Lung (1992) 170:41-50
New York Inc. 1992
Superoxide Anion Production by Rat Neutrophils at Various Stages of BleomycinInduced Lung Injury Elizabeth B. Tarnell, 1 Bonnie L. Oliver, 1 George M. Johnson, 2 Florence L. Watts, 3 and Roger S. Thrall ~ Departments of IMedicine, Pulmonary Division, 2Pediatrics, and 3Anesthesiology, University of Connecticut Health Center, Farmington, CT 06030, USA
Abstract. This study investigated the level of activation of neutrophils isolated from rats at various stages of bleomycin-induced lung injury. Neutrophils were collected from blood and bronchoalveolar lavage (BAL) fluid and their superoxide anion (O2)-generating capacity measured in response to phorbol myristate acetate (PMA) and opsonized zymosan (OZ) stimulation. When stimulated with PMA, BAL neutrophils isolated from animals 3 days after bleomycin treatment had a significantly greater capacity to produce 0 2 than BAL neutrophils from animals 7 days after bleomycin treatment. The 0 2 levels of 7 day BAL neutrophils more closely resembled the resting levels obtained with circulating neutrophils from both control and bleomycintreated animals. There were no differences observed in any of the neutrophils when stimulated with OZ. Myeloperoxidase levels were measured in plasma and BAL and found to be elevated only in plasma at 7 days after bleomycin. These data demonstrate that neutrophil activation does occur in this model and that the activation appears to be transient, in response to specific stimuli and compartmentalized between the lung and blood.
Key words: Superoxide--Neutrophils--Bleomycin-induced lung injury. Introduction
Free radical generation by phagocytic cells has been implicated in the pathogenesis of several forms of acute lung injury, some of which lead to pulmonary fibrosis. Evidence has accumulated supporting the role of free radicals in diseases such as the adult respiratory distress syndrome (ARDS) [2, 15, 41], radiation pneumonitis [18], hyperoxia [13, 16, 32], emphysema due to cigarette
Offprint requests to: R. S. Thrall
42
E . B . Tarnell et al.
smoking [6], exposure to a variety of drugs and toxins [4,10, 25, 29, 30], immune complexes, and in various experimental models [23, 33]. Mechanisms by which oxygen metabolites inflict tissue injury include protein denaturation resulting in enzyme inactivation, and lipid peroxidation, which contributes to plasma membrane instability [12, 24]. The lung uses a variety of antioxidant enzymes and substances to aid in its protection, and impairment of these defenses may alter susceptibility to injury [21, 27]. Bleomycin-induced lung injury in a variety of animals has been accepted as a model of acute lung injury that progresses to fibrosis [1, 3, 14, 28, 34, 35]. In the rat model, peak periods of acute inflammation are observed at 3 and 7 days after bleomycin administration, followed by chronic injury and scar formation. Because neutrophils have been shown to make up a large percentage of cells in bronchoalveolar lavage (BAL) and in lung tissue during the acute stages of bleomycin injury [36], their level of activation may play an important role in the pathogenesis of lung injury in this model. In addition, clinical studies have shown large influxes of neutrophils in BAL and lung tissue in acute stages of various adult human lung diseases such as bacterial pneumonia and ARDS. Whether these neutrophils are beneficial or harmful in all cases remains to be determined. Since uncomplicated bacterial pneumonias do not routinely progress to ARDS, we hypothesize that neutrophils can be activated to different levels in various lung diseases and that this may influence the extent and/or type of injury. To test our hypothesis we studied both in vitro and in vivo levels of neutrophil activation in the bleomycin model of lung injury. First, we directly studied the levels of in vitro O~- production by neutrophils isolated from peripheral blood and BAL and rats during the acute stages of bleomycin-induced lung injury. Second, we indirectly approximated in vivo activation of neutrophils by measuring myeloperoxidase (MPO) levels in the plasma and BAL of these rats. Finally, to determine if neutrophil activation levels were different in various models, we compared the levels of both O2 and MPO to those we observed previously in the oleic acid model of acute lung injury [39].
Materials and Methods
Animals Adult male pathogen-free Fischer 344 rats, weighing 175-200 g, were obtained from Charles River Laboratories (Worthington, MA). The animals were free from respiratory disease and housed in isolation from all other laboratory animals. No evidence of infection was observed in any of our animals based on histologic evaluation.
Reagents Bleomycin sulfate (Blenoxane) was a gift from Bristol Laboratories (Syracuse, NY). Bovine serum albumin (BSA), cytochrome C (type VI), superoxide dismutase (SOD), n-ethyl maleimide (NEM), phorbol 12-myristate 13-acetate (PMA), HEPES, zymosan, and ficoll were purchased from Sigma
Neutrophil Superoxide Production in Bleomycin Injury
43
Chemical Company (St. Louis, MO). Hypaque (50%) was obtained from Sterling Drug, Inc. (New York, NY) and heparin (10,000/zg/ml) was purchased from Elkins-Sinn, Inc. (Cherry Hill, NJ). Hanks' balanced salt solution (HBSS) without magnesium and calcium was obtained from Hazleton Biologics (Lenexa, KS). Ketamine hydrochloride was purchased from Parke, Davis Co. (Detroit,
M1).
Induction of Lung Injury Animals were anesthetized via an intraperitoneal injection of ketamine HCI. Each animal in the treatment group received a single intratracheal injection of 2.0/zl of bleomycin sulfate in 0.3 ml of sterile saline [35]. Control animals received sterile saline in a similar manner. Animals were sacrificed at 3 and 7 days after bleomycin or saline injection. It should be noted that these times represent the peak periods of inflammation. Insufficient numbers of neutrophits are recovered at earlier and later time points after bleomycin for the assays to be performed.
