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American Journal of Pathology, Vol. 132, No. 2, August 1988 Copynght © American Association of Pathologists

Ozone-Induced Lamellar Body Responses in a Rat Modelfor Alveolar Injury and Repair J. U. BALIS, MD,J. F. PATERSON, BA, E. M. HALLER, S. A. SHELLEY, PhD, and M. R. MONTGOMERY, PhD

From the Departments ofPathology and EnvironmentalOccupational Health, University ofSouth Florida and James A. Haley Veterans Hospital, Tampa, Florida

Exposure of adult rats to 3 ppm ozone for 8 hours results in diffuse alveolar damage with well-defined sequential stages of bronchiolo-alveolar injury and repair. This model is characterized by acute pulmonary edema showing high concentration of lavage fluid protein that is maximally elevated at 24 hours with return to control level at recovery (96 hours). Using techniques that enable optimal preservation of lamellar body ultrastructure, it was demonstrated morphometrically that expansion ofthe vacuolated lamellar body (LB) compartment is an early, transient LB response of the type II cell to acute injury. This change appears to be initiated by increased LB secretion. The reparative stage, 24-48 hours postexposure, begins with hypertrophy rather than hyperplasia of many type II cells, resulting in a 3-fold increase of mean type II cell volume at 48 hours. During this stage there is also significant expansion of the total LB compartment with corresponding increased LB storage of surfactant disaturated phosphatidylcholine (DSPC) per type II cell. At

recovery, 96 hours, the lungs contained twice the normal numbers-of type II cells, but the total size of lamellar body compartment per type II cell as well as the DSPC content of the isolated lamellar body pool returned to normal levels. In contrast, accumulating surfactant DSPC in lavage fluid increased progressively throughout the reparative and recovery stages presumably due in part to parallel increase in type II cell numbers at 48 and 96 hours. Additional changes of surfactant included abnormal secretion of densely coiled lamellar bodies that accumulated in alveolar spaces at the expense of tubular myelin. These observations indicate that acute oxidant injury to alveoli initiates progressive hypertrophy followed by hyperplasia of type H cells, in association with sequential development of characteristic lamellar body changes leading to increased storage and secretion of surfactant with reduced ability to form tubular myelin. (Am J Pathol 1988, 132:330-344)

THE LUNG SURFACTANT PHOSPHOLIPIDS are synthesized by the type II cells, stored in the lamellar bodies of these cells, secreted into the hypophase of the alveolar lining layer, organized extracellularly into various morphologic forms including tubular myelin, and spread rapidly onto the alveolar air-liquid interface, where they form a monomolecular surface film, composed predominantly of the strongly surface active disaturated phosphatidylcholine (DSPC). 1-5 Type II cells also produce and secrete surfactant-associated glycoproteins and proteolipids, which appear to promote the adsorption and spreading of DSPC on the alveolar surface6"1' as well as the reuse of surfactant lipids by the type II cells.5.2"3 The lamellar bodies are composed of a perilamellar limiting membrane, a thin peripheral zone containing amorphous material and occasionally small vesicles, and electron dense, membranelike lamellae that are

arranged in stacks or whorls. The biogenesis of lamellar bodies involves the participation of multivesicular bodies (MVB) and lysosomal granules in the formation of intermediate forms.2-4 Isolated lamellar bodies were shown recently to contain surfactant-associated glycoproteins and proteolipids.'4 These lung-specific proteins appear to be localized within the peripheral compartment and multivesicular component of lamellar bodies.'5"6 In addition to surfactant components, the lamellar bodies store, in an acidic environSupported by National Institutes of Health Grants HL34793 and ES03340 and by the Medical Research Service of Veterans Administration. Accepted for publication April 5, 1988. Address reprint requests to John U. Balis, MD, Department of Pathology, Box 11, University of South Florida, College of Medicine, Tampa, Florida 33612.

330

OZONE-INDUCED LAMELLAR BODY RESPONSES

Vol. 132 * No. 2

331

Table 1-Stereologic Estimates of Type Il Cells Numbers and Volumes in Rats Exposed to 3 ppm Ozone for 8 Hours Time postozone exposure

Control

Zero-time

24-hours

48-hours

96-hours

NV Left Lung

23 (3)

25(1)

24 (2)

43(10)

53 (9)*t

[X1o6] NV Total Lung

65 (8)

75 (4)

65 (4)

125(31)

126 (35)

382 (34) {92}

477 (44)

926 (59)* {160}

1167 (106)f {185}

672 (50) {191}

[X1O0G] VN

{135}

N, = Estimated mean number of type 11 cells (number of rats in each group = 3).

VN = Estimated mean volume of type 11 cells in cubic microns. *'1 Values, Mean with SEM shown in parenthesis, are significantly different control*; control, 0-time, and 24 hourst; and control, 0-time and 96 hours*. I } = Number of cells per group.

at P < 0.005

ment, 17 a diversity of lysosomal hydrolases, 18-27 whose pathophysiologic significance is unclear. The authors' working hypothesis is that lamellar bodies are multifunctional compartmented organelles, that incorporate basic components of both secretory pathway and lysosomal system. The basic reactions of alveolar epithelium to oxidative and other forms of acute lung injury are well characterized.2'2833 They include a sequence of events that begins with necrosis of type I epithelium followed by hyperplasia and hypertrophy of type II cells. However, the secretory functions of acutely injured versus regenerating type II cells have not been defined adequately. The present study uses short exposure (8 hours) of rats to 3 ppm ozone as a model for bronchiolo-alveo-

lar injury and repair. Using this model, it has been demonstrated that oxidative injury to type II cells results in characteristic lamellar body lesions, followed by increased storage and secretion ofseemingly defective surfactant. Portions of the study appeared previously in abstract form.34

