Activated Eosinophils Increase Vascular Permeability and Resistance in Isolated Perfused Rat Lungs 1- 3
KEISAKU FUJIMOTO, JAMES C. PARKER, and STEPHEN G. KAYES
Introduction Eosinophils are granule-containing polymorphonuclear leukocytes that have been implicated as primary effector cells in the pathophysiology of asthma and allergic diseases (1-3). Eosinophils can cause tissue injury by the generation of reactive oxygen metabolites, such as Oi, H 2 0 2 , ·OH, singlet O 2 , HOCl, HOI, and HOBr (4-6); by the release of toxic granule-contained proteins, such as major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil cationic protein (ECP) (7-9); and by the interactions of these toxic substances (10, 11). Additionally, leukotriene C 4 (LTC 4 ), a potent mediator of bronchoconstriction (12) and pulmonary vasoconstriction (13), is the predominant leukotriene elaborated by eosinophils (14). Many clinical studies, both morphologic and immunochemical, have suggested that eosinophils may also be associated with other types of lung injury under certain circumstances (3). For instance, a role has been suggested for eosinophils in the adult respiratory distress syndrome (ARDS). ARDS is characterized by increased pulmonary vascular permeability, and both bronchoalveolar lavage fluid and plasma from these patients have elevated ECP levels (15). However, whether eosinophils cause lung injury directly and increase pulmonary microvascular permeability has not been determined. We hypothesized that eosinophils may cause lung injury by releasing toxic oxygen metabolites and/or cytotoxic proteins. This hypothesis was tested using eosinophils collected from the lungs of rats previously infected with Toxocara canis, which leads to an eosinophil-rich pneumonitis. The effect of these cells following nonspecific activation with phorbol myristate acetate (PMA) in an isolated perfused rat lung was determined by measuring pulmonary vascular and airway pressures and the capillary filtration coefficient (Kf,C>. 1414
SUMMARY The effects of eosinophlls activated with phorbol myrlstate acetate (PMA) on Isolated perfused rat lungs were examined. Eosinophils were obtained from lungs of rats Infected with Toxocara canis by bronchoalveolar lavage, Incubated with PMA, and administered to an Isolated perfused rat lung preparation. Vascular endothelial permeability was assessed by measuring the capillary filtration coefficient (K".) In the perfused lungs. In lungs receiving either no eosinophlls (control) or nonactivated eoslnophlls, there were no changes In pUlmonary hemodynamics or K"., However, In lungs receiving 2 x 10' eoslnophlls activated with PMA, there was a transient 4.8-fold Increase In pulmonary vascular resistance that peaked at 30 min, primarily due to the constriction of small arteries and veins. After the Initial pressor response, K". was increased to 7.5 times control at 130 min and resulted in marked lung edema, Increased wet-dry weight ratios, and edema on histologic examination. Pulmonary arterial pressure and K1,. responses were dose related for eosinophil numbers between 1 x 10' and 4 x 10' cells. Peak airway pressure (Paw) during constant tidal volume ventilation also increased In lungs receiving activated eoslnophlls compared to the control and nonactivated eosinophil groups. These findings Indicate that activated eoslnophlls are potent effector cells and can cause pUlmonary vasoconstriction, bronchoconstriction, and vascular endothelial injury without widespread plugging of capillaries by aggregated eoslnophils. AM REV RESPIR DIS 1990; 142:1414-1421
Methods Preparation of Eosinophils Eosinophils were collected from the lungs of rats infected with Toxocara canis as described by Kayes and colleagues (16). Briefly, male Charles River Raleigh rats (Charles River Breeding Laboratories, Kingston, NY) (weighing 150 to 250 g) were infected with 2 x 103 infective eggs of T. canis by gastric intubation under ether anesthesia. The population of eosinophils markedly increased in the lungs of these rats and reached a peak on Day 14 after infection. At this time the eosinophils that accumulated in the lungs were collected by bronchoalveolar lavage (BAL) with 50 ml of Ca- and Mg-free Dulbecco's phosphatebuffered saline (DBSS, Grand Island Biological Company, Long Isand, NY) with 60 U heparin/ml (Upjohn Co., Kalamazoo, MI). The collected cells were suspended in Ca- and Mg-free DBSS with 0.1070 gelatin and 5 mM glucose (DBSS-GG) and the contaminating red cells lysed with hypotonic saline. A cytosmear of the collected eosinophils was prepared for differential counting and stained with Hemal® stain (Hemal Stain Co., Inc., Danbury, CT), and cell viability was> 95% using trypan blue dye exclusion. Lavaged cells were more than 80% eosinophils (mean ± SD, 88.3 ± 6.9%), and contaminating cells were almost exclusively mononuclear cells.
Isolated Perfused Rat Lung Preparation Male Charles River Raleigh rats (weighing 350
to 400 g) were used in all experiments. The method of isolation and perfusion of the lungs was modified from that developed by Marshall (17). Briefly, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/kg). The trachea was cannulated, and the lungs were ventilated with a gas mixture of 20% O 2 and 5% CO 2 using a piston-type respirator (Model 683; Rodent Ventilator; Harvard, South Natick, MA), which was set as follows: a tidal volume of 3 ml; a respiratory rate of 40/min; and 2.5 cm H 2 0 positive end-expiratory pressure to keep the lungs in Zone 3 conditions. The chest was opened, and heparin (300 U) was injected directly into the right ventricle and allowed to circulate for 5 min. The main pulmonary artery and the left atrium were cannulated
(Received in originalform November 20, 1989 and in revised form May 31, 1990) 1 From the Departments of Physiology and Structural and Cellular Biology, College of Medicine, University of South Alabama, Mobile, Alabama. 2 Supported by Grants HL-24571 and AI 19968 from the National Institutes of Health and by Grant 870034 from the American Heart Association, Al~ abama affiliate. 3 Correspondence and requests for reprints should be addressed to James C. Parker, Ph.D., Department of Physiology, College of Medicine, MSB 3024, University of South Alabama, Mobile, AL 36688.
1415
ACTIVATED EOSINOPHIL-INDUCED LUNG INJURY
through incisions in the right and left ventricle, respectively, without previous bleeding of the animal to prevent the increased neutrophil margination in the lung known to occur with decreased blood flow (18). The lungs were perfused with 5070 bovine albumin in Kreb's bicarbonate buffer using a roller pump (Minipuls 2; Gilson, Middleton, WI). The lungs and heart were then removed from the thoracic cavity, suspended from the weighing system, and perfused at a flow rate of 20 ml/min. The lungs were flushed with perfusate until the effluent was clear of red blood cells, and then the perfusate was recirculated (30 ml total recirculating volume). Arterial, venous, and airway pressures were measured by pressure transducers (Cobe, Lakewood, CO) via catheters connected to the cannulas in close proximity to the lungs. The perfusate was maintained at 37° C using a constant-temperature water bath. The flow rate of perfusate was maintained at 20 ml/min as long as the lungs remained in an isogravimetric state. When the lungs could not maintain an isogravimetric state, the flow rate was decreased until the lungs were isogravimetric. Lung weight was monitored by a springcounterbalanced force transducer (FT03D; Grass, Quincy, MA). Sensitivity of the recorder was calibrated so that 0.15 g caused a 2.0 cm deflection. Lung weight and vascular pressures were recorded continuously on a Grass model 7D polygraph.
