Lung (1992) 170:267-279
New York Inc. 1992
Intercellular Adhesion Molecule-1 Contributes to Pulmonary Oxygen Toxicity in Mice: Role of Leukocytes Revised Craig D. Wegner,J Walter W. Wolyniec,l April M. LaPlante,l Kristin Marschman, 1 Klaus Lubbe, 2 Nancy Haynes, 3 Robert Rothlein, 3 and L. Gordon Letts ~ Departments of IPharmacology, 2Molecular Biology, and 3Immunology, Boehringer Ingelheim Pharmaceuticals, Inc., 90 East Ridge, P.O. Box 368, Ridgefield, CT 06877, USA
Abstract. In immature or injured lungs, impaired alveolar gas exchange forces the use of elevated levels of inhaled oxygen to maintain life. But, at high concentrations oxygen induces lung injury, edema, and bronchopulmonary dysplasia, probably by stimulating the generation of reactive oxygen radicals and subsequent neutrophil infiltration. In addition to regulating neutrophil diapedesis, intercellular adhesion molecule- 1 (ICAM- 1) expression is marked on inflamed alveolar epithelium, suggesting a role for ICAM-1 in oxygeninduced, neutrophil-mediated parenchymal damage. To test this, we evaluated the rat anti-mouse ICAM-1 monoclonal antibody YN1/1.7 in 2 protocols of oxygen-induced toxicity in adult, male Balb-c mice: ->95% 02 for 84 hr and ->95% O2 for 60 hr followed by 48 hr at 21% (ambient) O2. YN1/1.7 treatment partially attenuated the neutrophil infiltration, lung damage (lavage lactate dehydrogenase [LDH] activity) and dysfunction (reductions in respiratory system compliance [Crs] and diffusion capacity of the lungs for carbon monoxide [DLco]in the 84 hr exposure protocol. In the milder 60 hr exposure protocol, YN1/1.7 completely blocked the oxygen-induced lung dysfunction (reductions in Crs and DLco). These results confirm the contribution of leukocytes in the pathogenesis of pulmonary oxygen toxicity and indicate that antagonism of ICAM-1 may provide a therapeutic approach to reducing hyperoxic lung injury and dysfunction. Key words: Cell adhesion--Neutrophils--Acute lung injury--Alveolitis--Bronchopulmonary dysplasia--Pulmonary function. Introduction
Bypass surgery, trauma, head injuries, emboli, septic shock, pneumonia, smoke inhalation, and premature birth frequently initiate acute lung injury, edema, and Offprint requests to: C. D. Wegner
268
C.D. Wegner et al.
inflammation that result in impaired alveolar gas exchange. In such patients, elevated levels of inspired oxygen are required to achieve acceptable blood oxygen saturation. However, prolonged exposure to high concentrations of oxygen can precipitate acute edematous lung injury (alveolar septal thickening), followed by fibrosis, pulmonary hypertension, and/or bronchopulmonary dysplasia [6, 25]. In normal, healthy and adult humans, normobaric inhalation of 95-100% oxygen for as little as 14-16 hr causes tracheobronchial irritation, impaired mucociliary clearance, cough, substernal pain, decreased vital capacity, alveolar-capillary "leak" (interstitial edema), and release of chemoattractants and growth factors from alveolar macrophages [7, 13, 35]. In animals, 72-84 hr of continuous exposure to pure oxygen results in the death of most individuals [2]. In the presence of acute lung injury, that is, in cases where elevated levels of oxygen may be required, oxygen at concentrations as low as 50% have been shown to potentiate the injury [46, 50]. In newborn mice, continuous exposure to 80% oxygen acutely increases the persistence and morbidity of pneumonia, and chronically induces bronchopulmonary dysplasia [11, 30]. Thus, agents that attenuate or delay the toxic effects of inhaled oxygen would be of obvious benefit. Results of many investigations indicate that the initiation of pulmonary