The Effect of Different Mineral Dusts on the Mechanism of Phagocytosis: A Scanning Electron Microscope Study KLARA
MILLER,
ROBERT I. M. HANDFIELD,
AND
ELLIOTT
KAC.AS’
Alveolar macrophages from normal rats were incubated with crocidolite asbestos fibers and quartz particles i/t r‘irrc~. The early events in phagocytosis were studied sequentially. Differences were found in the way in which macrophages recognized. attached. and phagocytosed the two dusts. Phagocytosis of quartz was extremely rapid: The macrophages typically developed long tenuous filopodia. Crocidolite was phagocytosed more slowly: Many macrophages developed large flattened pseudopodia. These different phagocytic mechanisms may reflect the different biochemical and cytotoxic properties of the dusts.
INTRODUCTION
Earlier scanning electron microscopy studies have illustrated the in viva effects of inhalation of quartz and crocidolite asbestos fibers on the surface morphology of rat alveolar macrophages (Miller and Kagan, 1976, 1977). Macrophages, after ingestion of asbestos, showed an increase in IgG receptor sites and an enhanced ability to spread across a glass substrate, and more extensive cytoplasmic processes were noted than in resident alveolar macrophages. But after ingestion of quartz, macrophages showed pronounced intracytoplasmic vacuolation, and many cells assumed bizarre shapes and exhibited a deterioration of the plasma membrane. The actual process relating to phagocytosis of these mineral dusts has not, however, been documented. It was thought important to observe how resident alveolar macrophages from normal rats recognized and phagocytosed quartz particles and crocidolite fibers in r’itt~~. Recognition by phagocytes may in many cases be due to physicochemical affinities between the cell and the recognized material (Wilkinson, 1976), and the ingestibility of particles can be influenced by variations in their net surface charges or their hydrophobic properties (Stossel et trl., 1972). Quartz (SiO,) has a crystalline tetrahedron structure and silicic acid is liberated when quartz is immersed in fluids. Crocidolite is a fibrous iron silicate and belongs to the amphibole group of asbestos minerals. In a recent scanning electron microscope study of the ingestion of microorganisms by peritoneal macrophages, different surface changes were observed during phagocytosis of Bacillus c’et~~~(sand Stmphylococclrs ~UYPUS (Walters et ul., 1976). This was considered due to both the morphology and the size of the microorganisms. The behavior of alveolar macrophages as regards attachment and ’ Present Washington,
address: American D.C. 20006.
National
Red
Cross.
Blood
Program,
National
Headquarters.
139 OOl3-9351/78/01.51-0139$02.00/O Copyright All
rights
cs of
1978 reproduction
by
Academic in any
Prws.
Inc.
form
reserved
140
hIII,LER,
HANDFIELD,
AND
KAGAN
phagocytosis of quartz and crocidolite might therefore also differ in a somewhat similar fashion. If different mineral dusts initiate different mechanisms of phagocytosis, this may have some bearing on their biochemical effect on tl macrophage and thus on the pathogenesis of dust diseases. The ability of macrophages to adhere to glass surface presents a reliable modr for studying the early events in phagocytosis and for obtaining sequential data Alveolar macrophages were therefore incubated with quartz particles or crocidolite fibers after their attachment to glass. S. N[I~~~IS (which is roughly similar in size to quartz particles) and glass fibers of similar configuration to crocidolite asbestos were also used in parallel experiments. Glass cover slips were taken for scanning electron microscopy at 2-minute intervals for 30 minutes and the various processes of phagocytosis were observed. MATERIALS
AND METHODS
Crocidolite asbestos. Two samples of crocidolite fibers were used, one consisting of fibers 20 pm long or more, and the other, 5 pm long or less. The fiber lengths were obtained by sedimentation. Qutrrtz. Quartz particles were obtained from Dowson & Dobson, Ltd. (l-3 pm in diameter). Glcrss fibers. The glass fibers were produced by milling Whatman GF/A glass fiber filter paper in a Glen Creston mill for 2 minutes. The fiber lengths varied from 5 to 100 ,u.m, and the diameter, from 0.5 to 2 ym. Stcrphylococcrrs ourem (I-pm diameter). An inoculum of S. ourells was cultured in brain/heart infusion agar (DIFCO) overnight at 37°C and the bacterial suspension was collected. Cell collection. Adult male outbred rats, Wistar strain, were used for all experiments. Animals were killed with an intraperitoneal overdose of sodium pentobarbital. A plastic cannula was inserted in the trachea, the rib cage was transected, and the alveolar macrophage population was recovered from the lungs by repeated washings with Ca?+- and Mg’+- free minimum essential medium (Joklik, Grand Island Biological Co., Grand Island, N.Y.) prewarmed to 37°C. Cell culture. The alveolar macrophage suspensions were centrifuged at 2008 for 20 minutes at ambient temperature, washed twice, and resuspended in Hepesbuffered RPMI-1640 (Grand Island Biological Co., Grand Island, N.Y.) supplemented with fresh L-glutamine (0.3 mgiml), gentamicin (IO/-&ml), streptomycin (lOpg/ml), and penicillin (200 U/ml). The cell concentration was adjusted to 1 x lob/ml, and l-ml aliquots were applied to circular sterile glass cover slips (12 mm in diameter). The cover slips were placed in disposable trays (Bellco Glass Inc., Vinelands, N.J.) and the cultures were incubated in a standard tissue culture incubator. Cell viability as evaluated by Trypan blue dye exclusion was never less than 95%. The cells were allowed to adhere to the glass substrate for 45 minutes at 37°C. They were then washed vigorously with RPMI-1640 and cultured for one hour in RPMI-1640 containing 10% fetal calf serum (GIBCO). The macrophages were washed again vigorously to remove all traces of serum and were incubated with one of the dusts (100 pg/ml of RPMI). Samples were taken at 2-minute intervals during 30 minutes for scanning electron microscopy.
