THE AMERICAN JOURNAL OF ANATOMY 191:401-407 (19911
Comparative Morphology and Morphometry of Alveolar Macrophages From Six Species P.J. HALEY, B.A. MUGGENBURG, D.N. WEISSMAN, AND D.E. BICE Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87185
ABSTRACT Pulmonary alveolar macrophages (PAM) were collected from normal, healthy mice, rats, dogs, cynomolgus monkeys, chimpanzees, and humans and evaluated for morphologic and morphometric characteristics. The PAM of mice, rats, and dogs were morphologically similar and had statistically similar frequency distributions for size. The cell size distribution for these three species was relatively homogeneous. The PAM of nonhuman primates and humans were morphologically heterogenous with sometimes prominent cytoplasmic vacuolation, irregular cell outlines, and increased numbers of multinucleated cells as compared to the PAM of rodents and dogs. The mean size of human PAM was statistically greater than that for all other species evaluated, including nonhuman primates. These data indicate that significant differences in PAM morphology and size exist among species. INTRODUCTION
mice were obtained from Simonsen Laboratories (Gilroy, CA). The rats were bred and raised at the Inhalation Toxicology Research Institute (ITRI). The rodents were maintained in a barrier rodent housing facility operated on a cleanldirty corridor system. All personnel showered before entering and wore clean laboratory-supplied clothing, face mask, and gloves. All materials that entered the facility were sterilized prior to use. All materials that could not be sterilized in the tunnel washer or autoclave were chemically sterilized. The air-supply system was a once-through, 100% outside air system and was pre- and HEPA-filtered. Each room was exhausted by a HEPA-filtered exhaust plenum system. Heating was by LP gas heat exchanger and cooling was by mechanical refrigeration. The animal rooms were maintained at 72°C and at 50% relative humidity under a 12-hr light. and dark cycle. The rodents were kept in polycarbonate cages with hardwood bedding (Sani-Chips, P.J. Murphy Forest Products), and were fed ad libitum with NIH-31 Autoclavable Mouse and Rat Diet (Zeigler Bros., Inc., Gardners, PA). Water was obtained from the treated, domestic water supply and was provided to each cage by a n automated watering system. The rodents were euthanized by a n intraperitoneal injection of sodium pentobarbital, and the heartllung block was removed. The lungs of mice were lavaged with four 1 ml aliquots, and the lungs of rats with two 7 ml aliquots of normal saline.
Pulmonary alveolar macrophages (PAM) play important roles in the maintenance of pulmonary homeostasis. These roles include removal of particles from the alveolar compartment (Harmsen et al., 1985; Morrow, 1988; Territo and Golde, 19791, phagocytosis and destruction of microbial organisms (Hunninghake et al., 1979; Territo and Golde, 1979), control of neoplastic Dogs cells (Hunninghake et al., 1979; Lemarbre et al., 1980), amplification of pulmonary inflammatory responses Four male Beagle dogs, 2 years old, were used in this (Hunninghake et al., 1979), and the generation and study. The dogs were born and raised in a closed colony regulation of local immune responses (Murphy and at ITRI that uses a generation breeding system and Herscowitz, 1984). Such studies have investigated the random mating, giving each parent equal representafunctional aspects of PAM, using PAM from numerous tion in subsequent generations and minimizing inspecies for in vivo experiments with the ultimate goal breeding. No dogs have been introduced into the colony being the extrapolation of the results to humans. While since 1965. The dogs were kept in indoorloutdoor kenspecies differences for macrophage functions have been nels with cement runs. The ambient air humidity was examined (Schlesinger, 1985; Snipes et al., 1983; Tho- low (less than 40%). Each dog was observed daily and mas, 19721, attempts to compare the morphology of given a n annual physical examination that included a PAM from different species have not been reported. We complete blood count, selected serum chemistry detercompared the morphologic and morphometric charac- minations, and chest radiographs. The dogs were proteristics of alveolar macrophages from normal, healthy vided water ad libitum and fed dry kibble once a day individuals of five different laboratory animal species (Wayne Dog Food, Continental Grain, Chicago, IL). All and humans in order to determine if significant mor- dogs were vaccinated at 6, 10, and 14 weeks and then phologic differences among species occur. MATERIALS AND METHODS Mice and Rats
Four male 10-week-old B6C3F1 mice, and four male 10-week-old F344/Crl rats were used in this study. The 0 1991 WILEY-LISS, INC
Received December 1, 1989. Accepted March 25, 1991.
