Spontaneous injury in isolated sheep lungs: role of resident polymorphonuclear leukocytes DAVID
B. PEARSE
AND
J. T. SYLVESTER
Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Medical Institutions at the Asthma and Allergy Center, Francis Scott Key Medical Center, Baltimore, Maryland 21224
PEARSE, DAVID B., AND J. T. SYLVESTER. Spontaneous injury in isolated sheep lungs: role of resident polymorphonuclear leukocytes. J. Appl. Physiol. 72(6): 24752481,1992.-Perfusion of isolated sheep lungs with homologous blood caused pulmonary hypertension and edema that was not altered by depletion of perfusate polymorphonuclear (PMN) leukocytes (D. B. Pearse et al., J. Appl. Physiol. 66: 1287-1296, 1989). The purpose of this study was to evaluate the role of resident PMN leukocytes in this injury. First, we quantified the content and activation of lung PMN leukocytes before and during perfusion of eight isolated sheep lungs with a constant flow (100 ml. kg-‘. min-‘) of homologous blood. From measurements of myeloperoxidase (MPO) activity, we estimated that the lungs contained 1.2 X 10” PMN leukocytes, which explained why the lung PMN leukocyte content, measured by MPO activity and histological techniques, did not increase significantly with perfusion, despite complete sequestration of 2.0 X 10’ PMN leukocytes from the perfusate. MPO activities in perfusate and lymph supernatants did not increase during perfusion, suggesting that lung PMN leukocytes were not activated. Second, we perfused lungs from 6 mechlorethamine-treated and 6 hydroxyurea-treated sheep with homologous leukopenic blood and compared them with 11 normal lungs perfused similarly. Despite marked reductions in lung PMN leukocyte concentration, there were no differences in pulmonary arterial pressure, lymph flow, or reservoir weight between groups. Extravascular lung water was greater in both groups of leukopenic lungs. These results suggest that resident PMN leukocytes did not contribute to lung injury in this model. pulmonary edema; pulmonary hypertension; lung lymph; trophil; lymphocyte; myeloperoxidase; mechlorethamine; droxyurea
neuhy-
SPECIES, including humans, the pulmonary vasculature is known to contain large numbers of polymorphonuclear (PMN) leukocytes (11). In sheep, for example, the number of PMN leukocytes in the lung was estimated to be three to four times larger than the number circulating in the blood (6, 25). These resident cells are not believed to be permanently adherent to pulmonary endothelium but rather to have a prolonged transit time through the lung (11). Although PMN leukocytes are believed to have an important role in a variety of lung injury models, the relative contribution of resident and circulating cells has not been determined. Perfusion of isolated sheep lungs with blood caused rapid sequestration of circulating leukocytes in the lung followed by pulmonary arterial hypertension, increased lymph flow, hemorrhage, and edema (18, 20). We re-
IN SEVERAL
0161-7567/92
$2.00 Copyright
cently examined the role of perfusate leukocytes in this injury by perfusing normal sheep lungs with leukopenic or normal blood from donor sheep (20). We found that perfusate leukocytes contributed to lung hemorrhage but were not necessary for the development of pulmonary hypertension or edema. The limited effect of depleting perfusate leukocytes could be explained if resident PMN leukocytes contributed to the injury. The purpose of the present study was to evaluate the role of resident PMN leukocytes in this model. METHODS
Preparation. Studies were performed in isolated sheep lungs perfused and ventilated in situ. Young sheep (1925 kg) were anesthetized with intramuscular ketamine (30 mg/kg) and atropine (0.5 mg). Anesthesia was maintained by intravenous injections of ketamine (3-6 mg/ kg) at 15 to 30-min intervals. A tracheostomy was performed and mechanical ventilation was begun with a tidal volume of 12 ml/kg and respiratory rate of 15/min. A sternotomy was performed, the superior thoracic duct was catheterized above the hilum of the left lung with silicone rubber tubing (0.03 in. ID), and the inferior thoracic duct was clamped. Heparin (10,000 U) was injected intravenously before exsanguination from a femoral arterial catheter. The left atrium and pulmonary artery were cannulated, and the ascending aorta and proximal pulmonary artery were ligated with a ligature passed through the transverse sinus and around the pulmonary arterial cannula. The pulmonary vasculature was then flushed with 1 liter of 3% dextran (mol wt 70,000) in normal saline solution at 39OC to remove residual blood. The lungs were connected to an extracorporeal circuit described previously (17) and perfused in situ at 100 ml kg-’ min-l with a mixture of blood (-1,000 ml) from donor sheep (leukopenic or nonleukopenic as specified below) and 3% dextran in Ringer lactate solution (-300 ml). The perfusate was warmed (38-39” C) with a heat exchanger (Travenol Miniprime) and continuously filtered of clots and bubbles with a filter. The time from onset of exsanguination to onset of perfusion was 20-40 min. The desired flow, measured by electromagnetic flow probe (model EP 300A, Carolina Medical Electronics), was achieved over 2-3 min by gradually increasing roller pump speed. The lungs were perfused for 180 min with time 0 defined as the time a flow of 100 ml kg-’ min-’ was achieved. Blood drained from the left atrium to a reservoir suspended from a force l
l
l
0 1992 the American
l
Physiological
Society
2475
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 13, 2019.
