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(Journal of Leukocyte Biology. 2001;70:868-872.)
© 2001 by Society for Leukocyte Biology

In vivo GM-CSF prevents dexamethasone suppression of killing of Aspergillus fumigatus conidia by bronchoalveolar macrophages

Elmer Brummer*, Anjum Maqbool* and David A. Stevens*

* California Institute for Medical Research and
{dagger} Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, and
{ddagger} Stanford University School of Medicine, Stanford, California

Correspondence: Dr. Elmer Brummer, Div. of Infectious Diseases, Dept. of Medicine, Santa Clara Valley Medical Center, 751 S. Bascom Ave., San Jose, CA 95128-2699. E-mail: e.brummer{at}juno.com


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ABSTRACT
 
Dexamethasone (DEX) is a potent immunosuppressive agent used in the treatment of several disorders. However, despite its beneficial effects, DEX puts patients at risk for opportunistic infections, especially pulmonary aspergillosis. Previously we reported that in vitro granulocyte-macrophage colony-stimulating factor (GM-CSF) blocks the immunosuppressive action of DEX on bronchoalveolar macrophages (BAMs). Here we report that BAMs freshly isolated from mice treated intraperitoneally with DEX for 24 h had significantly (P<0.01) reduced killing of conidia, i.e., 15 ± 5% conidia killed by BAMs from DEX-treated mice versus 35 ± 3% by BAMs from mice given saline, 38 ± 5% by BAMs from mice given GM-CSF, and 39 ± 1% by BAMs from mice given both DEX and GM-CSF. On the other hand, in another compartment GM-CSF could not block the DEX reduction of spleen weight and spleen cellularity. Unlike GM-CSF, granulocyte colony-stimulating factor did not block DEX suppression of BAMs. GM-CSF given 24 h before DEX resulted in blocking of DEX suppression of BAM conidiacidal activity. However, when DEX was given 24 h before GM-CSF, DEX suppression of BAM was not reversed. These data show that GM-CSF in vivo blocks the in vivo immunosuppressive effects of DEX on BAM killing of conidia and suggest a potential use of GM-CSF in patients at risk for aspergillosis due to immunosuppressive DEX treatment.

Key Words: alveolar macrophages • conidia • glucocorticoids


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INTRODUCTION
 
Airborne conidia of Aspergillus species, especially Aspergillus fumigatus, are ubiquitous in the environment and pose a risk for pulmonary infection in a variety of immunocompromised hosts, including patients immunosuppressed by corticosteroids [1 , 2 ] or patients with Cushing’s syndrome [3 , 4 ]. Once infection is established, it is highly lethal [5 , 6 ] and remains high despite improvements in diagnosis and new antifungal drugs [6 ]. Although efforts have been made to reduce the risk of exposure, such as the use of air filtration, effective prophylaxis is being sought, particularly for neutropenic patients with antifungal agents and immunomodulation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) not only stimulates production of cells of the granulocyte and macrophage lineages but also enhances the functional activities of neutrophils, monocytes, and macrophages [7 , 8 ]. GM-CSF has been reported to interfere in vitro with dexamethasone (DEX) suppression of human monocyte antifungal activity against hyphae of A. fumigatus [9 , 10 ] and killing of conidia by murine peritoneal and bronchoalveolar macrophages (BAMs) [11 ]. However, in vitro treatment of spleen cells with GM-CSF has failed to interfere with DEX suppression of spleen cell-proliferative responses to the mitogen concanavalin A [11 ]. This suggests that GM-CSF might prevent unwanted effects of DEX on macrophages but would not interfere with desired DEX suppression on lymphocyte responses.

Here we report our investigation of the effects of in vivo treatment with DEX, GM-CSF, or both DEX and GM-CSF on ex vivo ability of BAMs to kill conidia of A. fumigatus.


