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*
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy; and
Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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; and 6) the interaction between
CD40 on monocytes and CD40 ligand on activated T lymphocytes responding
to C. neoformans played a critical role in IL-12
secretion. These data highlight the mechanisms of IL-12 production by
human monocytes exposed to C. neoformans, indicating a
possible biphasic secretion of IL-12, dependent on the direct effect of
fungal insult, and characterized by consistent secretion of IL-12 that
is dependent on the interaction of CD40 with the CD40 ligand expressed
on activated T cells responding to C.
neoformans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 is usually produced rapidly after infection in a T
cell-independent and/or T cell-dependent pathway, the latter involving
the interaction between CD40 ligand (CD40L) on activated T cells and
CD40 on APC or on phagocytic cells (11, 12). Phagocytic cells are
believed to be the most important physiologic producers of IL-12,
which, in turn, induces production of IFN- from both T and NK cells
(9, 13). IL-12 has been shown to be important for the development of an
effective immune response against C. neoformans in mice (14, 15). Furthermore, IL-12 administration has been shown to enhance the
efficacy of antifungal drugs, suggesting that it may have therapeutic
use in cryptococcosis (16, 17).
Recent studies have demonstrated that both C. neoformans and
Candida albicans induce expression of IL-12 p40 mRNA in PBMC
of healthy HIV-seronegative donors. C. neoformans-mediated
induction occurs later and is more prolonged than that with C.
albicans (18). Priming PBMC with exogenous IFN- has been shown
to induce appreciable levels of IL-12 in the cell supernatants of PMBCs
exposed to fungi (19). Nevertheless, both C. neoformans and
C. albicans can induce production of IFN- after
interaction with lymphocytes and NK cells (20). The production of
IFN- by PBMC in response to either fungus peaks after day 7 of
incubation, suggesting that fungal-induced IFN- secretion could play
a role in regulating IL-12 in the late phase of the immune response
(21). However, other cytokine signals may also influence IL-12
production. In a previous study, we demonstrated that human monocytes
stimulated with C. neoformans produce IL-10, and that this
effect is quantitatively dependent on the presence of the
polysaccharide capsule (22). Since IL-10 is a potent down-regulator of
IL-12, this observation raises the possibility that IL-12 secretion by
monocytes exposed to C. neoformans is limited by IL-10 (23).
Furthermore, Ab opsonins may influence IL-12 expression through direct
effects on phagocytosis, FcR stimulation, and/or secondary effects on
cytokine production.
In this study, we explored several aspects of IL-12 production by
C. neoformans and C. albicans. In particular, we
studied the dependence of IL-12 production on: encapsulation of
C. neoformans, endogenous IFN-, and the presence of a
specific Ab to the capsular polysaccharide. Our results indicate that
IL-12 production in response to C. neoformans follows a
biphasic pattern. In the first phase, IL-12 is produced by monocytes as
a direct result of fungal stimulation and this process can be
up-regulated by Ab-mediated phagocytosis. The second phase involves a
more consistent production of IL-12 that involves the ligation of
specific activated T cells of MHC class II as well as CD40 molecules.
This study elucidates the mechanism by which C. neoformans
triggers IL-12 production and strongly suggests that IL-12 is produced
primarily through a T cell-dependent pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium and FCS were obtained from Eurobio Laboratories
(Paris, France). Human serum
(HS)3 was obtained from
Biosource International (Camarillo, CA). Mouse (IgG1) monoclonal
anti-human CD40 and mouse (IgG1) monoclonal anti-human CD40L
were obtained from Calbiochem (La Jolla, CA). Anti-glucuronoxylomannan
(GXM) mAb was purified from hybridoma 2H1 ascites fluid, as previously
described (24). Mouse (IgG1) monoclonal anti-human HLA-DR was
purchased from Boehringer Mannheim (Mannheim, Germany). Mouse (IgG1)
mAb to human CD16 (FcRIII) was obtained from Ancell (Bayport, MN).