Estimation of Pulmonary Capillary Pressure and Determination of Arterial and Venous Occlusion Pressures Pulmonary capillary pressure (Ppc) was estimated by rapidly clamping both inflow and outflow cannulas (19-21). Pulmonary arterial pressure (Ppa) falls and pulmonary venous pressure (Ppv) rises to a pressure equivalent to capillary pressure after simultaneous occlusion. Arterial occlusion (Pao) and venous occlusion (Pvo) pressures were also determined by separate rapid occlusions of the arterial inflow and venous outflow tubes, respectively. The inflection points between the rapid and slow components of the pressure-time curves following inflow or outflow occlusion were taken as the Pao or Pvo, respectively (19). A predicted capillary pressure (Pc,p) was also calculated in lungs in which perfusate flow was reduced below control values to prevent excessive edema formation. Pc,p is an estimate of the capillary pressure that would have existed if flow had not been decreased and describes the maximal capillary pressure change that could have occurred in intact lungs, such that (1) Pc,p = Ppv + Rv·Qcontrol where Ppv and Rv are measured in the injured state and Qcontrol is the initial perfusate flow. Estimation of Pulmonary Vascular Resistance From Ppc, Ppa, Ppv, Pao, Pvo, and perfu-
sate flow (Q) normalized to 100 g initial wet lung weight, resistances for each of the pulmonary vascular segments were calculated as follows: total pulmonary vascular resistance, RT = (Ppa - Ppv)/(Q/loo g); precapillary resistance, RA Yz (Ppa - Ppc)/(Q/loo g); postcapillary resistance, Rv = (Ppc- Ppv)/(Q/loo g); large and small artery resistances, respectively, RLA = (Ppa - Pao)/(Q/loo g) and RSA = (Pao - Ppc)/(Q/loo g); small and large vein resistances, respectively, Rsv = (Ppc - Pvo)/(Q/loo g) and RLV = (Pvo Pv)/(Q/loo g) (20, 21).
Measurement of the Capillary Filtration Coefficient The capillary filtration coefficient (Kf,c) was measured by the zero-time extrapolation method of Drake and coworkers (22). After a rapid increase in capillary pressure produced by raising the venous reservoir, there is a twocomponent increase in lung weight. The initial rapid weight gain is due to an increased lung vascular volume followed by a slow component of weight gain representing filtration of fluid into the pulmonary interstitium (23). The rate of slow weight gain (~WI M) was plotted on semilogarithmic graph paper, and the line of best fit was extrapolated to zero time (t = 0). Kf,c was then calculated using the equation Kf,c =
[(~W/~t) t = 0 -
(~WI ~t) init]/ ~Ppc
(2)
where ~Ppc is the increase in capillary pressure measured using the double occlusion before and after Ppc was elevated and (~WI ~t)init is the rate of lung weight gain before the pressure increase. The Kf,c was always determined from an initial isogravimetric state, where (~WI ~t)init = O. If an isogravimetric state was not attainable, the upper equation was used to calculate Kf,c, which was normalized to 100 g initial wet lung weight. When the zero-time extrapolation could not be obtained because of severe lung damage, Kf,c was calculated using the average rate of weight gain between 3.0 and 4.0 min after the pressure increase since this value was found empirically to predict the extrapolated zero-time rate of weight gain (24). When Kf,c might be underestimated as a result of the low flow rates used to prevent excess edema formation, Ppc was raised by increasing the flow rate. That is, the flow rate was increased from 15 to 50% of baseline flow while venous pressure was maintained low. The increased Ppc caused an increase in the rate of weight gain, and we calculated Kf,c using equation (2).
Measurement of Wet and Dry Lung Weights Initial lung weight was estimated by measuring the weight of the heart, mediastinal tissue, and lungs at the beginning of the experiment and subtracting the weight of all extrapulmonary tissue at the end of the experiment. Lungs excised at the end of the
experiment were weighed for final wet lung weight. The wet lungs were then dried in an oven at 60° C for 1 month and again weighed to calculate the lung wet/dry weight ratio.
Activation of Eosinophils Before the experiments, 2 x 106 eosinophils suspended in 2 ml DBSS-GG without Ca and Mg were incubated with 10 Ilg/ml of phorbol myristate acetate (PMA; Sigma Chemical, St. Louis, MO) at 37° C for 30 min and then centrifuged at 1,000 rpm for 10 min at room temperature (Beckman TJ-6 Centrifuge; Beckman Instruments, Fullerton, CAl. The supernatant was aspirated, and the pellet of activated eosinophils was resuspended in the last 50 III supernatant. Experimental Protocols Isolated perfused lungs were allowed to attain an initial isogravimetric state at a venous pressure of 3 cm H 2 0, and the baseline pulmonary hemodynamics, Paw, and Kf,c were measured. At 30, 90, and 130 min after the addition of eosinophils, the same variables were again measured. The lungs were then grouped as follows. Activated eosinophil group. In six lungs, 2 x 106 activated eosinophils were added to the reservoir 10 min after baseline measurements. To establish the requisite number of activated eosinophils capable of producing a measurable effect, we also examined the responses to 1 and 4 million activated eosinophils in additional lungs. Nonactivated eosinophil group. In six lungs, 2 x 106 eosinophils suspended in 2 ml DBSS-GG were incubated without PMA and the eosinophils added to the reservoir as in the activated eosinophil group. Control group. In six lungs, 50 III DBSSGG containing 10 Ilg/ml of PMA but without eosinophils was incubated as previously and then added to the reservoir as a PMA carryover control. Measurement of Eosinophil Peroxidase To examine whether the activated eosinophils continued to produce cytotoxic products after incubation with PMA, eosinophil peroxidase (EPa) released from eosinophils was also measured. Eosinophils (1 x 107 cells) suspended in DBSS-GG (1 ml) were incubated with 10 Ilg PMA at 37° C for 30 min and then the supernatant was removed. The pellet of activated eosinophils was again resuspended in the perfusate (1 mI). These activated eosinophils were then incubated without PMA at 37° C for 130 min and the supernatant removed. These supernatants were kept at - 80° C until the assay for EPa. The assay for EPa is a composite modification of that described by Strath and coworkers (26) and Cramer and colleagues (25). All reagents were obtained from Sigma Chemical Co. The main reaction cocktail (prepared fresh immediately before use) consisted of a 1:50 dilution of 5 mM orthophenylene diamine (OPD) diluted in 50 ml of 0.05 M'ITisHCl, 0.05 ml of 0.1% lWeen® 20 (peroxide-
