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Candida albicans hyphal form enhances tumor necrosis factor mRNA levels and protein secretion in murine ANA-1 macrophages
CELLULAR
IMMUNOLOGY
142, 137- 144 ( 1992)
Candida albicans Hyphal Form Enhances Tumor Necrosis Factor mRNA Levels and Protein Secretion in Murine ANA-l Macrophages ELISABETTABLASI,LUCIA PITZURRA,MANUELAPULITI, ANDREABARTOLI,AND FRANCESCO BISTONI Department @“Experimental Medicine and Biochemical Sciences,Microbiology Section, University of Perugia, Perugia, Italy Received November 21, 1991: accepted February 29, 1992
We havedemonstratedthat Candida albicans in its hyphal form (H-Candidu) acts as a stimulating agent in the cloned macrophage population ANA- I. Both tumor necrosis factor (TNF) mRNA levels and secreted biological activity augment in ANA-l macrophages exposed to HCandida. Such effectsare observed at an effector-to-target cell ratio of 1:1 and occur after 1 and 3 hr of coincubation, respectively. The phenomenon is independent of the metabolic status of the fungus, since viable as well as heat-killed H-Cundida are comparable in inducing TNF mRNA levels.The extent and kinetics of H-Cundida-mediated effectsare similar to thoseobservedfollowing to lipopolysaccharide (LPS). This implies that C. albicuns in exposure of ANA- I macrophages its hyphal form is a potent macrophagemodulator; whether it acts through the same mechanism(s) as LPS remains to be elucidated. o 1992 Academic PW, IIK.
INTRODUCTION Tumor necrosis factor (TNF), produced primarily by cells of the monocyte/macrophage lineage, has been found to display many biological activities, in addition to tumor cytotoxicity (1). TNF can activate neutrophils, enhance the cytolytic activity of macrophages, augment natural killer cell activity, promote T- and B-ceil proliferation, and modulate endothelial cell-surface antigens (for review see (2, 3)). In viva and in vitro studies provide evidence on the possible involvement of TNF as an immunopotentiating monokine against infectious agents. In fact, TNF has been shown to influence the interaction of macrophageswith protozoan parasites (4), bacteria (5) and fungi (6). In particular, Kindler et al. (7) claim that TNF, releasedby macrophages in the microenvironment of BCG-induced granulomas, is involved in a process of autoamplification that favors further macrophage accumulation and differentiation leading to bacterial elimination. Thus, pleiotropic immunoregulatory functions are ascribed to TNF suggestingthat it may play a pivotal role in the induction and expression of immune responses. Using an in vivo experimental model of fungal infection, we have shown that TNF, together with other cytokines, is produced in large amounts during Candida infection and likely mediates the observed immunomodulating effects (8). In addition, in vitro studies have proved that murine splenic macrophages or human blood mononuclear cells respond to Candidu with enhanced TNF production (9, 10). However, the mul137 00088749/92 $5.00 Copyright 0 1992 by Academic Press, k All rights of reproduction in any form reserved.
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tistep cascadeof events involved in the host-Candida relationship remains to be elucidated. The dimorphic properties of the fungus undoubtedly contribute to the complexity of the interaction. In fact, C. albicuns is capable of dimorphic transition from yeast to hyphal form, while colonizing the susceptible host. In particular, it is widely acceptedthat during the early stagesof infection phagocytes remove and destroy most of the fungal yeast forms. However, the Cundidu cells that escapethe phagocytosis processrapidly convert into hyphal form and invade the organism where they produce granulomas and causetissue damage (11). Initial evidence indicates that macrophages (12) as well as PMN and other natural effector cells (13, 14) can affect the hyphal form of the fungus in vitro. In this report, we show that C. ufbicuns hyphal form enhances TNF mRNA levels and protein secretion in the cloned macrophage population ANA- 1. The phenomenon occurs regardlessof the fact that either viable or heat-killed microorganisms have been used. Bacterial lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, has been included in our studies because it is one of the most potent TNF inducers (1, 15). Although slightly less effective than LPS, Cundidu most likely acts through the same mechanism(s), the kinetics of TNF production by Cundidu and LPS being comparable. These data suggestthe possibility that macrophages may play an immunopotentiating role in the late stagesof Cundidu infections as secretory cells. MATERIALS AND METHODS Cell Lines ANA-I murine macrophages, derived by immortalization of bone marrow cells from C57BL/6 mice with a recombinant retrovirus carrying v-&and v-myc oncogenes (16, 17), were cultured in RPM1 1640 medium supplemented with glutamine (4 mM), gentamicin (50 pg/ml), and 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Lagan, UK) (complete medium). L-929 fibroblast cells (CCL 1, American Type Culture Collection, Rockville, MD) were maintained in complete medium. Reagents TNF-a and rabbit anti-mouse TNF-(U monoclonal antibodies were obtained from Genzyme (Boston, MA). LPS from Escherichiu coli (serotype 0 128:B12) was purchased from Sigma Chemicals, (St. Louis, MO). Cundidu ulbicuns The C. ulbicuns strain used throughout the study (CA-6) was isolated from a clinical specimen. It was grown at 28°C with mild agitation in low-glucose Winge medium aspreviously described( 18). Under theseconditions, the organism grew asan essentially pure yeast-phasepopulation. To obtain the hyphal form of C. ufbicuns (H-Candida), the pure yeast-form population of C. ulbicuns was harvested from Winge medium, washed twice in saline, resuspended in complete medium, dispensed in 60-mm tissue culture plates (5 X lO’/ml, 5 ml/plate), and incubated at 37°C in 5% COZ. More than 98% of the microorganisms showed hyphal form after 3 hr incubation, as detailed elsewhere( 12). The C. ulbicuns preparations were tested for endotoxin contamination and only those containing endotoxins lessthan 0.05 rig/ml (as assessedby the Limulus umebocyte lysate assay)were used.
