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Surface morphology of the human alveolar macrophage
Experimental
SURFACE
Cell Research
MORPHOLOGY ALVEOLAR
109 (1977) 71-77
OF THE
HUMAN
MACROPHAGE
S. G. QUAN and D. W. GOLDE Division
of Hematology-Oncology,
Department of Medicine, Los Angeles, CA 90024, USA
UCLA
School
of Medicine,
SUMMARY The surface morphology of human alveolar macrophages from smokers and non-smokers was studied by scanning electron microscopy. Cells were examined after various time intervals in vitro and their morphologic response to the ingestion of carbon and iron particles was evaluated. Alveolar macrbphages obtained from cigarette smokers were found to spread faster, have more lamellipodia, filopodia, blebs and microvilli than those from non-smokers. When cells were cultured with carbon or iron there was approximately a tenfold increase in the number of blebs and membrane convolutions. These studies suggest that.functional and metabolic alterations resulting from exposure to smoke and particulate matter are reflected by prominent changes in surface morphology.
The alveolar macrophage is an important and specialized element in the continuum of cells in the mononuclear phagocyte system. These cells share many properties with macrophages in other tissues but have certain distinctive metabolic and fine structural characteristics [5, 221. Numerous investigators have reported on the surface morphology of macrophages obtained from animals [2-4, 13, 15, 17-19, 241; however, relatively little information is available on the human alveolar macrophage. Recently developed techniques have led to better preservation of the cell surface, permitting more detailed comparative studies with the scanning electron microscope @EM) [21]. Alveolar macrophages are a first line of defense for the lung against microbial invasion and they are highly active in clearing organic and inorganic particulate matter.
These functions relate importantly to surface membrane characteristics. Changes in cell form and surface morphology induced by environmental factors can effectively be evaluated by scanning electron microscopy. This report describes studies performed with the SEM on alveolar macrophages retrieved by bronchopulmonary lavage from normal human subjects. MATERIALS
AND METHODS
Collection of alveolar macrophages Informed consent was obtained from healthy young adult volunteers for fiberoptic bronchoscopy and bronchopulmonary lavage. A careful smoking history was taken. Fiberoptic bronchoscopy was performed via a transnasal approach with topical anesthesia. No systemic medications were administered. The tiberscope was positioned in a lower-lobe segment and lavage performed with 300 ml sterile phy&logic saline. The effluent was added to an equal volume of Hanks’ balanced salt solution with 20% fetal calf Exp Cd
Rc.s IO9 (1977)
72
Quan and Golde
serum and antibiotics, centrifuged at 3x lo? g for 10 min, and washed once with alpha medium (Flow Laboratories, Rockville, Md) containing 20% fetal calf serum (Grand Island Biological Company, Santa Clara, Calif.) and antibiotics. The cells were pelleted once again and resuspended in complete alpha medium. To investigate the effects of ingestion of foreign substances, macrophages were allowed to adhere to coverslips and cultured for 72 h with 10 pg/ml iron powder (99.9% pure, Kern Chemical Corp., Los Angeles, Calif.) and 10 pg/ml washed charcoal (Matheson, Coleman & Bell, Los Angeles, Calif.).
Electron microscopy Cells were allowed to settle on glass or plastic coverslius (Lux Scientific. Newberrv Park, Calif.) at room temperature for 10 min before-fixation. Some macronharres were cultured on coverslips in Petri dishes ?Lux Scientific) in a 37°C humidified incubator with 5% CO, for 1 h, 24 h, and 72 h and then processed. Coverslips were rinsed with buffered saline-to &move non-adherent cells and fixed for 30 min at room temperature with 3 % glutaraldehyde and 1% sucrose in a 0.1 M cacodvlate buffer, pH 7.3. The fixed cells were washed with buffer, then distilled water, and finally dehydrated through a graded series of ethanol and Freon TF. Critical point drying was performed in Freon 13 with the Bomar SPCYOO/EX dryer [6]. Cells were coated by vacuum evaporation with gold or carbon and gold and examined with an ETEC Autoscan RI microscope at 20 kV with either 45” or 75” tilt. Micrographs were recorded on Polaroid N/P film type 55.