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P.J. HALEY E T AL
yearly for parvovirus; a t 8, 12, 16, and 52 weeks, then yearly for canine distemper, canine adenovirus-2, and parainfluenza; and a t 12 weeks, 1 year, and every 3 years for rabies. The dogs were anesthetized with 4% halothane in oxygen by mask followed by endotracheal intubation. Anesthesia was maintained using 1.5% halothane in oxygen. A fiberoptic bronchoscope (Olympus BF3, Olympus Optical Co., Lake Success, NY) was introduced into individual lung lobes, and each diaphragmatic lobe was lavaged with five 10 ml aliquots of normal saline. Nonhuman Primates
Four young adult, male, wild-caught cynomolgus monkeys (Mucucu fusciculuris; Charles River Primate, Port Washington, NY) were housed in separate stainless-steel cages with a n automatic watering system and were fed dry Monkey Chow (Ralston Purina, St. Louis, MO) and fresh fruit twice daily. Each monkey received a complete physical examination that included chest radiographs, selected serum chemistry determination, and complete blood cell counts. Tuberculosis skin tests were performed on each animal quarterly. These monkeys had been in captivity for approximately 1 year before the study. Air supply for the primate housing facility a t ITRI was a once-through 100% outside air system that used a single air-handling unit, complete with pre- and HEPA-filtration. Cooling was by evaporative cooling and chilled water coils. Room humidity was controlled by room thermostats and a humidistat and direct steam injection system. For bronchoalveolar lavage (BAL), cynomolgus monkeys were tranquilized with ketamine hydrochloride (Ketaset, Bristol Labs, Syracuse, NY) a t 10 mgikg body weight intramuscularly (IM). After immobilization, anesthesia was induced with 4% halothane in oxygen by mask, after which the monkeys were intubated. A pediatric fiberoptic bronchoscope (Olympus BF Type 3C4, Olympus Optical Co., Lake Success, NY) was introduced into individual lung lobes and each diaphragmatic lobe was lavaged with five 7 ml aliquots of normal saline. Four male, approximately 10-year-old chimpanzees (Pun troglodytes) were selected from the colony at the Primate Research Institute, New Mexico State University, Holloman Air Force Base, Alamogordo, New Mexico, where they were kept in indoorloutdoor cement runs. Three of the chimpanzees were wild-caught and one was raised in captivity at the Primate Research Institute where they had been maintained for more than 5 years. Each chimpanzee was immobilized with ketamine hydrochloride (Ketaset, Bristol Laboratories, New York, NY) a t a dose of 10 mglkg body weight IM. Atropine sulfate was given subcutaneously, 0.07 mgikg body weight. Anesthesia was maintained with halothane gas in a mixture of 60% oxygen and 40% nitrous oxide, administered a t a 4% concentration following endotracheal intubation. A fiberoptic bronchoscope (Olympus BF-4B2, Olympus Corporation of America, New Hyde Park, NY) was introduced into individual lung lobes, and six 10 ml aliquots of normal saline were used to lavage each diaphragmatic lobe.
Humans
All human samples were obtained from nonsmoking, male individuals, 24-34 years old, living in the vicinity of New Orleans, Louisiana, with no recent history of pulmonary disease. Topical anesthesia was achieved by inhaling 5 cc of nebulized, 4% lidocaine, followed by gargling twice with 5 cc of 4% lidocaine, spraying 1 cc of 2% lidocaine on the vocal cords, and then depositing 1-2 cc of 1% lidocaine into the trachea. After local anesthesia, a fiberoptic bronchoscope (Olympus BF-lT, Olympus Optical Co., Lake Success, NY) was gently wedged into a middle lobe segmental bronchus, and five 50 ml boluses of sterile, normal saline were instilled and immediately recovered by gentle suction. Processing of Samples
Fluid and cells were placed on ice following the lavage procedure. Cells were separated from the BAL fluid by centrifugation (1,000 RPM; 10 min) and were washed three times in culture medium (RPMI 1640 with 25 mM Hepes buffer, Gibco, Tucson, AZ). Cytocentrifuge samples were prepared by resuspending the lung cells to a concentration of 2 x 106/ml and centrifuging 100 p1 aliquots at 400 RPM for 4 min in a Shandon, Cytospin 2 cytocentrifuge (Shandon Southern Instruments, Sewickley, PA). Cytocentrifuge preparations were stained with a rapid Romanofsky-type stain (Diff-Quick, American Scientific Products, McGraw Park, IL). Morphometry
A cytospin preparation of pulmonary cells from each animal was projected onto a digitizing tablet attached to a n image analysis system (Videoplan, Carl Zeiss, Inc., New York, NY). The criteria for selection of PAM included those cells with round to oval and sometimes indented nuclei, slightly diffuse and lacy nuclear chromatin with little clumping, and moderately to markedly abundant light-blue to slate-gray cytoplasm with variable numbers of cytoplasmic vacuoles. Lymphocytes were identified as having small, round nuclei with darkly staining, clumped chromatin, and a small rim of basophilic cytoplasm t h a t lacked vacuoles. Polymorphonuclear leukocytes (neutrophils and eosinophils) were identified by their segmented nuclei, heavily clumped nuclear chromatin, and the presence of red to orange cytoplasmic granules. The perimeters of the nucleus and cell membrane for each of 300 PAM for each animal were traced on the tablet; and the data were transformed, by Videoplan data acquisition software, into nuclear and total cell area and effective cell diameter. The cytoplasmic area of each cell was calculated by subtracting the nuclear area from the total cell area, after which the ratio of nuclear area to area of cytoplasm (N:C ratio) was calculated. Distribution frequency tables and a computerized algorithm were used to generate histograms based on a sample increment size of 20 and a curve smoothing value of 0.2. A total of 300 cells per individual and four individuals per species were evaluated to give a total of 1,200 data points per species. Statistics
The means of PAM nuclear areas, cytoplasmic areas, N:C ratios, and diameters for each species were com-
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MORPHOMETRIC AND MORPHOLOGIC CHARACTERISTICS OF PAM
TABLE 1. Differential cell counts of pulmonary cells obtained from six species by BAL Species' Mouse Rat Dog Cynomolgus monkey Chimpanzee Human
Neutrophil 0 0 0.5 (0.3)' 0 3.4 (1.1) 0.8 (0.75)
(% of 200 cells counted) Lymphocyte Macrophage 1.5 (0.4) 98.5 (0.7) 0.5 (0.2) 99.5 (0.2) 2.3 (0.6) 95.3 (0.9) 1.3 (0.3) 93.8 (3.0) 1.3 (1.0) 94.4 (2.1) 9.9 (2.6) 89.3 (2.5)
Eosinophil 0 0 2.0 (1.0) 5.0 (3.2) 1.0 (0.5) 0.6 (0.12)
'N = 4 for each species 'Standard error.
pared statistically using the Newman-Keuls multiple range test. The criterion for statistical significance was set a t P < 0.05.
other species evaluated (P < 0.05). No significant differences were noted in the N:C ratios of the PAM among the six species evaluated.
