Vol. 59, No. 5
INFECTION AND IMMUNITY, May 1991, p. 1747-1754 0019-9567/91/051747-08$02.00/0 Copyright © 1991, American Society for Microbiology
Murine Natural Killer Cells Are Fungicidal to Cryptococcus neoformans MICHELLE R. HIDORE,l NASRIN NABAVI,2 FRANK SONLEITNER,3 AND JUNEANN W. MURPHY'* Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731901; Department of Immunopharmacology, Hoffman-LaRoche, Nutley, New Jersey 071102; and Department of Zoology, University of Oklahoma, Norman, Oklahoma 730193 Received 10 December 1990/Accepted 22 February 1991 Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus
neoformnans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neofornans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans.
cocci; therefore, inhibition of cryptococcal growth has been used as an indication of NK cell-mediated damage to the cryptococcal cell. Our previous studies, in which CFU counts were used as an index of NK cell-mediated cryptococcal damage, have clearly demonstrated that murine NK cells inhibit proliferation of cryptococcal target cells (7, 9, 19-21). It is not clear from those investigations, however, whether the anticryptococcal effects of NK cells are fungistatic or fungicidal; therefore, we have referred to the anticryptococcal activity of NK cells as growth inhibition rather than killing. The issue of whether NK cells actually kill cryptococci is of primary importance in determining the potential role of these effector cells in host resistance to this pathogen, so the study presented here was designed to establish whether NK cells only prevent proliferation of C. neoformans or whether they actually kill the organism. To accurately assess NK cell-mediated effects on C. neoformans viability, it was necessary to determine the viability of both cryptococci that were bound to NK cells and cryptococci that were unbound in the cell suspension. Therefore, vital stains which could be used to assess cryptococcal viability at the individual cell level and simultaneously in the entire cell suspension were employed in this study. Vital stains that detect intact metabolic function have previously proven to be rapid and reliable indices of the viability of fungi such as C. neoformans, Candida albicans, Aspergillus fumigatus, and Rhizopus oryzae (2, 4, 13). The vital stains chosen for these studies measure the metabolic function of two different enzyme systems, mitochondrial
Previous studies in our laboratory have focused on murine natural killer (NK) cell interactions with a fungal target, Cryptococcus neoformans. C. neoformans is an encapsulated, yeastlike organism which causes human infection via the pulmonary route, ranging from an asymptomatic upper respiratory infection which is often spontaneously resolved by the host to a potentially fatal disseminated cryptococcosis which is generally manifested as meningitis. The low incidence of dissemination relative to the high frequency of exposure to this ubiquitous organism suggests a role for innate resistance mechanisms in host defense against cryptococci. Previous studies by us (7-10, 17, 19-21) and by others (14) indicate that NK cells function in conjunction with other natural resistance mechanisms in the first-line host defense against cryptococci. We have shown that binding of NK cells to C. neoformans target cells is prerequisite to the events that result in cryptococcal cell damage (9, 19, 21). Furthermore, we have demonstrated that murine NK cells not only inhibit the growth of C. neoformans in vitro but also contribute to clearance of the organism in vivo
(7, 8).
C. neoformans is structurally distinct from the standard tissue or tumor cell target used in the model of NK cellmediated cytotoxicity (17, 19, 21). Although tumor cell lysis is the standard measure of NK cell-mediated tumor cytotoxicity, the cryptococcal cell wall precludes lysis of crypto*
Corresponding author. 1747
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HIDORE ET AL.
dehydrogenases and esterases, within viable fungal cells. One of the vital stains, the yellow tetrazolium salt [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), readily enters C. neoformans cells and is then reduced by the mitochondrial dehydrogenases within viable cells to its purple formazan derivative. This results in the accumulation of clearly visible purple MTT-formazan crystals within viable cryptococci when viewed by light microscopy, whereas dead cells, which do not reduce MTT, do not accumulate the purple formazan crystals (13). The other vital stain used as a means of assessing viability of C. neoformans was the fluorogenic substrate fluorescein diacetate (FDA) (2, 4). FDA is a nonpolar compound that readily enters the cell and is then hydrolyzed by esterases within viable cryptococci to a polar compound which is unable to exit the cell and thus accumulates within viable cryptococci, resulting in fluorochromasia (4). To compare cryptococcal viability, as determined by the vital stains, with the ability of the cells to proliferate, three approaches for assessing proliferation of cryptococci at the individual cell level and in the total cell suspension were employed in this study. One method used for assessing C. neoformans proliferation, CFU counts on the surface of a solid medium (12), is a common spread plate technique for determining proliferation of cryptococci and has been our standard method for measuring NK cell-mediated anticryptococcal effects (20). To quantify proliferation of individual cryptococci associated with NK cells or free in the suspension, the procedure of Grimm et al. (5) was modified to assess cryptococcal CFU counts within agarose. This technique is equivalent to pour plate methods for enumerating viable organisms, with the exception that the colonies are detected microscopically. In addition to colony formation on the surface of or within solid medium, extinction dilution analysis, a method for assessing proliferation of a microorganism in broth culture, was also used (3, 22). Initial comparisons of the vital stains and the proliferation assays indicated that the techniques employed effectively distinguished between live and heat-killed cryptococci at the individual cell level and in total cell suspensions. Following an 18-h incubation with NK cells, C. neoformans viability was assessed by using the vital stains, and the results were compared with NK cell-mediated growth inhibition detected by the proliferation assays. At the single-cell level, the viability of cryptococci incubated with NK cells, as assessed by vital stains, was significantly diminished, compared with that of the control samples containing cryptococci without NK cells. In the total cell suspensions, vital stains confirmed
these results. At the individual cell level and in the total cell suspensions, the numbers of killed cryptococci in the NK cell-C. neoformans suspensions, as determined by the vital stains, were comparable to the numbers of cryptococci that were unable to proliferate. Together, our data clearly demonstrate that NK cells are fungicidal to C. neoformans
targets. MATERIALS AND METHODS Mice. Female CBA/J mice were obtained from Jackson Laboratories, Bar Harbor, Maine. The animals were maintained in the University of Oklahoma animal facility until they were used for these studies at 7 to 8 weeks of age. Reagents. Stock solutions (5 mg/ml) of the tetrazolium dye MTT in RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) and of fluorescein diacetate (FDA) in acetone were prepared. The MTT solution was stored in the dark at
INFECT. IMMUN. IFC.IMN
40C, and the FDA solution was stored at -200C. Both reagents were purchased from Sigma Chemical Co., St. Louis, Mo. Fungal target. C. neoformans isolate 184A was maintained on modified Sabouraud agar slants (18). After 3 days of growth at room temperature, blastoconidia were harvested, washed three times in sterile physiological saline, and adjusted to the desired cell concentration for each experiment with complete medium (CM) consisting of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U of penicillin per ml, and 100 pRg of streptomycin per ml. Cell concentrations were based on hemacytometer counts and confirmed by determining CFU on modified Sabouraud agar plates. Heat-killed C. neoformans cells were prepared by incubating cryptococci (10' cells per ml) in sterile physiological saline at 560C for 60 min. A sample of the heat-treated cells was plated on modified Sabouraud agar to confirm, by the absence of proliferation, that the cryptococcal cells had been killed. Effector cells. Murine, splenic, nylon wool-nonadherent cells were obtained by the method of Julius et al. (11) and then further enriched for NK cells on discontinuous Percoll gradients by a method that has been previously described in detail (15, 21). With this protocol, the cytolytic activity against YAC-1, the standard tumor cell target of murine NK cells, is routinely significantly enriched in the cell fractions from the low-density Percoll layers (fractions 1 and 2) compared with the anti-YAC-1 activity of the input nylon wool-nonadherent cell population. Typically, the nylon wool-nonadherent spleen cell populations that we collect contain approximately 75% Thy-l' cells, 2 to 3% mouse Ig' cells, 1 to 2% nonspecific esterase-positive cells, 9 to 10% asialo GM,' cells, and 6% large granular lymphocytes (cells with NK cell morphology and detectable granules in the cytoplasm), whereas Percoll fraction 1 and 2 cell populations contain 20% Thy-l' cells, no detectable mouse Ig + or nonspecific esterase-staining cells, 40% asialo GM,' cells, and 36% large granular lymphocytes (21). In a previous study, we showed with scanning electron microscopy that the Percoll fraction 1 and 2 effector cells which bound to C. neoformans target cells were asialo GM,-positive cells (21). For convenience throughout the remainder of this report, we have referred to the Percoll fraction 1 and 2 cells which are highly enriched for NK cells as NK cells. NK cell fractions were adjusted to the appropriate concentration for each assay in CM and used as' effector cells in these studies. Incubation of NK cells with C. neoformans target cells. The optimal conditions required for murine NK cells to mediate anticryptococcal effects have been previously described (9, 17, 21). Briefly, 106 NK cells suspended in 0. 1 ml of CM and 5 X 1O5 cryptococcal target cells in 0.1 ml of CM were added to quadruplicate wells of a flat-bottom, 96-well microtiter plate (Linbro Scientific Co., Hamden, N.Y.). Quadruplicate control wells contained 0. 1 ml of the cryptococcal target cell suspension and 0. 1 ml of CM. After the plate was incubated for 18 h at 370C in 7% C02, viability and proliferation of cryptococci in the experimental and control wells were assessed by the methods described below. The generation time for C. neoformans isolate 184A in control wells was 3.5 h when determined by CFU counts. FDA vital staining of C. neoformans cells. Fluorescence staining of viable cryptococci with the fluorogenic substrate FDA was used to measure NK cell-mediated anticryptococcal activity in single conjugates and in total cell suspensions simultaneously (2, 4). The contents of each well containing either NK cells and cryptococci or cryptococci alone were
VOL. 59, 1991
