Journal of Applied Physiology
Vol. 82, No. 1, pp. 226-232, January 1997
CELLULAR ASPECTS OF LUNG FUNCTION

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally

Brooks M. Hybertson¹, Stuart L. Bursten², Jonathan A. Leff¹, Young M. Lee¹, Eric K. Jepson¹, Chris R. Dewitt¹, John Zagorski³, Hyun G. Cho⁴, and John E. Repine¹

¹ The Webb-Waring Institute for Biomedical Research and the Department of Medicine at the University of Colorado Health Sciences Center, Denver, Colorado, 80262; ² Cell Therapeutics, Inc., Seattle, Washington 98119; ³ National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892; and ⁴ Yeungnam University, Kyungsan 712-749, Korea

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff, Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally. J. Appl. Physiol. 82(1): 226-232, 1997.Interleukin-1 (IL-1) is increased in lung lavages from patients with the acute respiratory distress syndrome, and administering IL-1 intratracheally causes neutrophil accumulation and a neutrophil-dependent oxidative leak in lungs of rats. In the present study, we found that rats pretreated intraperitoneally with lisofylline [(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species, did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1. However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbers of lung lavage neutrophils as rats given IL-1 intratracheally. Lungs of rats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lung leak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

cytokines; cytokine-induced neutrophil chemoattractant; acute respiratory distress syndrome; inflammation; phosphatidic acid; acute lung injury

INTRODUCTION

INTERLEUKIN-1 (IL-1) is a proinflammatory cytokine that is increased in lung lavages from patients with acute edematous lung injury [acute respiratory distress syndrome (ARDS)] (11, 22, 23). Administration of IL-1 also produces acute lung injury in relevant biological models (9, 10, 12-16, 19, 24, 25). For example, intratracheal insufflation of IL-1 (50 ng, recombinant human IL-1) causes a rapid (5 h) increase in lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, lung lavage neutrophil numbers, lung leak, lung histological abnormalities, lung oxidized glutathione levels, and exhaled hydrogen peroxide levels in rats (12, 13). IL-1-induced acute lung leak in rats is reduced by administering antioxidants, liposomal-prostaglandin E₁, IL-1 receptor antagonist, or polyclonal anti-CINC antibody (10, 12-16).

Phospholipid metabolites are emerging as critical signaling messengers, which may not only reflect but also contribute to acute inflammatory lung injury (3, 20). For example, treatment with lisofylline [(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], which inhibits the conversion of lysophosphatidic acid to phosphatidic acid (PA) in vitro, prevented lung injury in mice subjected to hemorrhage and resuscitation (1). LSF treatment abrogated mRNA increases of tumor necrosis factor-, IL-1, interferon-, and IL-6 in lung cells after blood loss and resuscitation (1), and also abrogated mRNA and protein release increases of tumor necrosis factor- and IL-1 in lipopolysaccharide-stimulated blood mononuclear cells but did not affect IL-8 expression (21). Moreover, acyl ratios (oleate+linoleate/palmitate) were increased in the serum of ARDS patients, and these increases were reduced by treatment with LSF (3). Because of the similarities between acute lung injury in ARDS patients and acute lung injury in rats given IL-1 intratracheally, we studied the effect of LSF treatment on lung leak and lung neutrophil responses in rats insufflated with IL-1 intratracheally.

MATERIALS AND METHODS

Reagent procurement. Recombinant human IL-1 (endotoxin level <1 EU/mg) was provided by Dr. Richard Chizzonite of Hoffmann-LaRoche (Nutley, NJ) or purchased from R&D Systems (Minneapolis, MN), frozen in aliquots, and thawed daily before use. Methoxyflurane was obtained from Pitman-Moore (Mundelein, IL), ketamine hydrochloride from Parke-Davis (Morris Plains, NJ), xylazine from Haver (New York, NY), ¹²⁵I-labeled bovine serum albumin from ICN Radiochemicals (Irvine, CA), and heparin sodium from Elkins-Sinn (Cherry Hill, NJ). Polyclonal goat anti-CINC antibody was provided by J. Zagorski (NIDR, NIH, Bethesda, MD). CINC [listed as "IL-8 (Rat)"] and rabbit anti-CINC antiserum were obtained from Peptides International (Louisville, KY). Anti-rabbit immunoglobulin (IgG)-horseradish peroxidase conjugate was obtained from Boehringer-Mannheim (Indianapolis, IN). LSF was obtained from Cell Therapeutics (Seattle, WA). All other reagents were purchased from Sigma Chemical (St. Louis, MO).