2476
RESIDENT
PMN
LEUKOCYTES
transducer -100 cm below the animal. The negative value of reservoir weight (AW) was measured as an index of fluid entry into the lung extravascular space during perfusion. This measurement was previously shown to accurately reflect extravascular fluid accumulation in this preparation (17,ZO). The lymph catheter in the superior thoracic duct was allowed to drain into a small beaker suspended from a force transducer -20 cm below the lungs to permit continuous determination of lung lymph flow (QL). This position ensured that the cannulated duct was always collapsed, thus maintaining a lymphatic outflow pressure of zero (17). Lymph collected in this fashion from our preparation was previously shown to be uncontaminated by lymph from unperfused organs above and below the diaphragm (17). During perfusion, the lungs were ventilated with warmed humidified gas at a tidal volume of 12 ml/kg body wt and a frequency of 10 breaths/min. Inspired 0, and CO, concentrations, measured with gas analyzers (Beckman OM-11 and LB-Z), were 28 and 5%, respectively. End-expiratory tracheal pressure was maintained at 3-4 mmHg. Tracheal, pulmonary arterial (Ppa), and left atria1 pressures (Pla) were measured with Statham P50 transducers referenced to the level of the left atrium. Ppa was determined at end expiration while the lung was mechanically ventilated.. Pla was kept subatmospheric. Pressures, flow, AW, QL, and inspired 0, and CO, concentrations were continuously recorded (Grass model 7D polygraph). Perfusate 0, and CO, tensions and pH were measured at regular intervals using standard electrode techniques (Radiometer BMS III-MK IV). Perfusate pH was adjusted to 7.35-7.45 within the first 30 min with 1 N NaHCO,. Perfusate glucose concentrations were monitored with Dextro-stix and kept within 90-130 mg/dl by periodic addition of 50% glucose in water. Blood, obtained from the reservoir immediately before perfusion and the left atrium at fixed intervals during perfusion, was analyzed for total leukocyte, PMN leukocyte, and platelet counts as previously described (20). In three experiments, lymph samples were also analyzed for total and PMN leukocyte counts. After 180 min of perfusion, flow was stopped and the lungs were removed from the chest for measurement of wet weight/dry weight (WW/ DW), blood-free dry lung weight (BFDLW), and extravascular lung water (EVLW) by the method of Pearce and co-workers (19). Effect of perfusion with normal blood on lung PMN leukocyte content and actiuation. To determine 1) the number of resident PMN leukocytes relative to the number of PMN leukocytes sequestered from the perfusate and 2) the effect of perfusion on the number of both resident and sequestered PMN leukocytes, we measured lung PMN leukocyte concentration in histological sections before and during perfusion of eight sheep lungs with normal homologous blood. Biopsies were obtained just ventral to the level of the hilum from random sites after the lungs were flushed of residual blood but before perfusion (time 0) and after 5, 30, 120, and 180 min of perfusion. The tissue was fixed in Karnofsky’s solution and imbedded in either paraffin or methacrylate for sectioning. The paraffin sections were stained with hematoxylin and eosin and the methacrylate sections with toluidine
IN