smoker macrophage surface had more lamellipodia and filopodia. The morphology of the human alveolar macrophages was found to change markedly with time in vitro. Cells settled for 10 min on coverslips were usually spherical and attached by lamellipodia (figs 1, 2). Macrophages allowed to settle on the substrate for 1 h became flatter with smoother lamellipodia. The smoker macrophages (fig. 3) showed no ruffles on the portion of the cell that was closely adhered to the substrate, but the raised central area had many folds and convolutions and few blebs. At 1 h, non-smoker macrophages continued to have many tilopodia, blebs, and creases on the cell surface, and had highly folded lamellipodia (fig. 4). Smoker macrophages in culture for 24 h showed two morphologies (figs 5, 6), either very flat, adherent cells or rounded, attached cells. Both types of cells were attached by filopodia, lamellipodia or a combination of the two. Nonsmoker macrophages could not be categorized into two morphologies. Very flat
RESULTS Studies were performed on 8 subjects. The alveolar macrophages ranged in size from 8 pm in diameter to over 100 pm in length. Cells were observed in two general forms: spherical-shaped cells with a raised nuclear or central area and elongated, flat, highly adherent cells. The macrophages attached to the substrate by lamellipodia and tilopodia, and had numerous folds and blebs on the cell surfaces. Macrophages from cigarette smokers attached to and spread faster on the substrate than did those from non-smokers (figs 1, 2). The membranes of smoker macrophages were highly convoluted. These cells were more expanded on the substrate and had thicker lamellipodia than non-smoker macrophages. The E.rp Cd
RPS 109 (I977)
1. Typical macrophage from cigarette smoker. Cell surfaces are completely covered with very deep folds, and few microvilli and blebs. The cell periphery is highly creased and has a thickened lamellipodia. Settled for 10 min. X2600. Fig. 2. Alveolar macrophage from a non-smoker settled for 10 min. Cell is rounded and attached to the substrate by several small and delicate lamellipodia. Surface has folds but few blebs and microvilli. ~6 100. Fig. 3. Macrophage from a smoker settled for 1 h and then fixed. Cells are very adherent, show extensive spreading, have few blebs and folds on the surface, and filopodia attachments between cells. x760. Fig. 4. Non-smoker macrophages showing cytoplasmic spreading and ruffling at cell edges. Cell surface is partially smooth but also has blebs, folds, and microvilli. Cells settled for 1 h. X 1400. Fig. 5. Smoker macrophages were observed in two forms when cultured for 24 h. Cells were either very flat and elongated or spherical, and adhered to the surface with lamellioodia and filooodia. x600. Fig. 6. The morphology of smoker macrophages clearlv seen at 75” tilt. Prominent tilopodia are at the base of cells cultured for 24 h. Portions of a flattened cell are in the foreground. x2300. Fig.