washed in phosphate-buffered saline (PBS) and resuspended in 0.2 ml of PBS containing 0.01 ml of the FDA stock solution, resulting in a final concentration of 0.05 mg of FDA per ml. After a 2-h incubation at 37°C in 7% CO2 in the dark, the cells were washed with PBS and examined with an Olympus BH-2 microscope equipped with phase contrast optics, epifluorescence illumination, and fluorescein fluorescence optics (Olympus Corp., Lake Success, N.Y.). For determination of cryptococcal viability at the single cell level, a minimum of 100 conjugates from each of the four experimental wells were assessed for the presence of green fluorescent (viable) cryptococci. The percentage of killed NK cell-bound cryptococci was calculated according to the following formula: percent killed C. neoformans in conjugates = [(mean number of conjugates - mean number of conjugates containing viable cryptococci)/mean number of conjugates] x 100. For determination of the total number of viable cryptococci in the wells, a minimum of 200 cryptococci (unbound and bound to NK cells) from each experimental well and each control well were scored for the presence of green fluorescence (viable cells). The percentage of killed cryptococci in the total cell suspensions was calculated according to the following formula: percent C. neoformans killed = [(mean percent viable cryptococci in control wells - percent viable cryptococci in experimental wells)/ mean percent viable cryptococci in control wells] x 100. MTT vital staining of C. neoformans cells. A second vital stain, the tetrazolium dye MTT, was used for assessing NK cell-mediated anticryptococcal effects in single conjugates and in total cell suspensions simultaneously (13). The contents of each well containing either NK cells and cryptococci or cryptococci alone were washed in serum-free RPMI 1640 medium and resuspended in 1.0 ml of RPMI 1640 containing 0.5 mg of MTT. After a 3-h incubation at 37°C in 7% CO2, the cells were washed in serum-free RPMI 1640 and examined by light microscopy and phase contrast optics. For determination of cryptococcal viability at the single cell level, a minimum of 100 conjugates from each of the 4 experimental wells were scored for the presence of viable cryptococci, i.e., cryptococci containing purple MTT-formazan crystals. For determination of the total number of viable cryptococci in the wells, a minimum of 200 cryptococci (NK cell bound and unbound) from each sample well and each control well were assessed for the presence of purple MTT-formazan crystals. The percentage of killed, NK cell-bound cryptococci and the percentage of killed cryptococci in the total cell suspension were determined according to the formulas given in the description of FDA vital staining. Analysis of C. neoformans proliferation in NK cell-C. neoformans conjugates. Proliferation of cryptococci in individual NK cell-C. neoformans conjugates was assessed by an agarose slide culture technique that was modified from the method of Grimm et al. (5). NK cells were mixed with cryptococcal target cells at an effector cell-to-target cell ratio of 2:1 in CM, and the mixture was incubated at 37°C in 7% CO2. After a 2-h incubation, the supernatants were removed from the cell mixtures, 1% (wt/vol) agarose in CM at 39°C was added, and the mixture was immediately spread on a microscope slide. After the agarose solidified, NK cell-C. neoformans conjugates were located by light microscopy and a microlocator slide (Baxter Scientific, McGaw Park, Ill.). After incubating the slides for 16 h in CM, the conjugates were relocated and proliferation of bound cryptococcal cells was determined by the formation of microcolonies containing numerous blastoconidia. A minimum of 100 conjugates in each of 4 samples were assessed for the presence
MURINE NK CELLS KILL CRYPTOCOCCI
1749
of proliferating and nonproliferating cryptococci in each experiment. The percentage of cryptococci that were bound to NK cells and were unable to proliferate was determined by the following formula: percent nonproliferating C. neoformans bound to NK cells = (mean number of NK cell-C. neoformans conjugates with nonproliferating cryptococci/ mean number of NK cell-C. neoformans conjugates) x 100. Analysis of C. neoformans proliferation on a solid medium. NK cell-mediated anticryptococcal effects were assessed by measuring cryptococcal proliferation on the surface of a solid growth medium, which is the standard CFU (spread plate) assay previously described by Murphy and McDaniel (20). Briefly, C. neoformans cells were incubated with or without NK cells for 18 h, and then the content of each experimental (C. neoformans and NK cells) and each control (C. neoformans only) well was serially diluted in sterile physiological saline. Each diluted sample was plated in duplicate on modified Sabouraud agar. After 3 days of incubation at room temperature, CFU were enumerated and the percentage of cryptococcal growth inhibition was determined by the following formula: % cryptococcal growth inhibition = [(mean control CFU - mean experimental CFU)/mean control CFU] x 100. Extinction dilution analysis of C. neoformans proliferation in liquid medium. In contrast to CFU counts which depended upon proliferation of C. neoformans on solid medium, extinction dilution analysis was used to assess NK cell-mediated anticryptococcal effects by measuring proliferation of cryptococci in broth culture following the 18-h incubation with NK cells. The contents of each experimental well and each control well were diluted, beginning with a 1:100 dilution and followed by seven serial fivefold dilutions in nutrient broth (pH 5.8) consisting of 1.0% (wt/vol) neopeptone, 1.0% (wt/vol) glucose, 0.2% (wt/vol) yeast extract, and 0.5% (wt/vol) sodium chloride. Then, 0.1 ml of the contents of each dilution was dispensed into each of five wells of a V-bottom, 96-well microtiter plate. After the plate was incubated for 3 days at room temperature, the number of replicates per dilution having cryptococcal growth, as assessed by the formation of a pellet of C. neoformans cells in the bottom of the well, was determined. The number of replicate wells with cryptococcal growth and the dilution were used to determine the number of proliferating cryptococcal cells in the experimental and control wells at the end of the 18-h incubation by using the most-probable-number method of statistical analysis described by Russek and Colwell (22). The percentage of cryptococcal inhibition was calculated by the following formula: percent C. neoformans inhibition = [(mean viable cryptococci in control wells mean viable cryptococci in experimental wells)/mean viable cryptococci in control wells] x 100. Statistical analysis. Means and standard errors of the means were calculated, and the two-tailed Student t test was used to analyze the data. RESULTS Comparisons of vital stains and proliferation assays for distinguishing between viable and nonviable C. neoformans at the individual cell level. The detection of viable cryptococci by the two vital staining methods was compared with results obtained when CFU counts on the surface of a solid medium and within agarose were used to detect proliferating cryptococci. For this comparison, a known number of proliferating cryptococci, determined on the basis of a CFU count, was mixed with a known number of heat-killed cryptococci,
INFECT. IMMUN.
HIDORE ET AL.
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TABLE 1. Detection of viable C. neoformans by MTT and FDA staining and comparison with proliferation of cryptococci as assessed by CFU counts in agarosea Theoretical % of viable
%
C. neoformansb 100 ± 7 75
Viable C. neoformans
%
bC
MTT stain
95 83
0 1
FDA stain
94 75
±
±
1 3
% Killed C. neorormans
In Conjugates
Experimental Method
110
Proliferating
210
310 410
j
j
510 610
efras
C. neoformans
90 71
±
±
2 3
50
55±1
59±1
50±1
25 0
33 2 4±1
28 ± 3 1±1
28 ± 1 0±0
Values represent the mean + the standard error of the mean of quadruplicate samples. Data are representative of three experiments. b Mixtures of proliferating and heat-killed cryptococci were based on CFU counts (spread plate). c As determined by CFU counts in agarose.
CFU Count
MTT
|
Stain j
a
determined by hemacytometer counts. Samples of each mixture were stained with MTT or FDA, and the efficacies of the stains for detecting viable C. neoformans were determined by comparing the percentage of stained cryptococci with the expected percentage of viable cells, determined on the basis of the original proportions of proliferating and heat-killed C. neoformans that were mixed together. In addition, samples of each cryptococcal cell mixture were resuspended in agarose and placed on a microscope slide. After the slides were incubated for 18 h at 37°C in CM, the efficacy of the agarose method for detecting proliferating C. neoformans cells was determined by comparing the percentage of proliferating cryptococci in agarose to the expected percentage of proliferating cells determined on the basis of the proportions of the original mixtures. The data in Table 1 show that given the standard error of the assays, detection of viable cryptococci by the MTT and FDA staining techniques was equivalent to detection of proliferating cryptococci by the agarose method. In addition, the percentage of viable cryptococci detected by each of the three methods was comparable to the expected percentages of viable cells based on CFU counts obtained by the spread plate technique (Table 1). Comparisons of methods for detecting C. neoformans proliferation in total cell suspensions. Since CFU counts on the surface of Sabouraud agar plates have been our standard assay for determining NK cell-mediated cryptococcal damage, detection of cryptococcal proliferation in broth culture by extinction dilution analysis was compared with detection of proliferating cryptococci by CFU counts. Although both CFU counts and extinction dilution analysis measure cryptococcal proliferation, these two assays differ in that CFU counts assess proliferation of cryptococci on a solid medium, whereas extinction dilution analysis measures cryptococcal proliferation in broth culture. A cryptococcal cell suspension which contained 3 x 105 cryptococci per ml by hemacytometer counts was diluted and assayed in parallel for detection of proliferation by CFU counts and extinction dilution analysis. The CFU counts showed that the suspension contained 3.5 x 105 + 0.3 x 105 cryptococci per ml, and the extinction dilution results demonstrated that the suspension contained 4.4 x 105 + 0.6 x 105 cryptococci per ml. Given the standard error of the assays, the CFU counting and the extinction dilution method were equivalent in their efficacies of detecting proliferating cryptococci. NK cell-mediated damage of individual C. neoformans target cells in conjugates. Once the efficacy of vital stains (MTT and FDA) for distinguishing between viable and dead C.
FDA Stain
1
FIG. 1. Percentage of killed C. neoformans target cells bound to NK cells. After incubating NK cells with cryptococcal target cells for 18 h, the percentage of killed cryptococci that were bound to the effector cells was assessed by the MTT and FDA vital stains, and the results were compared with those obtained by using the single cell proliferation assay in agarose. Data are representative of three experiments. Bars represent the mean + standard error of the mean
of quadruplicate samples.
neoformans cells was established, these techniques were then applied to the assessment of NK cell-mediated cryptococcal damage and the results were compared with those obtained by using proliferation assays (CFU counts and extinction dilution analysis). After NK cells were incubated with cryptococcal target cells, the viability of cryptococci in conjugates with NK cells was assessed at the single conjugate level. The percentages of cryptococci that were bound to NK cells and that could (i) stain with MTT, (ii) stain with FDA, or (iii) form microcolonies in agarose were determined, and from these data, the percentages of killed and nonproliferating C. neoformans cells bound to NK cells were calculated (Fig. 1). Approximately 45% of the cryptococci that were bound to NK cells were unable to stain with MTT or FDA, indicating that 45% of the bound cryptococci were killed. Similarly, 45% of the C. neoformans cells bound to NK cells were unable to proliferate (Fig. 1). The results show that the three techniques were comparable for determining the level of cryptococcal killing in single conjugates (Fig. 1). In addition, the vital stains demonstrated that dead C. neoformans cells (nonstaining cryptococci) that were not bound to NK cells were present in the cell suspensions. To further analyze the effects of NK cells on the total C. neoformans population, studies were designed to assess the percentage of killed cryptococci (unbound and bound to NK cells) present in the NK cell-C. neoformans suspension. Effects of NK cells on C. neoformans target cells in the total cryptococcal cell population. After NK cells were incubated with cryptococci for 18 h, the numbers of viable cryptococci in wells containing NK cells and cryptococci and in wells containing cryptococci alone were assessed with the vital staining assays, and the results were compared with those obtained by using proliferation assays. Data in Figure 2 demonstrate a strong correlation between the percentage of NK cell-mediated cryptococcal killing detected by MTT staining and FDA staining and the percentage of nonproliferating cryptococci detected by CFU counts. Approximately 26% of the total cryptococcal population, both NK cell
VOL. 59, 1991
MURINE NK CELLS KILL CRYPTOCOCCI 4
Experimental
% C. neoformans Killing
Method
3
~01 :0- .