oxygen toxicity occurs through a direct increase in the intracellular production of partially reduced oxygen species (superoxide, O) ; hydrogen peroxide, H202; and hydroxyl radical, OH) that overwhelm intracellular antioxidant defense mechanisms [9, 28]. In the first 16-48 hr of pure oxygen inhalation, while not resulting in notable morphologic injury, this heightened generation of intracellular oxygen radicals causes ultrastructural and lactate dehydrogenase (LDH) release-documented endothelial injury leading to intrapulmonary platelet and then neutrophil retention (margination) [4, 10, 33]; enhanced release of neutrophil chemoattractants, fibronectin, and fibroblast growth factors by alveolar macrophages [13, 21]; and type I alveolar epithelial injury [40], resulting in proliferation of type II pneumocytes and altered surfactant properties [20]. Once these effects are initiated, the lung injury is amplified and morphologic changes become pronounced, exponentially between 48 and 72 hr of pure oxygen breathing asociated with a marked infiltration of neutrophils [9, 10]. This association has led several investigators to suggest [18, 26, 29, 44], and some to demonstrate [31, 37], that the inhibition of neutrophil infiltration and mediated tissue injury would significantly delay or reduce pulmonary oxygen toxicity. Yet the role of neutrophils remains controversial. Das and colleagues found that the nonsteroidal anti-inflammatory drug ibuprofen inhibited the neutrophil influx but not the lung injury induced by pure oxygen breathing in rabbits [12]. Neutrophil depletion with cytotoxic agents in most [5, 27, 32, 42], but not all [37], studies has failed to protect against hyperoxic-induced microvascular and/ or alveolar epithelial injury. However, in addition to making animals neutropenic, these cytotoxic agents have many other effects including reduction of food intake which, in discord, has been shown to reduce lung antioxidants and accelerate oxygen-induced lung injury [19, 41]. In this report, we have determined the contribution of intercellular adhesion
ICAM-1 in Pulmonary Oxygen Toxicity
269
molecule-1 (ICAM-1), and, in the process, the role of leukocytes, to the lung inflammation and reduced lung function induced by pure oxygen breathing in adult mice. ICAM-1 has been shown previously to mediate partially neutrophil adherence to and migration through monolayers of cytokine-inflamed endothelium in vitro [39], be upregulated on endothelium and airway epithelium both in vitro and in vivo 4-24 hr after an inflammatory stimulus [16, 39, 47], and partially mediate granulocyte influx into lung airspaces in vivo [3, 47]. In addition, neutrophil adherence mediated by macrophage antigen 1 (Mac-l) (CD1 lb/ CD 18), a neutrophil receptor for ICAM-1 [39, 43], augments neutrophil respiratory burst [36].
Methods
Animals and Exposure Protocols: Animal care and use were performed in accordance with the principles stated in the "Guide for the Care and Use of Laboratory Animals," Department of Health, Education and Welfare (National Institutes of Health), publication 85-23, 1985. Protocols described below were approved by the Animal Care and Use Committee, Boehringer Ingelheim Pharmaceuticals, Inc. Male Balb-c mice (Charles River, Raleigh, NC) weighing 23-27 (n = 8-14 per group) were housed on wire mesh above bedding in clear Plexiglass chambers (34.3 x 44.5 × 40.6 cm; maximum 20 mice/chamber) and exposed to pure oxygen at atmospheric pressure. Food (laboratory mouse chow) and water were supplied ad libitum and the chambers cleaned once every 24 hr. Chamber oxygen, humidity, and temperature were monitored continually using a