74
Quan and Golde
adherent cells were not observed (figs 7, 8). At 24 h, cells from both smokers and nonsmokers continued to have some blebs and microvilli but these structures were less evident. Smoker macrophages cultured for 72 h had the typical morphology of cultured macrophages seen at the light microscope level. Macrophages from smokers at this time generally had a polar orientation and were very elongated with smooth and flattened cytoplasm (fig. 9). The cell surface had few microvilli and blebs. After 72 h in culture, non-smoker macrophages (fig. 10) were morphologically very different from smoker macrophages. Spherical cells were attached to the substrate by filopodia and lamellipodia as in the 24-h culture but there were numerous tilopodia attachments between cells. By 72 h, the cells were more adherent to the substrate and had many lamellipodia and blebs on the surface. The cytoplasmic periphery was very smooth. When iron or carbon was added to the cultures, a prominent change in cellular morphology occurred (figs 11, 12). Cells ingesting iron or carbon were more spherical than elongated and had many convolutions on the surface and filopodia at or near the cell periphery. This distinct change in morphology was more apparent in cells cultured with iron. DISCUSSION Human alveolar macrophages obtained from cigarette smokers differ from nonsmoker cells in having more extensive spreading, larger surface area, more and larger ruffles or lamellipodia, and more filopodia. These findings are similar to results reported by Polliack [19] comparing activated and control murine macrophages from the peritoneal cavity. In contrast, War-r & Martin [23] reported that human E.rp Cell
Res 109 (1977)
alveolar macrophages from smokers had few undulations on the surface and were smooth to cobblestone-like in appearance while non-smokers’ cells had highly undulating membranes. Variation in results may be due to differences in technique. The time of fixation after retrieval and adherence is particularly important. Also, there may be considerable heterogeneity in the population of macrophages. In man, alveolar macrophages may maintain their numbers by migration of precursor cells from the bone marrow and by in situ proliferation [ 10, 11, 231. The length of exposure in the lungs and the stage in the cells’ life span can result in functional and metabolic differences leading to variation in surface morphology. The substrate to which the cell adheres produces cell-surface alterations that may not stimulate the in vivo condition [4]1 For Fig. 7. Macrophages from a non-smoker cultured for 24 h. Cells are generally spherical and show prominent filopodia attachments. The cytoplasm is not well spread and cell attachment is by lamellipodia and filopodia. x 1200. Fig. 8. Non-smoker macrophages cultured for 24 h are attached to the substrate with both lamellipodia and filopodia as seen at 75” tilt. The surface is covered with blebs, folds, and few microvilli. The difference in morphology between smoker and non-smoker macrophages is most clearly seen at this angle. x 1700. Fig. 9. Smoker macrophages in culture for 72 h. Cells are elongated with smooth cytoplasm at the ends. There are few blebs and folds. x 1000. Fig. 10. Macrophages from a non-smoker cultured for 72 h. Cells have maintained their spherical morphology but have become more well spread. Cells show lamellipodia, blebs, and microvilli with many filopodia attachments between cells. x880. Fig. 11. 72-h culture of smoker macrophage with 10 pg/ml of iron. Cells are very different in morphology when compared with control macrophages (fig. 9). There are many blebs, undulations, tilopodia attachments between cells, and few microvilli. x 1300. Fig. 12. Smoker macrophage cultured with 10 pg/ml carbon for 72 h. Cells are spheroid with a ruffled cytoplasmic edge. Portions of the cytoplasm are very smooth and very adherent to the substrate. Surfaces have lamellipodia, few blebs and microvilli, and some filopodia. x690.
76
Quan and Golde
