Effects of Repetitive Bolus Injections of Zymosan-activated Plasma on Lung Mechanics and Airway Responsiveness in Awake Sheep1-3

JAMES R. SNAPPER, MARTHA J. BUTTERFIELD, 4 DANIEL B. RAYBURN, and PETER L. LEFFERTS

Introduction

Alterations in lung mechanics and airway responsiveness have been described in association with the adult respiratory distress syndrome in humans (1) as well as in a variety of animal models of acute lung injury (2-8). Wehave previously noted that endotoxin (7, 8) and phorbol myristate acetate (PMA) (3, 4, 9) caused qualitatively similar alterations in lung mechanics, pulmonary hemodynamics and lung fluid and solute exchange in chronically instrumented awake sheep. Endotoxin and PMA did not, on the other hand, have similar effects on airway responsiveness. Four and one-half hours after endotoxin, airway responsiveness to aerosol histamine was markedly increased (7), whereas at the same point in time after PMA, only a small increase in airway responsiveness was observed (3). These differences led to the current series of experiments in which a third stimulus, zymosan-activated plasma (ZAP), a complement-activated plasma, was studied. The effects of ZAP have been studied extensively by Gee and colleagues (10-12) in anesthetized sheep. ZAP has been given as either a continuous infusion (10, 13) or as single or repetitive bolus injections (10-12). ZAP causes reproducible increases in pulmonary arterial pressure, decreases in circulating blood leukocyte counts, hypoxemia, increases in lung lymph flow and lymph protein clearance (10-13) and pulmonary inflammation (13). We elected to employ the repetitive bolus injections of ZAP protocol of Gee and colleagues since this methodology reportedly causes reproducible alterations and most closely mimics the pulmonary dysfunction associated with endotoxin and PMA. In addition to measurements of changes in hemodynamics, oxygenation, lung fluid and solute exchange, and lymph and plasma eicosanoid concentrations, we studied, for the 578

SUMMARY Westudied the pulmonary effects of repetitive bolus Injections of autologous zymosanactivated plasma (ZAP) in nine chronically Instrumented awake sheep. Aerosol histamine responsiveness was determined 1 h before and 4.5 h after the first bolus injection of ZAP. Each sheep received In the pulmonary artery a total of eight 5-ml bolus Injections of ZAP separated by 30 min. On a separate day, with the order of experimentation varied to avoid sequential bias, six of the nine sheep also received "control" plasma (plasma prepared In the Identical fashion as ZAP but not Incubated with zymosan). ·Control" plasma caused reproducible transient increases In pulmonary artery pressure, but It did not cause alterations In any of the other measured variables. Repetitive bolus injections of ZAP caused reproducible alterations in lung mechanics, pUlmonary hemodynamics, lung fluid and solute exchange, oxygenation, and peripheral leukocyte counts. The Increases in thromboxane-B, concentrations in lung lymph and plasma were greatest after the first bolus inJection of ZAp, with the magnitude of these changes diminishing on succeeding injections of ZAP. Aerosol histamine responsiveness did not Increase after the eight bolus injections of ZAP. AM REV RESPIR DIS 1991; 143:578-5S4

first time, the effects of repetitive bolus injections of ZAP on lung mechanics and pulmonary responsiveness to aerosol histamine in nine chronically instrumented awake sheep. Control studies were done on an additional six sheep employing plasma treated in the exact manner as ZAP without the addition of zymosan. Methods Chronically Instrumented Awake Sheep Preparation Nine yearling sheep of both sexes weighing 30 to 40 kg were instrumented as previously described (8, 14). Through a left thoracotomy,catheters wereplaced directly into the left atrium and pulmonary artery. Through a second left thoracotomy, lymphatics that transverse the left hemidiaphragm were interrupted. Through a right thoracotomy, a silastic catheter was placed into the efferent duct that emerged from the caudal mediastinal lymph node. Through a second right thoracotomy, the tail of the caudal mediastinal node was resected below the inferior pulmonary ligament. Possible contaminating lymphatics transversing the right hemidiaphragm were interrupted. At the time of right thoracotomy, silastic balloons, which measured 4 x 3 em, with silastic catheters (ID, 0.157 ern) that extended from within the balloons were positioned within the pleural space. Through a neck incision, catheters wereplaced into the

aorta via the carotid artery and into the superior vena cava via the external jugular vein. A thermistor-tipped Swan-Ganz catheter was positioned in the pulmonary artery via the external jugular vein.A tracheostomy wasperformed. The sheep were allowed 5 to 7 days to recover from the operation before experimentation. A size 10 Shiley cuffed tracheostomy tube (Shiley,Inc., Irvine, CAl was inserted at the time of experimentation.

(Received in originaljorm January 5, 1990and in revised form September 4, 1990) 1 From the Center for Lung Research, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, and the Department of Respiratory Research, Walter Reed Army Institute of Research, Washington, D.C. , Supported by Grant No. HL-27274 and SCOR in Pulmonary Vascular Diseases Grant No. HL19153 from the National Heart, Lung, and Blood Institute. This work was done during the tenure of an Established Investigatorship awarded to J. R. Snapper by the American Heart Association with funds contributed in part by the Middle Tennessee Chapter of the American Heart Association. 3 Correspondence and requests for reprints should be addressed to James R. Snapper, M.D., B-1308, Medical Center North, Vanderbilt University Hospital, Nashville, TN 37232. 4 Recipient of a grant from the American Lung Association of Tennessee.