ISOLATED
LUNG
INJURY
blue. Lung PMN leukocyte concentration, expressed as PMN leukocytes per 100 lung cells, was determined by counting all cell nuclei in a l-cm2 grid in 10 random highpower fields on coded slides to allow determination of PMN leukocyte concentration in a blinded fashion. We normalized PMN leukocyte number to 100 lung cells to avoid error introduced by changes in alveolar dimension within and between lung sections. For comparison, lung PMN leukocyte concentration was also determined in lung biopsies from four intact anesthetized sheep. In four of the eight perfused lungs, myeloperoxidase (MPO) activity was measured spectrophotometrically using a modification of the method of Goldblum and coworkers (8). The lung was weighed, homogenized for 30 s in 5 ml 50 mM phosphate buffer (pH 7) on ice with a tissue homogenizer (Brinkman Polytron), and centrifuged at 15,000 g for 10 min at 4°C. The supernatant was decanted for MPO and hemoglobin (Hb) measurements. To solubilize MPO (4), the pellet was resuspended in 5 ml 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer, homogenized, and freezethawed three times. The Hb-free supernatant was decanted for MPO measurement, and the pellet was resuspended in HTAB. In separate experiments, we determined that nearly 95% of the extractable activity was released during the first and second extraction cycles. On the basis of these results, lung samples were processed with two extraction cycles. Because a small-molecular-weight dialyzable inhibitor of MPO activity has been found in skin (G. Rothstein, personal communication), 1 ml from 20 supernatant samples was dialyzed overnight against 0.5% HTAB in 50 mM phosphate buffer (pH 7) at 25’ C through tubing that excluded >lZ,OOO mol wt (Spectrum Medical Industries), and the MPO activities of the dialyzed and undialyzed fractions were compared. The MPO activities of the dialyzed and undialyzed fractions were not different by paired t test, indicating that a dialyzable inhibitor was not present in sheep lung (data not shown). To assessPMN leukocyte activation, we measured the MPO activity in plasma from blood obtained before exsanguination of the donor sheep (n = 3) and in perfusate supernatant at the times listed above (n = 7). MPO activity was also measured in cell-free lymph from the thoracic duct from the recipient sheep before exsanguination and in lung lymph after 30 min of perfusion (n = 7). In three of the eight perfusions, the MPO activity of the lymph cells at each time was also determined. Lung lymph and whole blood samples were centrifuged to obtain cell-free lymph and plasma. MPO activity was measured in these samples after 1:l dilution in 1% HTAB in 100 mM phosphate buffer (pH 7). The lymph cell fraction was resuspended in 1 ml 0.5% HTAB in 50 mM phosphate buffer (pH 7) and freeze-thawed three times. After repeat centrifugation, the supernatants were decanted for MPO measurement. To determine the specificity of the assay and the MPO activity per PMN leukocyte, MPO activity was measured in serial dilutions of lo7 isolated sheep PMN leukocytes and mononuclear leukocytes. Isolated PMN leukocytes from two sheep were prepared by the method of Parker and co-workers (16). Cell counts were performed using a
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 13, 2019.
