Downregulation of blood and bone marrow neutrophils decreases expression of acute lung injury in sheep PATRICK J. McKENNA, DAVID L. ROSOLIA, YOKO ISHIHARA, KURT H. ALBERTINE, NORMAN C. STAUB, AND MARLYS H. GEE Departments of Physiology and Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and The Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco, California 91143 McKenna, Patrick J., David L. Rosolia, Yoko Ishihara, Kurt H. Albertine, Norman C. Staub, and Marlys H. Gee. Downregulation of blood and bone marrow neutrophils decreases expression of acute lung injury in sheep. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1492H1498, 1992.--e have shown that infusion of zymosan-activated plasma (ZAP) in sheep causes acute lung injury and downregulates peripheral blood neutrophils in that elicited superoxide release is reduced for at least 24 h after the infusion. The present study was designed to test the following hypotheses: 1) peripheral blood neutrophils are representative of neutrophils marginated in the pulmonary circulation, 2) blood neutrophils are downregulated because neutrophils developing in bone marrow are similarly affected, and 3) downregulated neutrophils have a reduced capacity to produce tissue injury. In a series of experiments in 21 sheep, we showed that elicited superoxide release was similar in peripheral blood neutrophils and in marginated neutrophils washed out of the pulmonary vascular bed. Measurements of superoxide release from blood and bone marrow neutrophils collected 2-24 h after ZAP infusion revealed progressive downregulation with time and greater downregulation of superoxide release in bone marrow neutrophils compared with peripheral blood neutrophils. Finally, after downregulating peripheral blood neutrophils, subsequent infusion of ZAP in conscious sheep produced sequestration of neutrophils in the pulmonary circulation but failed to produce a sustained increase in lung lymph protein clearance. The results suggest that neutrophil downregulation, as measured in vitro, is expressed in vivo as reduced ability of neutrophils to produce tissue injury when challenged by an activating agent. complement; superoxide anion; lymph STUDIES, we have shown that intravenous infusion of zymosan-activated plasma (ZAP) in sheep results in a decrease in circulating leukocytes and neutrophils in systemic arterial blood of at least 50% and that the decrease in cell count represents sequestration of leukocytes in the pulmonary circulation (5,6,9). Furthermore, prolonged or repeated sequestration of ZAPactivated neutrophils produces a sustained, but moderate, increase in the flow of protein-rich lymph from the lung (5, 6, 9). The increase in lung microvascular permeability is inhibited by administration of superoxide dismutase, suggesting that pulmonary endothelial injury occurs as the result of release of reactive oxygen species from sequestered, activated neutrophils (6). Infusion of complement anaphylatoxins also changes the functional characteristics of neutrophils isolated from peripheral blood long after the period of sequestration (1). Sheep peripheral blood neutrophils isolated 24 h after infusion of ZAP are downregulated; i.e., stimulation of neutrophils with phorbol 12-myristate 13-acetate (PMA) resulted in a 40-60% reduction in the release of superoxide anion. In contrast, infusion of
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endotoxin resulted in priming of circulating neutrophils 24 h after endotoxin, as evidenced by increased PMAstimulated neutrophil superoxide anion release from sheep blood and bone marrow neutrophils (4). We have also tested two other stimuli that produce neutrophildependent lung injury, air embolization and PMA infusion, and found no change in elicited superoxide anion release from neutrophils isolated 24 h after either infusion. These data indicate that changes in neutrophil function measured after ZAP or endotoxin are stimulus specific (1). The present experiments were designed to test three hypotheses concerning ZAP-induced neutrophil downregulation in vivo. First, equating function in marginated and circulating pools of neutrophils is a critical assumption underlying this work and much of that derived from ex vivo studies of neutrophil function. Under normal conditions, we assume that the circulating population of cells is representative of the much larger population of marginated ceils, particularly those marginated in the pulmonary circulation, which number about three times the circulating population of cells (13). To test this hypothesis, we washed the pulmonary circulation in normal, control sheep nearly free of neutrophils to compare the functional capability of marginated neutrophils with those circulating in the same animal. Our results indicate that the behavior of marginated and circulating cells is similar. Second, because neutrophils circulate for an estimated 6-8 h it is unlikely that cells directly exposed to the complement anaphylatoxins (the activated sequestered neutrophils) are contributing to the downregulation measured 24 h later. One mechanism that could explain the sustained effect on peripheral blood cells is that neutrophilic cells developing in bone marrow are also downregulated, thus sustaining the effect in blood as bone marrow cells mature and enter the circulation. We tested this hypothesis by measuring PMA-stimulated superoxide anion release from blood and bone marrow segmented neutrophils isolated 2-24 h after the ZAP infusion. Our results show that neutrophilic cells in bone marrow are downregulated to a greater extent than peripheral blood cells, suggesting that recruitment of downregulated neutrophils from bone marrow sustains the response in peripheral blood. Third, we questioned whether neutrophil downregulation affects subsequent lung injury responses in vivo. We reasoned that because ZAP infusion apparently produces an acute lung injury that is dependent on release of reduced oxygen metabolites from activated neutrophils, the injury should be mitigated in the presence of
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 17, 2019.