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MATERIALS AND METHODS
 
Reagents
DEX (4 mg/mL) used for injection in these experiments was from American Reagent Laboratories, Shirley, NY. Recombinant murine (rm) GM-CSF was from Immunex, Seattle, WA. Optimal colony-forming activity was at 10 to 100 ng/mL of GM-CSF, and 1 µg/mL was equivalent to 2,000 IU/mL. rmGM-CSF from Endogen (Woburn, MA) gave the same results (data not shown), and these reagents could be used interchangeably. Recombinant human (rh) granulocyte colony-stimulating factor (G-CSF) was from R&D Systems, Minneapolis, MN, and studied at the same milligrams-per-kilogram dose as GM-CSF. Human G-CSF is active on murine cells and in vivo in the mouse [12 ]. (2,3-bis [2-methoxy-4-nitro-5 sulfophenyl]-2H-tetrazolium-5-carboxanilide) (XTT) and coenzyme Q (2,3-dimethoxy-5-methyl-1,4-benzoquinone) were obtained from Sigma Chemical Co., St. Louis, MO.

In vivo treatment
In simultaneous treatment protocols, groups of 8–10-week-old CD-1 male mice (Charles River Laboratories, Hollister, CA) were treated intraperitoneally (i.p.) with saline (0.2 mL/mouse), DEX, rmGM-CSF (0.5 µg/mouse), or DEX + rmGM-CSF. In preliminary dose-finding studies, DEX doses of 0.8–1.2 mg/mouse were studied. Because the dose-response curve was flat in that range, a dose of 0.8 mg was selected for further studies. In one experiment mice were treated with saline, DEX (0.8 mg/mouse), rhG-CSF (0.5 µg/mouse), or DEX + rhG-CSF. This dose of G-CSF is biologically active in vivo, stimulating neutrophils and tissue macrophages [13 ].

In one sequential treatment protocol, groups of mice first were given saline or 0.5 µg of rmGM-CSF (Endogen) i.p. and 24 h later were given DEX (0.8 mg/mouse) or saline i.p. The reverse protocol consisted of giving DEX (0.8 mg/mouse) or saline first and 24 h later giving an injection i.p. with 0.5 µg/mL of rmGM-CSF (Endogen) or saline. In all cases BAMs were collected and tested 24 h after the last treatment.

BAMs
BAMs were obtained from lungs of mice by lavage with phosphate-buffered saline + 10% fetal bovine serum (FBS) + 0.1% EDTA (pH 7.2) as previously described by our laboratory [14 ]. BAMs at 106/mL of RPMI-1640 + 10% FBS were plated at 0.1 mL per microtest plate well [Costar, Corning, NY (catalog no. 3696)]. Cultures were incubated for 2 h at 37°C in 5% CO2 + 95% air, and nonadherent cells were aspirated. Adherent cells constituted the BAM monolayer. Each experiment had quadruplicate wells for each experimental condition studied.

Aspergillus fumigatus
A clinical isolate of A. fumigatus, AF-10, was from the culture collection at the Infectious Diseases Research Laboratory, California Institute for Medical Research, San Jose. AF-10 was grown on Sabouraud dextrose agar (SDA) at 35°C until abundant conidia were produced. Conidia were collected in distilled water, filtered through gauze, counted, and suspended in RPMI-1640 + 10% FBS + 10% fresh mouse serum to the desired inoculum concentration. Over 95% of the inoculum consisted of single conidia.

Killing assay
Short-term assays allowed assessment of killing. A 2.5-h assay was used to measure conidiacidal activity [15 ]. Monolayers of BAMs were challenged with approximately 103 conidia/well and incubated at 37°C for 2.5 h, and then well contents were harvested with distilled water (5 washes/well) into a final volume of 1 mL. Microscopic examination of harvested wells showed that well contents were completely removed. Harvested material was plated at 0.1 mL per 90-mm-diameter SDA petri plate. Inoculated SDA plates were incubated for 24 h at 35°C and then at room temperature for 24 h, resulting in formation of discrete colonies for counting. Percent killing [reduction of inoculum colony forming units (CFUs)] was calculated using the formula [1 - (experimental CFUs/inoculum CFUs)] x 100.