example, macrophages on glass will spread whereas cells adhered to Millipore filters tend to have the same morphology as cells on the alveolar wall [ 141. Changes in the membrane conformation of alveolar macrophages can reflect a corresponding change in its environment. The number of lamellipodia, lilopodia, and the number of microvilli and blebs decreased with increasing culture time. When alveolar macrophages from smokers and nonsmokers were compared there was more extensive ruffling and spreading of the cytoplasm and lilopodia in the smoker macrophages when observed at 10 min, 1 h, and 24 h. After 72 h in culture, macrophages from cigarette smokers had a very different morphology than cells examined at earlier time intervals. Cells were markedly elongated, had very few convolutions and blebs, and a very flat cytoplasm at the polar ends. Nonsmoker macrophages were still not as adherent and resembled smoker macrophages cultured for 24 h. Therefore the time interval after isolation is a critical variable in the interpretation of macrophage surface morphology. The morphology of the macrophages was not static but responded rapidly to changes in environment. Cells fed carbon and iron became more rounded, and the number of membrane undulations, blebs, filopodia and lamellipodia increased at least tenfold. A similar effect has been reported with animal macrophages treated with asbestos and silica [13, 161. The concomitant increase in these structures and the decrease in the total surface area of the cells are related to cell spreading and concentration. With the increased number of microvilli, blebs, filopodia and lamellipodia, the cell may rapidly change form without the need for synthesis of new cell membrane [8]. It is possible that E.rj,
Cd
Rrs
109 (/977)
there is movement of the membrane toward the cell margin [7] rather than away from it [ 1, 121 which could explain the increased adherence. There is other evidence for the storage of membrane in microvilli, blebs, filopodia and lamellipodia [8, 9, 201. For example, cell surface measurements are approximately the same when the cell is dividing and completely covered with microvilli, and at interphase when there are relatively few microvilli. Active movement and configurational changes occur during cell mitosis but are relatively restricted at interphase. The same types of morphological changes have been observed in our study of human alveolar macrophages where cell movement is associated with an increase in surface structures. Scanning electron microscopy provides a useful tool in the study of the surface morphology of human alveolar macrophages. Changes in the conformation of the cell membrane are a reflection of the response of the cell to its environment. Our studies show clear morphological differences between macrophages from smokers and nonsmokers and suggest an important relationship between function and morphology. The authors wish to thank Mr Gerald E. Garner of Scannine Electron Analvsis Laboratories, Inc., for supurb t&hnical assistance. This study was supported by USPHS Grants RR 00865 and HL 20675, and a grant from Brown and Williamson Tobacco Corp., Philip Morris Inc., R. J. Reynolds Tobacco Co., United States Tobacco Co., and Tobacco Associates, Inc.
REFERENCES 1. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 62 (1970) 389. 2. Albrecht, R M, Hinsdill, R D, Sandok, P L, Mackenzie, A P & Sachs, I B, Exp cell res 70 (1972) 230. 3. Carr, I, Clarke, J A & Salsbury, A J, J microsc 89 (1969) 105. 4. Cart-, K & Carr, I, Z Zellforsch 105 (1970) 234. 5. Cohen, A B & Cline, M J, J clin invest 50 (1971) 1390.
Macrophage surface morphology 6.
Cohen, A L, Marlow, D P & Garner, G E, J microsc 7 (1%8) 33 1. De Petris, S & Raff, M C, Nature 241 (1973) 257. Erickson. C A & Trinkaus. J P. Exp cell res 99 (1976) 375. Follett. E A C & Goldman. R D. Exo cell res 59 (1970) i24. Golde, D W, Byers, L A & Finley, T N, Nature 247 ( 1974) 373. Golde, D W, Finley, T N & Cline, M J, N Eng j med 290 (1974) 875. Harris, A & Dunn, G, Exp cell res 73 (1972) 519. Hope, I & Friend, J W, Micron 1 (1969) 3 10. Kuln, C & Finke, E H, J ultrastruct res 38 (1972) 161. Leake, E S, Wright, M J & Myrvik, Q N, J reticuloendothel sot 17 (1975) 370. Miller, K & Kagan, E, J reticuloendothel sot 20 (1976) 159. Papadimitriou, J M, Finley-Jones, J-M & Walters, M N-I, Exp cell res 76 (1973) 353.
18.
Z:
19. 20.
9.
21.
10. 11. 12. 13. 14. 15. 16. 17.
22. 23. 24. 25.
77
Parakkal, P, Pinto, J & Hanifin, J M, J ultrastruct res 48 (1974) 216. Polliack, A & Gordon, S, Lab invest 33 (1975) 469. Porter, K R, Prescott, D & Frve, J, J cell biol 57 (1973) 815. Porter, K R, Kelley, D & Andrews, PM, Proc fifth ann stereoscan ~011. Kent Cambridge Scientific, Morton Grove, Ill (1972). Pratt, S A, Smith M H, Ladman, A J & Finley, T N, Lab invest 24 (1971) 331. Thomas, E D, Ramberg, R W, Sale, G E, Sparkes, R S & Golde, D W, Science 192 (1976) 1016. Warfel, A H & Elberg, S S, Science 170 (1970) 446. Warr, G A & Martin, R R, Life sci 18 (1976) 1177.