579

ZYMOSAN-ACTIVATED PLASMA IN SHEEP

Lung Mechanics To characterize the awake sheep's response to repetitive bolus injections of ZAP and the effects of ZAP on pulmonary responsiveness to aerosol histamine, nine chronically instrumented awake sheep were studied while they stood in a specially constructed pressurecompensated, integrated-flow, whole-body plethysmograph (8). The tracheostomy tube was connected to an external valve via flexible noncollapsible tubing. A loose-fitting sling was placed under the sheep to prevent the sheep from lying down while in the plethysmograph. A constant bias flow of humidified air was used to reduce the effective dead space of the tubing. Volume (V) was measured by pressure-compensating the integrated signal from the plethysmographic pressure transducer. Flow (V) was obtained by electrically differentiating the volume signal. Airway opening pressure (Pao) was measured in the trachea by a multiple side-hole catheter positioned 0.5 em beyond the distal end of the tracheostomy tube. Pleural pressure (Ppl) was obtained from the pleural balloons that were placed at the time of surgery. Transpulmonary pressure (Ptp) was the pressure difference between Ppl and Pao. All pressure signals from pressure transducers, catheters, and silastic pleural balloons were tuned to eliminate phasic distortion to 20 Hz. Before each set of lung mechanic measurements, the sheep's lungs were inflated to 40 em H 20 airway opening pressure using the bias flow and an occluded airway. Simultaneous VlV and VlPtp curves wererecorded during spontaneous respiration on a Tektronix dual beam storage oscilloscope (Tektronix, Inc., Beaverton, OR) and photographed for calculation of dynamic lung compliance (Cdyn) and resistance to airflow across the lung (RL). Cdyn was calculated as V divided by Ptp at points of zero flow and expressed in liters per em H 20 at body temperature pressure standard (BTPS). RLwas calculated using the method of von Neergaard and Wirz (15) by dividing Ptp by V at midtidal volume and was expressed as em H 20/L/s at BTPS. Functional residual capacity (FRC) was measured using the Boyle's law method of Dubois and coworkers (16).The airway was manuallyobstructed at end-expiration. While the sheep attempted to breathe against the obstruction, a graph of the change in plethysmographic volume against the change in Pao was traced on the oscilloscope and photographed for calculation of FRC. Specific conductance (SOL) was calculated by dividing the reciprocal of RL (conductance) by FRC and expressed as seconds per em H 20 at BTPS. Lung mechanics were calculated every 15 min. Aerosol Histamine Dose-Response Curves Histamine diphosphate (United States Biochemical; Cleveland, OH) was dissolved in 0.9070 saline. Aerosols wereproduced by a Collison nebulizer driven by 100% oxygen (this nebulizer delivers particles with a mass me-

dian diameter between 2 and 4 um); 0.9% NaCI without histamine served as control. Histamine aerosols (0.1, 0.3, 1.0,3.0, 10.0,30.0 mg histamine base/ml) were used to inflate the lungs fivetimes to an airway opening pressure of 40 em H 20. This standardized the dose and gave a uniform volume history. Cdyn, RL,and FRC were measured 1min after completion of inhalations. Measurements of lung mechanics were expressed as percent of the saline control values. Histamine concentrations were increased until dynamic compliance decreased by at least 35% of control. Responsiveness was assessed by the effective dose of histamine that would have induced a reduction to 65% of control Cdyn (EDss Cdyn), 100070 increase in RL(ED 200RL), or a 25% increase in FRC (ED 125FRC). These values werecalculated by linear interpolation, never extrapolation, between the last two doses. Animals not reacting to the maximal dose of histamine (30 mg/rnl) were excluded from study (17).

Pulmonary Hemodynamics Pulmonary artery pressure (Ppa) in em H 20, left atrial pressure (PLA) in cm H 20, and aortic blood pressure (Psa) in mm Hg were continuously monitored using Hewlett-Packard Model 1208C pressure transducers (HewlettPackard Company, Palo Alto, CA). Cardiac output (CO) was measured by thermal dilution using the Swan-Ganz catheter and an Edwards 9520A cardiac output computer (Edwards Laboratories, Santa Ana, CA). Pulmonary vascular resistance (PVR) was calculated as (Ppa - PLA)/CO.

Differential counts were done manually on Wright's stained smears.

Thromboxane B2 and Prostacyclin Metabolites Lung lymph and plasma thromboxane B2 (ThB2) and 6-keto-prostaglandin-F w (6-ketoPOF,n) concentrations were measured using previouslydescribed radioimmunoassay techniques (8). Rabbit antisera specific for 6-ketoPOFIn antiserum cross-reacted less than 0.2% with prostaglandin (PO) POE, and PGE 2, and less than 0.1070 with POF2n, POD 2, and ThB2. The ThB2 antiserum crossreacted less than 0.1% with 6-keto-POF,n, PGD 2, POE" and POE2. ZAP and "Control" Plasma Preparation Autologous ZAP was prepared on the day prior to experimentation and frozen. Plasma (100 ml) was removed from heparinized blood (1,000USP units of heparin sodium per 100 ml of blood) and centrifuged for 15 min at 2,000 rpm. The plasma was incubated at 370 C for 45 min with 10 mg/ml of zymosan (Sigma Chemical Co., St. Louis, MO). The zymosan was removed after incubation by centrifugation (4,000rpm for 15min) followed by passing the plasma through a Millipore filter (0.45 urn pore size) (Millipore Corp., Bedford, MA). On the day of experimentation, the ZAP was allowed to thaw and come to room temperature prior to experimentation. Autologous "control" plasma was prepared in the identical manner to that used to prepare ZAP except zymosan was not added to the plasma during the 45-min incubation period at 370 C. Each sheep received ZAP and control plasma prepared from its own blood.