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TNF
PRODUCTION
139
Heat-killed H-Candida was prepared by heating H-Candida three times at 121“C. Only batches that showed no regrowth in Sabouraud broth and no [3H]glucose uptake (12) were employed in the assayas heat-killed H-Candida. Experimental Protocol ANA-l macrophages and H-Cundidu were coincubated in 60-mm tissue culture plates at 37°C in 5% CO2. All experiments were performed in complete medium. The incubation times and the macrophage/Cundida cell ratios, hereafter referred to as E: T ratios, varied from experiment to experiment as detailed in the tables and figure legends. The cultures were then employed in biological assaysor Northern RNA blot analysis. Tumor Necrosis Factor Assay The quantitation of TNF activity was performed by a bioassay using L-929 cells, as described ( 19). Briefly, L-929 cells were seededinto 96-well flat-bottom plates (4 X lo4 cells/well) and incubated for 24 hr at 37°C. The spent medium was then removed and replaced with test sample or standard TNF preparations containing actinomycin D (3 pg/ml). After 20 hr of incubation, plates were stained with 0.5% crystal violet in 20% methanol for 15 min and washed in tap water. After drying, absorbance was determined at 450 nm using a Titertek Multiscan plate reader (Flow, Rockville, MD). All determinations of TNF activity in test samples were compared to commercially available preparations of TNF with known titers; the results were expressedas U/ml. RNA Extraction and Northern Blot Analysis Total cellular RNA was isolated from stimulated and unstimulated ANA-l cells, solubilized with guanidine isothyocyanate as previously described (20). Briefly, 10 pg of total RNA were electrophoresed in denaturing conditions, blotted onto nylon membranes (Amersham International, Amersham, UK), cross-linked by uv irradiation and heated for 1 hr at 60°C in 0.1X SSCand 0.5% SDS. Filters were prehybridized for 6 hr at 37°C in prehybridization buffer, containing formamide and 0.6% denatured salmon sperm DNA, before 2 X lo6 cpm/ml of the specific 32P-labeledprobe were added for 18 hr in hybridization buffer containing dextran sulfate. Filters were washed four times at room temperature for 5 min and four times at 60°C for 30 min in IX SSCand 0.5% SDS. The filters were autoradiographed by using Kodak X-ARS films (Eastman-Kodak Company, Rochester, NY) with intensifying screensat -80°C. 32Plabeled probes were obtained by Nick translation using a commercial kit (Amersham), according to the procedure suggestedby the manufacturer. The specific activity was always higher than lo* cpm/pg. For TNF mRNA detection, the 1.2-kb PstI + EcoRI fragment (cloned from the pUC 9 plasmid), kindly provided by Dr. L. Varesio, was used. All results presented in this paper were reproduced in four to six separate experiments. Values in each experiment, except the Northern blot analysis, represent the averageof triplicate determinations. Standard errors did not exceed 5% of the means. RESULTS To evaluate whether H-Cundidu might affect the secretory properties of ANA-1 macrophages, experiments were performed in which cells were cocultured with H-
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INCUBATIONTIME (hr) FIG. 1. Time course of TNF secretion by ANA- 1 macrophagesupon stimulation with LPS or H-Candida. Cells were incubated in the presence of LPS (0; 1 &ml) or H-Candida (0; E:T = 1:lO) for the indicated time. Cell-free culture supematantswere harvestedand amessedfor TNF activity, as describedunder Materials and Methods. Each value representsthe average of three determinations.
