Effect of gliotoxin on human polymorphonuclear neutrophils.

OBJECTIVES: Candida albicans is known to produce gliotoxin, which has several prominent biological effects, including immunosuppression. Interference with host defenses may arise from the effects of this toxin on leukocyte structure and function. METHODS: Flow cytometric analysis revealed that polymorphonuclear leukocytes (PMN) were more sensitive to gliotoxin than were mononuclear cells. Structural and various functional aspects of PMN exposed to gliotoxin were studied. RESULTS: Gliotoxin at (1 microgram/mL) did not affect the viability but did diminish PMN chemotaxis and reduced their ability to ingest particles. Other functional aberrations included decreased nitroblue tetrazolium dye reduction, decreased superoxide production, and release of lactoferrin suggesting by degranulation. Gliotoxin also affected the ability of PMN to kill Escherichia coli. CONCLUSIONS: This study suggests a previously unrecognized potential virulence factor of C. albicans that could contribute to persistence of yeast colonization or recurrence of symptomatic infection through diminished host resistance.

including those with acquired immunodeficiency syndrome (AIDS), but it can also occur in individuals without known immune defects. Virulence attributes of Candida albicans, particularly those that may suppress the immune system, remain incompletely investigated. Several reports describe products of Candida that may contribute to its virulence, including a protease and cell wall mannans that have been reported to interfere with neutrophil function, z In addition, a crude product of candidal hyphae was shown to prevent normal superoxide production by stimulated neutrophils. 3 A new dimension was added to study Candida virulence when we found that clinical isolates of this organism produced gliotoxin, 4 an epipolythiodioxopiperazine mycotoxin. This class of compounds is known for various biological actions, including antibacterial, antiviral, and immunosuppressive activities, s We subsequently analyzed vaginal samples of three women severely symptomatic for vaginal candidiasis and found that they contained significant levels of gliotoxin. 6 We reasoned that in vivo production of gliotoxin by C.
phonuclear leukocytes (PMN) seemed to be crucial as a prominent defense mechanism. This study was undertaken to evaluate effect of gliotoxin on human PMN functions.

MATERIAL AND METHODS
Crystalline gliotoxin was purchased from Sigma Chemical Company (St. Louis, MO). Purchased gliotoxin was found identical to the toxin produced by clinical isolates of C. albicans. 7 Stock solutions were prepared in methanol and further diluted in RPMI or phosphate-buffered saline with glucose (PBSG) to attain the desired final concentrations. Preparation of whole heparinized blood for flow cytometry used blood drawn from healthy volunteers. To 10 mL of whole blood, 14 mL of lysing reagent (NH4C1, 8 g/L; KHCO3, 1.0 g/L, tetrasodium EDTA, 37.0 mg/mL, pH 7.3) was added, mixed for 3 to 5 minutes, and centrifuged for 30 minutes at 300g at room temperature. The cell pellet was washed twice with phosphate-buffered saline (PBS) and resuspended in 1 mL of PBS. PMN preparations were obtained by gradient centrifugation of whole blood through Mono-Poly added, and the labeling proceeded in the dark at room temperature for 15 minutes, followed by flow cytometric analysis. Cell viability after gliotoxin exposure was demonstrated both by trypan blue dye exclusion and by flow cytometry of propidium iodide treated cells.
Leukocytes (1106 leukocytes/mL) were incubated with gliotoxin diluted in PBSG or RPMI at 37C. Cells were exposed to 0.5-500 lag/mL of gliotoxin, and samples were taken at different time intervals ranging from 15 to 90 minutes. Samples were further mixed with 5 mL of 0.0005% propidium iodide (Sigma) and analyzed by flow cytometry.
After determining the gliotoxin concentration (1 lag/mL), PMN chemotaxis was investigated in modified Boyden chambers. Gliotoxin-treated PMN (1 lag gliotoxin, 1106 PMN/mL) were activated with LPS-treated human serum (0.1 mg of LPS to mL of human serum was incubated for 60 minutes at 37C). At the end of 30 minutes of incubation, the pattern of migration of PMN across a polycarbonate filter (3 lain pore size) was evaluated after Giemsa staining by light microscopy. Controls consisted of PMN incubated without gliotoxin.
