31, 176-188 (1983)
2. Parenchymal DANIEL
Department of Anatomy, University of Kentucky, Lexington, Kentucky 40536, and Kentucky Tobacco and Health Research institute, Cooper and Alumni Drives, Lexington, Kentucky 40506 Received March 25, 1982 Cigarette smoke and hydrocortisone acetate (HCA) induce marked abnormalities in lungs of C57BL/6J male mice. In many pulmonary regions of smoke-exposed, HCA-treated animals, alveoli were highly congested with surfactant and flocculent material. In addition, prominent alveolar collapse and septal hypertrophy were common. These conditions resembled pulmonary alveolar proteinosis described in humans. Administration of HCA to sham-treated animals also produced lung abnormalities, however, considerably milder in severity, while stress (resulting from sham treatment) or HCA injections of mice alone failed to induce any pulmonary tissue disorder. Data reported indicate that the genesis of abnormal conditions which resemble pulmonary alveolar proteinosis is potentiated by cumulative effects of different treatments (i.e., smoke, HCA, and stress), most significant being the interaction between cigarette smoke and the steroid.
INTRODUCTION It is presently accepted that pulmonary macrophages (septal and alveolar) play a significant role in the maintenance of lung function and integrity. In this regard, these phagocytes increase in number far above basal levels when excess particulate material gains access into the lungs (Bowden and Adamson, 1978; Kavet et al., 1978). In accord, pulmonary macrophages increase in number markedly in response to particulate material conveyed during inhalation of whole smoke (Pratt et al., 1971; Matulionis and Traurig, 1977; Matulionis, 1979), apparently a reaction to increased clearance demands. In addition to clearance, pulmonary macrophages have been implicated in a host of other important activities, for example, turnover of intraalveolar surfactant, secretion of proteolytic enzymes, participation in the immunologic reaction, and decapacitation of bacteria (for review, see Hacking and Golde, 1979). However, data bearing on the question as to whether the highly elevated pulmonary macrophage population induced by smoke inhalation is involved in maintaining alveolar and lung parenchymal integrity do not exist. It is possible to prevent the influx and subsequent accumulation of these cells in lungs of normal and smoke-exposed animals by administration of hydrocortisone (Blusse Van oud Alblas and VanFurth, 1979; Matulionis, 1982). Therefore, the present study was undertaken to describe and record the morphological events that occur in lungs of smoke-exposed animals following the reduction of the pulmonary macrophage population by hydrocortisone treatment. 176 0013-9351/83 $3.00 Copyright All rights
@ 1983 by Academic Press, Inc. of reproduction in any form reserved.
RESPONSE TO SMOKE AND HYDROCORTISONE
Ninety-nine 7- to &week-old C57BW6.I male mice were used in the present study. The animals were exposed to cigarette smoke, sham treated, housed, and maintained by the Kentucky Tobacco and Health Research Institute as previously described (Matulionis, 1982). Smoke was generated from the Kentucky Reference 2Al cigarette (University of Kentucky, Lexington, KY.). Five groups of animals were used to determine the effects of hydrocortisone acetate (HCA) on the morphology of lung tissue. Group 1 consisted of absolute controls and those injected with a vehicle used to suspend HCA; group 2 was composed of sham-treated animals; group 3 received injections of HCA only; group 4 consisted of shamtreated mice injected with HCA; and group 5 contained smoke-exposed animals injected with HCA (Matulionis, 1982). Smoke and sham treatment (Matulionis, 1982) lasted for 35 days before administration of HCA and during 13.5 days of the steroid treatment. Groups 3 - 5 received 0.5 mg/g body wt of HCA (hydrocortisone 21 acetate; Sigma Chemical Company, St. Louis, MO.) suspended in 0.1 ml of vehicle (containing 0.5% carboxymethyl cellulose, 0.9% benzylalcohol, and 0.4% polysorbate 80 in saline) (Thompson and VanFurth, 1970). All injections were administered subcutaneously in the nuchal region. Specific composition, numbers, animal weights, and mortality rate of each group are indicated in Table 1. Technical circumstances related to smoke exposure of animals prevents the simultaneous comparison of lung response in animals exposed only to smoke to those exposed to smoke and treated with HCA. However, the pulmonary response to smoke exposure alone is documented in a previous study (Matulionis, 1979). Therefore, the results of that study (Matulionis, 1979) are used to compare and contrast the effects produced by smoke and HCA treatment. Prior to autopsy of lungs, the mice were anesthetized by intraperitoneal injecTABLE GROUPNUMBERS,TREATMENT,NUMBERP
Group composition and treatment Normal controls None Sham-treated mice None Normal controls injected with HCA Sham-treated mice Injected with HCA Smoke-exposed animals Injected with HCA Total
Number of animals/group prior to HCAb injection
Number of surviving animals/ group
Mean wt. of surviving animals 21 SEM
Mortality rate over 13.5-day period (%)
23.5 +- 0.5
21.2 2 0.8
21.5 f 0.4
20.0 2 0.4
19.0 t .9
a Three to six animals were assessed at 6.5, 8.5, 11.5, and 13.5 days after HCA injection. b Hydrocortisone acetate.
