INFECTION AND IMMUNITY, June 1979, p. 932-938 0019-9567/79/06-0932/07$02.00/0
Vol. 24, No. 3
Phagocytosis and Intracellular Killing of Pathogenic Yeasts by Human Monocytes and Neutrophils KENNETH E. SCHUITt
Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 14 November 1978
The kinetics of phagocytosis and killing of four fungal forms with varying virulence by two types of phagocytic cells was examined. Human monocytes ingested Saccharomyces cerevisiae, Candida tropicalis, and the blastospores of Candida albicans more rapidly than did human neutrophils. There was no difference in the rate of phagocytosis of C. albicans pseudohyphae by these two cell types. Intracellular killing of each of the four fungal forms was consistently and significantly more rapid by monocytes than by neutrophils. Neutrophils were unable to destroy ingested C. albicans pseudohyphae. These experiments suggest that the monocyte plays an important role in host defenses against fungal diseases and that the relative virulence of the pathogenic yeasts in human disease may be related to the ability of these organisms to survive after being ingested by circulating phagocytes.
Leukocytes are important in host defenses against infection because they are able to ingest and kill microorganisms. Although all circulating phagocytes (neutrophils, monocytes and eosinophils) serve this function, the neutrophil appears to have an especially important role in the destruction of bacteria (5, 14, 16). The role of these cells in the defense against fungal infection is less clear, as neutrophils and monocytes may be unable to kill fungi (12), or may do so poorly (1, 2, 7). Although fungal diseases such as disseminated candidiasis are becoming increasingly common (6), several aspects of the pathogenesis of these infections are poorly understood. For example, what is the relative role of monocytes and neutrophils in the destruction of the pathogenic yeasts? Also, what is the importance in disease production of the blastospore and pseudohyphal forms of Candida albicans? The purpose of this investigation was to compare the ability of human neutrophils and monocytes to ingest and kill several species of fungi which represent a spectrum of virulence in human disease and to study the interaction of these leukocytes with the blastospore and pseudohyphal forms of C. albicans. MATERIALS AND METHODS Leukocyte preparation. Leukocyte-rich plasma was prepared from heparinized blood obtained from normal adult volunteers of both sexes by mixing whole t Present address: Children's Hospital of Pittsburgh, Pittsburgh, PA 15213.
blood in a 1:1 ratio with a 3% solution of gelatin (Plasmagel, Laboratoire Roger Bellon, Neuilly, France) and allowing it to settle for 1 h. The leukocyte suspension was diluted with an equal volume of buffered saline, layered onto Ficoll-Hypaque (2:1 ratio), and centrifuged for 40 min at 1,400 x g. The mononuclear cell layer was removed with a Pasteur pipette, and the neutrophils (which were pelleted along with the erythrocytes at the bottom of the centrifuge tube) were suspended after carefully decanting the liquid portions of the gradient. The erythrocytes in both preparations were lysed by suspending the cells in 8 ml of distilled water for 30 s after which physiological osmolarity was restored by the addition of 4 ml of 3.6% NaCl. The neutrophils and monocytes were washed an additional time with buffered saline, suspended in McCoy medium, and counted in a hemocytometer. Fungi. Strains of C. albicans, Candida tropicalis, and Saccharomyces cerevesiae were obtained from the Department of Hospital Laboratories, North Carolina Memorial Hospital, Chapel Hill. All strains were maintained on Trypticase soy agar slants and subcultured every 2 weeks. For the experiments, fungi were grown on Trypticase soy agar