LSF and IL-1 administration. Male Sprague-Dawley rats weighing 300-350 g (Sasco, Omaha, NE) were fed a normal diet and acclimated to Denver altitude for at least 10 days before study. Initially, rats were injected intraperitoneally with either LSF in sterile saline (100 mg/kg, 0.6 ml) or sterile saline. After 15 min, each rat was anesthetized with inhaled methoxyflurane. Subsequently, the trachea was exposed, a Teflon catheter was inserted into the trachea, and 50 ng of IL-1 (in 0.5 ml sterile saline) were rapidly insufflated along with two 3-ml puffs of air. Sham-treated rats received identical anesthesia and surgery but were given only sterile saline intratracheally. The incision was closed and sutured, then rats were allowed to awaken and were returned to their cages with free access to food and water.

Lung lavage protein and neutrophil measurement. Five hours after intratracheal insufflation of IL-1, rats were anesthetized with ketamine (90 mg/kg) and xylazine (7 mg/kg) intraperitoneally, and the trachea was cannulated. Subsequently, saline (3.0 ml × 2) was slowly injected intratracheally and then withdrawn. The volume of lung lavage recovered (~4.5 ml) was similar from each rat. Recovered lavage was then centrifuged (1,200 g, room temperature) for 5 min, and the supernatant was collected for analysis of total protein and CINC levels. Lung lavage protein levels were determined by protein reduction of alkaline Cu (II) to Cu (I) and subsequent complexation of Cu (I) with bicinchoninic acid to form a chromophore that was detected spectrophotometrically at 562 nm (protein-determination kit, Sigma Chemical). After the leukocyte pellet was resuspended in 1.0 ml of saline, leukocytes were counted in a hemacytometer, and a cytospin preparation (Shandon Southern Products, Cheshire, UK) was Wright-stained and differentially counted to determine the percentage of neutrophils.

CINC assay. CINC concentrations in lung lavage supernatants were measured by sandwich enzyme-linked immunosorbent assay by using goat anti-CINC IgG as capture antibody, rabbit anti-CINC antisera as secondary antibody, and anti-rabbit IgG-horseradish peroxidase conjugate with o-phenylenediamine substrate for detection (27).

Lung leak measurement. In separate experiments, 4.5 h after IL-1 insufflation, rats were anesthetized with ketamine and xylazine intraperitoneally and then injected with ¹²⁵I-bovine serum albumin (1 µCi, 0.5 ml) intravenously. After an additional 30 min, the trachea was cannulated, and the rats were ventilated. Immediately thereafter, laparotomy and thoracotomy were performed, and heparin was injected into the right ventricle (200 U, 0.2 ml). Blood samples were obtained (1 ml), and the lungs were perfused blood free with phosphate-buffered saline before excision. The right lungs and blood samples were counted in a gamma counter (Beckman, Fullerton, CA), and the lung leak index was determined by computing the counts per minute of ¹²⁵I in the right lung divided by counts per minute of ¹²⁵I in 1.0 ml of blood (lung leak index).

Lung morphology evaluation. In separate experiments, 5 h after IL-1 insufflation, rats were anesthetized with ketamine and xylazine intraperitoneally; then the chest was opened, lungs were removed, and small blocks of tissue (~1 mm³) were fixed by immersion in cold glutaraldehyde solution (1 h, 4°C, 2.5% glutaraldehyde). Air was removed from the tissue by capping the sample vial and applying a small vacuum with a 50-ml syringe. After fixation, samples were rinsed and stored overnight in 0.1 M potassium phosphate buffer, pH 7.4. Next, samples were postfixed with 1% osmium tetroxide in 0.1 M potassium phosphate buffer for 2 h at room temperature, dehydrated through a graded series of ethanol to propylene oxide, and embedded in Epon 812 resin. Block sections, 60-80 nm thick, were cut with a diamond knife by using a Sorval MT-7000 ultramicrotome and then stained with uranyl acetate and lead citrate. Sections were examined by using a Hitachi H-600 transmission electron microscope at 75 kV.

