Pentoxifylline Reduces Injury to Isolated Lungs Perfused with Human Neutrophils1- 3
RUTH J. MCDONALD·
Introduction
N eutrophils and neutrophil-derived ox-
idants have been implicated in the development of acute lung injury such as that seen in the adult respiratory distress syndrome (ARDS), in bronchopulmonary dysplasia, and in animal models of lung injury, including the isolated perfused lung (1-3). Shasbyand coworkers (4) first described acute edematous injury in isolated rabbit lungs perfused with human neutrophils and the neutrophil stimulant phorbol myristate acetate (PMA). In this model, injury to the isolated rat lung is dependent upon neutrophil-derived oxidant production as oxygen metabolite scavengers prevent injury, and injury is also prevented when neutrophils that do not produce oxidants are substituted (3, 5). Pentoxifylline is a methylxanthine derivative that has recently been shown to reduce neutrophil-dependent injury to the lung in several animal models (6). Continuous infusion of pentoxifylline reduced neutrophil counts in lung lavage fluid and lung wet-to-dry-weight ratios in a guinea pig model of sepsis after the administration of Escherichia coli (7) or tumor necrosis factor (8). In addition, pentoxifylline was also effective at reducing endotoxin-induced pulmonary vascular protein accumulation in the dog, and this effect was associated with a reduction in neutrophil accumulation in the lung (9). The ability of pentoxifylline to reduce injury to the lung in these models may be due to one or more of its known effects on neutrophils or on other circulating cells.Although pentoxifylline is similar in its chemical structure to theophyline, pentoxifylline in vitro can decrease neutrophil adherence to pulmonary endothelial cells, decrease neutrophil aggregation, and reduce oxidant production by stimulated neutrophils (10-13). Lastly, pentoxifylline can increase erythrocyte deformability and reduce blood viscosity, increasing the filterability of blood and its cellular components (14). The ability of pentoxifylline to reduce lung injury in animal models may be due to these or other as yet undefined effects.
SUMMARY Neutrophlls and neutrophil-derived oxidants have been Implicated In tha davelopmant of acute lung Injury such as that seen In the adult re8plratory distress syndrome (ARDS), In bronchopulmonary dysplasia (BPD), and In animal model8 of lung Injury, Including the Isolated perfused lung. Both neutrophll-derlved oxidant production and retention of neutrophlls In the lung are reqUIl13d for Injury In thl8 model. Pentoxlfylllne can reduca lung Injury from sepsis In the guinea pig and endotoxin-Induced neutrophil sequeatratlon and lung Injury In the dog. It Is also known to Increase neutrophil deformabillty, which may affect retention In the pulmonary mlcrovaaculatul8. we evaluated neutrophil oxidant production, retention In Isolated lungs, and neutrophil-mediated acute lung Injury after phorbol myrl8tate acetate (PMA) In the presence of pentoxlfyiline. Pantoxlfyliine (2 mM) 81gnlflc8ntly reduced 8uperoxlde anion and hydrogen peroxide production In "nro from PMAstimulated neutrophlls whan pentoxlfyiline was directly added to the Incubation mlxturaa, but not when neutrophlls ware prelncubated with the agent. Pantoxlfylllne did not reduce retention of neutrophllsln Isolated lungs aa determined by Infusion of '''In-labeled neutrophlls and gamma countIng. Pantoxlfylllne pravented Incre_ In total lung walght, lung-to-body-walght retlo, and perfusate thromboxane concentrations when It was present In perfusate buffer, whether or not neutrophlls ware prelncubated In pentoxlfylllne prior to Infusion Into the lung. Pentoxlfyiline did not reduce InJuryto lungs perfused with glucose and glucose oxidase. Weconclude that pentoxlfylllne reduces neutrophil oxidant production and neutrophll-dependent lung Injury. AM REV RESPIR DIS 1991; 144:1347-1350
We proposed the isolated perfused lung as a good model in which to investigate the potential for pentoxifylline to directly reduce neutrophil-mediated lung injury. Our hypothesis was that pentoxifylline would reduce neutrophil-mediated injury to isolated lungs perfused with neutrophils and the neutrophil stimulant PMA, and allow evaluation of the possible mechanisms of this effect. Methods Isolation of Human Neutrophils Human neutrophils wereisoJated from citrate anticoagulated blood on plasma-Percoll gradients after sedimentation of erythrocytes with Dextran, washed twice in calcium- and magnesium-free Hank's balanced salt solution (HBSS) (GIBCO, Grand Island, NY) and resuspended in complete HBSS, as previously described (15). Neutrophils were isolated and kept at room temperature until use in isolated lung or in in vitro experiments.
Preparation of Isolated Lungs Isolated, ventilated, perfused rat lungs were prepared as previously described. Male Sprague-Dawley rats (Bainton & Kingman, Freemont, CA) weighing 350 to 400 g were anesthetized intraperitoneally with pentobarbital and ventilated through a tracheostomy with 95070 air and 5% CO 2 , The pulmonary
artery and left atrium were catheterized, and the lungs were perfused with modified KrebsHenseleit buffer and 4% Ficoll. The pulmonary circulation was rinsed free of blood with 50 ml of perfusate buffer, and then the system was closed for recirculation of perfusate. A stable baseline was assured by observation and determination of pH in the perfusate for 20 min prior to the initiation of the experimental protocol. Neutrophils (4rnlat 1 x 107/ml) were infused directly into the pulmonary artery over 5 min, and PMA (0.025 ug/ml final concentration; Sigma Chemical, St. Louis, MO) was added to the perfusate reservoir 5 min after the completion of the neutrophil infusion. Pulmonary artery pressure and weight gain were continuously monitored. (Received in originalform December 14, 1990 and in revisedform July 1, 1991) 1 From the Department of Pediatrics and the California Primate ResearchCenter, University of California, Davis, California. 2 Presented in part at the Annual Meeting of the American Thoracic Society, Boston, May 1990. 3 Correspondence and requests for reprints should be addressed to Ruth J. McDonald, M.D., Department of Pediatrics, University of California Davis Medical Center, 2516 Stockton Blvd., Sacramento, CA 95817. • Recipient of Clinical Investigator Award K08 HL-01980 from the National Heart, Lung, and Blood Institute.
1347
1348
Lungs were perfused for a total of 120 min, or until gross edema fluid was observed in the trachea and dripping from the pleural surfaces (15). In some experiments, pentoxifylline (PTXF, 2 mM; a gift from HoechstRoussel Pharmaceuticals, Inc., Somerville, NJ) was added to the perfusate buffer at the beginning of the experimental protocol. In an additional series of experiments, isolated lungs were prepared and perfused with chemically generated oxidants by the addition of 0.5% ~-D-glucose (G, Sigma) and 5 U glucose oxidase (GO, Sigma) as previously described (16).
