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Effect of blood leucocyte depletion on the inflammatory response of the lung to quartz
rOXI(‘OLOOY AND 4PPLIED PHARMACOLOGY 109, l27- 136 (I 99 1)
Effect of Blood Leucocyte Depletion on the Inflammatory Response of the Lung to Quartz’ ROGENE
F. HENDERSON,
Inhalatiorl
JACK R. HARREMA,
JON A. HOTCHKISS,
AND DIVA S. BOEHME-
Toxicology Research Institute, Lovelace Biomedicul and Environmental Research Instltutr. P.O. Bm 5890. Albuquerque, New .Meuico X718.5
Received October 12, 1990; accepted Februury 8. 1991 Effect of Blood Leucocyte Depletion on the Inflammatory Response of the Lung to Quartz HENDERSON,R. F., HARKEMA, J. R., HOTCHKISS,J. A., AND BOEHME, D. S. (199 1). To.xico/..lpppl. Pharmacol. 109, 127-136. This study determined the effect of blood leucocyte depletion on the early inflammatory response of the lung to a-quartz. F344/N rats were instilled intratracheally with either physiological saline or 2 or 5 mg of aquartz suspended in saline. One day prior to the instillation. half of the rats received an ip injection of rabbit antiserum that had been raised against rat neutrophils. The other half of the rats received an ip injection of normai rabbit serum. One day after the instillation of saline or quartz, the animals were euthanized and observed for changes in blood cell numbers. lung histopathology, and bronchoalveolar lavage fluid (BALF) content of indicators of an inflammatory response and cytotoxicity. The rabbit antiserum depleted the blood of most white blood cells of all types. BALF fluid from saline-instilled animals did not differ between the white blood cell-depleted and the nondepleted animals except for a 20% reduction in numbers of alveolar macrophages in the depleted animals. BALF fluid from the nondepleted, quartz-instilled animals had a dose-dependent increase in content of neutrophils and protein (indicator of an increase in the permeability of the alveolar/capillary barrier) as well as an increase in lactate dehydrogenase and glutatbione reductase (cytoplasmic enzymes whose presenceextracellularly indicates cytotoxicity). alkaline phosphatase (indicator of type II cell secretory activity), &glucuronidase. and acid proteinase (lysosomal enzymes) activities. The higher dose of quartz also elicited an increase in LTB, and PGEz content of BALF. GSH content of BALF was decreased by the quartz exposure. The depletion of blood white blood cells prevented the influx of neutrophils into the alveoli of the quartz-exposed rats and decreased the BALF markers of capillary permeability and cytotoxicity (protein content and extracellular cytoplasmic enzymes). The absence of neutrophils in the alveoli had no effect on the lysosomal content of BALF, indicating that the neutrophils were not the source ofthese enzymes in nondepleted rats exposed to aquartz. The quartz-induced elevation of LTB4 in BALF was not observed in depleted rats, suggesting that neutrophils may be the source of the increase in this leukotriene in the BALF. Both the GSH content and the alkaline phosphatase activity in BALF were enhanced in the absenceof alveolar neutrophils. The enhancement of GSH in BALF is consistent with the neutrophils being the source of reactive oxygen species that deplete GSH. The increased alkaline phosphatase activity in the BALF of both the depleted and nondepleted animals is consistent with the type II cell hypertrophy that was induced by quartz instillation and was neutrophil independent. The study provides information on the probable sources of some of the inflammatory mediators in BALF and suggeststhat neutrophils contribute to the acute toxicity accompanying the inflammatory response of the lung to quartz. The neutrophil is not required. however. for the production of type II cell hypertrophy. 8 1991 Academx press.inc.
Our laboratory (Henderson, 1988, 1989), as well as others (Beck et al., 1982; Moores et ’ The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper. for governmental purposes, is acknowledged
al., 1980) have documented the inflammatory response of the lung to inhaled or instilled particles by analysis of bronchoalveolar lavage fluid (BALF) from exposed animals. Cellular changes in BALF during acute inflammation include an influx of neutrophils (PMN) and 127
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activation of pulmonary alveolar macrophages (PAM). The neutrophil has been implicated as contributing to the lung injury incurred during an inflammatory response. Neutrophils damage cultured endothelial cells (Weiss et al., 198 1) and epithelial cells (Suttorp and Simon, 1986). The mechanism of neutrophil-induced injury may be due to toxic oxygen species released by this leucocyte (Mustafa and Tiemey, 1978). The oxidative killing mechanisms of neutrophils toward bacteria are well known (Cheson et al., 1977). The enzymatic activities found in the acellular supernatant of BALF provide important information concerning the degree of cytotoxicity present (measured by extracellular activities of such cytoplasmic enzymes as lactate dehydrogenase, LDH, or glutathione reductase, GR) and the release of lysosomal enzymes (such as P-glucuronidase and acid protease activities) during an inflammatory response to inhaled particles (Henderson, 1988, 1989). However, the cellular source of these enzymes is unknown. Eicosanoids are increased in BALF in response to inhaled particles, particularly LTB4 and PGF2, (Henderson et al., 1988a). The cellular source of these metabolites is also not known. Finally, reduced glutathione (GSH) is increased in a dose-dependent manner in BALF from animals exposed to diesel soot, but the source of the GSH is not known (Henderson et al., 1988b). To better understand the mechanisms of the inflammatory response to particles in the lung, it would be valuable to know the cellular source(s) of these mediators of inflammation. The primary purpose of this study was to obtain information on the source of the inflammatory mediators in BALF by eliminating one of the potential sources, the neutrophil. We hypothesized that the absence of the neutrophil would decrease markers of cell injury in BALF, but would have little effect on the lysosomal enzymes or the GSH. The toxic agent used to induce an inflammatory response was a-quartz and the animal model was the F344/N rat, because of the large amount of information known about the pulmonary inflammatory response to quartz in
ET AL.
that animal (Driscoll et al., 1990; Warheit et al., 199 1). The method for depleting the neutrophils was a rabbit anti-rat neutrophil antiserum that has been used in previous studies at our laboratory (Harkema, 1990). METHODS Animals. Young (1 l-l 5 weeks) female F344/N rats from the Institute’s barrier breeding facility were used in the study. Prior to the study the animals were housed two or three per cage in polycarbonate cages supplied with sterilized hardwood chip bedding and filter tops. Animal rooms were maintained at 20-22°C with a relative humidity of 20-50s and a 12-hr light/dark cycle starting at 6:00 AM. Water from bottles with sipper tubes and food (Lab Blox, Allied Mills, Chicago, IL) were provided ad libitum. Murine neutrophil antiserum. The method for depleting neutrophils was by intraperitoneal (ip) injection of a cytotoxic, rabbit anti-rat neutrophil antiserum (Accurate Scientific Corp., Westbury, NY). This antiserum was raised against normal rat neutrophils isolated from whole blood and preadsorbed with rat serum and red blood cells (RBCs). The procedures for producing this antiserum were based on standard methodologies described by Davis et al. ( 1969). In this study, the PMN-depleted animals received a 1 ml injection of the antiserum 24 hr prior to exposure to LYquartz or saline. An equal number of animals received an injection of 1 ml of normal rabbit serum at the same time prior to the exposures to a-quartz or to saline. Exposure to a-quartz. To achieve a high concentration of a-quartz in the lungs of rats in a short period of time, the rats received either 0.5 ml of physiological saline or 2 or 5 mg of a-quartz suspended in 0.5 ml saline instilled intratracheally according to the method of Brain (1976). A concentrated suspension of quartz was allowed to stand for 90 min to remove larger particles. The small-particle suspension was then decanted and aliquots were dried and weighed to determine the concentration of a-quartz. The suspension was then diluted with saline to achieve a concentration of 2.0 or 5.0 mg quartz/O.5 ml saline. Experimental design. The experimental design is outlined in Table 1. There were 24 animals treated with 2.0 mg of quartz, 24 treated with 5.0 mg of quartz, and 24 given saline intratracheally. Half of each of the quartztreated groups and half of the saline-treated animals were injected ip with the antiserum 24 hr before the intratracheal injections. The other half were injected ip with normal rabbit serum. In each of these subsets of 12 animals, I day after the exposure to quartz or saline, half of the animals received bronchoalveolar lavage (BAL) and the other half were observed for histopathology of the left lung and for the cellular content of the blood. In addition, the right lung of the animals designated for histologic evaluation was lavaged to obtain sufficient Iavage fluid for analysis of all the parameters of interest.
WBC TABLE
DESIGN
Animals injected with normal rabbit serum
0 (saline 2 5
BALF analysis only)
6 6 6
AND
I
EXPERIMENTAL
Quartz instilled (mg)
DEPLETION
Histopathology” 6 6 6
Animals injected with rabbit antiserum BALF analysis 6 6 6
Histopathology” 6 6 6
a BALF was obtained from the right lung of these animals as well as from the lungs of animals assigned for BALF analysis.