mAb to human CD4 (IgG1) FITC, mAb to human CD8 (IgG2a), Con A,
monoclonal anti-human IFN-, and unrelated mAb mouse IgG1 
were purchased from Sigma. Amphotericin B was purchased from Bristol
Myers Squibb (Sermoneta, Italy). LPS from Escherichia coli
055:135 was obtained from Difco Laboratories (Detroit, MI). RPMI 1640,
FCS, C. neoformans (approximately 5 x
107), Con A, HS, and mAb 2H1 were tested for endotoxin
contamination by Limulus amebocyte lysate assay (Sigma),
which had a sensitivity of approximately 0.05–0.1 ng of E.
coli LPS/ml. All reagents tested negative.
Preparation of peripheral blood monocytes (PBM) and lymphocytes
Heparinized venous blood, obtained from healthy donors, was diluted with RPMI 1640 plus 5% FCS (cRPMI), and the mononuclear cells were separated by density-gradient centrifugation on Ficoll-Hypaque (25). The mononuclear cells were washed twice in cRPMI and incubated for 1 h at a concentration of 2 x 106 to 3 x 106/ml in cell culture petri dishes (Nunc Inter Med, Roskilde, Denmark). The remaining adherent cells (approximately 2 x 104/well) were >98% viable, as evaluated by trypan blue dye exclusion. Nonadherent cells were E rosetted, as previously described (26). The cells recovered were T lymphocyte [T(E+)] cells, >98% CD3+, as evaluated by flow cytometry analysis.
Microorganisms
The two strains of C. neoformans used in this study were obtained from J. Orendi (Central Bureau Schimmel Cultures (CBS), Delft, The Netherlands). C. neoformans var. neoformans 6995 (CBS 6995, also known as NIH 37) is a thinly encapsulated isolate of serotype A. C. neoformans var. neoformans 7698 (CBS 7698; also known as NIH B-4131) is an acapsular mutant. C. albicans PCA-2 was kindly supplied by Dr. Kerridge (University of Cambridge, Cambridge, U.K.). This is an agerminative strain that grows as pure yeast form in vitro at both 28°C and 37°C in conventional mycologic media. The morphologic characteristics and growth conditions of two strains of C. neoformans and C. albicans isolates have been described (27). The cultures were maintained by serial passage on Sabouraud agar (Bio Merieux, Lyon, France) and harvested by suspending a single colony in RPMI 1640. The cells were washed twice, counted on a hemocytometer, and adjusted to the desired concentration. Cells of C. neoformans 6995 and 7698 were killed by autoclaving.
Preparation of coculture of monocytes and T lymphocytes
Monolayers of PBM (2 x 104) adherent in flat-bottom 96-well plates were incubated with or without heat-inactivated C. neoformans (2 x 105) for 2 h at 37°C in 5% CO2 in RPMI 1640 plus 10% HS and used throughout as APC. The PBM monolayers were washed to remove nonattached microorganisms, and autologous T(E+) cells (105) in RPMI 1640 plus 10% HS were added to the cultures. Supernatant fluids were harvested after various days of culture for IL-12 determination. In selected experiments, proliferating T cells responding to C. neoformans harvested from the above culture after 4 days of incubation were mixed and incubated for 18 h with autologous monocytes. These monocytes had been incubated with or without C. neoformans for 2 h. Supernatant fluids for IL-12 determination were harvested after 3 days of culture. In all experiments, the proliferating cell population was >98% CD3 positive, as evaluated by flow cytometry analysis. The viability of lymphocytes and the adherent cells after 3 and 7 days was >98% in each experimental group evaluated by trypan blue dye exclusion.
Cytokine determination
IL-12 and IFN- were determined with a human IL-12
ultrasensitive ELISA kit purchased from Biosource. The IL-12 kit is a
solid-phase ELISA based on the Ab sandwich principle; its sensitivity
is <0.8 pg/ml detectable cross-reaction with human IL-1ß, IL-2,
IL-3, IL-4, IL-5, IL-7, IL-8, IFN-, granulocyte-macrophage CSF,
leukocyte inhibiting factor stem cells factor, TNF-
, IL-10,
IL-13, and IL-15. The assay recognizes both natural and recombinant
human IL-12, as well as the free p40 subunit. Selected samples were
tested with a kit for human IL-12 p70 heterodimer (Genzyme, Cambridge,
MA). The detection limit of the assay was 2 pg/ml.