Cundidu (E:T = 1:10) or LPS for different times. Figure 1 shows the time course of TNF secretion in cell-free supernatants, as assessedby the TNF-sensitive L-929 cell line. ANA-l cells per se produced undetectable amounts of TNF. When H-Cundidu was used as a stimulating agent, significant TNF levels were found as early as 3 hr after stimulation and maximal levels were reached at 6 hr. Upon LPS stimulation, a pattern of TNF secretion comparable to that of H-Cundida-treated cells was observed. As shown in Table 1, both LPS and H-Cundidu induced TNF activity in a dosedependent manner. In fact, maximal activity was observed upon treatment with 1 pg/ ml LPS or at an E:T ratio of 1:10 for H-Cundidu. When lower doses of LPS (GO.1
TABLE 1 Dose-Response of TNF Secretion by ANA-I Macrophages Stimulated with C. albicans or LPS” Treatment None LPS (rg/ml)
H-Candida (E:T)
Dose
TNF Activity W/ml)
5 1 0.1 0.0 1 1:20 1:lO I:1 1O:l
<2 140 130 40 4 100 110 90 2
a Cells were incubated for 6 hr in the presence of the indicated amounts of LPS or H-Candida, then supematants were harvested and assayedas described under Materials and Methods. Each value represents the average of three determinations.
141
Candidu HYPHAL FORM ENHANCES TNF PRODUCTION TABLE 2 Neutralization of TNF Activity in Culture Supernatants of ANA-I Macrophages by Anti-Mouse TNF-a Antibodies“ Treatment
Addition of anti-TNF-cu antibody
TNF (U/ml)
+ +
<2 130 12 110 <2
None LPS H-Candida
u Cells were incubated with or without LPS (1 *g/ml) or H-Candidu (ET = 1:lO) for 6 hr. Supernatants were then harvested and assayedas described under Materials and Methods in the presence or absenceof anti-mouse TNF-ol antibodies (I: 1000dilution). Each value representsthe average of three determinations.
pg/ml) or E:T ratios of 1:1 or 10:1 were employed, TNF activity decreased.Table 2 shows that the biological activity of culture supernatants was completely neutralized by the addition of specific anti-TNF-cYantibodies, thus demonstrating that the cytotoxic activity releasedby ANA-l macrophages upon exposure to LPS or H-Candida was, in fact, TNF-a. We then examined whether LPS or H-Candida could affect TNF gene expression. For this purpose, TNF mRNA levels were measured in H-Candidu- or LPS-stimulated and unstimulated ANA-l macrophages (Fig. 2). Both treatments enhanced TNF mRNA levels in a time-dependent manner; the LPS-induced levels were higher than those induced by H-Cundida. Moreover, while enhancement of TNF mRNA was detected as early as 30 min after LPS stimulation, 1 hr was necessaryto observe a (hr)
INCUBATION TIME
ab
c
a
b
3
2
1
05
c
a
b
c
a b c
_ 1.2Kb
FIG. 2. Northern blot analysis of TNF mRNA in ANA- 1 macrophagesstimulated with LPS or H-Candida. ANA-l macrophages ( lo6 cells/ml) were incubated with (a) medium, (b) H-Cundidu (ET = 1:IO), or (c) LPS ( 1 &ml) for the indicated times and processedfor Northern blot analysis as described under Materials and Methods. Blotted RNA (10 pg per lane) was hybridized with ‘*P-labeled probes for TNF.
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similar increase in H-Candida-treated cells. When the blots were rehybridized with GAPDH probe no differences in the levels of mRNA were observed (data not shown). To investigate whether the metabolic status of the fungus could interfere with the described phenomenon, heat-killed H-Candida was tested in our system. Figure 3 shows that heat-killed H-Candida as well as viable H-Cundidu were comparable in enhancing TNF mRNA levels in ANA-l macrophages at E:T ratios of 1:1 or 1:10. Overall, these data indicate that C. ulbicuns in its hyphal form is able to modulate TNF mRNA levels and protein secretion in macrophages. DISCUSSION The results of this investigation establish that C. ulbicans hyphal form enhances TNF mRNA levels and protein secretion in the cloned macrophage population ANA1. The phenomenon, which is equally mediated by viable and killed microorganisms, is time- and dose-dependent and closely resembles that induced by LPS. Macrophages are the main source of TNF, a monokine with multiple biological activities, a number of which are involved in various aspects of the inflammatory processes(for review see (2, 21)). Macrophages release TNF in response to several agents, the most potent being bacterial endotoxin (LPS). TNF is detected in the blood after an injection of LPS (1) and is produced locally within the liver granulomas of BCG-infected mice (7). Experimental systemic infections with C. ulbicuns also result in a significant enhancement of TNF sera levels as well as in augmented production of this cytokine by splenic cells (8). These findings, together with the knowledge that TNF enhances in vitro immune functions including anti-Cundidu properties of phagocytes (6, 22), suggestthat TNF may play an antimicrobial role in vivo as an immunopotentiating cytokine in Cundidu infections as well. However, because of the dimorphic properties of C. ulbicuns, the host-fungal interaction is particularly complex and not yet clearly understood. In a recent report, we provided evidence that murine
NONE
E/T RATIO
VIABLE H-CANDIDA
KILLED H-CANDIDA
I:1
IO:1
/
I:10
11
10.1
j
I
/
i
1:lO
I
/
-1.2 Kb
FIG. 3. Northern blot analysis of TNF mRNA in ANA-l macrophages stimulated with viable or killed H-Can&u. ANA-l macrophages ( lo6 cells/ml) were incubated with medium, viable H-Candida, or killed H-Candida at the indicated E:T ratios for 6 hr, and then processedfor Northern blot analysis as described under Materials and Methods. Blotted RNA (10 pg per lane) was hybridized with 32P-labeledprobes for TNF.