Hlsiopatho1og.v. The left lung was intratracheally perfused with 10% buffered formalin at 30cm fixative pressure for at least 24 hr. Midsagittal sections of the left lung lobe were embedded in paraffin, sectioned at 4-6 pm in thickness, and stained with hematoxylin and eosin for light microscopic examination. .-tnalysis oiblood cells. Blood samples were taken from the heart at the time of death and total and differential blood cell counts were made. The total white and red blood cell counts were made on a Coulter S550 whole blood counter. Differential counts of leucocytes were determined by counting 100 cells from blood smears stained with a Wright-Giemsa stain. 5ronchoalvedar luvage. The lavage was performed on the excised lung taken from the rats under anesthesia (4% halothane in oxygen). For the total lung lavages, 5 ml of sterile physiological saline was gently instilled into the trachea and allowed to fill all lung lobes. The fluid was immediately withdrawn by gentle suction. This was repeated one more time and the lavage fluids combined. In the animals for which the left lung was used for evaluation of the histopathology, the left lung was clamped off and the right lung lavaged two times with 4 ml of saline as described above. The right lung was then clamped off and the clamp removed from the left lung bronchus. The left lung was then fixed with 10% formalin as described above. .Inul.vsi.r qfbronchoalveolar lavage. The bronchoalveolar lavage fluid was analyzed for various markers of cell damage and inflammation. An aliquot was removed and analyzed for total cell counts by a hemocytometer count. The ceils were then removed by centrifugation at 300g and the cell oellet was resusnended in 1 ml of saline. A cytospin prep&ation ofthe ceils was stained with a WrightGiemsa-type stain (DifF-Quik, Harleco) and a differential cell count made. Cell viability was determined by trypan blue dye exclusion. The acellular supematant was analyzed for the following parameters. The soluble protein concentration was measured as a marker for damage to the alveolar capillary
LUNG
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INFLAMMATION
barrier. The amount of activity of the cytoplasmic enzyme. lactate dehydrogenase (LDH), was determined as a marker for cytotoxicity. The glutathione reductase activity was also measured as a second cytoplasmic enzyme that would be expected to be involved in antioxidant activities. Two lysosomal enzyme activities. @-glucuronidase and acid proteins activities, were measured as markers of enzymes expected to be released from phagocytes. either during phagocytic activity or cell death. Alkaline phosphatase activity was measured as a possible marker of type II cell secretions (Miller d a/.. 1987). The arachidonic acid meas tabolites. LTBA. PGF2
RESULTS Haad cell counts. The blood cell counts in the animals of the various treatment groups are shown in Table 2. The counts of all types of white cells, not just the neutrophils, appeared to be depressed by the antiserum, although the number of monocytes and eosinophils in rat blood is normally low, and it is difficult to detect a depression in their numbers.
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ET AL.
TABLE 2 HEMATOLOGY DATA Animals injected with normal serum Parameter RBC ( 106/mm3) WBC ( 103/mm3) Hemoglobin (g/dl) Hematocrit (%) Neutrophils ( I03/mm3) Lymphocytes ( 103/mm3) Monocytes ( 103/mm3) Eosinophils ( 103/mm3)
Saline 8.8 4.1 15.1 47.7 0.55 3.5
* 0.2 +- 0.4 kO.3 + 1.1 t 0.09 + 0.3 00:
2 mg
quartz
9.0 ?I 0.2 4.1 + 0.4 15.4 + 0.3 48.8 f 0.9 0.3 r 0.04 3.7 k 0.4 Od Od
5w quartz 8.4 3.3 14.5 44.9 0.4 2.7
f 0.1 * 0.3 f 0.2 f 0.5 2 0.1 f 0.2 Od 0.1 + 0.04
Animals injected with antiserum
2 mg
Saline
quartz
8.9 f 0.3 0.5 + 0.1” 15.2 r+ 0.5 47.7 + 2.0 0‘Z.h 0.5 f 0.13” Ob Ob
9.3 f 0.2 0.7 -+ 0.2” 15.8 + 0.2 49.6 -t 0.9 0n.h 0.7 f 0.2” Ob Ob
5 mg quartz 8.1 f 0.5 + 14.1 + 43.1 +
0.4 0.2” 0.6 1.9
0 oB
0.5 z!c0.2” Ob Ob
Note. (R + SE, n = 5-6). n Differs from corresponding values for animals injected with normal serum p 5 0.05. ’ Values from all animals were zero. ’ Values from five animals were zero and from one animal was 0.1. d Values from four animals were zero and from two were 0.1.
BALF parameters. The alterations in parameters measured in the BALF and the lung weights of the animals in the study are listed in Table 3. The data indicate that the rabbit antiserum itself (as opposed to normal rabbit serum) had no effect on most of the parameters in animals not exposed to a-quartz. None of the values for the nondepleted, control animals (saline instilled) were statistically different from historical controls. There were no significant differences between the values of the various parameters in BALF from the control animals in the depleted and nondepleted rats with one exception. (Compare Columns 1 and 4 of the 6 columns of numbers.) There was a decrease in total nucleated cells in BALF from the depleted animals primarily due to a 20% decrease in alveolar macrophages. The administration of the a-quartz caused an acute inflammatory response in the animals that did not receive antiserum. (Compare Columns 2 and 3 with Column 1 in Table 3.) An inflammatory response was observed as indicated by an increase (in most cases dosedependent increases) in the BALF content of protein and neutrophils and in LDH, glutathione reductase, ,&glucuronidase, acid protease, and alkaline phosphatase activities. In
animals receiving the higher dose of a-quartz, there were also increases in BALF content of PGE2 and LTB4 and a decrease in GSSG and in the total glutathione equivalents. Thus, the instilled a-quartz did induce the expected inflammatory response and there was an indication of cytotoxicity accompanying the inflammation. The depletion of white blood cells by the antiserum altered the lung’s response to the instilled a-quartz. (Compare Columns 5 and 6 with Columns 2 and 3 in Table 3.) The most striking difference between the response of the depleted vs the nondepleted rats was the lack of an influx of neutrophils into the bronchoalveolar region in the depleted animals. The resident white blood cell of the alveoli, the macrophage, was decreased in number to half that of the saline-instilled, depleted, control animals and one-third that of the quartz-exposed, nondepleted rats. The indicators of cytoxicity, i.e., the cytoplasmic enzyme activities, LDH, and glutathione reductase, were elevated in the exposed, depleted animals but the elevated values were one-half to one-third those in the exposed, nondepleted rats. In contrast, the lysosomal enzymes, fi-glucuronidase and acid protease, were both elevated in response to a!-
WBC DEPLETION
AND LUNG
INFLAMMATION
131
TABLE 3 EFFFECT
OF WHITE
BLOOD
CELL DEPLETION
ON THE RESPONSE OF THE LUNG
Animals injected with normal rabbit serum BALF parameter Lung weight, g Total nucleated cells. IO’/ml PAM, 103/ml PMN, 1O’/ml Alkaline phosphatase, mIU/ml LDH, mlU/ml GR, mIU/ml BG, mIU/ml Protein, mg/ml Acid protease, fig Hb/ 4 hr/ml PC%. pg/ml PGL. m/ml LTB, , pg/ml Cell viability, R GSH, nmol/ml GSSG. nmol/ml GSH plus 2 GSSG’
TO CX-QUARTZ
Animals injected with rabbit antiserum
Saline control
2 mg quartz
5 mg quartz
Saline control
0.98 f 0.04
1.10 -+ 0.03
1.16 + 0.10
0.91 * 0.05
0.97 ? 0.02’
I .oo rt 0.04
621 + 84 504 f 33 116 & 58
3155 IL 145” 669 f 42 2470 t 130”
2431 f 106” 516 f 85 1850 f I IO’
410 + 20* 391 + 15” 20t 13
223 f 38”,” 222 +- 38”.b 1.3 + 0.6h
205 + 39”.’ 203 * 39,‘.’ 1.8 t O.h*
6.0 +- 0.8 71?7 11.7 t 1.4 0.13 IT 0.02 0.23 r 0.04
14.8 f 282 k 25.6 + 1.38 f 0.49 f
29.5 553 40.0 3.72 0.72
7.5 * 73 2 I I .4 f 0.1 1 ? 0.20 k
60.7 _t 24.3”,h 124 i 14”.” 17.0 * 1.5ti,” 1.25 f 0.23” 0.27 xk 0.03h
44. I t 0.h”:” 182 t I?“.’ 18.2 _t 0.4”.’ 2.68 -+ 0.04’0.33 f 0.(13’
27 +- 1 49 f 9 30 t 2 16+ I 87 + 2 2.35 t 0.45 0.29 + 0.07 2.94 rt 0.58
37-+ I” 54 I? 6 26 -+ 2 20 f 2 84? 3 1.78 + 0.15 0.19 + 0.03 2.16 + 0.20
33 f 2” 80 -?I9” 36?2” 19 * 4 65 f 4h 2.71 -+ 0.36 0.26 f 0.03 3.23 f 0.39
34 +- I ’ 71 *4 34 i- 3 18t I’ 83?3’ 3.34 i 0.33 0.20 zk 0.1I5 ’ 3.74 t 0.42’
0.5” 10” 1.2” 0.05” 0.03”
+ 2.8” 2k 24” It 1.9” 2 0.16“ + 0.04“
I.1 5 0.6 0.01 0.04
37 + I” 27 * 1 85 t 5” 63 +- 6 33* I 30 i 2 27 f 2” 23 t 3 93* 1 71 *9 1.53 f 0.18 2.57 t 0.43 0.05 -+ 0.0 1il 0.20 i- 0.03 1.63 + 0.19 2.97 + 0.49
2 mg quartz
5 mg quartz --
Note. (.F f SE. ,r = 5-6). ’ Statistically significant from the appropriate saline control (p < 0.05). ’ Differ from corresponding value in nondepleted animals, p s 0.05. ‘ Total glutathione equivalents. Values used are in nmol; thus, the nmol ofGSSG are multiplied by 2 to be equivalenr to the nmol of GSH necessary to form a nmol of GSSG.