for 48 h at 37°C, gently scraped from the surface, and washed twice before counting in a hemocytometer. Cultures of C. albicans grown on agar in this manner consisted entirely of blastospores and had little tendency to differentiate into pseudohyphae during the course of the experiments. The pseudohyphal form of C. albicans was prepared by incubating blastospores in McCoy medium containing 33% serum, 50 fyg of cysteine per ml, and 20 ,tg of sodium thioglycolate per ml. After 1 h, germ tube formation was well underway in more than 80% of the cells, but tangled mycelia had not formed. Phagocytosis and intracellular killing. Neutrophils or monocytes were allowed to adhere to glass
932
VOL. 24, 1979
LEUKOCYTES AND PATHOGENIC YEASTS
cover slips after which the nonadherent cells were removed by gentle, repeated rinses of buffered saline. Cell suspensions were diluted so that approximately 106 cells adhered, nearly all of which were monocytes or neutrophils, respectively, by morphological criteria. Leukocyte viability was measured by mixing an aliquot of the cell suspension with trypan blue. Suspensions with less than 95% viability were discarded. All experiments were performed in McCoy medium supplemented with 10% pooled human serum (Flow Laboratories). Pooled serum was stored in individual aliquots at -70'C until needed. Approximately 106 fungi (blastospores or pseudohyphae) were added to 1 ml of medium over the leukocyte-bearing cover slips. After 20, 60, 90, 120, 180, and 240 min of incubation, cover slips were rinsed well, and the attached cells were stained for 60 s with a 1.6 x 10-5 M solution of acridine orange. The cells were examined with a fluorescence microscope, and the number of cells which had ingested fungi was assessed by direct examination, whereas the viability of the intracellular fungi was measured on the basis of the fluorescing color of the fungus after the techniques of Smith and Rommel (17) (live fungi fluoresce a pale green; dead cells fluoresce a copper red). At least 200 cells were examined in each sample. The results of the phagocytosis experiments are expressed as the percent of the total number of phagocytes which contained two or more yeast cells at any one time; results of intracellular killing determinates are expressed as the percent of ingested yeasts which have been killed. Results were analyzed using the Student's t test. The accuracy of this technique for differentiating between live and dead fungi was examined in preliminary experiments in which fungi were killed with amphotericin B or by heating. The shift from green to red cells correlated well with the disappearance of viable cells as measured by standard quantitative plating techniques on agar. Secondly, incubation of phagocytic mixtures with 1 mM phenylbutazone (which decreased intracellular killing) sharply decreased the color shift of ingested fungi. Finally, the data obtained using this technique with C. albicans blastospores compared favorably with the percent killing of neutrophils at 60 min using other techniques such as vital staining (9, 13, 18). Electron microscopy. Leukocytes were separated from whole blood as indicated above and incubated in suspension with fungi. Monocytes or neutrophils mixed in a 1:1 ratio with S. cerevisiae and C. albicans (blastospores or pseudohyphae) were incubated in McCoy medium with constant oscillation. After 60 min of incubation, the phagocytic mixtures were chilled, washed twice in cold phosphate-buffered saline, and fixed in 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h. Cells were then washed three times in buffer, postfixed for 30 min in 1% osmium tetroxide, dehydrated, and embedded in Epon. Sections were cut with an LKBHuxley microtome, stained with uranyl acetate and lead citrate, and examined with an A.E.I. EM-60 microscope.