Lung tissue lipid and fatty acid analysis. In separate experiments, 5 h after IL-1 insufflation, rats were anesthetized with ketamine and xylazine intraperitoneally, blood (3 ml) was collected, the trachea was cannulated, and the lungs were ventilated. Subsequently, laparotomy and thoracotomy were performed, and heparin was injected into the right ventricle (200 U, 0.2 ml). Next, the lungs were perfused blood free with phosphate-buffered saline, excised, and then frozen. Lung tissue samples were homogenized and extracted, and lipids were analyzed by high-performance liquid chromatography (HPLC) as follows. Lipids were extracted from lung tissue homogenate samples as previously described (4, 7). Free fatty acids were separated from phospholipids and purified by normal-phase HPLC by using a Gilson System 45 chromatograph, a Waters µ-Porasil silica column, and a gradient mobile phase of 1-9% water in hexane-to-isopropanol (3:4, vol/vol) run at a flow rate of 1 ml/min (7, 26). Phospholipids were quantitated by ultraviolet absorption as previously described (5). Free fatty acids were quantitated after collection after HPLC separation (3-4.5 min fraction). Fatty acids were diluted with methanol, then derivatized with 9-anthroyldiazomethane (17, 28). A sample of the resultant solution (20 µl) containing derivatized anthroyl free fatty acids was then analyzed by reverse-phase HPLC. Two reverse-phase columns were used in series (Spherisorb C8, 5 µm, 4.6 mm × 25 cm, followed by Spherisorb C₁₈, 3 µm, 4.6 mm × 15 cm, both from Alltech, Deerfield, IL) to facilitate separation of both saturated and unsaturated fatty acids. The mobile phase was an acetonitrile and water gradient (70-100% acetonitrile) at 1 ml/min (2). Both ultraviolet absorption and fluorescence (excitation 305-395 nm, emission 430-470 nm) were used for detection of separated anthroyl fatty acids (5, 17). This analytical scheme allowed separation of free fatty acids, including laurate (C_12:0), myristate (C_14:0), myristoleate (C_14:1), palmitate (C_16:0), palmitoleate (C_16:1; n-9), heptadecanoate (C_17:0), stearate (C_18:0), oleate (C_18:1; n-9), petroselinate (C_18:1; n-6), linoleate (C_18:2, n-9,12), -linolenate (C_18:3; n-9,12,15), -linolenate (C_18:3, n-6,9,12), eicosatrienoate (C_18:3), and arachidonate (C_20:4, n-5, 8,11,14), with interassay coefficient of variation for the capacity factors <12%. Fatty acid standards (Avanti Polar Lipids, Alabaster, AL) were derivatized as above and used to quantitate samples, with the extraction efficiencies calculated by using heptadecanoate standards. The amount of each fatty acid standard recovered was directly proportional to the amount added in a range from ~40 ng to 10 µg. Intra-assay coefficients of variation for quantitation of analytes ranged from 12-15% for oleate, linoleate, and saturated fatty acids to 17-22% for polyunsaturated acids. The practical detection limit was ~34 ng (121 pmol). Fatty acid quantitation was verified by performing gas-chromatographic separation with mass-spectrometric detection of free fatty acids.

Statistical analyses. Data were analyzed by using a one-way analysis of variance with a Student-Newman-Keuls test of multiple comparisons. A P value <0.05 was considered statistically significant.

RESULTS

Effect of LSF on IL-1-induced lung leak. Rats pretreated with saline and then given IL-1 intratracheally had an increased (P < 0.05) lung leak index compared with saline-pretreated rats given saline intratracheally (Fig. 1A). In contrast, rats pretreated with LSF and then given IL-1 intratracheally had decreased (P < 0.05) lung leak compared with saline-pretreated rats given IL-1 intratracheally (Fig. 1A). Similarly, rats given IL-1 intratracheally had increased (P < 0.05) lung lavage fluid protein concentration compared with untreated control rats (Fig. 1B), and the IL-1-induced lavage fluid protein increase was attenuated (P < 0.05) by pretreatment with LSF (Fig. 1B).

Effect of LSF on IL-1-induced lung lavage neutrophil accumulation. The number of neutrophils recovered in lung lavages from rats pretreated with saline and then given IL-1 intratracheally was significantly greater (P < 0.05) than in saline-pretreated rats given saline intratracheally (Fig. 2). Rats pretreated with LSF and then given IL-1 intratracheally had the same (P > 0.05) lung lavage neutrophil counts as saline-pretreated rats given IL-1 intratracheally (Fig. 2).