Keceived January 18, 1977 Revised version received April 11, 1977 Accepted April 28, 1977
Exp Cd
Re.\ 109 (1977)
SURFACE
Cell Research
MORPHOLOGY ALVEOLAR
109 (1977) 71-77
OF THE
HUMAN
MACROPHAGE
S. G. QUAN and D. W. GOLDE Division
of Hematology-Oncology,
Department of Medicine, Los Angeles, CA 90024, USA
UCLA
School
of Medicine,
SUMMARY The surface morphology of human alveolar macrophages from smokers and non-smokers was studied by scanning electron microscopy. Cells were examined after various time intervals in vitro and their morphologic response to the ingestion of carbon and iron particles was evaluated. Alveolar macrbphages obtained from cigarette smokers were found to spread faster, have more lamellipodia, filopodia, blebs and microvilli than those from non-smokers. When cells were cultured with carbon or iron there was approximately a tenfold increase in the number of blebs and membrane convolutions. These studies suggest that.functional and metabolic alterations resulting from exposure to smoke and particulate matter are reflected by prominent changes in surface morphology.
The alveolar macrophage is an important and specialized element in the continuum of cells in the mononuclear phagocyte system. These cells share many properties with macrophages in other tissues but have certain distinctive metabolic and fine structural characteristics [5, 221. Numerous investigators have reported on the surface morphology of macrophages obtained from animals [2-4, 13, 15, 17-19, 241; however, relatively little information is available on the human alveolar macrophage. Recently developed techniques have led to better preservation of the cell surface, permitting more detailed comparative studies with the scanning electron microscope @EM) [21]. Alveolar macrophages are a first line of defense for the lung against microbial invasion and they are highly active in clearing organic and inorganic particulate matter.
These functions relate importantly to surface membrane characteristics. Changes in cell form and surface morphology induced by environmental factors can effectively be evaluated by scanning electron microscopy. This report describes studies performed with the SEM on alveolar macrophages retrieved by bronchopulmonary lavage from normal human subjects. MATERIALS
AND METHODS
Collection of alveolar macrophages Informed consent was obtained from healthy young adult volunteers for fiberoptic bronchoscopy and bronchopulmonary lavage. A careful smoking history was taken. Fiberoptic bronchoscopy was performed via a transnasal approach with topical anesthesia. No systemic medications were administered. The tiberscope was positioned in a lower-lobe segment and lavage performed with 300 ml sterile phy&logic saline. The effluent was added to an equal volume of Hanks’ balanced salt solution with 20% fetal calf Exp Cd
Rc.s IO9 (1977)
72
Quan and Golde
serum and antibiotics, centrifuged at 3x lo? g for 10 min, and washed once with alpha medium (Flow Laboratories, Rockville, Md) containing 20% fetal calf serum (Grand Island Biological Company, Santa Clara, Calif.) and antibiotics. The cells were pelleted once again and resuspended in complete alpha medium. To investigate the effects of ingestion of foreign substances, macrophages were allowed to adhere to coverslips and cultured for 72 h with 10 pg/ml iron powder (99.9% pure, Kern Chemical Corp., Los Angeles, Calif.) and 10 pg/ml washed charcoal (Matheson, Coleman & Bell, Los Angeles, Calif.).
Electron microscopy Cells were allowed to settle on glass or plastic coverslius (Lux Scientific. Newberrv Park, Calif.) at room temperature for 10 min before-fixation. Some macronharres were cultured on coverslips in Petri dishes ?Lux Scientific) in a 37°C humidified incubator with 5% CO, for 1 h, 24 h, and 72 h and then processed. Coverslips were rinsed with buffered saline-to &move non-adherent cells and fixed for 30 min at room temperature with 3 % glutaraldehyde and 1% sucrose in a 0.1 M cacodvlate buffer, pH 7.3. The fixed cells were washed with buffer, then distilled water, and finally dehydrated through a graded series of ethanol and Freon TF. Critical point drying was performed in Freon 13 with the Bomar SPCYOO/EX dryer [6]. Cells were coated by vacuum evaporation with gold or carbon and gold and examined with an ETEC Autoscan RI microscope at 20 kV with either 45” or 75” tilt. Micrographs were recorded on Polaroid N/P film type 55.