RESULTS Experiments were performed to compare the
933
kinetic aspects of ingestion and killing of four fungal forms by two types of phagocytic cells. The rate at which neutrophils and monocytes ingest C. albicans (blastospores and pseudohyphae), C. tropicalis, and S. cerevisiae is shown in Fig. 1. Each point represents the average of at least five experiments. With three of the fungi, the rate of phagocytosis by monocytes was greater than that by neutrophils during at least part of the 240-min experiment. The rates of ingestion by monocytes and neutrophils were consistently different for C. albicans blastospores (P < 0.025 at 60 min; P < 0.025 at 120 min and P < 0.05 at 240 min). Monocytes ingested S. cerevisiae more rapidly than did the neutrophils early in the course of the experiment (P < 0.025 at 60 min). However, although the percent of phagocytizing monocytes was greater than the neutrophils in the latter parts of the experiment, the differences were not statistically significant (P > 0.05 at 120 and 240 min). Similarly, monocytes initiated phagocytosis of C. tropicalis more rapidly than neutrophils early in the experiment (P < 0.025 at 60 min), but not at a significantly different rate later (P > 0.05 at 120 min; P > 0.10 at 240 min). In contrast, the ingestion of C. albicans pseudohyphae by neutrophils and monocytes was at no time different (P > 0.2 at 60 min; P > 0.10 at 120 min; and P > 0.05 at 240 min). Figure 2 shows the rates at which the four fungal forms were killed by monocytes and neutrophils. Each point represents the average of at least fivb experiments. In each case, the percent of dead intracellular organisms was consistently and significantly greater in the monocyte culture than with neutrophils (C. albicans blastospores: 60 min, P < 0.001; 120 min, P < 0.001; 240 min, P < 0.001; C. albicans pseudohyphae: 60 min, P < 0.025; 120 min, P < 0.01; 240 min, P < 0.001; S. cerevisiae: 60 min, P < 0.001; 120 min, P < 0.001; 240 min, P < 0.001; C. tropicalis: 60 min, P < 0.05; 120 min, P < 0.01; 240 min, P < 0.05). To confirm the kinetic data obtained by fluorescent microscopy, populations of leukocytes and fungi were examined with the electron microscope. Figure 3 shows the structure of intracellular fungi after 60 min of incubation. In Fig. 3A, the destruction of S. cerevisiae by a monocyte is well underway. Three yeast forms are sequestered within the cytoplasm of the monocyte. A well defined enclosing membrane cannot be seen. The yeast cell walls are swollen and poorly defined; and the cytoplasm is disrupted. On the other hand, after 60 min, S. cerevisiae appears intact inside a neutrophil (Fig. 3B) with an intact, well-formed cell wall and a homogeneously granular cytoplasm. The pseudohyphae of C. albicans are destroyed less efficiently by
934
INFECT. IMMUN.
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TIME IN MINUTES FIG. 1. Phagocytosis of yeasts by monocytes and neutrophils. Results phagocytes which have ingested two or more yeasts.
both monocytes (Fig. 3C) and neutrophils (Fig. 3D). In both examples shown here, after 60 min, the intracellular pseudohypae were intact. DISCUSSION The data in this report indicate that, in vitro, monocytes phagocytize and destroy fungi more efficiently, suggesting that the circulating monocyte has an important role in host defenses against fungi. The relative efficiency of monocytic fungicidal activity is especially pronounced in the case of C. albicans pseudohyphae. Although neutrophils were able to ingest these organisms, little or no intracellular killing occurred; monocytes were able to kill 50% of the ingested pseudohyphae by 180 min of incubation. On the other hand, the neutrophil appears to be the primary circulating cell in the defense against bacteria, as these cells destroy a variety of bacteria more rapidly than do monocytes (5, 14, 16). In the case of fungi, however, the situa-
are
the percent of
tion is less clear. Although neutrophils (1), monocytes (2), and macrophages (15) all destroy intracellular C. albicans poorly, reports are conflicting as to whether the rate of killing by monocytes or neutrophils is more rapid (8, 10, 11, 16). Calderone and co-workers have found that monocytes are the predominant cell ingesting Candida in endocardial vegetations (2). Fungal virulence is related in some cases to the ability to undergo dimorphic differentiation. For example, an organism such as C. albicans causes human disease more frequently than does C. tropicalis, an organism which in vitro usually is unable to differentiate into pseudohyphae. Considerable controversy exists concerning the relative pathogenicity of the yeast versus the pseudohyphal form of C. albicans. Several lines of evidence point to the pseudohyphal form as the more pathogenic, including the appearance of intracellular pseudohyphae in patients (4) and experimental animals (2) with candidiasis, the in vitro survival of pseudohyphae within leuko-