Effect of LSF on IL-1-induced lung lavage CINC increases. Saline-pretreated rats given IL-1 intratracheally had increased (P < 0.05) concentrations of CINC in their lung lavages compared with saline-pretreated rats insufflated with saline (Fig. 3). Rats pretreated with LSF and then given IL-1 intratracheally had the same (P > 0.05) CINC protein concentration levels in their lung lavages as saline-pretreated rats given IL-1 intratracheally (Fig. 3).

Effect of LSF on IL-1-induced lung morphological abnormalities. Neutrophil infiltration and lung tissue abnormalities were evident in lung sections from rats given IL-1 intratracheally (Fig. 4B), compared with lung sections from saline-pretreated rats (Fig. 4A). In particular, lung sections from rats given IL-1 intratracheally exhibited neutrophil accumulation, thinning and disruption of the basement membrane, blebbing of cells on the alveolar epithelial surface, and swelling of endothelial cells. By comparison, rats pretreated with LSF and then given IL-1 intratracheally had comparable pulmonary neutrophil infiltration but less injury compared with saline-pretreated rats given IL-1 intratracheally (Fig. 4C).

Effect of LSF on IL-1-induced unsaturated lung tissue PA levels. Saline-pretreated rats given IL-1 intratracheally had increased (P < 0.05) lung tissue unsaturated PA contents compared with saline-pretreated rats given saline intratracheally (Fig. 5A). This finding was confirmed by measuring the unsaturated fatty acid content of the hydrolyzed PA fraction, which revealed an increase (P < 0.05) in linoleoyl and linolenoyl mass of >300% in the isolated lung PA fraction. In contrast, rats pretreated with LSF before intratracheal IL-1 insufflation had decreased unsaturated PA in lung tissue, compared with saline-pretreated rats given IL-1 intratracheally (Fig. 5A). In these circumstances, linoleoyl or linolenoyl acyl chains did not increase in the hydrolyzed lung tissue PA fraction.

DISCUSSION

We found that intraperitoneal pretreatment with LSF decreased the development of acute lung leak in rats subsequently given IL-1 intratracheally. Rats given IL-1 intratracheally had elevated lung leak indexes and lung lavage fluid protein levels, and these increases were attenuated by pretreatment with LSF. Remarkably, however, LSF pretreatment decreased neither IL-1-induced CINC levels nor lavage-recoverable neutrophil accumulation in the lung. Because neutrophil-dependent processes cause lung leak after IL-1 administration intratracheally (10, 12-16, 19), this finding suggests that LSF decreases lung leak by some mechanism other than simply decreasing the number of neutrophils recruited to the lung. One possibility is that LSF protects the lung intrinsically against oxidative stress, an assumption that is supported by other data that reveal that LSF treatment reduces leak in isolated rat lungs perfused with xanthine oxidase-generated O₂ radicals (D. M. Guidot, personal communication).

Because IL-1-induced lung leak involves oxidative mechanisms and because LSF does not scavenge superoxide anion radical in vitro, LSF may confer protection, at least in part, by altering lung susceptibility to oxidants. This latter premise is also tenable because lipid membranes are prime targets for oxidant attack in the lung. In addition, IL-1 administration increases the relative concentration of unsaturated to saturated lung lipid species, making the lung more susceptible to oxidative stress, and lungs treated with LSF did not develop these IL-1-induced alterations in unsaturated acyl chain content and presumably might be more resistant to oxidative stress. An alternate possibility is that LSF inhibits neutrophil function, but this postulate is not supported by two related experiments. First, LSF treatment did not decrease neutrophil oxidative burst in vitro (1). Second, LSF-pretreated neutrophils injured isolated rat lungs given IL-8 intratracheally (8).

Although PA species have been implicated in lipid signaling pathways and cytokine expression (1, 6, 21), our work shows that LSF prevented IL-1-induced lung leak without reducing IL-1-induced lung lavage CINC level increases or pulmonary neutrophil accumulation. Similarly, it was previously found that LSF does not inhibit IL-8 expression in blood mononuclear cells stimulated with lipopolysaccharides (21). Thus, it appears that the mechanism of protection by LSF treatment against IL-1-induced lung injury was due, at least in part, to alteration of cell membrane fatty acid composition to a less oxidant-susceptible makeup. Chemokine (CINC) signaling and neutrophil accumulation in the lungs of IL-1-treated rats, on the other hand, were not decreased by treatment with LSF.