RESIDENT
PMN
LEUKOCYTES
hemocytometer, and purity was determined by differential counting of stained cytocentrifuged slides. The cells were 90% pure and 98% viable by trypan blue exclusion. Sheep mononuclear leukocytes were isolated from the thoracic duct lymph of two intact anesthetized sheep by centrifugation and resuspension in Hanks’ solution and from the blood of a third sheep using lymphocyte separation media (Organon Tednika). All of the mononuclear cell suspensions were 100% pure. Serial dilutions of PMN leukocytes and mononuclear cells were then subjected to the extraction procedure described above for the measurement of MPO activity. Effect of resident PMN leukocyte depletion. Resident PMN leukocytes were depleted by two different drugs. Mechlorethamine blocks leukocyte production by alkylating DNA, whereas hydroxyurea inhibits ribonucleoside diphosphate reductase, a rate-limiting enzyme in DNA synthesis that catalyzes the conversion of ribonucleotides to deoxyribonucleotides (5). Six sheep were treated with an intravenous bolus of 0.1 mg/kg mechlorethamine HCl w 72 h before their lungs were perfused with homologous leukopenic blood from another mechlorethaminetreated sheep. Six other sheep were treated with hydroxyurea (200 mg/kg iv daily for 6 days). Two days after the last injection, their lungs were perfused with homologous leukopenic blood from another hydroxyurea-treated sheep. These two groups of leukopenic lungs were compared with 11 untreated lungs perfused with leukopenic blood from mechlorethamine-treated donor sheep and 12 untreated lungs perfused with homologous normal blood. After perfusion, a biopsy was obtained for measurement of lung PMN leukocyte concentration, and the lungs were subjected to a gravimetric analysis of lung water as described above. To determine if mechlorethamine or hydroxyurea had any direct edemagenic effects on the lung, six unperfused lungs from mechlorethamine-treated sheep and three unperfused lungs from hydroxyurea-treated sheep were removed from the chest several minutes after exsanguination for measurement of lung water. These results were compared with those of seven unperfused lungs from normal donor sheep that were reported in a previous study (20). STATISTICS
The time courses of lung PMN leukocyte concentration and MPO activity, MPO activity in plasma and lymph, and perfusate and lymph leukocytes were analyzed by one-factor analysis of variance with repeated measures among groups. The gravimetric data (converted to arctangents) and the effects of mechlorethamine and hydroxyurea on lung PMN leukocyte concentration were compared by randomized one-factor analysis of variance. The relationships between isolated PMN leukocyte number and MPO activity and lung PMN leukocyte concentration and MPO activity were determined by linear regression. All other time course data were compared with a two-factor (group, time) split-plot analysis of variance (24). When significant (P 5 0.05) variance ratios were obtained, least significant differences were calculated to allow comparison of individual means.
IN
ISOLATED
LUNG 6 5
m
2477
INJURY
0 TOTAL LEUKOCYTES 0 PMN LEUKOCYTES
I
o
f 1SE
0 60
Cl PLASMA n LUNG LYMPH A SYSTEMIC LYMPH
0.4 c 5 ,0.3 OF: g g y 20.2 0.1 O'* I
ISCH
0
30 60 90 TIME @in)
120
150
180
FIG. 1. Time course of perfusate total leukocytes, perfusate polymorphonuclear (PMN) leukocytes, lung PMN leukocyte concentration, and myeloperoxidase (MPO) activity in lung, lymph, and plasma during perfusion of isolated sheep lungs with normal homologous blood. Values preceding 30-min ischemic period (ISCH) were obtained from intact anesthetized sheep (I). Values after ischemic period but preceding time 0 were obtained from reservoir blood or lung just before perfusion.
Values presented in the text are means t SE. Differences were considered significant when P IS 0.05. RESULTS
Effect of perfusion with normal blood on lung PMN leukocyte content and actiuation. Figure 1 shows the time
course of perfusate total leukocytes, PMN leukocytes, lung PMN leukocyte concentration, and MPO activity in lung, lymph, and plasma. The perfusate total and PMN leukocyte counts in these eight lungs fell from 4,540 t 610 and 1,790 t 380/mm3, respectively, before perfusion to 1,050 t 240 and 10 t lo/mm3 by 5 min and remained low. Despite this, analysis of variance revealed that there were no significant changes in lung PMN leukocyte concentration or MPO activity, which averaged 2.63 t 0.36% and 38.4 t 7.5 units/g blood-free dry lung, respectively. The lack of a significant increase in lung PMN leukocyte concentration between 0 and 5 min of perfusion was also confirmed by paired t test. Moreover, lung PMN leukocyte concentration in these perfused lungs was not different from that measured in four intact sheep (1.69 t 0.34%). Plasma MPO activity in the donor sheep before exsanguination was 0.48 t 0.06 unit/ml. This value was not
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 13, 2019.