smoker macrophage surface had more lamellipodia and filopodia. The morphology of the human alveolar macrophages was found to change markedly with time in vitro. Cells settled for 10 min on coverslips were usually spherical and attached by lamellipodia (figs 1, 2). Macrophages allowed to settle on the substrate for 1 h became flatter with smoother lamellipodia. The smoker macrophages (fig. 3) showed no ruffles on the portion of the cell that was closely adhered to the substrate, but the raised central area had many folds and convolutions and few blebs. At 1 h, non-smoker macrophages continued to have many tilopodia, blebs, and creases on the cell surface, and had highly folded lamellipodia (fig. 4). Smoker macrophages in culture for 24 h showed two morphologies (figs 5, 6), either very flat, adherent cells or rounded, attached cells. Both types of cells were attached by filopodia, lamellipodia or a combination of the two. Nonsmoker macrophages could not be categorized into two morphologies. Very flat
RESULTS Studies were performed on 8 subjects. The alveolar macrophages ranged in size from 8 pm in diameter to over 100 pm in length. Cells were observed in two general forms: spherical-shaped cells with a raised nuclear or central area and elongated, flat, highly adherent cells. The macrophages attached to the substrate by lamellipodia and tilopodia, and had numerous folds and blebs on the cell surfaces. Macrophages from cigarette smokers attached to and spread faster on the substrate than did those from non-smokers (figs 1, 2). The membranes of smoker macrophages were highly convoluted. These cells were more expanded on the substrate and had thicker lamellipodia than non-smoker macrophages. The E.rp Cd
RPS 109 (I977)
1. Typical macrophage from cigarette smoker. Cell surfaces are completely covered with very deep folds, and few microvilli and blebs. The cell periphery is highly creased and has a thickened lamellipodia. Settled for 10 min. X2600. Fig. 2. Alveolar macrophage from a non-smoker settled for 10 min. Cell is rounded and attached to the substrate by several small and delicate lamellipodia. Surface has folds but few blebs and microvilli. ~6 100. Fig. 3. Macrophage from a smoker settled for 1 h and then fixed. Cells are very adherent, show extensive spreading, have few blebs and folds on the surface, and filopodia attachments between cells. x760. Fig. 4. Non-smoker macrophages showing cytoplasmic spreading and ruffling at cell edges. Cell surface is partially smooth but also has blebs, folds, and microvilli. Cells settled for 1 h. X 1400. Fig. 5. Smoker macrophages were observed in two forms when cultured for 24 h. Cells were either very flat and elongated or spherical, and adhered to the surface with lamellioodia and filooodia. x600. Fig. 6. The morphology of smoker macrophages clearlv seen at 75” tilt. Prominent tilopodia are at the base of cells cultured for 24 h. Portions of a flattened cell are in the foreground. x2300. Fig.