Histologic Evaluation of Lung Damage Lung damage was determined as described previously [34, 35]. Lungs were removed after the animal was killed and inflated with 10% buffered formaldehyde at a constant pressure of 20 cmHzO. Tissue sections were processed in the usual manner for light microscopic examination with hematoxylin and eosin stain. Only qualitative histologic evaluations were made.
Cell Harvests Circulating Neutrophils. Blood was collected via cardiac aspiration with a heparinized syringe. Blood from 4 rats was pooled to yield enough cells for the superoxide assay. Blood was diluted with an equal volume of heparinized saline and neutrophils were isolated as described below. BAL Neutrophils. BAL
was performed with 50 ml of sterile heparinized saline in 5-7 ml aliquots, injected intratracheally to inflate the lungs fully. Fluid was then retrieved by aspiration and strained through gauze to remove mucus. Cells from 4-7 rats were pooled to obtain enough cells for the superoxide assay. Ceils were washed and neutrophils isolated as described below.
Isolation of Neutrophils Cell suspensions were placed over an equal volume of ficoll-Hypaque gradient (ficoli 9% and Hypaque 34% in a ratio of 2.4 : 1) and centrifuged at 350 g/4°C for 20 minutes. The monocyte-rich upper layer was removed, leaving the neutrophil-rich pellet. Hypotonic lysis of erythrocytes was performed. Neutrophils were washed with HBSS containing 10 mM of HEPES. Cells were counted using a standard AO hemacytometer. This method of isolation yielded a cell suspension of >85% neutrophils. Viability was determined to be >98% with nigrosin dye exclusion.
Superoxide Assay. Cells
were suspended in HBSS containing 10 mM HEPES at 3 x 106 cells/m1. Superoxide release was determined at 37°C using the superoxide dismutase-inhibitable reduction of cytochrome C at 550 nm for 5 min [9]. Absorbance was read on a Beckman Model 24 spectrophotometer. The reaction mixture contained 119 mM cytochrome C, 1 mM sodium azide, 1 mM calcium chloride, 2.6 mM magnesium chloride, and was initiated with the addition of the appropriate stimulus, 100 ng/ml PMA or 1 mg/ml opsonized zymosan (OZ). The reaction was stopped after 5 min by the addition of 0.5 mM NEM. Generation of O~- was calculated using 21.2 x 103 m/cm as the molar extinction coefficient for reduced-minus-oxidized cytochrome [26].
44
E.B. Tarnell et al.
Myeloperoxidase Assay Myeloperoxidase (MPO) activity, used as an indicator of neutrophil degranulation [17], was measured by a micromodification [40] of the colorimetric assay measuring oxidation of o-dianisidine hydrochloride at 450 nm [20]. Plasma or BAL samples were diluted with phosphate-buffered saline fourfold before the assay. Sample measurements were done in triplicate. The molar absorbancy of oxidized o-dianisidine, 1.13 x 104/cm, was used to calculate the moles of hydrogen peroxide decomposed. One unit of peroxidase activity was defined as that degrading 1 t~molof peroxide/rain at 25°C.
Statistical Analysis Statistical analysis was performed using 1-way analysis of variance followed by the Newman-Keuls procedure [42].
Results
Histologic Analysis of Lung Damage Histologic changes induced by b l e o m y c i n administration in the lungs of the experimental animals were similar to those described previously in this laboratory [35]. Severe intra-alveolar e d e m a and h e m o r r h a g e with a diffuse interstitial and inra-alveolar neutrophil cell infiltrate were o b s e r v e d 3-7 days after bleomycin administration.
Superoxide Generation in Circulating Neutrophils W h e n stimulated with PMA, circulating neutrophils (CN) isolated f r o m animals at 3 and 7 days after b l e o m y c i n p r o d u c e d O f levels similar to conrol (Fig. la). W h e n stimulated with OZ, C N f r o m animals at 7 days after t r e a t m e n t p r o d u c e d significantly lower (p < 0.01) 0 2 levels than C N f r o m 3 days, but neither s h o w e d a significant change f r o m control (Fig. lb).
Superoxide Generation in BAL Neutrophils The O{ levels p r o d u c e d by B A L neutrophils isolated 3 days after b l e o m y c i n administration w e r e significantly elevated a b o v e the levels p r o d u c e d b y either the B A L neutrophils obtained 7 days after b l e o m y c i n (p < 0.01) (Fig. 2a) or any of the C N (p < 0.01) f r o m Fig. la, which w e r e stimulated with PMA. After stimulation with OZ, 0 2 levels in B A L neutrophils were not significantly different b e t w e e n the 3 and 7 day animals (Fig. 2b), nor was there a significant difference b e t w e e n these B A L neutrophils or any of the OZ-stimulated C N in Fig. lb.
Neutrophil Superoxide Production in Bleomycin Injury
A
45
B PMA
OZ.
rE u3 cO -J w O o
40.
40,
30.
30,
II
7" E ¢,v LU O
20.
20.
I
X
o
nuJ
T 1
10-
10,
cO
CON
3 DAY
7 DAY
CON
3 DAY
7 DAY
TIME P O S T B L E O M Y C I N
Fig. 1. a. Superoxide generation in circulating neutrophils (CN) after stimulation with PMA at 3 and 7 days after bleomycin treatment. PMA stimulated CN produced O2- levels similar to control. b Superoxide generation in CN after stimulation with OZ. OZ-stimulated CN produced significantly lower (*p < 0.01) O~- levels at 7 days after bleomycin treatment compared to 3 days. Vertical bars represent mean + / - SEM of 4 experiments.