Candida HYPHAL
FORM
ENHANCES
TNF
PRODUCTION
143
macrophages inhibit the growth of H-Can&&z in vitro (12). Here, we demonstrate that ANA-l macrophages respond to the hyphal form of the fungus with augmented TNF mRNA levels and protein secretion, such effectsbeing time and dose dependent. In our opinion, these data provide two major contributions on the issue of hostCandidu interaction. On one hand, we provide the first evidence that H-Candidu is a macrophagestimulating signal, capable of enhancing TNF mRNA levels and secretion. On the other hand, we demonstrate that a cloned macrophage population responds to Cundidu with enhanced TNF production. Therefore, the capacity of reacting against the fungus with TNF production is fully ascribed to the macrophage. Such an assertion does not exclude the possibility that in vivo macrophagesmay interact with other cell types to fulfill the optimal immunoresponse against Cundida. Experiments are in progressto establish whether the coincubation of ANA- 1 macrophageswith other cell types would affect macrophage-mediated TNF production in responseto Candida. The TNF activity produced by ANA- 1 macrophagesin responseto H-Candida has been characterized as TNF-a, since neutralization of such activity in the supernatants is obtained by specific anti-TNF-cr monoclonal antibodies. Northern blot analysis further confirms this result. In fact, the detected mRNA bands have the expected 1.2kb size. With the purpose of quantifying the magnitude of the H-Candidu-induced phenomenon, LPS hasbeen included as a positive control in parallel cultures. Similarly to what has been previously described in other cell systems (23, 24), we have also found LPS to be the most effective inducer. In fact, the maximal levels of TNF observed in cells exposed to H-Candida at an E:T ratio of 1:1 never reach those produced by LPS-stimulated cells. Further variations of the ET ratio, up to 150, fail to determine any additional enhancement (data not shown). Moreover, the kinetics of TNF mRNA induction are comparable for both agents. Whether H-Cundidu and LPS act through an identical mechanism(s) is still unclear and further investigation is neededto address this issue. The fact that TNF induction is mediated both by viable and killed fungi indicates that heat-insensitive structures, likely expressedon the fungal surface, act as stimulating signals regardlessof the metabolic status of the fungus. Experiments are in progressto establish whether purified cell wall components, known to be active in other cell systems (25) may be responsible for the observed Candidu-induced macrophage stimulation. Overall, our data imply that a macrophage may play a pleiotropic function against Candida infections. While certainly effective as a scavengerin the phagocytic clearance of yeasts,macrophagesmay also sustain the precious role of secretory cells within the tissues,where hyphae undergo elongation and causefunctional damage(11). Therefore, in addition to being a first line defense element, macrophages may also exert potentiating activity at later stagespostinfection, when the dimorphic fungus, by transition, has overcome the now harmless phagocytic defense mechanisms. ACKNOWLEDGMENTS The authors thank Eileen Zannetti for excellent editorial and secretarial assistance. This work was supported by Contract No. 91.00088.PF41 within the Progetto Finalizzato FATMA from the Consiglio Nazionale delle Richerche, Italy.
REFERENCES 1. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson,
Sci. USA 12, 3666, 1975.
B., Proc. Nut/. Acud.
144 2. 3. 4. 5. 6. 7. 8.
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Le, J., and Vilcek, J., Lab. Invest. 56, 234, 1987. Sherry, B., and Cerami, C., J. Cell. Biol. 107, 1269, 1988. de Titto, E., Catterall, J. R., and Remington, J. S., J. Immunol. 137, 1342, 1986. Nakane, A., Minagawa, T., and Kato, K., Infect. Zmmun. 56,2563, 1988. Blasi, E., Farinelli, S., Varesio, L., and Bistoni, F., Infect. Immun. 58, 1073, 1990. Kindler, V., Sappino, A. P., Grau, G. E., Piguet, P. F., and Vassalli, P., Cell 56, 731, 1989. Vecchiarelh, A., Cenci, E., Puliti, M., Blasi, E., Puccetti, P., Cassone,A., and Bistoni, F., Cell. Zmmunol. 124,334, 1989.
9. Vecchiarelli, A., Puliti, M., Torosantucci, A., Cassone, A., and Bistoni, F., Cell. Immunol. 134, 65, 1991. 10. Djeu, J. Y., Blanchard, D. K., Richards, A. L., and Friedman, H., J. Immunol. 141,4047, 1988. 11. Odds, F. C., “Candida and Candidosis.” Bailliere Tindall, London, 1988. 12. Scaringi, L., Blasi, E., Cornacchione, P., Bietta, C., and Bistoni, F., Mycoses 34, 119, 1991. 13. Beno, D. W. A., and Mathews, H. L., Cell. Immunol. 128, 89, 1990. 14. Diamond, R. D., Krzesicki, R., and Jao, W., J. Clin. Invest. 61, 349, 1978. 15. Gifford, G. E., and Lohmann-Matthes, M. L., Int. J. Cancer 38, 135, 1986. 16. Blasi, E., Matheison, B. J., Varesio, L., Cleveland, J. L., Borchert, P. A., and Rapp, U., Nature (London) 318,667,
1985.