2
.
o
C 01
IE
-0
1,0
210
310
410
510
C
610
!0.10.
iiI-
1751
--
4 10,
CFU Count
o a ca . C
E
MTT Stain
I0. 20
C
0
cp
No. _C
4 I
.C
10-20-
FDA Stain
---
a2
Extinction Dilution
FIG. 2. Percentage of C. neoformans killing in the total cell suspension. After incubating NK cells with cryptococcal target cells for 18 h, the percentage of killed cryptococci in samples containing effector cells and target cells compared with that in samples containing cryptococci alone was assessed by using the MTT and FDA vital stains. C. neoformans viability, as assessed by vital stains, was compared with proliferation of cryptococci, as determined by CFU counts and extinction dilution analysis. Data are representative of three experiments. Bars represent the mean ± the standard error of the mean of quadruplicate samples.
bound and unbound, did not have the enzymatic activities necessary for labeling with MTT or FDA and had a reduced ability to proliferate, compared with the cryptococcal control suspensions. In contrast, the extinction dilution method of detecting proliferation of cryptococci in broth culture consistently indicated a percentage of nonproliferating cryptococci (61% + 5%) significantly (P < 0.001) greater than the level of cryptococcal killing indicated by MTT staining, FDA staining, or CFU counts on the surface of solid medium (25 to 27%) (Fig. 2). Kinetics of NK cell-C. neoformnans binding in comparison to NK cell-mediated killing of cryptococci. In previous studies, NK cell-C. neoformans conjugate formation and NK cellmediated anticryptococcal activity were determined in two different assays (19-21). NK cell-C. neoformans conjugate formation was assessed by enumerating conjugates by light microscopy after incubating NK cells with cryptococci at an effector cell-to-target cell ratio of 2:1, whereas NK cellmediated cryptococcal growth inhibition was determined by using CFU counts after incubating NK cells with cryptococci at an effector cell-to-target cell ratio of 50:1 (20, 21). Therefore, to compare the kinetics of NK cell-C. neoformans binding and NK cell-mediated killing of C. neoformans in single conjugates and in the total cryptococcal cell population within a single assay, effector and target cells were mixed at an effector cell-to-target cell ratio of 2:1 and incubated at 37°C. Samples were removed at 2-h intervals throughout the 18-h incubation and were stained with MTT (Fig. 3). Three parameters of NK cell-C. neoformans interactions were determined at each interval: (i) percentage of C. neoformans killed in the total cell suspension (Fig. 3A), (ii) percentage of dead C. neoformans in conjugate with NK cells (Fig. 3B), and (iii) percentage of effector cells conju-
*
.
*
2
4
6
.
.
.
*
*
10
12
14
16
i8
Ti;ne (h) FIG. 3. Kinetics of NK cell-C. neoformans conjugate formation and NK cell-mediated killing of cryptococci at the single conjugate level and in cell suspension. C. neoformans viability, as determined by MTT staining, and NK cell-C. neoformans conjugate formation were assessed at 2-h intervals after mixing the effector and target cells. Data are representative of two experiments. Bars represent the mean + the standard error of the mean of quadruplicate samples.
gated to C. neoformans cells (Fig. 3C). A gradual but steady increase in the percentage of C. neoformans killing in the total cell suspension (Fig. 3A) paralleled a similar increase in the percentage of NK cell-C. neoformans conjugates during the first 12 h of incubation (Fig. 3C). Optimal levels of both cryptococcal conjugate formation and C. neoformans killing in the total cell suspension occurred between 12 and 18 h after mixing the effector and target cells. In contrast to the steady increase in C. neoformans killing in the total cell suspension throughout the experiment (Fig. 3A), the percentage of killed target cells within NK cell-C. neoformans conjugates reached nearly optimal levels by 6 h after mixing the effector and target cells and remained relatively constant throughout the remainder of the 18-h incubation (Fig. 3B). DISCUSSION Previous studies in our laboratory have demonstrated that murine NK cells bind to and inhibit the growth of the fungal target cell C. neoformans in vitro and contribute to clearance of the organism in vivo (7-10, 17, 19-21). Considering the facts that NK cells kill tumor cell targets and that many similarities between NK cell-C. neoformans interactions and NK cell-tumor cell interactions have been demonstrated, it seemed likely that the NK cell-mediated growth inhibition that we have previously reported was actually a measure of cryptococcal killing. However, our previous studies were based on a single type of proliferative assay and therefore did not clearly establish whether the anticryptococcal effects of NK cells were fungistatic or fungicidal. The question of whether NK cells actually kill cryptococci is of primary importance in determining the potential role of NK cells in the complex interactions that result in host resistance to this pathogen. Therefore, the purpose of the study presented here was to determine whether NK cells kill cryptococcal target cells. To evaluate the fungicidal capacity of NK cells for cryptococci, the viability of both cryptococci that were bound to NK cells and cryptococci that were unbound had to be
1752
HIDORE ET AL.
determined; therefore, techniques for assessing cryptococcal viability at the single cell level and in the entire cell suspension were required. Vital stains have been reported to be reliable quantitative assays for determining the viability of C. neoformans cells (2, 4, 12, 13). Before these methods were applied to the assessment of NK cell-mediated anticryptococcal effects, initial experiments were performed to determine the efficacies of the vital stains for distinguishing between dead and live cryptococci at the single cell level and in the total cell suspension. In addition, detection of viable cryptococci by the vital stains was compared to detection of proliferating C. neoformans cells both at the individual cell level and in the total cell suspension. Two distinct approaches using vital stains that are indices of intact metabolic function within viable cells were employed to determine cryptococcal viability. MTT and FDA, the two vital stains used in this study, reflect the metabolic function of two different enzymes within viable cells, mitochondrial dehydrogenases and esterases, respectively. Our study demonstrated that the MTT and FDA vital stains effectively distinguished between live and dead cryptococci in suspensions containing known percentages of viable and heat-killed C. neoformans (Table 1). Furthermore, these studies also demonstrated that the agarose slide CFU assay for assessing proliferation of cryptococci was comparable to the vital stains and to CFU counts on the surface of solid medium for the detection of proliferating C. neoformans (Table 1). In the total cell suspension, this study demonstrated that CFU counts on the surface of a solid medium and extinction dilution analysis (broth culture) were reliable and comparable methods for the detection of proliferating C. neoformans cells in suspensions containing known proportions of viable and heat-killed cryptococci. Since MTT and FDA vital stains were demonstrated to be reliable indices of C. neoformans viability, these techniques were used to assess NK cell-mediated anticryptococcal effects at the single conjugate level and in the total cell suspensions. In addition, following incubation with NK cells, cryptococcal viability as determined by the vital stains was compared with proliferation of cryptococci at the individual cell level (CFU counts within agarose) and in the total cell suspensions (CFU counts on the surface of a solid medium and extinction dilution analysis). When the viability of individual cryptococci was assessed at the single conjugate level following an 18-h incubation with NK cells, approximately 45% of the C. neoformans target cells attached to NK cells were dead, as determined by the inability to stain with either of the two vital stains, MTT and FDA (Fig. 1). Our data are consistent with reports on the percentage of dead tumor cells in conjugates with NK cells, which ranges from 14 to 52%, depending on the type of tumor cell target (24). In correlation with the vital stains, approximately 45% of the cryptococci attached to NK cells were unable to proliferate in agarose, suggesting that the inability to proliferate was also indicative of cryptococcal cell death. Together, these data clearly indicate that NK cells kill a portion of the bound C. neoformans cells. The percentage of viable cryptococci in the total NK cell-C. neoformans cell suspension following an 18-h incubation was also assessed. Approximately 30% of the total population of cryptococci (both NK cell bound and unbound) in the samples (containing NK cells and cryptococci), compared with the control samples (containing cryptococci alone), were killed, i.e., they did not have the enzymatic activities required for staining with MTT or FDA (Fig. 1). In addition, approximately 30% of the total crypto-
INFECT. IMMUN.