Myeloperoxidase Levels Plasma levels of M P O were significantly (p < 0.01) elevated 7 days after bleomycin treatment c o m p a r e d to both control and 3 day levels (Fig. 3a). There were no significant differences between B A L levels of M P O at any time (Fig. 3b).
Discussion
We have d e m o n s t r a t e d that at the period of p e a k acute inflammation, 3 days after bleomycin treatment, B A L neutrophils are activated to produce high levels of O2 when stimulated in vitro by PMA. Later, at 7 days after b l e o m y c i n treatment, PMA-stimulated B A L neutrophils a p p e a r to be at " r e s t i n g " or con-
46
E . B . Tarnell et al.
B
A
o_zz
PMA L~ -J --I
LU
% 40. I
o 30 E LU C~ X O o~ uJ o_
~
30'
T
20
20,
T 10-
10,
3 DAY
7 DAY
TIME
3 DAY
POST
7 DAY
BLEOMYCIN
Fig. 2. a. Superoxide generation in BAL neutrophils isolated 3 and 7 days after bleomycin and stimulated with PMA. At 3 days, O~- levels were significantly (*p < 0.01) higher than at 7 days. This level of activity was also significantly higher (p < .01) than those observed in any CN from Fig. la. b Superoxide generation in BAL neutrophils isolated 3 and 7 days after bleomycin and stimulated with OZ. There was no significant difference between BAL ceils at 3 and 7 days after bleomycin treatment, nor was there a difference between these cells and OZ stimulated CN from Fig. lb. Vertical bars represent mean + / - SEM of 4 experiments.
trol levels of activity. In contrast, the phagocytic stimulus, OZ, did not increase O2 production in BAL neutrophils at either time point. Our data therefore suggest that activation of neutrophils does occur in the bleomycin model and that the activation may be in response to a specific stimulus. The activation also appears to be transient depending on the stage of lung injury. Plasma levels of MPO were significantly elevated 7 days after bleomycin treatment, further indicating in vivo neutrophil activation. MPO levels were not elevated in the BAL at any time after bleomycin treatment. Previous studies from our laboratory have shown that 0 2 levels in CN and BAL neutrophils obtained at peak periods of inflammation in the oleic acid model of acute lung injury were not elevated above "resting" levels [39]. In contrast, BAL neutrophils isolated from the bleomycin model in this study were activated above "resting" levels. In addition, different plasma and BAL MPO
Neutrophil Superoxide Production in Bleomycin Injury
A
47
B PLASMA
BAL
80m
. D ¢-.
60-
o
Q. 40-
lo
CON
3 DAY
7 DAY
TIME
POST
CON
3 DAY
7 DAY
BLEOMYCIN
Fig. 3. a. Myeloperoxidase (MPO) levels in plasma from control and bleomycin-treated animals. There was a significant (*p < 0.01) increase in the MPO level at 7 days compared to both control and 3 days after bleomycin treatment, b MPO levels in BAL fluid from control and bleomycintreated animals. There was no difference between MPO levels at any time point. Vertical bars represent mean + / - SEM of 6 samples.
levels were observed in the bleomycin and oleic acid models. Although both models demonstrated high levels of MPO, the activity was expressed at different sites. In the bleomycin model MPO levels were elevated in plasma only, but in the oleic acid model MPO levels were elevated in BAL. Together, these data demonstrate that neutrophils obtained at peak periods of acute inflammation in different animal models of lung injury can be stimulated or primed to attain different levels of activation. The role that these differences may play in the pathogenesis of these models is unclear. The 3 day bleomycin BAL neutrophils appeared to be activated or "primed" in vivo since in vitro stimulation with PMA generated high levels of O~. The low BAL and plasma MPO levels at 3 days after bleomycin suggest that the neutrophils did not degranulate within the air spaces of the lung or in the plasma. It is possible that degranulation occurred in the interstitium of the lung where most of the inflammation resides and MPO levels in BAL or plasma would not
48
E.B. Tarnell et al.
reflect it. Another explanation could be that the PMA-stimulated 3 day bleomycin BAL neutrophils were "primed" in vivo but did not receive the secondary stimulus to become activated and degranulate. Priming agents such as lipopolysaccharide [19] and platelet-activating factor [38] have been shown not to be stimulus-specific and indeed primed cells are responsive to a variety of various stimulators other than the priming agent. Thus, if the cells were primed in this manner one would have expected them to also be responsive to OZ. However, a priming mechanism is still possible if priming occurred through activation of protein kinase C that has been shown to internalize and downregulate plasma membrane receptors while leaving the cell hyperactive to nonreceptor antagonists such as PMA [5, 31]. Thus, the cell would not be responsive to receptormediated activators such as OZ but still responsive to PMA. This may also explain the decreased CN response to OZ at 7 days after bleomycin treatment, when MPO levels were elevated in plasma. The cells could have been activated in vivo via protein kinase C, which downregulates membrane receptors for OZ and this, in turn, results in decreased responsiveness to OZ. The activated cells degranulate and release high levels of MPO into the plasma. These 7 day cells are not "primed"; they have already been activated and degranulated and now are in a state of "desensitization" or are incapable of responding to further stimuli, hence their inability to generate an elevated response even to PMA. The role of the neutrophil and oxygen metabolites in lung injury is controversial. Their significance in both the oleic acid and bleomycin models has been studied previously [7, 11, 22, 37]. Jenkinson and colleagues [22] administered bleomycin to vitamin-E-deficient rats and found no increase in mortality, evidence of lipid peroxidation, or lung collagen content when compared with controls and suggested that perhaps lipid peroxidation was not critical to development of the lesion. Chandler et al. [7] have subsequently shown that iron deficiency in bleomycin-treated hamsters reduced the severity of pulmonary fibrosis, possibly by preventing iron-catalyzed oxygen radical formation. We have further shown that neutrophil depletion in the oleic acid model suppresses lung injury, although the mechanism by which neutrophils contribute to damage may not be due to free radicals since treatment with catalase, dimethylsulfoxide, or superoxide dismutase had no effect on injury [11]. In contrast, we [37] and others [8] have shown that neutrophil depletion in the bleomycin model enhanced injury, evidenced by increased collagen. The neutrophil has a wide variety of capabilities and whether it is beneficial or harmful may very well depend on how that cell is stimulated or activated. Direct in vivo evidence demonstrating this is difficult to obtain and we believe that this is one approach to help us understand better in vivo neutrophil function during active stages of inflammation. In summary, our data suggest that in the bleomycin model, in vivo activation of neutrophils does occur and may be in response to specific stimuli, is transient, and appears to be compartmentalized. Furthermore, when comparing these results with similar data from the oleic acid model, we have shown that neutrophils are capable of attaining varied levels of activation in response to different types of lung injury.