1416
FWIMOTO, PARKER, AND KAYES
TABLE 1 PULMONARY HEMODYNAMICS AFTER CHALLENGE WITH 2 x 10' ACTIVATED EOSINOPHILS IN ISOLATED PERFUSED RAT LUNGS'
Q
Ppa (em H2 O)
Ppv (em H2 O)
Pao (em H2 O)
Pvo (em H2 O)
20.0 20.0 20.0
8.2 ± 1.0 9.4 ± 1.0 9.1 ± 0.2
3.0 ± 0.3 3.1 ± 0.2 3.3 ± 0.4
6.4 ± 0.4 7.2 ± 0.8 7.2 ± 0.3
4.7 ± 0.3 5.3 ± 0.5 5.5 ± 1.0
5.2 ± 0.3 5.9 ± 0.9 5.9 ± 0.9
20.0 20.0 10.3 ± 2.1t
8.2 ± 1.0 9.3 ± 1.0 16.9 ± 1.6H
3.1 ± 0.3 3.1 ± 0.2 3.2 ± 0.3
6.0 ± 0.3 7.1 ± 1.3 14.5 ± 1.3H
4.7 ± 0.3 5.0 ± 0.5 5.6 ± 0.8+
5.1 ± 0.3 5.8 ± 1.1 9.4 ± 0.9H
5.1 ± 0.3 5.8 ± 1.1 15.8 ± 4.1H
20.0 20.0 19.4 ± 1.4
8.4 ± 1.0 9.5 ± 1.1 11.5 ± 1.0H
3.1 ± 0.2 3.1 ± 0.3 3.2 ± 0.2
6.1 ± 0.4 7.1 ± 1.0 8.1 ± 0.8+
4.7 ± 0.3 5.0 ± 0.7 5.2 ± 0.3
5.2 ± 0.4 5.9 ± 1.0 6.5 ± 0.7+
5.2 ± 0.4 5.9 ± 1.0 6.7 ± 1.0+
20.0 20.0 20.0
8.5 ± 1.1 9.4 ± 0.9 9.7 ± 0.4
3.4 ± 0.3 3.2 ± 0.3 3.2 ± 0.2
6.3 ± 0.3 7.0 ± 0.9 6.9 ± 0.6
5.0 ± 0.5 4.9 ± 0.6 5.5 ± 0.5
5.3 ± 0.5 5.7 ± 1.1 6.0 ± 0.9
5.3 ± 0.5 5.7 ± 1.1 6.0 ± 0.9
(mltm/n)
Baseline Controls Non-Ac.Eo. Ac.Eo. 30 min Controls Non-Ae.Eo. Ae.Eo. 90 min Controls Non-Ac.Eo. AC.Eo.
Ppe (em H2 O)
Ppe,p (em H2 O)
5.2 ± 0.3 5.9 ± 0.9 5.9 ± 0.9
130 min Controls lIIort-Ac.Eo. Ac.Eo.
Definition of abbrevialions: Non-Ac.Eo. = nonactivated eosinophils (2 x 10'); Ac.Eo. = activated eosinophils (2 x 10'); (;) = perfusate flow; Ppa = pulmonary arterial pressure; Ppv = pulmonary venous pressure; Pao = pulmonary arterial occlusion pressure; Pvo = pulmonary venous occlusion pressure; Ppc = pulmonary capillary pressure; Ppc,p = pulmonary predicted capillary pressure. • Values are mean :t: SO (n = 6). t p < 0.05 versus control group. t p < 0.05 versus baseline.
free), and 5.6 III of 30070 H 2 0 2 • The pH was adjusted to 8.0, and 100 III sample was added to 900 III reaction mixture. Because EPa can be inhibited by 3-amino-I,2,4-triazole (3-AT) (25), all assays were run with duplicate samples in which the amount ofnis-HCI was reduced by 5.0 ml and replaced with 5.0 ml of 20 mM 3-AT (final 3-AT concentration was 2 mM). Samples were incubated at 37° C in water for 30 min and the reaction stopped with 4 M H 2 S0 4 , Each sample (200 Ill) was transferred to an EIA-grade 96-well, flat-bottomed plate (Flow Labs, Inc., McLean, YA) and the absorbance read at 490 nm in an ELISA plate reader (Model EL308; Bio-Tec Instruments, Inc., Winooski, YT). Results were expressed as A 490 U/IO' cells.
Histologic Examination Since PMA can cause the aggregation of cells (27), there was a possibility that aggregated eosinophils may form emboli that could contribute to the lung injury. To determine the presence of emboli and the location of the eosinophils in the lung vasculature, lungs previously perfused with nonactivated or PMAactivated eosinophils were examined histologically. The lungs were removed at 30 and 130 min after the addition of cells, cut into pieces, and fixed in fresh Buin's (picric acid, 37% formalin, and glacial acetic acid, 70:25:5) for 48 h. Tissue samples were then rinsed and dehydrated in steps through graded alcohols to absolute alcohol, embedded in paraffin, sectioned at 5 11m, and stained with hematoxylin and eosin. A total of 15 microscopic sections from each experimental group of lungs were studied. Statistics The results from each group are expressed as
mean ± SD. Comparison of variables within groups were performed using analysis of variance (ANaYA) followed by a NewmanKeuls multiple range test. Comparisons between the groups were performed using a Student's unpaired t test. Differences were considered significant at p < 0.05.
Results
Pulmonary Hemodynamic Responses The hemodynamic responses of lungs to PMA alone (control) and activated and nonactivated eosinophils (2 x 106 cells) are summarized in table 1. There were no changes in any hemodynamic variables in the control and nonactivated eosinophil groups. However, in lungs challenged with activated eosinophils, the pulmonary arterial pressure (Ppa) began to increase at about 7 min after the administration of activated eosinophils and
reached a peak at 30 min. At the same time that the pulmonary arterial pressure increased, the lung weight decreased transiently and then gradually increased. At this time the flow rate was decreased to about 500/0 of the baseline flow rate to maintain an isogravimetric state. Pao, Pvo, Pc, and Pc,P were significantly increased at 30 min but returned to baseline values from 90 to 130 min after the administration of activated eosinophils. Figure 1 shows the changes in total pulmonary vascular resistance (RT) with respect to time of control, nonactivated, and activated eosinophil groups. There were no changes in the control and nonactivated eosinophil groups. In activated eosinophil-treated lungs, RT significantly increased at 30 min to 4.5 times the baseline value and then returned to the baseline value at 90 to 130 min. The intensity of this pressor response was
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Fig. 1. Time course of total pulmonary vascular resistance in rat lungs. Values are mean ± SD (n = 6) for the control group (open circles), nonactivated eosinophil group (open triangles), and. group with 2 x 10· eosinophils activated with phorbol myristate acetate (PMA), (closed circles). 'p < 0.05 versus baseline; t p < 0.05 versus control group. (\lin.)