Lung Fluid and Solute Exchange Lung lymph was collected continuously with flow (Qlymph) determined for each IS-min interval and pooled for 30-min intervals for Experimental Protocol measurement of total protein and eicosanoid Nine chronically instrumented awake sheep concentrations. Whole blood was collected for measurement of plasma proteins, arterial were studied. An aerosol histamine doseblood gas determinations, white blood cell response curve was completed a minimum of (WBC) count, and eicosanoid concentrations. 1 h before giving the first 5-ml rapid bolus of autologous ZAP into the pulmonary arTotal protein concentrations in lung lymph and blood plasma were measured by a moditery, In all sheep, lung mechanics had returned fied biuret method (18) with an automated . to within 10% of baseline values prior to givsystem (AutoAnalyzer II; Technicon Instru- ing ZAP. Repetitive rapid bolus injections of ments Corp., Tarrytown, NY). Lymph pro- 5 ml of autologous ZAP in the pulmonary tein clearance (CLP) was calculated by mul- artery were repeated every 30 min for a total tiplying Qlymph by the LIP ratio. of eight doses. Lung mechanics measurements weretaken at 5, 15,and 25 minutes after each Other Variables ZAP bolus. Ppa and PLA were measured continuously. Lung lymph was collected continuArterial blood samples were collected anaerobically every 30 min during baseline and at ously with Qlymph determined for each 152 and 22 min after each bolus injection of min interval and pooled for 30-min intervals, ZAP. Pao2and Paco2 and pH weremeasured arterial blood samples were collected anaerusing a Corning 158 pH/blood gas ana- obically for arterial blood gas determinations and whole blood leukocyte counts at 2 and lyzer (Corning Medical, Medfield, MA). The alveolar to arterial oxygen difference 22 min after each ZAP bolus. CO was deter(AAaPo2) while breathing room air was cal- mined in triplicate every 30 min before the culated using the alveolar gas equation as- first bolus injection of ZAP, and at 5 and 28 suming a fixed respiratory exchange ratio of min after each bolus injection of ZAP. Airway responsiveness to aerosol histamine was 0.8. Whole blood leukocyte counts weredone on an automated system (Coulter Counter again measured 4.5 h after the first 5-ml bomodel ZBI; Coulter Electronics, Hialeah, FL). lus injection of ZAP into the pulmonary ar-

580

SNAPPER, BUTTERFIELD, RAYBURN, AND LEFFERTS

tery (1 h after the eighth bolus injection of autologous ZAP). Six of the nine sheep in which aerosol histamine dose-response curves were done before and after repetitive bolus injections of ZAP had similar studies done with autologous control plasma. The identical protocol to that used for ZAP was followed for the control plasma. Instead of ZAP, each sheep had aerosol histamine dose-response curves done before and after the rapid bolus injection of 5 ml of control plasma. In order to avoid sequential bias, the order of experimentation was varied, with some sheep studied first with control plasma and some sheep studied first with ZAP. A minimum of4 days was allowed between experimentation in any given sheep.

Fig. 1. Effect of repetitive bolus injections of autologous ZAP on lung mechanics in chronically instrumented awake sheep. The upper panel contains data for Cdyn; data for RL are plotted on the lower panel. The horizontal axis is time in hours, with the vertical arrows indicating time points when the sheep were given bolus injections of ZAP.Data are mean values ± SEM (n = 9).

2

TIME (HOURS)

Statistics Data were analyzed using one- and two-way analyses of variance and Kramer's adaptation of Duncan's multiple range tests. For comparisons of the effects of ZAP with control plasma, only paired data from individual sheep were employed (n = 6). The effects of ZAP over time on the different variables measured were based on all sheep studied with repetitive bolus injections of ZAP (n = 9) or control plasma (n = 6). Wilcoxan's signed rank test was used to determine if ZAP or control plasma altered ED.sCdyn. The null hypothesis was rejected for p < 0.05 (19, 20).

Results

Lung Mechanics Repetitive bolus injections of autologous ZAP caused reproducible changes in lung mechanics. Mean Cdyn for the nine sheep studied with repetitive bolus ZAP was 0.064 ± 0.007 L/cm H 20 (mean ± SEM) prior to the first injection of ZAP. Cdyn decreased to a mean value of 0.035 ± 0.005 5 min after ZAP (p < 0.05). Cdyn had increased to a mean value of 0.047 ± 0.006 15 min after ZAP (p < 0.05). Cdyn had not returned to baseline by 30 min after ZAP (p < 0.05). Mean Cdyn immediately before the last seven injections of ZAP was 0.053 ± 0.007 (p < 0.05) (figure 1). ZAP caused reproducible increases in RL and decreases in SOL (p < 0.05). Mean RL prior to the first bolus injection of ZAP was 1.36 ± 0.40. RL increased to 3.77 ± 0.62 5 min after the eight bolus injections of ZAP (p < 0.05). RL had decreased to 2.55 ± 0.60 by 15 min (p < 0.05) and to 2.15 ± 0.58 immediately prior to the last seven injections of ZAP (figure 1). ZAP did not cause consistent changes in FRC (p > 0.05). Mean FRC was 1.09 ± 0.6 L immediately before the first bolus injection of ZAP, 0.99 ± 0.07 5 min after the eight bolus injections of ZAP, 1.03 ± 0.07 15 min after ZAP, and 1.03 ± 0.07 immediately before the last seven injec-