Antifungal assay
Longer-term assays allowed assessment of effector cell activity in fungal inhibition. A 48-h assay was used to determine inhibition of conidia germination and or hyphal growth by BAMs from the different groups of treated mice. The colorimeteric XTT + coenzyme Q system was used to measure the quantitative metabolic reduction of XTT by viable fungal cells [16 , 17 ]. The quantitative change of the yellow substrate solution to the soluble orange metabolized, reduced form of XTT was measured by absorbance at 405 nM using a microplate reader (Dynex Technologies Inc., Chantilly, VA). Previous work established the linear relationship between the number of germinated conidia with subsequent hyphal growth and degree of XTT metabolism [16 , 18 ].

Spleen cells
Spleens were removed from mice and weighed, and single cell suspensions were prepared. Spleen cells were counted with a hemocytometer, and the total number of spleen cells per spleen was calculated.

Statistics
Statistically significant differences between groups were determined by the Student’s t-test with significance set at P < 0.05. When several groups were compared with a single control group, Bonferroni’s adjustment to the t-test was used.


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RESULTS
 
Effect of GM-CSF on DEX suppression of BAM conidiacidal activity
BAMs from DEX-treated mice had significantly (P<0.01) decreased conidiacidal activity [15±5%, n (number of experiments]=6) compared with that of control BAMs (35±3%), BAMs from GM-CSF-treated mice (38±5%), or BAMs from GM-CSF + DEX-treated mice (39±1%) (Fig. 1 ). A typical experiment is detailed in Table 1 . When BAMs were omitted from the assay, conidial CFUs were insignificantly different from those in the time-zero inoculum. The conidiacidal activity of control BAMs and the effect of DEX treatment were not altered when the ex vivo studies were performed with a fivefold increase in effector/target ratio (n=1). These results clearly demonstrated the in vivo suppression of BAM conidiacidal activity by DEX and the blocking of this effect by GM-CSF. The specificity of the GM-CSF effect was demonstrated by concurrent study with G-CSF, which also failed to affect BAM conidiacidal activity when given alone but failed to alter the effect of DEX when both were given simultaneously in vivo (n=2).



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  		Figure 1. Effect of in vivo treatments on BAM killing of A. fumigatus conidia. Bars represent mean percents ± SD of conidia killed by BAMs from saline-, GM-CSF-, DEX-, or GM-CSF + DEX-treated mice.


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  		Table 1. Effect of GM-CSF on DEX Suppression of BAM Killing of A. fumigatus Conidia

Effect of GM-CSF on DEX suppression of BAM antihyphal activity
The effect of DEX ± GM-CSF on the antifungal activity of BAM was also measured using the XTT assay. The extent of conidia germination and subsequent hyphal growth in cocultures was determined by this method. Figure 2 shows that growth was significantly inhibited [65%; (P<0.01)] by control BAMs and BAMs from mice treated with GM-CSF + DEX (55%, P<0.05) compared with growth in cultures without BAMs. Although BAMs from DEX-treated mice inhibited growth by 29%, this difference was not significant (P>0.05) compared with growth without BAMs. Inhibition of BAMs by DEX (29%) was significantly (P<0.05) less than inhibition by control BAMs (65%) or BAMs (55%) from GM-CSF + DEX-treated mice (Fig. 2) .



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  		Figure 2. Inhibition of conidia germination and hyphal growth by BAMs. Shown are differences in mean absorbance (Delta Absorbance) ± SD for four determinations of metabolized XTT by hyphae derived from 48-h cocultures of BAMs and conidia.