RESULTS
DISCUSSION
The cells recovered by BAL from all species were greater than 90% PAM, with the exception of those from humans, which had a slightly lower percentage (Table 1).Included in the PAM counts were multinucleated cells, which accounted for less than 2% of PAM in all species except chimpanzees and humans, in which the values were 8.7% and 5.0% of the PAM, respectively. Epithelial cells, either singular or in aggregates, were rare in all species; and these cells accounted for less than 1% of the differential cell count. The PAM of mice, rats, and dogs were morphologically similar (Fig. 1). PAM from these species tended to be rounded and regular in outline with moderate amounts of light blue, lacy cytoplasm. Cytoplasmic vacuolation was slight in all three species, and the presence of intracellular dust or other phagocytized debris was unusual. Nuclei were round to oval and frequently eccentric. Nuclear chromatin stained deep purple in a linear-clumped pattern. Nucleoli were single, dark blue, and difficult to identify. The PAM of cynomolgus monkeys, chimpanzees, and humans were similar among the three species but were morphologically heterogenous. The cytoplasm was either lacy and light blue, or opaque and slate grey. Cytoplasmic vacuolation was prominent in many cells. In primates, the cellular shape and outlines tended to be more irregular, a s pseudopodia and cytoplasmic blebs were observed often. Large, multinucleated alveolar macrophages and dust-laden cells were seen frequently in BAL from both nonhuman primates and man. Nuclei were also more irregular in shape than those observed for mice, rats, or dogs. However, the chromatin staining patterns of the nuclei among all species were similar. The frequency distribution of PAM size for mouse, rat, and dog were statistically similar (Fig. 2), and the range of cell sizes for these three species was narrow (Table 2). Comparatively, the PAM of nonhuman primates and man were larger (P < 0.05) than those of mouse, rat, and dog, and had a greater range in cell size. The sizes of PAM from the cynomolgus monkey and chimpanzee were not statistically different from one another, but were different from those of the mouse, rat, dog, and human (P < 0.05). The PAM of humans were significantly larger than those of all
Morphologic variations of PAM among species may reflect differences in cellular maturation, activation, or inherent species differences in cell size. In addition, the values obtained for diameter, area, or volume of PAM may vary with the methods of sample processing. Davies et al. (1977) fixed rat PAM in glutaraldehydeosmium, embedded them in Epon, and sectioned them at 1 pm. Using this technique, the volumes of PAM for control rats were 623 pm3 in one study (Davies et al., 1978) and 871 pm3 in anothcr. Strom (1984) using a n electronic particle counter coupled to a multichannel analyzer, reported that alveolar macrophages of normal rats ranged in size from 400-5,000 pm3, with a mean of 1,170 pm3. The PAM of rats had reported diameters of 10.5 pm, 11.5 pm, and 9.8 pm (Davies et al., 1977, 1978). The larger diameter reported here for the PAM of rats (18 pm) is probably a result of the cell spreading and flattening that occur during cytocentrifugation. Because of this flattening artifact, it is important to reproduce the methods described here when attempting to reproduce these findings. Diameters of cells in suspension would of course be expected to be much smaller. It has been reported (Rebar e t al., 1980) that more than 95% of the PAM of dogs prepared using similar cytocentrifuge procedures are 10-15 pm in diameter, values that are slightly less than those reported in this study. Others, using Fischer 344 rats and cytocentrifuge techniques similar to those used here, reported a mean cytoplasmic area for the PAMs of control rats of 170 pm2 (Hotchkiss et al., 1989).This value is similar to the 187 pm2 value found in the present study. The sizes reported for human alveolar macrophages have varied greatly (Territo and Golde, 1979; Hunninghake et al., 19791, but values obtained in the present study are within the range of those reported in the literature. Our data demonstrated significant differences between species in morphologic and morphometric characteristics of PAM. The PAM of mice, rats, and dogs were similar in size to one another but were smaller than those of nonhuman primates and humans. The relationship of these morphologic differences to functional differences among species is unknown. I t should be noted that there was greater homogeneity, and possibly a lower level of background activation, of PAM
404
P.J. HALEY E T AL.
Fig. l a x
from rodents reared in barrier conditions a s compared to the other species. Outbred species, such as man, that live in varying conditions of environmental pollution are more likely to have greater PAM morphologic heterogeneity, dust accumulation, and possibly back-
ground cellular activation. Such differences between barrier-reared rodents and humans underscores the difficulty in extrapolating data concerning PAM from one species to the other as well as the problem of using primary human cells for scientific investigations.
MORPHOMETRIC AND MORPHOLOGIC CHARACTERISTICS OF PAM
405
Fig. 1. Cytocentrifuge preparations of BAL fluid from normal mouse (A), rat (B),dog ( C ) ,cynomolgus monkey (D), chimpanzee (E), and human (F). A few lymphocytes can be seen (small arrows) and a neutrophil is present in C (large arrow). Bars = 20 pm.