tions of ZAP. ZAP caused reproducible creased immediately from a mean condecreases in SOL (p < 0.05). Mean SOL trol value before each bolus injection of was 1.08 ± 0.21 s/cm H 20 prior to the ZAP of 19 ± 1 em H 20 to a mean peak first injection of ZAP, 0.35 ± 0.05 5 min pressure of 70 ± 3 (p < 0.05). Ppa had after the bolus injections of ZAP (p < decreased to 32 ± 1 by 5 min (p < 0.05), 0.05), 0.62 ± 0.13 15 min after ZAP to 21 ± 1 by 15 min after the bolus in(p < 0.05), and 0.79 ± 0.16 immediately jection of ZAP, and, by 30 min after before the last seven injections of ZAP. ZAP, Ppa had returned to baseline. Repetitive bolus injections of control Tachyphylaxis of the response was not plasma did not cause reproducible observed. ZAP caused small nonsignifichanges in lung mechanics (P > 0.05). cant decreases in PLA (p > 0.05). Mean Mean Cdyn for the six sheep studied P1A prior to ZAP was - 0.7 ± 1.1 cm with repetitive control plasma was 0.063 H 20 and decreased to -1.3 ± 1.3 at 5 ± 0.012 L/cm H 20 prior to the first in- min and to -1.3 ± 1.2 at 15 min after jection of control plasma. Mean Cdyn ZAP. CO, measured at 5 min after the was 0.065 ± 0.012 5 min after control bolus injection of ZAP by thermal diluplasma, 0.065 ± 0.012 15 min after con- tion, did not change significantly (p > trol plasma, and 0.066 ± 0.012 immedi- 0.05). Mean CO prior to ZAP was 4.4 ately before the last seven injections of ± 0.3 L/min and 4.4 ± 0.2 5 min after control plasma. Mean RL was 1.71 ± ZAP. PVR could only be calculated for 0.71 for the six sheep prior to the first control values prior to each bolus injecinjection of control plasma, 1.81 ± 0.79 tion of ZAP and for 5 min after ZAP at 5 min after control plasma, 1.81 ± (points where Ppa, PLA, and CO had all 0.85 at 15 min after control plasma, and been determined). Mean PVR increased 1.73 ± 0.79 immediately before the last from 4.3 ± 0.5 em H 20/L/min prior seven injections of control plasma. FRC to ZAP to 7.9 ± 0.65 min after ZAP was 1.00 ± 0.07 L immediately before (p < 0.05). the first injection of control plasma, 1.01 "Control" plasma caused reproducible ± 0.07 5 min after the injection of con- significant increases in Ppa and PVR. trol plasma, 1.02 ± 0.07 15 min after The peak increases were significantly less control plasma, and 1.04 ± 0.07 im- than those observed after ZAP (p < 0.05), mediately preceding the last seven injec- and Ppa returned to baseline values more tions of control plasma. Mean SOL was rapidly after control plasma than after 1.05 ± 0.30 s/cm H 20 immediately be- ZAP (p < 0.05). Ppa increased from a fore the first injection of control plas- mean control value before each bolus inma, 1.01 ± 0.28 5 min after control jection of control plasma of 17 ± 1 em plasma, 1.09 ± 0.32 15 min after con- H 20 immediately to a mean peak prestrol plasma, and 1.14 ± 0.35 immediate- sure of 52 ± 4 (p < 0.05). Ppa had dely before the last seven injections of creased to 23 ± 2 by 5 min (p < 0.05) control plasma. and to 18 ± 1 by 15 min, and by 15 min after control plasma, Ppa had returned Pulmonary Hemodynamics to baseline. Thchyphylaxisof the response Repetitive bolus injections of autologous was not observed. Control plasma caused ZAP caused reproducible alterations in no significant changes in PLA or CO pulmonary hemodynamics. Ppa in- (p > 0.05). Mean PLA prior to control

581

ZYMOSAN-ACTIVATED PLASMA IN SHEEP

plasma was 0.3 ± 0.6 cm H 20 and 0.3 ± 0.5 at 5 min and 0.3 ± 0.5 at 15 min after control plasma. Mean CO prior to control plasma was 4.5 ± 0.4 Lzrnin and 4.6 ± 0.4 5 min after the bolus injection of the control plasma. Mean PVR increased from 3.8 ± 0.4 ern H 201 Llmin prior to control plasma to 5.1 ± 0.8 5 min after control plasma (P < 0.05). The increases in PVR were significantly less after control plasma than after ZAP (p < 0.05).

Lung Fluid and Solute Exchange Repetitive bolus injections of autologous ZAP caused an almost 4-fold increase in Qlymph and CLP. Mean Qlymph for the nine sheep studied prior to the first injection of ZAP was 1.8 ± 0.3 mIl15 min. Mean Qlymph increased in the first 15 min after ZAP to 6.8 ± 0.9 (p < 0.05). Qlymph did not return to baseline during the 30-min interval between ZAP injections (p < 0.05). After the first ZAP injection, the mean Qlymph immediately preceding the next seven bolus injections of ZAP was 5.1 ± 0.8 (p < 0.05). Despite the marked transient pulmonary hypertension and increased Qlymph, ZAP had only small effects on LIP ratio. LIP ratios were calculated for 30-min intervals. The LIP ratio for the nine sheep before the first bolus injection of ZAP was 0.66 ± 0.01, and 4 h later after eight injections of ZAP it was 0.63 ± 0.02 (p > 0.05). Only the LIP ratio determined at 1 h after the first bolus injection of ZAP was significantly below the control values determined before ZAP. CLPwas calculated for each 15-min interval using the 15-min values for Qlymph and the LIP ratio determined over the 30-min collection period. CLP increased from a mean value for the nine sheep studied of 1.2 ± 0.2 mIl15 min before the first injection of ZAP to a mean value of 4.2 ± 0.5 15 min after the injection of ZAP (p < 0.05). CLP had not returned to baseline prior to the succeeding injections of ZAP (p < 0.05). Mean CLPprior to the next seven injections of ZAP for the nine sheep studied was 3.2 ± 0.5 (p < 0.05). Control plasma' did not cause consistent changes in Qlymph, LIP ratio or CLP (p > 0.05). Mean Qlymph for the six sheep studied prior to the first injection of control plasma was 1.3 ± 0.4 mIl15 min. Mean Qlymph increased in the first 15min after control plasma to 2.0 ± 0.8. Mean Qlymph immediately preceding the next injection of control plasma was 1.7 ± 0.8. Baseline LIP ratio prior to the