coccal population in the experimental samples, compared with the control samples, were unable to proliferate on a solid medium (CFU counts), again indicating that the inability to proliferate was also a measure of cell death. These data support the data obtained at the single conjugate level in that three different approaches for measuring both metabolic function and proliferation of cryptococci indicated that NK cells kill C. neoformans target cells in the total cell suspension. Although vital stains in conjunction with CFU counts demonstrated that approximately 30% of the cryptococci in the total cell suspension were killed, extinction dilution analysis consistently indicated that NK cells inhibited proliferation of approximately 61% of the cryptococci in the effector cell-target cell suspension compared with the number of proliferating C. neoformans in the control suspensions. Because CFU counts and extinction dilution analysis were equally effective in distinguishing between viable and heat-killed cryptococci in our initial studies, these data suggest that NK cell-mediated anticryptococcal effects alter the ability of the cryptococcal cells to proliferate in a broth culture (extinction dilution analysis) in comparison to their ability to proliferate on a solid medium (CFU counts). This hypothesis is supported by studies indicating that injured microorganisms initiate specific repair mechanisms that are most effective in determining survival of the organism when optimal culture conditions are available during repair (1, 16). Therefore, it is likely that cryptococci damaged by NK cells proceed with a repair process that favors survival of the damaged cryptococci only under optimal culture conditions. If transferring cryptococci from a cell suspension to a solid culture medium, such as Sabouraud agar, provides optimal culture conditions for repair and ultimately proliferation of damaged but not-yet-dead cryptococci, then CFU counts on the surface of solid medium are an index of cryptococci that are dead at the end of the 18-h incubation. In contrast, if transferring the cryptococci from the cell suspension into a second liquid culture, as occurs in extinction dilution analysis, is unfavorable for repair of injured cryptococci, then the cryptococci that were damaged but not killed at the end of the 18-h incubation might continue the process, leading to cell death as a result of inefficient repair in liquid medium. Therefore, extinction dilution analysis would detect both cryptococci that were killed at the end of 18 h and cryptococci that were damaged by 18 h and that then proceeded to cell death in the broth culture. Our data comparing killing of cryptococci in the total cell suspension as determined by CFU counts (30%) and by extinction dilution analysis (61%) are in accordance with this hypothesis, which predicts that the level of NK cell-mediated cryptococcal killing would be greater when assessed by extinction dilution analysis than when determined by CFU counts (Fig. 2). These data indicate that CFU counts, in conjunction with MTT and FDA vital stains, are reliable indices of NK cell-mediated cryptococcal killing at a particular time after mixing effector and target cells, whereas extinction dilution analysis may detect a combination of damaged and killed cryptococci at a particular time in culture but more accurately reflects total NK cell-mediated anticryptococcal effects. In previous studies, NK cell binding to cryptococci was assessed at the single cell level by light microscopy, whereas NK cell-mediated anticryptococcal effects were measured in the cell suspension by CFU counts (19-21). To compare the kinetics of NK cell binding with killing of cryptococci at the single conjugate level and in the cell suspension within a single assay, samples were stained with MTT at 2-h intervals
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after mixing the effector cells and target cells (Fig. 3). Comparisons of the kinetics of (i) cryptococcal killing in the total cell suspension, (ii) killing of cryptococci bound to NK cells, and (iii) NK cell binding to C. neoformans indicate that NK cell-C. neoformans binding precedes killing of cryptococcal target cells. The similarities in the kinetics of killing of C. neoformans in the total cell suspension as measured by MTT staining and the kinetics of NK cell-mediated cryptococcal growth inhibition demonstrate that growth inhibition assays actually reflect the level of NK cell-mediated killing of C. neoformans. Thus, the data previously reported as NK cell-mediated cryptococcal growth inhibition are indicative of C. neoformans killing by NK cells (7-9, 17, 19-21). When the viability of individual cryptococci was assessed at the single conjugate level following an 18-h incubation with NK cells, approximately 30% of the NK cells were
bound to C. neoformans target cells (Fig. 3C) and only 45% of the bound target cells were killed (Fig. 1). There are several factors that could be responsible for these observations. First, it is possible that all of the effector cells in the population are not capable of binding to cryptococci. Alternatively, all of the effector cells may be capable of cryptococcal binding, but at any given time in an unsynchronized cell population, cryptococci might be attached to only a portion of the potential target-binding cells and only a fraction of the attached cryptococci would be killed. A further explanation for the observation that 100% of the bound cryptococcal target cells were not killed is that only a percentage of the NK cells that bind to cryptococci are capable of killing the organism. However, the present study did not allow us to determine whether either of these possible interpretations of the data is correct. In addition to observing that cryptococci attached to NK cells were killed, we also noted that cryptococci not bound to effector cells in the total cell suspension were dead, as determined by the lack of metabolic functions and proliferation. The demonstration of unbound, killed cryptococci in cell suspensions suggests at least two possible interpretations of the data. First, it is possible that NK cells may detach from killed cryptococcal target cells and recycle to kill subsequent target cells. This phenomenon of a single effector cell recycling to kill numerous target cells has been demonstrated in the model of NK cell-mediated tumor cell cytotoxicity (24). Further support for recycling of effector cells in the C. neoformans model is provided by the kinetic pattern of killing of bound cryptococci (Fig. 3B) in comparison to the pattern of killing of cryptococci in the total cell suspension (Fig. 3A). If both initial binding events and effector cell recycling are occurring simultaneously, as would be expected in an unsynchronized cell suspension, this might result in the observed gradual increase in the total number of dead cryptococcal cells in the suspension throughout the incubation while the percentage of dead, NK cell-bound cryptococci reached optimal and relatively constant levels after 6 h of incubation (Fig. 3A and B). A second, but not mutually exclusive, explanation for the observation of unbound, killed cryptococci in the total cell suspensions is that NK cells may kill unbound target cells through the release of soluble cytotoxic factors. Since NK cells have been shown to secrete or exocytose numerous cytotoxic substances (6, 23), it is possible that killing of bound cryptococci results in the release of cytotoxic components that also kill unbound cryptococci in the suspension. The data presented here are not sufficient to establish whether effector cell recycling or killing of cryptococcal targets via soluble cytotoxic factors or both are responsible for our observa-
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tions. However, in conjunction with our previous data which indirectly suggest that recycling occurs in the NK cell-C. neoformans model and clearly demonstrate that soluble cytoplasmic components in addition to the cytotoxic granules isolated from NK cells inhibit cryptococcal growth (9, 10, 20, 21), we favor the hypothesis that both mechanisms (effector cell recycling and killing of unbound cryptococci by soluble cytotoxic components) may be involved in NK cell-C. neoformans interactions. In conclusion, measures of metabolic function, the MTT and FDA vital stains, demonstrated that NK cells kill bound C. neoformans targets at the individual conjugate level and in the total cell suspension. These results were confirmed by studies which demonstrated that NK cells inhibited proliferation of cryptococci at the individual conjugate level and in the total cell suspensions. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans mixtures. First, these data indicate the possibility that NK cells bind to and kill a cryptococcal target cell and then recycle to kill subsequent cryptococcal targets. A second, but not mutually exclusive, possibility suggested by these data is that NK cells release undefined soluble cytotoxic factors which kill both bound and unbound C. neoformans cells. Taken together, the study presented here firmly establishes that murine NK cells kill C. neoformans target cells. ACKNOWLEDGMENT This work was supported by Public Health Service grant AI-18895 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Beuchat, L. R. 1984. Injury and repair of yeasts and moulds, p. 293-308. In M. H. E. Andrew and A. D. Russell (ed.), The revival of injured microbes. Academic Press, Inc., Orlando, Fla. 2. Calich, V. L. G., A. Purchio, and C. R. Paula. 1978. A new fluorescent viability test for fungi cells. Mycopathologia 66:175177. 3. Cochran, W. G. 1950. Estimation of bacterial densities by means of the most probable number. Biometrics 5:105-116. 4. Correa, B., A. Purchio, C. R. Paula, and W. Gambale. 1986. Evaluation of a fluorescent method (fluorescein diacetate and ethidium bromide solution) in the study of viable Cryptococcus neoformans strains. Mycopathologia 96:91-96. 5. Grimm, E. A., J. A. Thomas, and B. Bonavida. 1979. Mechanism of cell-mediated cytotoxicity at the single cell level. II. Evidence for first-order kinetics of T cell-mediated cytotoxicity and for heterogeneity of lytic rate. J. Immunol. 123:2870-2877. 6. Herberman, R. B., C. W. Reynolds, and J. R. Ortaldo. 1986. Mechanism of cytotoxicity by natural killer (NK) cells. Annu. Rev. Immunol. 4:651-680. 7. Hidore, M. R., and J. W. Murphy. 1986. Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool nonadherent cells. Infect. Immun. 51:547-555. 8. Hidore, M. R., and J. W. Murphy. 1987. Natural cellular resistance of beige mice against Cryptococcus neoformans. J. Immunol. 137:3624-3631. 9. Hidore, M. R., and J. W. Murphy. 1989. Murine natural killer cell interactions with a fungal target, Cryptococcus neoformans. Infect. Immun. 57:1990-1997. 10. Hidore, M. R., N. Nabavi, C. W. Reynolds, P. A. Henkart, and J. W. Murphy. 1990. Cytoplasmic components of natural killer cells inhibit the growth of Cryptococcus neoformans. J. Leukocyte Biol. 48:15-26. 11. Julius, M. H., E. Simpson, and L. A. Herzenberg. 1973. A rapid method for isolation of functional thymus-derived lymphocytes. Eur. J. Immunol. 3:645-649. 12. Lapage, S. P., J. E. Shelton, and T. G. Mitchell. 1970. Methods
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in microbiology, p. 1-133. Norris Ribbons, London. 13. Levitz, S. M., and R. D. Diamond. 1985. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J. Infect. Dis. 152:938-945. 14. Lipscomb, M. F., T. Alvarellos, G. B. Toews, R. Tompkins, Z. Evans, G. Koo, and V. Kumar. 1987. Role of natural killer cells in resistance to Cryptococcus neoformans infections in mice. Am. J. Pathol. 128:354-361. 15. Luini, W., D. Baraschi, S. Alberti, A. Aleotti, and A. Tagliabue. 1981. Morphological characterization of a cell population responsible for natural killer activity. Immunology 43:663-668. 16. Mackey, B. M. 1984. Lethal and sublethal effects of refrigeration, freezing and freeze-drying on microorganisms, p. 45-75. In M. H. E. Andrew and A. D. Russell (ed.), The revival of injured microbes. Academic Press, Inc., Orlando, Fla. 17. Murphy, J. W. 1989. Natural host resistance mechanisms against systemic mycotic agents, p. 149-184. In C. W. Reynolds and R. H. Wiltrout (ed.), Functions of the natural immune system. Plenum Publishing Corp., New York. 18. Murphy, J. W., and G. C. Cozad. 1972. Immunological unre-
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19. 20. 21.
22.
23. 24.
sponsiveness induced by cryptococcal capsular polysaccharide assayed by the hemolytic plaque technique. Infect. Immun. 5:896-901. Murphy, J. W., M. R. Hidore, and N. Nabavi. 1991. Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans. Infect. Immun. 59:1476-1488. Murphy, J. W., and D. 0. McDaniel. 1982. In vitro reactivity of natural killer (NK) cells against Cryptococcus neoformans. J. Immunol. 128:1577-1583. Nabavi, N., and J. W. Murphy. 1985. In vitro binding of natural killer cells to Cryptococcus neoformans targets. Infect. Immun. 50:50-57. Russek, E., and R. R. Colwell. 1983. Computation of most probable numbers. Appl. Environ. Microbiol. 45:1646-1650. Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187-375. UllHberg, M., and M. Jondal. 1981. Recycling and target binding capacity of human natural killer cells. J. Exp. Med. 153:615628.
