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Infection and Immunity, October 2000, p. 5628-5634, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Defective Induction of Interleukin-12 in Human
Monocytes by Germ-Tube Forms of Candida albicans
Paola
Chiani,
Carla
Bromuro, and
Antonella
Torosantucci*
Department of Bacteriology and Medical
Mycology, Istituto Superiore di Sanità, Rome, Italy
Received 7 February 2000/Returned for modification 10 March
2000/Accepted 28 June 2000
 |
ABSTRACT |
Yeast (Y) to germ-tube (GT) transition of Candida
albicans is considered a putative virulence trait. On the other
hand, interleukin-12 (IL-12) is a key promoter of T-helper type 1 protective immunity against this human opportunistic pathogen. We
studied IL-12 production by human monocytes cocultured in vitro with Y
or GT forms of C. albicans. Following stimulation by Y
cells, monocytes produced appreciable levels of IL-12, which, upon
addition of gamma interferon (IFN-
), compared to those achievable by
lipopolysaccharide (100 ng/ml) stimulation (140 ± 65 and 185 ± 80 pg/ml, respectively [mean ± standard deviation in four
independent experiments]). In contrast, IL-12 production by GT
cell-stimulated monocytes was much lower or absent (<5 pg/ml) and
could not be brought to the level induced by Y cells by the addition of
IFN- (30 ± 10 pg/ml in the four independent experiments
above). Besides being observed as actual cytokine production, this
lower response was also observed as specific IL-12 p40 mRNA transcript
and was not associated with hyperproduction of the IL-12-competing
cytokine IL-10. Phagocytosis and killing experiments in the presence of cytochalasin D showed that IL-12 production by Y cell-stimulated monocytes was phagocytosis dependent and that GT cells of C. albicans were not phagocytized by the human monocytes.
Importantly, however, Y and GT cells were equally killed by the
monocytes. Thus, the virulence trait attributed to the Y-GT transition
of C. albicans might also be related to the lack of
induction by GT cells of a protective anticandidal immunity through
defective IL-12 production.
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INTRODUCTION |
Several studies have provided
convincing evidence that the induction of a T-helper type 1 (Th1)
response during host-parasite relationship is critically regulated by
interleukin-12 (IL-12), a heterodimeric cytokine produced by a variety
of cells involved in antigen presentation, phagocytosis, and overall
host defense against microbial invaders (2, 27, 29). There
is also a rather wide consensus that Th1 response plays a critical role in the protection against Candida albicans, a major cause
not only of severe opportunistic infections in immunocompromised
patients but also of largely prevalent mucosal infections in otherwise healthy subjects (18). Overall, IL-12 has been considered a pivotal cytokine orchestrating anticandidal defense at both mucosal and
systemic levels (7, 13-15, 22, 25, 26).
On the other hand, infections by C. albicans are facilitated
by the expression of virulence traits enabling this fungus to somewhat
evade or divert immune response (9). One of these factors is
C. albicans's capacity of converting from a unicellular yeast (Y) to a filamentous habit of growth giving rise to true hyphal
cells. Germ-tube (GT) formation is the critical step in this conversion
(18). Importantly, GT formation promptly occurs in
biological fluids so as to be prevalently found in infection, as
opposed to the Y cells which are more commonly found in the saprophytic
or commensal state (18). GT cells are phagocytized much less
efficiently than Y cells by normal phagocytic effectors (31). They also express neoantigens on the cell surface,
with loss of some dominant and protective immunodeterminants, which are
expressed on Y surface (3, 4, 17, 20), a phenomenon which is
readily appreciated in vivo (11).
Because of the critical role of IL-12 in the anticandidal response and
the supposed immunoevasion properties of GT cells of C. albicans, as outlined above, we have investigated the production of this cytokine by human monocytes, a primary source of IL-12, upon
stimulation by Y and GT forms of a virulent C. albicans
strain in comparison with an avirulent, non-GT-forming strain of the fungus. We also examined whether IL-12 production was associated with
phagocytosis and killing of the two forms of growth of the fungus by
the activated human cells.
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MATERIALS AND METHODS |
Monocyte cultures and cytokine assays.