2478
RESIDENT
PMN
LEUKOCYTES
IN
y=O.65+0.04x r=0.71 p
B. PEARSE
AND
J. T. SYLVESTER
Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Medical Institutions at the Asthma and Allergy Center, Francis Scott Key Medical Center, Baltimore, Maryland 21224
PEARSE, DAVID B., AND J. T. SYLVESTER. Spontaneous injury in isolated sheep lungs: role of resident polymorphonuclear leukocytes. J. Appl. Physiol. 72(6): 24752481,1992.-Perfusion of isolated sheep lungs with homologous blood caused pulmonary hypertension and edema that was not altered by depletion of perfusate polymorphonuclear (PMN) leukocytes (D. B. Pearse et al., J. Appl. Physiol. 66: 1287-1296, 1989). The purpose of this study was to evaluate the role of resident PMN leukocytes in this injury. First, we quantified the content and activation of lung PMN leukocytes before and during perfusion of eight isolated sheep lungs with a constant flow (100 ml. kg-‘. min-‘) of homologous blood. From measurements of myeloperoxidase (MPO) activity, we estimated that the lungs contained 1.2 X 10” PMN leukocytes, which explained why the lung PMN leukocyte content, measured by MPO activity and histological techniques, did not increase significantly with perfusion, despite complete sequestration of 2.0 X 10’ PMN leukocytes from the perfusate. MPO activities in perfusate and lymph supernatants did not increase during perfusion, suggesting that lung PMN leukocytes were not activated. Second, we perfused lungs from 6 mechlorethamine-treated and 6 hydroxyurea-treated sheep with homologous leukopenic blood and compared them with 11 normal lungs perfused similarly. Despite marked reductions in lung PMN leukocyte concentration, there were no differences in pulmonary arterial pressure, lymph flow, or reservoir weight between groups. Extravascular lung water was greater in both groups of leukopenic lungs. These results suggest that resident PMN leukocytes did not contribute to lung injury in this model. pulmonary edema; pulmonary hypertension; lung lymph; trophil; lymphocyte; myeloperoxidase; mechlorethamine; droxyurea
neuhy-
SPECIES, including humans, the pulmonary vasculature is known to contain large numbers of polymorphonuclear (PMN) leukocytes (11). In sheep, for example, the number of PMN leukocytes in the lung was estimated to be three to four times larger than the number circulating in the blood (6, 25). These resident cells are not believed to be permanently adherent to pulmonary endothelium but rather to have a prolonged transit time through the lung (11). Although PMN leukocytes are believed to have an important role in a variety of lung injury models, the relative contribution of resident and circulating cells has not been determined. Perfusion of isolated sheep lungs with blood caused rapid sequestration of circulating leukocytes in the lung followed by pulmonary arterial hypertension, increased lymph flow, hemorrhage, and edema (18, 20). We re-
IN SEVERAL
0161-7567/92
$2.00 Copyright
cently examined the role of perfusate leukocytes in this injury by perfusing normal sheep lungs with leukopenic or normal blood from donor sheep (20). We found that perfusate leukocytes contributed to lung hemorrhage but were not necessary for the development of pulmonary hypertension or edema. The limited effect of depleting perfusate leukocytes could be explained if resident PMN leukocytes contributed to the injury. The purpose of the present study was to evaluate the role of resident PMN leukocytes in this model. METHODS
Preparation. Studies were performed in isolated sheep lungs perfused and ventilated in situ. Young sheep (1925 kg) were anesthetized with intramuscular ketamine (30 mg/kg) and atropine (0.5 mg). Anesthesia was maintained by intravenous injections of ketamine (3-6 mg/ kg) at 15 to 30-min intervals. A tracheostomy was performed and mechanical ventilation was begun with a tidal volume of 12 ml/kg and respiratory rate of 15/min. A sternotomy was performed, the superior thoracic duct was catheterized above the hilum of the left lung with silicone rubber tubing (0.03 in. ID), and the inferior thoracic duct was clamped. Heparin (10,000 U) was injected intravenously before exsanguination from a femoral arterial catheter. The left atrium and pulmonary artery were cannulated, and the ascending aorta and proximal pulmonary artery were ligated with a ligature passed through the transverse sinus and around the pulmonary arterial cannula. The pulmonary vasculature was then flushed with 1 liter of 3% dextran (mol wt 70,000) in normal saline solution at 39OC to remove residual blood. The lungs were connected to an extracorporeal circuit described previously (17) and perfused in situ at 100 ml kg-’ min-l with a mixture of blood (-1,000 ml) from donor sheep (leukopenic or nonleukopenic as specified below) and 3% dextran in Ringer lactate solution (-300 ml). The perfusate was warmed (38-39” C) with a heat exchanger (Travenol Miniprime) and continuously filtered of clots and bubbles with a filter. The time from onset of exsanguination to onset of perfusion was 20-40 min. The desired flow, measured by electromagnetic flow probe (model EP 300A, Carolina Medical Electronics), was achieved over 2-3 min by gradually increasing roller pump speed. The lungs were perfused for 180 min with time 0 defined as the time a flow of 100 ml kg-’ min-’ was achieved. Blood drained from the left atrium to a reservoir suspended from a force l
l
l
0 1992 the American
l
Physiological
Society
2475
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 13, 2019.