quartz to the same extent in the depleted and nondepleted animals. Protein concentration in the BALF of the depleted animals was not significantly elevated, also in contrast to the nondepleted animals. The increases in LTB4 seen in the nondepleted animals did not occur in the depleted rats. In contrast, GSH levels were higher in the BALF from the depleted animals instilled with 5 mg quartz than in the nondepleted rats. The only other parameter that was increased in BALF to a greater degree in response to a-quartz in the depleted vs the nondepleted animals was the alkaline phosphatase activity. Histopatholo~~~. The principal alteration in the lungs from nondepleted rats that were instilled with quartz was a multifocal, coalescing acute alveolitis (Fig. 1C). This alveolitis was
characterized by a moderate to marked influx of neutrophils into alveolar septa, alveolar duct walls. alveolar airspaces, and interstitium around centriacinar pulmonary arteries and bronchioles. In the affected regions of the lung there was moderate thickening of the alveolar septa due to capillary congestion. In addition, there was a marked hypertrophy and vacuolization of type 2 pneumocytes and alveolar macrophages in all of these rats, and a mild alveolar macrophage hyperplasia in some of these animals. Quartz particles were present individually or in aggregates within alveolar airspaces or phagocytized within alveolar macrophages. Proteinaceous material and necrotic cellular debris were also present in some of the alveolar airspaces. Rats instilled with 5 mg of quartz had more severe alveohtis ( i.e.,
132
HENDERSON
ET
AL.
WBC
DEPLETION
AND
more neutrophils and other cellular changes in larger areas of the lung parenchyma) compared to animals instilled with 2 mg quartz. In contrast, antiserum-injected rats that were instilled with 2 or 5 mg of quartz had a conspicuous absence of acute alveolitis (Fig. 1D). Although there was a lack of neutrophil influx into the alveolar airspaces in these animals, hypertrophy and vacuolization of type 2 pneumocytes and alveolar macrophages were similar to those present in the nondepleted rats instilled with quartz. No hyperplasia of alveolar macrophages was evident in any of the depleted rats instilled with quartz. Nondepleted rats instilled with saline only had no significant alterations in the lung (Fig. 1A). Depleted rats instilled with saline only had a marked attenuation of the number of polymorphonuclear and mononuclear leukocytes within the lumens of pulmonary arterioles and veins and within capillaries in alveolar septa (Fig. 1B).
LUNG
INFLAMMATION
133
14% of normal levels while neutrophils were essentially eliminated. For the purpose of this study, the depletion of most white cells in the blood was not a major problem because the normal initial inflammatory response observed in rat lungs involves the influx of only neutrophils, and not lymphocytes (Henderson, 1988; also see Table 3, present study). Following the neutrophil influx, there is recruitment of monocytes, but the time frame of the current study would not allow observation of that recruitment. Therefore, the current study allowed us to determine what effect the lack of neutrophil influx has on the acute response to a-quartz in the lung. Neutrophil depletion was accompanied by a decrease in the BALF protein content and in indicators of cytotoxicity (the cytoplasmic enzyme activities, LDH and GR) in response to a-quartz. This suggests that the neutrophil influx plays a major role in increasing the permeability of the alveolar/capillary barrier and in producing cellular toxicity during the inflammatory response. This is in agreement DISCUSSION with earlier work indicating the cytotoxicity of neutrophils in association with reperfusion The purpose of this study was to obtain in- injury (Lucchesi. 1990), adult respiratory disformation on the source of the inflammatory tress syndrome (Swank and Moore. 1989). mediators in BALF by eliminating one of the potential sources, the neutrophil. In the past, immune complexes (Johnson and Ward. 1982), and in lung epithelial cells in culture highly toxic materials, such as nitrogen mus(Suttorp and Simon, 1986). tard (Shasby et al., 198 1) or hydroxyurea The neutrophil depletion did not, however. (O’Byrne et al., 1984), have been used to dehave any effect on the BALF levels of lysoplete animals of neutrophils to determine the somal enzymes (acid protease, P-glucuronirole of these cells in various processes. The dase), indicating that neutrophils are not the disadvantage of such treatments is the toxic source of the high levels of these enzyme acside effects that confound such experiments. tivities observed in response to quartz in this In the present study, neutrophils were depleted study and in other studies (Beck et al., 1982; by use of an antiserum prepared from rabbits Henderson et al., 1985; Dethloff et al., 1986). injected with rat neutrophils. The antiserum The likely source of these lysosomal enzymes was adsorbed with rat serum and rat RBCs to remove any antibodies developed against the is the activated macrophage, although other serum proteins or RBCs. The antiserum treat- sources, such as epithelial cells, cannot be exment of the rats did not result in any decrease cluded. The suggestion that the macrophage is the source of the lysosomal enzymes is in the blood RBCs (Table 2). The antiserum did, however, reduce the white blood cells in agreement with earlier studies in which ( WBCs) in the blood to approximately 10% of only particle-induced inflammatory responses caused an increase in lysosomal enzyme acthe normal level. Lymphocytes, the major tivity in BALF. while pulmonary inflammaWBC present, were reduced to approximately
134
HENDERSON
tion induced by pollutant gases did not (reviewed by Henderson, 1988). The concentrations of arachidonate metabolites in BALF were not greatly elevated in response to quartz despite the other indicators of an inflammatory response. The eicosanoid concentrations in BALF were not affected by the instillation of a-quartz in either the depleted or nondepleted animals except at the highest dose of quartz. Twenty-four hours after the instillation of 5 mg of quartz, PGE2 and LTB4 concentrations were elevated in BALF from the nondepleted animals but the LTB4 was not elevated in the depleted animals. This points to the neutrophil as a potential source of the LTB4 or as being a factor that enhances the release of the metabolite from other cells. Work reported by Ham et al. ( 1983) indicates that activated neutrophils release LTB4, and this release is inhibited by PGE2. The increase in PGEz at the same time as the increase in LTB4 in nondepleted rats exposed to the highest dose of a-quartz may represent a control mechanism to down regulate the inflammation. The antioxidant tripeptide, glutathione, was reduced in the BALF from the nondepleted rats exposed to the highest amount of quartz, while the depleted animals did not show such a reduction in BALF glutathione equivalents. The release of GSH from quartz-stimulated macrophages has been reported (Boehme et al., 199 1; Boehme and Henderson, 1990). One would expect the levels of GSH to be reduced as the antioxidant was consumed in reducing the reactive oxidant species known to be released by neutrophils (Cheson et al., 1977). However, the oxidized form of GSH (GSSG) did not increase in a manner concomitant to the decrease in GSH, rather the GSSG also decreased in the BALF. This suggests that other sulfhydryl species, such as mixed protein disulfides, may have been formed or that the neutrophils were destroying both the oxidized and reduced forms of the tripeptide. The data suggest that the neutrophil is a major source of the reactive oxidant species in the inflammatory response, because GSH was not de-
ET
AL.
creased in the BALF from depleted animals exposed to quartz. The only other BALF parameter measured that was enhanced by the neutrophil depletion was the BALF alkaline phosphatase activity. This enzyme in BALF has been suggested as a marker for type II cell secretory activity (Henderson, 1988). The early reports of Kuhn (1968) and Sorokin (1967) indicated that the type II cell was the only alveolar epithelial cell containing alkaline phosphatase and that the enzyme was at the apical portion of the cell. Phagocytes in the alveoli were reported to contain only traces of this enzyme (Sorokin, 1967). More recent work by Miller etal. (1987) confirms that alkaline phosphatase is unique to the type II cell in the alveolar region, and Edelson et al. (1988) reported no staining of alkaline phosphatase in phagocytic cells from adult rats. The work of Edelson et al. ( 1988) also indicates that alkaline phosphatase appears in the type II cell only at the end of the maturation and at the same time as the cell becomes able to secrete surface active material, suggesting that the enzyme may play a role in that process. Thus, it is logical to think that alkaline phosphatase in the epithelial lining fluid may come from the type II cell. The histopathology of the quartz-instilled rats, both in the depleted and nondepleted animals, showed type II cell hypertrophy, a response to quartz that was observed earlier by Miller et al. (1986). In the present study there was an increase in BALF alkaline phosphatase activity accompanying the type II cell hypertrophy in both the depleted and nondepleted animals, but the increase was much higher in the depleted animals. One possible explanation for this is that the neutrophils in the alveoli of the nondepleted animals either inactivated the enzyme or in some way decreased its release from the type II cells. Interestingly, the depletion of the neutrophils in the alveolar space did not affect the development of type II cell hypertrophy. This suggests that the activated macrophage or other alveolar epithelial cells are capable of releasing factors that lead to type II cell hy-
WBC DEPLETION
AND LUNG
pertrophy in the absence of recruited neutrophils. In summary, the study indicated that the source of lysosomal enzymes in the inflammatory response of the rat lung to a-quartz is not the neutrophil and is likely to be the macrophage. Elevations in LTB4 in BALF in response to a-quartz required neutrophils, while GSH and alkaline phosphatase concentrations were enhanced in the absence of neutrophils. The results of this study confirmed earlier research indicating that the neutrophil plays a significant role in the early toxic effects accompanying an inflammatory response in the lung, including contributing to the damage of alveolar cells and to the increase in alveolar/ capillary permeability. The neutrophil was not, however, necessary for the development of type II cell hypertrophy. This study did not address the question of the effect of neutrophils on the long term effects of quartz exposure, but the fact that type II cell hypertrophy occurred in the absence of neutrophils suggests that the quartz would have some long term effect on the lung even in the absence of the initial influx of neutrophils.