Human monocytes were
obtained from leukocyte buffy coats, diluted in RPMI 1640 (Gibco-BRL,
Grand Island, N.Y.) and separated by density gradient centrifugation on
Lympholyte-H. Monocytes in the mononuclear cell population were let to
adhere to plastic culture plates for 1 h at 37°C, under a 5%
CO2 atmosphere in RPMI 1640 supplemented with 10% fetal
calf serum, 2 mmol of L-glutamine, 100 U of penicillin per
ml, and 100 µg of streptomycin (Gibco-BRL) per ml (hereafter referred
to as complete medium [CM]). After removal of nonadherent cells,
monocytes were recovered by gentle scraping, resuspended at
106/ml in CM, and cultured in multiwell microplates with or
without Candida cells, gamma interferon (IFN-) (R&D
Systems, Minneapolis, Minn.), or cytochalasin D (Sigma Chemical Co.,
St. Louis, Mo.), as specified for each single experiment. Cultures were
incubated 18 h at 37°C under 5% CO2. Cytokine
production was evaluated by assaying culture supernatants by indirect
enzyme-linked immunosorbent assay (ELISA) (Endogen Inc., Woburn,
Mass.). In particular, for IL-12 measurements, two different ELISAs
were employed, one selectively recognizing only the p70 bioactive
heterodimer and one measuring both p70 and the free p40 monomer of the cytokine.
Fungal cells.
Both live and heat-inactivated Y and GT cells
were used throughout this study. Fungal cells were obtained either from
strain BP of C. albicans, which is virulent and competent
for GT formation, or from another strain (CA2), which is a
low-virulence mutant incapable of GT conversion (24, 25).
Live Y cells of both strains of C. albicans were grown for
18 h in Winge broth at 28°C. Cells were harvested by
centrifugation, extensively washed with phosphate-buffered saline
(PBS), resuspended at the desired concentration in RPMI, and
administered to freshly isolated monocyte cultures for IL-12
stimulation. During cocultivation with monocytes in CM at 37°C, more
than 90% of the cells of the germinative strain BP developed, within
60 to 90 min, GT forms, i.e., hyphal filaments emerging from Y cells
and 3 to 10 times as long as the Y cells of origin, growing by apical
elongation. Under the same conditions, the agerminative strain CA2 grew
by budding in a stable Y form.
For a direct comparison of Y and GT forms for IL-12 induction, Y cells
were differentiated in GT-inducing medium as reported elsewhere
(4). Briefly, washed Y cells as described above were resuspended at 2 × 106 cells/ml in Lee's medium and
incubated at 28 or 37°C for 90 min. At 28°C, both
Candida strains maintained the Y form in this medium. At
37°C strain BP, within 90 min of incubation, differentiated >90% GT
cells, as defined above, while strain CA2 maintained its Y form. Y and
GT cells were harvested by centrifugation, washed with H2O,
and resuspended in PBS at the desired concentration. For heat
inactivation, Y or GT cells prepared as above were resuspended in
H2O and treated at 70°C for 40 min. After inactivation
fungal cells were extensively washed and brought to the desired cell density in PBS.
Phagocytosis and killing assays.
To measure phagocytosis, we
used a short-term 3H-glucose uptake inhibition assay based
on the principle that free Candida cells efficiently take up
the radiolabeled sugar while monocyte-ingested Candida cells
do not. In these assays, monocytes (5 × 106/ml) were
cocultured in microplates with live Y or GT cells (from the CA2 or BP
strain, respectively) at various monocyte/fungal cell ratios, in the
presence or in the absence of cytochalasin D (Sigma). Wells containing
Candida Y or GT cells alone were also included in the
experiment as controls. Each condition was assayed in triplicate.
Cocultures were incubated 30 min at 37°C under 5% CO2,
after which 0.5 µCi of 3H-glucose (specific activity, 50 Ci/mmol; Amersham-Pharmacia, Little Chalfont, United Kingdom) was added
to the wells and the plates were incubated for an additional 1.5 h. Monocytes were lysed with Triton X-100 (0.2%; Sigma), and fungal
cells were harvested from the plates and counted in a 
-counter.
