Growth Inhibition of Candida albicans by Rabbit Alveolar Macrophages

INFCTION AND IMMUNITY, Mar. 1977, p. 910-915 Copyright © 1977 American Society for Microbiology

Vol. 15, No. 3 Printed in U.S.A.

Growth Inhibition of Candida albicans by Rabbit Alveolar Macrophages ELLENA M. PETERSON

AND

RICHARD A. CALDERONE

Department of Microbiology, Georgetown University, Schools of Medicine and Dentistry, Washington, D. C. 20007

Received for publication August 1976

Normal rabbit alveolar macrophages were infected in vitro with Candida albicans. Early after infection, germ tube formation by phagocytized C. albicans was inhibited in contrast to extracellular (nonphagocytized) C. albicans. Over an 8-h period, plate counts of C. albicans incubated with alveolar macrophages revealed a decrease in colony-forming units in contrast to C. albicans alone. In addition, an assay was developed which specifically measured C. albicans [3H]leucine incorporation in the presence of alveolar macrophages. Using this assay, we observed a 71 to 93% inhibition of macromolecular synthesis in C. albicans when incubated with alveolar macrophages. Autoradiographic studies showed that the inhibition of leucine incorporation was restricted to the ingested Candida. The major process by which large particles (i.e., >2.5 Am) such as Candida albicans and other fungi are cleared from the lung is probably through the mucociliary transport system (4). However, additional clearance mechanisms must exist for those large particles which evade the mucociliary action. The alveolar macrophage (AM) is a candiate for such a mechanism. They have been shown to play a critical role in the inactivation of bacteria and clearance of inert particles (5). In these capacities, the AM aids in the maintenance of an essentially sterile environment in the lung spaces. To date there have been no reports concerning the interaction of C. albicans with the AM, although studies with other fungi have indicated that the AM has a rather limited role in protection of the host against invasion (2, 3). In this study, we have investigated the in vitro relationship between C. albicans and rabbit AM. Our data indicate that the AM can inhibit the growth of ingested C. albicans. MATERIALS AND METHODS C. albicans. A clinical isolate of C. albicans (serotype b) was obtained from F. Blank, Skin and Cancer Hospital, Philadelphia, Pa. Cloned stock cultures, which were transferred monthly, were grown at 370C and maintained at 40C on brain heart infusion slants. Inoculum for all experiments was obtained in the following manner. C. albicans was grown at 370C for 18 h on brain heart infusion slants. Yeast cells were harvested by centrifugation (480 x g, 40C) and washed and suspended in 0.9% saline. Cell suspen-

sions were standardized by hemocytometer counts. Cultures used were at least 99% viable as judged by trypan blue exclusion. Macrophages. Normal AM were obtained from New Zealand white female rabbits (2.3 to 2.7 kg) using a modification of the procedure of Myrvik et al. (8). Animals were sacrificed by injecting a lethal solution of pentobarbitol into the marginal ear vein. The lungs, heart, and trachea were removed and superficially cleansed with sterile 0.9% saline. The lungs were then ravaged with 200 ml of 0.9% saline containing heparin (0.5 U/ml), penicillin (100 U/ml), and streptomycin (100 ,ug/ml). This method yielded a leukocyte population that was 93.1 + 1.9% viable as judged by trypan blue exclusion and 98.5 + 1.9% macrophages as determined by Wright stain differentials. Serum. Fresh, autologous serum was found to be necessary for optimal phagocytosis. Sera collected before each lavage were stored at -20°C and used within 1 week of collection. Sera were negative for precipitating antibody to a C. albicans cytoplasmic

antigen. Infection ratios. AM were suspended to a concentration of 2 x 106 cells/ml in medium 199 containing penicillin (100 U/ml), streptomycin (100 ,ug/ml), and 0.1% bovine serum albumin (wt/vol). Monolayers of AM (2 x 106 cells/cover slip) were prepared on glass cover slips (22 mm2) in tissue culture dishes (35 by 100 mm). After attachment of AM (1 h, 37°C, 5% C02), cover slips were washed with Hanks balanced salts solution (HBSS) and overlaid with 1 ml of a C. albicans cell suspension (varying in concentration from 5 x 105 cells/ml to 8 x 106 cells/ml), suspended in medium 199 containing penicillin (100 U/ml), streptomycin (100 ,ug/ml), and 25% serum. The infected monolayers were incubated on a rotary shaker (75 rpm) for 1 h at 37°C. The cover slips were then placed at 37°C in an atmosphere of 5% CO2 in 910