ACKNOWLEDGMENTS The authors thank the personnel at the Inhalation Toxicology Research Institute for their suggestions and
assistance in the preparation of this manuscript. The authors would like to thank, in particular, Ms. Deborah Swafford for her technical help. The authors would
406
'4
CELL AREA IN MICRONS*
CELL AREA IN MICRONS~
CELL AREA IN MICRONS~
CELL AREA IN MICRONS~
24
W
16
CELL AREA IN MICRONS~
Fig. 2. Histograms of cell area of PAM from normal mouse (A), rat (B),dog (C),cynornolgus monkey (D),chimpanzee (E),and human (F). N = 4 for each histogram. Cell size is in Fm'. Area to the left of the vertical line represents 90% of total cell numbers. The frequency dis-
CELL AREA IN MICRONS~
tribution of PAM size for mouse, rat, and dog were not different. The PAM of man were larger ( P < 0.05) than those of nonhuman primates, mouse, rat, and dog. PAM of nonhuman primates were different from mouse, rat, dog, and humans ( P < 0.05).
407
MORPHOMETRIC AND MORPHOLOGIC CHARACTERISTICS OF PAM
TABLE 2. Nuclear area,' cytoplasmic area, N:C ratio, and cell diametes for PAM from six species Cytoplasmic
Nuclear area
area
Species'
(pm2)
Mouse Rat Dog
65.7b (0.4)' 71.4b (0.5) 64.6b (1.1) 96.9" (0.8) 111.2a (0.8) 101.oa (0.9)
(pm2) 214.3' (2.2) 187.2' (2.1) 136.1' (2.6) 348.3' (4.2) 307.gb (3.9) 453.9" (5.5)
Cynomolgus m o n k e y Chimpanzee Human
N:C Ratio 0.35a.d (0.01) 0.42a2b(0.01) 0.44a (0.01) 0.30d." (0.01) 0.4OazC (0.01) 0.24e (0.01)
Diameter (pm)
19' (0.08) 18C,d (0.08) 16d(0.09) 23' (0.11) 23' (0.11) 26" (0.14)
'N = 4 for each species. 'Standard error of the mean is given in parentheses. Letter superscripts refer to statistical comparisons. *Values with different letter superscripts are statistically different at P 5 0.05.
also like to thank Drs. J. Mauderly, J. Benson, B. Snipes, N. Johnson, and T. Coons for their valuable comments and criticisms during the review of this work and manuscript preparation. Research was supported by the Office of Health and Environmental Research of the U.S. Department of Energy under Contract No. DE-AC04-76EV01013, in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. LITERATURE CITED Davies, P., C. Sornberger, and G. Huber 1977 The stereology of pulmonary alveolar macrophages after prolonged experimental exposure to tobacco smoke. Lab. Invest., 37r297-306. Davies, P., C. Sornberger, E. Engel, and G. Huber 1978 Stereology of lavaged populations of alveolar macrophages: effects of in vivo exposurc to tobncco smoke. Exp. Mol. Pathol., 29t170-182. Harmsen, A.G., B.A. Muggenburg, M.B. Snipes, and D.E. Bice 1985 The role of macrophages in particle translocation from lungs to lymph nodes. Science, 230t1277-1280. Hotchkiss, J.A., J.R. Harkema, D.T. Kirkpatrick, and R.F. Henderson 1989 Response of rat alveolar macrophages to ozone: quantitative assessment of population size, morphology, and proliferation following acute exposure. Exp. Lung Res., 15.1-16. Hunninghake, G.W., J.E. Gadek, 0. Kawanami, V.J. Ferrans, and R.G. Crystal 1979 Inflammatory and immune processes in the
human lung in health and disease: evaluation by bronchoalveolar lavage. Am. J. Pathol., 97:149-205. Lemarbre, P., J. Hoidal, R. Vesella, and J. Rinehart 1980 Human pulmonary macrophage tumor cell cytotoxicity. Blood, 55t612617. Morrow, P.E. 1988 Possible mechanisms to explain dust overloading of the lungs. Fundam. Appl. Toxicol., 10:369-384. Murphy, M.A., and H.B. Herscowitz 1984 Heterogeneity among alveolar macrophages in humoral and cell-mediated immune responses: separation of functional subpopulations by density gradient centrifugation on Percoll. J. Leukoc. Biol., 35t39-54. Rebar, A.H., D.B. DeNicola, and B.A. Muggenburg 1980 Bronchopulmonary lavage cytology in the dog: normal findings. Vet. Pathol., 17r294-304. Schlesinger, R.B. 1985 Comparative deposition of inhaled aerosols in experimental animals and humans: a review. J . Toxicol. Environ. Healt.h, 15:197-214. Snipes, M.B., B.B. Boecker, and R.O. McClellan 1983 Retention of . monodisperse or polydisperse aluminosilicate particles inhaled by dogs, rats, and mice. Toxicol. Appl. Pharmacol., 6Yt345-362. Strom, K.A. 1984 Response of pulmonary cellular defenses to the inhalation of high concentrations of diesel exhaust. J. Toxicol. Environ. Health, 13t919-944. Territo, M.C., and D.W. Golde 1979 The function of human alveolar macrophages. J. Reticuloendoth. SOC.,25t111-120. Thomas, R. 1972 An interspecies model for retention of inhaled particles. In: Assessment of Airborne Particles. T.T. Mercer, P.E. Morrow, W. Stober, eds. C.C. Thomas, Springfield, IL, pp. 405419.