NEUTROPHIL
FUNCTION
downregulated neutrophils. To test this hypothesis, we infused autologous ZAP into awake sheep equipped with lung lymph fistulas on three consecutive days. Our results show a similar degree of leukocyte and neutrophil sequestration in the lung with each infusion but a progressive decrease in the vascular injury from the first to the third day. METHODS Twenty-one adult male sheep weighing 30-40 kg were used. Most sheep were instrumented to provide chronic vascular access via catheters in a carotid artery and external jugular vein. Some of the sheep also had chronic lung lymph fistulas. Procedures to instrument the sheep with vascular and lymph catheters were done in an operating suite approved for recovery surgery in large animals using techniques previously described (1, 2, 4). For all invasive procedures, anesthesia was induced by intravenous injection of 0.5-1.0 g of sodium thiamylal and maintained by pentobarbital drip. We allowed a &day recovery before initiating an experiment. Each animal was studied in one protocol only. A flow-directed thermodilution catheter was placed in a pulmonary artery on the day of the experiment. This catheter and a catheter in the descending thoracic aorta were connected to disposable minipressure transducers (Cobe, CDX III; North Chicago, IL) for continuous recording of blood pressures (model 7, Grass Instruments, Quincy, MA). Pressures were referenced to the approximate level of the left atrium. The thermodilution catheter was connected to a cardiac output computer (Sorenson, model 3500, Salt Lake City, UT) that also provided a measurement of blood temperature. Cardiac output measurements consisted of the average obtained with three consecutive 7-ml injections of room temperature saline. Lung lymph flow rate was measured by 30-min collections of lymph in tared conical tubes treated with heparin to prevent clotting. Three-milliliter samples of systemic arterial blood were taken periodically to measure leukocyte count (hemacytometer), differentials (Wright stain), and blood gases (ABL 30 Acid/Base Blood Gas Analyzer, Radiometer, Copenhagen, Denmark). Forty-milliliter samples of blood in 0.01 M EDTA were also obtained periodically to isolate neutrophils. Experimental
Protocols
Lung wash studies. To collect marginated neutrophils from the pulmonary circulation, we anesthetized six normal sheep with pentobarbital. A loo-ml blood sample was obtained for isolation of circulating neutrophils and for plasma used in all subsequent gradients. Through a midsternal incision, the heart and lungs were removed en bloc. Saline infused through the pulmonary artery was used to wash blood from the lungs in the first wash, and saline with 0.1 M EDTA was used in a second wash to remove marginated neutrophils (12). Both washes were collected to measure cell count and differential and to isolate the neutrophils. Blood and bone marrow neutrophil studies. To determine the effects of intravascular complement activation on neutrophils in peripheral blood and bone marrow, two experiments were done. In the first experiment, four awake sheep equipped with vascular catheters were given a l-h intravenous infusion of ZAP at 3.8 ml/min. Twenty-four hours later a blood sample was obtained to isolate neutrophils, and the sheep were subsequently anesthetized with sodium thiamylal. The region over a dorsal iliac crest was shaved and cleaned with disinfectant, and a l-cm skin incision was made. Ten to thirty milliliters of bone marrow were aspirated under sterile conditions into 12-ml syringes treated with 500 U heparin and 180,000 IU streptokinase (Kabikinase,
AND
ACUTE
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INJURY
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SmithKline Beecham). To minimize clotting, 1 ml of this mixture was slowly infused into the marrow cavity at the beginning of each syringe. Midway through the collection, the syringe was removed and mixed by inversion. Bone marrow aspirates were immediately pooled in modified N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (HEPES) buffer containing (in mM) 140 NaCl, 10 HEPES, 10 KCl, 0.1 CaCl,, 0.2 MgCl,, 11.0 NaHCO:
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endotoxin resulted in priming of circulating neutrophils 24 h after endotoxin, as evidenced by increased PMAstimulated neutrophil superoxide anion release from sheep blood and bone marrow neutrophils (4). We have also tested two other stimuli that produce neutrophildependent lung injury, air embolization and PMA infusion, and found no change in elicited superoxide anion release from neutrophils isolated 24 h after either infusion. These data indicate that changes in neutrophil function measured after ZAP or endotoxin are stimulus specific (1). The present experiments were designed to test three hypotheses concerning ZAP-induced neutrophil downregulation in vivo. First, equating function in marginated and circulating pools of neutrophils is a critical assumption underlying this work and much of that derived from ex vivo studies of neutrophil function. Under normal conditions, we assume that the circulating population of cells is representative of the much larger population of marginated ceils, particularly those marginated in the pulmonary circulation, which number about three times the circulating population of cells (13). To test this hypothesis, we washed the pulmonary circulation in normal, control sheep nearly free of neutrophils to compare the functional capability of marginated neutrophils with those circulating in the same animal. Our results indicate that the behavior of marginated and circulating cells is similar. Second, because neutrophils circulate for an estimated 6-8 h it is unlikely that cells directly exposed to the complement anaphylatoxins (the activated sequestered neutrophils) are contributing to the downregulation measured 24 h later. One mechanism that could explain the sustained effect on peripheral blood cells is that neutrophilic cells developing in bone marrow are also downregulated, thus sustaining the effect in blood as bone marrow cells mature and enter the circulation. We tested this hypothesis by measuring PMA-stimulated superoxide anion release from blood and bone marrow segmented neutrophils isolated 2-24 h after the ZAP infusion. Our results show that neutrophilic cells in bone marrow are downregulated to a greater extent than peripheral blood cells, suggesting that recruitment of downregulated neutrophils from bone marrow sustains the response in peripheral blood. Third, we questioned whether neutrophil downregulation affects subsequent lung injury responses in vivo. We reasoned that because ZAP infusion apparently produces an acute lung injury that is dependent on release of reduced oxygen metabolites from activated neutrophils, the injury should be mitigated in the presence of
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 17, 2019.
