Received 4 December 1995/Returned for modification 5 January 1996/Accepted 20 April 1996. In this study, we ... (Falcon; Becton Dickinson, Oxnard, Calif.) at 2.
INFECTION AND IMMUNITY, July 1996, p. 2846–2849 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 64, No. 7
Purified Capsular Polysaccharide of Cryptococcus neoformans Induces Interleukin-10 Secretion by Human Monocytes ANNA VECCHIARELLI,1* CINZIA RETINI,1 CLAUDIA MONARI,1 CARLO TASCINI,1 FRANCESCO BISTONI,1 2 AND THOMAS R. KOZEL Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, 06122 Perugia, Italy,1 and Department of Microbiology, University of Nevada School of Medicine, Reno, Nevada 89557-00462 Received 4 December 1995/Returned for modification 5 January 1996/Accepted 20 April 1996
In this study, we demonstrated that purified capsular polysaccharide of Cryptococcus neoformans is a potent inducer of interleukin-10 (IL-10) secretion by human monocytes. Endogenous IL-10 was involved in regulating tumor necrosis factor alpha and IL-1b secretion by human monocytes in response to encapsulated C. neoformans strains. Our results suggest a new immunosuppressive effect exerted by glucuronoxylomannan through the induction of IL-10, a potent downregulator of proinflammatory cytokines. Paris, France), washed twice, counted on a hematocytometer, and adjusted to the desired concentration. All C. neoformans strains were resuspended in saline and inactivated by autoclaving. Heat-killed cryptococci were washed twice in RPMI 1640 medium before use. To prepare PBM, heparinized venous blood samples from healthy donors were diluted with RPMI 1640 medium. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation (12). Cells were washed twice in RPMI 1640 medium, placed into cell culture petri dishes (Nunc Inter Med, Roskilde, Denmark) at a concentration of 2 3 106 to 3 3 106/ml in RPMI 1640 medium supplemented with 5% fetal calf serum (Eurobio Laboratories), and incubated at 378C in a 5% CO2 atmosphere for 1 h. Nonadherent cells were removed by washing the dishes three to five times with warm RPMI 1640 medium. Adherent cells were carefully recovered with a rubber policeman. The latter cells were .98% esterase positive; cell viability was assessed by trypan blue dye exclusion test (.98% viable) at the beginning and end of all experiments. Harvested PBM were placed in 24-well flat-bottom tissue culture plates (Falcon; Becton Dickinson, Oxnard, Calif.) at 2 3 106 cells per ml and incubated for 3, 18, or 48 h without or with (i) LPS (10 mg/ml; Difco Laboratories, Detroit, Mich.) or (ii) cells of C. neoformans 3168, 6995, or 7698 alone at an effector/target ratio of 1:1 or cells of strain 7698 (effector/target ratio 5 1:1) plus purified GXM (500, 250, or 0.1 mg/ml). After incubation in RPMI 1640 medium plus 10% non-heat-inactivated human serum at 378C in a 5% CO2 atmosphere, supernatant fluids were harvested and stored at 2208C until assayed. Cytokine levels in culture supernatant fluids were measured by enzymelinked immunosorbent assay kits for human IL-10 (Biosource International, Camarillo, Calif.) and human IL-1b (Genzyme, Boston, Mass.) and by a bioassay for TNF, as previously described (13). GXM was prepared from a serotype A strain (strain ATCC 24064), as described elsewhere (1). Figure 1A shows that PBM produced higher levels of IL-10 in response to encapsulated C. neoformans strains than in response to acapsular C. neoformans 7698. In fact, 7698 was a poor stimulator of IL-10 in all determinations. Considering that C. neoformans cells fix complement, the results observed may be due to activation by the alternative complement pathway. To investigate the effects of purified capsular material on IL-10 secretion, PBM were incubated with different doses of