digital oxygen monitor (Gas Tech. Inc., model 6873; Lab Safety Supply, Janesville, WI) and wall-mount hygrometer/ thermometer (Oakton, model 3313-90; Cole Palmer Instrument Co., Chicago, IL), respectively. Chamber air was circulated by a 3" fan and carbon dioxide accumulation was eliminated by mixing soda lime (cat. S-200, 4-8 mesh, indicator grade; Fisher Scientific, Springfield, N J) in the bedding. The rat IgG2b monoclonal antibody YN1/1.7 directed against murine lymphocyte activation antigen-2 (MALA-2) [45], the homolog of human ICAM-1 [23], was prepared as described previously [22] and evaluated at 3 or 10mg/kg, intraperitoneally (i.p.), every 12 hr (twice daily) in two protocols of hyperoxia-induced lung injury: pure oxygen for 84 hr, and pure oxygen for 60 hr followed by 48 hr of ambient air. In both protocols, chamber oxygen ranged from 95 to 98%, humidity from 60 to 70%, and temperature remained constant at 26.5°C during the pure oxygen exposures. YNI/1.7treated animals were compared to saline-treated animals in both protocols as well as to 10 mg/kg, i.p. rat IgG (cat. 1-4131, from serum; Sigma Chemical Co., St. Louis, MO) in the second protocol. To avoid undue suffering by any animal, from 66 hr until the conclusion of each protocol, the mice were observed continually (around the clock) and any animal found in severe respiratory distress was sacrificed immediately by cervical dislocation. One saline-treated animal in the first protocol needed to be sacrificed at 77 hr of exposure. In both protocols, scientists performing the dosing, monitoring, and measurements were blinded to the treatment the mice were receiving.
Lung Function At the end of each protocol, the mice were anesthetized with 60-90 mg/kg i.p. sodium pentobarbital (Nembutal, cat. NDC 0074-3778-05; Abbott Labs., North Chicago, IL) and a tracheal caunula (Teflon catheter cut to 2 cm with Luer Plug, 18 gauge, cat. 2N112; Baxter Healthcare Corp., Deerfield, IL) inserted. Three tidal volume (1.2 ml) breaths were given by connecting a syringe to the tracheal cannula to eliminate areas of atelectasis and provide a constant volume history for the lung function measurements described below.
270
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Respiratory system impedance (Zrs) was measured by discrete frequency (4-40 Hz in 11 equal logarithmic steps) sinusoidal forced oscillations (amplitude of 1 ml/sec -+ 20% at each frequency) superimposed on tidal breathing using a technique previously described in detail [ 15, 24]. Advantages of this technique include that all transducers used are frequency response corrected, the impedance of the tracheal cannula is accurately eliminated, and the measurements are independent of the ventilatory'pattern (tidal volume and respiratory rate) of the animal. Respiratory system compliance (Crs) was computed from Zrs as described previously [24]. Diffusion capacity of the lungs (Dkco) was measured by the carbon monoxide single breath method [17]. In brief, 1.0 ml of gas containing 0.479% carbon monoxide, 0.477% neon, 20.22% oxygen, and 78.824% nitrogen (cat. NDC 11939-3935; Matheson Gas Products, Inc., East Rutherford, NJ) was injected into the lungs through the tracheal cannula. Ten seconds later, a 0.5 ml dead space/airway sample was withdrawn followed immediately by the withdrawing of the 0.5 ml alveolar sample. The alveolar gas sample was analyzed using a gas chromatograph (model 01111, Carle AGC series 100; Carle Chromatography, Loveland, CO). The values for DL0o obtained for the unexposed mice (see Results section) are similar to those previously reported for CBA/Ca Lac Cbi mice using a rebreathing method [14].