PHAGOCYTOSIS
FIG. I. Scanning electron mlcrograph showing rophage 4 minutes after addition of fibers. .x4000.
OF MINERAL
phagocytosis
141
DUSTS
of crocidolite
fibers
by alveolar
mac-
Scrrttnittg elrctmn tnicroscop~. Cover slips were removed with forceps, rinsed in 280 mOsmo1 phosphate buffer (pH 7.3, fixed in 1% glutaraldehyde in buffer for 30 minutes at room temperature, and then fixed in 2% glutaraldehyde at 4°C. After 24-hour samples were postfixed in 1% osmium tetroxide for 1 hour and then taken through graded solutions of aIcoho1 and amyI acetate. The specimens were subsequently dried by the critical point method, coated with carbon and gold, and examined in a JEOL JSM-US.
142
FIG. 2. Detail brane processes.
MILLER,
of Fig. 1 demonstrating ~9000.
HANDFIELD,
how
some fibers
AND
have
KAGAN
been enveloped
by macrophage
mem-
RESULTS
Both short and long crocidolite fibers were attached firmly to the macrophages after 4 minutes. In experiments where short fibers (5 pm) were used. the attachment occurred without any extensive spreading of pseudopodia or development of thin filopodia. The fibers appeared to be firmly attached to the still ruffled surface of the macrophage and were held in place by numerous cytoplasmic processes (Fig. 1). It was evident that some degree of phagocytosis had already taken place and, in some areas, portions of fibers were surrounded completely by membrane cytoplasm (Fig. 2). The macrophages were able to attach to and phagocytose a number of fibers simultaneously, and active phagocytosis of one fiber did not
PHAGOCI’TOSIS
OF
MINERAL
DUSTS
143
FIG. 3. Scanning electron micrograph demonstrating attachment of long crocidolite fiber (220 pm) by alveolar macrophage 4 minutes after addition of fibers. x2250.
prevent further attachment to other fibers. After 20 minutes, very few macrophages were found with attached crocidolite fibers and many of them exhibited a smooth surface membrane suggestive of a replete cell (Walters et at., 1976). In the case of long crocidolite fibers (20 Frn or longer) the processes of attachment and phagocytosis were somewhat different. Many macrophages developed large flattened pseudopodia with an elevated central mass and the fiber was generally attached to the flattened pseudopodia by small cytoplasmic tendrils (Fig. 3). The flattened pseudopodia often spread behind the central mass of the macrophage, possibly providing an anchoring foothold for stability. The fibers were attached along their long axes and eventually phagocytosed by the macrophage
144
h’lILLER.
HANDFIELD,
AND
KAGAh’
FIG. 4. Scanning electron micrograph demonstrating phagocytosis of long crocidolite pm) by alveolar macrophage 10 minutes after addition of fibers. x2250.
fiber (220
end first (Fig. 4). It would appear that a long fiber may be engulfed more easily over a small area than over an otherwise extensive surface. Long fibers in the process of being phagocytosed were often seen coated with cytoplasmic or cell debris. Scanning electron micrographs of crocidolite fibers prior to their addition to macrophage cultures showed no evidence of surface coating. The general appearance was that of smooth regular fibers. Thus the surface coating observed after their addition to the biological system may possibly be a result of previously unsuccessful phagocytosis by other macrophages which have died while trying to engulf these fibers (Fig. 5). Phagocytosis of one or more fibers did not prevent the cell from further active phagocytosis (Fig. 6).