INFLAMMATION
135
macrophages in response to particles in vitro. (submitted for publication). BOEHME, D. S., AND HENDERSON, R. F. (1990). Glutathione release by pulmonary alveolar macrophages in vitro: A possible index of particle cytotoxicity. Toy-icologist
10, 277.
BRAIN, J. D., KNUDSON, D. E., SOROKIN. S. P., AND DAVIS, M. A. (1976). Pulmonary distribution ofparticles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11, 13-33. CHESON, B. D., CURNUTTE. J. T.. AND BABIOR. B. M. (1977). The oxidative killing mechanisms of the neutrophil. In Pr0gres.s in Clinical Immunology (R. S. Schwartz, Ed.). Vol. 3. pp. I-65. Grune & Stratton. New York. DAVIS, R. C., COOPERBAND,S. R., AND MANNICK, .I. A. (1969). Preparation and in vifro assay of effective and ineffective antilymphocyte sera. Sur,qery 66, 58-64. DETHLOFF. L. A., GILMORE. L. B.. GLADEN. B. C.. GEORGE. G.. CHHABRA, R. S.. AND HOOK. G. E. R (1986). Effects of silica on the composition of the pulmonary extracellular lining. Torico/ ,Qppl. Pharmtrc,oi 84,66-83.
DRISCOLL,K. E.. MAURER, J. K., LINDENSCHMIDT,R. C’.. ROMBERGER. D.. RENNARD. S. 1.. AND CROSB\. L. ( 1990). Respiratory tract responsesto dust: Relationships between dust burden, lung injury, alveolar macrophagc fibronectin release. and the development of pulmonan; fibrosis. To.xicol. .-lppl. Pharmacol. 106, 88- 10 I EDELSON, J. D., SHANNON. J. M., AND MASON. K. J. ( 1988). Alkaline phosphatase: A marker of alveolar type II cell differentiation. .Am. Rer. Respir. DRY. 138, 126% 1275.
ACKNOWLEDGMENTS Research was conducted under OHER/U.S. Department of Energy Contract DE-AC0476EV0 10 13. The facilities used for this research were fully accredited by the American Association of Laboratory Animal Care. The authors gratefully acknowledge the assistance of all members of the ITRI staff and in particular that of James Waide. Dee Esparza, and Lois Herrera. Dr. Dea S. Boehme was an Associated Western Universities postdoctoral participant at the time of this study.
REFERENCES
HAM, E. A., SODERMAN.D. D.. ZANETTI, M. E.. Dot C;HERTY, H. W.. MCCAULEY. E.. KUEHL. F. A.. JR. (1983). Inhibition by prostaglandins of leukotriene B., release from activated neutrophils. Proc Natl. .-lead S(,i (‘St 80,4349-4353.
HARKEMA, J. R.. AND HOTCHKISS. J. A. (1991). 111riro effects of endotoxin on nasal epithelial mucosubstances: Quantitative histochemistry. Ep. Lung Res. 17. 74.S 761.
HENDERSON,R. F.. BENSON.J. M., HAHN. F. F., HOBRS. C. H., JONES,R. K.. MAIIDERLY. J. L.. McCrf-LLAU, R. 0.. AND PICKRELI.. J. A. (1985). New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis. Fundum .1pp/ To\,< II/ 5, 451-458.
BECK. B. D., BRAIN, J. D., AND BOHANNON, D. E. (1982). An in viva hamster bioassay to assessthe toxicity of particles for the lungs. To.uicol. Appl. Pharmacol. 66, 9-29.
BERGMEYER, H. U.. GAWEHN, K., AND GRASSL. M. (1974). Enzymes as biochemical reagents. In Methods o/Enzymatic Analysis (H. U. Bergmeyer. Ed.), pp. 425522. Academic Press, New York. BOEHME. D. S.. MAPLES, K. R.. AND HENDERSON, R. F. ( I99 1). Glutathione release by pulmonary alveolar
HENDERSON.R. F. ( 1988). Use of bronchoalveolar lavage to detect lung damage. In Toxicology (!I thy Lung (D. E. Gardner. J. D. Crapo. and E. J. Massaro. Eds.), pp. 739-268. Raven Press, New York. HENDERSON. R. F., LEUNG. H. W.. HARMSE~, A. G., AND MCCLELLAN. R. 0. (1988a). Species differences in release of arachidonate metabolites in response to inhaled diluted diesel exhaust. To.xico/. Left. 42, 37.5-332. HENDERSON,R. F., PICKRELL, J. A., JONES.R. K S~JN, J. D.. BENSON.J. M.. MAUDERLY. J. L.. &ND M(CL EL-
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LAN. R. 0. (198817). Response of rodents to inhaled diluted diesel exhaust: Biochemical and cytological changes in bronchoalveolar lavage fluid and in lung tis-
sue. Fundam. Appl. Toxicol. II, 546-567. HENDERSON, R. F. ( 1989). Bronchoalveolar lavage: A tool for assessing the health status of the lung. In Concepts in Inhalation Toxicology (R. 0. McClellan and R. F. Henderson, Eds.), pp. 4 15-444. Hemisphere, New York. JOHNSON, K. J.. AND WARD, P. A. (1982). Biology of disease. New concepts in the pathogenesis of immune complex-induced tissue injury. Lab. Invest. 47, 218. KUHN, C. (1968). Cytochemistry of pulmonary alveolar epithelial cells. Am. J. Pathol. 53, 809-833. LEVY. A. L. (1954). A paper chromatographic method for the quantitative estimation of amino acids. Nature 174, 126-127. LUCCHESI, B. R. (1990). Myocardial ischemia, reperfusion, and free radical injury. Am. J. Cardiol. 65, 141-231. MILLER, B. E., DETHLOFF, L. A., AND HOOK, G. E. R. (1986). Silica-induced hypertrophy of type II cells in the lungs of rats. Lab. Invest. 55, 153-163. MILLER, B. E., CHAPIN, R. E., PINKERTON, K. E., GILMORE, L. B., MARONPOT, R. R.. AND HOOK, G. E. R. (1987). Quantitation of silica-induced type II cell hyperplasia by using alkaline phosphatase histochemistry in glycol methacrylate embedded lung. Exp. Lung Res. 12, 135-148. MOORES, S. R., SYKES, S. E., MORGAN, A., EVANS. N.. EVANS, J. C., AND HOLMES, A. (1980). The short-term cellular and biochemical response of the lung to toxic dusts: An in vivo cytotoxicity test. In The In C’itro Effect of Mineral Dusts (R. C. Brown, I. P. Gormley, M. Chamberlain, and R. Davies. Eds.), pp. 297-303. Academic Press, New York. MWSTAFA, M. G., AND TIERNEY, D. F. (1978). Biochemical and metabolic changes in the lung with oxygen, ozone and nitrogen dioxide toxicity. Am. Rev. Respir. Dis. 118, 1061-1090. O’BYRNE, P. M.. WALTERS, E. H., GOLD. B. D., AIZAWA, H. A., FABBRI, L. M.. ALPERT, S. E., NADEL, J. A., AND HOLTZMAN, M. J. (1984). Neutrophil depletion inhibits airway hyperresponsiveness induced by ozone exposure. Am. Rev. Respir. Dis. 130, 2 14-2 19.
ET
AL.
PICKRELL, J. A., GREGORY, R. E., COLE, D. J., HAHN, F. F., AND HENDERSON, R. F. (1987). Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung. Exp. Mol. Pathol. 46, 168-179. REED. D. J., BABSON, J. R., BEATTY. P. W., BRODIE, A. E., ELLIS, W. W., AND POTTER, D. W. (1980). High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106, 55-62. SHASBY, D. M., SHASBY, S. S., BOWMAN, C. M., Fox, R. B., HARADA, R. M.. TATE, R. M., AND REPINE, J. E. (198 1). Angiotensin-converting enzyme concentrations in the lung lavage of normal rabbits and rabbits treated with nitrogen mustard exposed to hyperoxia.
Am. Rev. Respir. Dis. 124, 202-203. SOROKIN, S. P. (1967). A morphologic and cytochemical study on the great alveolar cell. J. Histochem. Cytochem. 14,884-867. SUTHERLAND, M. W., NELSON, J., HARRISON, G., AND FORMAN, H. J. (1985). Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages. Arch. Biochem. Biophys. 243, 325-33 I. SUTTORP, N., AND SIMON, L. M. (1986). Importance of the glutathione redox cycle for the resistance of lung epithelial cells against a polymorphonuclear leukocytemediated oxidant attack. Biochem. Pharmacol. 35, 2268-2770. SWANK, D. W., AND MOORE, S. B. (1989). Roles of the neutrophil and other mediators in adult respiratory distress syndrome. Maqjo Clin. Proc. 64, 1118- 1132. WARHEIT, D. B., CARAKOSTUS, M. C., HARTSKY, M. A., AND HANSEN, J. F. (1991). Development of a shortterm inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107,350-368. WEISS, S. J., YOUNG, J., LOBUGLIO. A. F., SLIVKA, A., AND NIMEH, N. F. (198 1). Role of hydrogen Peroxide in neutrophil-mediated destruction of cultured endothelial cells. J. Clin. Invest. 68, 7 14-72 1.