Percent phagocytosis was calculated by comparing the radiolabel
incorporation by fungal cells cocultured with monocytes with
incorporation by control fungal cells in the absence of monocytes.
Label incorporation by monocytes alone was always <100 cpm.
Alternatively, monocytes (5 × 10
6/ml) were cultured
for 4 h at 37°C under 5% CO
2 with heat-inactivated
Y or GT cells from
C. albicans strain BP at a
monocyte/
Candida ratio of 1:1. Samples
of the cultures were
smeared onto silanized microscope slides
and fixed 30 min at room
temperature with 4% formaldehyde in PBS.
Slides were washed with
absolute ethanol and stained by the periodic
acid-Schiff technique for
observation of fungal cell phagocytosis.
Monocytes were counterstained
with 1:10 diluted trypan blue. Slides
were examined under a Leitz
Diaplan
microscope.
Killing of Y or GT cells by monocytes was evaluated by cocolturing
monocytes (2 × 10
6/ml) with Y or GT cells at final
monocyte-to-
Candida ratios of
10:1 and 20:1, in the presence
or absence of cytochalasin D (1
µg/ml). Triplicate samples of
cocultures and control
Candida cultures
without monocytes
were incubated 4 h at 37°C under 5% CO
2. Monocytes
were finally lysed by addition of Triton X-100 (0.2%), and residual
live fungal cells were enumerated by CFU counts in duplicate Sabouraud
agar plates. Percent killing was calculated by comparison of CFU
counts
from
Candida-monocyte cocultures and CFU counts from control
cultures of
Candida cells
alone.
RT-PCR analysis of cytokine gene expression.
The reverse
transcriptase PCR (RT-PCR) analysis of cytokine gene expression was
carried out as described elsewhere (30). Aliquots of cDNA
yielding equivalent amounts of -actin amplified band were used for
the semiquantitative evaluation of cytokine mRNAs. PCR was performed in
a 10-µl volume in a Perkin-Elmer 9600 thermal cycler running for 25 (for -actin), 30 (for IL-10 and tumor necrosis factor alpha
[TNF-
]), or 40 (for IL-12 p40) cycles of 1-min denaturation at
94°C; 40-s annealing at 62°C, and 1-min extension at 72°C.
Cytokine-specific primer pairs were synthesized by Gibco-BRL according
to published sequences (1, 12). The PCR products were
visualized by electrophoresis and ethidium bromide staining, identified
by their predicted molecular sizes, and quantitatively evaluated by
densitometric scanning.
Statistical analysis.
Data were evaluated by the
nonparametric Mann-Whitney U test or by the parametric Student test, as indicated.
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RESULTS |
Differential stimulation of IL-12 production by Y and GT forms of
C. albicans.
To examine the production of bioactive IL-12
p70 by human monocytes following stimulation with Y or GT forms of
C. albicans, we performed four independent experiments with
freshly isolated human monocytes, incubated with live or
heat-inactivated Y or GT cells of the fungus, in the presence or
absence of IFN-. Data from preliminary experiments (not shown)
served to establish the optimal effector/target ratio and time of
incubation for optimal cytokine production. Live fungal cells
developing GT forms (>90% within 60 to 90 min) in
monocyte-Candida cocultures were obtained from the virulent,
GT-competent strain BP. Live, Y form-growing cells not developing GT
were obtained from the low-virulence, nongerminative strain CA2.
Since during cocultivation of monocytes with live fungal cells
uncontrolled alterations of monocyte/Candida ratio and
fungal growth-related toxicity could occur, we also used
predifferentiated and heat-inactivated Y cells (from the BP and CA2
strains) and GT cells (from the BP strain) as IL-12 stimulants. This
experimental approach also allowed us to detect any potential
strain-dependent difference in the IL-12 response.
As shown in Table
1, monocytes alone were
substantially unable to produce appreciable quantities of IL-12, even
in the presence
of IFN-
. However, their incubation with
heat-inactivated Y cells
from both strains of
C. albicans,
but not with inactivated GT
cells, greatly promoted heterodimer
production, which, in three
of the four experiments, reached values
above 100 pg/ml in the
presence of IFN-
, which is comparable to the
IL-12 production
seen for some donors by such a potent stimulation as
that achievable
with 100 ng of lipopolysaccharide (LPS) per ml.