Neutrophil Superoxide Production in Bleomycin Injury
49
Acknowledgment. The authors wish to thank Ms. Debra Parkinson for the excellent illustrations.
References 1. Adamson IYR, Bowden DH (1974) The pathogenesis ofbleomycin-induced pulmonary fibrosis in mice. Am J Pathol 77:185-198 2. Baldwin SR, Grum CM, Boxer LA, Simon RH, Ketan LH, Deuvall LJ (1986) Oxidant activity in expired breath of patients with adult respiratory distress syndrome. Lancet 1(8471): 11-13 3. Berend N, Feldsien D, Cederbaums D, Cherniack RM (1985) Structure-function correlation of early stages of lung injury induced by intratracheal bleomycin in the rabbit. Am Rev Respir Dis 132:582-589 4. Boyd MR, Catigrani GL, Sasame HA, Mitchell JR, Stiko AW (1979) Acute pulmonary injury in rats by nitrofurantoin and modification by vitamin E, dietary fat, and oxygen. Am Rev Respir Dis 120:93-99 5. Brown EJ (1989) The interaction of small oligomers of complement 3B (C3B) with phagocytes. High affinity binding and phorbal ester induced internalization by polymorphonuclear leukocytes. J Biol Chem 264:6196-6201 6. Carp H, Miller F, Hoidal JR, Janoff A (1982) Potential mechanism of emphysema: alpha 1proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity. Proc Natl Acad Sci USA 79:2041-2045 7. Chandler DB, Barton JC, Briggs DD, Butler TW, Kennedy JI, Grizzle WE, Fulmer JD (1988) Effect of iron deficiency on bleomycin-induced lung fibrosis in the hamster. Am Rev Respir Dis 137:85-89 8. Clark JG, Kuhn C (1982) Bleomycin-induced pulmonary fibrosis in hamsters: effect of neutrophil depletion on lung collagen synthesis. Am Rev Respir Dis 126:737-739 9. Cohen HJ, Chovaniec ME (1978) Superoxide generated by digitoxin in stimulated guinea pig granulocytes. J Clin Invest 61:1081-1087 10. Cooper JA, White DA, Mathay RA (1986) Drug-induced pulmonary disease. Am Rev Respir Dis 133:321-340 11. Eiermann GJ, Dickey BF, Thrall RS (1983) Polymorphonuclear leukocyte participation in acute oleic acid-induced lung injury. Am Rev Respir Dis 128:845-850 12. Fantone JC, Ward PA (1982) Role of oxygen-derived free radicals and metabolites in leukocyte dependent inflammatory reactions. Am J Pathol 107:397-418 13. Fisher AB (1980) Oxygen therapy: side effects and toxicity. Am Rev Respir Dis 122:61-69 14. Fleischman RW, Baker JR, Thompson GR, Schaeppi UH, Illievski VR, Cooney DA, Davis RD (1971) Bleomycin induced interstitial pneumonia in dogs. Thorax 26:675-682 15. Fowler AA, Hyers TM, Fisher BJ, Bechard DE, Centor RM, Webster RD (1987) The adult respiratory distress syndrome. Cell populations and soluble mediators in the air spaces of patients at high risk. Am Rev Respir Dis t36:1225-1231 16. Freeman BA, Crapo JD (1981) Hyperoxia increases oxygen radical production in rat lung and lung mitochondria. J Biol Chem 256:10986-10992 17. Goldblum SE, Wu KM, Jay M (1985) Lung myeloperoxide as a measure of pulmonary leukostatis in rabbits. J Appl Physiol 59:1978-1985 18. Gross NJ (1977) Pulmonary effects of radiation therapy. Ann Intern Med 86:81-92 19. Guthrie LA, McPhail LC, Henson PM, Johnston RB (1984) Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide producing enzyme. J Exp Med 160:1656-1661 20. Henson PM, Zanolin B, Schwartznan RDA, Hong SR (1978) Intracellular control of human neutrophil secretion I. C5a-induced stimulus-specific demonstration and the effectof cytochalasin B. J Immunol 121:851-855 21. Hoover RL, Robinson JM, Karnovsky MJ (1987) Adhesion of polymorphonuclear leukocytes to endothelium enhances the efficiency of detoxification of oxygen free radicals. Am J Pathol 126:258-268
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22. Jenkinson SG, Duncan CA, Lawrence RA, Collins JF (1987) Lack ofenhancement of bleomycin lung injury in vitamin E-deficient rats. J Crit Care 2:264-269 23. Johnson KJ, Ward PA (1982) Acute and progressive lung injury after contact with phorbol myristate acetate. Am J Pathol 107:29-35 24. Klebanoff SJ (1980) Oxygen metabolism and toxic properties of phagocytes. Ann Intern Med 93:480-489 25. Martin WJ, Gadek JE, Hunnighake GW, Crystal RG (1981) Oxidant injury of lung parenchymal cells. J Clin Invest 68:1277-1288 26. Massey V (1956) The microestimation of succinate and the extinction coefficient of cytochrome C. Biochim Biophys Acta 34:255-256 27. McCord JM, Fridovich I (1978) The biology and pathology