Lung Inflammation After the measurement of lung function, whole lung lavage was performed to assess neutrophil infiltration, lung microvascular fluid leak, cell damage, and neutrophil activation. Three 1.0 ml aliquots of bicarbonate-buffered (0.5 mM) normal saline (pH 7.4) were slowly infused and then gently aspirated through the tracheal cannula and combined (return volume ranged from 2.1 to 2.5 ml). The mice were then sacrificed by cervical dislocation. Total leukocytes per ml of lavage were determined with a Coulter counter (model ZM; Coulter Electronics, Hialeah, FL). Differential cell counts (200 cells counted) were performed on Wright-Giemsa-stained cytocentrifuge (Cytospin, model 2; Shandon, Oakland, CA) preparations. The lung lavage fluid then was centrifuged at 600 x g for 10 to remove cells and particulates, after which the supernatant was collected and aliquoted: 0.5 ml kept at 4°C for LDH activity measurement (within 24 hr) and the remaining portion frozen at -70°C for total protein and myeloperoxidase (MPO) activity measurements. Total protein in the lung lavage supernatant was measured by a modified Lowry method using a commercially available kit (BCA Protein Assay Reagent 23225; Pierce, Rockford, IL). Lactate dehydrogenase (LDH) activity was also measured using a commercially available kit (cat. 44564; Roche Diagnostic Systems, Inc., Nutley, N J). Myeloperoxidase activity was measured by a modification of a previously described microtiter plate colormetric assay [8]. In brief, a 100/xl sample of lavage supernatant was aded to 100/xl of the reaction mixture consisting of 100 mM potassium phosphate buffer (pH 6.0), 1 mM o-dianisidine dihydrochloride (DMB, indicator), 0.1% Triton, 1 mM hydrogen peroxide, and 2 mM 3-amino-l,2,4-triazole (AMT, inhibitor of eosinophil peroxidase). After 30 min at 24°C, the optical density at 450 nm was read on a microplate reader (model 340 ATTC; SLT Labinstruments, Hillsborough, NC) and compared to a standard curve constructed for human leukocyte MPO (cat. M-2146; Sigma Chemical Co., St. Louis, MO). Each of these assays were performed in triplicate.
LPS-Induced Neutrophil Influx A YNI/I.7 dose-response curve for inhibition of an inhaled lipopolysaccharide (LPS)-induced neutrophil infiltration was constructed to determine the YN1/1.7 dose(s) to be used in the oxygen toxicity experiments. LPS (S. Typhosa 0901, cat. 3124-25-6; Difco Labs., Detroit, MI), dissolved in phosphate-buffered (5 mM, pH 7.4) saline (0.5%), was ultrasonically nebulized (Ultra-Neb 99; DeVilbiss Co., Somerset, PA) and delivered at 1.5 L/min for 5 min to a 3.5 L chamber containing the mice. Four hours later, the numbers of neutrophils in the whole lung lavage were determined
1CAM-I in Pulmonary Oxygen Toxicity
271
using the techniques described above. No increase in lung lavage total protein content was induced at any of the LPS concentrations used.
Immunohistochernistry The effect of hyperoxia on alveolar ICAM-1 expression was determined by immunohistochemical staining with YN1/1.7 compared to rat IgG in lung sections taken at various times of pure oxygen exposure. Lungs were removed, inflated with O.C.T. (cat. 4583, Tissue Tek, Miles; Baxter, Bedford, MA) and then frozen in liquid nitrogen. After being cryosectioned, 5-10/xm sections were fixed in acetone for 10 min and either stained immediately or stored at 20°C.Staining was done with the Biotin-Strept Avidin system. Primary antibody (YN1/I.7 or rlgG) was incubated with tissue as undiluted culture supernatants (RPMI 1640medium with 10% fetal bovine serum) for 1 hr at room temperature. Blocking for nonspecific protein binding was accomplished by applying normal goat serum (BioGenex Labs., San Ramon, CA). Sections were immunostained by a 3-stage detection method using biotinilated rat antimouse IgG (BioGenex Labs.) linked with perioxidase-conjugated streptavidin (BioGenex Labs.) and visualized with 3-amino-9-ethylcarbazole(AEC; Lipshaw, Detroit, MI) as the substrate. The sections were counterstained with Mayer's hematoxylin (Lipshaw).