Retention of Neutrophils in Isolated Lungs Freshly isolated neutrophils were suspended in plasma and incubated with ["'In]tropolone for 5 min at room temperature, washed twice in a mixture of plasma and Ca2+-Mg'+free HBSS, and resuspended in complete HBSS at I x 1Q7 Iml. Labeled neutrophils were incubated in PTXF (2 mM) or buffer at room temperature for 20 min and then infused into isolated lungs, and subsequently PMA was added to the perfusate reservoir. At the end of the experimental protocol lungs were counted in a gamma counter, and the percentage of total counts infused remaining in the lung were used to determine the percentage of neutrophils retained in the lung (15). Determination of Neutrophil Oxidant Production Neutrophils (I x IQ6 Iml) wereincubated with PMA (0.025 ug/ml) for determination of superoxide anion and hydrogen peroxide production. Superoxide anion production was measured as the superoxide-dismutase-inhibitable reduction of cytochrome c at 37° C (17).Hydrogen peroxide production was measured by the ferrous ammonium sulfate and potassium thiocyanate method (18, 19). Neutrophils were incubated for 30 min at 37° C in HBSS, 10/0 normal pooled serum, I mM sodium azide, and PMA. Hydrogen peroxide concentrations were then determined on aliquots from triplicate incubations of mixtures with or without PTXF (2 mM) and performed in parallel on neutrophils preincubated in PTXF (2 mM), washed once, and resuspended in HBSS. Determination of Neutrophil Elastase Release Elastase release in vitro by PMA-stimulated neutrophils was determined by the measurement of human neutrophil elastase activity using the neutrophil-elastase-specific synthetic chromogenic substrate methoxysuccinylala-ala-pro-val-s-nitroanaline (Sigma). Neutrophils, at 2 x 106/ml in HBSS, wereincubated in triplicate with PMA (0.025 ug/ml) in the presence or absence of 2 mM PTX for 30 min at 37° C. Parallel incubations were performed on neutrophils preincubated in 2 mM PTXF. The mixtures were then centrifuged (5 min at 1,000 g), and the supernatants were frozen at - 70 c C until assay. Elastase release was determined as percent of total elastase present in identical aliquots of neutrophils suspended in HBSS, immediately placed on ice, sonicated (three lO-sbursts)
RUTH J. MCDONALD
(Vibra Cell Sonicator; Sonics & Materials, Inc., Danbury, CT), and frozen (20). In separate determinations, the presence of 2 mM PTXF did not alter measurable elastase activity of sonicated neutrophils or purified human neutrophil elastase using this substrate.
Determination of Lung Perfusate Thromboxane Concentration Thromboxane (TXB,) concentrations were determined on perfusate samples using radioimmunoassay, as previously described. Perfusate samples were taken at the end of the experimental protocol, placed on ice, centrifuged (1,000 g for 5 min), and frozen at - 70° C until assay. Ficoll was added to assay standards to assure that matrix composition was equivalent to that of perfusate samples (3). Determination of Lung Lavage Leukotriene C. Concentration Leukotriene C. (LTC.) was determined in lung lavage fluid by radioimmunoassay (Leukotriene C. PH] RIA Kit; Du Pont NEN Research Products, Boston, MA). At the end of the experimental protocol, lungs were weighed, and 6 ml of cold saline were instilled gently into the lung through the tracheal cannula. Lavage fluid was recovered by slow aspiration, with an averagereturn volume of 5 ml. Fluid was iced and centrifuged (1,000 g for 5 min), and the supernatants were frozen at -70° C until assay. Samples were assayed in duplicate with strict adherence to the manufacturer's recommended protocol. Statistics All data were analyzed using ANOVA on a Macintosh SE computer and Statview SE+Graphics statistical program (Abacus Concepts, Inc., Berkeley, CAl with a level of significance of p < 0.05. Results
Isolated lungs perfused with neutrophils and PMA had increases in total lung weight and lung-to-body-weight ratios when compared with lungs perfused with buffer alone, neutrophils alone, PMA alone, or PTXF alone. As expected, to-
tal lung weight was increased to 5.0 ± 0.8 g in lungs perfused with neutrophils and PMA (table 1).However, when PTXF was added to perfusates and neutrophils were added that had also been preincubated in PTXF, increases in lung weight and lung-to-body-weight ratios were reduced, with total lung weights of only 1.7 ± 0.1 g, no different from those of control lungs. Increases in pulmonary artery pressure did not occur in lungs perfused with buffer or PTXF alone. Small increases in pulmonary artery pressure occurred in lungs perfused with neutrophils alone and in lungs perfused with PMA alone or with added neutrophils (table 1). The presence of PTXF in the perfusate or pretreatment of neutrophils with PTXF did not prevent similar increases in pulmonary artery pressure prior to weight gain in all lungs perfused with PMA. In order to investigate the site of action of PTXF in this system, we next compared the ability of PTXF to reduce lung injury when the agent was added directly to the buffer and untreated neutrophils were added with the effects of preincubation of the neutrophils alone with PTXF and perfusion with buffer that did not contain PTXF. When pentoxifylline was present in the buffer only, the reduction in weight gain after the addition of neutrophils and PMA was similar to that seen in lungs where neutrophils had also been preincubated in PTXF and PTXF was added to the buffer. However, when neutrophils were preincubated in pentoxifylline, washed, and then added to isolated lungs, total lung weight was not reduced. Lung weights and lung-to-body-weight ratios were reduced in both groups where pentoxifylline was present in the buffer when compared with lungs perfused with neutrophils and PMA. Pulmonary artery pressure increases prior to weight gain were similar in these groups.
TABLE 1 PENTOXIFYLUNE REDUCES INJURY IN ISOLATED RAT LUNGS PERFUSED WITH NEUTROPHILS AND PMA*
Substances Added to Perfusate Buffer Neutrophils + PMA Neutrophils preincubated in PTXF + PTXF + PMA Neutrophils + PTXF + PMA Neutrophils preincubated in PTXF + PMA Neutrophils PMA PTXF
Pulmonary Artery Pressure Increases
Total Lung Weight (g)
Lung/Body Weight Ratio (x 10-Oj
1.6 ± 0.2 (9}t 5.0 ± O.S (10)
4.5 ± 0.2t 15.3 ± 2.7
0.3 ± 0.2t 3.3 ± 0.5
1.7 ± 0.1 (S}t 2.1 ± 0.3 (7)t
5.1 ± 0.3t 6.1 ± o.st
3.3 ± 0.6:1: 2.5 ± 0.4:1:
3.2 1.S 1.6 1.7
9.7 5.4 4.9 4.S
3.4 ± 1.4 ± 2.S ± O.S ±
± ± ± ±
0.9 0.1 0.1 0.1
(6):1: (S}t (S}t (6)t
± ± ± ±
2.9:1: O.4t 0.3t 0.2t
• Values are mean ± SE. Number of determinations is shown in parentheses. Value significantly different (p < 0.05) from value obtained after addition of neutrophils and PMA. Value not signifk:antly different (p > 0.05) from value obtained after addition of neutrophils and PMA.