Cryptococcus neoformans is a potentially pathogenic yeast that is ubiquitous in our environment. Cryptococcosis is unusual among healthy individuals, but encapsulated forms of this organism may produce disseminated disease in individuals with weakened immune systems (9). The capsule is an important virulence factor because it has tolerogenic and antiphagocytic properties (2, 3, 7, 8) which are related to one or more of the structural features of the major polysaccharide antigen of the capsule, glucuronoxylomannan (GXM). Recently, we reported that the secretion of cytokines by human monocytes is markedly influenced by encapsulation of C. neoformans as well as by the presence of purified capsular polysaccharide (14). Of particular interest was the ability of GXM to suppress lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor alpha (TNF-a). To date, little is known about the mechanism(s) by which C. neoformans influences cytokine secretion by monocytes. Since interleukin-10 (IL-10) has been reported to be a potent downregulator of TNF-a and IL-1b (4, 15), we hypothesized that inhibition of proinflammatory cytokine secretion could be mediated by endogenously produced IL-10 by human peripheral blood monocytes (PBM) secreted in response to C. neoformans and its capsular material. Therefore, the focus of this research was to examine the ability of capsular material to regulate IL-10 production by monocytes by using an acapsular (7698), thinly encapsulated (6995), or heavily encapsulated (3168) strain of C. neoformans and purified GXM alone or in combination with the acapsular strain. The following strains of C. neoformans were obtained from J. Orendi: a thinly encapsulated strain of C. neoformans var. neoformans serotype A (CBS 6995 5 NIH 37; National Institutes of Health, Bethesda, Md.), a heavily encapsulated serotype A strain (NCPF 3168; National Collection of Pathogenic Fungi, London, United Kingdom), and an acapsular mutant of C. neoformans var. neoformans (CBS 7698 5 NIH B-4131). The cultures were maintained by serial passage on Sabouraud agar (Bio Merieux, Lyon, France), harvested by suspending a single colony in RPMI 1640 medium (Eurobio Laboratories, * Corresponding author. Mailing address: Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. Phone: 39-75-585-3407. Fax: 39-75-585-3413. 2846
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FIG. 1. Time course of IL-10 production by PBM stimulated with encapsulated or acapsular C. neoformans alone or in combination with purified GXM (A) or with LPS (10 mg/ml) alone or in combination with purified GXM (B). Data are the means 6 standard deviations of three separate experiments with three different donors. NS, not stimulated.
GXM in the presence of acapsular C. neoformans 7698. Cytokine secretion exhibited a dose-dependent response to the incorporation of GXM into the medium. Kinetic studies showed that appreciable levels of IL-10 could be detected 3 h after the challenge of PBM with C. neoformans strains and reached a maximum within 48 h. This response occurred earlier and persisted longer when compared with the production of TNF-a and IL-1b, which were secreted at maximal levels 18 h after stimulation with C. neoformans (14). The presence of endogenous IL-10 at the initiation of macrophage activation (3
h) may be important for effective downregulation of TNF-a and IL-1b and could induce their decline within 48 h (14). Cryptococcal disease occurs after an invasion of live microorganisms; thus, experiments were performed to determine whether IL-10 was secreted from PBM in response to live as well as heat-inactivated C. neoformans cells. The IL-10 secretion by PBM treated for 18 h with live 7698, 6995, and 3168 in the presence of inhibitory concentrations of amphotericin B was 35.1 6 2.8, 50.3 6 4.0, and 58.1 6 3.5 pg/ml, respectively. The level of IL-10 secretion by unstimulated PBM was 9.8 6 2.1 pg/ml. The results prove that the magnitude of response to live microorganisms is similar to that obtained with killed microorganisms. It has been shown that LPS is a potent inducer of IL-10 from macrophages (4). As a consequence, we determined whether GXM was able to regulate LPS-induced IL-10 secretion in our experimental system. The results (Fig. 1B) show that the time course of IL-10 secretion reached a maximum at 18 to 48 h after LPS stimulation and that the addition of GXM was unable, under the conditions of this experiment, to modulate the amount of LPS-induced IL-10 secretion. Although GXM was
TABLE 1. Effects of polymyxin B addition on cytokine production by PBM treated with GXMa Stimulus
None LPS (10 mg/ml) GXM (500 mg/ml) FIG. 2. Time course of IL-10 production by PBM stimulated with GXM. Data are the means 6 standard deviations of three separate experiments with three different donors. NS, not stimulated.