74
Quan and Golde
adherent cells were not observed (figs 7, 8). At 24 h, cells from both smokers and nonsmokers continued to have some blebs and microvilli but these structures were less evident. Smoker macrophages cultured for 72 h had the typical morphology of cultured macrophages seen at the light microscope level. Macrophages from smokers at this time generally had a polar orientation and were very elongated with smooth and flattened cytoplasm (fig. 9). The cell surface had few microvilli and blebs. After 72 h in culture, non-smoker macrophages (fig. 10) were morphologically very different from smoker macrophages. Spherical cells were attached to the substrate by filopodia and lamellipodia as in the 24-h culture but there were numerous tilopodia attachments between cells. By 72 h, the cells were more adherent to the substrate and had many lamellipodia and blebs on the surface. The cytoplasmic periphery was very smooth. When iron or carbon was added to the cultures, a prominent change in cellular morphology occurred (figs 11, 12). Cells ingesting iron or carbon were more spherical than elongated and had many convolutions on the surface and filopodia at or near the cell periphery. This distinct change in morphology was more apparent in cells cultured with iron. DISCUSSION Human alveolar macrophages obtained from cigarette smokers differ from nonsmoker cells in having more extensive spreading, larger surface area, more and larger ruffles or lamellipodia, and more filopodia. These findings are similar to results reported by Polliack [19] comparing activated and control murine macrophages from the peritoneal cavity. In contrast, War-r & Martin [23] reported that human E.rp Cell
Res 109 (1977)
alveolar macrophages from smokers had few undulations on the surface and were smooth to cobblestone-like in appearance while non-smokers’ cells had highly undulating membranes. Variation in results may be due to differences in technique. The time of fixation after retrieval and adherence is particularly important. Also, there may be considerable heterogeneity in the population of macrophages. In man, alveolar macrophages may maintain their numbers by migration of precursor cells from the bone marrow and by in situ proliferation [ 10, 11, 231. The length of exposure in the lungs and the stage in the cells’ life span can result in functional and metabolic differences leading to variation in surface morphology. The substrate to which the cell adheres produces cell-surface alterations that may not stimulate the in vivo condition [4]1 For Fig. 7. Macrophages from a non-smoker cultured for 24 h. Cells are generally spherical and show prominent filopodia attachments. The cytoplasm is not well spread and cell attachment is by lamellipodia and filopodia. x 1200. Fig. 8. Non-smoker macrophages cultured for 24 h are attached to the substrate with both lamellipodia and filopodia as seen at 75” tilt. The surface is covered with blebs, folds, and few microvilli. The difference in morphology between smoker and non-smoker macrophages is most clearly seen at this angle. x 1700. Fig. 9. Smoker macrophages in culture for 72 h. Cells are elongated with smooth cytoplasm at the ends. There are few blebs and folds. x 1000. Fig. 10. Macrophages from a non-smoker cultured for 72 h. Cells have maintained their spherical morphology but have become more well spread. Cells show lamellipodia, blebs, and microvilli with many filopodia attachments between cells. x880. Fig. 11. 72-h culture of smoker macrophage with 10 pg/ml of iron. Cells are very different in morphology when compared with control macrophages (fig. 9). There are many blebs, undulations, tilopodia attachments between cells, and few microvilli. x 1300. Fig. 12. Smoker macrophage cultured with 10 pg/ml carbon for 72 h. Cells are spheroid with a ruffled cytoplasmic edge. Portions of the cytoplasm are very smooth and very adherent to the substrate. Surfaces have lamellipodia, few blebs and microvilli, and some filopodia. x690.
76
Quan and Golde