t
*
(mm Hg)
1.2:1: 0.4t 0.5:1: O.4t
1349
PENTOXIFYLLINE REDUCES INJURY IN ISOLATED WNGS
TABLE 2 PENTOXIFYLLINE (2 mM) REDUCES SUPEROXIDE ANION AND HYDROGEN PEROXIDE PRODUCTION BY PMA-STIMULATED NEUTROPHILS IN VITRO" H,O, Produced (PM)
Cytochrome c Reduced
Additions to Incubation Mixtures
(nM)
Neutrophils + PMA Neutrophils + PMA + PTXF Neutrophils preincubated in PTXF + PMA Neutrophils preincubated in PTXF + PTXF + PMA
68.7 42.8 65.9 35.8
± 6.1 (8) ± 5.6 (7)t ± 7.7 (6)*
± 4.2 (6)t
40.6 16.2 43.7 21.6
± 7.3 (6)
± 3.7 (6)t ± 7.8 (5)* ± 5.4 (5)t
" Values are mean ± SEM. Number of determinations is shown in parentheses. Value significantly different (p < 0.05) from value obtained with neutrophils and PMA. Value not significantly different (p > 0.05) from value obtained with neutrophils and PMA.
t
*
TABLE 3 PERFUSATE THROMBOXANE AND LUNG LAVAGE LTC. LEVELS IN ISOLATED RAT LUNGS PERFUSED WITH NEUTROPHILS, PMA, AND PTXF"
Substances Added to Perfusate Buffer Neutrophils Neutrophils Neutrophils Neutrophils Neutrophils PMA PTXF
Thromboxane
Leukotriene C.
Ipg/m/)
(ng/m/)
135 468 117 133 154 168 120 85
+ PMA preincubated in PTXF + PTXF + PMA + PTXF + PMA preincubated in PTXF + PMA
± 27 (7)t
± 100 (7) ± 41 (7)t ± 35 (6)t ± 21 (6)t
± 57 (6)t ± 31 (8)t ± 28 (6)t
3.3 4.3 1.9 3.3 2.3 2.1 2.1 2.0
± 0.6* ± 0.9
± 0.3* ± 0.7*
± 0.7* ± 0.3* ± 0.4*
± 0.6*
" Values are mean ± SE. Number of determinations is shown in parentheses. t Value significantly different (p < 0.05) from value obtained after addition of neutrophils and PMA. Value not significantly different (p > 0.05) from value obtained after addition of neutrophils and PMA.
*
Isolated lungs perfused with ll1In_ labeled neutrophils and PMA retained 87 ± 3070 of total counts infused when counted at the end of the experimental period. When pentoxifyIIine was present in the perfusate and neutrophils had been preincubated in it, 90 ± 8% of total counts infused remained (n = 5). PentoxifyIIine did not reduce retention of neutrophils in isolated lungs. PTXF reduced oxidant production by PMA stimulated neutrophils in vitro when present in the reaction mixtures (table 2). In the presence of 2 mM PTXF, superoxide anion production was reduced by 39%, and hydrogen peroxide concentrations were reduced by 61%. However, when neutrophils were preincubated in PTXF, washed, and then assayed for PMA-stimulated oxidant production there was no decrease in either superoxide anion or hydrogen peroxide cencentrations produced. Preincubation of neutrophils in PTXF did not further reduce
oxidant production by neutrophils when PTXF was already present in the incubation mixtures. In a separate study, we determined that the presence of pentoxifyIIine did not interfere with the detection of reagent grade hydrogen peroxide. A similar pattern of results was obtained when higher concentrations of PMA were used (data not shown). Elastase release after stimulation with PMA was not affected by the presence of PTXF or by preincubation of neutrophils in PTXF. Elastase release by neutrophils stimulated with PMA was 16 ± 1% oftotal; 12 ± 2% was released in the presence of PTXF, and 12 ± 1% was released by cells preincubated in PTXF, and these differences were not significant. Only when cells preincubated in PTXF were stimulated with PMA in the presence of PTXF was there a small but measurable reduction in elastase release, to 10 ± 1% (p < 0.05). Thromboxane levels were increased in
the perfusates of lungs perfused with neutrophils and PMA when compared with thromboxane levels in lungs perfused with buffer, neutrophils, PMA, or PTXF alone (table 3). The elevation in perfusate thromboxane did not occur when PTXF was present in the perfusate buffer andlor the neutrophils had been preincubated in PTXF. Lavage LTC4 concentrations were similar in lungs perfused with buffer, neutrophils, PMA, or PTXF alone (table 3). In addition, there was no difference in the lavage fluid LTC4 of lungs perfused with neutrophils and PMA, or lungs perfused with neutrophils that had been preincubated in PTXF, with or without PTXF in the perfusate buffer. Isolated lungs perfused with oxygen metabolites generated by the reaction of glucose oxidase and 13-D-glucose demonstrated increases in lung weight, lung-tobody-weight ratio, and pulmonary artery pressure when compared with lungs perfused with 13-D-glucose alone (table 4). The addition of 2 mM PTXF did not prevent increases in lung weight, lung-tobody-weight ratio, or pulmonary artery pressure in lungs perfused with GIGO. PTXF did prevent increases in perfusate thromboxane.
Discussion These results clearly show that PTXF can reduce injury, as detected by weight gain, in isolated lungs perfused with neutrophils and PMA. The greatest effect was seen in lungs perfused with PTXF when added neutrophils were first preincubated in PTXF, but similar results were seen when PTXF was added only to the perfusate buffer. In contrast, no reduction in injury occurred when neutrophils were added that had been preincubated in PTXF. PTXF alone had no detectable adverse effect on the lung. Neutrophil-dependent injury in the isolated lung model requires an intact respiratory burst in added neutrophils, and it can also be inhibited by agents that impair retention of neutrophils in the pulmonary vasculature. In this study, the presence of PTXF significantly reduced oxidant production by neutrophils in vitro. Superoxide anion production and
TABLE 4 EFFECTS OF PENTOXIFYLLINE IN ISOLATED RAT LUNGS PERFUSED WITH 13-D-GLUCOSE AND GLUCOSE OXIDASE Total Lung Weight Substances Added to Perfusate 13·o-glucose p-o-glucose p·o-glucose
+ glucose oxidase + glucose oxidase + PTXF
Lung/Body Weight Ratio (x 10-")
Pulmonary Artery Pressure Increases
Perfusate Thromboxane
(g)
(mmHrIJ
(pgIm/)
2.1 ± 0.1 (5)t 4.1 ± 0.1 (5) 4.3 ::t:: 0.4 (5)*
5.76::t:: 0.2t 11.5 ::t:: 0.4 11.8 ::t:: 1.0*
1.8 ::t:: 0.3t 4.8 ± 0.8 4.1 ± 0.8*
109 ± 14t 3,500 ::t:: 630
• Values are mean ± SE. Number of determinations is shown in parentheses. t Value significantly different {p < 0.05) from value obtained after addition of Il-D-glucose and glucose oxidase. Value not significantly different (p > 0.05) from value obtained after addition of Il-D-glucose and glucose oxidase.