of oxygen radicals. Ann Intern Med 89:122-127 28. McCullough B, Collins JF, Johanson WG, Grover FL (1978) Bleomycin-induced diffuse interstitial pulmonary fibrosis in baboons. J Clin Invest 61:79-88 29. Mustafa MG, Tierney DF (1978) Biochemical and metabolic changes in the lung with oxygen, ozone and nitrogen dioxide toxicity. Am Rev Respir Dis 118:1061-1088 30. Oberley LW, Buettner GR (1979) The production of hydroxyl radical by bleomycin and iron (II). FEBS Lett 97:47-49 31. O'Flaherty JT, Redman JF, Jacobson DP, Rossi AG (1990) Stimulation and priming of protein kinase C translocation by a Ca transient independent mechanism. Studies in human neutrophils challenged with platelet activating factor and other receptor agonists. J Biol Chem 265:21619-21623 32. Rister M, Baehner RL (1976) The alteration of superoxide dismutase, catalase, glutathione peroxidase and NADPH cytochrome C reductase in guinea pig polymorphonuclear leukocytes and alveolar macrophages during hyperoxia. J Clin Invest 58:1174-1184 33. Shasby DM, Vanbenthuysen KM, Tate RM, Shasby SS, McMurtry I, Repine JE (1982) Granulocytes mediate acute edematous lung injury in rabbits and in isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen radicals. Am Rev Respir Dis 125:443-447 34. Snider GL, Bartolome RL, Goldstein RH, O'Brien JJ, Lucey EC (1978) Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Am Rev Respir Dis 117:289-297 35. Thrall RS, McCormick JR, Jack RM, McReynolds RA, Ward PA (1979) Bleomycin-induced pulmonary fibrosis in the rat. Am J Pathol 95:117-130 36. Thrall RS, Phan SH, McCormick JR, Ward PA (1981) The development of bleomycin-induced pulmonary fibrosis in neutrophil-depleted and complement-depleted rats. Am J Pathol 105:76-81 37. Thrall RS, Barton RW, D'Amato DA, Sulavik SB (1982) Differential cellular analysis of bronchoalveolar lavage fluid obtained at various stages during the development of bleomycininduced pulmonary fibrosis in the rat. Am Rev Respir Dis 120:488-492 38. Vercellotti GM, Yin HQ, Gustafson KS, Nelson RD, Jacob HS (1988) Platelet activating actor primes neutrophil responses to agonists: role in promoting neutrophil mediated endothelial damage. Blood 71:1100-1107 39. Watts FL, Oliver BL, Johnson GM, Thrall RS (1990) Superoxide production by rat neutrophils in the oleic acid model of lung injury. Free Radical Biol Med 9:327-332 40. Webster RO, Henson PM (1978) Rapid measurement of neutrophil exocytosis. Inflammation 3:129-135 41. Weitand JD, Davis WB, Holder JF, Mohammed JR Jr, Dorinsky PM, Gadek JE it986) Lung neutrophils in the adult respiratory distress syndrome, clinical and pathophysiologic significance. Am Rev Respir Dis 133:218-225 42. Zar J (1984) Biostatistical analysis. Prentice Hall, Inglewood, CA, pp 162-260 Accepted for publication: 21 August 1991
New York Inc. 1992
Superoxide Anion Production by Rat Neutrophils at Various Stages of BleomycinInduced Lung Injury Elizabeth B. Tarnell, 1 Bonnie L. Oliver, 1 George M. Johnson, 2 Florence L. Watts, 3 and Roger S. Thrall ~ Departments of IMedicine, Pulmonary Division, 2Pediatrics, and 3Anesthesiology, University of Connecticut Health Center, Farmington, CT 06030, USA
Abstract. This study investigated the level of activation of neutrophils isolated from rats at various stages of bleomycin-induced lung injury. Neutrophils were collected from blood and bronchoalveolar lavage (BAL) fluid and their superoxide anion (O2)-generating capacity measured in response to phorbol myristate acetate (PMA) and opsonized zymosan (OZ) stimulation. When stimulated with PMA, BAL neutrophils isolated from animals 3 days after bleomycin treatment had a significantly greater capacity to produce 0 2 than BAL neutrophils from animals 7 days after bleomycin treatment. The 0 2 levels of 7 day BAL neutrophils more closely resembled the resting levels obtained with circulating neutrophils from both control and bleomycintreated animals. There were no differences observed in any of the neutrophils when stimulated with OZ. Myeloperoxidase levels were measured in plasma and BAL and found to be elevated only in plasma at 7 days after bleomycin. These data demonstrate that neutrophil activation does occur in this model and that the activation appears to be transient, in response to specific stimuli and compartmentalized between the lung and blood.