VOL. 15, 1977 air for 3 h. Subsequently, Gram stains were performed on the monolayers and the percent phagocytosis was determined. Plate counts. Monolayers of AM, prepared as described above (2 x 106 cells/cover slip), were infected with C. albicans (5 x 105 cells/ml). At designated time intervals, the cover slips were scraped with a rubber policeman and dilutions of the cell suspensions were made in distilled water. Pour plates were made using Sabouraud dextrose agar. Colony-forming units (CFU) were counted after an 18-h incubation period (370C) and compared with control cultures consisting of C. albicans incubated on cover slips without AM. Effect of cycloheximide on AM and C. albicans [3H]leucine incorporation. Tissue culture tubes (13 by 100 mm) contained a cell suspension of either AM (2 x 106 cells/ml) or C. albicans (5 x 105 cells/ml) in medium 199 with penicillin (100 U/ml), streptomycin (100 jig/ml), and 25% serum. Cycloheximide (10 ,ug/ml, Nutritional Biochemical Co.) was added to half of the tissue culture tubes. [3H]leucine (5 ,uCi/ ml; 60 Ci/mmol, New England Nuclear Corp., Boston, Mass.) was added to all cell suspensions. Subsequently,, all tubes were incubated at 37°C on a rotary mixer at 10 rpm. At designated intervals, duplicate cell suspensions were precipitated on ice with cold 15% trichloroacetic acid. Nonspecific trapping of label was determined by labeling cultures on ice and precipitating immediately with cold 15% trichloroacetic acid. Subsequently, all precipitated cultures were filtered onto glass fiber filters (Whatman GF/ A) and washed with 5% trichloroacetic acid followed by 95% ethanol. All radioactive measurements were made in an Intertechnique liquid scintillation counter which had approximately a 30% efficiency for 3H on GF/A filters in a scintillation liquid of the following composition: 0.1 g of p-bis[-(5-phenyloxazolyl)]benzene and 5 g of 2, 5-diphenyloxazole dissolved in 1.0 liter of scintillation grade toluene. Effect of AM on C. albicans [3Hlleucine incorporation. Tissue culture tubes (13 by 100 mm) containing a cell suspension of either AM (2 x 106 cells/ml), C. albicans (5 x 105 cells/ml), or both in medium 199 with penicillin (100 U/ml), streptomycin (100 ,g/ ml), and 25% serum were incubated at 37°C, 10 rpm. At designated intervals, cell suspensions (AM alone, C. albicans alone, or AM and C. albicans) were centrifuged (2,000 x g, 4°C, 10 min), washed with HBSS, and resuspended in 1 ml of HBSS. Cycloheximide (10 jag/ml) was added to all tissue culture tubes except for cultures of AM incubated alone which were used to check the metabolic state of the AM. All cultures were then incubated with [3H]leucine (5 uCi/ml) for 1 h at 37°C on a rotary mixer (10 rpm). Subsequently, cultures were precipitated with cold 15% trichloroacetic acid. Measurements of nonspecific trapping and counting procedures were as described in the previous section. Calculations to determine the inhibitory effect of AM on C. albicans incorporation were as follows: the counts per minute (cpm) obtained from cycloheximide-treated AM cultures were subtracted from the cpm obtained from cycloheximide-treated cultures of

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GROWTH INHIBITION OF C. ALBICANS

AM and C. albicans incubated together. This figure, representing C. albicans specific incorporation when incubated with AM, was compared with the cpm obtained from cultures of C. albicans incubated without AM. Autoradiography. AM monolayers were infected with C. albicans as described above. At designated time intervals, infected monolayers were washed with HBSS. Monolayers were then incubated (370C, 75 rpm) in 1 ml of HBSS containing cycloheximide (10 gg/ml) and [3H]leucine (5 juCi/ml). The monolayers were incubated for 1 h after which the cover slips were washed, fixed in 70% ethanol, and dipped into melted Kodak NTB-4 emulsion. Cover slips were exposed for 1 week (40C), developed, and then stained with Giemsa.