Effect of Blood Leucocyte Depletion on the Inflammatory Response of the Lung to Quartz’ ROGENE
F. HENDERSON,
Inhalatiorl
JACK R. HARREMA,
JON A. HOTCHKISS,
AND DIVA S. BOEHME-
Toxicology Research Institute, Lovelace Biomedicul and Environmental Research Instltutr. P.O. Bm 5890. Albuquerque, New .Meuico X718.5
Received October 12, 1990; accepted Februury 8. 1991 Effect of Blood Leucocyte Depletion on the Inflammatory Response of the Lung to Quartz HENDERSON,R. F., HARKEMA, J. R., HOTCHKISS,J. A., AND BOEHME, D. S. (199 1). To.xico/..lpppl. Pharmacol. 109, 127-136. This study determined the effect of blood leucocyte depletion on the early inflammatory response of the lung to a-quartz. F344/N rats were instilled intratracheally with either physiological saline or 2 or 5 mg of aquartz suspended in saline. One day prior to the instillation. half of the rats received an ip injection of rabbit antiserum that had been raised against rat neutrophils. The other half of the rats received an ip injection of normai rabbit serum. One day after the instillation of saline or quartz, the animals were euthanized and observed for changes in blood cell numbers. lung histopathology, and bronchoalveolar lavage fluid (BALF) content of indicators of an inflammatory response and cytotoxicity. The rabbit antiserum depleted the blood of most white blood cells of all types. BALF fluid from saline-instilled animals did not differ between the white blood cell-depleted and the nondepleted animals except for a 20% reduction in numbers of alveolar macrophages in the depleted animals. BALF fluid from the nondepleted, quartz-instilled animals had a dose-dependent increase in content of neutrophils and protein (indicator of an increase in the permeability of the alveolar/capillary barrier) as well as an increase in lactate dehydrogenase and glutatbione reductase (cytoplasmic enzymes whose presenceextracellularly indicates cytotoxicity). alkaline phosphatase (indicator of type II cell secretory activity), &glucuronidase. and acid proteinase (lysosomal enzymes) activities. The higher dose of quartz also elicited an increase in LTB, and PGEz content of BALF. GSH content of BALF was decreased by the quartz exposure. The depletion of blood white blood cells prevented the influx of neutrophils into the alveoli of the quartz-exposed rats and decreased the BALF markers of capillary permeability and cytotoxicity (protein content and extracellular cytoplasmic enzymes). The absence of neutrophils in the alveoli had no effect on the lysosomal content of BALF, indicating that the neutrophils were not the source ofthese enzymes in nondepleted rats exposed to aquartz. The quartz-induced elevation of LTB4 in BALF was not observed in depleted rats, suggesting that neutrophils may be the source of the increase in this leukotriene in the BALF. Both the GSH content and the alkaline phosphatase activity in BALF were enhanced in the absenceof alveolar neutrophils. The enhancement of GSH in BALF is consistent with the neutrophils being the source of reactive oxygen species that deplete GSH. The increased alkaline phosphatase activity in the BALF of both the depleted and nondepleted animals is consistent with the type II cell hypertrophy that was induced by quartz instillation and was neutrophil independent. The study provides information on the probable sources of some of the inflammatory mediators in BALF and suggeststhat neutrophils contribute to the acute toxicity accompanying the inflammatory response of the lung to quartz. The neutrophil is not required. however. for the production of type II cell hypertrophy. 8 1991 Academx press.inc.
Our laboratory (Henderson, 1988, 1989), as well as others (Beck et al., 1982; Moores et ’ The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper. for governmental purposes, is acknowledged
al., 1980) have documented the inflammatory response of the lung to inhaled or instilled particles by analysis of bronchoalveolar lavage fluid (BALF) from exposed animals. Cellular changes in BALF during acute inflammation include an influx of neutrophils (PMN) and 127
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activation of pulmonary alveolar macrophages (PAM). The neutrophil has been implicated as contributing to the lung injury incurred during an inflammatory response. Neutrophils damage cultured endothelial cells (Weiss et al., 198 1) and epithelial cells (Suttorp and Simon, 1986). The mechanism of neutrophil-induced injury may be due to toxic oxygen species released by this leucocyte (Mustafa and Tiemey, 1978). The oxidative killing mechanisms of neutrophils toward bacteria are well known (Cheson et al., 1977). The enzymatic activities found in the acellular supernatant of BALF provide important information concerning the degree of cytotoxicity present (measured by extracellular activities of such cytoplasmic enzymes as lactate dehydrogenase, LDH, or glutathione reductase, GR) and the release of lysosomal enzymes (such as P-glucuronidase and acid protease activities) during an inflammatory response to inhaled particles (Henderson, 1988, 1989). However, the cellular source of these enzymes is unknown. Eicosanoids are increased in BALF in response to inhaled particles, particularly LTB4 and PGF2, (Henderson et al., 1988a). The cellular source of these metabolites is also not known. Finally, reduced glutathione (GSH) is increased in a dose-dependent manner in BALF from animals exposed to diesel soot, but the source of the GSH is not known (Henderson et al., 1988b). To better understand the mechanisms of the inflammatory response to particles in the lung, it would be valuable to know the cellular source(s) of these mediators of inflammation. The primary purpose of this study was to obtain information on the source of the inflammatory mediators in BALF by eliminating one of the potential sources, the neutrophil. We hypothesized that the absence of the neutrophil would decrease markers of cell injury in BALF, but would have little effect on the lysosomal enzymes or the GSH. The toxic agent used to induce an inflammatory response was a-quartz and the animal model was the F344/N rat, because of the large amount of information known about the pulmonary inflammatory response to quartz in
ET AL.
that animal (Driscoll et al., 1990; Warheit et al., 199 1). The method for depleting the neutrophils was a rabbit anti-rat neutrophil antiserum that has been used in previous studies at our laboratory (Harkema, 1990). METHODS Animals. Young (1 l-l 5 weeks) female F344/N rats from the Institute’s barrier breeding facility were used in the study. Prior to the study the animals were housed two or three per cage in polycarbonate cages supplied with sterilized hardwood chip bedding and filter tops. Animal rooms were maintained at 20-22°C with a relative humidity of 20-50s and a 12-hr light/dark cycle starting at 6:00 AM. Water from bottles with sipper tubes and food (Lab Blox, Allied Mills, Chicago, IL) were provided ad libitum. Murine neutrophil antiserum. The method for depleting neutrophils was by intraperitoneal (ip) injection of a cytotoxic, rabbit anti-rat neutrophil antiserum (Accurate Scientific Corp., Westbury, NY). This antiserum was raised against normal rat neutrophils isolated from whole blood and preadsorbed with rat serum and red blood cells (RBCs). The procedures for producing this antiserum were based on standard methodologies described by Davis et al. ( 1969). In this study, the PMN-depleted animals received a 1 ml injection of the antiserum 24 hr prior to exposure to LYquartz or saline. An equal number of animals received an injection of 1 ml of normal rabbit serum at the same time prior to the exposures to a-quartz or to saline. Exposure to a-quartz. To achieve a high concentration of a-quartz in the lungs of rats in a short period of time, the rats received either 0.5 ml of physiological saline or 2 or 5 mg of a-quartz suspended in 0.5 ml saline instilled intratracheally according to the method of Brain (1976). A concentrated suspension of quartz was allowed to stand for 90 min to remove larger particles. The small-particle suspension was then decanted and aliquots were dried and weighed to determine the concentration of a-quartz. The suspension was then diluted with saline to achieve a concentration of 2.0 or 5.0 mg quartz/O.5 ml saline. Experimental design. The experimental design is outlined in Table 1. There were 24 animals treated with 2.0 mg of quartz, 24 treated with 5.0 mg of quartz, and 24 given saline intratracheally. Half of each of the quartztreated groups and half of the saline-treated animals were injected ip with the antiserum 24 hr before the intratracheal injections. The other half were injected ip with normal rabbit serum. In each of these subsets of 12 animals, I day after the exposure to quartz or saline, half of the animals received bronchoalveolar lavage (BAL) and the other half were observed for histopathology of the left lung and for the cellular content of the blood. In addition, the right lung of the animals designated for histologic evaluation was lavaged to obtain sufficient Iavage fluid for analysis of all the parameters of interest.
WBC TABLE
DESIGN
Animals injected with normal rabbit serum
0 (saline 2 5
BALF analysis only)
6 6 6
AND
I
EXPERIMENTAL
Quartz instilled (mg)
DEPLETION
Histopathology” 6 6 6
Animals injected with rabbit antiserum BALF analysis 6 6 6
Histopathology” 6 6 6
a BALF was obtained from the right lung of these animals as well as from the lungs of animals assigned for BALF analysis.