Key words: Superoxide--Neutrophils--Bleomycin-induced lung injury. Introduction
Free radical generation by phagocytic cells has been implicated in the pathogenesis of several forms of acute lung injury, some of which lead to pulmonary fibrosis. Evidence has accumulated supporting the role of free radicals in diseases such as the adult respiratory distress syndrome (ARDS) [2, 15, 41], radiation pneumonitis [18], hyperoxia [13, 16, 32], emphysema due to cigarette
Offprint requests to: R. S. Thrall
42
E . B . Tarnell et al.
smoking [6], exposure to a variety of drugs and toxins [4,10, 25, 29, 30], immune complexes, and in various experimental models [23, 33]. Mechanisms by which oxygen metabolites inflict tissue injury include protein denaturation resulting in enzyme inactivation, and lipid peroxidation, which contributes to plasma membrane instability [12, 24]. The lung uses a variety of antioxidant enzymes and substances to aid in its protection, and impairment of these defenses may alter susceptibility to injury [21, 27]. Bleomycin-induced lung injury in a variety of animals has been accepted as a model of acute lung injury that progresses to fibrosis [1, 3, 14, 28, 34, 35]. In the rat model, peak periods of acute inflammation are observed at 3 and 7 days after bleomycin administration, followed by chronic injury and scar formation. Because neutrophils have been shown to make up a large percentage of cells in bronchoalveolar lavage (BAL) and in lung tissue during the acute stages of bleomycin injury [36], their level of activation may play an important role in the pathogenesis of lung injury in this model. In addition, clinical studies have shown large influxes of neutrophils in BAL and lung tissue in acute stages of various adult human lung diseases such as bacterial pneumonia and ARDS. Whether these neutrophils are beneficial or harmful in all cases remains to be determined. Since uncomplicated bacterial pneumonias do not routinely progress to ARDS, we hypothesize that neutrophils can be activated to different levels in various lung diseases and that this may influence the extent and/or type of injury. To test our hypothesis we studied both in vitro and in vivo levels of neutrophil activation in the bleomycin model of lung injury. First, we directly studied the levels of in vitro O~- production by neutrophils isolated from peripheral blood and BAL and rats during the acute stages of bleomycin-induced lung injury. Second, we indirectly approximated in vivo activation of neutrophils by measuring myeloperoxidase (MPO) levels in the plasma and BAL of these rats. Finally, to determine if neutrophil activation levels were different in various models, we compared the levels of both O2 and MPO to those we observed previously in the oleic acid model of acute lung injury [39].
Materials and Methods
Animals Adult male pathogen-free Fischer 344 rats, weighing 175-200 g, were obtained from Charles River Laboratories (Worthington, MA). The animals were free from respiratory disease and housed in isolation from all other laboratory animals. No evidence of infection was observed in any of our animals based on histologic evaluation.
Reagents Bleomycin sulfate (Blenoxane) was a gift from Bristol Laboratories (Syracuse, NY). Bovine serum albumin (BSA), cytochrome C (type VI), superoxide dismutase (SOD), n-ethyl maleimide (NEM), phorbol 12-myristate 13-acetate (PMA), HEPES, zymosan, and ficoll were purchased from Sigma
Neutrophil Superoxide Production in Bleomycin Injury
43
Chemical Company (St. Louis, MO). Hypaque (50%) was obtained from Sterling Drug, Inc. (New York, NY) and heparin (10,000/zg/ml) was purchased from Elkins-Sinn, Inc. (Cherry Hill, NJ). Hanks' balanced salt solution (HBSS) without magnesium and calcium was obtained from Hazleton Biologics (Lenexa, KS). Ketamine hydrochloride was purchased from Parke, Davis Co. (Detroit,
M1).
Induction of Lung Injury Animals were anesthetized via an intraperitoneal injection of ketamine HCI. Each animal in the treatment group received a single intratracheal injection of 2.0/zl of bleomycin sulfate in 0.3 ml of sterile saline [35]. Control animals received sterile saline in a similar manner. Animals were sacrificed at 3 and 7 days after bleomycin or saline injection. It should be noted that these times represent the peak periods of inflammation. Insufficient numbers of neutrophits are recovered at earlier and later time points after bleomycin for the assays to be performed.
Histologic Evaluation of Lung Damage Lung damage was determined as described previously [34, 35]. Lungs were removed after the animal was killed and inflated with 10% buffered formaldehyde at a constant pressure of 20 cmHzO. Tissue sections were processed in the usual manner for light microscopic examination with hematoxylin and eosin stain. Only qualitative histologic evaluations were made.
Cell Harvests Circulating Neutrophils. Blood was collected via cardiac aspiration with a heparinized syringe. Blood from 4 rats was pooled to yield enough cells for the superoxide assay. Blood was diluted with an equal volume of heparinized saline and neutrophils were isolated as described below. BAL Neutrophils. BAL
was performed with 50 ml of sterile heparinized saline in 5-7 ml aliquots, injected intratracheally to inflate the lungs fully. Fluid was then retrieved by aspiration and strained through gauze to remove mucus. Cells from 4-7 rats were pooled to obtain enough cells for the superoxide assay. Ceils were washed and neutrophils isolated as described below.
Isolation of Neutrophils Cell suspensions were placed over an equal volume of ficoll-Hypaque gradient (ficoli 9% and Hypaque 34% in a ratio of 2.4 : 1) and centrifuged at 350 g/4°C for 20 minutes. The monocyte-rich upper layer was removed, leaving the neutrophil-rich pellet. Hypotonic lysis of erythrocytes was performed. Neutrophils were washed with HBSS containing 10 mM of HEPES. Cells were counted using a standard AO hemacytometer. This method of isolation yielded a cell suspension of >85% neutrophils. Viability was determined to be >98% with nigrosin dye exclusion.