Effect of treatments on spleen weight and cellularity
DEX treatment significantly (P<0.01; four experiments, each with five mice per group) decreased spleen weight to half normal (mean spleen weights±SD: saline-treated mice, 0.135±0.015 g; DEX-treated mice, 0.07±0.015 g). This effect was not blocked by simultaneous treatment with GM-CSF (0.075±0.01 g). Similarly, the cellularity (number of spleen cells recovered per spleen) of spleens from DEX-treated mice was significantly (P<0.01) reduced by more than fivefold compared with normal cellularity (Fig. 3 ). Concomitant treatment with GM-CSF and DEX did not reverse the effect of DEX on spleen cellularity (Fig. 3) .



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  		Figure 3. Effect of treatments on spleen cellularity. Bars indicate mean numbers of recovered spleen cells ± SD from five spleens in each treatment group. Results are representative of four experiments, as described in the text.

Effect of treating mice with GM-CSF and then DEX
GM-CSF was given i.p. to mice, and 24 h later DEX was administered. BAMs collected 24 h after the GM-CSF + DEX treatments killed 29 ± 1% of conidia (P<0.01) (Fig. 4 ). On the other hand, BAMs from mice treated first with saline and then DEX had significantly (P<0.01) decreased conidiacidal activity (8±4%). BAMs from mice given GM-CSF first and then saline killed 24 ± 4% of conidia (Fig. 4) .



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  		Figure 4. Effect of GM-CSF first and then DEX on BAM killing of A. fumigatus conidia. Bars represent mean percent killing ± SD by BAMs in two experiments. The horizontal axis indicates sequential treatments of the mice from which BAMs were then derived.

Spleens from mice given GM-CSF first and then DEX had significantly (P<0.01) decreased size and weight compared with spleens from mice given GM-CSF first and then saline (Fig. 5 ). This indicates that GM-CSF could not abrogate the effect of DEX in this compartment.



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  		Figure 5. Effect of GM-CSF first and then DEX or saline on spleen weight. Bars represent the mean weights ± SD of spleens (five per group) from mice treated with GM-CSF first and then saline (GM/Sal) or DEX (GM/Dex) or from mice treated with saline first and then DEX (Dex). gm, grams.

Effect of treatment with DEX first and then GM-CSF on BAM conidiacidal activity
In another experiment, BAMs from mice treated with saline first and then GM-CSF killed 27% (P<0.01) of conidia. By contrast, BAMs from mice given DEX first and then GM-CSF killed 11% of conidia, which was not significant (P>0.05) activity and which was similar to the 7% killing by BAMs from mice given DEX and then saline. This indicates that GM-CSF given later could not reverse the effect of DEX on BAMs.

Spleens from mice (four mice per group) treated with DEX and then GM-CSF (0.081±0.009 g) or saline (0.074±0.009 g) weighed significantly less that spleens from mice given saline and then GM-CSF (0.117±0.029 g). These and the above data (Fig. 5) show that the effect of DEX on spleen size and weight can not be reversed by GM-CSF given either before or after DEX exposure.


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DISCUSSION
 
The percent killing of A. fumigatus conidia by human [19 ] or murine [20 ] BAMs reported by others is similar to the level reported here of killing by BAMs from saline-treated mice. On the other hand, we are not aware of published reports about the effect of DEX in vivo on BAM conidiacidal activity ex vivo. The same can be said for the effects of GM-CSF or GM-CSF + DEX in vivo on ex vivo killing of conidia by murine BAMs.

The dose of DEX used in vivo here for suppression (0.8 mg/mouse, 23 mg/kg of body weight) was within the range of doses used in studies by others who investigated in vivo effects of DEX on interferon (IFN)-{gamma} production by spleen cells [21 ] or nitric oxide production by peritoneal macrophages [22 ]. Our regimen consisted of a single dose of DEX i.p. followed by testing 24 h later. By comparison, one group [21 ] administered DEX (0.02, 0.1, or 0.75 mg/kg) orally for 7 days prior to assays, and another group [22 ] treated mice with DEX (0.3, 3, 10, or 30 mg/kg) subcutaneously for 3, 6, or 16 days prior to testing peritoneal macrophages. Clearly our DEX regimen was sufficient to impair macrophage antifungal activity. Whether different doses, regimens, and routes of DEX administration would change our results remains under investigation.