*
None detected
1350
RUTH J. MCDONALD
hydrogen peroxide release fell dramati- has a direct effect on thromboxane cally in the presence of2 mM PTXF. The production by the lung. Thromboxane reduction in oxidant production was production in this model has been linked readily reversible; no decrease in in vitro to neutrophil oxidant production (3), and oxidant production was seen when cells the reduction in thromboxane may be the were resuspended in PTXF-free buffer result of the reduction in neutrophilprior to assay. The requirement that mediated injury to the lung in the presPTXF be present in the perfusate buffer ence of PTXF. A similar reduction in perin order to reduce injury to the lung is fusate thromboxane was seen in lungs a direct parallel to the finding that only perfused with GIGO and PTXF, althe presence of PTXF reduced oxidant though PTXF did not protect lungs from production by PMA-stimulated neutrochemically generated oxidants. No phils in vitro. Significant reductions in changes in lung lavage leukotriene C4 lung weight or in superoxide anion and were seen in this study. hydrogen peroxide production by neutroA reduction in PMA-stimulated neuphils and PMA were not seen when neu- trophil superoxide anion production in the presence of PTXF has been reported trophils were preincubated in PTXF and then used in the isolated lung or in vitro. by some investigators (11, 13) but not by PTXF did not reduce retention of neu- others (21). To our knowledge, the reverstrophils in isolated lungs. ible nature of this effect has not been preThe effects of PTXF on PMA-stimu- viously described. Inhibition of neutrolated elastase release were different from phil degranulation by PTXF has also those on oxidant production; inhibition been reported (11, 12),but the effect was of elastase release occurred only when small, consistent with the small decrease neutrophils were preincubated in PTXF in elastase release reported here. and PTXF was present in incubation Overall, the results of the present inmixtures. The magnitude of inhibition vestigation are most significant in that was small, however, and the importance very fewpharmacologic agents have preof this effect in reducing neutrophil- viously been shown to reduce neutrophildependent injury to the lung, and this mediated injury is unknown. Pentoxifylline prevented increases in is the first report of an effective agent that does not appear to be an oxygen lung weight but not increases in pulmonary artery pressure in isolated lungs per- metabolite scavenger (12). In the experifused with neutrophils and PMA. It is ments reported here, PTXF produced a unlikely that pentoxifylline prevented dramatic reduction in lung injury, and weight gain only by a reduction in pul- the effects of PTXF on neutrophil funcmonary artery pressure increases, as pul- tion appeared to be reversible. Because monary artery pressure increases did oc- PTXF is at present used in the treatment cur in lungs perfused with PTXF and of human disease (22) and has shown some beneficial effects in patients with PMA. In contrast to the dramatic protection COPD (23), further investigation of the potential for this unique pharmacologic seen in lungs perfused with neutrophils and PMA, no reduction in weight gain agent to reduce neutrophil-mediated inor increasesin pulmonary artery pressure jury to the lung may be appropriate. occurred when PTXF wasadded to lungs Acknowledgment perfused with GIGO. This finding sugThe writer thanks Linda Bruckner and Lester gests that PTXF prevents injury in lungs C. Pan for expert technical assistance. perfused with neutrophils and PMA through mechanisms unique to this modReferences el such as a direct effect on the added 1. Weiland JE, Davis WB, Holter JF. Mohammed neutrophils. Lack of protection from JR, Dorinsky PM, Gadek JE. Lung neutrophils GIGO-induced injury is also consistent in the adult respiratory distress syndrome. Am Rev with our results which show that PTXF Respir Dis 1986; 133:218-25. does not reduce H 202 concentrations in 2. Tate RM, Repine JE. Neutrophils and the adult
vitro. The presence of PTXF prevented increases in perfusate thromboxane levels; however, this does not mean that PTXF
respiratory distress syndrome. Am Rev Respir Dis 1983; 128:552-9. 3. McDonald RJ. Berger EM. Repine JE. Neutrophil derived oxygenmetabolites stimulate thromboxane release,pulmonary artery pressure increases,
and weight gains in isolated perfused rat lungs. Am Rev Respir Dis 1987; 135:957-9. 4. Shasby DM, VanBenthuysen KM, Thte RM, Shasby SSt McMurtry IF. Repine JE. Granulocytes mediate acute edematous lung injury in rabbits and isolated rabbit lungs perfused with phorbol myristate acetate. Role of oxygen radicals. Am Rev Respir Dis 1982; 125:443-7. 5. Fox RB. Prevention of granulocyte mediated oxidant lung injury in rats by a hydroxyl radical scavenger. dimethyl thiourea. J Clin Invest 1984; 74:1456-64. 6. Mandell GL. ARDS. neutrophils, and pentoxifylline. Am Rev Respir Dis 1988; 138:1103-5. 7. Ishizaka A, Wu ZH, Stephens KE, et 01. Attenuation of acute lung injury in septic guinea pigs by pentoxifylline. Am Rev Respir Dis 1988; 138:376-82. 8. Lilly CM, Sandhu JS, Ishizaka A, et 01. Pentoxifylline prevents tumor necrosis factor-induced lung injury. Am Rev Respir Dis 1989; 139:1361-8. 9. Welsh CH. Lien D, Worthen GS. Weil JV. Pentoxifylline decreases endotoxin-induced pulmonary neutrophil sequestration and extravascular protein accumulation in the dog. Am Rev Respir Dis 1988; 138:II06-4. 10. Bertocchi F, Proserpio p. Lampugnani MG, Dejana E. The effect of pentoxifylline on polymorphonuclear cell adhesion to cultured endothelial cells. A preliminary report. In: Mandell G, Novick WJ, eds. Pentoxifylline and leukocyte function. Somerville, New Jersey: Hoechst-Roussel Pharmaceuticals. 1988; 68-74. II. Hammerschmidt DE, Kotasek D, McCarthy T, Hugh P, Freyburger G, Vercellotti GM. Pent oxifylline inhibits granulocyte and platelet function, including granulocyte priming by platelet activating factor. J Lab Clin Med 1988; II2:254-63. 12. Sullivan GW. Carper HT, Novick WJJ, Mandell GL. Inhibition of the inflammatory action of interleukin-I and tumor necrosis factor (alpha) on neutrophil function by pentoxifylline. Infect Immun 1988; 56:1722-9. 13. Currie M8, Rao KMK, Padmanabhan J, Jones A, Crawford J, Cohen HJ. Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation, and superoxide production. J Leukoc BioI 1990; 47:244-50. 14. Nees S, Schonharting M. Role of different blood cell types in blood rheology. In: Mandell G, Novick WJ. eds. Pentoxifylline and leukocyte function. Somerville, New Jersey: Hoechst-Roussel Pharmaceuticals, 1988; 105-14. 15. McDonald RJ, Bruckner LV, Repine JE. Neutrophil elastase augments acute edematous injury in isolated rat lungs perfused with neutrophil cytoplasts, Am Rev Respir Dis 1989; 140:1825-7. 16. McDonald RJ, Berger EM, Repine JE. Alveolar macrophage antioxidants prevent hydrogenperoxide-mediated lung damage. Am Rev Respir Dis 1991; 143:1088-91. 17. McDonaldRJ. Berger EM, WhiteCW, White JG, Freeman BA. Repine JE. Effect of liposomeencapsulated or polyethyleneglycol conjugated superoxide dismutase on neutrophil bactericidal activity in vitro and bacterial clearance in vivo. Am Rev Respir Dis 1985; 131:633-8.