Superoxide Assay. Cells
were suspended in HBSS containing 10 mM HEPES at 3 x 106 cells/m1. Superoxide release was determined at 37°C using the superoxide dismutase-inhibitable reduction of cytochrome C at 550 nm for 5 min [9]. Absorbance was read on a Beckman Model 24 spectrophotometer. The reaction mixture contained 119 mM cytochrome C, 1 mM sodium azide, 1 mM calcium chloride, 2.6 mM magnesium chloride, and was initiated with the addition of the appropriate stimulus, 100 ng/ml PMA or 1 mg/ml opsonized zymosan (OZ). The reaction was stopped after 5 min by the addition of 0.5 mM NEM. Generation of O~- was calculated using 21.2 x 103 m/cm as the molar extinction coefficient for reduced-minus-oxidized cytochrome [26].
44
E.B. Tarnell et al.
Myeloperoxidase Assay Myeloperoxidase (MPO) activity, used as an indicator of neutrophil degranulation [17], was measured by a micromodification [40] of the colorimetric assay measuring oxidation of o-dianisidine hydrochloride at 450 nm [20]. Plasma or BAL samples were diluted with phosphate-buffered saline fourfold before the assay. Sample measurements were done in triplicate. The molar absorbancy of oxidized o-dianisidine, 1.13 x 104/cm, was used to calculate the moles of hydrogen peroxide decomposed. One unit of peroxidase activity was defined as that degrading 1 t~molof peroxide/rain at 25°C.
Statistical Analysis Statistical analysis was performed using 1-way analysis of variance followed by the Newman-Keuls procedure [42].
Results
Histologic Analysis of Lung Damage Histologic changes induced by b l e o m y c i n administration in the lungs of the experimental animals were similar to those described previously in this laboratory [35]. Severe intra-alveolar e d e m a and h e m o r r h a g e with a diffuse interstitial and inra-alveolar neutrophil cell infiltrate were o b s e r v e d 3-7 days after bleomycin administration.
Superoxide Generation in Circulating Neutrophils W h e n stimulated with PMA, circulating neutrophils (CN) isolated f r o m animals at 3 and 7 days after b l e o m y c i n p r o d u c e d O f levels similar to conrol (Fig. la). W h e n stimulated with OZ, C N f r o m animals at 7 days after t r e a t m e n t p r o d u c e d significantly lower (p < 0.01) 0 2 levels than C N f r o m 3 days, but neither s h o w e d a significant change f r o m control (Fig. lb).
Superoxide Generation in BAL Neutrophils The O{ levels p r o d u c e d by B A L neutrophils isolated 3 days after b l e o m y c i n administration w e r e significantly elevated a b o v e the levels p r o d u c e d b y either the B A L neutrophils obtained 7 days after b l e o m y c i n (p < 0.01) (Fig. 2a) or any of the C N (p < 0.01) f r o m Fig. la, which w e r e stimulated with PMA. After stimulation with OZ, 0 2 levels in B A L neutrophils were not significantly different b e t w e e n the 3 and 7 day animals (Fig. 2b), nor was there a significant difference b e t w e e n these B A L neutrophils or any of the OZ-stimulated C N in Fig. lb.
Neutrophil Superoxide Production in Bleomycin Injury
A
45
B PMA
OZ.
rE u3 cO -J w O o
40.
40,
30.
30,
II
7" E ¢,v LU O
20.
20.
I
X
o
nuJ
T 1
10-
10,
cO
CON
3 DAY
7 DAY
CON
3 DAY
7 DAY
TIME P O S T B L E O M Y C I N
Fig. 1. a. Superoxide generation in circulating neutrophils (CN) after stimulation with PMA at 3 and 7 days after bleomycin treatment. PMA stimulated CN produced O2- levels similar to control. b Superoxide generation in CN after stimulation with OZ. OZ-stimulated CN produced significantly lower (*p < 0.01) O~- levels at 7 days after bleomycin treatment compared to 3 days. Vertical bars represent mean + / - SEM of 4 experiments.
Myeloperoxidase Levels Plasma levels of M P O were significantly (p < 0.01) elevated 7 days after bleomycin treatment c o m p a r e d to both control and 3 day levels (Fig. 3a). There were no significant differences between B A L levels of M P O at any time (Fig. 3b).
Discussion
We have d e m o n s t r a t e d that at the period of p e a k acute inflammation, 3 days after bleomycin treatment, B A L neutrophils are activated to produce high levels of O2 when stimulated in vitro by PMA. Later, at 7 days after b l e o m y c i n treatment, PMA-stimulated B A L neutrophils a p p e a r to be at " r e s t i n g " or con-
46
E . B . Tarnell et al.