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Murine Natural Killer Cells Are Fungicidal to Cryptococcus neoformans MICHELLE R. HIDORE,l NASRIN NABAVI,2 FRANK SONLEITNER,3 AND JUNEANN W. MURPHY'* Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731901; Department of Immunopharmacology, Hoffman-LaRoche, Nutley, New Jersey 071102; and Department of Zoology, University of Oklahoma, Norman, Oklahoma 730193 Received 10 December 1990/Accepted 22 February 1991 Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus
neoformnans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neofornans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans.
cocci; therefore, inhibition of cryptococcal growth has been used as an indication of NK cell-mediated damage to the cryptococcal cell. Our previous studies, in which CFU counts were used as an index of NK cell-mediated cryptococcal damage, have clearly demonstrated that murine NK cells inhibit proliferation of cryptococcal target cells (7, 9, 19-21). It is not clear from those investigations, however, whether the anticryptococcal effects of NK cells are fungistatic or fungicidal; therefore, we have referred to the anticryptococcal activity of NK cells as growth inhibition rather than killing. The issue of whether NK cells actually kill cryptococci is of primary importance in determining the potential role of these effector cells in host resistance to this pathogen, so the study presented here was designed to establish whether NK cells only prevent proliferation of C. neoformans or whether they actually kill the organism. To accurately assess NK cell-mediated effects on C. neoformans viability, it was necessary to determine the viability of both cryptococci that were bound to NK cells and cryptococci that were unbound in the cell suspension. Therefore, vital stains which could be used to assess cryptococcal viability at the individual cell level and simultaneously in the entire cell suspension were employed in this study. Vital stains that detect intact metabolic function have previously proven to be rapid and reliable indices of the viability of fungi such as C. neoformans, Candida albicans, Aspergillus fumigatus, and Rhizopus oryzae (2, 4, 13). The vital stains chosen for these studies measure the metabolic function of two different enzyme systems, mitochondrial
Previous studies in our laboratory have focused on murine natural killer (NK) cell interactions with a fungal target, Cryptococcus neoformans. C. neoformans is an encapsulated, yeastlike organism which causes human infection via the pulmonary route, ranging from an asymptomatic upper respiratory infection which is often spontaneously resolved by the host to a potentially fatal disseminated cryptococcosis which is generally manifested as meningitis. The low incidence of dissemination relative to the high frequency of exposure to this ubiquitous organism suggests a role for innate resistance mechanisms in host defense against cryptococci. Previous studies by us (7-10, 17, 19-21) and by others (14) indicate that NK cells function in conjunction with other natural resistance mechanisms in the first-line host defense against cryptococci. We have shown that binding of NK cells to C. neoformans target cells is prerequisite to the events that result in cryptococcal cell damage (9, 19, 21). Furthermore, we have demonstrated that murine NK cells not only inhibit the growth of C. neoformans in vitro but also contribute to clearance of the organism in vivo
(7, 8).
C. neoformans is structurally distinct from the standard tissue or tumor cell target used in the model of NK cellmediated cytotoxicity (17, 19, 21). Although tumor cell lysis is the standard measure of NK cell-mediated tumor cytotoxicity, the cryptococcal cell wall precludes lysis of crypto*
Corresponding author. 1747
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dehydrogenases and esterases, within viable fungal cells. One of the vital stains, the yellow tetrazolium salt [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), readily enters C. neoformans cells and is then reduced by the mitochondrial dehydrogenases within viable cells to its purple formazan derivative. This results in the accumulation of clearly visible purple MTT-formazan crystals within viable cryptococci when viewed by light microscopy, whereas dead cells, which do not reduce MTT, do not accumulate the purple formazan crystals (13). The other vital stain used as a means of assessing viability of C. neoformans was the fluorogenic substrate fluorescein diacetate (FDA) (2, 4). FDA is a nonpolar compound that readily enters the cell and is then hydrolyzed by esterases within viable cryptococci to a polar compound which is unable to exit the cell and thus accumulates within viable cryptococci, resulting in fluorochromasia (4). To compare cryptococcal viability, as determined by the vital stains, with the ability of the cells to proliferate, three approaches for assessing proliferation of cryptococci at the individual cell level and in the total cell suspension were employed in this study. One method used for assessing C. neoformans proliferation, CFU counts on the surface of a solid medium (12), is a common spread plate technique for determining proliferation of cryptococci and has been our standard method for measuring NK cell-mediated anticryptococcal effects (20). To quantify proliferation of individual cryptococci associated with NK cells or free in the suspension, the procedure of Grimm et al. (5) was modified to assess cryptococcal CFU counts within agarose. This technique is equivalent to pour plate methods for enumerating viable organisms, with the exception that the colonies are detected microscopically. In addition to colony formation on the surface of or within solid medium, extinction dilution analysis, a method for assessing proliferation of a microorganism in broth culture, was also used (3, 22). Initial comparisons of the vital stains and the proliferation assays indicated that the techniques employed effectively distinguished between live and heat-killed cryptococci at the individual cell level and in total cell suspensions. Following an 18-h incubation with NK cells, C. neoformans viability was assessed by using the vital stains, and the results were compared with NK cell-mediated growth inhibition detected by the proliferation assays. At the single-cell level, the viability of cryptococci incubated with NK cells, as assessed by vital stains, was significantly diminished, compared with that of the control samples containing cryptococci without NK cells. In the total cell suspensions, vital stains confirmed
these results. At the individual cell level and in the total cell suspensions, the numbers of killed cryptococci in the NK cell-C. neoformans suspensions, as determined by the vital stains, were comparable to the numbers of cryptococci that were unable to proliferate. Together, our data clearly demonstrate that NK cells are fungicidal to C. neoformans
targets. MATERIALS AND METHODS Mice. Female CBA/J mice were obtained from Jackson Laboratories, Bar Harbor, Maine. The animals were maintained in the University of Oklahoma animal facility until they were used for these studies at 7 to 8 weeks of age. Reagents. Stock solutions (5 mg/ml) of the tetrazolium dye MTT in RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) and of fluorescein diacetate (FDA) in acetone were prepared. The MTT solution was stored in the dark at
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40C, and the FDA solution was stored at -200C. Both reagents were purchased from Sigma Chemical Co., St. Louis, Mo. Fungal target. C. neoformans isolate 184A was maintained on modified Sabouraud agar slants (18). After 3 days of growth at room temperature, blastoconidia were harvested, washed three times in sterile physiological saline, and adjusted to the desired cell concentration for each experiment with complete medium (CM) consisting of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U of penicillin per ml, and 100 pRg of streptomycin per ml. Cell concentrations were based on hemacytometer counts and confirmed by determining CFU on modified Sabouraud agar plates. Heat-killed C. neoformans cells were prepared by incubating cryptococci (10' cells per ml) in sterile physiological saline at 560C for 60 min. A sample of the heat-treated cells was plated on modified Sabouraud agar to confirm, by the absence of proliferation, that the cryptococcal cells had been killed. Effector cells. Murine, splenic, nylon wool-nonadherent cells were obtained by the method of Julius et al. (11) and then further enriched for NK cells on discontinuous Percoll gradients by a method that has been previously described in detail (15, 21). With this protocol, the cytolytic activity against YAC-1, the standard tumor cell target of murine NK cells, is routinely significantly enriched in the cell fractions from the low-density Percoll layers (fractions 1 and 2) compared with the anti-YAC-1 activity of the input nylon wool-nonadherent cell population. Typically, the nylon wool-nonadherent spleen cell populations that we collect contain approximately 75% Thy-l' cells, 2 to 3% mouse Ig' cells, 1 to 2% nonspecific esterase-positive cells, 9 to 10% asialo GM,' cells, and 6% large granular lymphocytes (cells with NK cell morphology and detectable granules in the cytoplasm), whereas Percoll fraction 1 and 2 cell populations contain 20% Thy-l' cells, no detectable mouse Ig + or nonspecific esterase-staining cells, 40% asialo GM,' cells, and 36% large granular lymphocytes (21). In a previous study, we showed with scanning electron microscopy that the Percoll fraction 1 and 2 effector cells which bound to C. neoformans target cells were asialo GM,-positive cells (21). For convenience throughout the remainder of this report, we have referred to the Percoll fraction 1 and 2 cells which are highly enriched for NK cells as NK cells. NK cell fractions were adjusted to the appropriate concentration for each assay in CM and used as' effector cells in these studies. Incubation of NK cells with C. neoformans target cells. The optimal conditions required for murine NK cells to mediate anticryptococcal effects have been previously described (9, 17, 21). Briefly, 106 NK cells suspended in 0. 1 ml of CM and 5 X 1O5 cryptococcal target cells in 0.1 ml of CM were added to quadruplicate wells of a flat-bottom, 96-well microtiter plate (Linbro Scientific Co., Hamden, N.Y.). Quadruplicate control wells contained 0. 1 ml of the cryptococcal target cell suspension and 0. 1 ml of CM. After the plate was incubated for 18 h at 370C in 7% C02, viability and proliferation of cryptococci in the experimental and control wells were assessed by the methods described below. The generation time for C. neoformans isolate 184A in control wells was 3.5 h when determined by CFU counts. FDA vital staining of C. neoformans cells. Fluorescence staining of viable cryptococci with the fluorogenic substrate FDA was used to measure NK cell-mediated anticryptococcal activity in single conjugates and in total cell suspensions simultaneously (2, 4). The contents of each well containing either NK cells and cryptococci or cryptococci alone were