RESULTS In the tissue culture media used throughout these experiments, C. albicans rapidly converted from a yeast to a filamentous form. When phagocytized early after infection, yeast cells were inhibited in germ tube formation in contrast to C. albicans, which remained extracellular (Fig. 1). Some of the phagocytized C. albicans, although initially retarded in germ tube formation, eventually germinated and grew out of AM. At the infection ratio used initially (2 C. albicans: 1 AM), much of the C. albicans remained extracellular. Therefore, in order to effectively study the inhibitory properties of AM on phagocytized C. albicans, the amount of extracellular C. albicans had to be minimized. The percent phagocytosis using different ratios of C. albicans to AM was determined. The data in Table 1 show that a ratio of ,"

r 0

/

Or

FIG. 1. Gram stain of phagocytized C. albicans (arrows) 2 h after AM infection. Note extensive germ tube formation by extracellular C. albicans. Infection ratio of2 Candida to 1 AM. Gram stain (x460).

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TABLE 1. Phagocytosis of C. albicans (yeast) versus infection ratio Yeast:AM

% AM with intracellular yeasta

1:4 1:2 1:1 2:1

a

15.7 14.2 30.8 31.8

Mean no. of yeast/infected AM

% Yeast phagocytized

1.13 1.57

44.5

1.68 1.86 2.11 4:1 48.0 AM containing yeast/500 AM x 100.

71

51.6

30 25

mide was included in the labeling mixture to reduce AM-43H]leucine incorporation. Ingestion itself does not affect the efficiency of grain development, for intra- and extracellular prelabeled C. albicans gave similar grain counts. These in vitro studies show that the AM is capable of efficiently ingesting and subsequently either terminating or markedly inhibiting the growth of C. albicans cells. DISCUSSION C. albicans, as well as many other fungi, by virtue of their relatively large cell size (i.e., >2.5 gm in diameter), are probably most often cleared from the lung by mucociliary transport (4). However, it is reasonable to assume that some C. albicans (albeit a small percentage) evade the mucociliary action and enter the alveolar spaces, where they encounter the AM. Since the AM seems to play an important role

1 C. albicans: 4 AM resulted in 71% phagocytosis of the total inoculum. At this low infection ratio, phagocytized Candida could still be visualized. This infection ratio was used in all subsequent experiments. Plate counts to quantitate the effect of AM on the growth of C. albicans were complicated by an extensive amount of germ tube clumping. Even so, over an 8-h period, growth of C. albicans (CFU/ml), when incubated in the presence of AM, was reduced when compared with control cultures of C. albicans (Fig. 2). However, a more accurate reflection of C. 4 albicans inhibition in the presence of AM would be achieved by measuring a C. albicans specific metabolic activity ([3H]leucine incorporation). We first had to find an agent that would minimize AM leucine incorporation but not affect C. albicans macromolecular synthesis. Thus, cycloheximide was included in the assay to reduce AM incorporation which would otherwise contribute to the background counts. At the concentration of cycloheximide used, 3 AM were drastically affected in their ability to LA incorporate [3H]leucine, whereas C. albicans x was not significantly affected (Fig. 3). Studies were also undertaken to determine the effect of cycloheximide on the uptake of [3H]leucine by AM. No significant differences in leucine uptake were found between control AM and cycloheximide-treated AM (data not shown). The results of the [3H]leucine incorporation assay are shown in Table 2. We observed the incorporation to be inhibited between 71 and 93% in the presence of AM. Maximal inhibition was maintained for at least the first 7 h of infection. Determinations were not made beyond this time. The differences between rabbits in the amount of inhibition is probably a reflection of the degree of heterogeneity among rabbits. Autoradiography, using [3H]leucine, estab4 8 2 6 lished that the reduction in incorporation was specific to the phagocytized C. albicans (Fig. 4 Hours and 5). Although intracellular yeast could be FIG. 2. Growth (CFUlml) of C. albicans incuseen within labeled macrophages, cyclohexi- bated with (-) and without (0) AM.

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in the inactivation of bacteria, we sought to investigate their effect on C. albicans. In retrospect, C. albicans was a good choice for assessing the inhibitory properties of the AM since once phagocytized, the fungus was unable to germinate in contrast to extracellular

913

Candida, which readily germinated. Throughout this study, we restricted our observations to the first 8 h of infection. Within this time period, we did not observe blastospore formation by extracellular germ tubes. Therefore, it can be assumed that the phagocytized yeast represented the original inoculum and thus a true growth inhibition rather than phagocytized blastospores produced by extracellular (nonphagocytized) germ tubes. Various techniques have been used by investigators to determine whether phagocytic cells are capable of inhibiting or inactivating ingested Candida sp. Stanley and Hurley utilized the property of germ tube formation by C. albicans to conclude that peritoneal macrophages could not inhibit the engulfed yeast (11). Ozato

E C._

CD 0-

S._

,,;e,~~~~~~~n ft.%}; w

30

60

90

Minutes

FIG. 3. Effect of cycloheximide (10 mg/ml) on [3H]leucine (5 uCi/ml) incorporation of C. albicans and AM. Symbols: (O) C. albicans incubated without cycloheximide; (-) C. albicans incubated with cycloheximide; (0) AM incubated without cycloheximide; (A) AM incubated with cycloheximide.