Polymyxin B addition
IL-10 (pg/ml)
2 1 2 1 2 1
10 6 2 961 4,540 6 289 13 6 1 96 6 5 95 6 5
a The IL-10 level was determined in the supernatant fluid of PBM treated with LPS or GXM for 18 h in the presence (1) or absence (2) of polymyxin B (50 mg/ml).
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INFECT. IMMUN. TABLE 2. Effects of anti-human IL-10 monoclonal antibody on cytokine secretion by PBMa
In vitro stimulus
None LPS (1 mg/ml) LPS (10 mg/ml) 7698 6995 3168
Monoclonal antibody addition
18 h
48 h
18 h
48 h
2 2 1 2 1 2 1 2 1 2 1
861 103 6 5 186 6 3 255 6 18 253 6 19 180 6 15 200 6 18 70 6 5 130 6 10 90 6 8 253 6 20
461 NTb NT 225 6 10 231 6 15 15 6 3 15 6 3 861 18 6 1 361 260
18 6 1 184 6 9 211 6 11 451 6 31 428 6 39 220 6 20 200 6 18 120 6 10 1,200 6 86 300 6 18 1,850 6 191
21 6 2 NT NT 534 6 49 504 6 31 205 6 18 280 6 20 39 6 4 60 6 7 20 6 3 50 6 5
TNF-a (U/ml)
IL-1b (pg/ml)
a Anti-human IL-10 monoclonal antibodies were added (1) or not added (2) at the time of cell culture. Data are the means 6 standard deviations of four separate experiments with four different donors. b NT, not tested.
able to modulate LPS-induced TNF production by human monocytes, it was not able to downregulate LPS-induced IL-1b production in these latter cells. As a matter of fact, GXM seems to be responsible for different effects observed on LPStreated monocytes; this phenomenon is currently under investigation. The results presented in Fig. 1 suggested that GXM mediates secretion of IL-10 by PBM. As a consequence, we evaluated the ability of purified GXM to induce IL-10 secretion. The results (Fig. 2) show a dose-dependent induction of GXM secretion that reached a maximum after 18 h. The ability of GXM to induce IL-10 secretion is most likely not due to contamination of the GXM preparation with LPS. The incorporation of polymyxin B into the reaction mixture completely blocked LPS-induced IL-10 secretion but had no effect on GXM-induced secretion of IL-10 (Table 1). It has been well documented that because of its downregulation of proinflammatory cytokine secretion (4, 15) and its enhancing effects on IL-1 receptor antagonist production (5), IL-10 is a powerful anti-inflammatory agent. In a previous paper, we described the ability of capsular material to downregulate proinflammatory cytokine secretion by PBM (14). To verify the possible involvement of endogenous IL-10 production in the regulation of TNF-a and IL-1b secretion in the presence of the cryptococcal capsule, experiments in which anti-IL-10 monoclonal antibody (10 mg/ml; Genzyme) was added to cultures of PBM stimulated with LPS or challenged with C. neoformans 7698, 6995, or 3168 were performed. Our results are consistent with the recent data of Levitz et al. showing that C. neoformans induces IL-10 production by peripheral blood mononuclear cells and that IL-10 is able to inhibit TNF-a and IL-1b release by these cells (10). The results (Table 2) show that proinflammatory cytokines induced by LPS (10 mg/ml) were unaffected by the addition of anti-IL-10 antibody when a smaller amount of LPS was used in our system. These results are in agreement with those of de Waal Malefyt et al., who reported an autoregulatory role for IL-10 by inhibiting LPS-induced IL-1b or TNF-a production from human monocytes (4). Anti-IL-10 treatment of PBM challenged with encapsulated C. neoformans 6995 or 3168 resulted in substantial increases in TNF-a and IL-1b secretion. The most significant effects were found on the production of TNF-a and IL-1b after 18 h of stimulation by C. neoformans 3168. The production of proinflammatory cytokines in response to C. neoformans 7698 was not regulated by the addition of anti-IL-10. This result suggests