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washed in phosphate-buffered saline (PBS) and resuspended in 0.2 ml of PBS containing 0.01 ml of the FDA stock solution, resulting in a final concentration of 0.05 mg of FDA per ml. After a 2-h incubation at 37°C in 7% CO2 in the dark, the cells were washed with PBS and examined with an Olympus BH-2 microscope equipped with phase contrast optics, epifluorescence illumination, and fluorescein fluorescence optics (Olympus Corp., Lake Success, N.Y.). For determination of cryptococcal viability at the single cell level, a minimum of 100 conjugates from each of the four experimental wells were assessed for the presence of green fluorescent (viable) cryptococci. The percentage of killed NK cell-bound cryptococci was calculated according to the following formula: percent killed C. neoformans in conjugates = [(mean number of conjugates - mean number of conjugates containing viable cryptococci)/mean number of conjugates] x 100. For determination of the total number of viable cryptococci in the wells, a minimum of 200 cryptococci (unbound and bound to NK cells) from each experimental well and each control well were scored for the presence of green fluorescence (viable cells). The percentage of killed cryptococci in the total cell suspensions was calculated according to the following formula: percent C. neoformans killed = [(mean percent viable cryptococci in control wells - percent viable cryptococci in experimental wells)/ mean percent viable cryptococci in control wells] x 100. MTT vital staining of C. neoformans cells. A second vital stain, the tetrazolium dye MTT, was used for assessing NK cell-mediated anticryptococcal effects in single conjugates and in total cell suspensions simultaneously (13). The contents of each well containing either NK cells and cryptococci or cryptococci alone were washed in serum-free RPMI 1640 medium and resuspended in 1.0 ml of RPMI 1640 containing 0.5 mg of MTT. After a 3-h incubation at 37°C in 7% CO2, the cells were washed in serum-free RPMI 1640 and examined by light microscopy and phase contrast optics. For determination of cryptococcal viability at the single cell level, a minimum of 100 conjugates from each of the 4 experimental wells were scored for the presence of viable cryptococci, i.e., cryptococci containing purple MTT-formazan crystals. For determination of the total number of viable cryptococci in the wells, a minimum of 200 cryptococci (NK cell bound and unbound) from each sample well and each control well were assessed for the presence of purple MTT-formazan crystals. The percentage of killed, NK cell-bound cryptococci and the percentage of killed cryptococci in the total cell suspension were determined according to the formulas given in the description of FDA vital staining. Analysis of C. neoformans proliferation in NK cell-C. neoformans conjugates. Proliferation of cryptococci in individual NK cell-C. neoformans conjugates was assessed by an agarose slide culture technique that was modified from the method of Grimm et al. (5). NK cells were mixed with cryptococcal target cells at an effector cell-to-target cell ratio of 2:1 in CM, and the mixture was incubated at 37°C in 7% CO2. After a 2-h incubation, the supernatants were removed from the cell mixtures, 1% (wt/vol) agarose in CM at 39°C was added, and the mixture was immediately spread on a microscope slide. After the agarose solidified, NK cell-C. neoformans conjugates were located by light microscopy and a microlocator slide (Baxter Scientific, McGaw Park, Ill.). After incubating the slides for 16 h in CM, the conjugates were relocated and proliferation of bound cryptococcal cells was determined by the formation of microcolonies containing numerous blastoconidia. A minimum of 100 conjugates in each of 4 samples were assessed for the presence
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of proliferating and nonproliferating cryptococci in each experiment. The percentage of cryptococci that were bound to NK cells and were unable to proliferate was determined by the following formula: percent nonproliferating C. neoformans bound to NK cells = (mean number of NK cell-C. neoformans conjugates with nonproliferating cryptococci/ mean number of NK cell-C. neoformans conjugates) x 100. Analysis of C. neoformans proliferation on a solid medium. NK cell-mediated anticryptococcal effects were assessed by measuring cryptococcal proliferation on the surface of a solid growth medium, which is the standard CFU (spread plate) assay previously described by Murphy and McDaniel (20). Briefly, C. neoformans cells were incubated with or without NK cells for 18 h, and then the content of each experimental (C. neoformans and NK cells) and each control (C. neoformans only) well was serially diluted in sterile physiological saline. Each diluted sample was plated in duplicate on modified Sabouraud agar. After 3 days of incubation at room temperature, CFU were enumerated and the percentage of cryptococcal growth inhibition was determined by the following formula: % cryptococcal growth inhibition = [(mean control CFU - mean experimental CFU)/mean control CFU] x 100. Extinction dilution analysis of C. neoformans proliferation in liquid medium. In contrast to CFU counts which depended upon proliferation of C. neoformans on solid medium, extinction dilution analysis was used to assess NK cell-mediated anticryptococcal effects by measuring proliferation of cryptococci in broth culture following the 18-h incubation with NK cells. The contents of each experimental well and each control well were diluted, beginning with a 1:100 dilution and followed by seven serial fivefold dilutions in nutrient broth (pH 5.8) consisting of 1.0% (wt/vol) neopeptone, 1.0% (wt/vol) glucose, 0.2% (wt/vol) yeast extract, and 0.5% (wt/vol) sodium chloride. Then, 0.1 ml of the contents of each dilution was dispensed into each of five wells of a V-bottom, 96-well microtiter plate. After the plate was incubated for 3 days at room temperature, the number of replicates per dilution having cryptococcal growth, as assessed by the formation of a pellet of C. neoformans cells in the bottom of the well, was determined. The number of replicate wells with cryptococcal growth and the dilution were used to determine the number of proliferating cryptococcal cells in the experimental and control wells at the end of the 18-h incubation by using the most-probable-number method of statistical analysis described by Russek and Colwell (22). The percentage of cryptococcal inhibition was calculated by the following formula: percent C. neoformans inhibition = [(mean viable cryptococci in control wells mean viable cryptococci in experimental wells)/mean viable cryptococci in control wells] x 100. Statistical analysis. Means and standard errors of the means were calculated, and the two-tailed Student t test was used to analyze the data. RESULTS Comparisons of vital stains and proliferation assays for distinguishing between viable and nonviable C. neoformans at the individual cell level. The detection of viable cryptococci by the two vital staining methods was compared with results obtained when CFU counts on the surface of a solid medium and within agarose were used to detect proliferating cryptococci. For this comparison, a known number of proliferating cryptococci, determined on the basis of a CFU count, was mixed with a known number of heat-killed cryptococci,
INFECT. IMMUN.
HIDORE ET AL.
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TABLE 1. Detection of viable C. neoformans by MTT and FDA staining and comparison with proliferation of cryptococci as assessed by CFU counts in agarosea Theoretical % of viable
%
C. neoformansb 100 ± 7 75
Viable C. neoformans
%
bC
MTT stain
95 83
0 1
FDA stain
94 75
±
±
1 3
% Killed C. neorormans
In Conjugates
Experimental Method
110
Proliferating
210
310 410
j
j
510 610
efras
C. neoformans
90 71
±
±
2 3
50
55±1
59±1
50±1
25 0
33 2 4±1
28 ± 3 1±1
28 ± 1 0±0
Values represent the mean + the standard error of the mean of quadruplicate samples. Data are representative of three experiments. b Mixtures of proliferating and heat-killed cryptococci were based on CFU counts (spread plate). c As determined by CFU counts in agarose.
CFU Count
MTT
|
Stain j
a
determined by hemacytometer counts. Samples of each mixture were stained with MTT or FDA, and the efficacies of the stains for detecting viable C. neoformans were determined by comparing the percentage of stained cryptococci with the expected percentage of viable cells, determined on the basis of the original proportions of proliferating and heat-killed C. neoformans that were mixed together. In addition, samples of each cryptococcal cell mixture were resuspended in agarose and placed on a microscope slide. After the slides were incubated for 18 h at 37°C in CM, the efficacy of the agarose method for detecting proliferating C. neoformans cells was determined by comparing the percentage of proliferating cryptococci in agarose to the expected percentage of proliferating cells determined on the basis of the proportions of the original mixtures. The data in Table 1 show that given the standard error of the assays, detection of viable cryptococci by the MTT and FDA staining techniques was equivalent to detection of proliferating cryptococci by the agarose method. In addition, the percentage of viable cryptococci detected by each of the three methods was comparable to the expected percentages of viable cells based on CFU counts obtained by the spread plate technique (Table 1). Comparisons of methods for detecting C. neoformans proliferation in total cell suspensions. Since CFU counts on the surface of Sabouraud agar plates have been our standard assay for determining NK cell-mediated cryptococcal damage, detection of cryptococcal proliferation in broth culture by extinction dilution analysis was compared with detection of proliferating cryptococci by CFU counts. Although both CFU counts and extinction dilution analysis measure cryptococcal proliferation, these two assays differ in that CFU counts assess proliferation of cryptococci on a solid medium, whereas extinction dilution analysis measures cryptococcal proliferation in broth culture. A cryptococcal cell suspension which contained 3 x 105 cryptococci per ml by hemacytometer counts was diluted and assayed in parallel for detection of proliferation by CFU counts and extinction dilution analysis. The CFU counts showed that the suspension contained 3.5 x 105 + 0.3 x 105 cryptococci per ml, and the extinction dilution results demonstrated that the suspension contained 4.4 x 105 + 0.6 x 105 cryptococci per ml. Given the standard error of the assays, the CFU counting and the extinction dilution method were equivalent in their efficacies of detecting proliferating cryptococci. NK cell-mediated damage of individual C. neoformans target cells in conjugates. Once the efficacy of vital stains (MTT and FDA) for distinguishing between viable and dead C.
FDA Stain
1
FIG. 1. Percentage of killed C. neoformans target cells bound to NK cells. After incubating NK cells with cryptococcal target cells for 18 h, the percentage of killed cryptococci that were bound to the effector cells was assessed by the MTT and FDA vital stains, and the results were compared with those obtained by using the single cell proliferation assay in agarose. Data are representative of three experiments. Bars represent the mean + standard error of the mean
of quadruplicate samples.
neoformans cells was established, these techniques were then applied to the assessment of NK cell-mediated cryptococcal damage and the results were compared with those obtained by using proliferation assays (CFU counts and extinction dilution analysis). After NK cells were incubated with cryptococcal target cells, the viability of cryptococci in conjugates with NK cells was assessed at the single conjugate level. The percentages of cryptococci that were bound to NK cells and that could (i) stain with MTT, (ii) stain with FDA, or (iii) form microcolonies in agarose were determined, and from these data, the percentages of killed and nonproliferating C. neoformans cells bound to NK cells were calculated (Fig. 1). Approximately 45% of the cryptococci that were bound to NK cells were unable to stain with MTT or FDA, indicating that 45% of the bound cryptococci were killed. Similarly, 45% of the C. neoformans cells bound to NK cells were unable to proliferate (Fig. 1). The results show that the three techniques were comparable for determining the level of cryptococcal killing in single conjugates (Fig. 1). In addition, the vital stains demonstrated that dead C. neoformans cells (nonstaining cryptococci) that were not bound to NK cells were present in the cell suspensions. To further analyze the effects of NK cells on the total C. neoformans population, studies were designed to assess the percentage of killed cryptococci (unbound and bound to NK cells) present in the NK cell-C. neoformans suspension. Effects of NK cells on C. neoformans target cells in the total cryptococcal cell population. After NK cells were incubated with cryptococci for 18 h, the numbers of viable cryptococci in wells containing NK cells and cryptococci and in wells containing cryptococci alone were assessed with the vital staining assays, and the results were compared with those obtained by using proliferation assays. Data in Figure 2 demonstrate a strong correlation between the percentage of NK cell-mediated cryptococcal killing detected by MTT staining and FDA staining and the percentage of nonproliferating cryptococci detected by CFU counts. Approximately 26% of the total cryptococcal population, both NK cell
VOL. 59, 1991
MURINE NK CELLS KILL CRYPTOCOCCI 4
Experimental
% C. neoformans Killing
Method
3
~01 :0- .