Comparative Morphology and Morphometry of Alveolar Macrophages From Six Species P.J. HALEY, B.A. MUGGENBURG, D.N. WEISSMAN, AND D.E. BICE Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87185
ABSTRACT Pulmonary alveolar macrophages (PAM) were collected from normal, healthy mice, rats, dogs, cynomolgus monkeys, chimpanzees, and humans and evaluated for morphologic and morphometric characteristics. The PAM of mice, rats, and dogs were morphologically similar and had statistically similar frequency distributions for size. The cell size distribution for these three species was relatively homogeneous. The PAM of nonhuman primates and humans were morphologically heterogenous with sometimes prominent cytoplasmic vacuolation, irregular cell outlines, and increased numbers of multinucleated cells as compared to the PAM of rodents and dogs. The mean size of human PAM was statistically greater than that for all other species evaluated, including nonhuman primates. These data indicate that significant differences in PAM morphology and size exist among species. INTRODUCTION
mice were obtained from Simonsen Laboratories (Gilroy, CA). The rats were bred and raised at the Inhalation Toxicology Research Institute (ITRI). The rodents were maintained in a barrier rodent housing facility operated on a cleanldirty corridor system. All personnel showered before entering and wore clean laboratory-supplied clothing, face mask, and gloves. All materials that entered the facility were sterilized prior to use. All materials that could not be sterilized in the tunnel washer or autoclave were chemically sterilized. The air-supply system was a once-through, 100% outside air system and was pre- and HEPA-filtered. Each room was exhausted by a HEPA-filtered exhaust plenum system. Heating was by LP gas heat exchanger and cooling was by mechanical refrigeration. The animal rooms were maintained at 72°C and at 50% relative humidity under a 12-hr light. and dark cycle. The rodents were kept in polycarbonate cages with hardwood bedding (Sani-Chips, P.J. Murphy Forest Products), and were fed ad libitum with NIH-31 Autoclavable Mouse and Rat Diet (Zeigler Bros., Inc., Gardners, PA). Water was obtained from the treated, domestic water supply and was provided to each cage by a n automated watering system. The rodents were euthanized by a n intraperitoneal injection of sodium pentobarbital, and the heartllung block was removed. The lungs of mice were lavaged with four 1 ml aliquots, and the lungs of rats with two 7 ml aliquots of normal saline.
Pulmonary alveolar macrophages (PAM) play important roles in the maintenance of pulmonary homeostasis. These roles include removal of particles from the alveolar compartment (Harmsen et al., 1985; Morrow, 1988; Territo and Golde, 19791, phagocytosis and destruction of microbial organisms (Hunninghake et al., 1979; Territo and Golde, 1979), control of neoplastic Dogs cells (Hunninghake et al., 1979; Lemarbre et al., 1980), amplification of pulmonary inflammatory responses Four male Beagle dogs, 2 years old, were used in this (Hunninghake et al., 1979), and the generation and study. The dogs were born and raised in a closed colony regulation of local immune responses (Murphy and at ITRI that uses a generation breeding system and Herscowitz, 1984). Such studies have investigated the random mating, giving each parent equal representafunctional aspects of PAM, using PAM from numerous tion in subsequent generations and minimizing inspecies for in vivo experiments with the ultimate goal breeding. No dogs have been introduced into the colony being the extrapolation of the results to humans. While since 1965. The dogs were kept in indoorloutdoor kenspecies differences for macrophage functions have been nels with cement runs. The ambient air humidity was examined (Schlesinger, 1985; Snipes et al., 1983; Tho- low (less than 40%). Each dog was observed daily and mas, 19721, attempts to compare the morphology of given a n annual physical examination that included a PAM from different species have not been reported. We complete blood count, selected serum chemistry detercompared the morphologic and morphometric charac- minations, and chest radiographs. The dogs were proteristics of alveolar macrophages from normal, healthy vided water ad libitum and fed dry kibble once a day individuals of five different laboratory animal species (Wayne Dog Food, Continental Grain, Chicago, IL). All and humans in order to determine if significant mor- dogs were vaccinated at 6, 10, and 14 weeks and then phologic differences among species occur. MATERIALS AND METHODS Mice and Rats
Four male 10-week-old B6C3F1 mice, and four male 10-week-old F344/Crl rats were used in this study. The 0 1991 WILEY-LISS, INC
Received December 1, 1989. Accepted March 25, 1991.
