted at the same NaCl concentration as that of the rat platelet group II (data not shown). In addition, both heparin-binding forms of PLA2 activity hydrolyzed 2-[1-14C]AA-GPE preferentially to 2-[1-14C]AA-GPC as the substrate. As shown in Table 3, the activity of the first peak of heparin-binding activity in both normal and smoke-treated lungs against 2-[1-14C]AA-GPE was <1.5 times that with 2-[1-14C]AA-GPC as the substrate, which is consistent with what is seen with group I PLA2 where this ratio is generally <2 (16). Group V secretory PLA2 has phospholipid head-group specificity and heparin binding characteristics that are more similar to group Ib than to group IIa secretory PLA2 (12a). Thus the first peak may include both group Ib and V secretory PLA2. In contrast, the activities of peak 2 against 2-[1-14C]AA-GPE were five times greater than those seen with 2-[1-14C]AA-GPC as the substrate, a property of group II PLA2. Both heparin-binding PLA2 activities of lungs were almost completely inhibited by preincubation with 5 mM DTT at 37°C for 30 min (data not shown). All of these properties are consistent with identification of the two heparin-bound activities as group I and group II phospholipases A2.

                              
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Table 3.   PLA2 specific activities from heparin-Sepharose chromatography of sheep lung

Characterization of the PLA2 activity detected in the heparin-nonbinding fractions of sheep lungs. To characterize the PLA2 activity in the heparin-nonbinding fractions, the active pools from normal and smoke-exposed sheep lungs were loaded onto a Mono Q FPLC column preequilibrated with 50 mM Tris · HCl, pH 7.4, and 1 mM EDTA. The PLA2 activities were eluted as a major peak in fractions 9-11 with increasing salt concentrations (Fig. 4). The specific activity of the heparin-nonbinding fractions from the smoke-exposed sheep lungs was higher than that of corresponding fractions from the normal lungs. To examine whether or not this activity is cPLA2, as a positive control, we partially purified cPLA2 from porcine spleen (13) and human monocytic U937 cells. These enzymes were eluted in the same fractions from the Mono Q column (data not shown).


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Fig. 4.   Ion-exchange chromatography by Mono Q fast-performance liquid chromatography (FPLC) of heparin-nonbinding PLA2 activity of sheep lung. Heparin-nonbinding fractions, obtained in experiments described in Fig. 2, were directly loaded to a Mono Q column that was preequilibrated with 50 mM Tris · HCl (pH 7.4) and 1 mM EDTA. Proteins were eluted at a flow rate of 1.0 ml/min, with a 20-ml linear gradient of 0-1.0 M NaCl. Fractions (1.0 ml) were collected, and 40 µl from each fraction was assayed for PLA2 activity for 30 min at 37°C with 2-[1-14C]AA-GPE as substrate.

The heparin-nonbinding activity was further characterized by examining the biochemical properties, including measurement of apparent molecular mass by gel-filtration chromatography, substrate specificity, Ca2+ requirement, pH dependency, and DTT sensitivity as well as the immunochemical properties such as immunoprecipitation and immunoblotting. To examine the apparent molecular mass, the active pool (3 ml) was concentrated to 200 µl with a Centricon 10 (Amicon) and injected onto Superose 12 gel-filtration FPLC preequilibrated with 50 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 0.5 M NaCl and eluted with the same buffer. The molecular mass was estimated to be 70-80 kDa by comparison with molecular-mass standards for gel-filtration chromatography (Pharmacia LKB Biotechnology) (Fig. 5). In a parallel experiment, the partially purified porcine spleen cPLA2, which has a true molecular mass >80 kDa (13), was found to elute in the same fractions (data not shown).


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Fig. 5.   Gel-filtration chromatography of the Mono Q-binding PLA2 activity with Superose 12 FPLC. Pooled sample (3 ml) eluted by Mono Q column (Fig. 4) was concentrated to ~200 µl with a Centricon 10. Concentrated sample was introduced onto a Superose 12 FPLC column, preequilibrated with 50 mM Tris · HCl (pH 7.4), 1 mM EDTA, and 0.5 M NaCl. Sixty microliters of each 1-ml fraction were assayed for PLA2 activity with 1-[1-14C]AA-GPE as substrate. Major peak of activity eluted with estimated molecular mass of 70-80 kDa. A mixture of molecular-mass standards, including RNase A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), and blue dextran 2000 (2,000 kDa), was injected (arrows) onto column under the same conditions as PLA2 sample.

The pH dependency of this lung PLA2 activity revealed an optimal activity at pH 9.0 at 5 mM Ca2+ with both 2-[1-14C]AA-GPE and 2-[1-14C]AA-GPC as substrates. Fractions showed full PLA2 activity at 10-5 M Ca2+ (data not shown).