Flc:. 5. Scanning electron micrograph demonstrating phagocytose a crocidolite fiber IO minutes after addition fiber. x 1440.
several alveolar of fibers. Note
macrophages attempting to dead macrophage at end of
Several cells concurrently attached to and attempted to phacytose very long fibers. In one instance the fiber was so long (50 pm) that it was physically incapable of being incorporated into a macrophage. However, each macrophage had enveloped part of its cell membrane around the fiber in a sleeve-like fashion (Fig. 7). The fine structure of the fiber was evident through the cell membrane under higher magnification. After 30 minutes of incubation, long fibers were still present in the culture system and macrophages were attempting to phagocytose them. The glass fibers used in these experiments were both thicker and shorter than the crocidolite fibers. but the processes of attachment and phagocytosis seemed quite similar. The smaller fibers were attached to the ruffled surface of the mac-
146
MILLER,
FIG. 6. Scanning electron micrograph lar macrophage 16 minutes after addition x 1800.
HANDFIELD,
AND
KAGAIi
demonstrating phagocytosis of long crocidolite fiber by alveoof fibers. Note presence of short fiber inside the macrophage.
rophages by short cytoplasmic projections, and the cells exhibited few pseudopodia (Fig. 8). Where longer fibers were present cytoplasmic projections from the macrophages had spread on the glass substrate. As with crocidolite fibers, attachment of one fiber did not prevent the macrophage from attaching many other fibers to its surface, and the processes of attachment and phagocytosis could take place simultaneously (Fig. 9). Fibers were generally phagocytosed end first with a lip or sleeve of cytoplasm
PHAGOCTTOSIS
OF
MINERAL
DCSTS
147
FIG. 7. Scanning electron micrograph demonstrating two macrophages concurrently attached to a very long crocidolite fiber (SO pm) 30 minutes after addition of fibers. x720.
firmly around the fiber (Fig. 10). After 20 minutes of incubation, only very occasional long fibers were found attached to the macrophages. A very different situation occurred when quartz particles were added to the macrophage cultures. The particles were phagocytosed extremely rapidly, within 2 to 4 minutes. After 4 minutes, only an occasional large quartz particle was found in the process of being engulfed by the macrophage. The macrophages responded to the presence of quartz particles with extensive formation of long tenuous filopodia. These filopodia appeared to be membranous extensions of the marginal cytoplasm or pseudopodia of the cell (Fig. 11). They
148
MILLER,
FIG. 8. Scanning electron micrograph after addition of fibers. x 5400.
HANDFIELD,
AND
K4GAN
demonstrating
glass fiber
attached
by macrophage
4 minutes
were longer and more numerous than any observed with any of the other objects phagocytosed in this model system. The quartz particles seemed to be attached mainly to the filopodia, and ingested particles appeared to lie in ridges raised between these extensions (Fig. 12). Very rarely were any particles found lying on the central mass of the macrophages. and both attachment and phagocytosis appeared to take place around the marginal cytoplasm of the cell. Attachment and phagocytosis of S. oweus took place in a similar way. As the microorganisms were alive, many of them were possibly undergoing meiosis when
PHAGOCYTOSIS
OF
MINERAL
DUSTS
149
FIG. 9. Scanning electron micrograph demonstrating macrophage with glass fibers 16 minutes after addition of fibers. Note simultaneous process of both attachment and phagocytosis. x3600.
added to the macrophage culture and were thus larger than 1 pm (R. Sher, unpublished data). S. aureus bacteria was also phagocytosed extremely rapidly and none were found after 4 minutes. Formation of filopodia was common, but never to the same extent as with the quartz experiments, and the development of many short filopodia, particularly during phagocytosis, was often observed (Fig. 13). Attachment took place at the edge of the cell by a thin layer of protoplasm partially covering the microorganism. During phagocytosis, as they sank deeper into the macrophage, a lip of cytoplasm began to surround the bacteria.
150
FIG. IO. Scanning electron lar macrophage. x 16,200.
MILLER,
micrograph
HANDFIELD,
demonstrating
AND
detail
KAGAN
of phagocytosis
of glass fiber
by alveo-
DISCUSSION
Macrophages interact with a variety of materials in the absence of serum factors, thus probably reflecting a nonimmunologic recognition (Rabinovitch, 1970). They also possess membrane factors which mediate this nonspecific binding and ingestion (Griffin et al., 1975). It is well known that alveolar macrophages quickly recognize, attach to, and phagocytose foreign inorganic particles when these are added to an in ivitro culture system. The phagocytic behavior as observed by scanning electron microscopy has not, however, been reported. Resident alveolar macrophages are usually rounded, with blunt surface ruffles
PHAGOCYTOSIS
FIG. I I. macrophages
Scanning of quartz
electron particles
micrograph 2 minutes
OF
demonstrating after
addition
MINERAL
151
DPSTS
attachment of particles.
and Note
phagocytosis extended
filopodia.
by
alveolar x 1800.