Statistics Analysis of variance was evaluated using Tukey's multiple comparison studentized range, honest significant difference (HSD), and T-test least significant difference (LDS) for the LPS and oxygen toxicity experiments, respectively. A p value of
New York Inc. 1992
Intercellular Adhesion Molecule-1 Contributes to Pulmonary Oxygen Toxicity in Mice: Role of Leukocytes Revised Craig D. Wegner,J Walter W. Wolyniec,l April M. LaPlante,l Kristin Marschman, 1 Klaus Lubbe, 2 Nancy Haynes, 3 Robert Rothlein, 3 and L. Gordon Letts ~ Departments of IPharmacology, 2Molecular Biology, and 3Immunology, Boehringer Ingelheim Pharmaceuticals, Inc., 90 East Ridge, P.O. Box 368, Ridgefield, CT 06877, USA
Abstract. In immature or injured lungs, impaired alveolar gas exchange forces the use of elevated levels of inhaled oxygen to maintain life. But, at high concentrations oxygen induces lung injury, edema, and bronchopulmonary dysplasia, probably by stimulating the generation of reactive oxygen radicals and subsequent neutrophil infiltration. In addition to regulating neutrophil diapedesis, intercellular adhesion molecule- 1 (ICAM- 1) expression is marked on inflamed alveolar epithelium, suggesting a role for ICAM-1 in oxygeninduced, neutrophil-mediated parenchymal damage. To test this, we evaluated the rat anti-mouse ICAM-1 monoclonal antibody YN1/1.7 in 2 protocols of oxygen-induced toxicity in adult, male Balb-c mice: ->95% 02 for 84 hr and ->95% O2 for 60 hr followed by 48 hr at 21% (ambient) O2. YN1/1.7 treatment partially attenuated the neutrophil infiltration, lung damage (lavage lactate dehydrogenase [LDH] activity) and dysfunction (reductions in respiratory system compliance [Crs] and diffusion capacity of the lungs for carbon monoxide [DLco]in the 84 hr exposure protocol. In the milder 60 hr exposure protocol, YN1/1.7 completely blocked the oxygen-induced lung dysfunction (reductions in Crs and DLco). These results confirm the contribution of leukocytes in the pathogenesis of pulmonary oxygen toxicity and indicate that antagonism of ICAM-1 may provide a therapeutic approach to reducing hyperoxic lung injury and dysfunction. Key words: Cell adhesion--Neutrophils--Acute lung injury--Alveolitis--Bronchopulmonary dysplasia--Pulmonary function. Introduction
Bypass surgery, trauma, head injuries, emboli, septic shock, pneumonia, smoke inhalation, and premature birth frequently initiate acute lung injury, edema, and Offprint requests to: C. D. Wegner
268
C.D. Wegner et al.
inflammation that result in impaired alveolar gas exchange. In such patients, elevated levels of inspired oxygen are required to achieve acceptable blood oxygen saturation. However, prolonged exposure to high concentrations of oxygen can precipitate acute edematous lung injury (alveolar septal thickening), followed by fibrosis, pulmonary hypertension, and/or bronchopulmonary dysplasia [6, 25]. In normal, healthy and adult humans, normobaric inhalation of 95-100% oxygen for as little as 14-16 hr causes tracheobronchial irritation, impaired mucociliary clearance, cough, substernal pain, decreased vital capacity, alveolar-capillary "leak" (interstitial edema), and release of chemoattractants and growth factors from alveolar macrophages [7, 13, 35]. In animals, 72-84 hr of continuous exposure to pure oxygen results in the death of most individuals [2]. In the presence of acute lung injury, that is, in cases where elevated levels of oxygen may be required, oxygen at concentrations as low as 50% have been shown to potentiate the injury [46, 50]. In newborn mice, continuous exposure to 80% oxygen acutely increases the persistence and morbidity of pneumonia, and chronically induces bronchopulmonary dysplasia [11, 30]. Thus, agents