1417
ACTIVATED EOSINOPHIL-INDUCED LUNG INJURY
(CiIIIII:I0Nmba/10Gt ) ~
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dependent upon the number of activated eosinophils, as shown in figure 2. The RT increased to 4.5 times baseline with 1 x 106 cells and to 15.5 times baseline with 4 x 106 cells. The changes in segmental pulmonary vascular resistances are shown in table 2. There were no changes in any segmental vascular resistances in control and nonactivated eosinophil groups. In the activated eosinophil group, both precapillary and postcapillary resistances were significantly increased at 30 min, but the ratio of upstream and downstream resistance did not change significantly. The increases relative to baseline values were greater in the small artery (740070) and vein (1380070) resistances relative to the large artery (260070) and vein (220070) resistances. Thus, the increase in RT was primarily due to the increases in small arterial and venous vascular resistances.
Capillary Filtration Coefficient Figure 3 shows the changes in Kf,c with respect to time in control, nonactivated, and activated eosinophil groups. There were no changes in Kf,c in the control and nonactivated eosinophil groups. In the activated eosinophil-treated lungs, Kf,c began to increase at 90 min and was significantly increased from a baseline value of 0.21 ± 0.09 to 1.53 ± 0.80 mIl min/em H 2 0/IOO g at 130 min (p < 0.01). Figure 4 shows the effects of different cell numbers of activated eosinophils on Kf,c. Although 1 million activated eosinophils produced no change at 90 min, at 130 min this caused a significant increase in Kf,c from 0.26 ± 0.09 to 0.91 ± 0.45 mllmin/cm H 2 0/IOO g. Kf,c was measured by raising the reservoir, as mentioned. When 4 million activated eosinophils were added, the flow rate fell to 15070 of baseline flow as a result of the
extensive vasoconstriction at 30 min. Therefore, Kf,c was measured by increasing the flow rate from 15 to 50070 of baseline flow rate in these experiments. The lungs challenged with 4 x 106 activated eosinophils showed a remarkable increase in Kf,c to eight times control at 30 min. At 90 min, Kf,c was further increased in some experiments, but the experiments were terminated because of accumulation of airway fluid.
Peak Airway Pressure Figure 5 shows the changes in peak airway pressure (Paw) with respect to time. Paw increased significantly at 30 min and increased further every time Kf,c was measured in all groups. There were no significant differences in the increases in Paw between control and nonactivated eosinophil groups, but in the activated eosinophil-treated group the increased Paw was significantly greater than in the other two groups at any time period measured, especially at 30 min. A graded response of Paw to increasing cell numbers was not observed. Addition of 4 x 106 activated eosinophils did not result in a larger increase in Paw than did 2 x 106 cells (figure 6). Lung Weight The lung weight changes are summarized in table 3. There were no significant differences in the initial lung weights between any groups. In the 2 and 4 million activated eosinophil-treated lungs, the final wet lung weights (wet LW) and wet! dry lung weight ratios (W/D) were sig-
TABLE 2 EFFECT OF 2 x 10' ACTIVATED EOSINOPHILS ON THE DISTRIBUTION OF VASCULAR RESISTANCE'
Baseline Controls Non-Ac.Eo. AC.Eo. 30 min Controls Non-Ac.Eo. AC.Eo 90 min Controls Non-Ac.Eo. AC.Eo. 130 min Controls Non-Ac.Eo. Ac.Eo.
RA
Rv
RAtRv
RLA
RSA
Rsv
RLV
2.5 ± 0.7 2.7 ± 0.7 2.6 ± 0.4
1.8 ± 0.4 2.2 ± 0.7 2.2 ± 0.9
1.4 ± 0.2 1.4 ± 0.5 1.3 ± 0.5
1.5 ± 0.8 1.7 ± 0.4 1.4 ± 0.2
1.0 ± 0.2 1.0 ± 0.4 1.2 ± 0.6
0.4 ± 0.1 0.5 ± 0.3 0.5 ± 0.3
1.4 ± 0.4 1.6 ± 0.4 1.7 ± 0.8
2.6 ± 0.6 2.7 ± 0.8 12.5 ± 4.3t
1.7 ± 0.4 2.1 ± 0.8 10.5 ± 4.3t
1.5 ± 0.2 1.4 ± 0.7 1.2 ± 0.2
1.8 ± 0.6 1.6 ± 0.8 3.6 ± 1.4t
0.8 ± 0.1 1.1 ± 0.4 8.9 ± 3.6t
0.4 ± 0.3 0.7 ± 0.6 6.9 ± 4.0t
1.3 ± 0.2 1.5 ± 0.3 3.7 ± 1.0t
2.7 ± 0.5 2.8 ± 0.7 4.3 ± 1.4
1.8 ± 0.3 2.2 ± 0.8 3.0 ± 1.3
1.5 ± 0.1 1.4 ± 0.5 1.6 ± 0.4
1.8 ± 0.6 1.8 ± 0.7 2.9 ± 1.3
1.0 ± 0.4 1.0 ± 0.4 1.5 ± 0.7
0.5 ± 0.2 0.8 ± 0.5 1.3 ± 1.0
1.3 ± 0.2 1.5 ± 0.5 1.6 ± 0.3
2.7 ± 0.5 2.9 ± 0.6 3.0 ± 0.3
1.7 ± 0.2 2.1 ± 0.8 2.4 ± 1.1
1.6 ± 0.2 1.6 ± 0.6 1.4 ± 0.5
1.8 ± 0.7 1.8 ± 0.4 2.2 ± 0.3
1.0 ± 0.4 1.0 ± 0.4 0.8 ± 0.5
0.4 ± 0.1 0.7 ± 0.5 0.7 ± 0.6
1.3 ± 0.2 1.3 ± 0.4 1.8 ± 0.5
Definition of abbreviations: Non-Ac.Eo. = nonactivated eosinophils (2 x 10' cells) group; Ac.Eo. = activated eosinophils (2 x 10' cells) group; RA = precapillary resistance; Rv = postcapillary resistance; RLA = large arterial resistance; RSA = small arterial resistance; Rsv = small venous resistance; RLV = large venous resistance . • Values are mean ± SD (n = 6). Resistance measurements are in cm H,O/Umin/100 g. t p < 0.05 versus baseline; p < 0.05 versus control group.