and few pseudopodia (Leake et al., 1975; Miller and Kagan 1976). Five minutes after the addition of crocidolite fibers to the culture system, characteristic morphological changes have occurred. The macrophages have developed large flattened pseudopodia or finger-like processes, possibly in response to ingestible particles (Griffin et al., 1975). However, the macrophages react in this manner only in the presence of crocidolite and glass fibers, and thus the shape and size of these fibers may determine the morphologic events of phagocytosis (Stossel. 1976). When quartz particles are added to alveolar macrophage cultures recognition is
152
MILLER,
HAKDFIELD,
AND
KAGAN
FIG. 12. Scanning electron micrograph demonstrating phagocytosis of quartz 4 minutes after addition of particles. Note phagocytosed particles near filopodia. x5400.
almost instantaneous and phagocytosis is extremely rapid. Whether this is due to the size and shape of the quartz or to its physicochemical properties is not known. The long lilopodia that develop may indicate cytoplasmic and surface motion and could be a chemotactic response (Walters et al.. 1976). Thus, differences have been found in the mechanism of phagocytosis when quartz or crocidolite dusts have been added to alveolar macrophage cultures. It is not known whether these differences are due simply to the size and shape of the minerals or to different physicochemical interactions. These two mineral dusts have quite different biological effects on the macrophage and it is interesting to note that quartz, acting as a semiconductor, has been said to exert an effect on the
PHAGOCYTOSIS
FIG. 13. Scanning electron micrograph minutes after addition of bacteria. Inset
OF
MINERAL
DUSTS
153
demonstrating phagocytosis of S. UI,YPIIS by macrophage 4 shows thin layer of protoplasm attaching bacteria. x4000.
macrophage before phagocytosis takes place (Klosterkdtter and Robock, 1975). While this in vitro study does not explain the dissimilar nature and distribution of the pathological lesions in silicosis and asbestosis, it does demonstrate that these dusts interact in a different fashion with alveolar macrophage membranes. The early events of macrophage recognition and attachment to crocidolite asbestos and quartz may thus have some bearing on the role of the macrophage in the pathogenesis of dust diseases.
154
MILLER,
HANDFIELD.
ASD
KAGAN
ACKNOWLEDGMENTS We thank Professor I. Webster for his constant advice and encouragement, and Mr. R.E.G. Rendall for providing the dusts used in this study. We also thank Mrs. M. Hengstberger and the Physical Research Unit of the Council for Scientific and Industrial Research. Pretoria. for the scanning electron facilities made available to us. The work was carried out by one of us (K.M.) in partial fulfillment of the requirements for a Ph.D. degree of the University of the Witwatersrand. Johannesburg.
REFERENCES Griffin,
F. M., Bianco. C., and Silverstein, S. C. (1975). Characterization of the macrophage receptor for complement and demonstration of its functional independence from the receptor for the Fc portion of immunoglobulin G. J. Exp. Med. 141, 1269- 1277. Griffin. F. M., Griffin. J. A., Leider. J. E.. and Silverstein, S. C. (1975). Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane. J. Et-p. Med. 142, l263- 1282. Klosterkotter, W., and Robock, K. (1975). New aspects on dust and pneumoconiosis research. Amo. Ied. Hyg. Ass. J. 39, 659-668. Leake, E. S.. Wright, M. J., and Myrvik, Q. N. (1975). Differences in surface morphology of alveolar macrophages attached to glass and to Millipore filters: A scanning electron microscope study. .I. R~ti~rrloer~tk~thel. Sot. 17. 370-379. Miller. K., and Kagan. E. (1976). The ill i,ii~j effects of asbestos on macrophage membrane structure and population characteristics of macrophages: A scanning electron microscope study. J. Rrtic.rr/or,ldot/lr,/. SW. 20, 159- 172. Miller, K., and Kagan, E. (1977). The iri vh’o effects of quartz on alveolar macrophage membrane topography and on the characterstics of the intrapulmonary cell population. J. Rrticu/orrfdotk~,/. SOCK. 21. 307-315. Rabinovitch, M. t 1970). Phagocytic recognition. Irr “Mononuclear Phagocytes” (R. van Furth. Ed.). pp. 2999315. Blackwell Scientific. Oxford. Stossel, T. P. ( 1976). The mechanism of phagocytosis. J. Rrtic,ll/(,rndothr/. Sot. 19, 237-245. Stossel. T. P., Mason, R. J.. Hartwig. J.. and Vaughan, M. (1972). Quantitative studies of phagocytosis by polymorphonuclear leukocytes: Use of emulsions to measure the initial rate of phagocytosis. ./. C/i/r. I~rrrsf. 51, 615-624. Walters, M. N-I., Papadimitriou, J. M.. and Robertson. T. A. (1976). The surface morphology of the phagocytosis of micro-organisms by peritoneal macrophages. J. Pot/lo/. 118, X1-226. Wilkinson, P. C. ( 1976). Recognition and response in mononuclear and granular phagocytes. C/i,r. E.rp. It,~mr,nol. 25, 355-366.