RUTH J. MCDONALD·
Introduction
N eutrophils and neutrophil-derived ox-
idants have been implicated in the development of acute lung injury such as that seen in the adult respiratory distress syndrome (ARDS), in bronchopulmonary dysplasia, and in animal models of lung injury, including the isolated perfused lung (1-3). Shasbyand coworkers (4) first described acute edematous injury in isolated rabbit lungs perfused with human neutrophils and the neutrophil stimulant phorbol myristate acetate (PMA). In this model, injury to the isolated rat lung is dependent upon neutrophil-derived oxidant production as oxygen metabolite scavengers prevent injury, and injury is also prevented when neutrophils that do not produce oxidants are substituted (3, 5). Pentoxifylline is a methylxanthine derivative that has recently been shown to reduce neutrophil-dependent injury to the lung in several animal models (6). Continuous infusion of pentoxifylline reduced neutrophil counts in lung lavage fluid and lung wet-to-dry-weight ratios in a guinea pig model of sepsis after the administration of Escherichia coli (7) or tumor necrosis factor (8). In addition, pentoxifylline was also effective at reducing endotoxin-induced pulmonary vascular protein accumulation in the dog, and this effect was associated with a reduction in neutrophil accumulation in the lung (9). The ability of pentoxifylline to reduce injury to the lung in these models may be due to one or more of its known effects on neutrophils or on other circulating cells.Although pentoxifylline is similar in its chemical structure to theophyline, pentoxifylline in vitro can decrease neutrophil adherence to pulmonary endothelial cells, decrease neutrophil aggregation, and reduce oxidant production by stimulated neutrophils (10-13). Lastly, pentoxifylline can increase erythrocyte deformability and reduce blood viscosity, increasing the filterability of blood and its cellular components (14). The ability of pentoxifylline to reduce lung injury in animal models may be due to these or other as yet undefined effects.
SUMMARY Neutrophlls and neutrophil-derived oxidants have been Implicated In tha davelopmant of acute lung Injury such as that seen In the adult re8plratory distress syndrome (ARDS), In bronchopulmonary dysplasia (BPD), and In animal model8 of lung Injury, Including the Isolated perfused lung. Both neutrophll-derlved oxidant production and retention of neutrophlls In the lung are reqUIl13d for Injury In thl8 model. Pentoxlfylllne can reduca lung Injury from sepsis In the guinea pig and endotoxin-Induced neutrophil sequeatratlon and lung Injury In the dog. It Is also known to Increase neutrophil deformabillty, which may affect retention In the pulmonary mlcrovaaculatul8. we evaluated neutrophil oxidant production, retention In Isolated lungs, and neutrophil-mediated acute lung Injury after phorbol myrl8tate acetate (PMA) In the presence of pentoxlfyiline. Pantoxlfyliine (2 mM) 81gnlflc8ntly reduced 8uperoxlde anion and hydrogen peroxide production In "nro from PMAstimulated neutrophlls whan pentoxlfyiline was directly added to the Incubation mlxturaa, but not when neutrophlls ware prelncubated with the agent. Pantoxlfylllne did not reduce retention of neutrophllsln Isolated lungs aa determined by Infusion of '''In-labeled neutrophlls and gamma countIng. Pantoxlfylllne pravented Incre_ In total lung walght, lung-to-body-walght retlo, and perfusate thromboxane concentrations when It was present In perfusate buffer, whether or not neutrophlls ware prelncubated In pentoxlfylllne prior to Infusion Into the lung. Pentoxlfyiline did not reduce InJuryto lungs perfused with glucose and glucose oxidase. Weconclude that pentoxlfylllne reduces neutrophil oxidant production and neutrophll-dependent lung Injury. AM REV RESPIR DIS 1991; 144:1347-1350
We proposed the isolated perfused lung as a good model in which to investigate the potential for pentoxifylline to directly reduce neutrophil-mediated lung injury. Our hypothesis was that pentoxifylline would reduce neutrophil-mediated injury to isolated lungs perfused with neutrophils and the neutrophil stimulant PMA, and allow evaluation of the possible mechanisms of this effect. Methods Isolation of Human Neutrophils Human neutrophils wereisoJated from citrate anticoagulated blood on plasma-Percoll gradients after sedimentation of erythrocytes with Dextran, washed twice in calcium- and magnesium-free Hank's balanced salt solution (HBSS) (GIBCO, Grand Island, NY) and resuspended in complete HBSS, as previously described (15). Neutrophils were isolated and kept at room temperature until use in isolated lung or in in vitro experiments.
Preparation of Isolated Lungs Isolated, ventilated, perfused rat lungs were prepared as previously described. Male Sprague-Dawley rats (Bainton & Kingman, Freemont, CA) weighing 350 to 400 g were anesthetized intraperitoneally with pentobarbital and ventilated through a tracheostomy with 95070 air and 5% CO 2 , The pulmonary
artery and left atrium were catheterized, and the lungs were perfused with modified KrebsHenseleit buffer and 4% Ficoll. The pulmonary circulation was rinsed free of blood with 50 ml of perfusate buffer, and then the system was closed for recirculation of perfusate. A stable baseline was assured by observation and determination of pH in the perfusate for 20 min prior to the initiation of the experimental protocol. Neutrophils (4rnlat 1 x 107/ml) were infused directly into the pulmonary artery over 5 min, and PMA (0.025 ug/ml final concentration; Sigma Chemical, St. Louis, MO) was added to the perfusate reservoir 5 min after the completion of the neutrophil infusion. Pulmonary artery pressure and weight gain were continuously monitored. (Received in originalform December 14, 1990 and in revisedform July 1, 1991) 1 From the Department of Pediatrics and the California Primate ResearchCenter, University of California, Davis, California. 2 Presented in part at the Annual Meeting of the American Thoracic Society, Boston, May 1990. 3 Correspondence and requests for reprints should be addressed to Ruth J. McDonald, M.D., Department of Pediatrics, University of California Davis Medical Center, 2516 Stockton Blvd., Sacramento, CA 95817. • Recipient of Clinical Investigator Award K08 HL-01980 from the National Heart, Lung, and Blood Institute.
1347
1348
Lungs were perfused for a total of 120 min, or until gross edema fluid was observed in the trachea and dripping from the pleural surfaces (15). In some experiments, pentoxifylline (PTXF, 2 mM; a gift from HoechstRoussel Pharmaceuticals, Inc., Somerville, NJ) was added to the perfusate buffer at the beginning of the experimental protocol. In an additional series of experiments, isolated lungs were prepared and perfused with chemically generated oxidants by the addition of 0.5% ~-D-glucose (G, Sigma) and 5 U glucose oxidase (GO, Sigma) as previously described (16).