2
.
o
C 01
IE
-0
1,0
210
310
410
510
C
610
!0.10.
iiI-
1751
--
4 10,
CFU Count
o a ca . C
E
MTT Stain
I0. 20
C
0
cp
No. _C
4 I
.C
10-20-
FDA Stain
---
a2
Extinction Dilution
FIG. 2. Percentage of C. neoformans killing in the total cell suspension. After incubating NK cells with cryptococcal target cells for 18 h, the percentage of killed cryptococci in samples containing effector cells and target cells compared with that in samples containing cryptococci alone was assessed by using the MTT and FDA vital stains. C. neoformans viability, as assessed by vital stains, was compared with proliferation of cryptococci, as determined by CFU counts and extinction dilution analysis. Data are representative of three experiments. Bars represent the mean ± the standard error of the mean of quadruplicate samples.
bound and unbound, did not have the enzymatic activities necessary for labeling with MTT or FDA and had a reduced ability to proliferate, compared with the cryptococcal control suspensions. In contrast, the extinction dilution method of detecting proliferation of cryptococci in broth culture consistently indicated a percentage of nonproliferating cryptococci (61% + 5%) significantly (P < 0.001) greater than the level of cryptococcal killing indicated by MTT staining, FDA staining, or CFU counts on the surface of solid medium (25 to 27%) (Fig. 2). Kinetics of NK cell-C. neoformnans binding in comparison to NK cell-mediated killing of cryptococci. In previous studies, NK cell-C. neoformans conjugate formation and NK cellmediated anticryptococcal activity were determined in two different assays (19-21). NK cell-C. neoformans conjugate formation was assessed by enumerating conjugates by light microscopy after incubating NK cells with cryptococci at an effector cell-to-target cell ratio of 2:1, whereas NK cellmediated cryptococcal growth inhibition was determined by using CFU counts after incubating NK cells with cryptococci at an effector cell-to-target cell ratio of 50:1 (20, 21). Therefore, to compare the kinetics of NK cell-C. neoformans binding and NK cell-mediated killing of C. neoformans in single conjugates and in the total cryptococcal cell population within a single assay, effector and target cells were mixed at an effector cell-to-target cell ratio of 2:1 and incubated at 37°C. Samples were removed at 2-h intervals throughout the 18-h incubation and were stained with MTT (Fig. 3). Three parameters of NK cell-C. neoformans interactions were determined at each interval: (i) percentage of C. neoformans killed in the total cell suspension (Fig. 3A), (ii) percentage of dead C. neoformans in conjugate with NK cells (Fig. 3B), and (iii) percentage of effector cells conju-
*
.
*
2
4
6
.
.
.
*
*
10
12
14
16
i8
Ti;ne (h) FIG. 3. Kinetics of NK cell-C. neoformans conjugate formation and NK cell-mediated killing of cryptococci at the single conjugate level and in cell suspension. C. neoformans viability, as determined by MTT staining, and NK cell-C. neoformans conjugate formation were assessed at 2-h intervals after mixing the effector and target cells. Data are representative of two experiments. Bars represent the mean + the standard error of the mean of quadruplicate samples.
gated to C. neoformans cells (Fig. 3C). A gradual but steady increase in the percentage of C. neoformans killing in the total cell suspension (Fig. 3A) paralleled a similar increase in the percentage of NK cell-C. neoformans conjugates during the first 12 h of incubation (Fig. 3C). Optimal levels of both cryptococcal conjugate formation and C. neoformans killing in the total cell suspension occurred between 12 and 18 h after mixing the effector and target cells. In contrast to the steady increase in C. neoformans killing in the total cell suspension throughout the experiment (Fig. 3A), the percentage of killed target cells within NK cell-C. neoformans conjugates reached nearly optimal levels by 6 h after mixing the effector and target cells and remained relatively constant throughout the remainder of the 18-h incubation (Fig. 3B). DISCUSSION Previous studies in our laboratory have demonstrated that murine NK cells bind to and inhibit the growth of the fungal target cell C. neoformans in vitro and contribute to clearance of the organism in vivo (7-10, 17, 19-21). Considering the facts that NK cells kill tumor cell targets and that many similarities between NK cell-C. neoformans interactions and NK cell-tumor cell interactions have been demonstrated, it seemed likely that the NK cell-mediated growth inhibition that we have previously reported was actually a measure of cryptococcal killing. However, our previous studies were based on a single type of proliferative assay and therefore did not clearly establish whether the anticryptococcal effects of NK cells were fungistatic or fungicidal. The question of whether NK cells actually kill cryptococci is of primary importance in determining the potential role of NK cells in the complex interactions that result in host resistance to this pathogen. Therefore, the purpose of the study presented here was to determine whether NK cells kill cryptococcal target cells. To evaluate the fungicidal capacity of NK cells for cryptococci, the viability of both cryptococci that were bound to NK cells and cryptococci that were unbound had to be
1752
HIDORE ET AL.
determined; therefore, techniques for assessing cryptococcal viability at the single cell level and in the entire cell suspension were required. Vital stains have been reported to be reliable quantitative assays for determining the viability of C. neoformans cells (2, 4, 12, 13). Before these methods were applied to the assessment of NK cell-mediated anticryptococcal effects, initial experiments were performed to determine the efficacies of the vital stains for distinguishing between dead and live cryptococci at the single cell level and in the total cell suspension. In addition, detection of viable cryptococci by the vital stains was compared to detection of proliferating C. neoformans cells both at the individual cell level and in the total cell suspension. Two distinct approaches using vital stains that are indices of intact metabolic function within viable cells were employed to determine cryptococcal viability. MTT and FDA, the two vital stains used in this study, reflect the metabolic function of two different enzymes within viable cells, mitochondrial dehydrogenases and esterases, respectively. Our study demonstrated that the MTT and FDA vital stains effectively distinguished between live and dead cryptococci in suspensions containing known percentages of viable and heat-killed C. neoformans (Table 1). Furthermore, these studies also demonstrated that the agarose slide CFU assay for assessing proliferation of cryptococci was comparable to the vital stains and to CFU counts on the surface of solid medium for the detection of proliferating C. neoformans (Table 1). In the total cell suspension, this study demonstrated that CFU counts on the surface of a solid medium and extinction dilution analysis (broth culture) were reliable and comparable methods for the detection of proliferating C. neoformans cells in suspensions containing known proportions of viable and heat-killed cryptococci. Since MTT and FDA vital stains were demonstrated to be reliable indices of C. neoformans viability, these techniques were used to assess NK cell-mediated anticryptococcal effects at the single conjugate level and in the total cell suspensions. In addition, following incubation with NK cells, cryptococcal viability as determined by the vital stains was compared with proliferation of cryptococci at the individual cell level (CFU counts within agarose) and in the total cell suspensions (CFU counts on the surface of a solid medium and extinction dilution analysis). When the viability of individual cryptococci was assessed at the single conjugate level following an 18-h incubation with NK cells, approximately 45% of the C. neoformans target cells attached to NK cells were dead, as determined by the inability to stain with either of the two vital stains, MTT and FDA (Fig. 1). Our data are consistent with reports on the percentage of dead tumor cells in conjugates with NK cells, which ranges from 14 to 52%, depending on the type of tumor cell target (24). In correlation with the vital stains, approximately 45% of the cryptococci attached to NK cells were unable to proliferate in agarose, suggesting that the inability to proliferate was also indicative of cryptococcal cell death. Together, these data clearly indicate that NK cells kill a portion of the bound C. neoformans cells. The percentage of viable cryptococci in the total NK cell-C. neoformans cell suspension following an 18-h incubation was also assessed. Approximately 30% of the total population of cryptococci (both NK cell bound and unbound) in the samples (containing NK cells and cryptococci), compared with the control samples (containing cryptococci alone), were killed, i.e., they did not have the enzymatic activities required for staining with MTT or FDA (Fig. 1). In addition, approximately 30% of the total crypto-
INFECT. IMMUN.