LEUKOCYTES AND PATHOGENIC YEASTS
VOL. 24, 1979
935
ease) was able to survive rather well after phagocytosis by neutrophils. C. tropicalis, an opportunistic pathogen, was killed by monocytes at a slower but statistically not significant rate than C. albicans pseudohyphae (P > 0.05). Finally, the blastospores of C. albicans were destroyed at a rate intermediate between the pseudohyphae and S. cerevisiae. The mechanism of the prolonged intracellular survival of virulent fungi is unknown. The techniques used in this study make it possible to study phagocytosis and intracellular killing simultaneously, using small volumes of blood. Rapid manipulation of the cells is also possible because of the ease with which the cover slips can be transferred from one solution to another. Data collected using this technique compare favorably with the results of experiments that use more traditional techniques of measuring fungicidal function (9, 13, 18). One inherent weakness in this system, however, is the difficulty of differentiating completely be-
cytes (1, 12), and the metabolic inhibition and ultimate cell death of macrophages infected with pseudohyphae (15). The present investigation likewise supports the hypothesis that pseudohyphae are uniquely resistant, as human neutrophils were unable to kill ingested pseudohyphae. Monocytes were able to kill the pseudohyphae more efficiently than were neutrophils, but were able to kill ingested blastospores more rapidly than pseudohyphae (52% dead at 60 min versus 34%, respectively). Indeed, it is possible that the circulating phagocyte acts as a vehicle in the dissemination of organisms to individuals with compromised host defenses, as the prolonged survival of intracellular pseudohyphae would allow them to be distributed throughout the body. The relative virulence of fungi in human disease thus may be related to the ability of phagocytes to kill ingested fungi. Both monocytes and neutrophils killed a strict nonpathogen, S. cerevisiae, most rapidly, whereas the pseudohyphae of C. albicans (capable of causing dis-
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LEUKOCYTES AND PATHOGENIC YEASTS
VOL. 24, 1979
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Vol. 24, No. 3
Phagocytosis and Intracellular Killing of Pathogenic Yeasts by Human Monocytes and Neutrophils KENNETH E. SCHUITt
Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 14 November 1978
The kinetics of phagocytosis and killing of four fungal forms with varying virulence by two types of phagocytic cells was examined. Human monocytes ingested Saccharomyces cerevisiae, Candida tropicalis, and the blastospores of Candida albicans more rapidly than did human neutrophils. There was no difference in the rate of phagocytosis of C. albicans pseudohyphae by these two cell types. Intracellular killing of each of the four fungal forms was consistently and significantly more rapid by monocytes than by neutrophils. Neutrophils were unable to destroy ingested C. albicans pseudohyphae. These experiments suggest that the monocyte plays an important role in host defenses against fungal diseases and that the relative virulence of the pathogenic yeasts in human disease may be related to the ability of these organisms to survive after being ingested by circulating phagocytes.
Leukocytes are important in host defenses against infection because they are able to ingest and kill microorganisms. Although all circulating phagocytes (neutrophils, monocytes and eosinophils) serve this function, the neutrophil appears to have an especially important role in the destruction of bacteria (5, 14, 16). The role of these cells in the defense against fungal infection is less clear, as neutrophils and monocytes may be unable to kill fungi (12), or may do so poorly (1, 2, 7). Although fungal diseases such as disseminated candidiasis are becoming increasingly common (6), several aspects of the pathogenesis of these infections are poorly understood. For example, what is the relative role of monocytes and neutrophils in the destruction of the pathogenic yeasts? Also, what is the importance in disease production of the blastospore and pseudohyphal forms of Candida albicans? The purpose of this investigation was to compare the ability of human neutrophils and monocytes to ingest and kill several species of fungi which represent a spectrum of virulence in human disease and to study the interaction of these leukocytes with the blastospore and pseudohyphal forms of C. albicans. MATERIALS AND METHODS Leukocyte preparation. Leukocyte-rich plasma was prepared from heparinized blood obtained from normal adult volunteers of both sexes by mixing whole t Present address: Children's Hospital of Pittsburgh, Pittsburgh, PA 15213.