MILLER,
ROBERT I. M. HANDFIELD,
AND
ELLIOTT
KAC.AS’
Alveolar macrophages from normal rats were incubated with crocidolite asbestos fibers and quartz particles i/t r‘irrc~. The early events in phagocytosis were studied sequentially. Differences were found in the way in which macrophages recognized. attached. and phagocytosed the two dusts. Phagocytosis of quartz was extremely rapid: The macrophages typically developed long tenuous filopodia. Crocidolite was phagocytosed more slowly: Many macrophages developed large flattened pseudopodia. These different phagocytic mechanisms may reflect the different biochemical and cytotoxic properties of the dusts.
INTRODUCTION
Earlier scanning electron microscopy studies have illustrated the in viva effects of inhalation of quartz and crocidolite asbestos fibers on the surface morphology of rat alveolar macrophages (Miller and Kagan, 1976, 1977). Macrophages, after ingestion of asbestos, showed an increase in IgG receptor sites and an enhanced ability to spread across a glass substrate, and more extensive cytoplasmic processes were noted than in resident alveolar macrophages. But after ingestion of quartz, macrophages showed pronounced intracytoplasmic vacuolation, and many cells assumed bizarre shapes and exhibited a deterioration of the plasma membrane. The actual process relating to phagocytosis of these mineral dusts has not, however, been documented. It was thought important to observe how resident alveolar macrophages from normal rats recognized and phagocytosed quartz particles and crocidolite fibers in r’itt~~. Recognition by phagocytes may in many cases be due to physicochemical affinities between the cell and the recognized material (Wilkinson, 1976), and the ingestibility of particles can be influenced by variations in their net surface charges or their hydrophobic properties (Stossel et trl., 1972). Quartz (SiO,) has a crystalline tetrahedron structure and silicic acid is liberated when quartz is immersed in fluids. Crocidolite is a fibrous iron silicate and belongs to the amphibole group of asbestos minerals. In a recent scanning electron microscope study of the ingestion of microorganisms by peritoneal macrophages, different surface changes were observed during phagocytosis of Bacillus c’et~~~(sand Stmphylococclrs ~UYPUS (Walters et ul., 1976). This was considered due to both the morphology and the size of the microorganisms. The behavior of alveolar macrophages as regards attachment and ’ Present Washington,
address: American D.C. 20006.
National
Red
Cross.
Blood
Program,
National
Headquarters.
139 OOl3-9351/78/01.51-0139$02.00/O Copyright All
rights
cs of
1978 reproduction
by
Academic in any
Prws.
Inc.
form
reserved
140
hIII,LER,
HANDFIELD,
AND
KAGAN
phagocytosis of quartz and crocidolite might therefore also differ in a somewhat similar fashion. If different mineral dusts initiate different mechanisms of phagocytosis, this may have some bearing on their biochemical effect on tl macrophage and thus on the pathogenesis of dust diseases. The ability of macrophages to adhere to glass surface presents a reliable modr for studying the early events in phagocytosis and for obtaining sequential data Alveolar macrophages were therefore incubated with quartz particles or crocidolite fibers after their attachment to glass. S. N[I~~~IS (which is roughly similar in size to quartz particles) and glass fibers of similar configuration to crocidolite asbestos were also used in parallel experiments. Glass cover slips were taken for scanning electron microscopy at 2-minute intervals for 30 minutes and the various processes of phagocytosis were observed. MATERIALS
AND METHODS
Crocidolite asbestos. Two samples of crocidolite fibers were used, one consisting of fibers 20 pm long or more, and the other, 5 pm long or less. The fiber lengths were obtained by sedimentation. Qutrrtz. Quartz particles were obtained from Dowson & Dobson, Ltd. (l-3 pm in diameter). Glcrss fibers. The glass fibers were produced by milling Whatman GF/A glass fiber filter paper in a Glen Creston mill for 2 minutes. The fiber lengths varied from 5 to 100 ,u.m, and the diameter, from 0.5 to 2 ym. Stcrphylococcrrs ourem (I-pm diameter). An inoculum of S. ourells was cultured in brain/heart infusion agar (DIFCO) overnight at 37°C and the bacterial suspension was collected. Cell collection. Adult male outbred rats, Wistar strain, were used for all experiments. Animals were killed with an intraperitoneal overdose of sodium pentobarbital. A plastic cannula was inserted in the trachea, the rib cage was transected, and the alveolar macrophage population was recovered from the lungs by repeated washings with Ca?+- and Mg’+- free minimum essential medium (Joklik, Grand Island Biological Co., Grand Island, N.Y.) prewarmed to 37°C. Cell culture. The alveolar macrophage suspensions were centrifuged at 2008 for 20 minutes at ambient temperature, washed twice, and resuspended in Hepesbuffered RPMI-1640 (Grand Island Biological Co., Grand Island, N.Y.) supplemented with fresh L-glutamine (0.3 mgiml), gentamicin (IO/-&ml), streptomycin (lOpg/ml), and penicillin (200 U/ml). The cell concentration was adjusted to 1 x lob/ml, and l-ml aliquots were applied to circular sterile glass cover slips (12 mm in diameter). The cover slips were placed in disposable trays (Bellco Glass Inc., Vinelands, N.J.) and the cultures were incubated in a standard tissue culture incubator. Cell viability as evaluated by Trypan blue dye exclusion was never less than 95%. The cells were allowed to adhere to the glass substrate for 45 minutes at 37°C. They were then washed vigorously with RPMI-1640 and cultured for one hour in RPMI-1640 containing 10% fetal calf serum (GIBCO). The macrophages were washed again vigorously to remove all traces of serum and were incubated with one of the dusts (100 pg/ml of RPMI). Samples were taken at 2-minute intervals during 30 minutes for scanning electron microscopy.