first bolus injection of control plasma was 0.63 ± 0.05. The LIP ratio after the eighth injection of control plasma was 0.62 ± 0.05. Mean CLP for the six sheep studied with control plasma was 0.7 ± 0.4 mIl15 min prior to the first injection of control plasma, 0.8 ± 0.4 15 min after the injection of control plasma, and 0.7 ± 0.3 immediately prior to the succeeding injections of ZAP.

Oxygenation ZAP caused Pao, to decrease and dAaPo2 to increase. Mean Pa02 prior to the first bolus injection of ZAP was 76.3 ± 4.0 mm Hg and 55.7 ± 4.65 min after the eight bolus injections of ZAP (p < 0.05). Pa02 and dAaP0 2 did not return to baseline after the first bolus injection of ZAP, with the mean Pa02 66.8 ± 3.7 preceding the last seven injections of ZAP (p < 0.05). Mean dAaP0 2 was 27.2 ± 3.3 immediately before the first bolus injection of ZAP, 48.6 ± 4.25 min after the eight injections of ZAP (p < 0.05), and 37.8 ± 3.5 immediately before the last seven injections of ZAP (p < 0.05). Control plasma had no consistent effect on Pa02 or dMiP02 (p > 0.05). Mean Pa02 was 75.2 ± 5.0 prior to the first injection of control plasma, 72.6 ± 3.7 5 min after the eight injections of control plasma, and 71.6 ± 3.7 immediately before the last seven injections of control plasma. Mean dAaP0 2 was 29.7 ± 3.9 immediately before the first bolus injection of control plasma, 33.0 ± 3.45 min after control plasma, and 34.1 ± 4.0 immediately before the last seven injections of control plasma.

White Cell Counts and Differentials Baseline total WBC was 9,055 ± 959

(cells/rum") before the first bolus injection of ZAP. Total WBC decreased consistently to a mean value of 3,686 ± 926 after the eight bolus injections of ZAP (p < 0.05). WBC increased prior to the successive injections of bolus ZAP (p < 0.05). Mean WBC 30 min after the eighth injection of ZAP had increased to 13,769 ± 1,667 (p < 0.05) (figure 2). Approximately 90070 of the decrease in WBC could be accounted for by a decrease in total polymorphonuclear cells. Baseline total polymorphonuclear cell count was 5,713 ± 605 (cells/nun") before the first bolus injection of ZAP. Total polymorphonuclear cell count decreased consistently to a mean value of 1,170 ± 583 after the eight bolus injections of ZAP (p < 0.05). The increase in WBC could be accounted for by an increase in total polymorphonuclear cell count. Total polymorphonuclear cell count increased 30 min after the eighth injection of ZAP to 10,236 ± 1,617 (p < 0.05) (figure 2). ZAP caused a small, but consistent, decrease in total lymphocyte count (p < 0.05). Total lymphocyte count prior to the first bolus injection of ZAP was 3,342 ± 435. Mean total lymphocyte count after the eight bolus injections of ZAP was 2,516 ± 471 (p < 0.05). Totallymphocyte count did not increase significantly prior to the succeeding bolus injections of ZAP (p > 0.05). Total lymphocyte count prior to the eighth bolus injection of ZAP was 3,443 ± 555 (p > 0.05) (figure 2). Control plasma had no consistent effect on WBC, total polymorphonuclear cells or total lymphocytes (p > 0.05). Mean WBC before the first bolus injection of control plasma was 9,038 ± 532 (cells/mm J),9,953 ± 8205 min after the

16

Totol

Fig. 2. Effect of repetitive bolus injections of autologous ZAP on blood leukocyte counts in chronically instrumented awake sheep. The upper panel contains data for total wec; data for total polymorphonuclear cell counts are plotted on the middle panel; data for total lymphocyte counts are plotted on the lower panel. The horizontal axis is time in hours, with the vertical arrows indicating time points when the sheep were given bolus injections of ZAP. Data are mean values ± SEM (n = 9).

wec

(cells 1l103/mm 3)

Totol Polymorphonuclear

Cells 3/mm3)

(cells l I 0

Total Lymphocytes 3 (cells l I 0 3/mm j

:J

--i----+---l-----.;e-----+--f---+--'--'--,

1-1

o

TIME (HOURS)

582

eight bolus injections of control plasma. Mean WBC prior to the eighth bolus injection of control plasma was 10,291 ± 1,016. Mean total polymorphonuclear cell count before the first bolus injection of control plasma was 5,799 ± 750 (cells/rum"), 6,160 ± 1,165 5 min after the eight bolus injections of control plasma. Mean total polymorphonuclear cell count prior to the eighth bolus injection of control plasma was 6,389 ± 1,613. Total lymphocyte count prior to the first bolus injection of control plasma was 3,239 ± 454, 3,792 ± 716 5 min after the eight bolus injections ofcontrol plasma, and 3,902 ± 949 prior to the eighth bolus injection of control plasma.