tions of 0.25-0.3 cc of 10% Nembutal and exsanguinated by severing the abdominal aorta. Lung tissues were obtained at 6.5, 8.5, 11.5, and 13.5 days after HCA injection. In addition, lungs from three control animals were sampled on the day that groups 3-5 were injected with HCA. From animals autopsied at 6.5 and 13.5 days after HCA treatment, a l-mm transverse slice was obtained from the inferior aspect of the cephalic two-thirds of the left lung, sectioned into l-mm cubes, and processed for electron microscopy (Matulionis, 1975). The remaining portions of the left lungs from these animals and the cephalic two-thirds of left lungs from animals autopsied at 0.0, 8.5, and 11.5 days after HCA treatment were prepared for light microscopy by routine partin methods, serially sectioned at 4 pm through approximately 360 pm of tissue, and stained with periodic acid-SchifY (PAS) reagent - hematoxylin. At each sampling time, the following parameters were quantified: volume densities of alveolar space surrounded by abnormally hypertrophied or collapsed lung parenchyma, alveolar space surrounded by normal lung parenchyma, abnormally hypertrophied or collapsed parenchyma, and normal lung parenchyma. Quantitation of the above parameters was done with light microscopy at two levels within the 360~pm area of the cephalic two-thirds of the left lung in each animal. Pulmonary parenchyma was classified as hypertrophied when it deviated markedly from the normal appearing tissue (Fig. 1). If assessment of the parenchyma, in regard to its normalcy, was problematic, that area was scored as normal. A Zeiss Kpl-8x integrating eyepiece with a graticule containing 25 asymmetrically arranged test points within a circle was used to estimate the volume densities. The magnification of the microscope was adjusted (80x) to allow the graticule within the eyepiece to circumscribe the lung tissue section in two to four visual fields. At each of the two levels, 50 to 100 test points (hits) were recorded by superimposing the graticule on the tissue and data sheets via a camera lucida. The volume densities were estimated by the equation Vvi = Ppi (Weibel, 1979) where Vvi is the volume fraction (%) occupied by a structure (volumetric density) and Ppi is equal to the fraction of test points intercepted by the structure. All data were statistically assessed by analysis of variance. RESULTS
The general health condition of all animals prior to HCA injections was good. However, after the steroid injections, animals of groups 4 (sham treated, HCA injected) and 5 (smoke exposed, HCA injected) became lethargic and appeared “sickly.” Those of group 5 also exhibited signs of respiratory distress. The poor health condition of groups 4 and 5 animals was reflected in a high mortality rate (Table 1). In addition, even though the health condition of group 3 animals (HCA-injected controls) appeared good, 27% of mice from this group died over a 13.5-day period after the onset of HCA injections. Detailed consideration and discussion of animal mortality, weight, and other parameters (i.e., pulmonary macrophage and leukocytic response to smoke exposure and steroid treatment) have been reported earlier (Matulionis, 1982) and will not be further discussed here.
FIG. 1. Pulmonary tissue of a group 4 (sham-HCA-treated) animal depicting normal (N) and abnormally hypertrophied (H) regions 6.5 days after steroid injection. Alveoli, A; alveolar septa, S. X 180. FIG. 2. Lungs of group 3 animal revealing normal-appearing tissue. Alveoli, A, alveolar septa, s. x168. FIG. 3. Lungs of a group 5 animal, 8.5 days following HCA treatment, showing a markedly congested abnormal region. Alveolar spaces (A) are reduced and many are filled with material (arrows) which stained positively with PAS. X 180.
of Pulmonary Tissue
Light microscopic examination of pulmonary tissue from animals of groups 1 - 3 revealed normal lung architecture (Fig. 2). For the most part, alveolar spaces were clearly visible, patent, and free of noticeable debris (Fig. 2). The thickness of alveolar septa were relatively uniform and easily discernible (Fig. 2). However, it should be noted that on occasion, especially near the surface of the lungs, the septa appeared somewhat thickened and the alveolar spaces more distorted than those depicted in Fig. 2. Such conditions were attributed to mechanical damage resulting from tissue manipulation during autopsy and/or tissue processing. Assessment of lung tissue from group 4 animals (sham treated, HCA injected) revealed two morphologically distinct conditions (Fig. 1). One appeared typically normal resembling that of control animals described above. The other condition (abnormal in nature) was characterized by reduced alveolar size, thickened septa, and atelectatic regions (Fig. 1). This type of abnormal condition was most prevalent during the first three sampling periods (6.5, 8.5, and 11.5 days) and was only occasionally noted 13.5 days after HCA treatment. Light microscopic assessment of lungs from smoke-exposed, HCA-treated ani-
mals (group 5) revealed large areas of prominent multifocal to diffuse thickenings of alveolar septa with a marked reduction of alveolar space (Fig. 3). Many alveoli were congested with PAS-positive material (Fig. 3). The architecture of these areas was altered to such a degree that the tissue no longer resembled lungs. However, in other legions (not depicted) lung tissue appeared normal or showed only slight hypertrophy of septa and reduction of alveolar space. Electron
Ultrastructural morphology of lungs from groups l-3 animals was normal. Normal or near normal morphology was also observed in many regions of lung sections from all group 4 animals. However, in this group many areas of the lungs appeared collapsed (Fig. 4). In addition, alveoli contained surfactant (Fig. 4) in excess of that seen in alveoli of control animals. However, in contrast to control animals, pulmonary macrophages (septal and alveolar) were encountered only rarely. The cytology of parenchymal cells was typical. The ultrastructure of many areas of the lungs of group 5 animals (smoke exposed, HCA treated) was markedly different than that of group 4 or control mice. In many areas alveolar collapse was prominent. Also, in these regions the septa seemed to be thickened. In the most normal appearing regions alveoli were free of surfactant (Fig. 5). However, other alveoli contained surfactant material in excess of that of group 4 animals (Fig. 5). In more abnormal regions of the lung sections, alveoli contained large heterogeneous inclusions in addition to surfactant (Fig. 6). In even more abnormal areas which were presumed to be those containing intraalveolar PAS-positive material (Fig. 3), the air spaces were totally filled with a flocculent electron-opaque material which seemed to suspend the surfactant (Fig. 7). Still other spaces in these highly abnormal regions were completely obliterated by surfactant (Fig. 8). Pulmonary macrophages, as in lungs of group 4 animals, were rarely encountered, an effect produced by HCA treatment (Matulionis, 1982). Those that were noted contained numerous inclusions, some of which were morphologically similar to the intraalveolar surfactant. The aberrant manifestations discerned in the smoke-exposed, HCA-treated mice (group 5) were never observed in any animal of the other groups. Therefore, excessive amounts of intraalveolar surfactant and other materials, as well as the alveolar collapse and septal thickening appears to be a combined effect of smoke exposure and steroid treatment. Morphometry
Space and Pulmonary
Volume density (percentage volume occupied by measured structure per unit volume) of space which was surrounded by normal parenchymal tissue, and that which was encompassed by abnormally thickened septal, or collapsed tissue was quantified. Further, pulmonary parenchyma, defined as tissue comprising the alveolar septa (excluding bronchi, bronchioles, large blood vessels, and connective tissue trabeculae) was divided into normal-appearing, collapsed, and/or hypertrophied septa, and quantified morphometrically. The volume density (30.0 * 0.9% mean value over time) of alveolar space surrounded by normal tissue was similar (P = 0.10) in animals of Groups l-3 (Fig.
FIG. 4. Electron micrograph of lung tissue from a group 4 animal 13.5 days after HCA treatment depicting relatively normal (upper area) and collapsed regions (lower area). Surfactant material in form of tubular myelin (T) and myelin figure (M) are observed in alveoli (A). x7420. FIG. 5. Ultrastructure of lung tissue from a group 5 animal 6.5 days post-HCA treatment depicting one alveolus free of material (Af) and others which contain surfactant (Sr). x7420. FIG. 6. Ultrastructure of alveolus from a group 5 animal (different from that shown in Fig. 5) 6.5 days following steroid treatment showing surfactant (Sr) and large heterogeneous inclusions (Hi). x7420. 181
FIG. 7. Electron micrograph of an alveolus from a group 5 animal 13.5 days after HCA treatment filled with flocculent material (Fm) which suspends tubular myelin (T) and myelin figures (M) type of surfactant. x7240. FIG. 8. Electron micrograph of a portion of an alveolus from a group 5 animal 13.5 days following HCA treatment. Note that tubular myelin (T) and myelin figure (M) type of surfactant tills the alveolus completely. x 13,750.
9). Likewise, the volume density of space surrounded by abnormal parenchyma was similar in these groups (P = 0.76), however, considerably lower (1.6 + 0.4% mean value over time) than that surrounded by normal tissue (Fig. 9). The mean values (over time) of alveolar space in lungs of group 4 animals surrounded by normal and abnormal parenchyma were 27.5 f 3 and 6.5 + 1.2%, respectively. These values were significantly different from those of groups l-3 (P = 0.0001) (Fig. 9). The most striking decrease of alveolar space surrounded by normal lung parenchyma (14.5 ? 2% mean value over time) and an increase in that surrounded by abnormal tissue (15.25 ? 1.1%) was noted in lungs of group 5 mice (Fig. 9). Alveolar space surrounded by normal parenchyma as a percentage of total lung was significantly reduced in this group of animals (I’ < 0.0001) when compared to group 4 and control values (Fig. 9). As anticipated, the volume density of abnormal parenchyma per day correlated well with an increase of alveolar space surrounded by this type of tissue. Normal and abnormal lung parenchyma in groups l-3 animals seemed to vary considerably at different sampling points (Figs. 10 and 11, respectively). However,
RESPONSE TO SMOKE
FIG. 9. Volume densities of normal (surrounded by typical septa) and abnormal (surrounded by collapsed or hypertrophied septa) alveolar space. The different treatments or groups of animals in this and the subsequent graphs are identified in the rectangle above the abscissa. The mean volume density over time of normal alveolar space for groups l-3 was 30 i O.%, for group 4,275 + 3%, and group 5, 14.5 + 2%, and that of alveolar space surrounded by collapsed or hypertrophied septa for groups l-3 was 1.6 -C 0.4%, for group 4,6.5 k 1.2%, and group 5, 15.25 5 1.1%. Vertical lines represent 1 SEM.
analysis of variance revealed that the volume density variations were not significant over time (P > 0.5). A considerably larger amount of normal than abnormal tissue (33 * 1.1 and 9.8 * 1.3%, respectively) was observed in the control animals (Figs. 10, 11). The volume density of normal and abnormal lung parenchyma in group 4 animals was similar (Figs. 10, 11) (25.0 2 1.4 and 26.5 + 2%, respectively) but the normal tissue was significantly lower (P = 0.0001) and the abnormal tissue significantly higher (P = 0.0001) (Figs. 10, 11) when compared to control values. The most marked decrease in normal lung parenchyma (12.8 +- 2.3% mean value over time) with a concomitant increase in volume density of abnormal tissue (38.0 t 3.8%) was observed in group 5 animals (Figs, 10, 11). The transformation of normal to abnormal parenchyma was highly significant (P = 0.0001) and indicates increase in pulmonary mass at the expense of alveolar space. DISCUSSION Three manifestations appear in lungs of smoke-exposed mice which have been treated with hydrocortisone acetate (HCA): (1) marked accumulation of surfactant
DAYS AFTER lsr MA ItVJECTlOAt FIG. 10. Volume densities of normal parenchyma in lungs of the five groups of animals studied. The mean volume densities of normal parenchyma over time in animals of groups 1- 3, group 4, and group 5 were 33 -C 1.1, 25 f 1.4, and 12.8 f 2.3%, respectively. The vertical lines represent 1 SEM.
and other material within alveolar spaces; (2) a decrease in alveolar space surrounded by normal septal tissue; and (3) an increase in abnormally thickenedcollapsed alveolar parenchyma. In general, these conditions resemble pulmonary alveolar proteinosis described in humans (Rosen ef al., 1958). The intraalveolar material appearing as tubular myelin and myelin figures ob-
L-y? s ._.... /-*
% 20 f 0
a 1 5
FIG. 11. Volume densities of abnormal parenchyma (collapsed or hypertrophied septa) in lungs of the five different groups of animals. Mean volume densities over time of groups l-3, group 4, and group 5 were 9.8 f 1.3, 26.5 -C 2, and 38 k 3.2%, respectively. Vertical lines represent 1 SEM.
served in lungs of group 5 mice conforms to the ultrastructure of surfactant described by several investigators (Mason et al., 1972; Gil and Weibel, 1969; Nichols, 1976a). Tubular myelin in the alveolar space has been designated as stored form (Mason, 1976) or effete breakdown products of surfactant (Kistler et al., 1976). Myelin figures appear to originate from the Type II alveolar cells and represent extruded lamellar bodies (Nichols, 1976a) suggesting their kinship to surfactant. The explanation for the accumulation of intraalveolar surfactant observed in the present study is related to the pulmonary macrophage. A number of studies indicate that these cells are intimately involved in clearance of excess and/or effete surfactant (Nichols, 1976b; Brain et al., 1978; Johansson et al., 1980; Wiernik, et af., 1981). On the other hand, it has been demonstrated that HCA treatment markedly reduces the pulmonary macrophage population size in normal and smoke-exposed mice (Blusse van Oud Alblas et al., 1981; Matulionis, 1982). The degree of reduction in the smoke-exposed animals is such that the highly elevated population of these cells (resulting from smoke inhalation) (Matulionis and Traurig, 1977; Matulionis, 1979) is lowered to or below control levels (Matulionis, 1982). In view of these observations and the fact that pulmonary macrophages have been implicated in the phagocytosis of excess and/or effete intraalveolar surfactant, it follows that reduction of the macrophage numbers would lead to surfactant accumulation in the air spaces. In addition to surfactant, alveolar spaces were often filled with flocculent electron-opaque material (Fig. 7). Since capillary damage was not observed, this material might represent accumulation of normal transudate that is normally cleared from the lungs by the macrophage system; in the absence of the phagocytic cells, it remains in the alveolar spaces. A similar hypothesis was proposed by investigators assessing the manifestation of alveolar lipoproteinosis in rats following inhalation of silica (Heppleston et al., 1970), pulmonary alveolar proteinosis in humans (Rosen et al., 1958; Ramiez and Harlan, 1968), and lipidosis induced by drugs in animals (Lullmann-Rauch et al., 1972). Functionally decapacitated pulmonary macrophages have been implicated in these abnormal conditions leading to diminished alveolar clearance (Harris, 1979; Golde, 1979) and thus accumulation of intraalveolar material (including surfactant). It is not possible to state that the macrophages observed in this study were functionally defective; however, their near absence would strongly suggest reduced alveolar clearance resulting in a condition resembling pulmonary alveolar proteinosis. Abnormal conditions noted are apparently not the sole result of HCA treatment since in sham and control animals injected with the steroid, severe abnormalities failed to develop. Further, cigarette smoke alone does not lead to the massive accumulation of intraalveolar surfactant and flocculent material nor of septal collapse and hypertrophy (Matulionis, 1979). Thus, it appears that a single insult, i.e., smoke exposure or HCA treatment, fails to evoke pulmonary abnormalities. In the present study, three different “insults,” smoke, HCA, and stress (resulting from manipulation of animals during smoke or sham treatment) were involved. Apparently, severity of the abnormalities is potentiated by cumulative effects of the different insults. Accordingly, lungs of group 5 animals, which were exposed to all three “insults,” were most severely altered, those of group 4, exposed to HCA treatment and stress showed minimal alterations, while those of groups 2 and 3,
receiving only one “insult” (stress or HCA, respectively), were normal. Onset or potentiation of a disease by combined effects of insults has been also observed by Haschek and Witschi (1979). They reported that mice develop only slight pulmonary fibrosis when treated with butylated hydroxytoluene. Extensive interstitial fibrosis manifests when animals are also exposed to oxygen, whereas oxygen alone had no effect. Likewise, urethan-initiated lung adenoma formation is promoted by insulting the animal with a second compound (butylated hydroxytoluene) (Witschi and Lock, 1979). The present study indicates that cigarette smoke when coupled with steroid treatment of animals causes the manifestation of lung abnormalities which resemble pulmonary alveolar proteinosis. This observation might be worthy of consideration in health management of human smokers that are subjected to cortisone therapy. Concomitant with accumulation of intraalveolar material, reduction of air space, increase in collapsed alveoli, and hypertrophied septa were noted. Smoke alone produces interstitial inflammation, but never such prominent alterations of space and septal tissue (Matulionis, 1979). HCA treatment alone (group 3) or coupled with stress (group 4) had no or slight (respectively) effect on alveolar space and parenchymal tissue. Thus, genesis of these abnormal conditions again appears to be promoted by combined effects of the different treatments (smoke, HCA, and stress), since lungs of group 5 mice exhibited most prominent deviations from the norm, those of group 4 minimal changes, and those of groups 2 and 3 no abnormalities. Factors which caused thickening of alveolar septa are not known. However, a speculative explanation for alveolar collapse can be postulated. Petrov and Filippeko (198 1) provide evidence to show that pulmonary atelectasis occurs if normal surfactant balance is disturbed. Surfactant levels in human smokers are lower than in nonsmokers (Finley and Ladman, 1972). Likewise, smoke exposure decreases the yield of lavageable surfactant in rats (Le Mesurieret al., 1980, 1981). Although large quantities of surfactant were observed in lungs of group 5 animals, the physical properties of this material are apparently altered by cigarette smoke rendering it incompetent to maintain the stability of the alveoli (Cook and Webb, 1966; Finley and Ladman, 1972; Le Mesurier, 1981). The observations recorded in this report indicate that abnormal conditions resembling pulmonary alveolar proteinosis result from a combined effect of primarily cigarette smoke exposure and steroid administration with stress playing an as yet undetermined role. Even though it is recognized that smoke inhalation occurs via different routes in animals and humans, resulting in altered amounts of particulate matter deposited in the lungs, the results lend themselves to an interesting speculative question. Could susceptibilities to respiration problems of human smokers who are in stressful situations and on cortisone treatment (for example, kidney transplant patients) be decreased by abstinence from smoking and/or removal of stress? ACKNOWLEDGMENTS The author wishes to express his gratitude to Linda Simmerman for her expert technical assistance and to John A. Turbek for statistical analysis of the data. This investigation was supported by the University of Kentucky, Tobacco and Health Research Grants 24133 and 4AOO7.
RESPONSE TO SMOKE AND HYDROCORTISONE
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