402
P.J. HALEY E T AL
yearly for parvovirus; a t 8, 12, 16, and 52 weeks, then yearly for canine distemper, canine adenovirus-2, and parainfluenza; and a t 12 weeks, 1 year, and every 3 years for rabies. The dogs were anesthetized with 4% halothane in oxygen by mask followed by endotracheal intubation. Anesthesia was maintained using 1.5% halothane in oxygen. A fiberoptic bronchoscope (Olympus BF3, Olympus Optical Co., Lake Success, NY) was introduced into individual lung lobes, and each diaphragmatic lobe was lavaged with five 10 ml aliquots of normal saline. Nonhuman Primates
Four young adult, male, wild-caught cynomolgus monkeys (Mucucu fusciculuris; Charles River Primate, Port Washington, NY) were housed in separate stainless-steel cages with a n automatic watering system and were fed dry Monkey Chow (Ralston Purina, St. Louis, MO) and fresh fruit twice daily. Each monkey received a complete physical examination that included chest radiographs, selected serum chemistry determination, and complete blood cell counts. Tuberculosis skin tests were performed on each animal quarterly. These monkeys had been in captivity for approximately 1 year before the study. Air supply for the primate housing facility a t ITRI was a once-through 100% outside air system that used a single air-handling unit, complete with pre- and HEPA-filtration. Cooling was by evaporative cooling and chilled water coils. Room humidity was controlled by room thermostats and a humidistat and direct steam injection system. For bronchoalveolar lavage (BAL), cynomolgus monkeys were tranquilized with ketamine hydrochloride (Ketaset, Bristol Labs, Syracuse, NY) a t 10 mgikg body weight intramuscularly (IM). After immobilization, anesthesia was induced with 4% halothane in oxygen by mask, after which the monkeys were intubated. A pediatric fiberoptic bronchoscope (Olympus BF Type 3C4, Olympus Optical Co., Lake Success, NY) was introduced into individual lung lobes and each diaphragmatic lobe was lavaged with five 7 ml aliquots of normal saline. Four male, approximately 10-year-old chimpanzees (Pun troglodytes) were selected from the colony at the Primate Research Institute, New Mexico State University, Holloman Air Force Base, Alamogordo, New Mexico, where they were kept in indoorloutdoor cement runs. Three of the chimpanzees were wild-caught and one was raised in captivity at the Primate Research Institute where they had been maintained for more than 5 years. Each chimpanzee was immobilized with ketamine hydrochloride (Ketaset, Bristol Laboratories, New York, NY) a t a dose of 10 mglkg body weight IM. Atropine sulfate was given subcutaneously, 0.07 mgikg body weight. Anesthesia was maintained with halothane gas in a mixture of 60% oxygen and 40% nitrous oxide, administered a t a 4% concentration following endotracheal intubation. A fiberoptic bronchoscope (Olympus BF-4B2, Olympus Corporation of America, New Hyde Park, NY) was introduced into individual lung lobes, and six 10 ml aliquots of normal saline were used to lavage each diaphragmatic lobe.
Humans
All human samples were obtained from nonsmoking, male individuals, 24-34 years old, living in the vicinity of New Orleans, Louisiana, with no recent history of pulmonary disease. Topical anesthesia was achieved by inhaling 5 cc of nebulized, 4% lidocaine, followed by gargling twice with 5 cc of 4% lidocaine, spraying 1 cc of 2% lidocaine on the vocal cords, and then depositing 1-2 cc of 1% lidocaine into the trachea. After local anesthesia, a fiberoptic bronchoscope (Olympus BF-lT, Olympus Optical Co., Lake Success, NY) was gently wedged into a middle lobe segmental bronchus, and five 50 ml boluses of sterile, normal saline were instilled and immediately recovered by gentle suction. Processing of Samples
Fluid and cells were placed on ice following the lavage procedure. Cells were separated from the BAL fluid by centrifugation (1,000 RPM; 10 min) and were washed three times in culture medium (RPMI 1640 with 25 mM Hepes buffer, Gibco, Tucson, AZ). Cytocentrifuge samples were prepared by resuspending the lung cells to a concentration of 2 x 106/ml and centrifuging 100 p1 aliquots at 400 RPM for 4 min in a Shandon, Cytospin 2 cytocentrifuge (Shandon Southern Instruments, Sewickley, PA). Cytocentrifuge preparations were stained with a rapid Romanofsky-type stain (Diff-Quick, American Scientific Products, McGraw Park, IL). Morphometry
A cytospin preparation of pulmonary cells from each animal was projected onto a digitizing tablet attached to a n image analysis system (Videoplan, Carl Zeiss, Inc., New York, NY). The criteria for selection of PAM included those cells with round to oval and sometimes indented nuclei, slightly diffuse and lacy nuclear chromatin with little clumping, and moderately to markedly abundant light-blue to slate-gray cytoplasm with variable numbers of cytoplasmic vacuoles. Lymphocytes were identified as having small, round nuclei with darkly staining, clumped chromatin, and a small rim of basophilic cytoplasm t h a t lacked vacuoles. Polymorphonuclear leukocytes (neutrophils and eosinophils) were identified by their segmented nuclei, heavily clumped nuclear chromatin, and the presence of red to orange cytoplasmic granules. The perimeters of the nucleus and cell membrane for each of 300 PAM for each animal were traced on the tablet; and the data were transformed, by Videoplan data acquisition software, into nuclear and total cell area and effective cell diameter. The cytoplasmic area of each cell was calculated by subtracting the nuclear area from the total cell area, after which the ratio of nuclear area to area of cytoplasm (N:C ratio) was calculated. Distribution frequency tables and a computerized algorithm were used to generate histograms based on a sample increment size of 20 and a curve smoothing value of 0.2. A total of 300 cells per individual and four individuals per species were evaluated to give a total of 1,200 data points per species. Statistics
The means of PAM nuclear areas, cytoplasmic areas, N:C ratios, and diameters for each species were com-
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MORPHOMETRIC AND MORPHOLOGIC CHARACTERISTICS OF PAM
TABLE 1. Differential cell counts of pulmonary cells obtained from six species by BAL Species' Mouse Rat Dog Cynomolgus monkey Chimpanzee Human
Neutrophil 0 0 0.5 (0.3)' 0 3.4 (1.1) 0.8 (0.75)
(% of 200 cells counted) Lymphocyte Macrophage 1.5 (0.4) 98.5 (0.7) 0.5 (0.2) 99.5 (0.2) 2.3 (0.6) 95.3 (0.9) 1.3 (0.3) 93.8 (3.0) 1.3 (1.0) 94.4 (2.1) 9.9 (2.6) 89.3 (2.5)
Eosinophil 0 0 2.0 (1.0) 5.0 (3.2) 1.0 (0.5) 0.6 (0.12)
'N = 4 for each species 'Standard error.
pared statistically using the Newman-Keuls multiple range test. The criterion for statistical significance was set a t P < 0.05.
other species evaluated (P < 0.05). No significant differences were noted in the N:C ratios of the PAM among the six species evaluated.