Particle ingestion by PMN was evaluated by uptake of fluorescein-conjugated styrene beads (Flow Cytometry Standards Corp., NC). The PMN (l106/mL) were incubated for 15 minutes with lag/mL gliotoxin and mixed with beads to achieve a particle-to-cell ratio of 100:1. The mixture was gently mixed on a rotating wheel, and aliquots were removed at timed intervals. Aliquots were centrifuged in medium containing 2% BSA, and the cells recovered were resuspended in MEM with 20 mM 2-N-morpholine propane sulfonic acid with 10% fetal bovine serum. The fluorescence due to ingested beads was measured by flow cytometry.
Nitroblue tetrazolium (NBT) reduction was used to measure respiratory burst activity. The PMN (1106 cells/mL) were pretreated with lag/mL gliotoxin (control cells remained untreated) with stimulant consisting of the supernatant from zymosan (Sigma) treated serum. One tenth of a milliliter of NBT was added to the mixture and incubated for 10-20 minutes, and the development of color was monitored spectrophotometrically at 570 with 630 used as the reference wavelength.
Production of superoxide anion by gliotoxintreated or untreated cells was determined spectro- (B) Human leukocytes exposed to 500 pg/mL-concentration of gliotoxin for 30 minutes showed a shift in PMN population from right to left on the forward scatter axis and downward on the side scatter scale, consistent with these cells becoming smaller and less granular.
photometrically by reduction of cytochrome C as described elsewhere. 8 Experiments were conducted in sets of three tubes in which phorbol rnyristate acetate (PMA, 10 laL/5 mL) was used as an activator for PMN superoxide production. The first tube was a cell-free control that showed baseline superoxide production from PMA, the second tube contained superoxide dismutase (2 lag) with cells and PMA to establish assay specificity, and the third tube contained cells and PMA and was the positive test. Cell concentration was l xl07 PMN in mL of HEPES (N-2-Hydroxyethylpiperazine-N- Intracellular killing of Escherichia coli by PMN was determined by suspending 2.5x106 PMN in 0.1 mL of fetal calf serum and 0.3 mL HBS and con-ditioning them for 5 minutes at 37C. Subsequently, 2107 E. coli contained in 0.1 mL of HBS were added to each well. Aliquots were removed at timed intervals and centrifuged at 150g for 5 minutes, and the supernatant fluid was removed. The supernatant-containing bacteria that had not been ingested (100 laL) were added to 9.9 mL of sterile water, and dilutions were plated on nutrient agar.
The pellet that contained the PMN was suspended in sterile water to lyse the cells and release viable intracellular bacteria, which were enumerated by viable plate count.

RESULTS
The effect of gliotoxin on the various types of leukocytes in peripheral blood was tested by incubating 500 lag gliotoxin per mL of whole blood lysate for 30 minutes at 37C. As illustrated by Figure 1, the granulocyte population appeared more affected (decreased forward scatter and decreased side scatter) than either the mononuclear cell population or the lymphocytes population. Thus, it appeared that gliotoxin may have caused a decrease in granulocyte size and granularity. To determine if the gliotoxin-treated leukocytes were dying, viability was measured by propidium iodide uptake. Gliotoxin (0.5-500 lag/mL for 15-90 minutes at 37C) did not appear to damage lymphocytes, whereas granulocytes and monocytes were stained with propidium Gliotoxin effect on leukocyte viability. Human leukocytes were exposed to 500 IJg/mL of gliotoxin for 15 and 30 minutes. Viability was measured by propidium iodide uptake. PMN were the most affected when compared with lymphocytes and monocyte populations of the peripheral blood.
iodide ( Figure 2). After 30 minutes of exposure to gliotoxin, 24% of granulocytes were stained with propidium iodide and 9% of monocytes were stained.
Because the granulocyte population, which consists mainly of PMN, appeared to be the most susceptible to gliotoxin, subsequent studies focused mainly on PMN that were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation. Isolated PMN exposed to gliotoxin (500 lag/mL for 15-90 minutes at 37C) showed an increase in propidium iodide uptake that was both gliotoxin dose-dependent and time-dependent, as shown by Figure 3. These results were corroborated by trypan blue dye exclusion that showed that minimum cytocidal gliotoxin concentration appeared to be greater than 1 lag/mL.  whereas only 13% of cells treated with pg/mL of gliotoxin/mL for 90 minutes ingested fluorescent beads. Actively phagocytic active cells increase their oxidative metabolism resulting in the generation of reactive oxygen species such as superoxide. The increased reducing capacity of phagocytically active leukocytes may be measured by nitroblue tetrazolium dye reduction. Figure 6  complement-stimulated gliotoxin-treated (1 pg/mL of gliotoxin for 10 minutes at 37C) PMN had diminished capacity for dye reduction compared with stimulated control PMN.