example, macrophages on glass will spread whereas cells adhered to Millipore filters tend to have the same morphology as cells on the alveolar wall [ 141. Changes in the membrane conformation of alveolar macrophages can reflect a corresponding change in its environment. The number of lamellipodia, lilopodia, and the number of microvilli and blebs decreased with increasing culture time. When alveolar macrophages from smokers and nonsmokers were compared there was more extensive ruffling and spreading of the cytoplasm and lilopodia in the smoker macrophages when observed at 10 min, 1 h, and 24 h. After 72 h in culture, macrophages from cigarette smokers had a very different morphology than cells examined at earlier time intervals. Cells were markedly elongated, had very few convolutions and blebs, and a very flat cytoplasm at the polar ends. Nonsmoker macrophages were still not as adherent and resembled smoker macrophages cultured for 24 h. Therefore the time interval after isolation is a critical variable in the interpretation of macrophage surface morphology. The morphology of the macrophages was not static but responded rapidly to changes in environment. Cells fed carbon and iron became more rounded, and the number of membrane undulations, blebs, filopodia and lamellipodia increased at least tenfold. A similar effect has been reported with animal macrophages treated with asbestos and silica [13, 161. The concomitant increase in these structures and the decrease in the total surface area of the cells are related to cell spreading and concentration. With the increased number of microvilli, blebs, filopodia and lamellipodia, the cell may rapidly change form without the need for synthesis of new cell membrane [8]. It is possible that E.rj,
Cd
Rrs
109 (/977)
there is movement of the membrane toward the cell margin [7] rather than away from it [ 1, 121 which could explain the increased adherence. There is other evidence for the storage of membrane in microvilli, blebs, filopodia and lamellipodia [8, 9, 201. For example, cell surface measurements are approximately the same when the cell is dividing and completely covered with microvilli, and at interphase when there are relatively few microvilli. Active movement and configurational changes occur during cell mitosis but are relatively restricted at interphase. The same types of morphological changes have been observed in our study of human alveolar macrophages where cell movement is associated with an increase in surface structures. Scanning electron microscopy provides a useful tool in the study of the surface morphology of human alveolar macrophages. Changes in the conformation of the cell membrane are a reflection of the response of the cell to its environment. Our studies show clear morphological differences between macrophages from smokers and nonsmokers and suggest an important relationship between function and morphology. The authors wish to thank Mr Gerald E. Garner of Scannine Electron Analvsis Laboratories, Inc., for supurb t&hnical assistance. This study was supported by USPHS Grants RR 00865 and HL 20675, and a grant from Brown and Williamson Tobacco Corp., Philip Morris Inc., R. J. Reynolds Tobacco Co., United States Tobacco Co., and Tobacco Associates, Inc.
REFERENCES 1. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 62 (1970) 389. 2. Albrecht, R M, Hinsdill, R D, Sandok, P L, Mackenzie, A P & Sachs, I B, Exp cell res 70 (1972) 230. 3. Carr, I, Clarke, J A & Salsbury, A J, J microsc 89 (1969) 105. 4. Cart-, K & Carr, I, Z Zellforsch 105 (1970) 234. 5. Cohen, A B & Cline, M J, J clin invest 50 (1971) 1390.
Macrophage surface morphology 6.
Cohen, A L, Marlow, D P & Garner, G E, J microsc 7 (1%8) 33 1. De Petris, S & Raff, M C, Nature 241 (1973) 257. Erickson. C A & Trinkaus. J P. Exp cell res 99 (1976) 375. Follett. E A C & Goldman. R D. Exo cell res 59 (1970) i24. Golde, D W, Byers, L A & Finley, T N, Nature 247 ( 1974) 373. Golde, D W, Finley, T N & Cline, M J, N Eng j med 290 (1974) 875. Harris, A & Dunn, G, Exp cell res 73 (1972) 519. Hope, I & Friend, J W, Micron 1 (1969) 3 10. Kuln, C & Finke, E H, J ultrastruct res 38 (1972) 161. Leake, E S, Wright, M J & Myrvik, Q N, J reticuloendothel sot 17 (1975) 370. Miller, K & Kagan, E, J reticuloendothel sot 20 (1976) 159. Papadimitriou, J M, Finley-Jones, J-M & Walters, M N-I, Exp cell res 76 (1973) 353.
18.
Z:
19. 20.
9.
21.
10. 11. 12. 13. 14. 15. 16. 17.
22. 23. 24. 25.
77
Parakkal, P, Pinto, J & Hanifin, J M, J ultrastruct res 48 (1974) 216. Polliack, A & Gordon, S, Lab invest 33 (1975) 469. Porter, K R, Prescott, D & Frve, J, J cell biol 57 (1973) 815. Porter, K R, Kelley, D & Andrews, PM, Proc fifth ann stereoscan ~011. Kent Cambridge Scientific, Morton Grove, Ill (1972). Pratt, S A, Smith M H, Ladman, A J & Finley, T N, Lab invest 24 (1971) 331. Thomas, E D, Ramberg, R W, Sale, G E, Sparkes, R S & Golde, D W, Science 192 (1976) 1016. Warfel, A H & Elberg, S S, Science 170 (1970) 446. Warr, G A & Martin, R R, Life sci 18 (1976) 1177.
Keceived January 18, 1977 Revised version received April 11, 1977 Accepted April 28, 1977
Exp Cd
Re.\ 109 (1977)