Blood and Lymph Concentrations of Cyclooxygenase Products ZAP caused a marked increase in blood and lung lymph concentrations of lXB2 • The increase in ThB2 concentrations was greatest after the first injection of ZAP and decreased on succeeding injections of ZAP (p < 0.05). Mean blood lXB2 prior to the first injection of ZAP was 0.014 ± 0.003 ng/ml and 2.615 ± 0.7025 min after the first bolus injection of ZAP (p < 0.05). Blood concentrations of lXB2 had returned to near baseline immediately before the succeeding injections of ZAP (0.055 ± 0.015). Mean blood lXB2 prior to the eighth injection of ZAP was 0.029 ± 0.007, and it was 0.303 ± 0.086 5 min after the eighth bolus injection of ZAP (p < 0.05). Lung lymph lXB2 , determined every 30 min, reached slightly higher concentrations than did those achieved in blood after ZAP. Mean lung lymph concentrations of lXB2 before the first bolus injection of ZAP was 0.018 ± 0.004 ng/ml. Mean lung lymph lXB2 during the 30-min period after the first bolus injection of ZAP was 3.432 ± 1.161 (p < 0.05) and 0.492 ± 0.206 during the 30-min period after the eighth ZAP injection (p < 0.05). ZAP caused no consistent changes in either blood or lung lymph concentrations of 6-keto-PGF ta (p > 0.05). Mean blood 6-keto-PGF tu concentration before ZAP was 0.010 ± 0.002 ng/ml, 0.014 ± 0.0035 min after the first bolus injection of ZAP, and 0.011 ± 0.003immediately prior to the last seven injections of ZAP. Mean lung lymph 6-keto-PGF ta concentration before ZAP was 0.018 ± 0.010, 0.022 ± 0.010 for the 30-min period after the first bolus injection of ZAP and 0.015 ± 0.004 for the 30-min periods after the last seven injections of ZAP. Control plasma had no consistent effect on blood or lung lymph concentra-

SNAPPER, BUTTERFIELD, RAYBURN, AND LEFFERTS

tion of lXB2 or 6-keto-PGF ta (p > 0.05). Blood concentrations of lXB2 were 0.022 ± 0.003 ng/ml immediately before the first injection of control plasma, 0.024 ± 0.002 5 min after control plasma, and 0.020 ± 0.002immediately before the last seveninjections of control plasma. Mean lung lymph lXB2 concentration was 0.019 ± 0.007 ng/ml before control plasma, 0.021 ± 0.009 over the 30-min immediately after the first bolus injection of control plasma, and 0.024 ± 0.004 in the 30-min period after the last seven bolus injections of control plasma. Mean blood 6-keto-PGF tu before control plasma was 0.014 ± 0.003 ng/ml, 0.011 ± 0.003 5 min after control plasma, and 0.011 ± 0.003 immediately prior to the last seven injections of control plasma. Lung lymph 6-keto-PGF tu concentrations were 0.019 ± 0.006 ng/ml prior to the first injection of control plasma, 0.015 ± 0.006 after the first injection of control plasma, and 0.017 ± 0.008 after the last seven injections of control plasma.

Aerosol Histamine Responsiveness Lung mechanics prior to the second histamine dose response curve, begun 1 h after the eighth bolus injection of ZAP and 4.5 h after the first bolus injection of ZAP, weresignificantly different from those obtained prior to the first aerosol histamine dose-response curve completed 1 h prior to beginning the repetitive bolus injections of ZAP. Cdyn before the first aerosol histamine dose-response curve was 0.070 ± 0.008 L/cm H 2 0 and 0.056 ± 0.007 after ZAP and prior to the second aerosol histamine doseresponse curve (p < 0.05). Control plasma had no significant effect on lung

mechanics. Cdyn prior to the first aerosol histamine dose-response curve was 0.070 ± 0.008 and 0.066 ± 0.008 L/cm H 2 0 after control plasma and before the second aerosol histamine dose-response curve (p > 0.05). No statistically significant correlation was observed between the percent change in baseline lung mechanics between the two aerosol histamine dose-response curves (before and after ZAP or before and after control plasma) and the change in aerosol histamine responsiveness noted between the two aerosol histamine dose-response curves. ZAP or control plasma did not increase airway responsiveness to aerosol histamine. Mean aerosol histamine doseresponse curves from the nine sheep studied before and after ZAP and the six sheep studied before and after control' plasma are plotted in figure 3. The data from before and after ZAP and control plasma summarized as ED 6sCdyn are plotted in figure 4. The mean of the log of ED 6sCdyn before ZAP was 0.70 ± 0.08 mg/ml and increased 4.5 h after ZAP to 0.80 ± 0.08 (p 0.05). 1\\'0 of the nine sheep studied with ZAP and one of six sheep studied with control plasma increased RLwith histamine challenge. ZAP and control plasma did not change ED 20 0RL in these animals. No sheep increased FRC with histamine challenge. Discussion

Repetitivebolus injections of autologous

ZAP

CONTROL PLASMA

--~....

100

80

l(,Conlrol 60 Cdyn 40

N=9

20

o

N=6

---,------r-----,,--,---, 0:1 0:'3 1:0 3',0 10.0 HISTAMINE CONCENTRATION

r,

C

(mg/ml)

'D

0:1 0:3 3:0 10:-0 HISTAMINE CONCENTRATION (mg/ml)

Fig. 3. Mean aerosol histamine dose-response curves from nine chronically instrumented awake sheep studied before (closed circles) and after (open circles) repetitive bolus injections of ZAP (left panel) or control plasma (right panel). The vertical axis is percent saline control Cdyn, and the horizontal axis is the dose of histamine aerosolized in mglml of histamine base. "C' indicates the saline control point.