Hlsiopatho1og.v. The left lung was intratracheally perfused with 10% buffered formalin at 30cm fixative pressure for at least 24 hr. Midsagittal sections of the left lung lobe were embedded in paraffin, sectioned at 4-6 pm in thickness, and stained with hematoxylin and eosin for light microscopic examination. .-tnalysis oiblood cells. Blood samples were taken from the heart at the time of death and total and differential blood cell counts were made. The total white and red blood cell counts were made on a Coulter S550 whole blood counter. Differential counts of leucocytes were determined by counting 100 cells from blood smears stained with a Wright-Giemsa stain. 5ronchoalvedar luvage. The lavage was performed on the excised lung taken from the rats under anesthesia (4% halothane in oxygen). For the total lung lavages, 5 ml of sterile physiological saline was gently instilled into the trachea and allowed to fill all lung lobes. The fluid was immediately withdrawn by gentle suction. This was repeated one more time and the lavage fluids combined. In the animals for which the left lung was used for evaluation of the histopathology, the left lung was clamped off and the right lung lavaged two times with 4 ml of saline as described above. The right lung was then clamped off and the clamp removed from the left lung bronchus. The left lung was then fixed with 10% formalin as described above. .Inul.vsi.r qfbronchoalveolar lavage. The bronchoalveolar lavage fluid was analyzed for various markers of cell damage and inflammation. An aliquot was removed and analyzed for total cell counts by a hemocytometer count. The ceils were then removed by centrifugation at 300g and the cell oellet was resusnended in 1 ml of saline. A cytospin prep&ation ofthe ceils was stained with a WrightGiemsa-type stain (DifF-Quik, Harleco) and a differential cell count made. Cell viability was determined by trypan blue dye exclusion. The acellular supematant was analyzed for the following parameters. The soluble protein concentration was measured as a marker for damage to the alveolar capillary
LUNG
129
INFLAMMATION
barrier. The amount of activity of the cytoplasmic enzyme. lactate dehydrogenase (LDH), was determined as a marker for cytotoxicity. The glutathione reductase activity was also measured as a second cytoplasmic enzyme that would be expected to be involved in antioxidant activities. Two lysosomal enzyme activities. @-glucuronidase and acid proteins activities, were measured as markers of enzymes expected to be released from phagocytes. either during phagocytic activity or cell death. Alkaline phosphatase activity was measured as a possible marker of type II cell secretions (Miller d a/.. 1987). The arachidonic acid meas tabolites. LTBA. PGF2
RESULTS Haad cell counts. The blood cell counts in the animals of the various treatment groups are shown in Table 2. The counts of all types of white cells, not just the neutrophils, appeared to be depressed by the antiserum, although the number of monocytes and eosinophils in rat blood is normally low, and it is difficult to detect a depression in their numbers.
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ET AL.
TABLE 2 HEMATOLOGY DATA Animals injected with normal serum Parameter RBC ( 106/mm3) WBC ( 103/mm3) Hemoglobin (g/dl) Hematocrit (%) Neutrophils ( I03/mm3) Lymphocytes ( 103/mm3) Monocytes ( 103/mm3) Eosinophils ( 103/mm3)
Saline 8.8 4.1 15.1 47.7 0.55 3.5
* 0.2 +- 0.4 kO.3 + 1.1 t 0.09 + 0.3 00:
2 mg
quartz
9.0 ?I 0.2 4.1 + 0.4 15.4 + 0.3 48.8 f 0.9 0.3 r 0.04 3.7 k 0.4 Od Od
5w quartz 8.4 3.3 14.5 44.9 0.4 2.7
f 0.1 * 0.3 f 0.2 f 0.5 2 0.1 f 0.2 Od 0.1 + 0.04
Animals injected with antiserum
2 mg
Saline
quartz
8.9 f 0.3 0.5 + 0.1” 15.2 r+ 0.5 47.7 + 2.0 0‘Z.h 0.5 f 0.13” Ob Ob
9.3 f 0.2 0.7 -+ 0.2” 15.8 + 0.2 49.6 -t 0.9 0n.h 0.7 f 0.2” Ob Ob
5 mg quartz 8.1 f 0.5 + 14.1 + 43.1 +
0.4 0.2” 0.6 1.9
0 oB
0.5 z!c0.2” Ob Ob
Note. (R + SE, n = 5-6). n Differs from corresponding values for animals injected with normal serum p 5 0.05. ’ Values from all animals were zero. ’ Values from five animals were zero and from one animal was 0.1. d Values from four animals were zero and from two were 0.1.
BALF parameters. The alterations in parameters measured in the BALF and the lung weights of the animals in the study are listed in Table 3. The data indicate that the rabbit antiserum itself (as opposed to normal rabbit serum) had no effect on most of the parameters in animals not exposed to a-quartz. None of the values for the nondepleted, control animals (saline instilled) were statistically different from historical controls. There were no significant differences between the values of the various parameters in BALF from the control animals in the depleted and nondepleted rats with one exception. (Compare Columns 1 and 4 of the 6 columns of numbers.) There was a decrease in total nucleated cells in BALF from the depleted animals primarily due to a 20% decrease in alveolar macrophages. The administration of the a-quartz caused an acute inflammatory response in the animals that did not receive antiserum. (Compare Columns 2 and 3 with Column 1 in Table 3.) An inflammatory response was observed as indicated by an increase (in most cases dosedependent increases) in the BALF content of protein and neutrophils and in LDH, glutathione reductase, ,&glucuronidase, acid protease, and alkaline phosphatase activities. In
animals receiving the higher dose of a-quartz, there were also increases in BALF content of PGE2 and LTB4 and a decrease in GSSG and in the total glutathione equivalents. Thus, the instilled a-quartz did induce the expected inflammatory response and there was an indication of cytotoxicity accompanying the inflammation. The depletion of white blood cells by the antiserum altered the lung’s response to the instilled a-quartz. (Compare Columns 5 and 6 with Columns 2 and 3 in Table 3.) The most striking difference between the response of the depleted vs the nondepleted rats was the lack of an influx of neutrophils into the bronchoalveolar region in the depleted animals. The resident white blood cell of the alveoli, the macrophage, was decreased in number to half that of the saline-instilled, depleted, control animals and one-third that of the quartz-exposed, nondepleted rats. The indicators of cytoxicity, i.e., the cytoplasmic enzyme activities, LDH, and glutathione reductase, were elevated in the exposed, depleted animals but the elevated values were one-half to one-third those in the exposed, nondepleted rats. In contrast, the lysosomal enzymes, fi-glucuronidase and acid protease, were both elevated in response to a!-
WBC DEPLETION
AND LUNG
INFLAMMATION
131
TABLE 3 EFFFECT
OF WHITE
BLOOD
CELL DEPLETION
ON THE RESPONSE OF THE LUNG
Animals injected with normal rabbit serum BALF parameter Lung weight, g Total nucleated cells. IO’/ml PAM, 103/ml PMN, 1O’/ml Alkaline phosphatase, mIU/ml LDH, mlU/ml GR, mIU/ml BG, mIU/ml Protein, mg/ml Acid protease, fig Hb/ 4 hr/ml PC%. pg/ml PGL. m/ml LTB, , pg/ml Cell viability, R GSH, nmol/ml GSSG. nmol/ml GSH plus 2 GSSG’
TO CX-QUARTZ
Animals injected with rabbit antiserum
Saline control
2 mg quartz
5 mg quartz
Saline control
0.98 f 0.04
1.10 -+ 0.03
1.16 + 0.10
0.91 * 0.05
0.97 ? 0.02’
I .oo rt 0.04
621 + 84 504 f 33 116 & 58
3155 IL 145” 669 f 42 2470 t 130”
2431 f 106” 516 f 85 1850 f I IO’
410 + 20* 391 + 15” 20t 13
223 f 38”,” 222 +- 38”.b 1.3 + 0.6h
205 + 39”.’ 203 * 39,‘.’ 1.8 t O.h*
6.0 +- 0.8 71?7 11.7 t 1.4 0.13 IT 0.02 0.23 r 0.04
14.8 f 282 k 25.6 + 1.38 f 0.49 f
29.5 553 40.0 3.72 0.72
7.5 * 73 2 I I .4 f 0.1 1 ? 0.20 k
60.7 _t 24.3”,h 124 i 14”.” 17.0 * 1.5ti,” 1.25 f 0.23” 0.27 xk 0.03h
44. I t 0.h”:” 182 t I?“.’ 18.2 _t 0.4”.’ 2.68 -+ 0.04’0.33 f 0.(13’
27 +- 1 49 f 9 30 t 2 16+ I 87 + 2 2.35 t 0.45 0.29 + 0.07 2.94 rt 0.58
37-+ I” 54 I? 6 26 -+ 2 20 f 2 84? 3 1.78 + 0.15 0.19 + 0.03 2.16 + 0.20
33 f 2” 80 -?I9” 36?2” 19 * 4 65 f 4h 2.71 -+ 0.36 0.26 f 0.03 3.23 f 0.39
34 +- I ’ 71 *4 34 i- 3 18t I’ 83?3’ 3.34 i 0.33 0.20 zk 0.1I5 ’ 3.74 t 0.42’
0.5” 10” 1.2” 0.05” 0.03”
+ 2.8” 2k 24” It 1.9” 2 0.16“ + 0.04“
I.1 5 0.6 0.01 0.04
37 + I” 27 * 1 85 t 5” 63 +- 6 33* I 30 i 2 27 f 2” 23 t 3 93* 1 71 *9 1.53 f 0.18 2.57 t 0.43 0.05 -+ 0.0 1il 0.20 i- 0.03 1.63 + 0.19 2.97 + 0.49
2 mg quartz
5 mg quartz --
Note. (.F f SE. ,r = 5-6). ’ Statistically significant from the appropriate saline control (p < 0.05). ’ Differ from corresponding value in nondepleted animals, p s 0.05. ‘ Total glutathione equivalents. Values used are in nmol; thus, the nmol ofGSSG are multiplied by 2 to be equivalenr to the nmol of GSH necessary to form a nmol of GSSG.