1418
FUJIMOTO, PARKER, AND KAYES
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KEISAKU FUJIMOTO, JAMES C. PARKER, and STEPHEN G. KAYES
Introduction Eosinophils are granule-containing polymorphonuclear leukocytes that have been implicated as primary effector cells in the pathophysiology of asthma and allergic diseases (1-3). Eosinophils can cause tissue injury by the generation of reactive oxygen metabolites, such as Oi, H 2 0 2 , ·OH, singlet O 2 , HOCl, HOI, and HOBr (4-6); by the release of toxic granule-contained proteins, such as major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil cationic protein (ECP) (7-9); and by the interactions of these toxic substances (10, 11). Additionally, leukotriene C 4 (LTC 4 ), a potent mediator of bronchoconstriction (12) and pulmonary vasoconstriction (13), is the predominant leukotriene elaborated by eosinophils (14). Many clinical studies, both morphologic and immunochemical, have suggested that eosinophils may also be associated with other types of lung injury under certain circumstances (3). For instance, a role has been suggested for eosinophils in the adult respiratory distress syndrome (ARDS). ARDS is characterized by increased pulmonary vascular permeability, and both bronchoalveolar lavage fluid and plasma from these patients have elevated ECP levels (15). However, whether eosinophils cause lung injury directly and increase pulmonary microvascular permeability has not been determined. We hypothesized that eosinophils may cause lung injury by releasing toxic oxygen metabolites and/or cytotoxic proteins. This hypothesis was tested using eosinophils collected from the lungs of rats previously infected with Toxocara canis, which leads to an eosinophil-rich pneumonitis. The effect of these cells following nonspecific activation with phorbol myristate acetate (PMA) in an isolated perfused rat lung was determined by measuring pulmonary vascular and airway pressures and the capillary filtration coefficient (Kf,C>. 1414
SUMMARY The effects of eosinophlls activated with phorbol myrlstate acetate (PMA) on Isolated perfused rat lungs were examined. Eosinophils were obtained from lungs of rats Infected with Toxocara canis by bronchoalveolar lavage, Incubated with PMA, and administered to an Isolated perfused rat lung preparation. Vascular endothelial permeability was assessed by measuring the capillary filtration coefficient (K".) In the perfused lungs. In lungs receiving either no eosinophlls (control) or nonactivated eoslnophlls, there were no changes In pUlmonary hemodynamics or K"., However, In lungs receiving 2 x 10' eoslnophlls activated with PMA, there was a transient 4.8-fold Increase In pulmonary vascular resistance that peaked at 30 min, primarily due to the constriction of small arteries and veins. After the Initial pressor response, K". was increased to 7.5 times control at 130 min and resulted in marked lung edema, Increased wet-dry weight ratios, and edema on histologic examination. Pulmonary arterial pressure and K1,. responses were dose related for eosinophil numbers between 1 x 10' and 4 x 10' cells. Peak airway pressure (Paw) during constant tidal volume ventilation also increased In lungs receiving activated eoslnophlls compared to the control and nonactivated eosinophil groups. These findings Indicate that activated eoslnophlls are potent effector cells and can cause pUlmonary vasoconstriction, bronchoconstriction, and vascular endothelial injury without widespread plugging of capillaries by aggregated eoslnophils. AM REV RESPIR DIS 1990; 142:1414-1421
Methods Preparation of Eosinophils Eosinophils were collected from the lungs of rats infected with Toxocara canis as described by Kayes and colleagues (16). Briefly, male Charles River Raleigh rats (Charles River Breeding Laboratories, Kingston, NY) (weighing 150 to 250 g) were infected with 2 x 103 infective eggs of T. canis by gastric intubation under ether anesthesia. The population of eosinophils markedly increased in the lungs of these rats and reached a peak on Day 14 after infection. At this time the eosinophils that accumulated in the lungs were collected by bronchoalveolar lavage (BAL) with 50 ml of Ca- and Mg-free Dulbecco's phosphatebuffered saline (DBSS, Grand Island Biological Company, Long Isand, NY) with 60 U heparin/ml (Upjohn Co., Kalamazoo, MI). The collected cells were suspended in Ca- and Mg-free DBSS with 0.1070 gelatin and 5 mM glucose (DBSS-GG) and the contaminating red cells lysed with hypotonic saline. A cytosmear of the collected eosinophils was prepared for differential counting and stained with Hemal® stain (Hemal Stain Co., Inc., Danbury, CT), and cell viability was> 95% using trypan blue dye exclusion. Lavaged cells were more than 80% eosinophils (mean ± SD, 88.3 ± 6.9%), and contaminating cells were almost exclusively mononuclear cells.
Isolated Perfused Rat Lung Preparation Male Charles River Raleigh rats (weighing 350
to 400 g) were used in all experiments. The method of isolation and perfusion of the lungs was modified from that developed by Marshall (17). Briefly, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/kg). The trachea was cannulated, and the lungs were ventilated with a gas mixture of 20% O 2 and 5% CO 2 using a piston-type respirator (Model 683; Rodent Ventilator; Harvard, South Natick, MA), which was set as follows: a tidal volume of 3 ml; a respiratory rate of 40/min; and 2.5 cm H 2 0 positive end-expiratory pressure to keep the lungs in Zone 3 conditions. The chest was opened, and heparin (300 U) was injected directly into the right ventricle and allowed to circulate for 5 min. The main pulmonary artery and the left atrium were cannulated
(Received in originalform November 20, 1989 and in revised form May 31, 1990) 1 From the Departments of Physiology and Structural and Cellular Biology, College of Medicine, University of South Alabama, Mobile, Alabama. 2 Supported by Grants HL-24571 and AI 19968 from the National Institutes of Health and by Grant 870034 from the American Heart Association, Al~ abama affiliate. 3 Correspondence and requests for reprints should be addressed to James C. Parker, Ph.D., Department of Physiology, College of Medicine, MSB 3024, University of South Alabama, Mobile, AL 36688.
1415
ACTIVATED EOSINOPHIL-INDUCED LUNG INJURY
through incisions in the right and left ventricle, respectively, without previous bleeding of the animal to prevent the increased neutrophil margination in the lung known to occur with decreased blood flow (18). The lungs were perfused with 5070 bovine albumin in Kreb's bicarbonate buffer using a roller pump (Minipuls 2; Gilson, Middleton, WI). The lungs and heart were then removed from the thoracic cavity, suspended from the weighing system, and perfused at a flow rate of 20 ml/min. The lungs were flushed with perfusate until the effluent was clear of red blood cells, and then the perfusate was recirculated (30 ml total recirculating volume). Arterial, venous, and airway pressures were measured by pressure transducers (Cobe, Lakewood, CO) via catheters connected to the cannulas in close proximity to the lungs. The perfusate was maintained at 37° C using a constant-temperature water bath. The flow rate of perfusate was maintained at 20 ml/min as long as the lungs remained in an isogravimetric state. When the lungs could not maintain an isogravimetric state, the flow rate was decreased until the lungs were isogravimetric. Lung weight was monitored by a springcounterbalanced force transducer (FT03D; Grass, Quincy, MA). Sensitivity of the recorder was calibrated so that 0.15 g caused a 2.0 cm deflection. Lung weight and vascular pressures were recorded continuously on a Grass model 7D polygraph.