coccal population in the experimental samples, compared with the control samples, were unable to proliferate on a solid medium (CFU counts), again indicating that the inability to proliferate was also a measure of cell death. These data support the data obtained at the single conjugate level in that three different approaches for measuring both metabolic function and proliferation of cryptococci indicated that NK cells kill C. neoformans target cells in the total cell suspension. Although vital stains in conjunction with CFU counts demonstrated that approximately 30% of the cryptococci in the total cell suspension were killed, extinction dilution analysis consistently indicated that NK cells inhibited proliferation of approximately 61% of the cryptococci in the effector cell-target cell suspension compared with the number of proliferating C. neoformans in the control suspensions. Because CFU counts and extinction dilution analysis were equally effective in distinguishing between viable and heat-killed cryptococci in our initial studies, these data suggest that NK cell-mediated anticryptococcal effects alter the ability of the cryptococcal cells to proliferate in a broth culture (extinction dilution analysis) in comparison to their ability to proliferate on a solid medium (CFU counts). This hypothesis is supported by studies indicating that injured microorganisms initiate specific repair mechanisms that are most effective in determining survival of the organism when optimal culture conditions are available during repair (1, 16). Therefore, it is likely that cryptococci damaged by NK cells proceed with a repair process that favors survival of the damaged cryptococci only under optimal culture conditions. If transferring cryptococci from a cell suspension to a solid culture medium, such as Sabouraud agar, provides optimal culture conditions for repair and ultimately proliferation of damaged but not-yet-dead cryptococci, then CFU counts on the surface of solid medium are an index of cryptococci that are dead at the end of the 18-h incubation. In contrast, if transferring the cryptococci from the cell suspension into a second liquid culture, as occurs in extinction dilution analysis, is unfavorable for repair of injured cryptococci, then the cryptococci that were damaged but not killed at the end of the 18-h incubation might continue the process, leading to cell death as a result of inefficient repair in liquid medium. Therefore, extinction dilution analysis would detect both cryptococci that were killed at the end of 18 h and cryptococci that were damaged by 18 h and that then proceeded to cell death in the broth culture. Our data comparing killing of cryptococci in the total cell suspension as determined by CFU counts (30%) and by extinction dilution analysis (61%) are in accordance with this hypothesis, which predicts that the level of NK cell-mediated cryptococcal killing would be greater when assessed by extinction dilution analysis than when determined by CFU counts (Fig. 2). These data indicate that CFU counts, in conjunction with MTT and FDA vital stains, are reliable indices of NK cell-mediated cryptococcal killing at a particular time after mixing effector and target cells, whereas extinction dilution analysis may detect a combination of damaged and killed cryptococci at a particular time in culture but more accurately reflects total NK cell-mediated anticryptococcal effects. In previous studies, NK cell binding to cryptococci was assessed at the single cell level by light microscopy, whereas NK cell-mediated anticryptococcal effects were measured in the cell suspension by CFU counts (19-21). To compare the kinetics of NK cell binding with killing of cryptococci at the single conjugate level and in the cell suspension within a single assay, samples were stained with MTT at 2-h intervals
VOL. 59, 1991
after mixing the effector cells and target cells (Fig. 3). Comparisons of the kinetics of (i) cryptococcal killing in the total cell suspension, (ii) killing of cryptococci bound to NK cells, and (iii) NK cell binding to C. neoformans indicate that NK cell-C. neoformans binding precedes killing of cryptococcal target cells. The similarities in the kinetics of killing of C. neoformans in the total cell suspension as measured by MTT staining and the kinetics of NK cell-mediated cryptococcal growth inhibition demonstrate that growth inhibition assays actually reflect the level of NK cell-mediated killing of C. neoformans. Thus, the data previously reported as NK cell-mediated cryptococcal growth inhibition are indicative of C. neoformans killing by NK cells (7-9, 17, 19-21). When the viability of individual cryptococci was assessed at the single conjugate level following an 18-h incubation with NK cells, approximately 30% of the NK cells were
bound to C. neoformans target cells (Fig. 3C) and only 45% of the bound target cells were killed (Fig. 1). There are several factors that could be responsible for these observations. First, it is possible that all of the effector cells in the population are not capable of binding to cryptococci. Alternatively, all of the effector cells may be capable of cryptococcal binding, but at any given time in an unsynchronized cell population, cryptococci might be attached to only a portion of the potential target-binding cells and only a fraction of the attached cryptococci would be killed. A further explanation for the observation that 100% of the bound cryptococcal target cells were not killed is that only a percentage of the NK cells that bind to cryptococci are capable of killing the organism. However, the present study did not allow us to determine whether either of these possible interpretations of the data is correct. In addition to observing that cryptococci attached to NK cells were killed, we also noted that cryptococci not bound to effector cells in the total cell suspension were dead, as determined by the lack of metabolic functions and proliferation. The demonstration of unbound, killed cryptococci in cell suspensions suggests at least two possible interpretations of the data. First, it is possible that NK cells may detach from killed cryptococcal target cells and recycle to kill subsequent target cells. This phenomenon of a single effector cell recycling to kill numerous target cells has been demonstrated in the model of NK cell-mediated tumor cell cytotoxicity (24). Further support for recycling of effector cells in the C. neoformans model is provided by the kinetic pattern of killing of bound cryptococci (Fig. 3B) in comparison to the pattern of killing of cryptococci in the total cell suspension (Fig. 3A). If both initial binding events and effector cell recycling are occurring simultaneously, as would be expected in an unsynchronized cell suspension, this might result in the observed gradual increase in the total number of dead cryptococcal cells in the suspension throughout the incubation while the percentage of dead, NK cell-bound cryptococci reached optimal and relatively constant levels after 6 h of incubation (Fig. 3A and B). A second, but not mutually exclusive, explanation for the observation of unbound, killed cryptococci in the total cell suspensions is that NK cells may kill unbound target cells through the release of soluble cytotoxic factors. Since NK cells have been shown to secrete or exocytose numerous cytotoxic substances (6, 23), it is possible that killing of bound cryptococci results in the release of cytotoxic components that also kill unbound cryptococci in the suspension. The data presented here are not sufficient to establish whether effector cell recycling or killing of cryptococcal targets via soluble cytotoxic factors or both are responsible for our observa-
MURINE NK CELLS KILL CRYPTOCOCCI
1753
tions. However, in conjunction with our previous data which indirectly suggest that recycling occurs in the NK cell-C. neoformans model and clearly demonstrate that soluble cytoplasmic components in addition to the cytotoxic granules isolated from NK cells inhibit cryptococcal growth (9, 10, 20, 21), we favor the hypothesis that both mechanisms (effector cell recycling and killing of unbound cryptococci by soluble cytotoxic components) may be involved in NK cell-C. neoformans interactions. In conclusion, measures of metabolic function, the MTT and FDA vital stains, demonstrated that NK cells kill bound C. neoformans targets at the individual conjugate level and in the total cell suspension. These results were confirmed by studies which demonstrated that NK cells inhibited proliferation of cryptococci at the individual conjugate level and in the total cell suspensions. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans mixtures. First, these data indicate the possibility that NK cells bind to and kill a cryptococcal target cell and then recycle to kill subsequent cryptococcal targets. A second, but not mutually exclusive, possibility suggested by these data is that NK cells release undefined soluble cytotoxic factors which kill both bound and unbound C. neoformans cells. Taken together, the study presented here firmly establishes that murine NK cells kill C. neoformans target cells. ACKNOWLEDGMENT This work was supported by Public Health Service grant AI-18895 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Beuchat, L. R. 1984. Injury and repair of yeasts and moulds, p. 293-308. In M. H. E. Andrew and A. D. Russell (ed.), The revival of injured microbes. Academic Press, Inc., Orlando, Fla. 2. Calich, V. L. G., A. Purchio, and C. R. Paula. 1978. A new fluorescent viability test for fungi cells. Mycopathologia 66:175177. 3. Cochran, W. G. 1950. Estimation of bacterial densities by means of the most probable number. Biometrics 5:105-116. 4. Correa, B., A. Purchio, C. R. Paula, and W. Gambale. 1986. Evaluation of a fluorescent method (fluorescein diacetate and ethidium bromide solution) in the study of viable Cryptococcus neoformans strains. Mycopathologia 96:91-96. 5. Grimm, E. A., J. A. Thomas, and B. Bonavida. 1979. Mechanism of cell-mediated cytotoxicity at the single cell level. II. Evidence for first-order kinetics of T cell-mediated cytotoxicity and for heterogeneity of lytic rate. J. Immunol. 123:2870-2877. 6. Herberman, R. B., C. W. Reynolds, and J. R. Ortaldo. 1986. Mechanism of cytotoxicity by natural killer (NK) cells. Annu. Rev. Immunol. 4:651-680. 7. Hidore, M. R., and J. W. Murphy. 1986. Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool nonadherent cells. Infect. Immun. 51:547-555. 8. Hidore, M. R., and J. W. Murphy. 1987. Natural cellular resistance of beige mice against Cryptococcus neoformans. J. Immunol. 137:3624-3631. 9. Hidore, M. R., and J. W. Murphy. 1989. Murine natural killer cell interactions with a fungal target, Cryptococcus neoformans. Infect. Immun. 57:1990-1997. 10. Hidore, M. R., N. Nabavi, C. W. Reynolds, P. A. Henkart, and J. W. Murphy. 1990. Cytoplasmic components of natural killer cells inhibit the growth of Cryptococcus neoformans. J. Leukocyte Biol. 48:15-26. 11. Julius, M. H., E. Simpson, and L. A. Herzenberg. 1973. A rapid method for isolation of functional thymus-derived lymphocytes. Eur. J. Immunol. 3:645-649. 12. Lapage, S. P., J. E. Shelton, and T. G. Mitchell. 1970. Methods
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in microbiology, p. 1-133. Norris Ribbons, London. 13. Levitz, S. M., and R. D. Diamond. 1985. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J. Infect. Dis. 152:938-945. 14. Lipscomb, M. F., T. Alvarellos, G. B. Toews, R. Tompkins, Z. Evans, G. Koo, and V. Kumar. 1987. Role of natural killer cells in resistance to Cryptococcus neoformans infections in mice. Am. J. Pathol. 128:354-361. 15. Luini, W., D. Baraschi, S. Alberti, A. Aleotti, and A. Tagliabue. 1981. Morphological characterization of a cell population responsible for natural killer activity. Immunology 43:663-668. 16. Mackey, B. M. 1984. Lethal and sublethal effects of refrigeration, freezing and freeze-drying on microorganisms, p. 45-75. In M. H. E. Andrew and A. D. Russell (ed.), The revival of injured microbes. Academic Press, Inc., Orlando, Fla. 17. Murphy, J. W. 1989. Natural host resistance mechanisms against systemic mycotic agents, p. 149-184. In C. W. Reynolds and R. H. Wiltrout (ed.), Functions of the natural immune system. Plenum Publishing Corp., New York. 18. Murphy, J. W., and G. C. Cozad. 1972. Immunological unre-
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19. 20. 21.
22.
23. 24.
sponsiveness induced by cryptococcal capsular polysaccharide assayed by the hemolytic plaque technique. Infect. Immun. 5:896-901. Murphy, J. W., M. R. Hidore, and N. Nabavi. 1991. Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans. Infect. Immun. 59:1476-1488. Murphy, J. W., and D. 0. McDaniel. 1982. In vitro reactivity of natural killer (NK) cells against Cryptococcus neoformans. J. Immunol. 128:1577-1583. Nabavi, N., and J. W. Murphy. 1985. In vitro binding of natural killer cells to Cryptococcus neoformans targets. Infect. Immun. 50:50-57. Russek, E., and R. R. Colwell. 1983. Computation of most probable numbers. Appl. Environ. Microbiol. 45:1646-1650. Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187-375. UllHberg, M., and M. Jondal. 1981. Recycling and target binding capacity of human natural killer cells. J. Exp. Med. 153:615628.