that attenuate or delay the toxic effects of inhaled oxygen would be of obvious benefit. Results of many investigations indicate that the initiation of pulmonary oxygen toxicity occurs through a direct increase in the intracellular production of partially reduced oxygen species (superoxide, O) ; hydrogen peroxide, H202; and hydroxyl radical, OH) that overwhelm intracellular antioxidant defense mechanisms [9, 28]. In the first 16-48 hr of pure oxygen inhalation, while not resulting in notable morphologic injury, this heightened generation of intracellular oxygen radicals causes ultrastructural and lactate dehydrogenase (LDH) release-documented endothelial injury leading to intrapulmonary platelet and then neutrophil retention (margination) [4, 10, 33]; enhanced release of neutrophil chemoattractants, fibronectin, and fibroblast growth factors by alveolar macrophages [13, 21]; and type I alveolar epithelial injury [40], resulting in proliferation of type II pneumocytes and altered surfactant properties [20]. Once these effects are initiated, the lung injury is amplified and morphologic changes become pronounced, exponentially between 48 and 72 hr of pure oxygen breathing asociated with a marked infiltration of neutrophils [9, 10]. This association has led several investigators to suggest [18, 26, 29, 44], and some to demonstrate [31, 37], that the inhibition of neutrophil infiltration and mediated tissue injury would significantly delay or reduce pulmonary oxygen toxicity. Yet the role of neutrophils remains controversial. Das and colleagues found that the nonsteroidal anti-inflammatory drug ibuprofen inhibited the neutrophil influx but not the lung injury induced by pure oxygen breathing in rabbits [12]. Neutrophil depletion with cytotoxic agents in most [5, 27, 32, 42], but not all [37], studies has failed to protect against hyperoxic-induced microvascular and/ or alveolar epithelial injury. However, in addition to making animals neutropenic, these cytotoxic agents have many other effects including reduction of food intake which, in discord, has been shown to reduce lung antioxidants and accelerate oxygen-induced lung injury [19, 41]. In this report, we have determined the contribution of intercellular adhesion
ICAM-1 in Pulmonary Oxygen Toxicity
269
molecule-1 (ICAM-1), and, in the process, the role of leukocytes, to the lung inflammation and reduced lung function induced by pure oxygen breathing in adult mice. ICAM-1 has been shown previously to mediate partially neutrophil adherence to and migration through monolayers of cytokine-inflamed endothelium in vitro [39], be upregulated on endothelium and airway epithelium both in vitro and in vivo 4-24 hr after an inflammatory stimulus [16, 39, 47], and partially mediate granulocyte influx into lung airspaces in vivo [3, 47]. In addition, neutrophil adherence mediated by macrophage antigen 1 (Mac-l) (CD1 lb/ CD 18), a neutrophil receptor for ICAM-1 [39, 43], augments neutrophil respiratory burst [36].
Methods
Animals and Exposure Protocols: Animal care and use were performed in accordance with the principles stated in the "Guide for the Care and Use of Laboratory Animals," Department of Health, Education and Welfare (National Institutes of Health), publication 85-23, 1985. Protocols described below were approved by the Animal Care and Use Committee, Boehringer Ingelheim Pharmaceuticals, Inc. Male Balb-c mice (Charles River, Raleigh, NC) weighing 23-27 (n = 8-14 per group) were housed on wire mesh above bedding in clear Plexiglass chambers (34.3 x 44.5 × 40.6 cm; maximum 20 mice/chamber) and exposed to pure oxygen at atmospheric pressure. Food (laboratory mouse chow) and water were supplied ad libitum and the chambers cleaned