Vol. 60, No. 10

INFECTION AND IMMUNrIy, Oct. 1992, p. 4200-4204

0019-9567/92/104200-05$02.00/0 Copyright © 1992, American Society for Microbiology

Killing of Coccidioides immitis by Human Peripheral Blood Mononuclear Cells NEIL M. AMPEL,* GEORGE C. BEJARANO, AND JOHN N. GALGIANI Medical and Research Services, Tucson Veterans Affairs Medical Center, and Department ofInternal Medicine, University ofArzona Health Sciences Center, Tucson, Arizona 85723 Received 13 April 1992/Accepted 30 July 1992

The ability of human peripheral blood mononuclear cells (MNL) obtained from healthy donors to kill the fungus Coccidioides immits was examined in vitro with an assay that uses a single fungal particle per well. MNL killed 25.0%o 3.5% of a coccidioidal arthroconidial target, compared with the 4.7% 2.9%o killed by polymorphonudear leukocytes obtained from the same donors (P = 0.012). Arthroconidial killing by MNL was not dependent on donor delayed dermal hypersensitivity to spherulin. Killing of another fungal target, Candida glabrata, was not significantly different between MNL and polymorphonuclear leukocytes (P = 0.783). Depletion of monocytes from MNL with Sephadex G-10 resulted in a significant reduction in arthroconidial killing (21.4% 13.6% versus 2.4% 3.4%; P = 0.025), while enrichment of monocytes by Percoll density gradient centrifugation or plastic adherence resulted in significantly increased arthroconidial killing compared with that by MNL (P = 0.005 and 0.001, respectively). Killing of 96-h spherules by MNL was 7.3% 3.1%, significantly less than the 21.4% 2.8% killing of arthroconidia in the same experiments (P = 0.016). Incubation of MNL with human recombinant gamma interferon or tumor necrosis factor alpha did not result in increased MNL killing of coccidioidal arthroconidia under various conditions. These results suggest that MNL have an inherent ability to kill coccidioidal arthroconidia in vitro which is not dependent on prior host exposure to C. immitis. This activity appears to reside in peripheral blood monocytes.

Coccidioidomycosis is an infection endemic to the desert regions of the southwestern United States, as well as to areas of Mexico, Central America, and Argentina. It is estimated that 25,000 to 100,000 cases of coccidioidomycosis occur in the United States each year (10). The causative organism, Coccidioides immitis, is a dimorphic fungus with a unique life cycle. In the soil, the organism exists as a mold with septated hyphae. Barrel-shaped arthroconidia break off from the hyphae and evolve into globular structures called spherules when inhaled by a susceptible host. Within the host, spherules progressively enlarge and undergo internal division, forming smaller structures called endospores. Large spherules may rupture to release packets of endospores, resulting in new foci of infection within the host (11). Cellular immunity has long been assumed to play a role in the host defense against coccidioidomycosis. In animal studies, there is evidence that macrophages, when activated by soluble T-lymphocyte products, such as gamma interferon (IFN-y), are capable of killing certain forms of C. immitis (3, 6). In humans, delayed dermal hypersensitivity, a marker for cellular immunity, decreases with increasing severity of disease (7, 9, 22). Moreover, in patients with presumed defects in cellular immunity, such as transplant recipients (8), those who receive corticosteroids (2), and individuals infected with the human immunodeficiency virus (14), there appears to be an increased risk for development of severe, disseminated coccidioidomycosis. However, there is limited information on which cells in humans are responsible for directly controlling C. immitis infection. Frey and Drutz have shown that polymorphonuclear leukocytes (PMN) were able to ingest and kill arthroconidia and endospores but not spherules in vitro (16). Galgiani and coworkers have demonstrated that PMN were *

capable of inhibiting in vitro uptake of the chitin precursor N-acetylglucosamine by arthroconidia but did not appear to kill the organisms (18). Studies by Petkus and Baum have indicated that human peripheral blood natural killer (NK) cells have the ability to inhibit the growth of young spherules and endospores in vitro (23). In recent work, Beaman has shown that human peripheral blood monocytes, when exposed to human IFN-y or tumor necrosis factor a (TNF-a), were capable of reducing the growth of coccidioidal endospores in vitro (4). We have previously demonstrated that the human peripheral blood mononuclear cells were also able to reduce in vitro uptake of N-acetylglucosamine by arthroconidia. Further, we showed that these cells were also able to kill coccidioidal arthroconidia by employing an assay which uses approximately one arthroconidium per well (1). In the present study, we expanded these data and attempted to determine which cell within the mononuclear cell fraction is responsible for this effector function. (This work was presented in part at the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, on 2 October 1991, in Chicago, Ill.)

MATERIALS AND METHODS Subjects. All subjects were healthy volunteers without evidence of active coccidioidomycosis. All were tested for dermal hypersensitivity to spherulin (Berkeley Biologics, Berkeley, Calif.). An induration with a diameter of 25 mm 24 or 48 h after placement of spherulin was considered indicative of a positive test. The study was approved by the Human Subjects Committee of the University of Arizona. Peripheral blood cell isolation. From 30 to 60 ml of blood was obtained by venipuncture from each volunteer. PMN and mononuclear leukocytes (MNL) were isolated from blood by the method of Ferrante and Thong (13). Cells were