Because acute lung injury in ARDS patients is associated with pulmonary cytokine production, neutrophil accumulation, and release of reactive oxygen metabolites, our findings are relevant to potential mechanisms and the treatment of patients with ARDS. Therapeutic strategies that stabilize lung tissue against inflammation-related injury without causing systemic suppression of the inflammatory response could provide pulmonary protection while preserving related host defense mechanisms against infection.

ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Jacqueline Smith Evans in manuscript preparation.

FOOTNOTES

Address for reprint requests: B. M. Hybertson, Webb-Waring Institute for Biomedical Research, 4200 East Ninth Ave., Box C-322, Denver, CO 80262 (E-mail: Brooks.Hybertson{at}uchsc.edu).

Received 10 May 1996; accepted in final form 19 September 1996.

REFERENCES

1.	Abraham, E., S. Bursten, R. Shenkar, J. Allbee, R. Tuder, P. Woodson, D. M. Guidot, G. Rice, J. W. Singer, and J. E. Repine. Phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice. J. Exp. Med. 181: 569-575, 1995. [Abstract/Free Full Text]
2.	Aveldano, M. I., M. VanRollins, and L. A. Horrocks. Separation and quantitation of free fatty acids and fatty acid methyl esters by reverse phase high-pressure liquid chromatography. J. Lipid Res. 24: 83-93, 1983. [Abstract]
3.	Bursten, S. L., D. A. Federighi, P. E. Parsons, W. E. Harris, E. E. Moore, F. A. Moore, J. W. Singer, and J. E. Repine. Ratio of serum linoleate, oleate, and palmitate as predictors of adult respiratory distress syndrome in patients with sepsis and trauma. Crit. Care. Med. 24: 1129-1136, 1996. [Medline]
4.	Bursten, S. L., and W. E. Harris. Rapid activation of phosphatidate phosphohydrolase in mesangial cells by lipid A. Biochemistry 30: 6195-6203, 1991. [Medline]
5.	Bursten, S. L., and W. E. Harris. Interleukin-1 stimulates phosphatidic acid-mediated phospholipase D activity in human mesangial cells. Am. J. Physiol. 266 (Cell Physiol. 35): C1093-C1104, 1994. [Abstract/Free Full Text]
6.	Bursten, S. L., R. Weeks, J. West, T. Le, T. Wilson, D. Porubek, J. A. Bianco, J. W. Singer, and G. C. Rice. Potential role for phosphatidic acid in mediating the inflammatory responses to TNF alpha and IL-1 beta. Circ. Shock 44: 14-29, 1994. [Medline]
7.	Folch, J., M. Lees, and G. H. Sloane-Stanley. Simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509, 1957. [Free Full Text]
8.	Guidot, D. M., G. C. Rice, J. W. Singer, S. Bursten, J. A. Bianco, E. Abraham, and J. E. Repine. Inhibition of lyso-phosphatidic acid acyl transferase (LPAAT) activity by CT1501R suppresses neutrophil adherence and chemotaxis in vitro and decreases interleukin-8 (IL-8)-induced injury in isolated rat lungs perfused with human neutrophils (Abstract). Clin. Res. 42: 145, 1994.
9.	Guidot, D. M., E. E. Stevens, M. J. Repine, A. E. Lucca-Broco, and J. E. Repine. Intratracheal but not intravascular interleukin-1 causes acute edematous injury in isolated neutrophil-perfused rat lungs through an oxygen radical mediated mechanism. J. Lab. Clin. Med. 12: 605-609, 1994.
10.	Hybertson, B. M., J. A. Leff, C. J. Beehler, P. C. Barry, and J. E. Repine. Effect of vitamin E deficiency and supercritical fluid aerosolized vitamin E supplementation of interleukin1-induced oxidative lung injury in rats. Free Radical Biol. Med. 18: 537-542, 1995. [Medline]
11.	Jacobs, R. F., D. R. Tabor, A. W. Burks, and G. D. Campbell. Elevated interleukin-1 release by human alveolar macrophages during the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 140: 1686-1692, 1989. [Medline]
12.	Koh, Y., B. M. Hybertson, E. K. Jepson, O. J. Cho, and J. E. Repine. Cytokine-induced neutrophil chemoattractant is necessary for interleukin-1-induced lung leak in rats. J. Appl. Physiol. 79: 472-478, 1995. [Abstract/Free Full Text]