B
A
o_zz
PMA L~ -J --I
LU
% 40. I
o 30 E LU C~ X O o~ uJ o_
~
30'
T
20
20,
T 10-
10,
3 DAY
7 DAY
TIME
3 DAY
POST
7 DAY
BLEOMYCIN
Fig. 2. a. Superoxide generation in BAL neutrophils isolated 3 and 7 days after bleomycin and stimulated with PMA. At 3 days, O~- levels were significantly (*p < 0.01) higher than at 7 days. This level of activity was also significantly higher (p < .01) than those observed in any CN from Fig. la. b Superoxide generation in BAL neutrophils isolated 3 and 7 days after bleomycin and stimulated with OZ. There was no significant difference between BAL ceils at 3 and 7 days after bleomycin treatment, nor was there a difference between these cells and OZ stimulated CN from Fig. lb. Vertical bars represent mean + / - SEM of 4 experiments.
trol levels of activity. In contrast, the phagocytic stimulus, OZ, did not increase O2 production in BAL neutrophils at either time point. Our data therefore suggest that activation of neutrophils does occur in the bleomycin model and that the activation may be in response to a specific stimulus. The activation also appears to be transient depending on the stage of lung injury. Plasma levels of MPO were significantly elevated 7 days after bleomycin treatment, further indicating in vivo neutrophil activation. MPO levels were not elevated in the BAL at any time after bleomycin treatment. Previous studies from our laboratory have shown that 0 2 levels in CN and BAL neutrophils obtained at peak periods of inflammation in the oleic acid model of acute lung injury were not elevated above "resting" levels [39]. In contrast, BAL neutrophils isolated from the bleomycin model in this study were activated above "resting" levels. In addition, different plasma and BAL MPO
Neutrophil Superoxide Production in Bleomycin Injury
A
47
B PLASMA
BAL
80m
. D ¢-.
60-
o
Q. 40-
lo
CON
3 DAY
7 DAY
TIME
POST
CON
3 DAY
7 DAY
BLEOMYCIN
Fig. 3. a. Myeloperoxidase (MPO) levels in plasma from control and bleomycin-treated animals. There was a significant (*p < 0.01) increase in the MPO level at 7 days compared to both control and 3 days after bleomycin treatment, b MPO levels in BAL fluid from control and bleomycintreated animals. There was no difference between MPO levels at any time point. Vertical bars represent mean + / - SEM of 6 samples.
levels were observed in the bleomycin and oleic acid models. Although both models demonstrated high levels of MPO, the activity was expressed at different sites. In the bleomycin model MPO levels were elevated in plasma only, but in the oleic acid model MPO levels were elevated in BAL. Together, these data demonstrate that neutrophils obtained at peak periods of acute inflammation in different animal models of lung injury can be stimulated or primed to attain different levels of activation. The role that these differences may play in the pathogenesis of these models is unclear. The 3 day bleomycin BAL neutrophils appeared to be activated or "primed" in vivo since in vitro stimulation with PMA generated high levels of O~. The low BAL and plasma MPO levels at 3 days after bleomycin suggest that the neutrophils did not degranulate within the air spaces of the lung or in the plasma. It is possible that degranulation occurred in the interstitium of the lung where most of the inflammation resides and MPO levels in BAL or plasma would not
48
E.B. Tarnell et al.
reflect it. Another explanation could be that the PMA-stimulated 3 day bleomycin BAL neutrophils were "primed" in vivo but did not receive the secondary stimulus to become activated and degranulate. Priming agents such as lipopolysaccharide [19] and platelet-activating factor [38] have been shown not to be stimulus-specific and indeed primed cells are responsive to a variety of various stimulators other than the priming agent. Thus, if the cells were primed in this manner one would have expected them to also be responsive to OZ. However, a priming mechanism is still possible if priming occurred through activation of protein kinase C that has been shown to internalize and downregulate plasma membrane receptors while leaving the cell hyperactive to nonreceptor antagonists such as PMA [5, 31]. Thus, the cell would not be responsive to receptormediated activators such as OZ but still responsive to PMA. This may also explain the decreased CN response to OZ at 7 days after bleomycin treatment, when MPO levels were elevated in plasma. The cells could have been activated in vivo via protein kinase C, which downregulates membrane receptors for OZ and this, in turn, results in decreased responsiveness to OZ. The activated cells degranulate and release high levels of MPO into the plasma. These 7 day cells are not "primed"; they have already been activated and degranulated and now are in a state of "desensitization" or are incapable of responding to further stimuli, hence their inability to generate an elevated response even to PMA. The role of the neutrophil and oxygen metabolites in lung injury is controversial. Their significance in both the oleic acid and bleomycin models has been studied previously [7, 11, 22, 37]. Jenkinson and colleagues [22] administered bleomycin to vitamin-E-deficient rats and found no increase in mortality, evidence of lipid peroxidation, or lung collagen content when compared with controls and suggested that perhaps lipid peroxidation was not critical to development of the lesion. Chandler et al. [7] have subsequently shown that iron deficiency in bleomycin-treated hamsters reduced the severity of pulmonary fibrosis, possibly by preventing iron-catalyzed oxygen radical formation. We have further shown that neutrophil depletion in the oleic acid model suppresses lung injury, although the mechanism by which neutrophils contribute to damage may not be due to free radicals since treatment with catalase, dimethylsulfoxide, or superoxide dismutase had no effect on injury [11]. In contrast, we [37] and others [8] have shown that neutrophil depletion in the bleomycin model enhanced injury, evidenced by increased collagen. The neutrophil has a wide variety of capabilities and whether it is beneficial or harmful may very well depend on how that cell is stimulated or activated. Direct in vivo evidence demonstrating this is difficult to obtain and we believe that this is one approach to help us understand better in vivo neutrophil function during active stages of inflammation. In summary, our data suggest that in the bleomycin model, in vivo activation of neutrophils does occur and may be in response to specific stimuli, is transient, and appears to be compartmentalized. Furthermore, when comparing these results with similar data from the oleic acid model, we have shown that neutrophils are capable of attaining varied levels of activation in response to different types of lung injury.
Neutrophil Superoxide Production in Bleomycin Injury
49
Acknowledgment. The authors wish to thank Ms. Debra Parkinson for the excellent illustrations.
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