Retention of Neutrophils in Isolated Lungs Freshly isolated neutrophils were suspended in plasma and incubated with ["'In]tropolone for 5 min at room temperature, washed twice in a mixture of plasma and Ca2+-Mg'+free HBSS, and resuspended in complete HBSS at I x 1Q7 Iml. Labeled neutrophils were incubated in PTXF (2 mM) or buffer at room temperature for 20 min and then infused into isolated lungs, and subsequently PMA was added to the perfusate reservoir. At the end of the experimental protocol lungs were counted in a gamma counter, and the percentage of total counts infused remaining in the lung were used to determine the percentage of neutrophils retained in the lung (15). Determination of Neutrophil Oxidant Production Neutrophils (I x IQ6 Iml) wereincubated with PMA (0.025 ug/ml) for determination of superoxide anion and hydrogen peroxide production. Superoxide anion production was measured as the superoxide-dismutase-inhibitable reduction of cytochrome c at 37° C (17).Hydrogen peroxide production was measured by the ferrous ammonium sulfate and potassium thiocyanate method (18, 19). Neutrophils were incubated for 30 min at 37° C in HBSS, 10/0 normal pooled serum, I mM sodium azide, and PMA. Hydrogen peroxide concentrations were then determined on aliquots from triplicate incubations of mixtures with or without PTXF (2 mM) and performed in parallel on neutrophils preincubated in PTXF (2 mM), washed once, and resuspended in HBSS. Determination of Neutrophil Elastase Release Elastase release in vitro by PMA-stimulated neutrophils was determined by the measurement of human neutrophil elastase activity using the neutrophil-elastase-specific synthetic chromogenic substrate methoxysuccinylala-ala-pro-val-s-nitroanaline (Sigma). Neutrophils, at 2 x 106/ml in HBSS, wereincubated in triplicate with PMA (0.025 ug/ml) in the presence or absence of 2 mM PTX for 30 min at 37° C. Parallel incubations were performed on neutrophils preincubated in 2 mM PTXF. The mixtures were then centrifuged (5 min at 1,000 g), and the supernatants were frozen at - 70 c C until assay. Elastase release was determined as percent of total elastase present in identical aliquots of neutrophils suspended in HBSS, immediately placed on ice, sonicated (three lO-sbursts)
RUTH J. MCDONALD
(Vibra Cell Sonicator; Sonics & Materials, Inc., Danbury, CT), and frozen (20). In separate determinations, the presence of 2 mM PTXF did not alter measurable elastase activity of sonicated neutrophils or purified human neutrophil elastase using this substrate.
Determination of Lung Perfusate Thromboxane Concentration Thromboxane (TXB,) concentrations were determined on perfusate samples using radioimmunoassay, as previously described. Perfusate samples were taken at the end of the experimental protocol, placed on ice, centrifuged (1,000 g for 5 min), and frozen at - 70° C until assay. Ficoll was added to assay standards to assure that matrix composition was equivalent to that of perfusate samples (3). Determination of Lung Lavage Leukotriene C. Concentration Leukotriene C. (LTC.) was determined in lung lavage fluid by radioimmunoassay (Leukotriene C. PH] RIA Kit; Du Pont NEN Research Products, Boston, MA). At the end of the experimental protocol, lungs were weighed, and 6 ml of cold saline were instilled gently into the lung through the tracheal cannula. Lavage fluid was recovered by slow aspiration, with an averagereturn volume of 5 ml. Fluid was iced and centrifuged (1,000 g for 5 min), and the supernatants were frozen at -70° C until assay. Samples were assayed in duplicate with strict adherence to the manufacturer's recommended protocol. Statistics All data were analyzed using ANOVA on a Macintosh SE computer and Statview SE+Graphics statistical program (Abacus Concepts, Inc., Berkeley, CAl with a level of significance of p < 0.05. Results
Isolated lungs perfused with neutrophils and PMA had increases in total lung weight and lung-to-body-weight ratios when compared with lungs perfused with buffer alone, neutrophils alone, PMA alone, or PTXF alone. As expected, to-
tal lung weight was increased to 5.0 ± 0.8 g in lungs perfused with neutrophils and PMA (table 1).However, when PTXF was added to perfusates and neutrophils were added that had also been preincubated in PTXF, increases in lung weight and lung-to-body-weight ratios were reduced, with total lung weights of only 1.7 ± 0.1 g, no different from those of control lungs. Increases in pulmonary artery pressure did not occur in lungs perfused with buffer or PTXF alone. Small increases in pulmonary artery pressure occurred in lungs perfused with neutrophils alone and in lungs perfused with PMA alone or with added neutrophils (table 1). The presence of PTXF in the perfusate or pretreatment of neutrophils with PTXF did not prevent similar increases in pulmonary artery pressure prior to weight gain in all lungs perfused with PMA. In order to investigate the site of action of PTXF in this system, we next compared the ability of PTXF to reduce lung injury when the agent was added directly to the buffer and untreated neutrophils were added with the effects of preincubation of the neutrophils alone with PTXF and perfusion with buffer that did not contain PTXF. When pentoxifylline was present in the buffer only, the reduction in weight gain after the addition of neutrophils and PMA was similar to that seen in lungs where neutrophils had also been preincubated in PTXF and PTXF was added to the buffer. However, when neutrophils were preincubated in pentoxifylline, washed, and then added to isolated lungs, total lung weight was not reduced. Lung weights and lung-to-body-weight ratios were reduced in both groups where pentoxifylline was present in the buffer when compared with lungs perfused with neutrophils and PMA. Pulmonary artery pressure increases prior to weight gain were similar in these groups.
TABLE 1 PENTOXIFYLUNE REDUCES INJURY IN ISOLATED RAT LUNGS PERFUSED WITH NEUTROPHILS AND PMA*
Substances Added to Perfusate Buffer Neutrophils + PMA Neutrophils preincubated in PTXF + PTXF + PMA Neutrophils + PTXF + PMA Neutrophils preincubated in PTXF + PMA Neutrophils PMA PTXF
Pulmonary Artery Pressure Increases
Total Lung Weight (g)
Lung/Body Weight Ratio (x 10-Oj
1.6 ± 0.2 (9}t 5.0 ± O.S (10)
4.5 ± 0.2t 15.3 ± 2.7
0.3 ± 0.2t 3.3 ± 0.5
1.7 ± 0.1 (S}t 2.1 ± 0.3 (7)t
5.1 ± 0.3t 6.1 ± o.st
3.3 ± 0.6:1: 2.5 ± 0.4:1:
3.2 1.S 1.6 1.7
9.7 5.4 4.9 4.S
3.4 ± 1.4 ± 2.S ± O.S ±
± ± ± ±
0.9 0.1 0.1 0.1
(6):1: (S}t (S}t (6)t
± ± ± ±
2.9:1: O.4t 0.3t 0.2t
• Values are mean ± SE. Number of determinations is shown in parentheses. Value significantly different (p < 0.05) from value obtained after addition of neutrophils and PMA. Value not signifk:antly different (p > 0.05) from value obtained after addition of neutrophils and PMA.