Viability of encapsulated or acapsular C. neoformans-treated cells
Viability of treated cells was measured with a colorimetric reaction that is based on the capacity of mitochondrial dehydrogenase of living cells to reduce MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Aldrich Chemical, Milan, Italy) into formazan. The quantity of formazan produced and measured at an OD of 540 nm in a microplate reader (Sorin Biomedica, Saluggia, Italy) correlated with the number of living cells (28).
Statistical analysis
Statistical significance was calculated using Student’s paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) show that monocytes
exposed to acapsular C. neoformans produced appreciable
levels of IL-12. In contrast, significantly lower levels of IL-12 were
secreted in response to the encapsulated strain. This result suggested
that encapsulation of C. neoformans could regulate IL-12
production by human monocytes. Capsular material of C.
neoformans and GXM, its principal constituent, has been shown to
be antiphagocytic and to have immunosuppressive properties (25, 29, 30). The mAb to GXM 2H1 (mAb 2H1) has been shown to be opsonic and can
mitigate deleterious effects caused by GXM (2, 24, 31). In our
experimental system, the addition of mAb 2H1 (10 µg/ml) enhanced
phagocytosis of encapsulated C. neoformans by three- to
fourfold. Measurement of IL-12 in the presence and absence of mAb 2H1
revealed that addition of mAb 2H1 to suspensions of PBMC and C.
neoformans facilitates IL-12 secretion by monocytes (Fig. 1). To
examine whether the kinetic of IL-12 production was affected by the use
of dead C. neoformans, we conducted an experiment with live
C. neoformans in a concentration of amphotericin B that was
fungistatic (32). Use of live or dead C. neoformans resulted
in similar amounts of IL-12 production. Amphotericin B at the
concentration used in our assay did not affect IL-12 production. To
investigate whether IL-12 levels increased when a larger number of
monocytes was used, we tested the capability of 10 x
106/ml monocytes to secrete IL-12 in response to
encapsulated C. neoformans. The results showed that IL-12
levels were dependent on the number of monocytes used, such that
10 x 106/ml monocytes were able to secrete bioactive
IL-12 p70 in response to encapsulated C. neoformans (12
pg/ml versus 0 pg/ml of unstimulated monocytes). Recently, it has been
reported that soluble CD16/FcRIII induces IL-12 release by dendritic
cells (33), raising the possibility that mAb 2H1 could increase IL-12
via FcRIII ligation. Experiments were done to examine this hypothesis.
In our experimental system, anti-CD16 mAb were used to block
FcRIII. The data obtained showed that anti-CD16 mAb induced a
significant (p < 0.05) increase of IL-12
production (8 ± 1 pg/ml) with respect to unstimulated cells
(undetectable levels) after 3 days of incubation. In addition, when
monocytes were incubated with the encapsulated strain (6995) in the
presence of anti-CD16 mAb, a significant increase
(p < 0.05) of IL-12 production was observed
(9 ± 2 pg/ml) with respect to cells treated with 6995 (4 ±
1 pg/ml). In contrast, the effect of anti-CD16 mAb was lost when
IL-12 was determined after 7 days of incubation.
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show that IL-12 secretion in response
to the encapsulated or acapsular strains of C. neoformans
was delayed as compared with C. albicans. The kinetic study
showed that maximum secretion occurred within 5–7 days of incubation.
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as a
soluble factor and CD40L as a molecule that interacts with receptor
CD40 on monocytes. Both possibilities were investigated. In a first
series of experiments, we evaluated IFN- secretion in fungal-treated
monocyte cultures plus T lymphocytes. The results reported in Fig. 3 show that appreciable levels of IFN-
were secreted within 5–7 days of incubation. The kinetic of IL-12
production was comparable with that of IFN-, raising the possibility
that the delayed onset of consistent levels of IL-12 secreted in
response to C. neoformans might hinge on endogenous IFN-.