ZYMOSAN-ACTIVATED PLASMA IN SHEEP

583 30.0

Fig. 4. Effect of repetitive bolus injections of ZAP (left panel) and control plasma (right panel) on pulmonary responsiveness to aerosol histamine expressed as ED. 5Cdyn. Individual values from a given sheep are joined by solid lines. Mean values for the nine sheep are the dashes immediately to the left and the right of each set of data.

10,0 ED65Cdyn (mg/ml)

3,0

1.0

-~-...

-...

BEFORE AFTER ZAP

ZAP were chosen for these studies to ensure a maximal effect on lung fluid and solute exchange and on pulmonary inflammation (10-13). Alternatively, ZAP could have been given as an aerosol, single bolus injection, or as a continuous infusion (10-13). Our own (13)experience and that of Gee and colleagues (10-12) in anesthetized sheep suggests that repetitive bolus injections of ZAP would be most likely to cause pathophysiologic changes analogous to those caused by endotoxin (21) and phorbol myristate acetate (PMA) (4). We were able, in chronically instrumented awake sheep, to confirm the previous reported effects of repetitive bolus injections of ZAP in anesthetized sheep on pulmonary hemodynamics and lung fluid and on solute exchange (10-13). The sawtoothed pattern of changes in the observed variables is unique to the experimental protocol employed for the ZAP studies, but if one looks only at the peak response that occurs after each bolus injection of ZAP, the changes are analogous to those observed after infusion of endotoxin (2, 5, 8, 21) or PMA (4, 9). Endotoxin (2, 5, 8, 21), unlike PMA (4, 9) or ZAP, causes significant changes in 6-keto-prostaglandin Pta concentrations in lung lymph and plasma. Repetitive bolus injections of ZAP caused marked reproducible changes in lung mechanics. The changes in lung mechanics like oxygenation, but unlike the changes observed for hemodynamics, did not return to baseline immediately prior to succeeding injections of ZAP. Although lung lymph concentrations of lXB 2 were slightly greater than those observed in plasma, arguing for intrapulmonary production of thromboxane, the fact that thromboxane concentrations were decreasing, whereas the changes in lung mechanics (as well as in pulmonary hemodynamics, oxygenation, and peripheral leukopenia) were unchanged argues against thromboxane as

-~-...

-,-

BEFORE AFTER CONTROL PLASMA

a mediator of these changes. It is possible that the local dose-response relationship between thromboxane and these variables rapidly reaches a plateau, and that even the small transients observed in TxB2 concentrations after the later injections of ZAP cause a maximal response. We attempted to use what we felt was the most appropriate control- repetitive bolus injections of autologous control plasma (autologous plasma handled in the exact manner as ZAP without the addition of zymosan). "Control" plasma caused marked reproducible increases in pulmonary artery pressure. These increases were less and more transient than those observed with ZAP. It is interesting that, despite the marked increases in pulmonary artery pressure observed with control plasma, as opposed to ZAP, control plasma had no effect on any of the other observed variables. Control plasma did not cause reproducible changes in lung fluid and solute exchange, lung mechanics, oxygenation, concentrations of cyclooxygenase products of arachidonic acid, leukocyte counts, airway responsiveness to aerosol histamine, or lung histology (22). The absence of any effect of the marked transient increases in pulmonary artery pressure on lung fluid and solute exchange suggest that the observed changes in pulmonary vascular resistance occurred at a site proximal to the pulmonary capillary or that the changes in hydrostatic pressure were so transient as to not be translated into increased transudation of fluid and solute into the pulmonary interstitium. Although it has been argued that marked transient increases in pulmonary artery pressure might alter pulmonary microvascular permeability, no such mechanism appears to be stimulated by repetitive injections of control plasma. We do not have an easy explanation for why the control plasma caused changes in pulmonary artery pressure. The handling of the plasma alone could

activate complement or a component of the complement pathway. Simple complement activation seems unlikely since ZAP (assuming that the mechanisms of action of ZAP involve complement activation in sheep) had quite different effects in the sheep. The effects of ZAP were dramatic, and one could argue that no control was needed. Alternatively, bolus injections of normal saline could have been used. We felt that the control plasma was the most appropriate control and that the results, though unexplained, were interesting and should be reported. ZAP, unlike endotoxin (7) or PMA (3), did not increase pulmonary responsiveness to aerosol histamine. ZAP actually caused a small statistical decrease in pulmonary responsiveness to aerosol histamine. Although lung mechanics had not returned to baseline immediately prior to the second aerosol histamine doseresponse curve, it is unlikely that these changes in baseline lung mechanics, in and of themselves, directly contributed to the small observed decrease in pulmonary responsiveness to aerosol histamine. These changes, in theory, would be more likely to increase rather than decrease aerosol histamine responsiveness.Thromboxane has been proposed as a possible mediator of increased, not decreased, airway responsiveness in a variety of species and experimental conditions (23-25). Cyclooxygenase inhibitors can cause small decreases in airway responsiveness to aerosol histamine in sheep (17). It is possible that the repetitive bolus injections of ZAP functionally depleted the ability of a specific cell type (mast cells or pulmonary intravascular macrophages [26], for example) to produce thromboxane. This effect, in theory, could account for small decrease in pulmonary responsiveness to aerosol histamine. AI. though alterations such as secretions might lead to altered aerosol deposition and could also account for the decrease in pulmonary responsiveness to aerosol histamine, the magnitude of the decrease in pulmonary responsiveness to aerosol histamine, though statistically significant, is very small. We do not want to overemphasize the very small changes in airway responsiveness observed after repetitive bolus injections of ZAP. Of far more interest would be an understanding of why three stimuli that cause similar pulmonary dysfunction (ZAP, PMA, and endotoxin) have very different effects on pulmonary responsiveness to aerosol histamine. These differences led to the separate studies, de-