t
*
(mm Hg)
1.2:1: 0.4t 0.5:1: O.4t
1349
PENTOXIFYLLINE REDUCES INJURY IN ISOLATED WNGS
TABLE 2 PENTOXIFYLLINE (2 mM) REDUCES SUPEROXIDE ANION AND HYDROGEN PEROXIDE PRODUCTION BY PMA-STIMULATED NEUTROPHILS IN VITRO" H,O, Produced (PM)
Cytochrome c Reduced
Additions to Incubation Mixtures
(nM)
Neutrophils + PMA Neutrophils + PMA + PTXF Neutrophils preincubated in PTXF + PMA Neutrophils preincubated in PTXF + PTXF + PMA
68.7 42.8 65.9 35.8
± 6.1 (8) ± 5.6 (7)t ± 7.7 (6)*
± 4.2 (6)t
40.6 16.2 43.7 21.6
± 7.3 (6)
± 3.7 (6)t ± 7.8 (5)* ± 5.4 (5)t
" Values are mean ± SEM. Number of determinations is shown in parentheses. Value significantly different (p < 0.05) from value obtained with neutrophils and PMA. Value not significantly different (p > 0.05) from value obtained with neutrophils and PMA.
t
*
TABLE 3 PERFUSATE THROMBOXANE AND LUNG LAVAGE LTC. LEVELS IN ISOLATED RAT LUNGS PERFUSED WITH NEUTROPHILS, PMA, AND PTXF"
Substances Added to Perfusate Buffer Neutrophils Neutrophils Neutrophils Neutrophils Neutrophils PMA PTXF
Thromboxane
Leukotriene C.
Ipg/m/)
(ng/m/)
135 468 117 133 154 168 120 85
+ PMA preincubated in PTXF + PTXF + PMA + PTXF + PMA preincubated in PTXF + PMA
± 27 (7)t
± 100 (7) ± 41 (7)t ± 35 (6)t ± 21 (6)t
± 57 (6)t ± 31 (8)t ± 28 (6)t
3.3 4.3 1.9 3.3 2.3 2.1 2.1 2.0
± 0.6* ± 0.9
± 0.3* ± 0.7*
± 0.7* ± 0.3* ± 0.4*
± 0.6*
" Values are mean ± SE. Number of determinations is shown in parentheses. t Value significantly different (p < 0.05) from value obtained after addition of neutrophils and PMA. Value not significantly different (p > 0.05) from value obtained after addition of neutrophils and PMA.
*
Isolated lungs perfused with ll1In_ labeled neutrophils and PMA retained 87 ± 3070 of total counts infused when counted at the end of the experimental period. When pentoxifyIIine was present in the perfusate and neutrophils had been preincubated in it, 90 ± 8% of total counts infused remained (n = 5). PentoxifyIIine did not reduce retention of neutrophils in isolated lungs. PTXF reduced oxidant production by PMA stimulated neutrophils in vitro when present in the reaction mixtures (table 2). In the presence of 2 mM PTXF, superoxide anion production was reduced by 39%, and hydrogen peroxide concentrations were reduced by 61%. However, when neutrophils were preincubated in PTXF, washed, and then assayed for PMA-stimulated oxidant production there was no decrease in either superoxide anion or hydrogen peroxide cencentrations produced. Preincubation of neutrophils in PTXF did not further reduce
oxidant production by neutrophils when PTXF was already present in the incubation mixtures. In a separate study, we determined that the presence of pentoxifyIIine did not interfere with the detection of reagent grade hydrogen peroxide. A similar pattern of results was obtained when higher concentrations of PMA were used (data not shown). Elastase release after stimulation with PMA was not affected by the presence of PTXF or by preincubation of neutrophils in PTXF. Elastase release by neutrophils stimulated with PMA was 16 ± 1% oftotal; 12 ± 2% was released in the presence of PTXF, and 12 ± 1% was released by cells preincubated in PTXF, and these differences were not significant. Only when cells preincubated in PTXF were stimulated with PMA in the presence of PTXF was there a small but measurable reduction in elastase release, to 10 ± 1% (p < 0.05). Thromboxane levels were increased in
the perfusates of lungs perfused with neutrophils and PMA when compared with thromboxane levels in lungs perfused with buffer, neutrophils, PMA, or PTXF alone (table 3). The elevation in perfusate thromboxane did not occur when PTXF was present in the perfusate buffer andlor the neutrophils had been preincubated in PTXF. Lavage LTC4 concentrations were similar in lungs perfused with buffer, neutrophils, PMA, or PTXF alone (table 3). In addition, there was no difference in the lavage fluid LTC4 of lungs perfused with neutrophils and PMA, or lungs perfused with neutrophils that had been preincubated in PTXF, with or without PTXF in the perfusate buffer. Isolated lungs perfused with oxygen metabolites generated by the reaction of glucose oxidase and 13-D-glucose demonstrated increases in lung weight, lung-tobody-weight ratio, and pulmonary artery pressure when compared with lungs perfused with 13-D-glucose alone (table 4). The addition of 2 mM PTXF did not prevent increases in lung weight, lung-tobody-weight ratio, or pulmonary artery pressure in lungs perfused with GIGO. PTXF did prevent increases in perfusate thromboxane.
Discussion These results clearly show that PTXF can reduce injury, as detected by weight gain, in isolated lungs perfused with neutrophils and PMA. The greatest effect was seen in lungs perfused with PTXF when added neutrophils were first preincubated in PTXF, but similar results were seen when PTXF was added only to the perfusate buffer. In contrast, no reduction in injury occurred when neutrophils were added that had been preincubated in PTXF. PTXF alone had no detectable adverse effect on the lung. Neutrophil-dependent injury in the isolated lung model requires an intact respiratory burst in added neutrophils, and it can also be inhibited by agents that impair retention of neutrophils in the pulmonary vasculature. In this study, the presence of PTXF significantly reduced oxidant production by neutrophils in vitro. Superoxide anion production and
TABLE 4 EFFECTS OF PENTOXIFYLLINE IN ISOLATED RAT LUNGS PERFUSED WITH 13-D-GLUCOSE AND GLUCOSE OXIDASE Total Lung Weight Substances Added to Perfusate 13·o-glucose p-o-glucose p·o-glucose
+ glucose oxidase + glucose oxidase + PTXF
Lung/Body Weight Ratio (x 10-")
Pulmonary Artery Pressure Increases
Perfusate Thromboxane
(g)
(mmHrIJ
(pgIm/)
2.1 ± 0.1 (5)t 4.1 ± 0.1 (5) 4.3 ::t:: 0.4 (5)*
5.76::t:: 0.2t 11.5 ::t:: 0.4 11.8 ::t:: 1.0*
1.8 ::t:: 0.3t 4.8 ± 0.8 4.1 ± 0.8*
109 ± 14t 3,500 ::t:: 630
• Values are mean ± SE. Number of determinations is shown in parentheses. t Value significantly different {p < 0.05) from value obtained after addition of Il-D-glucose and glucose oxidase. Value not significantly different (p > 0.05) from value obtained after addition of Il-D-glucose and glucose oxidase.