that the very low levels of IL-10 secreted in response to the acapsular strain (Fig. 1) are not sufficient to regulate TNF-a or IL-1b. The pathobiological significance of IL-10 production in vivo during C. neoformans infection should be investigated. However, during cryptococcal disease, patients develop a GXM concentration in the range of 250 to 500 mg/ml in their body fluids (6, 11). In conclusion, our results have identified a biological activity for purified capsular material of C. neoformans that further expands the known spectrum of its suppressive effects on cellmediated immunity. We are grateful to Eileen Mahoney Zannetti for excellent technical assistance. This study was supported by VIII Progetto AIDS (contract 9305-44) in Italy and by U.S. Public Health Service grant AI-14209 (to T.R.K.). REFERENCES 1. Cherniak, R., E. Reiss, M. E. Slodki, R. D. Plattner, and S. O. Blumer. 1980. Structure and antigenic activity of the capsular polysaccharide of Cryptococcus neoformans. Mol. Immunol. 17:1025–1032. 2. Clerici, M., F. T. Hakim, D. J. Venzon, S. Blatt, C. W. Hendrix, T. A. Wynn, and G. M. Shearer. 1993. Changes in interleukin-2 and interleukin-4 production in asymptomatic, human immunodeficiency virus-seropositive individuals. J. Clin. Invest. 91:759–765. 3. Collins, H. L., and G. J. Bancroft. 1992. Cytokine enhancement of complement-dependent phagocytosis by macrophages: synergy of tumor necrosis factor-a and granulocyte-macrophage colony-stimulating factor for phagocytosis of Cryptococcus neoformans. Eur. J. Immunol. 22:1447–1454. 4. de Waal Malefyt, R., J. Abrams, B. Bennett, C. G. Figdor, and J. E. de Vries. 1991. Interleukin-10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174: 1209–1220. 5. de Waal Malefyt, R., H. Yssel, M. G. Roncarolo, H. Spits, and J. E. de Vries. 1992. Interleukin-10. Curr. Opin. Immunol. 4:314–320. 6. Eng, R. H. K., E. Bishburg, and S. M. Smith. 1986. Cryptococcal infections in patients with acquired immune deficiency syndrome. Am. J. Med. 81:19– 23. 7. Fromtling, R. A., H. J. Shadomy, and E. J. Jacobson. 1982. Decreased virulence in stable acapsular mutants of Cryptococcus neoformans. Mycopathologia 79:23–29. 8. Hill, J. O. 1992. CD41 T cells cause multinucleate giant cells to form around Cryptococcus neoformans and confine the yeast within the primary site of infection in the respiratory tract. J. Exp. Med. 175:1685–1695. 9. Kwong-Chung, K. J., and J. E. Bennett. 1992. Medical mycology, p. 397–446. Lea and Febinger, Malvern, Pa. 10. Levitz, S. M., A. Tabuni, S.-H. Nong, and D. T. Golenbock. 1996. Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide. Infect. Immun. 64:945–951. 11. Pettoello-Mantovani, M., A. Casadevall, P. Smarnworawong, and H. Goldstein. 1994. Enhancement of HIV type 1 infectivity in vitro by capsular
VOL. 64, 1996 polysaccharide of Cryptococcus neoformans and Haemophilus influenzae. AIDS Res. Hum. Retroviruses 10:1079–1084. 12. Vecchiarelli, A., M. Dottorini, T. Beccari, C. Cociani, T. Todisco, and F. Bistoni. 1993. Inhibition of candidacidal activity of polymorphonuclear cells by alveolar macrophage-derived factor from lung cancer patients. Am. Rev. Respir. Dis. 147:414–419. 13. Vecchiarelli, A., M. Dottorini, D. Pietrella, C. Monari, C. Retini, T. Todisco, and F. Bistoni. 1994. Role of alveolar macrophages as antigen presenting cells in
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Cryptococcus neoformans infection. Am. J. Respir. Cell Mol. Biol. 11:130–137. 14. Vecchiarelli, A., C. Retini, D. Pietrella, C. Monari, C. Tascini, T. Beccari, and T. R. Kozel. 1995. Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1b secretion from human monocytes. Infect. Immun. 63:2919–2923. 15. Wang, P., P. Wu, M. I. Siegel, R. W. Egan, and M. Motasim Billah. 1994. IL-10 inhibits transcription of cytokine genes in human peripheral blood mononuclear cells. J. Immunol. 153:811–816.