blood in a 1:1 ratio with a 3% solution of gelatin (Plasmagel, Laboratoire Roger Bellon, Neuilly, France) and allowing it to settle for 1 h. The leukocyte suspension was diluted with an equal volume of buffered saline, layered onto Ficoll-Hypaque (2:1 ratio), and centrifuged for 40 min at 1,400 x g. The mononuclear cell layer was removed with a Pasteur pipette, and the neutrophils (which were pelleted along with the erythrocytes at the bottom of the centrifuge tube) were suspended after carefully decanting the liquid portions of the gradient. The erythrocytes in both preparations were lysed by suspending the cells in 8 ml of distilled water for 30 s after which physiological osmolarity was restored by the addition of 4 ml of 3.6% NaCl. The neutrophils and monocytes were washed an additional time with buffered saline, suspended in McCoy medium, and counted in a hemocytometer. Fungi. Strains of C. albicans, Candida tropicalis, and Saccharomyces cerevesiae were obtained from the Department of Hospital Laboratories, North Carolina Memorial Hospital, Chapel Hill. All strains were maintained on Trypticase soy agar slants and subcultured every 2 weeks. For the experiments, fungi were grown on Trypticase soy agar for 48 h at 37°C, gently scraped from the surface, and washed twice before counting in a hemocytometer. Cultures of C. albicans grown on agar in this manner consisted entirely of blastospores and had little tendency to differentiate into pseudohyphae during the course of the experiments. The pseudohyphal form of C. albicans was prepared by incubating blastospores in McCoy medium containing 33% serum, 50 fyg of cysteine per ml, and 20 ,tg of sodium thioglycolate per ml. After 1 h, germ tube formation was well underway in more than 80% of the cells, but tangled mycelia had not formed. Phagocytosis and intracellular killing. Neutrophils or monocytes were allowed to adhere to glass
932
VOL. 24, 1979
LEUKOCYTES AND PATHOGENIC YEASTS
cover slips after which the nonadherent cells were removed by gentle, repeated rinses of buffered saline. Cell suspensions were diluted so that approximately 106 cells adhered, nearly all of which were monocytes or neutrophils, respectively, by morphological criteria. Leukocyte viability was measured by mixing an aliquot of the cell suspension with trypan blue. Suspensions with less than 95% viability were discarded. All experiments were performed in McCoy medium supplemented with 10% pooled human serum (Flow Laboratories). Pooled serum was stored in individual aliquots at -70'C until needed. Approximately 106 fungi (blastospores or pseudohyphae) were added to 1 ml of medium over the leukocyte-bearing cover slips. After 20, 60, 90, 120, 180, and 240 min of incubation, cover slips were rinsed well, and the attached cells were stained for 60 s with a 1.6 x 10-5 M solution of acridine orange. The cells were examined with a fluorescence microscope, and the number of cells which had ingested fungi was assessed by direct examination, whereas the viability of the intracellular fungi was measured on the basis of the fluorescing color of the fungus after the techniques of Smith and Rommel (17) (live fungi fluoresce a pale green; dead cells fluoresce a copper red). At least 200 cells were examined in each sample. The results of the phagocytosis experiments are expressed as the percent of the total number of phagocytes which contained two or more yeast cells at any one time; results of intracellular killing determinates are expressed as the percent of ingested yeasts which have been killed. Results were analyzed using the Student's t test. The accuracy of this technique for differentiating between live and dead fungi was examined in preliminary experiments in which fungi were killed with amphotericin B or by heating. The shift from green to red cells correlated well with the disappearance of viable cells as measured by standard quantitative plating techniques on agar. Secondly, incubation of phagocytic mixtures with 1 mM phenylbutazone (which decreased intracellular killing) sharply decreased the color shift of ingested fungi. Finally, the data obtained using this technique with C. albicans blastospores compared favorably with the percent killing of neutrophils at 60 min using other techniques such as vital staining (9, 13, 18). Electron microscopy. Leukocytes were separated from whole blood as indicated above and incubated in suspension with fungi. Monocytes or neutrophils mixed in a 1:1 ratio with S. cerevisiae and C. albicans (blastospores or pseudohyphae) were incubated in McCoy medium with constant oscillation. After 60 min of incubation, the phagocytic mixtures were chilled, washed twice in cold phosphate-buffered saline, and fixed in 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h. Cells were then washed three times in buffer, postfixed for 30 min in 1% osmium tetroxide, dehydrated, and embedded in Epon. Sections were cut with an LKBHuxley microtome, stained with uranyl acetate and lead citrate, and examined with an A.E.I. EM-60 microscope.