Estimation of Pulmonary Capillary Pressure and Determination of Arterial and Venous Occlusion Pressures Pulmonary capillary pressure (Ppc) was estimated by rapidly clamping both inflow and outflow cannulas (19-21). Pulmonary arterial pressure (Ppa) falls and pulmonary venous pressure (Ppv) rises to a pressure equivalent to capillary pressure after simultaneous occlusion. Arterial occlusion (Pao) and venous occlusion (Pvo) pressures were also determined by separate rapid occlusions of the arterial inflow and venous outflow tubes, respectively. The inflection points between the rapid and slow components of the pressure-time curves following inflow or outflow occlusion were taken as the Pao or Pvo, respectively (19). A predicted capillary pressure (Pc,p) was also calculated in lungs in which perfusate flow was reduced below control values to prevent excessive edema formation. Pc,p is an estimate of the capillary pressure that would have existed if flow had not been decreased and describes the maximal capillary pressure change that could have occurred in intact lungs, such that (1) Pc,p = Ppv + Rv·Qcontrol where Ppv and Rv are measured in the injured state and Qcontrol is the initial perfusate flow. Estimation of Pulmonary Vascular Resistance From Ppc, Ppa, Ppv, Pao, Pvo, and perfu-
sate flow (Q) normalized to 100 g initial wet lung weight, resistances for each of the pulmonary vascular segments were calculated as follows: total pulmonary vascular resistance, RT = (Ppa - Ppv)/(Q/loo g); precapillary resistance, RA Yz (Ppa - Ppc)/(Q/loo g); postcapillary resistance, Rv = (Ppc- Ppv)/(Q/loo g); large and small artery resistances, respectively, RLA = (Ppa - Pao)/(Q/loo g) and RSA = (Pao - Ppc)/(Q/loo g); small and large vein resistances, respectively, Rsv = (Ppc - Pvo)/(Q/loo g) and RLV = (Pvo Pv)/(Q/loo g) (20, 21).
Measurement of the Capillary Filtration Coefficient The capillary filtration coefficient (Kf,c) was measured by the zero-time extrapolation method of Drake and coworkers (22). After a rapid increase in capillary pressure produced by raising the venous reservoir, there is a twocomponent increase in lung weight. The initial rapid weight gain is due to an increased lung vascular volume followed by a slow component of weight gain representing filtration of fluid into the pulmonary interstitium (23). The rate of slow weight gain (~WI M) was plotted on semilogarithmic graph paper, and the line of best fit was extrapolated to zero time (t = 0). Kf,c was then calculated using the equation Kf,c =
[(~W/~t) t = 0 -
(~WI ~t) init]/ ~Ppc
(2)
where ~Ppc is the increase in capillary pressure measured using the double occlusion before and after Ppc was elevated and (~WI ~t)init is the rate of lung weight gain before the pressure increase. The Kf,c was always determined from an initial isogravimetric state, where (~WI ~t)init = O. If an isogravimetric state was not attainable, the upper equation was used to calculate Kf,c, which was normalized to 100 g initial wet lung weight. When the zero-time extrapolation could not be obtained because of severe lung damage, Kf,c was calculated using the average rate of weight gain between 3.0 and 4.0 min after the pressure increase since this value was found empirically to predict the extrapolated zero-time rate of weight gain (24). When Kf,c might be underestimated as a result of the low flow rates used to prevent excess edema formation, Ppc was raised by increasing the flow rate. That is, the flow rate was increased from 15 to 50% of baseline flow while venous pressure was maintained low. The increased Ppc caused an increase in the rate of weight gain, and we calculated Kf,c using equation (2).
Measurement of Wet and Dry Lung Weights Initial lung weight was estimated by measuring the weight of the heart, mediastinal tissue, and lungs at the beginning of the experiment and subtracting the weight of all extrapulmonary tissue at the end of the experiment. Lungs excised at the end of the
experiment were weighed for final wet lung weight. The wet lungs were then dried in an oven at 60° C for 1 month and again weighed to calculate the lung wet/dry weight ratio.
Activation of Eosinophils Before the experiments, 2 x 106 eosinophils suspended in 2 ml DBSS-GG without Ca and Mg were incubated with 10 Ilg/ml of phorbol myristate acetate (PMA; Sigma Chemical, St. Louis, MO) at 37° C for 30 min and then centrifuged at 1,000 rpm for 10 min at room temperature (Beckman TJ-6 Centrifuge; Beckman Instruments, Fullerton, CAl. The supernatant was aspirated, and the pellet of activated eosinophils was resuspended in the last 50 III supernatant. Experimental Protocols Isolated perfused lungs were allowed to attain an initial isogravimetric state at a venous pressure of 3 cm H 2 0, and the baseline pulmonary hemodynamics, Paw, and Kf,c were measured. At 30, 90, and 130 min after the addition of eosinophils, the same variables were again measured. The lungs were then grouped as follows. Activated eosinophil group. In six lungs, 2 x 106 activated eosinophils were added to the reservoir 10 min after baseline measurements. To establish the requisite number of activated eosinophils capable of producing a measurable effect, we also examined the responses to 1 and 4 million activated eosinophils in additional lungs. Nonactivated eosinophil group. In six lungs, 2 x 106 eosinophils suspended in 2 ml DBSS-GG were incubated without PMA and the eosinophils added to the reservoir as in the activated eosinophil group. Control group. In six lungs, 50 III DBSSGG containing 10 Ilg/ml of PMA but without eosinophils was incubated as previously and then added to the reservoir as a PMA carryover control. Measurement of Eosinophil Peroxidase To examine whether the activated eosinophils continued to produce cytotoxic products after incubation with PMA, eosinophil peroxidase (EPa) released from eosinophils was also measured. Eosinophils (1 x 107 cells) suspended in DBSS-GG (1 ml) were incubated with 10 Ilg PMA at 37° C for 30 min and then the supernatant was removed. The pellet of activated eosinophils was again resuspended in the perfusate (1 mI). These activated eosinophils were then incubated without PMA at 37° C for 130 min and the supernatant removed. These supernatants were kept at - 80° C until the assay for EPa. The assay for EPa is a composite modification of that described by Strath and coworkers (26) and Cramer and colleagues (25). All reagents were obtained from Sigma Chemical Co. The main reaction cocktail (prepared fresh immediately before use) consisted of a 1:50 dilution of 5 mM orthophenylene diamine (OPD) diluted in 50 ml of 0.05 M'ITisHCl, 0.05 ml of 0.1% lWeen® 20 (peroxide-