PHAGOCYTOSIS
FIG. I. Scanning electron mlcrograph showing rophage 4 minutes after addition of fibers. .x4000.
OF MINERAL
phagocytosis
141
DUSTS
of crocidolite
fibers
by alveolar
mac-
Scrrttnittg elrctmn tnicroscop~. Cover slips were removed with forceps, rinsed in 280 mOsmo1 phosphate buffer (pH 7.3, fixed in 1% glutaraldehyde in buffer for 30 minutes at room temperature, and then fixed in 2% glutaraldehyde at 4°C. After 24-hour samples were postfixed in 1% osmium tetroxide for 1 hour and then taken through graded solutions of aIcoho1 and amyI acetate. The specimens were subsequently dried by the critical point method, coated with carbon and gold, and examined in a JEOL JSM-US.
142
FIG. 2. Detail brane processes.
MILLER,
of Fig. 1 demonstrating ~9000.
HANDFIELD,
how
some fibers
AND
have
KAGAN
been enveloped
by macrophage
mem-
RESULTS
Both short and long crocidolite fibers were attached firmly to the macrophages after 4 minutes. In experiments where short fibers (5 pm) were used. the attachment occurred without any extensive spreading of pseudopodia or development of thin filopodia. The fibers appeared to be firmly attached to the still ruffled surface of the macrophage and were held in place by numerous cytoplasmic processes (Fig. 1). It was evident that some degree of phagocytosis had already taken place and, in some areas, portions of fibers were surrounded completely by membrane cytoplasm (Fig. 2). The macrophages were able to attach to and phagocytose a number of fibers simultaneously, and active phagocytosis of one fiber did not
PHAGOCI’TOSIS
OF
MINERAL
DUSTS
143
FIG. 3. Scanning electron micrograph demonstrating attachment of long crocidolite fiber (220 pm) by alveolar macrophage 4 minutes after addition of fibers. x2250.
prevent further attachment to other fibers. After 20 minutes, very few macrophages were found with attached crocidolite fibers and many of them exhibited a smooth surface membrane suggestive of a replete cell (Walters et at., 1976). In the case of long crocidolite fibers (20 Frn or longer) the processes of attachment and phagocytosis were somewhat different. Many macrophages developed large flattened pseudopodia with an elevated central mass and the fiber was generally attached to the flattened pseudopodia by small cytoplasmic tendrils (Fig. 3). The flattened pseudopodia often spread behind the central mass of the macrophage, possibly providing an anchoring foothold for stability. The fibers were attached along their long axes and eventually phagocytosed by the macrophage
144
h’lILLER.
HANDFIELD,
AND
KAGAh’
FIG. 4. Scanning electron micrograph demonstrating phagocytosis of long crocidolite pm) by alveolar macrophage 10 minutes after addition of fibers. x2250.
fiber (220
end first (Fig. 4). It would appear that a long fiber may be engulfed more easily over a small area than over an otherwise extensive surface. Long fibers in the process of being phagocytosed were often seen coated with cytoplasmic or cell debris. Scanning electron micrographs of crocidolite fibers prior to their addition to macrophage cultures showed no evidence of surface coating. The general appearance was that of smooth regular fibers. Thus the surface coating observed after their addition to the biological system may possibly be a result of previously unsuccessful phagocytosis by other macrophages which have died while trying to engulf these fibers (Fig. 5). Phagocytosis of one or more fibers did not prevent the cell from further active phagocytosis (Fig. 6).