RESULTS Experiments were performed to compare the
933
kinetic aspects of ingestion and killing of four fungal forms by two types of phagocytic cells. The rate at which neutrophils and monocytes ingest C. albicans (blastospores and pseudohyphae), C. tropicalis, and S. cerevisiae is shown in Fig. 1. Each point represents the average of at least five experiments. With three of the fungi, the rate of phagocytosis by monocytes was greater than that by neutrophils during at least part of the 240-min experiment. The rates of ingestion by monocytes and neutrophils were consistently different for C. albicans blastospores (P < 0.025 at 60 min; P < 0.025 at 120 min and P < 0.05 at 240 min). Monocytes ingested S. cerevisiae more rapidly than did the neutrophils early in the course of the experiment (P < 0.025 at 60 min). However, although the percent of phagocytizing monocytes was greater than the neutrophils in the latter parts of the experiment, the differences were not statistically significant (P > 0.05 at 120 and 240 min). Similarly, monocytes initiated phagocytosis of C. tropicalis more rapidly than neutrophils early in the experiment (P < 0.025 at 60 min), but not at a significantly different rate later (P > 0.05 at 120 min; P > 0.10 at 240 min). In contrast, the ingestion of C. albicans pseudohyphae by neutrophils and monocytes was at no time different (P > 0.2 at 60 min; P > 0.10 at 120 min; and P > 0.05 at 240 min). Figure 2 shows the rates at which the four fungal forms were killed by monocytes and neutrophils. Each point represents the average of at least fivb experiments. In each case, the percent of dead intracellular organisms was consistently and significantly greater in the monocyte culture than with neutrophils (C. albicans blastospores: 60 min, P < 0.001; 120 min, P < 0.001; 240 min, P < 0.001; C. albicans pseudohyphae: 60 min, P < 0.025; 120 min, P < 0.01; 240 min, P < 0.001; S. cerevisiae: 60 min, P < 0.001; 120 min, P < 0.001; 240 min, P < 0.001; C. tropicalis: 60 min, P < 0.05; 120 min, P < 0.01; 240 min, P < 0.05). To confirm the kinetic data obtained by fluorescent microscopy, populations of leukocytes and fungi were examined with the electron microscope. Figure 3 shows the structure of intracellular fungi after 60 min of incubation. In Fig. 3A, the destruction of S. cerevisiae by a monocyte is well underway. Three yeast forms are sequestered within the cytoplasm of the monocyte. A well defined enclosing membrane cannot be seen. The yeast cell walls are swollen and poorly defined; and the cytoplasm is disrupted. On the other hand, after 60 min, S. cerevisiae appears intact inside a neutrophil (Fig. 3B) with an intact, well-formed cell wall and a homogeneously granular cytoplasm. The pseudohyphae of C. albicans are destroyed less efficiently by
934
INFECT. IMMUN.