*
None detected
1350
RUTH J. MCDONALD
hydrogen peroxide release fell dramati- has a direct effect on thromboxane cally in the presence of2 mM PTXF. The production by the lung. Thromboxane reduction in oxidant production was production in this model has been linked readily reversible; no decrease in in vitro to neutrophil oxidant production (3), and oxidant production was seen when cells the reduction in thromboxane may be the were resuspended in PTXF-free buffer result of the reduction in neutrophilprior to assay. The requirement that mediated injury to the lung in the presPTXF be present in the perfusate buffer ence of PTXF. A similar reduction in perin order to reduce injury to the lung is fusate thromboxane was seen in lungs a direct parallel to the finding that only perfused with GIGO and PTXF, althe presence of PTXF reduced oxidant though PTXF did not protect lungs from production by PMA-stimulated neutrochemically generated oxidants. No phils in vitro. Significant reductions in changes in lung lavage leukotriene C4 lung weight or in superoxide anion and were seen in this study. hydrogen peroxide production by neutroA reduction in PMA-stimulated neuphils and PMA were not seen when neu- trophil superoxide anion production in the presence of PTXF has been reported trophils were preincubated in PTXF and then used in the isolated lung or in vitro. by some investigators (11, 13) but not by PTXF did not reduce retention of neu- others (21). To our knowledge, the reverstrophils in isolated lungs. ible nature of this effect has not been preThe effects of PTXF on PMA-stimu- viously described. Inhibition of neutrolated elastase release were different from phil degranulation by PTXF has also those on oxidant production; inhibition been reported (11, 12),but the effect was of elastase release occurred only when small, consistent with the small decrease neutrophils were preincubated in PTXF in elastase release reported here. and PTXF was present in incubation Overall, the results of the present inmixtures. The magnitude of inhibition vestigation are most significant in that was small, however, and the importance very fewpharmacologic agents have preof this effect in reducing neutrophil- viously been shown to reduce neutrophildependent injury to the lung, and this mediated injury is unknown. Pentoxifylline prevented increases in is the first report of an effective agent that does not appear to be an oxygen lung weight but not increases in pulmonary artery pressure in isolated lungs per- metabolite scavenger (12). In the experifused with neutrophils and PMA. It is ments reported here, PTXF produced a unlikely that pentoxifylline prevented dramatic reduction in lung injury, and weight gain only by a reduction in pul- the effects of PTXF on neutrophil funcmonary artery pressure increases, as pul- tion appeared to be reversible. Because monary artery pressure increases did oc- PTXF is at present used in the treatment cur in lungs perfused with PTXF and of human disease (22) and has shown some beneficial effects in patients with PMA. In contrast to the dramatic protection COPD (23), further investigation of the potential for this unique pharmacologic seen in lungs perfused with neutrophils and PMA, no reduction in weight gain agent to reduce neutrophil-mediated inor increasesin pulmonary artery pressure jury to the lung may be appropriate. occurred when PTXF wasadded to lungs Acknowledgment perfused with GIGO. This finding sugThe writer thanks Linda Bruckner and Lester gests that PTXF prevents injury in lungs C. Pan for expert technical assistance. perfused with neutrophils and PMA through mechanisms unique to this modReferences el such as a direct effect on the added 1. Weiland JE, Davis WB, Holter JF. Mohammed neutrophils. Lack of protection from JR, Dorinsky PM, Gadek JE. Lung neutrophils GIGO-induced injury is also consistent in the adult respiratory distress syndrome. Am Rev with our results which show that PTXF Respir Dis 1986; 133:218-25. does not reduce H 202 concentrations in 2. Tate RM, Repine JE. Neutrophils and the adult
vitro. The presence of PTXF prevented increases in perfusate thromboxane levels; however, this does not mean that PTXF
respiratory distress syndrome. Am Rev Respir Dis 1983; 128:552-9. 3. McDonald RJ. Berger EM. Repine JE. Neutrophil derived oxygenmetabolites stimulate thromboxane release,pulmonary artery pressure increases,
and weight gains in isolated perfused rat lungs. Am Rev Respir Dis 1987; 135:957-9. 4. Shasby DM, VanBenthuysen KM, Thte RM, Shasby SSt McMurtry IF. Repine JE. Granulocytes mediate acute edematous lung injury in rabbits and isolated rabbit lungs perfused with phorbol myristate acetate. Role of oxygen radicals. Am Rev Respir Dis 1982; 125:443-7. 5. Fox RB. Prevention of granulocyte mediated oxidant lung injury in rats by a hydroxyl radical scavenger. dimethyl thiourea. J Clin Invest 1984; 74:1456-64. 6. Mandell GL. ARDS. neutrophils, and pentoxifylline. Am Rev Respir Dis 1988; 138:1103-5. 7. Ishizaka A, Wu ZH, Stephens KE, et 01. Attenuation of acute lung injury in septic guinea pigs by pentoxifylline. Am Rev Respir Dis 1988; 138:376-82. 8. Lilly CM, Sandhu JS, Ishizaka A, et 01. Pentoxifylline prevents tumor necrosis factor-induced lung injury. Am Rev Respir Dis 1989; 139:1361-8. 9. Welsh CH. Lien D, Worthen GS. Weil JV. Pentoxifylline decreases endotoxin-induced pulmonary neutrophil sequestration and extravascular protein accumulation in the dog. Am Rev Respir Dis 1988; 138:II06-4. 10. Bertocchi F, Proserpio p. Lampugnani MG, Dejana E. The effect of pentoxifylline on polymorphonuclear cell adhesion to cultured endothelial cells. A preliminary report. In: Mandell G, Novick WJ, eds. Pentoxifylline and leukocyte function. Somerville, New Jersey: Hoechst-Roussel Pharmaceuticals. 1988; 68-74. II. Hammerschmidt DE, Kotasek D, McCarthy T, Hugh P, Freyburger G, Vercellotti GM. Pent oxifylline inhibits granulocyte and platelet function, including granulocyte priming by platelet activating factor. J Lab Clin Med 1988; II2:254-63. 12. Sullivan GW. Carper HT, Novick WJJ, Mandell GL. Inhibition of the inflammatory action of interleukin-I and tumor necrosis factor (alpha) on neutrophil function by pentoxifylline. Infect Immun 1988; 56:1722-9. 13. Currie M8, Rao KMK, Padmanabhan J, Jones A, Crawford J, Cohen HJ. Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation, and superoxide production. J Leukoc BioI 1990; 47:244-50. 14. Nees S, Schonharting M. Role of different blood cell types in blood rheology. In: Mandell G, Novick WJ. eds. Pentoxifylline and leukocyte function. Somerville, New Jersey: Hoechst-Roussel Pharmaceuticals, 1988; 105-14. 15. McDonald RJ, Bruckner LV, Repine JE. Neutrophil elastase augments acute edematous injury in isolated rat lungs perfused with neutrophil cytoplasts, Am Rev Respir Dis 1989; 140:1825-7. 16. McDonald RJ, Berger EM, Repine JE. Alveolar macrophage antioxidants prevent hydrogenperoxide-mediated lung damage. Am Rev Respir Dis 1991; 143:1088-91. 17. McDonaldRJ. Berger EM, WhiteCW, White JG, Freeman BA. Repine JE. Effect of liposomeencapsulated or polyethyleneglycol conjugated superoxide dismutase on neutrophil bactericidal activity in vitro and bacterial clearance in vivo. Am Rev Respir Dis 1985; 131:633-8.