17. Cox, G. W., Matheison, B. J., Gandino, L., Blasi, E., Radzioch, D., and Varesio, L., J. Nut/. Cancer Inst. 81, 1492, 1989. 18. Marconi, P., Bistoni, F., Boncio, L., Bersiani, A., Bravi, P., and Pitzurra, M., Ann. Scluvo 18, 61, 1976. 19. Rubin, B. Y., Anderson, S. L., Sullivan, S. A., Williamson, B. D., Car-swell,B. D., and Old, L. Y., Proc. Natl. Acad. Sci. USA 82,6637, 1985. 20. Chomezynski, P., and Sacchi, N., Anal. Biochem. 162, 156, 1987. 21. Beutler, B., and Cerami, A., N. Engl. J. Med. 316, 379, 1987. 22. Djeu, J. Y., Blanchard, D. K., Halkias, D., and Friedman, H., J. Immunol. 137, 2980, 1986. 23. Lepe-Zuniga, J. L., and Klostergaard, J., Lymphokine Rex 9, 309, 1990. 24. Haas, J. G., Baeuerle, P. A., Riethmuller, G., and Ziegler-Heitbrock, H. W., Proc. Nutl. Acad. Sci. USA 87,9563, 1990. 25. Cassone,A., Curr. Top. Med. Mycol. 3, 249, 1989.
IMMUNOLOGY
142, 137- 144 ( 1992)
Candida albicans Hyphal Form Enhances Tumor Necrosis Factor mRNA Levels and Protein Secretion in Murine ANA-l Macrophages ELISABETTABLASI,LUCIA PITZURRA,MANUELAPULITI, ANDREABARTOLI,AND FRANCESCO BISTONI Department @“Experimental Medicine and Biochemical Sciences,Microbiology Section, University of Perugia, Perugia, Italy Received November 21, 1991: accepted February 29, 1992
We havedemonstratedthat Candida albicans in its hyphal form (H-Candidu) acts as a stimulating agent in the cloned macrophage population ANA- I. Both tumor necrosis factor (TNF) mRNA levels and secreted biological activity augment in ANA-l macrophages exposed to HCandida. Such effectsare observed at an effector-to-target cell ratio of 1:1 and occur after 1 and 3 hr of coincubation, respectively. The phenomenon is independent of the metabolic status of the fungus, since viable as well as heat-killed H-Cundida are comparable in inducing TNF mRNA levels.The extent and kinetics of H-Cundida-mediated effectsare similar to thoseobservedfollowing to lipopolysaccharide (LPS). This implies that C. albicuns in exposure of ANA- I macrophages its hyphal form is a potent macrophagemodulator; whether it acts through the same mechanism(s) as LPS remains to be elucidated. o 1992 Academic PW, IIK.
INTRODUCTION Tumor necrosis factor (TNF), produced primarily by cells of the monocyte/macrophage lineage, has been found to display many biological activities, in addition to tumor cytotoxicity (1). TNF can activate neutrophils, enhance the cytolytic activity of macrophages, augment natural killer cell activity, promote T- and B-ceil proliferation, and modulate endothelial cell-surface antigens (for review see (2, 3)). In viva and in vitro studies provide evidence on the possible involvement of TNF as an immunopotentiating monokine against infectious agents. In fact, TNF has been shown to influence the interaction of macrophageswith protozoan parasites (4), bacteria (5) and fungi (6). In particular, Kindler et al. (7) claim that TNF, releasedby macrophages in the microenvironment of BCG-induced granulomas, is involved in a process of autoamplification that favors further macrophage accumulation and differentiation leading to bacterial elimination. Thus, pleiotropic immunoregulatory functions are ascribed to TNF suggestingthat it may play a pivotal role in the induction and expression of immune responses. Using an in vivo experimental model of fungal infection, we have shown that TNF, together with other cytokines, is produced in large amounts during Candida infection and likely mediates the observed immunomodulating effects (8). In addition, in vitro studies have proved that murine splenic macrophages or human blood mononuclear cells respond to Candidu with enhanced TNF production (9, 10). However, the mul137 00088749/92 $5.00 Copyright 0 1992 by Academic Press, k All rights of reproduction in any form reserved.