quartz to the same extent in the depleted and nondepleted animals. Protein concentration in the BALF of the depleted animals was not significantly elevated, also in contrast to the nondepleted animals. The increases in LTB4 seen in the nondepleted animals did not occur in the depleted rats. In contrast, GSH levels were higher in the BALF from the depleted animals instilled with 5 mg quartz than in the nondepleted rats. The only other parameter that was increased in BALF to a greater degree in response to a-quartz in the depleted vs the nondepleted animals was the alkaline phosphatase activity. Histopatholo~~~. The principal alteration in the lungs from nondepleted rats that were instilled with quartz was a multifocal, coalescing acute alveolitis (Fig. 1C). This alveolitis was
characterized by a moderate to marked influx of neutrophils into alveolar septa, alveolar duct walls. alveolar airspaces, and interstitium around centriacinar pulmonary arteries and bronchioles. In the affected regions of the lung there was moderate thickening of the alveolar septa due to capillary congestion. In addition, there was a marked hypertrophy and vacuolization of type 2 pneumocytes and alveolar macrophages in all of these rats, and a mild alveolar macrophage hyperplasia in some of these animals. Quartz particles were present individually or in aggregates within alveolar airspaces or phagocytized within alveolar macrophages. Proteinaceous material and necrotic cellular debris were also present in some of the alveolar airspaces. Rats instilled with 5 mg of quartz had more severe alveohtis ( i.e.,
132
HENDERSON
ET
AL.
WBC
DEPLETION
AND
more neutrophils and other cellular changes in larger areas of the lung parenchyma) compared to animals instilled with 2 mg quartz. In contrast, antiserum-injected rats that were instilled with 2 or 5 mg of quartz had a conspicuous absence of acute alveolitis (Fig. 1D). Although there was a lack of neutrophil influx into the alveolar airspaces in these animals, hypertrophy and vacuolization of type 2 pneumocytes and alveolar macrophages were similar to those present in the nondepleted rats instilled with quartz. No hyperplasia of alveolar macrophages was evident in any of the depleted rats instilled with quartz. Nondepleted rats instilled with saline only had no significant alterations in the lung (Fig. 1A). Depleted rats instilled with saline only had a marked attenuation of the number of polymorphonuclear and mononuclear leukocytes within the lumens of pulmonary arterioles and veins and within capillaries in alveolar septa (Fig. 1B).
LUNG
INFLAMMATION
133
14% of normal levels while neutrophils were essentially eliminated. For the purpose of this study, the depletion of most white cells in the blood was not a major problem because the normal initial inflammatory response observed in rat lungs involves the influx of only neutrophils, and not lymphocytes (Henderson, 1988; also see Table 3, present study). Following the neutrophil influx, there is recruitment of monocytes, but the time frame of the current study would not allow observation of that recruitment. Therefore, the current study allowed us to determine what effect the lack of neutrophil influx has on the acute response to a-quartz in the lung. Neutrophil depletion was accompanied by a decrease in the BALF protein content and in indicators of cytotoxicity (the cytoplasmic enzyme activities, LDH and GR) in response to a-quartz. This suggests that the neutrophil influx plays a major role in increasing the permeability of the alveolar/capillary barrier and in producing cellular toxicity during the inflammatory response. This is in agreement DISCUSSION with earlier work indicating the cytotoxicity of neutrophils in association with reperfusion The purpose of this study was to obtain in- injury (Lucchesi. 1990), adult respiratory disformation on the source of the inflammatory tress syndrome (Swank and Moore. 1989). mediators in BALF by eliminating one of the potential sources, the neutrophil. In the past, immune complexes (Johnson and Ward. 1982), and in lung epithelial cells in culture highly toxic materials, such as nitrogen mus(Suttorp and Simon, 1986). tard (Shasby et al., 198 1) or hydroxyurea The neutrophil depletion did not, however. (O’Byrne et al., 1984), have been used to dehave any effect on the BALF levels of lysoplete animals of neutrophils to determine the somal enzymes (acid protease, P-glucuronirole of these cells in various processes. The dase), indicating that neutrophils are not the disadvantage of such treatments is the toxic source of the high levels of these enzyme acside effects that confound such experiments. tivities observed in response to quartz in this In the present study, neutrophils were depleted study and in other studies (Beck et al., 1982; by use of an antiserum prepared from rabbits Henderson et al., 1985; Dethloff et al., 1986). injected with rat neutrophils. The antiserum The likely source of these lysosomal enzymes was adsorbed with rat serum and rat RBCs to remove any antibodies developed against the is the activated macrophage, although other serum proteins or RBCs. The antiserum treat- sources, such as epithelial cells, cannot be exment of the rats did not result in any decrease cluded. The suggestion that the macrophage is the source of the lysosomal enzymes is in the blood RBCs (Table 2). The antiserum did, however, reduce the white blood cells in agreement with earlier studies in which ( WBCs) in the blood to approximately 10% of only particle-induced inflammatory responses caused an increase in lysosomal enzyme acthe normal level. Lymphocytes, the major tivity in BALF. while pulmonary inflammaWBC present, were reduced to approximately
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tion induced by pollutant gases did not (reviewed by Henderson, 1988). The concentrations of arachidonate metabolites in BALF were not greatly elevated in response to quartz despite the other indicators of an inflammatory response. The eicosanoid concentrations in BALF were not affected by the instillation of a-quartz in either the depleted or nondepleted animals except at the highest dose of quartz. Twenty-four hours after the instillation of 5 mg of quartz, PGE2 and LTB4 concentrations were elevated in BALF from the nondepleted animals but the LTB4 was not elevated in the depleted animals. This points to the neutrophil as a potential source of the LTB4 or as being a factor that enhances the release of the metabolite from other cells. Work reported by Ham et al. ( 1983) indicates that activated neutrophils release LTB4, and this release is inhibited by PGE2. The increase in PGEz at the same time as the increase in LTB4 in nondepleted rats exposed to the highest dose of a-quartz may represent a control mechanism to down regulate the inflammation. The antioxidant tripeptide, glutathione, was reduced in the BALF from the nondepleted rats exposed to the highest amount of quartz, while the depleted animals did not show such a reduction in BALF glutathione equivalents. The release of GSH from quartz-stimulated macrophages has been reported (Boehme et al., 199 1; Boehme and Henderson, 1990). One would expect the levels of GSH to be reduced as the antioxidant was consumed in reducing the reactive oxidant species known to be released by neutrophils (Cheson et al., 1977). However, the oxidized form of GSH (GSSG) did not increase in a manner concomitant to the decrease in GSH, rather the GSSG also decreased in the BALF. This suggests that other sulfhydryl species, such as mixed protein disulfides, may have been formed or that the neutrophils were destroying both the oxidized and reduced forms of the tripeptide. The data suggest that the neutrophil is a major source of the reactive oxidant species in the inflammatory response, because GSH was not de-
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creased in the BALF from depleted animals exposed to quartz. The only other BALF parameter measured that was enhanced by the neutrophil depletion was the BALF alkaline phosphatase activity. This enzyme in BALF has been suggested as a marker for type II cell secretory activity (Henderson, 1988). The early reports of Kuhn (1968) and Sorokin (1967) indicated that the type II cell was the only alveolar epithelial cell containing alkaline phosphatase and that the enzyme was at the apical portion of the cell. Phagocytes in the alveoli were reported to contain only traces of this enzyme (Sorokin, 1967). More recent work by Miller etal. (1987) confirms that alkaline phosphatase is unique to the type II cell in the alveolar region, and Edelson et al. (1988) reported no staining of alkaline phosphatase in phagocytic cells from adult rats. The work of Edelson et al. ( 1988) also indicates that alkaline phosphatase appears in the type II cell only at the end of the maturation and at the same time as the cell becomes able to secrete surface active material, suggesting that the enzyme may play a role in that process. Thus, it is logical to think that alkaline phosphatase in the epithelial lining fluid may come from the type II cell. The histopathology of the quartz-instilled rats, both in the depleted and nondepleted animals, showed type II cell hypertrophy, a response to quartz that was observed earlier by Miller et al. (1986). In the present study there was an increase in BALF alkaline phosphatase activity accompanying the type II cell hypertrophy in both the depleted and nondepleted animals, but the increase was much higher in the depleted animals. One possible explanation for this is that the neutrophils in the alveoli of the nondepleted animals either inactivated the enzyme or in some way decreased its release from the type II cells. Interestingly, the depletion of the neutrophils in the alveolar space did not affect the development of type II cell hypertrophy. This suggests that the activated macrophage or other alveolar epithelial cells are capable of releasing factors that lead to type II cell hy-
WBC DEPLETION
AND LUNG
pertrophy in the absence of recruited neutrophils. In summary, the study indicated that the source of lysosomal enzymes in the inflammatory response of the rat lung to a-quartz is not the neutrophil and is likely to be the macrophage. Elevations in LTB4 in BALF in response to a-quartz required neutrophils, while GSH and alkaline phosphatase concentrations were enhanced in the absence of neutrophils. The results of this study confirmed earlier research indicating that the neutrophil plays a significant role in the early toxic effects accompanying an inflammatory response in the lung, including contributing to the damage of alveolar cells and to the increase in alveolar/ capillary permeability. The neutrophil was not, however, necessary for the development of type II cell hypertrophy. This study did not address the question of the effect of neutrophils on the long term effects of quartz exposure, but the fact that type II cell hypertrophy occurred in the absence of neutrophils suggests that the quartz would have some long term effect on the lung even in the absence of the initial influx of neutrophils.