Consistent with this premise, neutralization of endogenous IFN- with
mAb to IFN- greatly reduced IL-12 secretion by monocytes in a
dose-dependent manner (Fig. 4).
Additional evidence for IFN- involvement in IL-12 secretion was
provided by priming monocytes with IFN- (500 U/ml) for 18 h
before the addition of encapsulated C. neoformans (E:T
= 1:2). This treatment greatly enhanced IL-12 production (about
100-fold).
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show that binding of mAb to MHC
class II and/or to CD40 results in a further increase of IL-12
secretion by monocytes responding to both the acapsular and
encapsulated strains. Simultaneous triggering of MHC class II molecules
and CD40 results in further stimulation of IL-12. We next studied the
contribution of activated T cells responding to C.
neoformans for IL-12 induction by human monocytes. Activated T
cells responding to Cryptococcus-laden monocytes, >70% CD4
positive (CD8 positive <30%) on phenotypic analysis, were mixed with
autologous freshly isolated monocytes pretreated overnight with
C. neoformans (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In addition,
IL-12 release was increased in the presence of specific activated T
cells, whereas the opposite occurred using anti-CD40L. Analysis of
molecular signals that regulate IL-12 production showed that in human
monocytes the ligation of CD40 and/or MHC class II molecules greatly
enhanced IL-12 secretion. Previously, it has been reported that IL-12 p40 transcripts are produced by PBMC from normal subjects in response to C. neoformans and C. albicans; however, IL-12 levels are not detectable in supernatant fluids of monocyte-enriched cells (18). Our results show appreciable levels of IL-12 production in response to C. albicans and the acapsular strain of C. neoformans. This apparent discrepancy could be due to the different strains of fungi used in our assay. The strain of C. albicans used in our study is different from that employed by Harrison and Levitz in that it is an agerminative strain. The strain of C. neoformans, which elicited IL-12 in our study, was an acapsular mutant. In addition, the low levels of IL-12 observed in response to the encapsulated strain could be ascribed to differences of the strain used in our assay. Alternatively, it could be due to the different assay used to measure IL-12 levels. Our kit recognized total IL-12 as well as free p40 subunit, whereas the kit used by Harrison and Levitz detected p70 heterodimer and not free p40 (18). Nevertheless, we were able to detect IL-12 as p70 heterodimer in experiments with large numbers of monocytes. However, recently a protective role for IL-12 p40 subunit in C. neoformans infection has been suggested (35).
The addition of mAb 2H1 enhanced IL-12 production by monocytes exposed
to the encapsulated C. neoformans, and we hypothesized that
Fc cross-linking could play a role. Consistent with this premise, mAb
anti-CD16 enhanced IL-12 release in response to 6995 despite little
or no phagocytosis. The enhancement of IL-12 due to Fc ligation
observed after 3 days of incubation was undetectable after 7 days.
These data suggest that in our experimental system, Fc-induced IL-12
release is a transient effect that regulates the early phase, but not
the late phase of IL-12 production; hence, it has little or no effect
on lymphocyte activation. In fact, IL-12 production by monocytes was
still higher in the presence of autologous T lymphocytes. It is
conceivable that the release of IL-12 by monocytes was greatly
increased upon interaction with Ag-specific T cells. The kinetic
production of IL-12 showed a strict correlation between the amount of
IL-12 released and the presence of specific activated T cells. In fact,
maximum levels of IL-12 were observed after 5–7 days of incubation, as
a consequence of specific T cell-blastogenic response (25). These
results strongly suggest that in the first step of the immune response,
low levels of IL-12 are secreted by monocytes in response to the
encapsulated strain; the presence of endogenous IL-12 facilitates
IFN- release from human activated T cells, as previously reported
(36, 37). However, the relatively low production of IL-12 observed by
us could be a result of its consumption by activated T cells.