138
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ET AL.
tistep cascadeof events involved in the host-Candida relationship remains to be elucidated. The dimorphic properties of the fungus undoubtedly contribute to the complexity of the interaction. In fact, C. albicuns is capable of dimorphic transition from yeast to hyphal form, while colonizing the susceptible host. In particular, it is widely acceptedthat during the early stagesof infection phagocytes remove and destroy most of the fungal yeast forms. However, the Cundidu cells that escapethe phagocytosis processrapidly convert into hyphal form and invade the organism where they produce granulomas and causetissue damage (11). Initial evidence indicates that macrophages (12) as well as PMN and other natural effector cells (13, 14) can affect the hyphal form of the fungus in vitro. In this report, we show that C. ufbicuns hyphal form enhances TNF mRNA levels and protein secretion in the cloned macrophage population ANA- 1. The phenomenon occurs regardlessof the fact that either viable or heat-killed microorganisms have been used. Bacterial lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, has been included in our studies because it is one of the most potent TNF inducers (1, 15). Although slightly less effective than LPS, Cundidu most likely acts through the same mechanism(s), the kinetics of TNF production by Cundidu and LPS being comparable. These data suggestthe possibility that macrophages may play an immunopotentiating role in the late stagesof Cundidu infections as secretory cells. MATERIALS AND METHODS Cell Lines ANA-I murine macrophages, derived by immortalization of bone marrow cells from C57BL/6 mice with a recombinant retrovirus carrying v-&and v-myc oncogenes (16, 17), were cultured in RPM1 1640 medium supplemented with glutamine (4 mM), gentamicin (50 pg/ml), and 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Lagan, UK) (complete medium). L-929 fibroblast cells (CCL 1, American Type Culture Collection, Rockville, MD) were maintained in complete medium. Reagents TNF-a and rabbit anti-mouse TNF-(U monoclonal antibodies were obtained from Genzyme (Boston, MA). LPS from Escherichiu coli (serotype 0 128:B12) was purchased from Sigma Chemicals, (St. Louis, MO). Cundidu ulbicuns The C. ulbicuns strain used throughout the study (CA-6) was isolated from a clinical specimen. It was grown at 28°C with mild agitation in low-glucose Winge medium aspreviously described( 18). Under theseconditions, the organism grew asan essentially pure yeast-phasepopulation. To obtain the hyphal form of C. ufbicuns (H-Candida), the pure yeast-form population of C. ulbicuns was harvested from Winge medium, washed twice in saline, resuspended in complete medium, dispensed in 60-mm tissue culture plates (5 X lO’/ml, 5 ml/plate), and incubated at 37°C in 5% COZ. More than 98% of the microorganisms showed hyphal form after 3 hr incubation, as detailed elsewhere( 12). The C. ulbicuns preparations were tested for endotoxin contamination and only those containing endotoxins lessthan 0.05 rig/ml (as assessedby the Limulus umebocyte lysate assay)were used.
Cundida HYPHAL
FORM
ENHANCES
TNF
PRODUCTION
139
Heat-killed H-Candida was prepared by heating H-Candida three times at 121“C. Only batches that showed no regrowth in Sabouraud broth and no [3H]glucose uptake (12) were employed in the assayas heat-killed H-Candida. Experimental Protocol ANA-l macrophages and H-Cundidu were coincubated in 60-mm tissue culture plates at 37°C in 5% CO2. All experiments were performed in complete medium. The incubation times and the macrophage/Cundida cell ratios, hereafter referred to as E: T ratios, varied from experiment to experiment as detailed in the tables and figure legends. The cultures were then employed in biological assaysor Northern RNA blot analysis. Tumor Necrosis Factor Assay The quantitation of TNF activity was performed by a bioassay using L-929 cells, as described ( 19). Briefly, L-929 cells were seededinto 96-well flat-bottom plates (4 X lo4 cells/well) and incubated for 24 hr at 37°C. The spent medium was then removed and replaced with test sample or standard TNF preparations containing actinomycin D (3 pg/ml). After 20 hr of incubation, plates were stained with 0.5% crystal violet in 20% methanol for 15 min and washed in tap water. After drying, absorbance was determined at 450 nm using a Titertek Multiscan plate reader (Flow, Rockville, MD). All determinations of TNF activity in test samples were compared to commercially available preparations of TNF with known titers; the results were expressedas U/ml. RNA Extraction and Northern Blot Analysis Total cellular RNA was isolated from stimulated and unstimulated ANA-l cells, solubilized with guanidine isothyocyanate as previously described (20). Briefly, 10 pg of total RNA were electrophoresed in denaturing conditions, blotted onto nylon membranes (Amersham International, Amersham, UK), cross-linked by uv irradiation and heated for 1 hr at 60°C in 0.1X SSCand 0.5% SDS. Filters were prehybridized for 6 hr at 37°C in prehybridization buffer, containing formamide and 0.6% denatured salmon sperm DNA, before 2 X lo6 cpm/ml of the specific 32P-labeledprobe were added for 18 hr in hybridization buffer containing dextran sulfate. Filters were washed four times at room temperature for 5 min and four times at 60°C for 30 min in IX SSCand 0.5% SDS. The filters were autoradiographed by using Kodak X-ARS films (Eastman-Kodak Company, Rochester, NY) with intensifying screensat -80°C. 32Plabeled probes were obtained by Nick translation using a commercial kit (Amersham), according to the procedure suggestedby the manufacturer. The specific activity was always higher than lo* cpm/pg. For TNF mRNA detection, the 1.2-kb PstI + EcoRI fragment (cloned from the pUC 9 plasmid), kindly provided by Dr. L. Varesio, was used. All results presented in this paper were reproduced in four to six separate experiments. Values in each experiment, except the Northern blot analysis, represent the averageof triplicate determinations. Standard errors did not exceed 5% of the means. RESULTS To evaluate whether H-Cundidu might affect the secretory properties of ANA-1 macrophages, experiments were performed in which cells were cocultured with H-
BLASI ET AL.