FIG. 4. Autoradiography. C. albicans (arrows) 4 h after AM infection. Cells incubated in media containing 5 pCi of [3H]leucine per ml and 10 pg of cycloheximide per ml. Note absence ofgrain development associated with intracellular yeast. Giemsa stain (x820).

TABLE 2. Alveolar macrophage inhibition of PHileucine incorporation in C. albicans Leucine incorporation (cpm x 10-3/ml) by C. albicans Rabbit no.

2.5 2.5 4 4 7 AM and/or C. albicans were incubated with 1 2 2 3 3

a

Time (h) of incubationa

Alveolar macrophages

Presentb 29.7 27.5

Absent 111.7

106.3 205.6 143.6 309.6

[3H]leucine

% Inhibition"

73 74 71 93 85

60.7 10.2 45.6 (5 ,Ci/ml) and cycloheximide (10 ,ug/ml) for 1 h

before harvest. b Minus cpm of cycloheximide-treated AM. e cpm with AM/cpm without AM x 100 = percent inhibition.

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PETERSON AND CALDERONE

INFECT. IMMUN.

-,

#.

FIG. 5. Autoradiography. Extracellular C. albicans (arrows) adjacent to an AM. Picture taken from same slide as Fig. 4. Note extensive grain development associated with the extracellular C. albicans. Giemsa stain (x1,250).

and Uesaka, using autoradiography to study the interaction of C. albicans and peritoneal macrophages, came to a similar conclusion (9). The possession of a candidacidal mechanism by human leukocytes was revealed by vital staining (1, 6, 7). In this report we described still another approach to determining Candida interactions with phagocytic cells. In an attempt to quantitate this apparent growth inhibition by AM, we first used plate counts. However, growth (CFU/ml) of C. albicans could only be approximated since germ tubes tended to clump in culture. Even so, the plate count experiments supported our observations that an inhibition of C. albicans growth by AM existed. Since inhibition could only be approximated by plate counts, we used leucine incorporation as the basis for a simpler, more sensititve assay which would measure growth inhibition of C. albicans by AM. Since we wanted to measure C. albicans specific protein synthesis, we also found it necessary to use an inhibitor of protein synthesis, cycloheximide, to reduce AM background incorporation. This agent was suitable for our assay, for it did not affect C. albicans incorporation. Some Saccharomyces sp. are also resistant to cycloheximide and this resistance has been shown to be associated with the 60S ribosomal subunit (10). Using this more sensitive assay, we found a 71 to 93% inhibition of leucine incorporation in C. albicans incubated with AM. To interpret

our results, it was necessary to establish the effects of cycloheximide on leucine uptake by AM. We found that cycloheximide did not affect leucine uptake by AM. One might further question whether the labeled leucine, once within the AM, is able to enter the phagocytic vacuole membranes. If an internal membrane exclusion does exist within the AM, then this could lead to starvation of the phagocytized organism, and thus serve as an effective inactivation mechanism. Within the population of the AM obtained from a single rabbit, we have observed a great amount of heterogeneity in their response to C. albicans. Whereas some AM are able to restrict C. albicans to its yeast form, still others, after 4 h, allow the yeast to germinate and even outgrow the AM. We do not know if this heterogeneity is a phenomenon that is unique to an in vitro system. In vivo, where, presumably, optimal conditions for phagocytosis and clearance exist, this heterogeneity may be minimized. The importance of the AM in the clearance and/or inactivation of C. albicans in vivo remains to be established. However, from this in vitro system, the AM can contribute to the restriction if not inactivation of those Candida which descend to the alveoli. ACKNOWLEDGMENTS We would like to thank M. P. Oeschger and T. Sreevalsan for their invaluable advice and discussions.

VOL. 15, 1977 E. P. was supported by Public Health Service training grant AI 00298-11 from the National Institute of Allergy and Infectious Diseases. This investigation was supported by grants from the Washington Heart Association and

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