a

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SNAPPER, BUTTERFIELD, RAYBURN, AND LEFFERTS

scribed in the accompanying manuscript, examining the effects of ZAP, PMA and endotoxin on lung structure and pulmonary inflammation (22). References 1. Simpson DL, Goodman M, Spector SL, Petty TL. Long-term follow-up and bronchial reactivity testing in survivors of the adult respiratory distress syndrome. Am Rev Respir Dis 1978; 117:449-54. 2. Brigham KL, Meyrick B. Endotoxin and lung injury. Am Rev Respir Dis 1986; 133:913-27. 3. Dyer EL, Lefferts PL, Snapper JR. Phorbol myristate acetate lung injury and airway responsiveness to aerosol histamine in awake sheep. J Allergy Clin Immunol 1986; 78:44-50. 4. Dyer EL, Snapper JR. Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate. J Appl Physiol1986; 60:576-89. 5. Hinson JM Jr, Hutchison AA, Ogletree ML, Brigham KL, Snapper JR. Effect of granulocyte depletion of altered lung mechanics after endotoxemia in sheep. J Appl Physiol 1983; 55:92-9. 6. Holtzman MJ, Fabbri LM, O'Byrne PM, et a/. Importance of airway inflammation for hyperresponsiveness induced by ozone. Am Rev Respir Dis 1983; 127:686-90. 7. Hutchison AA, Hinson JM, Brigham KL, Snapper JR. Effect of endotoxin on airway responsiveness to aerosol histamine in sheep. J Appl Physiol 1983; 54:1463-8. 8. Snapper JR, Hutchison AA, Ogletree ML, Brigham KL. Effects of cyclooxygenase inhibitors on the alterations in lung mechanics caused by endotoxemia in the unanesthetized sheep. J Clin Invest 1983; 72:63-76.

9. Loyd JE, Newman JH, English P, Ogletree ML, Meyrick BO, Brigham KL. Lung vascular effects of phorbol myristate acetate in awake sheep. J Appl Physiol 1983; 54:267-76. 10. Gee MH, Perkowski SZ, Tahamont MV, Flynn JT. Arachidonate cyclooxygenase metabolites as mediators of complement-initiated lung injury. Fed Proc 1985; 44:46-52. 11. Gee MH, Tahamont MV, Perkowski SZ, Flynn JT. Arachidonic acid metabolites as mediators of lung injury during intravascular complement activation. Prog Biochem Pharmacol1985; 20:108-19. 12. Perkowski SV, Havill AM, Flynn JT, Gee MH. Role of intrapulmonary release of eicosanoids and superoxide anion as mediators of pulmonary dysfunction and endothelial injury in sheep with intermittent complement activation. Circ Res 1983; 53:574-85. B. Meyrick BO, Brigham KL. The effect of a single infusion of zymosan-activated plasma on pulmonary microcirculation of sheep: structurefunction relationships. Am J Pathol 1984; 114: 32-45. 14. Staub N, Bland R, Brigham K, Remling R, Erdman J, Wooverton W. Preparation of chronic lymph fistulas in sheep. J Surg Res 1975; 19:315-20. 15. von Neergaard K, Wirz K. Die messung der stromungswiderstande in den atemwegen des Menschen, inshesmdere ber Asthma und emphysem. Zat Klin Med 1927; 105:51-82. 16. Dubois AB, Botelho SY, Bedell GN, Marshall R, Comroe JH Jr. A rapid plethysmographic method measuring thoracic gas volume: a comparison with nitrogen washout method for measuring functional residual capacity in normal subjects. J Clin Invest 1956; 35:322-6. 17. Snapper JR, Lefferts PL, Stecenko AA, Hinson JM Jr, Dyer EL. Bronchial responsiveness to

nonantigenic bronchoconstrictors in awake sheep. J Appl Physiol 1986; 61:752-9. 18. Failing J, Buckley M, Zak D. Automated determination of serum proteins. Am J Clin Pathol 1960; 33:83-8. 19. Snedecor GW, Cochran WG. Statistical Methods. 6th ed. Ames: Iowa State University, 1967. 20. Steel RGD, Torrie JH. Principles and procedures of statistics: a biometrical approach. 2nd ed. New York: McGraw-Hill, 1980. 21. Meyrick B, Brigham KL. Acute effects of Escherichiacoli endotoxin on the pulmonary microcirculation of anesthetized sheep: structure-function relationships. Lab Invest 1983; 48:458-70. 22. Simpson JF, Butterfield MJ, Lefferts PL, Dyer EL, Snapper JR, Meyrick B. Role of pulmonary inflammation in altered airway responsiveness in three models of acute lung injury. Am Rev Respir Dis 1991; 143:585-9. 23. Chung KF, Aizawa H, Leikauf GD, Ueki IF, Evans TW, Nadel JA. Airway hyperresponsiveness induced by platelet-activating factor: role of thromboxane generation. J Pharmacol Exp Ther 1986; 236:580-4. 24. Jones GL, Lane CG, O'Byrne PM. A thromboxane A 1 receptor antagonist, L-655,240, inhibits airway hyperresponsiveness after inhaled ozone in dogs. Am Rev Respir Dis 1988; 137:99. 25. McFarlane cs, Ford-Hutchison AW,Letts LG. Inhibition of thromboxane (1XA1}-induced airway responsiveness to aerosol acetylcholine by the selective TxA1 antagonist L-655,240 in the conscious primate. Am Rev Respir Dis 1988; 137:100. 26. Warner AE, Barry BE, Brain JD. Pulmonary intravascular macrophages in sheep morphology and function of a novel constituent of, the mononuclear phagocyte system. Lab Invest 1986; 55:276-88.