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macrophages in response to particles in vitro. (submitted for publication). BOEHME, D. S., AND HENDERSON, R. F. (1990). Glutathione release by pulmonary alveolar macrophages in vitro: A possible index of particle cytotoxicity. Toy-icologist
10, 277.
BRAIN, J. D., KNUDSON, D. E., SOROKIN. S. P., AND DAVIS, M. A. (1976). Pulmonary distribution ofparticles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11, 13-33. CHESON, B. D., CURNUTTE. J. T.. AND BABIOR. B. M. (1977). The oxidative killing mechanisms of the neutrophil. In Pr0gres.s in Clinical Immunology (R. S. Schwartz, Ed.). Vol. 3. pp. I-65. Grune & Stratton. New York. DAVIS, R. C., COOPERBAND,S. R., AND MANNICK, .I. A. (1969). Preparation and in vifro assay of effective and ineffective antilymphocyte sera. Sur,qery 66, 58-64. DETHLOFF. L. A., GILMORE. L. B.. GLADEN. B. C.. GEORGE. G.. CHHABRA, R. S.. AND HOOK. G. E. R (1986). Effects of silica on the composition of the pulmonary extracellular lining. Torico/ ,Qppl. Pharmtrc,oi 84,66-83.
DRISCOLL,K. E.. MAURER, J. K., LINDENSCHMIDT,R. C’.. ROMBERGER. D.. RENNARD. S. 1.. AND CROSB\. L. ( 1990). Respiratory tract responsesto dust: Relationships between dust burden, lung injury, alveolar macrophagc fibronectin release. and the development of pulmonan; fibrosis. To.xicol. .-lppl. Pharmacol. 106, 88- 10 I EDELSON, J. D., SHANNON. J. M., AND MASON. K. J. ( 1988). Alkaline phosphatase: A marker of alveolar type II cell differentiation. .Am. Rer. Respir. DRY. 138, 126% 1275.
ACKNOWLEDGMENTS Research was conducted under OHER/U.S. Department of Energy Contract DE-AC0476EV0 10 13. The facilities used for this research were fully accredited by the American Association of Laboratory Animal Care. The authors gratefully acknowledge the assistance of all members of the ITRI staff and in particular that of James Waide. Dee Esparza, and Lois Herrera. Dr. Dea S. Boehme was an Associated Western Universities postdoctoral participant at the time of this study.
REFERENCES
HAM, E. A., SODERMAN.D. D.. ZANETTI, M. E.. Dot C;HERTY, H. W.. MCCAULEY. E.. KUEHL. F. A.. JR. (1983). Inhibition by prostaglandins of leukotriene B., release from activated neutrophils. Proc Natl. .-lead S(,i (‘St 80,4349-4353.
HARKEMA, J. R.. AND HOTCHKISS. J. A. (1991). 111riro effects of endotoxin on nasal epithelial mucosubstances: Quantitative histochemistry. Ep. Lung Res. 17. 74.S 761.
HENDERSON,R. F.. BENSON.J. M., HAHN. F. F., HOBRS. C. H., JONES,R. K.. MAIIDERLY. J. L.. McCrf-LLAU, R. 0.. AND PICKRELI.. J. A. (1985). New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis. Fundum .1pp/ To\,< II/ 5, 451-458.
BECK. B. D., BRAIN, J. D., AND BOHANNON, D. E. (1982). An in viva hamster bioassay to assessthe toxicity of particles for the lungs. To.uicol. Appl. Pharmacol. 66, 9-29.
BERGMEYER, H. U.. GAWEHN, K., AND GRASSL. M. (1974). Enzymes as biochemical reagents. In Methods o/Enzymatic Analysis (H. U. Bergmeyer. Ed.), pp. 425522. Academic Press, New York. BOEHME. D. S.. MAPLES, K. R.. AND HENDERSON, R. F. ( I99 1). Glutathione release by pulmonary alveolar
HENDERSON.R. F. ( 1988). Use of bronchoalveolar lavage to detect lung damage. In Toxicology (!I thy Lung (D. E. Gardner. J. D. Crapo. and E. J. Massaro. Eds.), pp. 739-268. Raven Press, New York. HENDERSON. R. F., LEUNG. H. W.. HARMSE~, A. G., AND MCCLELLAN. R. 0. (1988a). Species differences in release of arachidonate metabolites in response to inhaled diluted diesel exhaust. To.xico/. Left. 42, 37.5-332. HENDERSON,R. F., PICKRELL, J. A., JONES.R. K S~JN, J. D.. BENSON.J. M.. MAUDERLY. J. L.. &ND M(CL EL-
136
HENDERSON
LAN. R. 0. (198817). Response of rodents to inhaled diluted diesel exhaust: Biochemical and cytological changes in bronchoalveolar lavage fluid and in lung tis-
sue. Fundam. Appl. Toxicol. II, 546-567. HENDERSON, R. F. ( 1989). Bronchoalveolar lavage: A tool for assessing the health status of the lung. In Concepts in Inhalation Toxicology (R. 0. McClellan and R. F. Henderson, Eds.), pp. 4 15-444. Hemisphere, New York. JOHNSON, K. J.. AND WARD, P. A. (1982). Biology of disease. New concepts in the pathogenesis of immune complex-induced tissue injury. Lab. Invest. 47, 218. KUHN, C. (1968). Cytochemistry of pulmonary alveolar epithelial cells. Am. J. Pathol. 53, 809-833. LEVY. A. L. (1954). A paper chromatographic method for the quantitative estimation of amino acids. Nature 174, 126-127. LUCCHESI, B. R. (1990). Myocardial ischemia, reperfusion, and free radical injury. Am. J. Cardiol. 65, 141-231. MILLER, B. E., DETHLOFF, L. A., AND HOOK, G. E. R. (1986). Silica-induced hypertrophy of type II cells in the lungs of rats. Lab. Invest. 55, 153-163. MILLER, B. E., CHAPIN, R. E., PINKERTON, K. E., GILMORE, L. B., MARONPOT, R. R.. AND HOOK, G. E. R. (1987). Quantitation of silica-induced type II cell hyperplasia by using alkaline phosphatase histochemistry in glycol methacrylate embedded lung. Exp. Lung Res. 12, 135-148. MOORES, S. R., SYKES, S. E., MORGAN, A., EVANS. N.. EVANS, J. C., AND HOLMES, A. (1980). The short-term cellular and biochemical response of the lung to toxic dusts: An in vivo cytotoxicity test. In The In C’itro Effect of Mineral Dusts (R. C. Brown, I. P. Gormley, M. Chamberlain, and R. Davies. Eds.), pp. 297-303. Academic Press, New York. MWSTAFA, M. G., AND TIERNEY, D. F. (1978). Biochemical and metabolic changes in the lung with oxygen, ozone and nitrogen dioxide toxicity. Am. Rev. Respir. Dis. 118, 1061-1090. O’BYRNE, P. M.. WALTERS, E. H., GOLD. B. D., AIZAWA, H. A., FABBRI, L. M.. ALPERT, S. E., NADEL, J. A., AND HOLTZMAN, M. J. (1984). Neutrophil depletion inhibits airway hyperresponsiveness induced by ozone exposure. Am. Rev. Respir. Dis. 130, 2 14-2 19.
ET
AL.
PICKRELL, J. A., GREGORY, R. E., COLE, D. J., HAHN, F. F., AND HENDERSON, R. F. (1987). Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung. Exp. Mol. Pathol. 46, 168-179. REED. D. J., BABSON, J. R., BEATTY. P. W., BRODIE, A. E., ELLIS, W. W., AND POTTER, D. W. (1980). High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106, 55-62. SHASBY, D. M., SHASBY, S. S., BOWMAN, C. M., Fox, R. B., HARADA, R. M.. TATE, R. M., AND REPINE, J. E. (198 1). Angiotensin-converting enzyme concentrations in the lung lavage of normal rabbits and rabbits treated with nitrogen mustard exposed to hyperoxia.
Am. Rev. Respir. Dis. 124, 202-203. SOROKIN, S. P. (1967). A morphologic and cytochemical study on the great alveolar cell. J. Histochem. Cytochem. 14,884-867. SUTHERLAND, M. W., NELSON, J., HARRISON, G., AND FORMAN, H. J. (1985). Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages. Arch. Biochem. Biophys. 243, 325-33 I. SUTTORP, N., AND SIMON, L. M. (1986). Importance of the glutathione redox cycle for the resistance of lung epithelial cells against a polymorphonuclear leukocytemediated oxidant attack. Biochem. Pharmacol. 35, 2268-2770. SWANK, D. W., AND MOORE, S. B. (1989). Roles of the neutrophil and other mediators in adult respiratory distress syndrome. Maqjo Clin. Proc. 64, 1118- 1132. WARHEIT, D. B., CARAKOSTUS, M. C., HARTSKY, M. A., AND HANSEN, J. F. (1991). Development of a shortterm inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107,350-368. WEISS, S. J., YOUNG, J., LOBUGLIO. A. F., SLIVKA, A., AND NIMEH, N. F. (198 1). Role of hydrogen Peroxide in neutrophil-mediated destruction of cultured endothelial cells. J. Clin. Invest. 68, 7 14-72 1.