Flc:. 5. Scanning electron micrograph demonstrating phagocytose a crocidolite fiber IO minutes after addition fiber. x 1440.
several alveolar of fibers. Note
macrophages attempting to dead macrophage at end of
Several cells concurrently attached to and attempted to phacytose very long fibers. In one instance the fiber was so long (50 pm) that it was physically incapable of being incorporated into a macrophage. However, each macrophage had enveloped part of its cell membrane around the fiber in a sleeve-like fashion (Fig. 7). The fine structure of the fiber was evident through the cell membrane under higher magnification. After 30 minutes of incubation, long fibers were still present in the culture system and macrophages were attempting to phagocytose them. The glass fibers used in these experiments were both thicker and shorter than the crocidolite fibers. but the processes of attachment and phagocytosis seemed quite similar. The smaller fibers were attached to the ruffled surface of the mac-
146
MILLER,
FIG. 6. Scanning electron micrograph lar macrophage 16 minutes after addition x 1800.
HANDFIELD,
AND
KAGAIi
demonstrating phagocytosis of long crocidolite fiber by alveoof fibers. Note presence of short fiber inside the macrophage.
rophages by short cytoplasmic projections, and the cells exhibited few pseudopodia (Fig. 8). Where longer fibers were present cytoplasmic projections from the macrophages had spread on the glass substrate. As with crocidolite fibers, attachment of one fiber did not prevent the macrophage from attaching many other fibers to its surface, and the processes of attachment and phagocytosis could take place simultaneously (Fig. 9). Fibers were generally phagocytosed end first with a lip or sleeve of cytoplasm
PHAGOCTTOSIS
OF
MINERAL
DCSTS
147
FIG. 7. Scanning electron micrograph demonstrating two macrophages concurrently attached to a very long crocidolite fiber (SO pm) 30 minutes after addition of fibers. x720.
firmly around the fiber (Fig. 10). After 20 minutes of incubation, only very occasional long fibers were found attached to the macrophages. A very different situation occurred when quartz particles were added to the macrophage cultures. The particles were phagocytosed extremely rapidly, within 2 to 4 minutes. After 4 minutes, only an occasional large quartz particle was found in the process of being engulfed by the macrophage. The macrophages responded to the presence of quartz particles with extensive formation of long tenuous filopodia. These filopodia appeared to be membranous extensions of the marginal cytoplasm or pseudopodia of the cell (Fig. 11). They
148
MILLER,
FIG. 8. Scanning electron micrograph after addition of fibers. x 5400.
HANDFIELD,
AND
K4GAN
demonstrating
glass fiber
attached
by macrophage
4 minutes
were longer and more numerous than any observed with any of the other objects phagocytosed in this model system. The quartz particles seemed to be attached mainly to the filopodia, and ingested particles appeared to lie in ridges raised between these extensions (Fig. 12). Very rarely were any particles found lying on the central mass of the macrophages. and both attachment and phagocytosis appeared to take place around the marginal cytoplasm of the cell. Attachment and phagocytosis of S. oweus took place in a similar way. As the microorganisms were alive, many of them were possibly undergoing meiosis when
PHAGOCYTOSIS
OF
MINERAL
DUSTS
149
FIG. 9. Scanning electron micrograph demonstrating macrophage with glass fibers 16 minutes after addition of fibers. Note simultaneous process of both attachment and phagocytosis. x3600.
added to the macrophage culture and were thus larger than 1 pm (R. Sher, unpublished data). S. aureus bacteria was also phagocytosed extremely rapidly and none were found after 4 minutes. Formation of filopodia was common, but never to the same extent as with the quartz experiments, and the development of many short filopodia, particularly during phagocytosis, was often observed (Fig. 13). Attachment took place at the edge of the cell by a thin layer of protoplasm partially covering the microorganism. During phagocytosis, as they sank deeper into the macrophage, a lip of cytoplasm began to surround the bacteria.
150
FIG. IO. Scanning electron lar macrophage. x 16,200.
MILLER,
micrograph
HANDFIELD,
demonstrating
AND
detail
KAGAN
of phagocytosis
of glass fiber
by alveo-
DISCUSSION
Macrophages interact with a variety of materials in the absence of serum factors, thus probably reflecting a nonimmunologic recognition (Rabinovitch, 1970). They also possess membrane factors which mediate this nonspecific binding and ingestion (Griffin et al., 1975). It is well known that alveolar macrophages quickly recognize, attach to, and phagocytose foreign inorganic particles when these are added to an in ivitro culture system. The phagocytic behavior as observed by scanning electron microscopy has not, however, been reported. Resident alveolar macrophages are usually rounded, with blunt surface ruffles
PHAGOCYTOSIS
FIG. I I. macrophages
Scanning of quartz
electron particles
micrograph 2 minutes
OF
demonstrating after
addition
MINERAL
151
DPSTS
attachment of particles.
and Note
phagocytosis extended
filopodia.
by
alveolar x 1800.