The antibacterial mediator, superoxide anion, was decreased when cells were pretreated with gliotoxin, compared with untreated cells as shown by Figure 7.
The final aspect of neutrophilic cell function studied was intracellular bacterial killing. Viable E. coli was used as the test organism, and PMN pretreated with gliotoxin or untreated control cells were allowed to ingest and kill the test bacterium. ing of specific virulence factors and their role in pathogenesis remains limited. In mucocutaneous candidiasis, PMN seemed to be crucial as a prominent defense mechanism. 9 Patients with few neutrophils or dysfunctional phagocytes are more susceptible to disease with Candida than individuals with normal neutrophil function. Interestingly, the results of this study suggest that gliotoxin is more deleterious to PMN than mononuclear cells or lymphocytes from peripheral blood, as evidenced by propidium iodide staining. This cytocidal effect, however, employed concentrations of gliotoxin that probably would not occur in vivo. 1 Both because of the importance of PMN in candidal infections and because of their apparent susceptibility to gliotoxin, we sought to identify functional alterations in gliotoxin-treated PMN. Our approach involved examining the various elements of the phagocytic response, including directed migration, particle engulfment, respiratory increment, and bactericidal effects. Because of the opportunity for the interaction between mucosal leukocytes and mucosal organisms, the inhibition of phagocytosis could play a role in the persistence or recur-rence of candidiasis. As shown in the present study, all aspects of phagocytic function were altered to some degree by incubation with gliotoxin. The possibility that gliotoxin may have a role in vaginal infection is dependent on whether the organism can actually produce the toxin in vivo. In previous studies we found that gliotoxin can be produced by Candida inoculated into fluid obtained on vaginal swabs from patients. 7 We also showed that women with yeast vaginitis had detectable levels of gliotoxin, and these levels were sufficient to cause the altered phagocytic function as reported in the present study. Since we were able to isolate the gliotoxin from the yeast vaginitis patients, we predict that it is not degraded but may be neutralized with the use of antimycotic agent.
While this investigation suggests a potential role for gliotoxin in mucocutaneous candidiasis, the mechanism of the deleterious effects on phagocytic function was not directly investigated. Several observations may be enlightening with regard to the potential role of gliotoxin in vivo. Engulfment of particles requires ligation of the particles to the phagocyte followed by membrane invagination. The apparent structural changes in leukocytes as shown by flow cytometry in concert with microscopic observation of cell blebbing (data not shown) suggest that membrane changes that decrease the effectiveness of particle ingestion may occur in response to gliotoxin exposure. Sporidesmin, another fungal toxin that is a chemical congener of gliotoxin, has been reported to cause disappearance of liver cell microvilli, 11 further suggest. ing membrane perturbation among compounds of this type. Clearly, more definitive studies in this area are warranted.
Several investigators have suggested mechanisms whereby gliotoxin exerts its immunosuppressive effects, and these theories provide a good basis for a more mechanistic study of biological activity. For example, the disulfide component of gliotoxin could cross link membrane receptors.
Petty z indicated that there may be a disulfide link between Fc receptors for antibody, and the interpolation of gliotoxin between these receptors could alter the ability to trigger appropriate phagocytic activity.
Moreover, this study suggested that while gliotoxin is able to reduce the respiratory burst activity of stimulated PMN, the toxin alone in the absence of a stimulant caused a relatively slight increment in NBT reduction (Fig. 6) A final potential mechanism of gliotoxin action is induction of apoptosis, a cell process that is not inconsistent with the above-mentioned mechanisms of cytotoxicity. The observation of membrane changes noted above coupled with reports in the literature that attribute apoptotic effects to gliotoxin underscore this as a potential mechanism of action. The present study indicates that there may be hitherto unrecognized reasons why vaginal yeast infection in some women can be chronic or recurrent and provides a new factor that deserves consideration in future studies of this condition.