(as determined by the Duncan's Multiple Range Test) from

Materials and Methods Animal Model Adult, specific pathogen-free, male Fischer 344 rats weighing 260 ± 0 g were used in all experiments. The rats were obtained from Charles River, barrier maintained during shipment, housed in protective filter cages, and acclimated in the authors' facility for at

Table 2-Type II Cell Morphometric Estimates in Rats Exposed to 3 ppm Ozone for 8 Hours. Values are Expressed in Square Microns

Time postexposure (hours)

Control N

Cell

Cytoplasm Nucleus

Mitochondria Total LBt compartment

Vacuolated LB

Compound LB Composite LB Electron-dense MVBX Electron-lucent MVB

=

271

0 N 285

24

48

96

288

N 264

76.7* (2.55) 18.5*

99.2* (2.91) 79.7* (2.34) 19.5*

(0.91)

(1.36)

(1.32)

5.3

1.7

6.9* (0.27) 17.6* (0.93) 1.6

(0.24)

(0.22)

6.2* (0.23) 16.5* (0.87) 1.0 (0.15) 3.8 (0.43)

69.6* (2.49) 55.7* (2.00) 14.0* (1.04) 5.5 (0.23) 11.8 (0.84) 0.9 (0.13) 2.4 (0.44) 0.3 (0.03) 0.2 (0.02) 0.2 (0.02)

=

N

=

424

58.0

53.2

95.2*

(1.92)

(1.79)

(3.33)

43.0 (1.41) 15.0

40.0 (1.35) 13.3

(0.93) 4.9

(0.20) 9.2 (0.46) 0.8 (0.12) 0.9 (0.15)

(0.22) 8.1

(0.43) 0.7

2.6

(0.11)

(0.35)

N

=

0.2

0.2

0.6*

0.4

(0.03)

(0.03)

(0.07)

(0.05)

0.2

0.1*

(0.02)

(0.02)

0.3 (0.03) 0.3 (0.03)

(0.03)

0.2

0.3

(0.02)

(0.03)

0.4 0.1

(0.02)

=

*Values, Mean with SEM shown in parenthesis, are significantly different from control at P < 0.05 as determined by Duncan's Multiple Range Test.

t Lamellar body. t Multivesicular body.

332

BALIS ET AL

AJP * August 1988

Table 3-Morphometric Estimates of Acute Lamellar Body Changes in Type 11 Cells from Centnacinar and Peripheral Lung Regions of Rats at Zero Time After Exposure to 3 ppm Ozone for 8 Hours

were killed during the morning hours, 9-10 A.M., by an overdose of ketamine hydrochloride and subsequent exsanguination.

Lung region

Centriacinar Control Zero-time Total LB compartment Volume density of LBt Vacuolated LB compartment % Vacuolated LB

N= 176

N= 187

9.80 (0.525)

7.42t (0.476) 0.15 (0.010)

0.17

(0.009) 0.81 (0.152) 8.71 (1.574)

Peripheral Control Zero-time N= 190

N= 138

8.58

8.51

(0.009)

(0.714)

0.15

0.17

(0.009)

(0.013)

1.11

0.74

2.44*

(0.177) 15.62* (2.213)

(0.126)

(0.443) 27.34t (3.503)

9.96 (1.753)

*'t Values, Mean with SEM shown in parenthesis, are significantly different from control at P < 0.05 and P < 0.001, respectively, as determined by Student's t-test for unpaired data. t Lamellar body. least 1 week with free access to water and food. Groups of 6 rats were exposed to ozone for 8 hours in a specially designed lucite exposure chamber.35 Ozone was generated by passing 100% oxygen through an ozone generator (OREC Model 03V1-0, Ozone Research and Equipment Company, Phoenix, AZ). The ozone was mixed with compressed air to produce 3.0 ± 0.2 ppm ozone concentration. The total gas flow was maintained at 120 1/min that resulted in approximately 1 air exchange per minute. The ozone concentration within the chamber was monitored continuously with a Mast Model 727-3 ozone monitor (Mast Development Company, Davenport, IA) connected to a stripchart recorder. At the end of the 8-hour ozone exposure, the animals were returned to room air. Some animals were killed immediately (zero time) while others were maintained in room air for 24, 48, and 96 hours postexposure. In all experiments, the animals, including control rats maintained in room air,

Preparation of Lungs for Morphologic Studies The lungs were initially fixed in situ by intratracheal instillation of prewarmed (37 C) 0.1 M phosphate buffered 2.5% glutaraldehyde at 20 cm water pressure for 15 minutes. Subsequently, the trachea was ligated, and the lungs were excised and fixed for an additional 4 hours at 37 C. Following fixation, the left lung was weighed and its length measured. The lung was then sliced into 5 mm slices perpendicular to the long axis of the lung, and 3 of these slices, randomly selected, were mounted in 3% agar on a plastic disk with the lung slices oriented to produce sections parallel to the main bronchus in the slice. Sections, 500 ,uthick, were obtained using a Brinkman MacIllwain tissue chopper. Following washing, the sections were postfixed at 4 C in buffered 1% reduced osmium tetroxide for 7 hours, washed thoroughly in 0.9% saline to remove excess osmium, stained en bloc at 4 C for 90 minutes with buffered uranyl acetate (1.5%), rapidly dehydrated in a graded series of acetone, and flat embedded in EM bed 812. Tissue shrinkage was measured as described previously,36 using sequential photographs of lung slices after fixation and flat embedding. The above procedure of tissue processing resulted in