and few pseudopodia (Leake et al., 1975; Miller and Kagan 1976). Five minutes after the addition of crocidolite fibers to the culture system, characteristic morphological changes have occurred. The macrophages have developed large flattened pseudopodia or finger-like processes, possibly in response to ingestible particles (Griffin et al., 1975). However, the macrophages react in this manner only in the presence of crocidolite and glass fibers, and thus the shape and size of these fibers may determine the morphologic events of phagocytosis (Stossel. 1976). When quartz particles are added to alveolar macrophage cultures recognition is
152
MILLER,
HAKDFIELD,
AND
KAGAN
FIG. 12. Scanning electron micrograph demonstrating phagocytosis of quartz 4 minutes after addition of particles. Note phagocytosed particles near filopodia. x5400.
almost instantaneous and phagocytosis is extremely rapid. Whether this is due to the size and shape of the quartz or to its physicochemical properties is not known. The long lilopodia that develop may indicate cytoplasmic and surface motion and could be a chemotactic response (Walters et al.. 1976). Thus, differences have been found in the mechanism of phagocytosis when quartz or crocidolite dusts have been added to alveolar macrophage cultures. It is not known whether these differences are due simply to the size and shape of the minerals or to different physicochemical interactions. These two mineral dusts have quite different biological effects on the macrophage and it is interesting to note that quartz, acting as a semiconductor, has been said to exert an effect on the
PHAGOCYTOSIS
FIG. 13. Scanning electron micrograph minutes after addition of bacteria. Inset
OF
MINERAL
DUSTS
153
demonstrating phagocytosis of S. UI,YPIIS by macrophage 4 shows thin layer of protoplasm attaching bacteria. x4000.
macrophage before phagocytosis takes place (Klosterkdtter and Robock, 1975). While this in vitro study does not explain the dissimilar nature and distribution of the pathological lesions in silicosis and asbestosis, it does demonstrate that these dusts interact in a different fashion with alveolar macrophage membranes. The early events of macrophage recognition and attachment to crocidolite asbestos and quartz may thus have some bearing on the role of the macrophage in the pathogenesis of dust diseases.
154
MILLER,
HANDFIELD.
ASD
KAGAN
ACKNOWLEDGMENTS We thank Professor I. Webster for his constant advice and encouragement, and Mr. R.E.G. Rendall for providing the dusts used in this study. We also thank Mrs. M. Hengstberger and the Physical Research Unit of the Council for Scientific and Industrial Research. Pretoria. for the scanning electron facilities made available to us. The work was carried out by one of us (K.M.) in partial fulfillment of the requirements for a Ph.D. degree of the University of the Witwatersrand. Johannesburg.
REFERENCES Griffin,
F. M., Bianco. C., and Silverstein, S. C. (1975). Characterization of the macrophage receptor for complement and demonstration of its functional independence from the receptor for the Fc portion of immunoglobulin G. J. Exp. Med. 141, 1269- 1277. Griffin. F. M., Griffin. J. A., Leider. J. E.. and Silverstein, S. C. (1975). Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane. J. Et-p. Med. 142, l263- 1282. Klosterkotter, W., and Robock, K. (1975). New aspects on dust and pneumoconiosis research. Amo. Ied. Hyg. Ass. J. 39, 659-668. Leake, E. S.. Wright, M. J., and Myrvik, Q. N. (1975). Differences in surface morphology of alveolar macrophages attached to glass and to Millipore filters: A scanning electron microscope study. .I. R~ti~rrloer~tk~thel. Sot. 17. 370-379. Miller. K., and Kagan. E. (1976). The ill i,ii~j effects of asbestos on macrophage membrane structure and population characteristics of macrophages: A scanning electron microscope study. J. Rrtic.rr/or,ldot/lr,/. SW. 20, 159- 172. Miller, K., and Kagan, E. (1977). The iri vh’o effects of quartz on alveolar macrophage membrane topography and on the characterstics of the intrapulmonary cell population. J. Rrticu/orrfdotk~,/. SOCK. 21. 307-315. Rabinovitch, M. t 1970). Phagocytic recognition. Irr “Mononuclear Phagocytes” (R. van Furth. Ed.). pp. 2999315. Blackwell Scientific. Oxford. Stossel, T. P. ( 1976). The mechanism of phagocytosis. J. Rrtic,ll/(,rndothr/. Sot. 19, 237-245. Stossel. T. P., Mason, R. J.. Hartwig. J.. and Vaughan, M. (1972). Quantitative studies of phagocytosis by polymorphonuclear leukocytes: Use of emulsions to measure the initial rate of phagocytosis. ./. C/i/r. I~rrrsf. 51, 615-624. Walters, M. N-I., Papadimitriou, J. M.. and Robertson. T. A. (1976). The surface morphology of the phagocytosis of micro-organisms by peritoneal macrophages. J. Pot/lo/. 118, X1-226. Wilkinson, P. C. ( 1976). Recognition and response in mononuclear and granular phagocytes. C/i,r. E.rp. It,~mr,nol. 25, 355-366.