Immunoprecipitation of PLA2 activity with anti-cPLA2 antiserum. The effect of anti-PLA2 antiserum on PLA2 activity of Mono Q active fractions was evaluated by measuring 2-[1-14C]AA-GPE- and 2-[1-14C]AA-GPC-hydrolyzing activities after immunoprecipitation with anti-cPLA2 antibody. As shown in Fig 6, incubation of 450 µl of a Mono Q active fraction with 50 µl of packed protein A-Sepharose beads saturated with rabbit anti-porcine spleen 85-kDa PLA2 antiserum resulted in a loss of PLA2 activity in the supernatant. As expected incubation of partially purified porcine spleen cPLA2 with 50 µl of packed protein A-Sepharose beads saturated with the anti-cPLA2 antiserum resulted in a loss of activity in the supernatants in a time-dependent manner, with an ~90% reduction in supernatant activity after 4 h of incubation. Incubation of preimmune serum-saturated beads had no effect on PLA2 activity.


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Fig. 6.   Immunoprecipitation of heparin-nonbinding PLA2 activity with anti-cytosolic PLA2 (cPLA2) antiserum. Packed protein A-Sepharose beads preequilibrated with 20 mM Tris · HCl (pH 7.5) buffer containing 5 mg/ml of BSA were mixed with rabbit antiserum or preimmune serum (2:1 vol/vol) for 24 h at 4°C with constant shaking. Beads were washed 6 times with 1.0 ml of 20 mM Tris · HCl (pH 7.5) containing 1 mM EDTA and 5 mg/ml of BSA. Fifty microliters of washed beads were incubated with 450 µl of Mono Q FPLC active fractions from sheep lungs or partially purified cPLA2 from porcine spleen for 4, 8, or 16 h at 4°C with constant shaking. To estimate rabbit antiserum-specific binding of 100-kDa PLA2, samples were centrifuged, and supernatants were assayed for PLA2 activity with 1-[1-14C]AA-GPE as substrate.

Western blot analysis of PLA2 derived from sheep lungs by using anti-cPLA2 antiserum. Western blot analysis was performed on lysates from normal and smoke-exposed sheep lungs removed from the animals 4 h after a 30-breath exposure to cotton smoke. As shown in Fig. 7, anti-cPLA2 antiserum reacted strongly with 100-kDa bands from normal and smoke-exposed sheep lungs as well as with partially purified pig spleen cPLA2 used as a standard. It is usual for cPLA2 to migrate at a higher molecular mass than that predicted by its amino acid sequence. Although cPLA2 activity was higher in the smoke-exposed lungs, there was no increase in cPLA2 protein, indicating that the increased activity was not related to increased synthesis of protein or cells containing cPLA2 that infiltrated the lungs.


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Fig. 7.   Western blot analysis of protein from sheep lungs. Two samples (25 and 50 µg) were prepared from active fractions of Mono Q column and loaded onto SDS-PAGE as described in METHODS. Arrow, cPLA2 protein that migrates at 100 kDa. Porcine spleen cPLA2 (purified cPLA2) was used as a standard.

Correlation between PLA2 activity and MPO activity. MPO activity was measured in lung homogenates as an index of neutrophil infiltration. There were significant differences in MPO activity between smoke-exposed and normal sheep lungs. Although there were increases in both PLA2 and MPO activities after acrolein-carbon and smoke exposure, shorter exposures to smoke (15 breaths) were associated with a significant increase in PLA2 without a significant increase in MPO activity above normal levels (Fig. 8). Thus the increase in PLA2 activity cannot be entirely accounted for by infiltration of MPO-positive cells into the tissue. It could derive partially from parenchymal lung cells and from endogenous normally present intravascular neutrophils.



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Fig. 8.   Myeloperoxidase (MPO; A) and PLA2 (B) activities in sheep lung extracts after exposure to acrolein or varying number of breaths of cotton smoke. Each aliquot of 100,000-g supernatants from normal and smoke-exposed lungs was assayed for MPO and PLA2 activities by methods described in text. Values are means ± SE; n = 9 animals/group. * P < 0.01 compared with nontreated group. ** P < 0.001 compared with nontreated group.

Sheep neutrophils contain cPLA2. Supernatants (100,000 g) from sheep neutrophils were applied to a Mono Q column, and bound proteins were eluted with a 20-ml linear gradient of 0-1.0 M NaCl (Fig. 9). Extracts (100,000 g) from U937 cells were also loaded on the same column. The main PLA2 activity in each sample measured with 2-[1-14C]AA-GPE as the substrate eluted with the same profiles as those predicted for cPLA2. Thus sheep neutrophils contain cPLA2.