1416
FWIMOTO, PARKER, AND KAYES
TABLE 1 PULMONARY HEMODYNAMICS AFTER CHALLENGE WITH 2 x 10' ACTIVATED EOSINOPHILS IN ISOLATED PERFUSED RAT LUNGS'
Q
Ppa (em H2 O)
Ppv (em H2 O)
Pao (em H2 O)
Pvo (em H2 O)
20.0 20.0 20.0
8.2 ± 1.0 9.4 ± 1.0 9.1 ± 0.2
3.0 ± 0.3 3.1 ± 0.2 3.3 ± 0.4
6.4 ± 0.4 7.2 ± 0.8 7.2 ± 0.3
4.7 ± 0.3 5.3 ± 0.5 5.5 ± 1.0
5.2 ± 0.3 5.9 ± 0.9 5.9 ± 0.9
20.0 20.0 10.3 ± 2.1t
8.2 ± 1.0 9.3 ± 1.0 16.9 ± 1.6H
3.1 ± 0.3 3.1 ± 0.2 3.2 ± 0.3
6.0 ± 0.3 7.1 ± 1.3 14.5 ± 1.3H
4.7 ± 0.3 5.0 ± 0.5 5.6 ± 0.8+
5.1 ± 0.3 5.8 ± 1.1 9.4 ± 0.9H
5.1 ± 0.3 5.8 ± 1.1 15.8 ± 4.1H
20.0 20.0 19.4 ± 1.4
8.4 ± 1.0 9.5 ± 1.1 11.5 ± 1.0H
3.1 ± 0.2 3.1 ± 0.3 3.2 ± 0.2
6.1 ± 0.4 7.1 ± 1.0 8.1 ± 0.8+
4.7 ± 0.3 5.0 ± 0.7 5.2 ± 0.3
5.2 ± 0.4 5.9 ± 1.0 6.5 ± 0.7+
5.2 ± 0.4 5.9 ± 1.0 6.7 ± 1.0+
20.0 20.0 20.0
8.5 ± 1.1 9.4 ± 0.9 9.7 ± 0.4
3.4 ± 0.3 3.2 ± 0.3 3.2 ± 0.2
6.3 ± 0.3 7.0 ± 0.9 6.9 ± 0.6
5.0 ± 0.5 4.9 ± 0.6 5.5 ± 0.5
5.3 ± 0.5 5.7 ± 1.1 6.0 ± 0.9
5.3 ± 0.5 5.7 ± 1.1 6.0 ± 0.9
(mltm/n)
Baseline Controls Non-Ac.Eo. Ac.Eo. 30 min Controls Non-Ae.Eo. Ae.Eo. 90 min Controls Non-Ac.Eo. AC.Eo.
Ppe (em H2 O)
Ppe,p (em H2 O)
5.2 ± 0.3 5.9 ± 0.9 5.9 ± 0.9
130 min Controls lIIort-Ac.Eo. Ac.Eo.
Definition of abbrevialions: Non-Ac.Eo. = nonactivated eosinophils (2 x 10'); Ac.Eo. = activated eosinophils (2 x 10'); (;) = perfusate flow; Ppa = pulmonary arterial pressure; Ppv = pulmonary venous pressure; Pao = pulmonary arterial occlusion pressure; Pvo = pulmonary venous occlusion pressure; Ppc = pulmonary capillary pressure; Ppc,p = pulmonary predicted capillary pressure. • Values are mean :t: SO (n = 6). t p < 0.05 versus control group. t p < 0.05 versus baseline.
free), and 5.6 III of 30070 H 2 0 2 • The pH was adjusted to 8.0, and 100 III sample was added to 900 III reaction mixture. Because EPa can be inhibited by 3-amino-I,2,4-triazole (3-AT) (25), all assays were run with duplicate samples in which the amount ofnis-HCI was reduced by 5.0 ml and replaced with 5.0 ml of 20 mM 3-AT (final 3-AT concentration was 2 mM). Samples were incubated at 37° C in water for 30 min and the reaction stopped with 4 M H 2 S0 4 , Each sample (200 Ill) was transferred to an EIA-grade 96-well, flat-bottomed plate (Flow Labs, Inc., McLean, YA) and the absorbance read at 490 nm in an ELISA plate reader (Model EL308; Bio-Tec Instruments, Inc., Winooski, YT). Results were expressed as A 490 U/IO' cells.
Histologic Examination Since PMA can cause the aggregation of cells (27), there was a possibility that aggregated eosinophils may form emboli that could contribute to the lung injury. To determine the presence of emboli and the location of the eosinophils in the lung vasculature, lungs previously perfused with nonactivated or PMAactivated eosinophils were examined histologically. The lungs were removed at 30 and 130 min after the addition of cells, cut into pieces, and fixed in fresh Buin's (picric acid, 37% formalin, and glacial acetic acid, 70:25:5) for 48 h. Tissue samples were then rinsed and dehydrated in steps through graded alcohols to absolute alcohol, embedded in paraffin, sectioned at 5 11m, and stained with hematoxylin and eosin. A total of 15 microscopic sections from each experimental group of lungs were studied. Statistics The results from each group are expressed as
mean ± SD. Comparison of variables within groups were performed using analysis of variance (ANaYA) followed by a NewmanKeuls multiple range test. Comparisons between the groups were performed using a Student's unpaired t test. Differences were considered significant at p < 0.05.
Results
Pulmonary Hemodynamic Responses The hemodynamic responses of lungs to PMA alone (control) and activated and nonactivated eosinophils (2 x 106 cells) are summarized in table 1. There were no changes in any hemodynamic variables in the control and nonactivated eosinophil groups. However, in lungs challenged with activated eosinophils, the pulmonary arterial pressure (Ppa) began to increase at about 7 min after the administration of activated eosinophils and
reached a peak at 30 min. At the same time that the pulmonary arterial pressure increased, the lung weight decreased transiently and then gradually increased. At this time the flow rate was decreased to about 500/0 of the baseline flow rate to maintain an isogravimetric state. Pao, Pvo, Pc, and Pc,P were significantly increased at 30 min but returned to baseline values from 90 to 130 min after the administration of activated eosinophils. Figure 1 shows the changes in total pulmonary vascular resistance (RT) with respect to time of control, nonactivated, and activated eosinophil groups. There were no changes in the control and nonactivated eosinophil groups. In activated eosinophil-treated lungs, RT significantly increased at 30 min to 4.5 times the baseline value and then returned to the baseline value at 90 to 130 min. The intensity of this pressor response was
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Fig. 1. Time course of total pulmonary vascular resistance in rat lungs. Values are mean ± SD (n = 6) for the control group (open circles), nonactivated eosinophil group (open triangles), and. group with 2 x 10· eosinophils activated with phorbol myristate acetate (PMA), (closed circles). 'p < 0.05 versus baseline; t p < 0.05 versus control group. (\lin.)