140
INCUBATIONTIME (hr) FIG. 1. Time course of TNF secretion by ANA- 1 macrophagesupon stimulation with LPS or H-Candida. Cells were incubated in the presence of LPS (0; 1 &ml) or H-Candida (0; E:T = 1:lO) for the indicated time. Cell-free culture supematantswere harvestedand amessedfor TNF activity, as describedunder Materials and Methods. Each value representsthe average of three determinations.
Cundidu (E:T = 1:10) or LPS for different times. Figure 1 shows the time course of TNF secretion in cell-free supernatants, as assessedby the TNF-sensitive L-929 cell line. ANA-l cells per se produced undetectable amounts of TNF. When H-Cundidu was used as a stimulating agent, significant TNF levels were found as early as 3 hr after stimulation and maximal levels were reached at 6 hr. Upon LPS stimulation, a pattern of TNF secretion comparable to that of H-Cundida-treated cells was observed. As shown in Table 1, both LPS and H-Cundidu induced TNF activity in a dosedependent manner. In fact, maximal activity was observed upon treatment with 1 pg/ ml LPS or at an E:T ratio of 1:10 for H-Cundidu. When lower doses of LPS (GO.1
TABLE 1 Dose-Response of TNF Secretion by ANA-I Macrophages Stimulated with C. albicans or LPS” Treatment None LPS (rg/ml)
H-Candida (E:T)
Dose
TNF Activity W/ml)
5 1 0.1 0.0 1 1:20 1:lO I:1 1O:l
<2 140 130 40 4 100 110 90 2
a Cells were incubated for 6 hr in the presence of the indicated amounts of LPS or H-Candida, then supematants were harvested and assayedas described under Materials and Methods. Each value represents the average of three determinations.
141
Candidu HYPHAL FORM ENHANCES TNF PRODUCTION TABLE 2 Neutralization of TNF Activity in Culture Supernatants of ANA-I Macrophages by Anti-Mouse TNF-a Antibodies“ Treatment
Addition of anti-TNF-cu antibody
TNF (U/ml)
+ +
<2 130 12 110 <2
None LPS H-Candida
u Cells were incubated with or without LPS (1 *g/ml) or H-Candidu (ET = 1:lO) for 6 hr. Supernatants were then harvested and assayedas described under Materials and Methods in the presence or absenceof anti-mouse TNF-ol antibodies (I: 1000dilution). Each value representsthe average of three determinations.
pg/ml) or E:T ratios of 1:1 or 10:1 were employed, TNF activity decreased.Table 2 shows that the biological activity of culture supernatants was completely neutralized by the addition of specific anti-TNF-cYantibodies, thus demonstrating that the cytotoxic activity releasedby ANA-l macrophages upon exposure to LPS or H-Candida was, in fact, TNF-a. We then examined whether LPS or H-Candida could affect TNF gene expression. For this purpose, TNF mRNA levels were measured in H-Candidu- or LPS-stimulated and unstimulated ANA-l macrophages (Fig. 2). Both treatments enhanced TNF mRNA levels in a time-dependent manner; the LPS-induced levels were higher than those induced by H-Cundida. Moreover, while enhancement of TNF mRNA was detected as early as 30 min after LPS stimulation, 1 hr was necessaryto observe a (hr)
INCUBATION TIME
ab
c
a
b
3
2
1
05
c
a
b
c
a b c
_ 1.2Kb
FIG. 2. Northern blot analysis of TNF mRNA in ANA- 1 macrophagesstimulated with LPS or H-Candida. ANA-l macrophages ( lo6 cells/ml) were incubated with (a) medium, (b) H-Cundidu (ET = 1:IO), or (c) LPS ( 1 &ml) for the indicated times and processedfor Northern blot analysis as described under Materials and Methods. Blotted RNA (10 pg per lane) was hybridized with ‘*P-labeled probes for TNF.