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Fig. 9.   Mono Q chromatography of 100,000-g supernatants of sheep neutrophils and U937 cells. Activity was eluted with a 20-ml linear gradient of 0-1.0 M NaCl. U937 cell supernatant PLA2 activity is cPLA2. This activity elutes with the same characteristics as activity of sheep neutrophils.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that there is an increase in cPLA2 activity in sheep lungs after exposure to synthetic smoke of acrolein-carbon or cotton smoke. In contrast, at a time when cPLA2 activity is increased with smoke inhalation, there is no apparent change in the small-molecular-mass secretory PLA2 activities that bind to heparin. We have previously identified cPLA2 in normal lungs (13), reported increased levels of PLA2 activity in patients with acute respiratory distress syndrome, and suggested that increased PLA2 activity may contribute to the pathophysiology of this syndrome (14). Small-molecular-mass forms of PLA2 have been purified by others from soluble fractions of guinea pig lungs (2) and rat lungs (23).

Acute lung damage can be caused by smoke inhalation alone without surface burns (40). Chemical toxins in smoke, not heat, produce a delayed-onset noncardiogenic pulmonary edema. In our experiments, we used two models of smoke inhalation: exposure to smoke derived from burning cotton and exposure to acrolein-carbon for 10 min. Acrolein-carbon exposure results in an accumulation of extravascular lung water in a dose-dependent fashion (10). Acrolein-carbon inhalation resulted in increases in cPLA2 activity equivalent to those observed with exposure to burning cotton. This increase in phospholipase activity may explain the rapid increases in bronchoconstrictor eicosanoids, PGF2alpha , thromboxane B2, and leukotriene C4 that are seen immediately after acrolein exposure, before neutrophil infiltration into the lung (20, 21). Enhanced cPLA2 activity will generate increased amounts of AA that can then be converted by cyclooxygenases and lipoxygenases to eicosanoids.

The BALF of smokers contains an increased number of cells, with higher percentages of macrophages and a lower percentage of lymphocytes than normal lung BALF (24). Macrophages can be a source of cPLA2. In the present study, the percentage of alveolar macrophages in the BALF was unchanged (90% of total cells; data not shown); however, lung tissue levels of MPO activity were increased after exposure to cotton or acrolein-carbon smoke. This increased MPO content is likely due to infiltrating polymorphonuclear neutrophils (PMNs). PMNs infiltrate the lung in response to an inflammatory stimulus. PMNs can contribute to the generation of reactive oxygen species, proteolytic enzymes, AA metabolites, and platelet-activating factor, which contribute to the increases in pulmonary microvascular permeability and pulmonary edema seen after smoke inhalation. Neutrophil depletion has been reported to prevent pulmonary edema associated with smoke inhalation (1). In this study, we found that sheep neutrophils contain cPLA2. With short periods of smoke exposure, cPLA2 activity increases more than MPO activity, and there is no increased amount of cPLA2 protein found on Western blot. Thus the increase in cPLA2 activity reflects increased endogenous cPLA2 activity in resident lung cells, a conclusion also consistent with the immediate increase in eicosanoids measured after acrolein exposure by others (20, 21). The increase in cPLA2 activity is likely related to phosphorylation of the protein, perhaps due to activation of one of the mitogen-activated protein kinase signaling pathways (31). In addition to the bronchoconstrictive, vasoconstrictive, and chemotactic effects of eicosanoids, the metabolism of AA by cyclooxygenases and lipoxygenases generates reactive oxygen species that can also contribute to the lung injury (36). Increased cellular cPLA2 activity also enhances the susceptibility of the cell to oxidant-induced injury (30).

In conclusion, our findings reveal an increase in cPLA2 activity in the lungs of sheep exposed to cotton smoke or synthetic smoke containing acrolein-carbon, an important component of cotton smoke. This PLA2 activity may serve as an important mediator of lung injury. cPLA2 likely contributes to high vascular permeability, bronchoconstriction, vasoconstriction, cellular injury, and inflammation by direct effects on membranes, by indirect effects mediated by free fatty acids and lysophospholipids released by cPLA2, and as a result of the eicosanoids and reactive oxygen species produced as a result of the metabolism of released AA.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and and Kidney Diseases Grants DK-39773 and DK 38452; National Institute of Neurological Disorders and Stroke Grant NS-10828; National Heart, Lung, and Blood Institute Grant HL-36829; and Shriners Burn Institute Grant 15872.


    FOOTNOTES

D. K. Kim was supported by a Paul Dudley White Fellowship from the Massachusetts Chapter of the American Heart Association.

Address for reprint requests and other correspondence: J. V. Bonventre, Suite 4002, Massachusetts General Hospital-East, 149 13th St., Charlestown, MA 02129 (E-mail: joseph_bonventre{at}hms.harvard.edu).

Received 6 May 1997; accepted in final form 22 April 1999.


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Am J Physiol Lung Cell Mol Physiol 277(3):L533-L542
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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