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0
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TIME IN MINUTES FIG. 1. Phagocytosis of yeasts by monocytes and neutrophils. Results phagocytes which have ingested two or more yeasts.
both monocytes (Fig. 3C) and neutrophils (Fig. 3D). In both examples shown here, after 60 min, the intracellular pseudohypae were intact. DISCUSSION The data in this report indicate that, in vitro, monocytes phagocytize and destroy fungi more efficiently, suggesting that the circulating monocyte has an important role in host defenses against fungi. The relative efficiency of monocytic fungicidal activity is especially pronounced in the case of C. albicans pseudohyphae. Although neutrophils were able to ingest these organisms, little or no intracellular killing occurred; monocytes were able to kill 50% of the ingested pseudohyphae by 180 min of incubation. On the other hand, the neutrophil appears to be the primary circulating cell in the defense against bacteria, as these cells destroy a variety of bacteria more rapidly than do monocytes (5, 14, 16). In the case of fungi, however, the situa-
are
the percent of
tion is less clear. Although neutrophils (1), monocytes (2), and macrophages (15) all destroy intracellular C. albicans poorly, reports are conflicting as to whether the rate of killing by monocytes or neutrophils is more rapid (8, 10, 11, 16). Calderone and co-workers have found that monocytes are the predominant cell ingesting Candida in endocardial vegetations (2). Fungal virulence is related in some cases to the ability to undergo dimorphic differentiation. For example, an organism such as C. albicans causes human disease more frequently than does C. tropicalis, an organism which in vitro usually is unable to differentiate into pseudohyphae. Considerable controversy exists concerning the relative pathogenicity of the yeast versus the pseudohyphal form of C. albicans. Several lines of evidence point to the pseudohyphal form as the more pathogenic, including the appearance of intracellular pseudohyphae in patients (4) and experimental animals (2) with candidiasis, the in vitro survival of pseudohyphae within leuko-
LEUKOCYTES AND PATHOGENIC YEASTS
VOL. 24, 1979
935
ease) was able to survive rather well after phagocytosis by neutrophils. C. tropicalis, an opportunistic pathogen, was killed by monocytes at a slower but statistically not significant rate than C. albicans pseudohyphae (P > 0.05). Finally, the blastospores of C. albicans were destroyed at a rate intermediate between the pseudohyphae and S. cerevisiae. The mechanism of the prolonged intracellular survival of virulent fungi is unknown. The techniques used in this study make it possible to study phagocytosis and intracellular killing simultaneously, using small volumes of blood. Rapid manipulation of the cells is also possible because of the ease with which the cover slips can be transferred from one solution to another. Data collected using this technique compare favorably with the results of experiments that use more traditional techniques of measuring fungicidal function (9, 13, 18). One inherent weakness in this system, however, is the difficulty of differentiating completely be-
cytes (1, 12), and the metabolic inhibition and ultimate cell death of macrophages infected with pseudohyphae (15). The present investigation likewise supports the hypothesis that pseudohyphae are uniquely resistant, as human neutrophils were unable to kill ingested pseudohyphae. Monocytes were able to kill the pseudohyphae more efficiently than were neutrophils, but were able to kill ingested blastospores more rapidly than pseudohyphae (52% dead at 60 min versus 34%, respectively). Indeed, it is possible that the circulating phagocyte acts as a vehicle in the dissemination of organisms to individuals with compromised host defenses, as the prolonged survival of intracellular pseudohyphae would allow them to be distributed throughout the body. The relative virulence of fungi in human disease thus may be related to the ability of phagocytes to kill ingested fungi. Both monocytes and neutrophils killed a strict nonpathogen, S. cerevisiae, most rapidly, whereas the pseudohyphae of C. albicans (capable of causing dis-
1001
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FIG. 2. Intracellular killing of yeasts by monocytes and neutrophils. Results are expressed as the percent of intracellular yeasts which are dead.
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FIG. 3. Interaction of yeasts and leukocytes. Sixty-minute incubation. (A) Monocyte and S. cerevisiae (x12,500); (B) neutrophil and S. cerevisiae (x13,500); (C) monocyte and C. albicans pseudohypha (x10,000); (D) neutrophil and C. albicans pseudohypha (x8,500). 936
937
LEUKOCYTES AND PATHOGENIC YEASTS
VOL. 24, 1979
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