1417
ACTIVATED EOSINOPHIL-INDUCED LUNG INJURY
(CiIIIII:I0Nmba/10Gt ) ~
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dependent upon the number of activated eosinophils, as shown in figure 2. The RT increased to 4.5 times baseline with 1 x 106 cells and to 15.5 times baseline with 4 x 106 cells. The changes in segmental pulmonary vascular resistances are shown in table 2. There were no changes in any segmental vascular resistances in control and nonactivated eosinophil groups. In the activated eosinophil group, both precapillary and postcapillary resistances were significantly increased at 30 min, but the ratio of upstream and downstream resistance did not change significantly. The increases relative to baseline values were greater in the small artery (740070) and vein (1380070) resistances relative to the large artery (260070) and vein (220070) resistances. Thus, the increase in RT was primarily due to the increases in small arterial and venous vascular resistances.
Capillary Filtration Coefficient Figure 3 shows the changes in Kf,c with respect to time in control, nonactivated, and activated eosinophil groups. There were no changes in Kf,c in the control and nonactivated eosinophil groups. In the activated eosinophil-treated lungs, Kf,c began to increase at 90 min and was significantly increased from a baseline value of 0.21 ± 0.09 to 1.53 ± 0.80 mIl min/em H 2 0/IOO g at 130 min (p < 0.01). Figure 4 shows the effects of different cell numbers of activated eosinophils on Kf,c. Although 1 million activated eosinophils produced no change at 90 min, at 130 min this caused a significant increase in Kf,c from 0.26 ± 0.09 to 0.91 ± 0.45 mllmin/cm H 2 0/IOO g. Kf,c was measured by raising the reservoir, as mentioned. When 4 million activated eosinophils were added, the flow rate fell to 15070 of baseline flow as a result of the
extensive vasoconstriction at 30 min. Therefore, Kf,c was measured by increasing the flow rate from 15 to 50070 of baseline flow rate in these experiments. The lungs challenged with 4 x 106 activated eosinophils showed a remarkable increase in Kf,c to eight times control at 30 min. At 90 min, Kf,c was further increased in some experiments, but the experiments were terminated because of accumulation of airway fluid.
Peak Airway Pressure Figure 5 shows the changes in peak airway pressure (Paw) with respect to time. Paw increased significantly at 30 min and increased further every time Kf,c was measured in all groups. There were no significant differences in the increases in Paw between control and nonactivated eosinophil groups, but in the activated eosinophil-treated group the increased Paw was significantly greater than in the other two groups at any time period measured, especially at 30 min. A graded response of Paw to increasing cell numbers was not observed. Addition of 4 x 106 activated eosinophils did not result in a larger increase in Paw than did 2 x 106 cells (figure 6). Lung Weight The lung weight changes are summarized in table 3. There were no significant differences in the initial lung weights between any groups. In the 2 and 4 million activated eosinophil-treated lungs, the final wet lung weights (wet LW) and wet! dry lung weight ratios (W/D) were sig-
TABLE 2 EFFECT OF 2 x 10' ACTIVATED EOSINOPHILS ON THE DISTRIBUTION OF VASCULAR RESISTANCE'
Baseline Controls Non-Ac.Eo. AC.Eo. 30 min Controls Non-Ac.Eo. AC.Eo 90 min Controls Non-Ac.Eo. AC.Eo. 130 min Controls Non-Ac.Eo. Ac.Eo.
RA
Rv
RAtRv
RLA
RSA
Rsv
RLV
2.5 ± 0.7 2.7 ± 0.7 2.6 ± 0.4
1.8 ± 0.4 2.2 ± 0.7 2.2 ± 0.9
1.4 ± 0.2 1.4 ± 0.5 1.3 ± 0.5
1.5 ± 0.8 1.7 ± 0.4 1.4 ± 0.2
1.0 ± 0.2 1.0 ± 0.4 1.2 ± 0.6
0.4 ± 0.1 0.5 ± 0.3 0.5 ± 0.3
1.4 ± 0.4 1.6 ± 0.4 1.7 ± 0.8
2.6 ± 0.6 2.7 ± 0.8 12.5 ± 4.3t
1.7 ± 0.4 2.1 ± 0.8 10.5 ± 4.3t
1.5 ± 0.2 1.4 ± 0.7 1.2 ± 0.2
1.8 ± 0.6 1.6 ± 0.8 3.6 ± 1.4t
0.8 ± 0.1 1.1 ± 0.4 8.9 ± 3.6t
0.4 ± 0.3 0.7 ± 0.6 6.9 ± 4.0t
1.3 ± 0.2 1.5 ± 0.3 3.7 ± 1.0t
2.7 ± 0.5 2.8 ± 0.7 4.3 ± 1.4
1.8 ± 0.3 2.2 ± 0.8 3.0 ± 1.3
1.5 ± 0.1 1.4 ± 0.5 1.6 ± 0.4
1.8 ± 0.6 1.8 ± 0.7 2.9 ± 1.3
1.0 ± 0.4 1.0 ± 0.4 1.5 ± 0.7
0.5 ± 0.2 0.8 ± 0.5 1.3 ± 1.0
1.3 ± 0.2 1.5 ± 0.5 1.6 ± 0.3
2.7 ± 0.5 2.9 ± 0.6 3.0 ± 0.3
1.7 ± 0.2 2.1 ± 0.8 2.4 ± 1.1
1.6 ± 0.2 1.6 ± 0.6 1.4 ± 0.5
1.8 ± 0.7 1.8 ± 0.4 2.2 ± 0.3
1.0 ± 0.4 1.0 ± 0.4 0.8 ± 0.5
0.4 ± 0.1 0.7 ± 0.5 0.7 ± 0.6
1.3 ± 0.2 1.3 ± 0.4 1.8 ± 0.5
Definition of abbreviations: Non-Ac.Eo. = nonactivated eosinophils (2 x 10' cells) group; Ac.Eo. = activated eosinophils (2 x 10' cells) group; RA = precapillary resistance; Rv = postcapillary resistance; RLA = large arterial resistance; RSA = small arterial resistance; Rsv = small venous resistance; RLV = large venous resistance . • Values are mean ± SD (n = 6). Resistance measurements are in cm H,O/Umin/100 g. t p < 0.05 versus baseline; p < 0.05 versus control group.
1418
FUJIMOTO, PARKER, AND KAYES
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