2476
RESIDENT
PMN
LEUKOCYTES
transducer -100 cm below the animal. The negative value of reservoir weight (AW) was measured as an index of fluid entry into the lung extravascular space during perfusion. This measurement was previously shown to accurately reflect extravascular fluid accumulation in this preparation (17,ZO). The lymph catheter in the superior thoracic duct was allowed to drain into a small beaker suspended from a force transducer -20 cm below the lungs to permit continuous determination of lung lymph flow (QL). This position ensured that the cannulated duct was always collapsed, thus maintaining a lymphatic outflow pressure of zero (17). Lymph collected in this fashion from our preparation was previously shown to be uncontaminated by lymph from unperfused organs above and below the diaphragm (17). During perfusion, the lungs were ventilated with warmed humidified gas at a tidal volume of 12 ml/kg body wt and a frequency of 10 breaths/min. Inspired 0, and CO, concentrations, measured with gas analyzers (Beckman OM-11 and LB-Z), were 28 and 5%, respectively. End-expiratory tracheal pressure was maintained at 3-4 mmHg. Tracheal, pulmonary arterial (Ppa), and left atria1 pressures (Pla) were measured with Statham P50 transducers referenced to the level of the left atrium. Ppa was determined at end expiration while the lung was mechanically ventilated.. Pla was kept subatmospheric. Pressures, flow, AW, QL, and inspired 0, and CO, concentrations were continuously recorded (Grass model 7D polygraph). Perfusate 0, and CO, tensions and pH were measured at regular intervals using standard electrode techniques (Radiometer BMS III-MK IV). Perfusate pH was adjusted to 7.35-7.45 within the first 30 min with 1 N NaHCO,. Perfusate glucose concentrations were monitored with Dextro-stix and kept within 90-130 mg/dl by periodic addition of 50% glucose in water. Blood, obtained from the reservoir immediately before perfusion and the left atrium at fixed intervals during perfusion, was analyzed for total leukocyte, PMN leukocyte, and platelet counts as previously described (20). In three experiments, lymph samples were also analyzed for total and PMN leukocyte counts. After 180 min of perfusion, flow was stopped and the lungs were removed from the chest for measurement of wet weight/dry weight (WW/ DW), blood-free dry lung weight (BFDLW), and extravascular lung water (EVLW) by the method of Pearce and co-workers (19). Effect of perfusion with normal blood on lung PMN leukocyte content and actiuation. To determine 1) the number of resident PMN leukocytes relative to the number of PMN leukocytes sequestered from the perfusate and 2) the effect of perfusion on the number of both resident and sequestered PMN leukocytes, we measured lung PMN leukocyte concentration in histological sections before and during perfusion of eight sheep lungs with normal homologous blood. Biopsies were obtained just ventral to the level of the hilum from random sites after the lungs were flushed of residual blood but before perfusion (time 0) and after 5, 30, 120, and 180 min of perfusion. The tissue was fixed in Karnofsky’s solution and imbedded in either paraffin or methacrylate for sectioning. The paraffin sections were stained with hematoxylin and eosin and the methacrylate sections with toluidine
IN