In our experimental system, appreciable levels of IFN- were
secreted by T cells cocultured with Cryptococcus-laden
monocytes within 3 to 7 days of incubation. However, the possibility
that a larger number of cells could lead to a consistent increase of
IFN-, as observed for IL-12, should be considered. GXM-binding mAb
is likely to have enhanced IFN- production as a consequence of
increased secretion of IL-12. On the other hand, endogenous IFN-, in
turn, is likely to have enhanced late secretion of IL-12, as
demonstrated by the fact that addition of Abs to IFN- greatly
reduced IL-12 secretion. Given the low number and functional defect of
T cells in AIDS patients with cryptococcosis, we speculate that the
latter mechanism of IL-12 induction may be compromised.
The role of specific activated T cells on IL-12 production by monocytes was determined in monocytes exposed to C. neoformans and challenged with specific activated T cells responding to C. neoformans. We observed a strong up-regulation of IL-12, showing that the contribution of Ag-specific T cells appears to be essential for optimal production of Cryptococcus-induced IL-12. Recent studies documented that IL-12 produced in a T cell-dependent pathway is mediated through CD40L/CD40 interaction (38, 39, 40). Additional blocking of anti-CD40L mAb strongly reduced IL-12 production by human monocytes, suggesting that the interaction of CD40L with CD40 is the major trigger for IL-12 production in our experimental system. Given these results, it seems possible that the molecular signals that contribute to triggering IL-12 production by monocytes exposed to C. neoformans are dependent on binding of CD40 and/or MHC class II molecules. Indeed, the ligation by T cells of MHC class II molecules and CD40, either separately or even more so in combination, stimulated IL-12 release.
In a previous study, we reported that human monocytes stimulated with C. neoformans produce IL-10 that is quantitatively dependent on the presence of capsular material (22). In fact, acapsular isolates are poorer inducers of IL-10 than the encapsulated strains (22, 27). Nevertheless, it has been documented that IL-10 is a potent down-regulator of IL-12 (39, 41), raising the possibility that IL-12 secretion by monocytes exposed to C. neoformans could be limited by IL-10 (23). In a similar manner, mAb 2H1, which inhibits IL-10 production (2), could improve IL-12 secretion.
In conclusion, our results strongly suggest that IL-12 produced in response to C. neoformans follows a biphasic pattern: the first phase in which IL-12 is produced by monocytes directly by fungal insult and could be up-regulated by the phagocytic process and/or endogenous IL-10. A more consistent production of IL-12 occurs in a later phase involving the ligation by specific activated T cells of MHC class II as well as CD40 molecules. The current study elucidates the mechanism triggered by C. neoformans in IL-12-producing cells and strongly suggests that IL-12 is produced primarily in a T cell-dependent pathway.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anna Vecchiarelli, Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail address:
3 Abbreviations used in this paper: HS, human serum; CD40L, CD40 ligand; GXM, glucuronoxylomannan; PBM, peripheral blood monocyte.
Received for publication July 6, 1998. Accepted for publication October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by T cells. Nature 378:88.[Medline]
production by NK cell stimulatory factor (NKSF): characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173:869. are required for initiating the protective Th1 response to pulmonary cryptococcosis in resistant C. B-17 mice. Am. J. Respir. Cell Mol. Biol. 17:733. interferon by natural killer cells. Infect. Immun. 65:3594.[Abstract]
restores deficient IL-12 production by peripheral blood mononuclear cells from HIV-seropositive donors. J. Immunol. 158:459.[Abstract]
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and interleukin-1ß secretion from human monocytes. Infect. Immun. 63:2919.[Abstract]
RIII induces maturation of dendritic cells and production of several cytokines including IL-12. Adv. Exp. Med. Biol. 417:345.[Medline]
and interleukin-10. J. Exp. Med. 183:2559.. J. Immunol. 157:1127.[Abstract]
). Curr. Opin. Immunol. 9:17.[Medline]
production by suppressing natural killer cell stimulatory factor/interleukin 12 synthesis in accessory cells. J. Exp. Med. 178:1041.This article has been cited by other articles:
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