RESULTS
DISCUSSION
The cells recovered by BAL from all species were greater than 90% PAM, with the exception of those from humans, which had a slightly lower percentage (Table 1).Included in the PAM counts were multinucleated cells, which accounted for less than 2% of PAM in all species except chimpanzees and humans, in which the values were 8.7% and 5.0% of the PAM, respectively. Epithelial cells, either singular or in aggregates, were rare in all species; and these cells accounted for less than 1% of the differential cell count. The PAM of mice, rats, and dogs were morphologically similar (Fig. 1). PAM from these species tended to be rounded and regular in outline with moderate amounts of light blue, lacy cytoplasm. Cytoplasmic vacuolation was slight in all three species, and the presence of intracellular dust or other phagocytized debris was unusual. Nuclei were round to oval and frequently eccentric. Nuclear chromatin stained deep purple in a linear-clumped pattern. Nucleoli were single, dark blue, and difficult to identify. The PAM of cynomolgus monkeys, chimpanzees, and humans were similar among the three species but were morphologically heterogenous. The cytoplasm was either lacy and light blue, or opaque and slate grey. Cytoplasmic vacuolation was prominent in many cells. In primates, the cellular shape and outlines tended to be more irregular, a s pseudopodia and cytoplasmic blebs were observed often. Large, multinucleated alveolar macrophages and dust-laden cells were seen frequently in BAL from both nonhuman primates and man. Nuclei were also more irregular in shape than those observed for mice, rats, or dogs. However, the chromatin staining patterns of the nuclei among all species were similar. The frequency distribution of PAM size for mouse, rat, and dog were statistically similar (Fig. 2), and the range of cell sizes for these three species was narrow (Table 2). Comparatively, the PAM of nonhuman primates and man were larger (P < 0.05) than those of mouse, rat, and dog, and had a greater range in cell size. The sizes of PAM from the cynomolgus monkey and chimpanzee were not statistically different from one another, but were different from those of the mouse, rat, dog, and human (P < 0.05). The PAM of humans were significantly larger than those of all
Morphologic variations of PAM among species may reflect differences in cellular maturation, activation, or inherent species differences in cell size. In addition, the values obtained for diameter, area, or volume of PAM may vary with the methods of sample processing. Davies et al. (1977) fixed rat PAM in glutaraldehydeosmium, embedded them in Epon, and sectioned them at 1 pm. Using this technique, the volumes of PAM for control rats were 623 pm3 in one study (Davies et al., 1978) and 871 pm3 in anothcr. Strom (1984) using a n electronic particle counter coupled to a multichannel analyzer, reported that alveolar macrophages of normal rats ranged in size from 400-5,000 pm3, with a mean of 1,170 pm3. The PAM of rats had reported diameters of 10.5 pm, 11.5 pm, and 9.8 pm (Davies et al., 1977, 1978). The larger diameter reported here for the PAM of rats (18 pm) is probably a result of the cell spreading and flattening that occur during cytocentrifugation. Because of this flattening artifact, it is important to reproduce the methods described here when attempting to reproduce these findings. Diameters of cells in suspension would of course be expected to be much smaller. It has been reported (Rebar e t al., 1980) that more than 95% of the PAM of dogs prepared using similar cytocentrifuge procedures are 10-15 pm in diameter, values that are slightly less than those reported in this study. Others, using Fischer 344 rats and cytocentrifuge techniques similar to those used here, reported a mean cytoplasmic area for the PAMs of control rats of 170 pm2 (Hotchkiss et al., 1989).This value is similar to the 187 pm2 value found in the present study. The sizes reported for human alveolar macrophages have varied greatly (Territo and Golde, 1979; Hunninghake et al., 19791, but values obtained in the present study are within the range of those reported in the literature. Our data demonstrated significant differences between species in morphologic and morphometric characteristics of PAM. The PAM of mice, rats, and dogs were similar in size to one another but were smaller than those of nonhuman primates and humans. The relationship of these morphologic differences to functional differences among species is unknown. I t should be noted that there was greater homogeneity, and possibly a lower level of background activation, of PAM
404
P.J. HALEY E T AL.
Fig. l a x
from rodents reared in barrier conditions a s compared to the other species. Outbred species, such as man, that live in varying conditions of environmental pollution are more likely to have greater PAM morphologic heterogeneity, dust accumulation, and possibly back-
ground cellular activation. Such differences between barrier-reared rodents and humans underscores the difficulty in extrapolating data concerning PAM from one species to the other as well as the problem of using primary human cells for scientific investigations.
MORPHOMETRIC AND MORPHOLOGIC CHARACTERISTICS OF PAM
405
Fig. 1. Cytocentrifuge preparations of BAL fluid from normal mouse (A), rat (B),dog ( C ) ,cynomolgus monkey (D), chimpanzee (E), and human (F). A few lymphocytes can be seen (small arrows) and a neutrophil is present in C (large arrow). Bars = 20 pm.