ISOLATED
LUNG
INJURY
blue. Lung PMN leukocyte concentration, expressed as PMN leukocytes per 100 lung cells, was determined by counting all cell nuclei in a l-cm2 grid in 10 random highpower fields on coded slides to allow determination of PMN leukocyte concentration in a blinded fashion. We normalized PMN leukocyte number to 100 lung cells to avoid error introduced by changes in alveolar dimension within and between lung sections. For comparison, lung PMN leukocyte concentration was also determined in lung biopsies from four intact anesthetized sheep. In four of the eight perfused lungs, myeloperoxidase (MPO) activity was measured spectrophotometrically using a modification of the method of Goldblum and coworkers (8). The lung was weighed, homogenized for 30 s in 5 ml 50 mM phosphate buffer (pH 7) on ice with a tissue homogenizer (Brinkman Polytron), and centrifuged at 15,000 g for 10 min at 4°C. The supernatant was decanted for MPO and hemoglobin (Hb) measurements. To solubilize MPO (4), the pellet was resuspended in 5 ml 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer, homogenized, and freezethawed three times. The Hb-free supernatant was decanted for MPO measurement, and the pellet was resuspended in HTAB. In separate experiments, we determined that nearly 95% of the extractable activity was released during the first and second extraction cycles. On the basis of these results, lung samples were processed with two extraction cycles. Because a small-molecular-weight dialyzable inhibitor of MPO activity has been found in skin (G. Rothstein, personal communication), 1 ml from 20 supernatant samples was dialyzed overnight against 0.5% HTAB in 50 mM phosphate buffer (pH 7) at 25’ C through tubing that excluded >lZ,OOO mol wt (Spectrum Medical Industries), and the MPO activities of the dialyzed and undialyzed fractions were compared. The MPO activities of the dialyzed and undialyzed fractions were not different by paired t test, indicating that a dialyzable inhibitor was not present in sheep lung (data not shown). To assessPMN leukocyte activation, we measured the MPO activity in plasma from blood obtained before exsanguination of the donor sheep (n = 3) and in perfusate supernatant at the times listed above (n = 7). MPO activity was also measured in cell-free lymph from the thoracic duct from the recipient sheep before exsanguination and in lung lymph after 30 min of perfusion (n = 7). In three of the eight perfusions, the MPO activity of the lymph cells at each time was also determined. Lung lymph and whole blood samples were centrifuged to obtain cell-free lymph and plasma. MPO activity was measured in these samples after 1:l dilution in 1% HTAB in 100 mM phosphate buffer (pH 7). The lymph cell fraction was resuspended in 1 ml 0.5% HTAB in 50 mM phosphate buffer (pH 7) and freeze-thawed three times. After repeat centrifugation, the supernatants were decanted for MPO measurement. To determine the specificity of the assay and the MPO activity per PMN leukocyte, MPO activity was measured in serial dilutions of lo7 isolated sheep PMN leukocytes and mononuclear leukocytes. Isolated PMN leukocytes from two sheep were prepared by the method of Parker and co-workers (16). Cell counts were performed using a
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 13, 2019.