142
BLASI ET AL.
similar increase in H-Candida-treated cells. When the blots were rehybridized with GAPDH probe no differences in the levels of mRNA were observed (data not shown). To investigate whether the metabolic status of the fungus could interfere with the described phenomenon, heat-killed H-Candida was tested in our system. Figure 3 shows that heat-killed H-Candida as well as viable H-Cundidu were comparable in enhancing TNF mRNA levels in ANA-l macrophages at E:T ratios of 1:1 or 1:10. Overall, these data indicate that C. ulbicuns in its hyphal form is able to modulate TNF mRNA levels and protein secretion in macrophages. DISCUSSION The results of this investigation establish that C. ulbicans hyphal form enhances TNF mRNA levels and protein secretion in the cloned macrophage population ANA1. The phenomenon, which is equally mediated by viable and killed microorganisms, is time- and dose-dependent and closely resembles that induced by LPS. Macrophages are the main source of TNF, a monokine with multiple biological activities, a number of which are involved in various aspects of the inflammatory processes(for review see (2, 21)). Macrophages release TNF in response to several agents, the most potent being bacterial endotoxin (LPS). TNF is detected in the blood after an injection of LPS (1) and is produced locally within the liver granulomas of BCG-infected mice (7). Experimental systemic infections with C. ulbicuns also result in a significant enhancement of TNF sera levels as well as in augmented production of this cytokine by splenic cells (8). These findings, together with the knowledge that TNF enhances in vitro immune functions including anti-Cundidu properties of phagocytes (6, 22), suggestthat TNF may play an antimicrobial role in vivo as an immunopotentiating cytokine in Cundidu infections as well. However, because of the dimorphic properties of C. ulbicuns, the host-fungal interaction is particularly complex and not yet clearly understood. In a recent report, we provided evidence that murine
NONE
E/T RATIO
VIABLE H-CANDIDA
KILLED H-CANDIDA
I:1
IO:1
/
I:10
11
10.1
j
I
/
i
1:lO
I
/
-1.2 Kb
FIG. 3. Northern blot analysis of TNF mRNA in ANA-l macrophages stimulated with viable or killed H-Can&u. ANA-l macrophages ( lo6 cells/ml) were incubated with medium, viable H-Candida, or killed H-Candida at the indicated E:T ratios for 6 hr, and then processedfor Northern blot analysis as described under Materials and Methods. Blotted RNA (10 pg per lane) was hybridized with 32P-labeledprobes for TNF.
Candida HYPHAL
FORM
ENHANCES
TNF
PRODUCTION
143
macrophages inhibit the growth of H-Can&&z in vitro (12). Here, we demonstrate that ANA-l macrophages respond to the hyphal form of the fungus with augmented TNF mRNA levels and protein secretion, such effectsbeing time and dose dependent. In our opinion, these data provide two major contributions on the issue of hostCandidu interaction. On one hand, we provide the first evidence that H-Candidu is a macrophagestimulating signal, capable of enhancing TNF mRNA levels and secretion. On the other hand, we demonstrate that a cloned macrophage population responds to Cundidu with enhanced TNF production. Therefore, the capacity of reacting against the fungus with TNF production is fully ascribed to the macrophage. Such an assertion does not exclude the possibility that in vivo macrophagesmay interact with other cell types to fulfill the optimal immunoresponse against Cundida. Experiments are in progressto establish whether the coincubation of ANA- 1 macrophageswith other cell types would affect macrophage-mediated TNF production in responseto Candida. The TNF activity produced by ANA- 1 macrophagesin responseto H-Candida has been characterized as TNF-a, since neutralization of such activity in the supernatants is obtained by specific anti-TNF-cr monoclonal antibodies. Northern blot analysis further confirms this result. In fact, the detected mRNA bands have the expected 1.2kb size. With the purpose of quantifying the magnitude of the H-Candidu-induced phenomenon, LPS hasbeen included as a positive control in parallel cultures. Similarly to what has been previously described in other cell systems (23, 24), we have also found LPS to be the most effective inducer. In fact, the maximal levels of TNF observed in cells exposed to H-Candida at an E:T ratio of 1:1 never reach those produced by LPS-stimulated cells. Further variations of the ET ratio, up to 150, fail to determine any additional enhancement (data not shown). Moreover, the kinetics of TNF mRNA induction are comparable for both agents. Whether H-Cundidu and LPS act through an identical mechanism(s) is still unclear and further investigation is neededto address this issue. The fact that TNF induction is mediated both by viable and killed fungi indicates that heat-insensitive structures, likely expressedon the fungal surface, act as stimulating signals regardlessof the metabolic status of the fungus. Experiments are in progressto establish whether purified cell wall components, known to be active in other cell systems (25) may be responsible for the observed Candidu-induced macrophage stimulation. Overall, our data imply that a macrophage may play a pleiotropic function against Candida infections. While certainly effective as a scavengerin the phagocytic clearance of yeasts,macrophagesmay also sustain the precious role of secretory cells within the tissues,where hyphae undergo elongation and causefunctional damage(11). Therefore, in addition to being a first line defense element, macrophages may also exert potentiating activity at later stagespostinfection, when the dimorphic fungus, by transition, has overcome the now harmless phagocytic defense mechanisms. ACKNOWLEDGMENTS The authors thank Eileen Zannetti for excellent editorial and secretarial assistance. This work was supported by Contract No. 91.00088.PF41 within the Progetto Finalizzato FATMA from the Consiglio Nazionale delle Richerche, Italy.
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