once every 24 hr. Chamber oxygen, humidity, and temperature were monitored continually using a digital oxygen monitor (Gas Tech. Inc., model 6873; Lab Safety Supply, Janesville, WI) and wall-mount hygrometer/ thermometer (Oakton, model 3313-90; Cole Palmer Instrument Co., Chicago, IL), respectively. Chamber air was circulated by a 3" fan and carbon dioxide accumulation was eliminated by mixing soda lime (cat. S-200, 4-8 mesh, indicator grade; Fisher Scientific, Springfield, N J) in the bedding. The rat IgG2b monoclonal antibody YN1/1.7 directed against murine lymphocyte activation antigen-2 (MALA-2) [45], the homolog of human ICAM-1 [23], was prepared as described previously [22] and evaluated at 3 or 10mg/kg, intraperitoneally (i.p.), every 12 hr (twice daily) in two protocols of hyperoxia-induced lung injury: pure oxygen for 84 hr, and pure oxygen for 60 hr followed by 48 hr of ambient air. In both protocols, chamber oxygen ranged from 95 to 98%, humidity from 60 to 70%, and temperature remained constant at 26.5°C during the pure oxygen exposures. YNI/1.7treated animals were compared to saline-treated animals in both protocols as well as to 10 mg/kg, i.p. rat IgG (cat. 1-4131, from serum; Sigma Chemical Co., St. Louis, MO) in the second protocol. To avoid undue suffering by any animal, from 66 hr until the conclusion of each protocol, the mice were observed continually (around the clock) and any animal found in severe respiratory distress was sacrificed immediately by cervical dislocation. One saline-treated animal in the first protocol needed to be sacrificed at 77 hr of exposure. In both protocols, scientists performing the dosing, monitoring, and measurements were blinded to the treatment the mice were receiving.
Lung Function At the end of each protocol, the mice were anesthetized with 60-90 mg/kg i.p. sodium pentobarbital (Nembutal, cat. NDC 0074-3778-05; Abbott Labs., North Chicago, IL) and a tracheal caunula (Teflon catheter cut to 2 cm with Luer Plug, 18 gauge, cat. 2N112; Baxter Healthcare Corp., Deerfield, IL) inserted. Three tidal volume (1.2 ml) breaths were given by connecting a syringe to the tracheal cannula to eliminate areas of atelectasis and provide a constant volume history for the lung function measurements described below.
270
C . D . Wegner et al.
Respiratory system impedance (Zrs) was measured by discrete frequency (4-40 Hz in 11 equal logarithmic steps) sinusoidal forced oscillations (amplitude of 1 ml/sec -+ 20% at each frequency) superimposed on tidal breathing using a technique previously described in detail [ 15, 24]. Advantages of this technique include that all transducers used are frequency response corrected, the impedance of the tracheal cannula is accurately eliminated, and the measurements are independent of the ventilatory'pattern (tidal volume and respiratory rate) of the animal. Respiratory system compliance (Crs) was computed from Zrs as described previously [24]. Diffusion capacity of the lungs (Dkco) was measured by the carbon monoxide single breath method [17]. In brief, 1.0 ml of gas containing 0.479% carbon monoxide, 0.477% neon, 20.22% oxygen, and 78.824% nitrogen (cat. NDC 11939-3935; Matheson Gas Products, Inc., East Rutherford, NJ) was injected into the lungs through the tracheal cannula. Ten seconds later, a 0.5 ml dead space/airway sample was withdrawn followed immediately by the withdrawing of the 0.5 ml alveolar sample. The alveolar gas sample was analyzed using a gas chromatograph (model 01111, Carle AGC series 100; Carle Chromatography, Loveland, CO). The values for DL0o obtained for the unexposed mice (see Results section) are similar to those previously reported for CBA/Ca Lac Cbi mice using a rebreathing method [14].