13.	Leff, J. A., J. W. Baer, M. E. Bodman, J. M. Kirkman, P. F. Shanley, L. M. Patton, C. J. Beehler, J. M. McCord, and J. E. Repine. Interleukin-1-induced lung neutrophil accumulation and oxygen metabolite-mediated lung leak in rats. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L2-L8, 1994. [Abstract/Free Full Text]
14.	Leff, J. A., J. W. Baer, J. M. Kirkman, M. E. Bodman, P. F. Shanley, O. J. Cho, M. J. Ostro, and J. E. Repine. Liposome-entrapped PGE1 posttreatment decreases IL-1-induced neutrophil accumulation and lung leak in rats. J. Appl. Physiol. 76: 151-157, 1994. [Abstract/Free Full Text]
15.	Leff, J. A., M. E. Bodman, O. J. Cho, S. Rohrbach, O. K. Reiss, J. L. Vannice, and J. E. Repine. Post-insult treatment with interleukin-1 receptor antagonist decreases oxidative lung injury in rats given intratracheal interleukin-1. Am. J. Respir. Crit. Care Med. 150: 109-112, 1994. [Abstract]
16.	Leff, J. A., C. P. Wilke, B. M. Hybertson, P. F. Shanley, C. J. Beehler, and J. E. Repine. Postinsult treatment with N-acetyl-L-cysteine decreases IL-1-induced neutrophil influx and lung leak in rats. Am. J. Physiol. 265: L501-L506, 1993. [Abstract/Free Full Text]
17.	Nakaya, T., T. Tomomoto, and M. Imoto. The syntheses and the reactions of 9-anthroyldiazomethane and -naphthyldiazomethane. Bull. Chem. Soc. Jpn. 40: 691-692, 1967.
19.	Repine, J. E. Interleukin-1-mediated acute lung injury and tolerance to oxidative injury. Environ. Health Persp. 102: 75-78, 1994.
20.	Rice, G. C., P. A. Brown, R. J. Nelson, J. A. Bianco, J. W. Singer, and S. Bursten. Protection from endotoxic shock in mice by pharmacologic inhibition of phosphatidic acid. Proc. Natl. Acad. Sci. USA 91: 3857-3861, 1994. [Abstract/Free Full Text]
21.	Rice, G. C., J. Rosen, R. Weeks, J. Michnick, S. Bursten, J. A. Bianco, and J. W. Singer. CT-1501R selectively inhibits induced inflammatory monokines in human whole blood ex vivo. Shock 1: 254-266, 1994. [Medline]
22.	Siler, T. M., J. E. Swierkosz, T. M. Hyers, A. A. Fowler, and R. O. Webster. Immunoreactive interleukin-1 in bronchoalveolar lavage fluid of high-risk patients and patients with the adult respiratory distress syndrome. Exp. Lung Res. 15: 881-894, 1989. [Medline]
23.	Suter, P. M., S. Suter, E. Girardin, P. Roux-Lombard, G. E. Grau, and J. Dayer. High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am. Rev. Respir. Dis. 145: 1016-1022, 1992. [Medline]
24.	Tang, G., J. E. White, P. D. Lumb, D. A. Lawrence, and M. F. Tsan. Role of endogenous cytokines in endotoxin- and interleukin-1-induced pulmonary inflammatory response and oxygen tolerance. Am. J. Respir. Cell Mol. Biol. 12: 339-344, 1995. [Abstract]
25.	Ulich, T. R., L. R. Watson, S. M. Yin, K. Z. Guo, P. Wang, H. Thang, and J. del Castillo. The intratracheal administration of endotoxin and cytokines. I. Characterization of LPS-induced IL-1 and TNF mRNA expression and the LPS-, IL-1-, and TNF-induced inflammatory infiltrate. Am. J. Pathol. 138: 1485-1496, 1991. [Abstract]
26.	Van Kessel, W. S., W. M. Hax, R. A. Demel, and J. De Gier. High-performance liquid chromatographic separation and direct ultraviolet detection of phospholipids. Biochim. Biophys. Acta 486: 524-530, 1977. [Medline]
27.	Wittwer, A. J., L. S. Carr, J. Zagorski, G. J. Dolecki, B. A. Crippes, and J. E. De Larco. High-level expression of cytokine-induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: purification and production of blocking antibodies. J. Cell. Physiol. 156: 421-427, 1993. [Medline]
28.	Yoshida, T., A. Uetake, H. Yamaguchi, N. Nimura, and T. Kinoshita. New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives. Anal. Biochem. 173: 70-74, 1988. [Medline]