NEUTROPHIL
FUNCTION
downregulated neutrophils. To test this hypothesis, we infused autologous ZAP into awake sheep equipped with lung lymph fistulas on three consecutive days. Our results show a similar degree of leukocyte and neutrophil sequestration in the lung with each infusion but a progressive decrease in the vascular injury from the first to the third day. METHODS Twenty-one adult male sheep weighing 30-40 kg were used. Most sheep were instrumented to provide chronic vascular access via catheters in a carotid artery and external jugular vein. Some of the sheep also had chronic lung lymph fistulas. Procedures to instrument the sheep with vascular and lymph catheters were done in an operating suite approved for recovery surgery in large animals using techniques previously described (1, 2, 4). For all invasive procedures, anesthesia was induced by intravenous injection of 0.5-1.0 g of sodium thiamylal and maintained by pentobarbital drip. We allowed a &day recovery before initiating an experiment. Each animal was studied in one protocol only. A flow-directed thermodilution catheter was placed in a pulmonary artery on the day of the experiment. This catheter and a catheter in the descending thoracic aorta were connected to disposable minipressure transducers (Cobe, CDX III; North Chicago, IL) for continuous recording of blood pressures (model 7, Grass Instruments, Quincy, MA). Pressures were referenced to the approximate level of the left atrium. The thermodilution catheter was connected to a cardiac output computer (Sorenson, model 3500, Salt Lake City, UT) that also provided a measurement of blood temperature. Cardiac output measurements consisted of the average obtained with three consecutive 7-ml injections of room temperature saline. Lung lymph flow rate was measured by 30-min collections of lymph in tared conical tubes treated with heparin to prevent clotting. Three-milliliter samples of systemic arterial blood were taken periodically to measure leukocyte count (hemacytometer), differentials (Wright stain), and blood gases (ABL 30 Acid/Base Blood Gas Analyzer, Radiometer, Copenhagen, Denmark). Forty-milliliter samples of blood in 0.01 M EDTA were also obtained periodically to isolate neutrophils. Experimental
Protocols
Lung wash studies. To collect marginated neutrophils from the pulmonary circulation, we anesthetized six normal sheep with pentobarbital. A loo-ml blood sample was obtained for isolation of circulating neutrophils and for plasma used in all subsequent gradients. Through a midsternal incision, the heart and lungs were removed en bloc. Saline infused through the pulmonary artery was used to wash blood from the lungs in the first wash, and saline with 0.1 M EDTA was used in a second wash to remove marginated neutrophils (12). Both washes were collected to measure cell count and differential and to isolate the neutrophils. Blood and bone marrow neutrophil studies. To determine the effects of intravascular complement activation on neutrophils in peripheral blood and bone marrow, two experiments were done. In the first experiment, four awake sheep equipped with vascular catheters were given a l-h intravenous infusion of ZAP at 3.8 ml/min. Twenty-four hours later a blood sample was obtained to isolate neutrophils, and the sheep were subsequently anesthetized with sodium thiamylal. The region over a dorsal iliac crest was shaved and cleaned with disinfectant, and a l-cm skin incision was made. Ten to thirty milliliters of bone marrow were aspirated under sterile conditions into 12-ml syringes treated with 500 U heparin and 180,000 IU streptokinase (Kabikinase,
AND
ACUTE
LUNG
INJURY
H1493
SmithKline Beecham). To minimize clotting, 1 ml of this mixture was slowly infused into the marrow cavity at the beginning of each syringe. Midway through the collection, the syringe was removed and mixed by inversion. Bone marrow aspirates were immediately pooled in modified N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (HEPES) buffer containing (in mM) 140 NaCl, 10 HEPES, 10 KCl, 0.1 CaCl,, 0.2 MgCl,, 11.0 NaHCO:
