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Pulmonary tissue and cigarette smoke
ENVIRONMENTAL
RESEARCH
27, 361-371 (1982)
Pulmonary
Tissue
and Cigarette
Smoke
1. Cellular Response to Hydrocortisone DANIEL Department Kentucky
H. MATULIONIS
of Anatomy, University of Kentucky, Lexington, Tobacco and Health Research Institute. Cooper Lexington. Kentucky 40506
Kentucky 40536. and and Alumni Drives.
Received April 7, 1981 Hydrocortisone acetate (HCA) administration significantly reduces the population of pulmonary macrophages and blood leukocytes in control, sham-treated, and smoke-exposed C57BL/6J mice. This treatment impedes markedly the influx of macrophages from bone marrow into the lungs. The small number of new phagocytes noted in lungs following HCA treatment appears to arise by proliferation of in situ pulmonary macrophages. Mortality rate of sham-treated and smoke-exposed mice was approximately twice that of control animals following HCA treatment. While severe pulmonary disorders were noted in lungs of HCAtreated, smoke-exposed animals, considerably milder abnormalities were seen in lungs of sham-treated mice. The data reported indicate that physical stress generated by manipulation during sham and smoke treatment, exposure to cigarette smoke, and reduction of pulmonary macrophages and leukocyte populations by HCA administration are factors which adversely affect pulmonary integrity and survival time of the animals.
INTRODUCTION
The importance of pulmonary macrophages in maintaining the sterility and normal function of the lungs has long been recognized and is reviewed elsewhere (Martin and Wart-, 1977; Huber et nl., 1977). The number of phagocytes increases markedly when particulate materials gain access to the lungs in excess of basal levels (Bowden and Adamson, 1978; Kavet et al., 1978; Matulionis, 1979a). Inhalation of whole cigarette smoke, conveying numerous particles, evokes a prominent phagocytic (Matulionis, 1979a; Brody and Craighead, 1975; Matulionis and Traurig, 1977; Pratt et al., 1971) and leukocytic (Matulionis, 1979a) response in the lungs; however, macrophages accumulate only in pulmonary tissue with no corresponding increase in other tissues (Matulionis, 1979a). The markedly elevated population of lung macrophages does not arise from in situ proliferation of macrophages, but are, for the most part, of bone marrow-blood monocyte origin (Matulionis, 1979b). Thus, interference with the influx of phagocytic cells from the bone marrow-monocyte system should decrease the macrophage population in lung tissue. Generation of macrophages by bone marrow is influenced adversely by hydrocortisone acetate (HCA) (Thompson and VanFurth, 1973). Further, it has been shown that HCA administration markedly reduces the pulmonary macrophage population in normal mice (Blusse Van oud Alblas and VanFurth, 1979). The number of phagocytes is also reduced at the sites of inflammation and wound healing by the steroid treatment (Leibovich and Ross, 1975). In addition, it has been reported that in animals exposed to particles of iron oxide, peripheral blood 361 0013-9351/82/020361-11$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
362
DANIEL
H.
MATULIONIS
leukocytes are reduced significantly following treatment with hydrocortisone acetate (Kavet et al., 1978). However, data regarding the ability or inability of this steroid to decrease the influx of macrophages into the lungs and reduce the blood leukocyte population in animals maintained on a “smoking schedule” are not available. Therefore, the goal of the present study is to determine whether subcutaneous administration of hydrocortisone acetate decreases the elevated pulmonary macrophage population (septal and alveolar) and blood leukocytes in animals exposed to cigarette smoke. This study is an initial segment of a broader investigation designed to develop an animal model that can be utilized to assess the genesis of pulmonary disorders related to cigarette smoking. MATERIALS
AND METHODS
Lung tissue from 86 7- to 8-week-old C57BL/6J mice was used in this study. The mice were housed, maintained, exposed to cigarette smoke, and sham-treated by the Kentucky Tobacco and Health Research Institute (KTHRI) as previously detailed, (Matulionis, 1979a) with one modification: the animals were housed one per cage. Briefly, smoke exposure of animals occurred in the KTHRI single-port reverse smoking machine. The machine operates in the following fashion (Benner et al., 1973). A lit cigarette is placed in a holder connected to the smoke exposure chamber. Snouts of mice, which are restrained in specially constructed holders, are inserted into the exposure chamber. The puff is generated by an air cylinder which moves over the cigarette creating a slightly above-ambient pressure around the cigarette. Smoke exits into the exposure column from the outlet of the cigarette holder and is diluted with air to a desired concentration (20% smoke). Following the smoke puff, the air cylinder is retracted, allowing the cigarette to burn at atmospheric pressure. The smoke remains in the exposure chamber for 15 set after which it is flushed with room air. Forty-live seconds are allowed to elapse before the next smoke puff is delivered into the exposure chamber. Sham treatment consisted of exposing animals to air instead of smoke. Five groups of animals were established for this study. At the end of the 13.5day-long experiment, Group 1 consisted of 22 untreated-absolute controls; Group 2 was composed of 12 animals each injected subcutaneously (SC) with a vehicle (containing 0.5% carboxymethyl cellulose, 0.9% benzyl alcohol, and 0.4% polysarbate 80 in saline) (Thompson and VanFurth, 1970) used to suspend the HCA; Group 3 contained 19 mice which received SC injections of HCA; Group 4 consisted of 14 sham-treated mice injected with the steroid (HCA); and Group 5 contained 19 smoke-exposed animals which were also injected with HCA (Table 1). All injections were administered SC in the nuchal region. Smoke and sham treatment (Matulionis, 1979a) occurred for 35 days prior to administration of HCA. Animals of Groups 3, 4, and 5 received 0.5 mg/g body wt of HCA (hydrocortisone 21acetate; Sigma Chemical Co., St. Louis, MO.) suspended in 0.1 ml of vehicle, while those of Group 2 were injected with only 0.1 ml of the vehicle (Table 1). HCA and vehicle injections were repeated at 4-day intervals over a period of 12 days. Each animal received four injections. A pharmacological dose of steroid was used to assure a response to the drug since a major goal of the present study was to determine whether it is possible to decrease the markedly elevated pulmonary macrophage population that is induced by cigarette smoke. Presently, experi-
CELLULAR
TREATMENTS,
Group 1 2 3 4 5
NUMBERS,=
Group composition and treatment Normal controls None Normal controls Injected with vehicle Normal controls Injected with HCA Sham-treated mice Injected with HCA Smoke-exposed animals Injected with HCA Total
363
RESPONSE TO SMOKE AND HYDROCORTISONE TABLE 1 WEIGHTS, AND MORTALITY RATES OF MICE
Number of surviving animals/group
Mean wt of surviving animals ?I SEM
Mortality rate over 13.5day period (%I
22
22
23.2 2 0.4
0
12
12
23.8 5 0.5
0
26
19
21.5 2 0.4
21
26
14
20.0 f 0.4
53
36
19
19.0 k 0.9
52
122
86
Number of animals/group prior to HCA” injection
a Three to six animals were assessed at 6.5, 8.5, 11.5, and 13.5 days after HCA injection. b Hydrocortisone acetate.
ments are m progress to evaluate the pulmonary cell response to various doses of HCA. Three days after the first injection of the steroid all animals were injected intraperitoneally with rH]thymidine according to the schedule (9 /.&i/g body wt over a 3-day period) described earlier (Matulionis, 1979a). Lung tissues were obtained at autopsy at 6.5, 8.5, 11.5, and 13.5 days after the initial HCA (or vehicle) injections or 3.5, 8, and 10 days after the first rH]thymidine injection. Tissue sampling began at 6.5 days following HCA administration to allow suffrcient time for the steroid effect to take place. Three days were allowed to elapse after rH]thymidine injections before tissue samples were obtained, since it has been shown (Matulionis, 1979a) that emigration of labeled macrophages into the lungs begins at this time. At each sampling time, lung tissue from three to six animals of each group was processed for autoradiography and scintillation counting. Details of methods used for autoradiography, scintillation counting, and quantitation of pulmonary macrophage populations, as well as the specific regions of the lungs sampled, have been reported earlier (Matulionis, 1979a). Prior to autopsy, the mice were anesthetized by intraperitoneal injections of 0.25 to 0.3 cc of 10% Nembutal. Cardiac blood samples were collected from the right ventricle, the animals were exsanguinated by severing the abdominal aorta, and lung tissue was collected. Total white blood cell counts (WBC) were determined by standard techniques using the ZBi Coulter counter (Table 2). RESULTS General Observations
The general health status, lung tissue, and white blood cell counts assessed for animals in Group 2 (vehicle treated) were normal in every respect and similar to those in Group 1 (absolute controls). In view of these findings, Group 2 animals
364
DANIEL
WHITE Days
post HCA” treatment
Group
6.5 8.5 11.5 13.5 Group
1.08 0.90 1.12 1.45
mean
a Hydrocortisone b Mean c 1 SEM.
+ k + +
BLOOD
1 0.07b 0.06 0.05 0.06
1.138 k 0.115
H.
MATULIONIS
TABLE 2 CELL COUNTS
Group 0.45 0.50 0.57 0.63 0.5375
3 * ” * k
0.05 0.04 0.03 0.06
-r- 0.30
(x 1000)
Group 2.66 1.96 1.44 0.99
f f k +
4 0.21 0.78 0.08 0.10
1.76 k 0.36
Group 3.25 2.26 1.97 1.30
2 + + +
5 0.53 0.23 0.20 0.16
2.20 k 0.41
acetate.
will be incorporated and be considered simultaneously with those of Group 1, and henceforth both groups will be referred to as Group 1. Unavoidable technical circumstances related to smoke exposure of animals prevents the simultaneous assessment of cell response in lungs of animals exposed to smoke only and in those that were both smoke exposed and HCA treated. However, a detailed study has been reported recently (Matulionis, 1979a) which documents such a response to smoke alone. Therefore, results of the previous study by Matulionis (1979a), which describes the effects of smoke exposure only, will be used to compare and contrast effects produced by combined smoke exposure and HCA treatment. The mean weights of animals in Groups 1, 3, 4, and 5, averaged over the four sampling periods, are shown in Table 1. Weights of animals in Groups 1, 4, and 5 were statistically similar to those of control, sham-treated, and smoke-exposed mice, respectively, which had not been injected with the hydrocortisone acetate (HCA) (Matulionis, 1979a). Thus the weight difference between the absolute control (Group 1) and the HCA-injected, sham-treated (Group 4), and smoke-exposed (Group 5) animals does not reflect the effects of HCA. Group 3 animals (absolute control-HCA-injected animals) weighed somewhat less than those of Group 1 (absolute controls). This observation is surprising since both groups consumed similar amounts of food per day (Group 1 consumed 4.7 + 0.2 g; Group 3, 4.4 * 0.3 g). The perceptible health status of Group 1 and 3 animals, as well as those animals exposed to smoke alone (Matulionis, 1979a), was good throughout the period of investigation. However, animals of Groups 4 and 5 were “sickly” in appearance, lethargic, and exhibited considerable quivering. Those of Group 5 also exhibited signs of respiratory distress. The poor health status of Group 4 and 5 animals was reflected by a high mortality rate. Slightly more than 50% of animals from these groups died during the last 13.5 days of the experiment (Table 1). Smoke exposure or sham treatment alone, i.e., without HCA treatment (Matulionis, 1979a), causes no increase in mortality over that experienced by control animals. It is of interest to note that more animals in Group 4 died during the first half of the 13.5-day period, while most animals of Group 5 expired during the last half. Twenty-seven percent of animals in Group 3, but none of Group 1, died during the 13.5-day period (Table 1).
CELLULAR
RESPONSE
TO
SMOKE
AND
HYDROCORTISONE
365
The mean WBC count of animals in Group 3 (537.5 + 30/ml) was considerably lower than that of the controls (Group 1) (1138 t 1U/ml) and did not appreciably fluctuate throughout the experimental period (Table 2). By contrast, WBC counts of animals in Groups 4 and 5 were initially elevated but decreased by more than 2.5-fold from 6.5 to 13.5 days after HCA administration (Table 2). Pulmonary
Cell Response to HCA
The total pool of rH]thymidine-labeled cells in lung sections was assessed in terms of labeled macrophage cells and labeled nonmacrophages (leukocytes, fibroblasts, type II cells, endothelial cells, etc.). However, it was not possible to quantitate each of the latter cell types individually, thus the results reported in regard to labeled nonmacrophage cells are based on comparative overall observations. Assessment of autoradiographic preparations of lung tissue taken from animals of Group 1 throughout the sampling period revealed a relatively stable pool of rH]thymidine-labeled cells (Fig. 1, bar 1). However, a slight decrease of the pool of labeled cells was observed at 13.5 days post HCA treatment (Fig. 1). Labeled leukocytes were the most numerous in the labeled pool of cells. The total number of labeled cells in lungs of animals from Group 3 was markedly reduced (by an average of 38.0 +- 6.0%) when compared to the number seen in untreated control animals (Fig. 1, bar 3). Furthermore, a significant decline in the population of labeled cells was noted at the last two sampling times (P < 0.0001) (Fig. 1, bar 3). No one cell type stood out as the predominantly labeled cell in lungs of Group 3 animals. HCA treatment appears to suppress, in normal animals, cell division in the lungs or to interfere with emigration of cells, which have recently synthesized DNA, from other sites to the lungs. The total pool of labeled cells in Group 4 (21.80 ? 4.2/lung area) (Fig. 1, bar 4) and Group 5 (33.9 +- 7.Ulung area) (Fig. 1, bar 5) was considerably larger than that of either Group 1 (9.3 ? 0.15 lung area) (Fig. 1, bar 1) or Group 3 (4.7 ? O.S/lung area) (Fig. 1, bar 3) at the first sampling time, but declined to, or below, absolute control values by 13.5 days following HCA treatment. Neutrophils, followed by lymphocytes, were the most numerous labeled nonmacrophage cells noted per unit area (0.028 mm2) in lung tissue of animals in Groups 4 and 5 at the first two sampling times. These two cell types composed the largest proportion of the total pool of labeled cells at 6.5 and 8.5 days after HCA treatment. Other nonmacrophage [3H]thymidine-labeled cells were (in order of decreasing numbers) endothelial cells, septal tibroblasts, type II alveolar cells, monocyte-like cells, and unidentifiable cells. At 11.5 and 13.5 days after HCA treatment, the population of labeled neutrophils and lymphocytes declined to such a degree that they could no longer be identified as the predominantly labeled cells. Scintillation counting data revealed that the marked decline of labeled cells was paralleled closely by a decrease in disintegration (per min/mg) of protein of lung tissue. Stress (sham treatment) and/or an experimental insult (cigarette smoke) appears to stimulate the generation of large numbers of cells which take residence in the lungs. Subsequently these cells are rapidly depleted. The marked decrease in the total labeled cell pool appears to reflect a decrease in actual population size rather than
366
I
IANIEL
H.
MATULIONIS
I.
,.
I.
L
Days
Post
HCA
Trt.
1. Total number of [3H]thymidine-labeled cells per unit area (0.028 mm*) of lung tissue at various times following HCA treatment (Trt.) is indicated by the upper limit of each bar. The white region of the bars represents the total number of labeled nonmacrophage cells, while the black areas signify the number of labeled macrophages in the total pool of labeled cells. Bars 1, 3, 4, and 5 represent labeled cell populations in lungs of controls (Group l), controls treated with HCA (Group 3), sham, HCA-treated animals (Group 41, and smoke, HCA-injected mice (Group 5) respectively. The vertical lines outside the bars represent 2 one standard error of the mean of the total number of labeled cells/area, while those within the bar that of the total number of labeled nonmacrophage cells. FIG.
depletion rate of labeled cells, since it paralleled, for the most part, decreases in actual WBC counts (Table 2). Labeled macrophage population in the total pool of labeled cells was small in all groups of animals (Fig. 1). However, the contribution of labeled macrophages to the total pool of labeled cells was most prominent in lungs of Group 1 animals (Fig. 1, bar 1). Furthermore, since the population of nonmacrophage labeled cells decreased markedly in lungs of Group 4 and 5 animals (P < O.OOOl), the labeled macrophage became a more obvious component of the total pool of labeled cells at 13.5 days following HCA treatment.
CELLULAR
Response
of Pulmonary
RESPONSE
TO
Macrophages
SMOKE
AND
HYDROCORTISONE
367
to HCA
Regarding the pulmonary macrophage population, both rH]thymidine-labeled and unlabeled macrophages were quantitated. The mean number of labeled and unlabeled macrophages (alveolar and septal) in lungs of control animals (Group 1) over the four sampling periods was 1.23 + 0.05 (mean + one standard deviation)/ unit area (0.028 mm2), while the mean number of labeled macrophages was 0.58 + O.OS/unit area (Fig. 2). In control animals which received HCA injections (Group 3) the mean total number of macrophages was reduced to 0.8 -+ 0.04 cell per area (Fig. 3). Labeled macrophages in the same group of animals numbered only 0.28 ? 0.03 per a similar area of lung tissue (Fig. 3). The above data clearly indicates that steroid treatment of normal animals reduces the population size of pulmonary macrophages (P < 0.0003). In mice which were subjected to sham and HCA treatment (Group 4), the macrophage population was stable over the sampling period (Fig. 4) and the mean number (0.8 + 0.04) was similar to that in animals of Group 3 (Fig. 3). However, the mean number of labeled macrophages (0.2 ? 0.06), over the same time, was slightly less than that recorded for animals in Group 3 (compare Fig. 4 to Fig. 3). This observation suggests that the stress of sham treatment further impedes the generation of new macrophages or the influx of macrophages from other sites to the lungs of animals which received the steroid treatment. In animals exposed to cigarette smoke and treated with HCA (Group 5), the total number of macrophages per unit area of lung tissue was 3.8 (+ 0.6) 6.5 days after HCA injection (Fig. 5). Over the next 2 days, the macrophage population decreased more than twofold (Fig. 5). During the remaining sampling period, the total macrophage population was relatively stable but was significantly (P < 0.007) above that of Group 1 values (compare Fig. 5 to Fig. 2). The number of labeled pulmonary macrophages in Group 5 animals declined slightly but insignificantly from the first to the last sampling date (Fig. 5). By comparing the pulmonary macrophage population reported previously from smoke-exposed animals which were nof treated with HCA (Matulionis, 1979a) with that of animals ex-
FIG. 2. Total number (the upper line) of pulmonary macrophages (Mac.) in control animals (Group 1) not treated with hydrocortisone acetate (HCA), which was quantitated at the same time as that of animals receiving HCA treatment. The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm’), and the area between the lower line and the abscissa the number of rH]thymidine-labeled macrophages. The vertical lines represent 2 one standard error of the mean in this and subsequent graphs.
368
DANIEL
H.
MATULIONIS
FIG. 3. Total number (the upper line) of pulmonary macrophages in control animals treated with hydrocortisone acetate (HCA) (Group 3). The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm*) and the area between the lower line and the abscissa the number of rH]thymidine-labeled macrophages.
posed to smoke and treated with the steroid (Group 5), it is evident that hydrocortisone acetate treatment diminished the markedly elevated pool of lung phagocytes which is induced by cigarette smoke. Pathologic
Conditions
Induced
by HCA
Abnormal conditions noted in Group 4 and 5 animals are quantitated, described in detail, and discussed in a report which is presently in preparation. Thus, only several general observations will be noted here. The right ventricular hypertrophy was present in all animals of Group 5 and in several mice in Group 4. However, the enlarged heart was noted only during the first two sampling periods in Group 4 animals, while such a condition was present at all sampling periods in mice of Group 5. Lungs of animals in Group 5 were seen to have gray to brown to black areas on their surface. Light microscopic examination of lungs from smoke-exposed, HCA-treated animals (Group 5) revealed prominent multifocal to diffuse thickenings of alveolar septa and a marked reduction of alveolar space. In some instances, alveolar walls were collapsed and atelectatic. Occasionally pools of colloid-like and frequently flocculant material were observed in many of the alveolar spaces. The above conditions, although in a considerably milder form, were observed on occasion in lungs of Group 4 animals only at 6.5 and 8.5 days after HCA injection, and were never seen in lungs of Group 1 and 3 mice. It should be emphasized that animals treated with HCA and subjected to cigarette smoke were afflicted with the most severe pulmonary abnormalities.
b$y-/ 6b.S
1l.s l3.s
03 Days
Fort
HCA
Trt.
FIG. 4. Total number (the upper line) of pulmonary macrophages in sham animals treated with hydrocortisone acetate (HCA) (Group 4). The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm?, and the area between the lower line and abscissa the number of rH]thymidine-labeled macrophages.
CELLULAR
RESPONSE
TO
I * 4.5
SMOKE
AND
as Days
HYDROCORTISONE
1LS
Post
HCA
369
135
Trt.
FIG. 5. Total number (the upper line) of pulmonary macrophages in smoke-exposed animals treated with hydrocortisone acetate (HCA) (Group 5). The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm?, and the area between the lower line and abscissa the number of rH]thymidine-labeled macrophages.
DISCUSSION
HCA treatment of mice which had previously been exposed to cigarette smoke reduces markedly the number of pulmonary macrophages. The total population of phagocytic cells was reduced fivefold when compared with data of an earlier experiment which described the effect of smoke on the macrophage system of animals not treated with HCA (Matulionis, 1979a). More strikingly, the pool of labeled pulmonary macrophages quantified earlier from mice exposed only to cigarette smoke (Matulionis, 1979a) was 11 times larger than that of animals treated with both smoke and HCA (Group 5). The population of labeled macrophages in animals of Group 5 was, however, not completely suppressed but was similar to that of Group 1 (controls) (compare Fig. 5 to Fig. 2). This observation suggests that macrophage generation is not completely arrested by HCA. Data which indicate that a small percentage of in situ pulmonary macrophages are capable of division (Matulionis, 1979b; Blusse Van oud Alblas and VanFurth, 1979; VanFurth, 1970; Bowden et al., 1969) may account for the formation of new macrophages, since HCA does not appear to hinder cell division (Thompson and VanFurth, 1973). Further, it seems that the generation of new macrophages (though modest in number) can be, to a degree, elevated by demands for increased phagocytes or particulate clearance of material conveyed by cigarette smoke, since in normal HCA-treated animals the population of the new phagocytes is reduced more than in smoke-exposed mice. Even though there is evidence for in situ division of pulmonary macrophages, it must be stressed that their mitotic activity does not contribute significantly to the marked increase in the pulmonary macrophage population size in response to cigarette smoke (Matulionis, 1979b)
370
DANIEL
H.
MATULIONIS
and that HCA administration conspicuously reduces this population in the lungs of “smoked” animals. In addition to reducing the macrophage population, steroid treatment induced a marked leukopenia in normal animals (Group 3). The mean number of leukocytes (cardiac blood) in Group 3 animals (HCA-injected controls) was approximately 50% of that seen in Group 1 (no-drug animals). However, leukocyte counts of sham-treated and smoke-exposed mice injected with HCA were approximately 2.5 times higher than those of control animals at 6.5 days after steroid treatment but dropped progressively to values recorded for absolute controls at 13.5 days after the HCA injection. A number of investigators observed that in normal mice (Thompson and VanFurth, 1970) and guinea pigs (Brahim and Bahadue, 1979) treated with HCA, peripheral blood lymphocytes decreased in number. However, the population of polymorphonuclear leukocytes in normal Swiss mice increased 5 to 10 times 12 to 14 days after steroid treatment (Thompson and VanFurth, 1970). Likewise, Leibovich and Ross (1975) noted a similar type of response to circulating leukocytes in guinea pigs during wound healing after HCA treatment. Apparently the granulocytic response to HCA in normal animals is different from that in animals which are subjected to smoke exposure or stress (sham treatment). The insults induce a leukocytic response which is subsequently suppressed by hydrocortisone. A similar increase, followed by decline of leukocytes, was observed by Kavet et al. (1978) in hamsters exposed to iron oxide aerosols and treated with HCA. These investigators suggested that iron oxide exposure induced an inflammatory response while HCA reduced it and blocked recruitment of cells. The present study supports the above suggestions. The simultaneous reduction of leukocyte and macrophage populations by HCA in smoke-exposed and sham-treated animals prevents the implication of either the white blood cells or the phagocytes as being the sole causal factor in the increased mortality rate and abnormal pulmonary condition noted in the lungs of these animals. Furthermore, it cannot be said that cigarette smoke caused the elevation of mortality rate (52%) of HCA-injected mice since sham animals, treated with the steroid, showed a similar mortality rate (53%). In addition, increased mortality cannot be related to emaciation since the weights of smoke-exposed and shamtreated animals injected with the steroid were similar to their counterparts which were not treated with the steroid (Matulionis, 1979a). The high death rate might be related to cumulative adverse effects of steroid (causing reduction of macrophages and leukocytes) and stress produced during sham and smoke treatment, while the lower mortality rate (27%) of control animals which received only steroid treatment, and were not stressed, reflects the adverse effect of HCA only. Striking abnormal conditions were noted in lungs of smoke-exposed, HCAtreated mice. Similar conditions, though to a much lesser extent and severity, were also noted in sham-treated, HCA-injected animals during the first two sampling periods. However, lungs of control animals injected with the steroid were free of pathology and similar to those of untreated and vehicle-injected control animals. These results indicate that HCA treatment alone does not induce abnormal conditions. Such conditions develop only when animals are additionally stressed and their defense mechanisms break down. The abnormalities are de-
CELLULAR
RESPONSE
scribed in detail, quantitated, publication.
TO
SMOKE
AND
HYDROCORTISONE
371
and discussed in a report which is being prepared for ACKNOWLEDGMENTS
The author wishes to express his gratitude to Linda Simmerman for her expert technical assistance. This investigation was supported by a University of Kentucky Tobacco and Health Research Institute Grant, project 24133.
REFERENCES Benner, J. F., Owens, S., Hancock, R., and Griffith, R. B. (1973). Smoking machine development and inhalation atmosphere monitoring. Proc. Univ. KY. Tob. Health Res. Inst. Conf. Rep. No. 4, 494-506. Blusse Van oud Alblas, A., and VanFurth, R. (1979). Origin, kinetics, and characteristics of pulmonary macrophages in the normal steady state. J. Exp. Med. 149, 1504- 1518. Bowden, D. H., and Adamson, J. Y. R. (1978). Adaptive response of the pulmonary macrophagic system to carbon. Lab. Invest. 38, 422-429. Bowden, D. H., Adamson, J. Y. R., Grantham, W. C., and Wyatt, J. P. (1969). Origin of the lung macrophage: Evidence derived from radiation injury. Arch. Pathol. 88, 540-546. Brahim, F., and Bahadue, G. (1979). Influx of long-lived lymphocytes into guinea pig marrow during hydrocortisone administration. J. Reticuloendothel. Sot. 25, 397-404. Brody, A. R., and Craighead, J. E. (1975). Cytoplasmic inclusions in pulmonary macrophages of cigarette smokers. Lab. Invest. 32, 125- 132. Huber, G. L., LaForce, F. M., and Johanson, W. G. (1977). Experimental models and pulmonary antimicrobial defenses. In “Respiratory Defense Mechanisms” (J. D. Brain, D. Proctor, and L. Reid, Eds.), pp. 979-1022. Dekker, New York. Kavet, R. I., Brain, J. D., and Levens. D. J. (1978). Characteristics of pulmonary macrophages lavaged from hamsters exposed to iron oxide aerosols. Lab. Invest. 38, 312-319. Leibovich, S. J.. and Ross, R. (1975). The role of the macrophage in wound repair. Amer. J. Pathol. 78,71-100. Martin, R. R., and Warr, G. A. (1977). Cigarette smoking and human pulmonary macrophages. Hosp. Pratt.
12, 97-
104.
Matulionis, D. H. (1979a). Reaction of macrophages to cigarette smoke. I. Recruitment of pulmonary macrophages. Arch. Environ. Health 34, 293-297. Matulionis, D. H. (1979b). Reaction of macrophages to cigarette smoke. II. Immigration of macrophages to the lungs. Arch. Environ. Health 34, 298-301. Matulionis, D. H., and Traurig, H. H. (1977). In sita response of lung macrophages and hydrolase activities to cigarette smoke. Lab. Invest. 37, 314-325. Pratt, S. A., Smith, M. H., Ladman, A. J., and Finley, T. N. (1971). The ultrastructure of alveolar macrophages from human cigarette smokers and nonsmokers. Lab. Invest. 24, 331-338. Thompson, J., and VanFurth, R. (1970). The effect of glucocorticosteroids on the kinetics of mononuclear phagocytes. J. Exp. Med. 131, 429-442. Thompson, J., and VanFurth, R. (1973). The effect of glucocorticosteroids on the proliferation and kinetics of promonocytes and monocytes of bone marrow. J. Exp. Med. 136, 10-21. VanFurth, R. (1970). Origin and kinetics of monocytes and macrophages. Semin. Hemarol. 7, 125- 141.
RESEARCH
27, 361-371 (1982)
Pulmonary
Tissue
and Cigarette
Smoke
1. Cellular Response to Hydrocortisone DANIEL Department Kentucky
H. MATULIONIS
of Anatomy, University of Kentucky, Lexington, Tobacco and Health Research Institute. Cooper Lexington. Kentucky 40506
Kentucky 40536. and and Alumni Drives.
Received April 7, 1981 Hydrocortisone acetate (HCA) administration significantly reduces the population of pulmonary macrophages and blood leukocytes in control, sham-treated, and smoke-exposed C57BL/6J mice. This treatment impedes markedly the influx of macrophages from bone marrow into the lungs. The small number of new phagocytes noted in lungs following HCA treatment appears to arise by proliferation of in situ pulmonary macrophages. Mortality rate of sham-treated and smoke-exposed mice was approximately twice that of control animals following HCA treatment. While severe pulmonary disorders were noted in lungs of HCAtreated, smoke-exposed animals, considerably milder abnormalities were seen in lungs of sham-treated mice. The data reported indicate that physical stress generated by manipulation during sham and smoke treatment, exposure to cigarette smoke, and reduction of pulmonary macrophages and leukocyte populations by HCA administration are factors which adversely affect pulmonary integrity and survival time of the animals.
INTRODUCTION
The importance of pulmonary macrophages in maintaining the sterility and normal function of the lungs has long been recognized and is reviewed elsewhere (Martin and Wart-, 1977; Huber et nl., 1977). The number of phagocytes increases markedly when particulate materials gain access to the lungs in excess of basal levels (Bowden and Adamson, 1978; Kavet et al., 1978; Matulionis, 1979a). Inhalation of whole cigarette smoke, conveying numerous particles, evokes a prominent phagocytic (Matulionis, 1979a; Brody and Craighead, 1975; Matulionis and Traurig, 1977; Pratt et al., 1971) and leukocytic (Matulionis, 1979a) response in the lungs; however, macrophages accumulate only in pulmonary tissue with no corresponding increase in other tissues (Matulionis, 1979a). The markedly elevated population of lung macrophages does not arise from in situ proliferation of macrophages, but are, for the most part, of bone marrow-blood monocyte origin (Matulionis, 1979b). Thus, interference with the influx of phagocytic cells from the bone marrow-monocyte system should decrease the macrophage population in lung tissue. Generation of macrophages by bone marrow is influenced adversely by hydrocortisone acetate (HCA) (Thompson and VanFurth, 1973). Further, it has been shown that HCA administration markedly reduces the pulmonary macrophage population in normal mice (Blusse Van oud Alblas and VanFurth, 1979). The number of phagocytes is also reduced at the sites of inflammation and wound healing by the steroid treatment (Leibovich and Ross, 1975). In addition, it has been reported that in animals exposed to particles of iron oxide, peripheral blood 361 0013-9351/82/020361-11$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
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leukocytes are reduced significantly following treatment with hydrocortisone acetate (Kavet et al., 1978). However, data regarding the ability or inability of this steroid to decrease the influx of macrophages into the lungs and reduce the blood leukocyte population in animals maintained on a “smoking schedule” are not available. Therefore, the goal of the present study is to determine whether subcutaneous administration of hydrocortisone acetate decreases the elevated pulmonary macrophage population (septal and alveolar) and blood leukocytes in animals exposed to cigarette smoke. This study is an initial segment of a broader investigation designed to develop an animal model that can be utilized to assess the genesis of pulmonary disorders related to cigarette smoking. MATERIALS
AND METHODS
Lung tissue from 86 7- to 8-week-old C57BL/6J mice was used in this study. The mice were housed, maintained, exposed to cigarette smoke, and sham-treated by the Kentucky Tobacco and Health Research Institute (KTHRI) as previously detailed, (Matulionis, 1979a) with one modification: the animals were housed one per cage. Briefly, smoke exposure of animals occurred in the KTHRI single-port reverse smoking machine. The machine operates in the following fashion (Benner et al., 1973). A lit cigarette is placed in a holder connected to the smoke exposure chamber. Snouts of mice, which are restrained in specially constructed holders, are inserted into the exposure chamber. The puff is generated by an air cylinder which moves over the cigarette creating a slightly above-ambient pressure around the cigarette. Smoke exits into the exposure column from the outlet of the cigarette holder and is diluted with air to a desired concentration (20% smoke). Following the smoke puff, the air cylinder is retracted, allowing the cigarette to burn at atmospheric pressure. The smoke remains in the exposure chamber for 15 set after which it is flushed with room air. Forty-live seconds are allowed to elapse before the next smoke puff is delivered into the exposure chamber. Sham treatment consisted of exposing animals to air instead of smoke. Five groups of animals were established for this study. At the end of the 13.5day-long experiment, Group 1 consisted of 22 untreated-absolute controls; Group 2 was composed of 12 animals each injected subcutaneously (SC) with a vehicle (containing 0.5% carboxymethyl cellulose, 0.9% benzyl alcohol, and 0.4% polysarbate 80 in saline) (Thompson and VanFurth, 1970) used to suspend the HCA; Group 3 contained 19 mice which received SC injections of HCA; Group 4 consisted of 14 sham-treated mice injected with the steroid (HCA); and Group 5 contained 19 smoke-exposed animals which were also injected with HCA (Table 1). All injections were administered SC in the nuchal region. Smoke and sham treatment (Matulionis, 1979a) occurred for 35 days prior to administration of HCA. Animals of Groups 3, 4, and 5 received 0.5 mg/g body wt of HCA (hydrocortisone 21acetate; Sigma Chemical Co., St. Louis, MO.) suspended in 0.1 ml of vehicle, while those of Group 2 were injected with only 0.1 ml of the vehicle (Table 1). HCA and vehicle injections were repeated at 4-day intervals over a period of 12 days. Each animal received four injections. A pharmacological dose of steroid was used to assure a response to the drug since a major goal of the present study was to determine whether it is possible to decrease the markedly elevated pulmonary macrophage population that is induced by cigarette smoke. Presently, experi-
CELLULAR
TREATMENTS,
Group 1 2 3 4 5
NUMBERS,=
Group composition and treatment Normal controls None Normal controls Injected with vehicle Normal controls Injected with HCA Sham-treated mice Injected with HCA Smoke-exposed animals Injected with HCA Total
363
RESPONSE TO SMOKE AND HYDROCORTISONE TABLE 1 WEIGHTS, AND MORTALITY RATES OF MICE
Number of surviving animals/group
Mean wt of surviving animals ?I SEM
Mortality rate over 13.5day period (%I
22
22
23.2 2 0.4
0
12
12
23.8 5 0.5
0
26
19
21.5 2 0.4
21
26
14
20.0 f 0.4
53
36
19
19.0 k 0.9
52
122
86
Number of animals/group prior to HCA” injection
a Three to six animals were assessed at 6.5, 8.5, 11.5, and 13.5 days after HCA injection. b Hydrocortisone acetate.
ments are m progress to evaluate the pulmonary cell response to various doses of HCA. Three days after the first injection of the steroid all animals were injected intraperitoneally with rH]thymidine according to the schedule (9 /.&i/g body wt over a 3-day period) described earlier (Matulionis, 1979a). Lung tissues were obtained at autopsy at 6.5, 8.5, 11.5, and 13.5 days after the initial HCA (or vehicle) injections or 3.5, 8, and 10 days after the first rH]thymidine injection. Tissue sampling began at 6.5 days following HCA administration to allow suffrcient time for the steroid effect to take place. Three days were allowed to elapse after rH]thymidine injections before tissue samples were obtained, since it has been shown (Matulionis, 1979a) that emigration of labeled macrophages into the lungs begins at this time. At each sampling time, lung tissue from three to six animals of each group was processed for autoradiography and scintillation counting. Details of methods used for autoradiography, scintillation counting, and quantitation of pulmonary macrophage populations, as well as the specific regions of the lungs sampled, have been reported earlier (Matulionis, 1979a). Prior to autopsy, the mice were anesthetized by intraperitoneal injections of 0.25 to 0.3 cc of 10% Nembutal. Cardiac blood samples were collected from the right ventricle, the animals were exsanguinated by severing the abdominal aorta, and lung tissue was collected. Total white blood cell counts (WBC) were determined by standard techniques using the ZBi Coulter counter (Table 2). RESULTS General Observations
The general health status, lung tissue, and white blood cell counts assessed for animals in Group 2 (vehicle treated) were normal in every respect and similar to those in Group 1 (absolute controls). In view of these findings, Group 2 animals
364
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WHITE Days
post HCA” treatment
Group
6.5 8.5 11.5 13.5 Group
1.08 0.90 1.12 1.45
mean
a Hydrocortisone b Mean c 1 SEM.
+ k + +
BLOOD
1 0.07b 0.06 0.05 0.06
1.138 k 0.115
H.
MATULIONIS
TABLE 2 CELL COUNTS
Group 0.45 0.50 0.57 0.63 0.5375
3 * ” * k
0.05 0.04 0.03 0.06
-r- 0.30
(x 1000)
Group 2.66 1.96 1.44 0.99
f f k +
4 0.21 0.78 0.08 0.10
1.76 k 0.36
Group 3.25 2.26 1.97 1.30
2 + + +
5 0.53 0.23 0.20 0.16
2.20 k 0.41
acetate.
will be incorporated and be considered simultaneously with those of Group 1, and henceforth both groups will be referred to as Group 1. Unavoidable technical circumstances related to smoke exposure of animals prevents the simultaneous assessment of cell response in lungs of animals exposed to smoke only and in those that were both smoke exposed and HCA treated. However, a detailed study has been reported recently (Matulionis, 1979a) which documents such a response to smoke alone. Therefore, results of the previous study by Matulionis (1979a), which describes the effects of smoke exposure only, will be used to compare and contrast effects produced by combined smoke exposure and HCA treatment. The mean weights of animals in Groups 1, 3, 4, and 5, averaged over the four sampling periods, are shown in Table 1. Weights of animals in Groups 1, 4, and 5 were statistically similar to those of control, sham-treated, and smoke-exposed mice, respectively, which had not been injected with the hydrocortisone acetate (HCA) (Matulionis, 1979a). Thus the weight difference between the absolute control (Group 1) and the HCA-injected, sham-treated (Group 4), and smoke-exposed (Group 5) animals does not reflect the effects of HCA. Group 3 animals (absolute control-HCA-injected animals) weighed somewhat less than those of Group 1 (absolute controls). This observation is surprising since both groups consumed similar amounts of food per day (Group 1 consumed 4.7 + 0.2 g; Group 3, 4.4 * 0.3 g). The perceptible health status of Group 1 and 3 animals, as well as those animals exposed to smoke alone (Matulionis, 1979a), was good throughout the period of investigation. However, animals of Groups 4 and 5 were “sickly” in appearance, lethargic, and exhibited considerable quivering. Those of Group 5 also exhibited signs of respiratory distress. The poor health status of Group 4 and 5 animals was reflected by a high mortality rate. Slightly more than 50% of animals from these groups died during the last 13.5 days of the experiment (Table 1). Smoke exposure or sham treatment alone, i.e., without HCA treatment (Matulionis, 1979a), causes no increase in mortality over that experienced by control animals. It is of interest to note that more animals in Group 4 died during the first half of the 13.5-day period, while most animals of Group 5 expired during the last half. Twenty-seven percent of animals in Group 3, but none of Group 1, died during the 13.5-day period (Table 1).
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365
The mean WBC count of animals in Group 3 (537.5 + 30/ml) was considerably lower than that of the controls (Group 1) (1138 t 1U/ml) and did not appreciably fluctuate throughout the experimental period (Table 2). By contrast, WBC counts of animals in Groups 4 and 5 were initially elevated but decreased by more than 2.5-fold from 6.5 to 13.5 days after HCA administration (Table 2). Pulmonary
Cell Response to HCA
The total pool of rH]thymidine-labeled cells in lung sections was assessed in terms of labeled macrophage cells and labeled nonmacrophages (leukocytes, fibroblasts, type II cells, endothelial cells, etc.). However, it was not possible to quantitate each of the latter cell types individually, thus the results reported in regard to labeled nonmacrophage cells are based on comparative overall observations. Assessment of autoradiographic preparations of lung tissue taken from animals of Group 1 throughout the sampling period revealed a relatively stable pool of rH]thymidine-labeled cells (Fig. 1, bar 1). However, a slight decrease of the pool of labeled cells was observed at 13.5 days post HCA treatment (Fig. 1). Labeled leukocytes were the most numerous in the labeled pool of cells. The total number of labeled cells in lungs of animals from Group 3 was markedly reduced (by an average of 38.0 +- 6.0%) when compared to the number seen in untreated control animals (Fig. 1, bar 3). Furthermore, a significant decline in the population of labeled cells was noted at the last two sampling times (P < 0.0001) (Fig. 1, bar 3). No one cell type stood out as the predominantly labeled cell in lungs of Group 3 animals. HCA treatment appears to suppress, in normal animals, cell division in the lungs or to interfere with emigration of cells, which have recently synthesized DNA, from other sites to the lungs. The total pool of labeled cells in Group 4 (21.80 ? 4.2/lung area) (Fig. 1, bar 4) and Group 5 (33.9 +- 7.Ulung area) (Fig. 1, bar 5) was considerably larger than that of either Group 1 (9.3 ? 0.15 lung area) (Fig. 1, bar 1) or Group 3 (4.7 ? O.S/lung area) (Fig. 1, bar 3) at the first sampling time, but declined to, or below, absolute control values by 13.5 days following HCA treatment. Neutrophils, followed by lymphocytes, were the most numerous labeled nonmacrophage cells noted per unit area (0.028 mm2) in lung tissue of animals in Groups 4 and 5 at the first two sampling times. These two cell types composed the largest proportion of the total pool of labeled cells at 6.5 and 8.5 days after HCA treatment. Other nonmacrophage [3H]thymidine-labeled cells were (in order of decreasing numbers) endothelial cells, septal tibroblasts, type II alveolar cells, monocyte-like cells, and unidentifiable cells. At 11.5 and 13.5 days after HCA treatment, the population of labeled neutrophils and lymphocytes declined to such a degree that they could no longer be identified as the predominantly labeled cells. Scintillation counting data revealed that the marked decline of labeled cells was paralleled closely by a decrease in disintegration (per min/mg) of protein of lung tissue. Stress (sham treatment) and/or an experimental insult (cigarette smoke) appears to stimulate the generation of large numbers of cells which take residence in the lungs. Subsequently these cells are rapidly depleted. The marked decrease in the total labeled cell pool appears to reflect a decrease in actual population size rather than
366
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I.
,.
I.
L
Days
Post
HCA
Trt.
1. Total number of [3H]thymidine-labeled cells per unit area (0.028 mm*) of lung tissue at various times following HCA treatment (Trt.) is indicated by the upper limit of each bar. The white region of the bars represents the total number of labeled nonmacrophage cells, while the black areas signify the number of labeled macrophages in the total pool of labeled cells. Bars 1, 3, 4, and 5 represent labeled cell populations in lungs of controls (Group l), controls treated with HCA (Group 3), sham, HCA-treated animals (Group 41, and smoke, HCA-injected mice (Group 5) respectively. The vertical lines outside the bars represent 2 one standard error of the mean of the total number of labeled cells/area, while those within the bar that of the total number of labeled nonmacrophage cells. FIG.
depletion rate of labeled cells, since it paralleled, for the most part, decreases in actual WBC counts (Table 2). Labeled macrophage population in the total pool of labeled cells was small in all groups of animals (Fig. 1). However, the contribution of labeled macrophages to the total pool of labeled cells was most prominent in lungs of Group 1 animals (Fig. 1, bar 1). Furthermore, since the population of nonmacrophage labeled cells decreased markedly in lungs of Group 4 and 5 animals (P < O.OOOl), the labeled macrophage became a more obvious component of the total pool of labeled cells at 13.5 days following HCA treatment.
CELLULAR
Response
of Pulmonary
RESPONSE
TO
Macrophages
SMOKE
AND
HYDROCORTISONE
367
to HCA
Regarding the pulmonary macrophage population, both rH]thymidine-labeled and unlabeled macrophages were quantitated. The mean number of labeled and unlabeled macrophages (alveolar and septal) in lungs of control animals (Group 1) over the four sampling periods was 1.23 + 0.05 (mean + one standard deviation)/ unit area (0.028 mm2), while the mean number of labeled macrophages was 0.58 + O.OS/unit area (Fig. 2). In control animals which received HCA injections (Group 3) the mean total number of macrophages was reduced to 0.8 -+ 0.04 cell per area (Fig. 3). Labeled macrophages in the same group of animals numbered only 0.28 ? 0.03 per a similar area of lung tissue (Fig. 3). The above data clearly indicates that steroid treatment of normal animals reduces the population size of pulmonary macrophages (P < 0.0003). In mice which were subjected to sham and HCA treatment (Group 4), the macrophage population was stable over the sampling period (Fig. 4) and the mean number (0.8 + 0.04) was similar to that in animals of Group 3 (Fig. 3). However, the mean number of labeled macrophages (0.2 ? 0.06), over the same time, was slightly less than that recorded for animals in Group 3 (compare Fig. 4 to Fig. 3). This observation suggests that the stress of sham treatment further impedes the generation of new macrophages or the influx of macrophages from other sites to the lungs of animals which received the steroid treatment. In animals exposed to cigarette smoke and treated with HCA (Group 5), the total number of macrophages per unit area of lung tissue was 3.8 (+ 0.6) 6.5 days after HCA injection (Fig. 5). Over the next 2 days, the macrophage population decreased more than twofold (Fig. 5). During the remaining sampling period, the total macrophage population was relatively stable but was significantly (P < 0.007) above that of Group 1 values (compare Fig. 5 to Fig. 2). The number of labeled pulmonary macrophages in Group 5 animals declined slightly but insignificantly from the first to the last sampling date (Fig. 5). By comparing the pulmonary macrophage population reported previously from smoke-exposed animals which were nof treated with HCA (Matulionis, 1979a) with that of animals ex-
FIG. 2. Total number (the upper line) of pulmonary macrophages (Mac.) in control animals (Group 1) not treated with hydrocortisone acetate (HCA), which was quantitated at the same time as that of animals receiving HCA treatment. The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm’), and the area between the lower line and the abscissa the number of rH]thymidine-labeled macrophages. The vertical lines represent 2 one standard error of the mean in this and subsequent graphs.
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FIG. 3. Total number (the upper line) of pulmonary macrophages in control animals treated with hydrocortisone acetate (HCA) (Group 3). The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm*) and the area between the lower line and the abscissa the number of rH]thymidine-labeled macrophages.
posed to smoke and treated with the steroid (Group 5), it is evident that hydrocortisone acetate treatment diminished the markedly elevated pool of lung phagocytes which is induced by cigarette smoke. Pathologic
Conditions
Induced
by HCA
Abnormal conditions noted in Group 4 and 5 animals are quantitated, described in detail, and discussed in a report which is presently in preparation. Thus, only several general observations will be noted here. The right ventricular hypertrophy was present in all animals of Group 5 and in several mice in Group 4. However, the enlarged heart was noted only during the first two sampling periods in Group 4 animals, while such a condition was present at all sampling periods in mice of Group 5. Lungs of animals in Group 5 were seen to have gray to brown to black areas on their surface. Light microscopic examination of lungs from smoke-exposed, HCA-treated animals (Group 5) revealed prominent multifocal to diffuse thickenings of alveolar septa and a marked reduction of alveolar space. In some instances, alveolar walls were collapsed and atelectatic. Occasionally pools of colloid-like and frequently flocculant material were observed in many of the alveolar spaces. The above conditions, although in a considerably milder form, were observed on occasion in lungs of Group 4 animals only at 6.5 and 8.5 days after HCA injection, and were never seen in lungs of Group 1 and 3 mice. It should be emphasized that animals treated with HCA and subjected to cigarette smoke were afflicted with the most severe pulmonary abnormalities.
b$y-/ 6b.S
1l.s l3.s
03 Days
Fort
HCA
Trt.
FIG. 4. Total number (the upper line) of pulmonary macrophages in sham animals treated with hydrocortisone acetate (HCA) (Group 4). The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm?, and the area between the lower line and abscissa the number of rH]thymidine-labeled macrophages.
CELLULAR
RESPONSE
TO
I * 4.5
SMOKE
AND
as Days
HYDROCORTISONE
1LS
Post
HCA
369
135
Trt.
FIG. 5. Total number (the upper line) of pulmonary macrophages in smoke-exposed animals treated with hydrocortisone acetate (HCA) (Group 5). The area between the upper and lower lines represents the number of unlabeled pulmonary macrophages per unit area of lung tissue (0.028 mm?, and the area between the lower line and abscissa the number of rH]thymidine-labeled macrophages.
DISCUSSION
HCA treatment of mice which had previously been exposed to cigarette smoke reduces markedly the number of pulmonary macrophages. The total population of phagocytic cells was reduced fivefold when compared with data of an earlier experiment which described the effect of smoke on the macrophage system of animals not treated with HCA (Matulionis, 1979a). More strikingly, the pool of labeled pulmonary macrophages quantified earlier from mice exposed only to cigarette smoke (Matulionis, 1979a) was 11 times larger than that of animals treated with both smoke and HCA (Group 5). The population of labeled macrophages in animals of Group 5 was, however, not completely suppressed but was similar to that of Group 1 (controls) (compare Fig. 5 to Fig. 2). This observation suggests that macrophage generation is not completely arrested by HCA. Data which indicate that a small percentage of in situ pulmonary macrophages are capable of division (Matulionis, 1979b; Blusse Van oud Alblas and VanFurth, 1979; VanFurth, 1970; Bowden et al., 1969) may account for the formation of new macrophages, since HCA does not appear to hinder cell division (Thompson and VanFurth, 1973). Further, it seems that the generation of new macrophages (though modest in number) can be, to a degree, elevated by demands for increased phagocytes or particulate clearance of material conveyed by cigarette smoke, since in normal HCA-treated animals the population of the new phagocytes is reduced more than in smoke-exposed mice. Even though there is evidence for in situ division of pulmonary macrophages, it must be stressed that their mitotic activity does not contribute significantly to the marked increase in the pulmonary macrophage population size in response to cigarette smoke (Matulionis, 1979b)
370
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and that HCA administration conspicuously reduces this population in the lungs of “smoked” animals. In addition to reducing the macrophage population, steroid treatment induced a marked leukopenia in normal animals (Group 3). The mean number of leukocytes (cardiac blood) in Group 3 animals (HCA-injected controls) was approximately 50% of that seen in Group 1 (no-drug animals). However, leukocyte counts of sham-treated and smoke-exposed mice injected with HCA were approximately 2.5 times higher than those of control animals at 6.5 days after steroid treatment but dropped progressively to values recorded for absolute controls at 13.5 days after the HCA injection. A number of investigators observed that in normal mice (Thompson and VanFurth, 1970) and guinea pigs (Brahim and Bahadue, 1979) treated with HCA, peripheral blood lymphocytes decreased in number. However, the population of polymorphonuclear leukocytes in normal Swiss mice increased 5 to 10 times 12 to 14 days after steroid treatment (Thompson and VanFurth, 1970). Likewise, Leibovich and Ross (1975) noted a similar type of response to circulating leukocytes in guinea pigs during wound healing after HCA treatment. Apparently the granulocytic response to HCA in normal animals is different from that in animals which are subjected to smoke exposure or stress (sham treatment). The insults induce a leukocytic response which is subsequently suppressed by hydrocortisone. A similar increase, followed by decline of leukocytes, was observed by Kavet et al. (1978) in hamsters exposed to iron oxide aerosols and treated with HCA. These investigators suggested that iron oxide exposure induced an inflammatory response while HCA reduced it and blocked recruitment of cells. The present study supports the above suggestions. The simultaneous reduction of leukocyte and macrophage populations by HCA in smoke-exposed and sham-treated animals prevents the implication of either the white blood cells or the phagocytes as being the sole causal factor in the increased mortality rate and abnormal pulmonary condition noted in the lungs of these animals. Furthermore, it cannot be said that cigarette smoke caused the elevation of mortality rate (52%) of HCA-injected mice since sham animals, treated with the steroid, showed a similar mortality rate (53%). In addition, increased mortality cannot be related to emaciation since the weights of smoke-exposed and shamtreated animals injected with the steroid were similar to their counterparts which were not treated with the steroid (Matulionis, 1979a). The high death rate might be related to cumulative adverse effects of steroid (causing reduction of macrophages and leukocytes) and stress produced during sham and smoke treatment, while the lower mortality rate (27%) of control animals which received only steroid treatment, and were not stressed, reflects the adverse effect of HCA only. Striking abnormal conditions were noted in lungs of smoke-exposed, HCAtreated mice. Similar conditions, though to a much lesser extent and severity, were also noted in sham-treated, HCA-injected animals during the first two sampling periods. However, lungs of control animals injected with the steroid were free of pathology and similar to those of untreated and vehicle-injected control animals. These results indicate that HCA treatment alone does not induce abnormal conditions. Such conditions develop only when animals are additionally stressed and their defense mechanisms break down. The abnormalities are de-
CELLULAR
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scribed in detail, quantitated, publication.
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371
and discussed in a report which is being prepared for ACKNOWLEDGMENTS
The author wishes to express his gratitude to Linda Simmerman for her expert technical assistance. This investigation was supported by a University of Kentucky Tobacco and Health Research Institute Grant, project 24133.
REFERENCES Benner, J. F., Owens, S., Hancock, R., and Griffith, R. B. (1973). Smoking machine development and inhalation atmosphere monitoring. Proc. Univ. KY. Tob. Health Res. Inst. Conf. Rep. No. 4, 494-506. Blusse Van oud Alblas, A., and VanFurth, R. (1979). Origin, kinetics, and characteristics of pulmonary macrophages in the normal steady state. J. Exp. Med. 149, 1504- 1518. Bowden, D. H., and Adamson, J. Y. R. (1978). Adaptive response of the pulmonary macrophagic system to carbon. Lab. Invest. 38, 422-429. Bowden, D. H., Adamson, J. Y. R., Grantham, W. C., and Wyatt, J. P. (1969). Origin of the lung macrophage: Evidence derived from radiation injury. Arch. Pathol. 88, 540-546. Brahim, F., and Bahadue, G. (1979). Influx of long-lived lymphocytes into guinea pig marrow during hydrocortisone administration. J. Reticuloendothel. Sot. 25, 397-404. Brody, A. R., and Craighead, J. E. (1975). Cytoplasmic inclusions in pulmonary macrophages of cigarette smokers. Lab. Invest. 32, 125- 132. Huber, G. L., LaForce, F. M., and Johanson, W. G. (1977). Experimental models and pulmonary antimicrobial defenses. In “Respiratory Defense Mechanisms” (J. D. Brain, D. Proctor, and L. Reid, Eds.), pp. 979-1022. Dekker, New York. Kavet, R. I., Brain, J. D., and Levens. D. J. (1978). Characteristics of pulmonary macrophages lavaged from hamsters exposed to iron oxide aerosols. Lab. Invest. 38, 312-319. Leibovich, S. J.. and Ross, R. (1975). The role of the macrophage in wound repair. Amer. J. Pathol. 78,71-100. Martin, R. R., and Warr, G. A. (1977). Cigarette smoking and human pulmonary macrophages. Hosp. Pratt.
12, 97-
104.
Matulionis, D. H. (1979a). Reaction of macrophages to cigarette smoke. I. Recruitment of pulmonary macrophages. Arch. Environ. Health 34, 293-297. Matulionis, D. H. (1979b). Reaction of macrophages to cigarette smoke. II. Immigration of macrophages to the lungs. Arch. Environ. Health 34, 298-301. Matulionis, D. H., and Traurig, H. H. (1977). In sita response of lung macrophages and hydrolase activities to cigarette smoke. Lab. Invest. 37, 314-325. Pratt, S. A., Smith, M. H., Ladman, A. J., and Finley, T. N. (1971). The ultrastructure of alveolar macrophages from human cigarette smokers and nonsmokers. Lab. Invest. 24, 331-338. Thompson, J., and VanFurth, R. (1970). The effect of glucocorticosteroids on the kinetics of mononuclear phagocytes. J. Exp. Med. 131, 429-442. Thompson, J., and VanFurth, R. (1973). The effect of glucocorticosteroids on the proliferation and kinetics of promonocytes and monocytes of bone marrow. J. Exp. Med. 136, 10-21. VanFurth, R. (1970). Origin and kinetics of monocytes and macrophages. Semin. Hemarol. 7, 125- 141.