Lung Inflammation After the measurement of lung function, whole lung lavage was performed to assess neutrophil infiltration, lung microvascular fluid leak, cell damage, and neutrophil activation. Three 1.0 ml aliquots of bicarbonate-buffered (0.5 mM) normal saline (pH 7.4) were slowly infused and then gently aspirated through the tracheal cannula and combined (return volume ranged from 2.1 to 2.5 ml). The mice were then sacrificed by cervical dislocation. Total leukocytes per ml of lavage were determined with a Coulter counter (model ZM; Coulter Electronics, Hialeah, FL). Differential cell counts (200 cells counted) were performed on Wright-Giemsa-stained cytocentrifuge (Cytospin, model 2; Shandon, Oakland, CA) preparations. The lung lavage fluid then was centrifuged at 600 x g for 10 to remove cells and particulates, after which the supernatant was collected and aliquoted: 0.5 ml kept at 4°C for LDH activity measurement (within 24 hr) and the remaining portion frozen at -70°C for total protein and myeloperoxidase (MPO) activity measurements. Total protein in the lung lavage supernatant was measured by a modified Lowry method using a commercially available kit (BCA Protein Assay Reagent 23225; Pierce, Rockford, IL). Lactate dehydrogenase (LDH) activity was also measured using a commercially available kit (cat. 44564; Roche Diagnostic Systems, Inc., Nutley, N J). Myeloperoxidase activity was measured by a modification of a previously described microtiter plate colormetric assay [8]. In brief, a 100/xl sample of lavage supernatant was aded to 100/xl of the reaction mixture consisting of 100 mM potassium phosphate buffer (pH 6.0), 1 mM o-dianisidine dihydrochloride (DMB, indicator), 0.1% Triton, 1 mM hydrogen peroxide, and 2 mM 3-amino-l,2,4-triazole (AMT, inhibitor of eosinophil peroxidase). After 30 min at 24°C, the optical density at 450 nm was read on a microplate reader (model 340 ATTC; SLT Labinstruments, Hillsborough, NC) and compared to a standard curve constructed for human leukocyte MPO (cat. M-2146; Sigma Chemical Co., St. Louis, MO). Each of these assays were performed in triplicate.
LPS-Induced Neutrophil Influx A YNI/I.7 dose-response curve for inhibition of an inhaled lipopolysaccharide (LPS)-induced neutrophil infiltration was constructed to determine the YN1/1.7 dose(s) to be used in the oxygen toxicity experiments. LPS (S. Typhosa 0901, cat. 3124-25-6; Difco Labs., Detroit, MI), dissolved in phosphate-buffered (5 mM, pH 7.4) saline (0.5%), was ultrasonically nebulized (Ultra-Neb 99; DeVilbiss Co., Somerset, PA) and delivered at 1.5 L/min for 5 min to a 3.5 L chamber containing the mice. Four hours later, the numbers of neutrophils in the whole lung lavage were determined
1CAM-I in Pulmonary Oxygen Toxicity
271
using the techniques described above. No increase in lung lavage total protein content was induced at any of the LPS concentrations used.
Immunohistochernistry The effect of hyperoxia on alveolar ICAM-1 expression was determined by immunohistochemical staining with YN1/1.7 compared to rat IgG in lung sections taken at various times of pure oxygen exposure. Lungs were removed, inflated with O.C.T. (cat. 4583, Tissue Tek, Miles; Baxter, Bedford, MA) and then frozen in liquid nitrogen. After being cryosectioned, 5-10/xm sections were fixed in acetone for 10 min and either stained immediately or stored at 20°C.Staining was done with the Biotin-Strept Avidin system. Primary antibody (YN1/I.7 or rlgG) was incubated with tissue as undiluted culture supernatants (RPMI 1640medium with 10% fetal bovine serum) for 1 hr at room temperature. Blocking for nonspecific protein binding was accomplished by applying normal goat serum (BioGenex Labs., San Ramon, CA). Sections were immunostained by a 3-stage detection method using biotinilated rat antimouse IgG (BioGenex Labs.) linked with perioxidase-conjugated streptavidin (BioGenex Labs.) and visualized with 3-amino-9-ethylcarbazole(AEC; Lipshaw, Detroit, MI) as the substrate. The sections were counterstained with Mayer's hematoxylin (Lipshaw).
Statistics Analysis of variance was evaluated using Tukey's multiple comparison studentized range, honest significant difference (HSD), and T-test least significant difference (LDS) for the LPS and oxygen toxicity experiments, respectively. A p value of