Corresponding author. 4200

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washed in RPMI 1640 (GIBCO, Grand Island, N.Y.) and suspended in RPMI 1640 containing 10% heat-inactivated autologous serum (cell culture medium) for further use. Cell counting, identification, and viability. Total number and viability were determined by examining the cells by light microscopy by using Trypan blue exclusion (1), and cell differential analysis was performed by using either a modified Giemsa stain (Diff-Quik; Geometric Data, Wayne, Pa.) or nonspecific esterase (Sigma, St. Louis, Mo.) (20). Separation of mononuclear cells into various fractions. (i) Percoll density gradient centrifugation. MNL were further separated by Percoll density gradient centrifugation (Pharmacia, Piscataway, N.J.) with a method previously described (26). Briefly, a continuous density gradient was achieved by centrifuging isotonic Percoll at 18,000 rpm on a Sorvall RC-2 fixed-rotor centrifuge (38,724 x g) for 25 min at 5°C. MNL were then layered onto the Percoll gradient and centrifuged at 2,900 rpm on a Beckman J6 swinging-bucket centrifuge (1,500 x g) for 25 min at 5°C. The two resultant cell bands were then removed by sterile Pasteur pipet, washed with RPMI 1640, and suspended in cell culture medium. (ii) Sephadex G-10. MNL were eluted through a Sephadex G-10 column to deplete the cell population of monocytes by an established technique (19). Sephadex G-10 (Pharmacia) was allowed to swell in phosphate-buffered saline and then autoclaved. A slurry of this was mixed with Hanks' balanced salt solution with 10% heat-inactivated fetal bovine serum (GIBCO) and placed into a 35-ml syringe whose end was plugged with nylon wool. After being washed MNL were layered on top of the column and allowed to elute through with cell culture medium. After a total of 30 ml of elutant was collected, the cells in it were washed, counted, suspended in cell culture medium, and then used in subsequent experiments. (iii) Adherence. MNL were allowed to adhere to plastic to enrich for monocytes. Approximately 50 x 106 MNL in cell culture medium were placed into a 75-cm2 sterile tissue culture flask (Costar, Van Nuys, Calif.) and incubated at 37°C in 5% CO2 and 95% air for 1 h. The nonadherent cells were then removed by decanting the contents of the flask into a sterile 50-ml polypropylene centrifuge tube, and this was placed on ice. Ten milliliters of EDTA-trypsin (GIBCO) was added to the flask bottom, and the flask was then incubated at room temperature for 10 min. Cells were removed from the flask bottom by agitation and gentle use of a cell scraper (Costar). Cells so obtained were added to 10 ml of RPMI 1640 and placed on ice. This sequence was repeated two or three times. Both adherent and nonadherent cells were next washed in RPMI and counted, their viability was determined, and they were then used in the killing experiments. C. immiits. The Silveira strain of C. immitis was used in all instances with strict adherence to containment. The spin bar technique of Sun and Huppert (25) was used to isolate arthroconidia from stock mycelial cultures grown for 8 to 12 weeks on glucose-yeast extract agar. Arthroconidia were washed in sterile, pyrogen-free water (Kendall-McGaw, Irvine, Calif.) and stored at 4°C until use. Coccidioidal spherules were prepared as described previously (17). Arthroconidia were added to Converse medium at a concentration of 7 x 108 fungal particles per liter in a 2-liter screw-top Erlenmeyer flask and tightly sealed. The flasks were then agitated at 150 rpm and 40°C in a gyratory incubator (Psychrotherm, New Brunswick, N.J.). After 24, 48, 72, and 96 h, the resultant spherule suspensions were


decanted into sealed buckets and centrifuged at 4,000 x g for 40 min at room temperature. The supematant was decanted, and the spherules were washed and stored in sterile, pyrogen-free water at 4°C. Candida glabrata. A strain of C. glabrata obtained from the clinical microbiology laboratory of the Tucson Veterans Affairs Medical Center was used in some experiments as another fungal killing target. C. glabrata was grown in Sabouraud dextrose broth and then washed and diluted in sterile, pyrogen-free water to appropriate concentrations. The suspension was stored at 4°C until used in the fungal killing assay described below. Fungal killing assay. The assay used to measure killing of coccidioidal arthroconidia, spherules, and C. glabrata was performed as previously described (1, 12). Briefly, 2 x 104 cells in cell culture medium were added to 48 wells of a 96-well U-bottom microtiter plate (Dynatech Laboratories, Alexandria, Va.) at a volume of 50 ,ul, and cell culture medium alone was added to the other 48 wells. A suspension containing C. immitis arthroconidia or spherules or C. glabrata at a concentration of approximately 20 particles per ml in RPMI 1640 was subsequently added in a volume of 50 ,ul to all of the wells. This yielded approximately one target particle per well. The plate was sealed, centrifuged, and incubated for 18 h at 37°C in 5% CO2 and 95% air. Subsequently, 100 ,l of Sabouraud dextrose broth was added to all of the wells. After further incubation for 10 days, the number of wells containing fungal growth was determined and recorded. The amount of fungal killing was determined by the following formula: % killing = [(control wells containing growth/48) - wells with cells containing growth/48)] x 100 where wells with cells are those wells containing MNL, PMN, or other effector cells. Cytokine activation. Various concentrations of recombinant human IFN--y (Genzyme, Boston, Mass.), recombinant human TNF-a (Genzyme), and lipopolysaccharide (Escherichia coli 0128:B12; Sigma) were added to wells containing MNL in the fungal killing assay to ascertain whether these agents increase MNL killing. Statistical analysis. The mean + the standard error of the mean was used where applicable. For continuous variables which were not paired, the Mann-Whitney U test was employed. For continuous, paired values, the Wilcoxon signed-rank test was used (24). A P value of

Killing of Coccidioides immitis by human peripheral blood mononuclear cells.

The ability of human peripheral blood mononuclear cells (MNL) obtained from healthy donors to kill the fungus Coccidioides immitis was examined in vit...
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