RESIDENT
PMN
LEUKOCYTES
hemocytometer, and purity was determined by differential counting of stained cytocentrifuged slides. The cells were 90% pure and 98% viable by trypan blue exclusion. Sheep mononuclear leukocytes were isolated from the thoracic duct lymph of two intact anesthetized sheep by centrifugation and resuspension in Hanks’ solution and from the blood of a third sheep using lymphocyte separation media (Organon Tednika). All of the mononuclear cell suspensions were 100% pure. Serial dilutions of PMN leukocytes and mononuclear cells were then subjected to the extraction procedure described above for the measurement of MPO activity. Effect of resident PMN leukocyte depletion. Resident PMN leukocytes were depleted by two different drugs. Mechlorethamine blocks leukocyte production by alkylating DNA, whereas hydroxyurea inhibits ribonucleoside diphosphate reductase, a rate-limiting enzyme in DNA synthesis that catalyzes the conversion of ribonucleotides to deoxyribonucleotides (5). Six sheep were treated with an intravenous bolus of 0.1 mg/kg mechlorethamine HCl w 72 h before their lungs were perfused with homologous leukopenic blood from another mechlorethaminetreated sheep. Six other sheep were treated with hydroxyurea (200 mg/kg iv daily for 6 days). Two days after the last injection, their lungs were perfused with homologous leukopenic blood from another hydroxyurea-treated sheep. These two groups of leukopenic lungs were compared with 11 untreated lungs perfused with leukopenic blood from mechlorethamine-treated donor sheep and 12 untreated lungs perfused with homologous normal blood. After perfusion, a biopsy was obtained for measurement of lung PMN leukocyte concentration, and the lungs were subjected to a gravimetric analysis of lung water as described above. To determine if mechlorethamine or hydroxyurea had any direct edemagenic effects on the lung, six unperfused lungs from mechlorethamine-treated sheep and three unperfused lungs from hydroxyurea-treated sheep were removed from the chest several minutes after exsanguination for measurement of lung water. These results were compared with those of seven unperfused lungs from normal donor sheep that were reported in a previous study (20). STATISTICS
The time courses of lung PMN leukocyte concentration and MPO activity, MPO activity in plasma and lymph, and perfusate and lymph leukocytes were analyzed by one-factor analysis of variance with repeated measures among groups. The gravimetric data (converted to arctangents) and the effects of mechlorethamine and hydroxyurea on lung PMN leukocyte concentration were compared by randomized one-factor analysis of variance. The relationships between isolated PMN leukocyte number and MPO activity and lung PMN leukocyte concentration and MPO activity were determined by linear regression. All other time course data were compared with a two-factor (group, time) split-plot analysis of variance (24). When significant (P 5 0.05) variance ratios were obtained, least significant differences were calculated to allow comparison of individual means.
IN
ISOLATED
LUNG 6 5
m
2477
INJURY
0 TOTAL LEUKOCYTES 0 PMN LEUKOCYTES
I
o
f 1SE
0 60
Cl PLASMA n LUNG LYMPH A SYSTEMIC LYMPH
0.4 c 5 ,0.3 OF: g g y 20.2 0.1 O'* I
ISCH
0
30 60 90 TIME @in)
120
150
180
FIG. 1. Time course of perfusate total leukocytes, perfusate polymorphonuclear (PMN) leukocytes, lung PMN leukocyte concentration, and myeloperoxidase (MPO) activity in lung, lymph, and plasma during perfusion of isolated sheep lungs with normal homologous blood. Values preceding 30-min ischemic period (ISCH) were obtained from intact anesthetized sheep (I). Values after ischemic period but preceding time 0 were obtained from reservoir blood or lung just before perfusion.
Values presented in the text are means t SE. Differences were considered significant when P IS 0.05. RESULTS
Effect of perfusion with normal blood on lung PMN leukocyte content and actiuation. Figure 1 shows the time
course of perfusate total leukocytes, PMN leukocytes, lung PMN leukocyte concentration, and MPO activity in lung, lymph, and plasma. The perfusate total and PMN leukocyte counts in these eight lungs fell from 4,540 t 610 and 1,790 t 380/mm3, respectively, before perfusion to 1,050 t 240 and 10 t lo/mm3 by 5 min and remained low. Despite this, analysis of variance revealed that there were no significant changes in lung PMN leukocyte concentration or MPO activity, which averaged 2.63 t 0.36% and 38.4 t 7.5 units/g blood-free dry lung, respectively. The lack of a significant increase in lung PMN leukocyte concentration between 0 and 5 min of perfusion was also confirmed by paired t test. Moreover, lung PMN leukocyte concentration in these perfused lungs was not different from that measured in four intact sheep (1.69 t 0.34%). Plasma MPO activity in the donor sheep before exsanguination was 0.48 t 0.06 unit/ml. This value was not
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 13, 2019.
2478
RESIDENT
PMN
LEUKOCYTES
IN
y=O.65+0.04x r=0.71 p