We used a second method to measure the antifungal activity of BAMs from treated mice; namely, the XTT assay. This method measured the sum of events, i.e., inhibition of conidia germination, killing of conidia, and outgrowth of viable conidia taking place in cocultures of BAMs from the different groups of treated mice.

These ex vivo results reflected what could take place in the bronchoalveolar compartment of lungs after exposure to conidia. In this respect, Waldorf et al. [23 ] reported a significant difference in killing of conidia ingested in vivo by BAMs from control mice (70%) versus BAMs from cortisone acetate-treated mice (39%) assayed ex vivo.

Use of GM-CSF in AIDS patients [24 ], bone marrow transplantation patients [25 ], and myelocytic leukemia patients [26 ] has been reported. The multiple doses used in these clinical situations (1.9 µg/kg/day) were much smaller that the single dose use here (14.2 µg/kg) but not as large as multiple doses (50 µg/kg daily for 3 days) used in a study with rats [27 ]. Additional studies are necessary to find the optimal dose of GM-CSF to block DEX suppression of BAMs as reported here.

Although G-CSF would not be expected to affect macrophages directly, G-CSF in vivo has secondary effects that can profoundly affect cellular immunity [28 ]. GM-CSF but not G-CSF (at the same milligrams-per-kilogram dose) blocked the suppression of BAM by DEX. This result suggests some degree of GM-CSF specificity in blocking DEX suppression of BAMs for conidiacidal activity. We speculate that GM-CSF acts directly on BAMs, which have receptors for GM-CSF, thus blocking the suppression of BAMs by DEX. GM-CSF studies on BAMs have shown that GM-CSF regulates BAM proliferation, morphology, and differentiation [29 ].

G-CSF in vitro has been shown to block DEX suppression of human neutrophil antifungal activity for hyphae of A. fumigatus [30 ]. The effect of GM-CSF was not investigated in that study. Because neutrophils have receptors for GM-CSF and GM-CSF enhances neutrophil antifungal activity against A. fumigatus [18 ], it might be possible to block DEX suppression of both BAM and neutrophils by GM-CSF.

We confirmed that DEX decreases spleen weight and cellularity, as has been reported by others [22 ]. In our study, GM-CSF did not prevent this effect of DEX on spleen weight or cellularity. This observation is important because it suggests that DEX used for depressing lymphocyte-mediated immunity would still be effective despite GM-CSF use, under circumstances in which GM-CSF could restore some relevant antimicrobial activity otherwise accompanying DEX immunosuppression. Tsutsui and Kamiyama [21 ] have reported that although DEX decreases spleen cellularity severalfold, the percentages of CD3+, CD4+, CD8+, and B220+ cells do not change. It remains to be determined whether concomitant DEX and GM-CSF treatment results in changes in percentages of lymphocyte phenotypes.

We investigated the effect of sequential treatments, e.g., GM-CSF first and then DEX or vice versa, on BAM conidiacidal activity. The results were similar to those of sequential studies done in vitro [11 ]; namely, GM-CSF given first blocked DEX given later, but GM-CSF did not block DEX when DEX was given first. These results support previous observations that prior signaling (IFN-{gamma} activation of macrophages) protects macrophages from immunosuppression by hydrocortisone acetate [31 ]. Additional insights into the mechanism of the interactions we describe here, with respect to the effects on cytokine networks, are currently being sought.

In summary, protection in the pulmonary compartment against DEX suppression by GM-CSF but not in other systemic compartments has potential for improving the design of therapeutic protocols for patients at risk of pulmonary aspergillosis.

Received January 17, 2001; revised July 23, 2001; accepted August 1, 2001.


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