ACKNOWLEDGMENTS The authors thank the personnel at the Inhalation Toxicology Research Institute for their suggestions and
assistance in the preparation of this manuscript. The authors would like to thank, in particular, Ms. Deborah Swafford for her technical help. The authors would
406
'4
CELL AREA IN MICRONS*
CELL AREA IN MICRONS~
CELL AREA IN MICRONS~
CELL AREA IN MICRONS~
24
W
16
CELL AREA IN MICRONS~
Fig. 2. Histograms of cell area of PAM from normal mouse (A), rat (B),dog (C),cynornolgus monkey (D),chimpanzee (E),and human (F). N = 4 for each histogram. Cell size is in Fm'. Area to the left of the vertical line represents 90% of total cell numbers. The frequency dis-
CELL AREA IN MICRONS~
tribution of PAM size for mouse, rat, and dog were not different. The PAM of man were larger ( P < 0.05) than those of nonhuman primates, mouse, rat, and dog. PAM of nonhuman primates were different from mouse, rat, dog, and humans ( P < 0.05).
407
MORPHOMETRIC AND MORPHOLOGIC CHARACTERISTICS OF PAM
TABLE 2. Nuclear area,' cytoplasmic area, N:C ratio, and cell diametes for PAM from six species Cytoplasmic
Nuclear area
area
Species'
(pm2)
Mouse Rat Dog
65.7b (0.4)' 71.4b (0.5) 64.6b (1.1) 96.9" (0.8) 111.2a (0.8) 101.oa (0.9)
(pm2) 214.3' (2.2) 187.2' (2.1) 136.1' (2.6) 348.3' (4.2) 307.gb (3.9) 453.9" (5.5)
Cynomolgus m o n k e y Chimpanzee Human
N:C Ratio 0.35a.d (0.01) 0.42a2b(0.01) 0.44a (0.01) 0.30d." (0.01) 0.4OazC (0.01) 0.24e (0.01)
Diameter (pm)
19' (0.08) 18C,d (0.08) 16d(0.09) 23' (0.11) 23' (0.11) 26" (0.14)
'N = 4 for each species. 'Standard error of the mean is given in parentheses. Letter superscripts refer to statistical comparisons. *Values with different letter superscripts are statistically different at P 5 0.05.
also like to thank Drs. J. Mauderly, J. Benson, B. Snipes, N. Johnson, and T. Coons for their valuable comments and criticisms during the review of this work and manuscript preparation. Research was supported by the Office of Health and Environmental Research of the U.S. Department of Energy under Contract No. DE-AC04-76EV01013, in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. LITERATURE CITED Davies, P., C. Sornberger, and G. Huber 1977 The stereology of pulmonary alveolar macrophages after prolonged experimental exposure to tobacco smoke. Lab. Invest., 37r297-306. Davies, P., C. Sornberger, E. Engel, and G. Huber 1978 Stereology of lavaged populations of alveolar macrophages: effects of in vivo exposurc to tobncco smoke. Exp. Mol. Pathol., 29t170-182. Harmsen, A.G., B.A. Muggenburg, M.B. Snipes, and D.E. Bice 1985 The role of macrophages in particle translocation from lungs to lymph nodes. Science, 230t1277-1280. Hotchkiss, J.A., J.R. Harkema, D.T. Kirkpatrick, and R.F. Henderson 1989 Response of rat alveolar macrophages to ozone: quantitative assessment of population size, morphology, and proliferation following acute exposure. Exp. Lung Res., 15.1-16. Hunninghake, G.W., J.E. Gadek, 0. Kawanami, V.J. Ferrans, and R.G. Crystal 1979 Inflammatory and immune processes in the
human lung in health and disease: evaluation by bronchoalveolar lavage. Am. J. Pathol., 97:149-205. Lemarbre, P., J. Hoidal, R. Vesella, and J. Rinehart 1980 Human pulmonary macrophage tumor cell cytotoxicity. Blood, 55t612617. Morrow, P.E. 1988 Possible mechanisms to explain dust overloading of the lungs. Fundam. Appl. Toxicol., 10:369-384. Murphy, M.A., and H.B. Herscowitz 1984 Heterogeneity among alveolar macrophages in humoral and cell-mediated immune responses: separation of functional subpopulations by density gradient centrifugation on Percoll. J. Leukoc. Biol., 35t39-54. Rebar, A.H., D.B. DeNicola, and B.A. Muggenburg 1980 Bronchopulmonary lavage cytology in the dog: normal findings. Vet. Pathol., 17r294-304. Schlesinger, R.B. 1985 Comparative deposition of inhaled aerosols in experimental animals and humans: a review. J . Toxicol. Environ. Healt.h, 15:197-214. Snipes, M.B., B.B. Boecker, and R.O. McClellan 1983 Retention of . monodisperse or polydisperse aluminosilicate particles inhaled by dogs, rats, and mice. Toxicol. Appl. Pharmacol., 6Yt345-362. Strom, K.A. 1984 Response of pulmonary cellular defenses to the inhalation of high concentrations of diesel exhaust. J. Toxicol. Environ. Health, 13t919-944. Territo, M.C., and D.W. Golde 1979 The function of human alveolar macrophages. J. Reticuloendoth. SOC.,25t111-120. Thomas, R. 1972 An interspecies model for retention of inhaled particles. In: Assessment of Airborne Particles. T.T. Mercer, P.E. Morrow, W. Stober, eds. C.C. Thomas, Springfield, IL, pp. 405419.