However, if Y
cells of the virulent, germinative strain were added in
their
viable state to the phagocyte cultures, in which they promptly
(60 to 90 min) differentiate to GT cells, IL-12 production was
much
lower, even in the presence of IFN-
. This event clearly
paralleled
the lower cytokine production following stimulation
by the inactivated,
predifferentiated GT cells (Table
1).
Conversely, the quantity of IL-12 heterodimer produced upon monocyte
stimulation by Y cells from the avirulent, non-GT-forming
strain of
C. albicans was detectable, even in the viable state,
and
was clearly superior to that produced upon stimulation by
viable cells
of the virulent, GT-forming strain, with or without
IFN-
as a
costimulator (Table
1). Differences between the levels
of IL-12
produced by stimulation with live Y cells of the two
different
Candida strains were highly significant (
P < 0.01, Mann-Whitney
U test), in parallel with the statistically
significant difference
between the levels of IL-12 stimulated by
inactivated Y and GT
cells of the germinative strain BP. On the
contrary, no statistically
significant difference in IL-12 production
was found between inactivated
Y cells of the germinative and the
agerminative strains (Table
1).
IL-12 production was also assessed as cytokine gene expression. As
shown in Fig.
1, the inability of GT
cells to induce IL-12
p70 clearly paralleled a similar inability to
stimulate the expression
of the IL-12 p40 gene transcript (Fig.
1C) and
actual subunit
production (Fig.
1A). As a control, Y and GT cells were
equally
able to trigger gene transcription and actual production of
TNF-
(Fig.
1B and C), indicating that the lower amount of IL-12
produced
under GT cell stimulation was specific and was not due to some
adsorption of the protein to GT cells or its increased degradation.
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FIG. 1.
Gene expression and actual production of IL-12 p40
subunit and TNF- by human monocytes stimulated with Y or GT cells of
C. albicans. (A and B) Production of IL-12 p40 subunit and
TNF- by Y- or GT-stimulated monocytes. Monocytes
(106/ml) from four different donors were cultured in four
different experiments (Exp1 to Exp4) in the presence of
heat-inactivated Y or GT cells of the germinative strain BP of C. albicans, at a monocyte/Candida ratio of 1:2, or in
medium only as a control (Contr). Production of IL-12 p40 (A) and
TNF- (B) was evaluated by ELISA after 18 h of culture. (C)
Analysis of IL-12 p40 and TNF- mRNA expression in
Candida-stimulated monocytes. Monocytes (5 × 106) were either unstimulated (C) or stimulated with Y
cells (Y) or GT cells (GT) of C. albicans, as described
above. After 3 or 18 h, as indicated, IL-12 p40 and TNF- gene
expression was evaluated by semiquantitative RT-PCR analysis, as
described in the text. Lane M, molecular weight markers. A
statistically significant difference (P < 0.01
[Mann-Whitney U test]) was measured between Il-12 p40 production by
monocytes stimulated with Y cells and those stimulated with GT cells.
Differences of TNF- production were not statistically significant.
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Recognizing that IL-12 expression is regulated by other cytokines,
among which IL-10 plays a prominent role (
10), we also
measured gene expression and actual production of IL-10 upon
stimulation
of monocytes with the two different forms of growth of the
fungus.
Both Y and GT cells induced a significant IL-10 gene
transcription
and actual cytokine production, and this was clearly
downregulated
in the presence of IFN-
(Fig.
2). High-level IL-12 production,
mediated
by IFN-
addition, was indeed coupled with significantly
reduced
IL-10 production, suggesting a complex interplay of these
three
cytokines in the monocyte response to
Candida.
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FIG. 2.
Comparison of IL-10 and IL-12 p70 production and IL-10
gene expression by human monocytes cocultured with Y or GT cells of
C. albicans. (A) IL-10 and IL-12 p70 production upon
stimulation with Y or GT cells of C. albicans. Monocytes
were cultured for 18 h in medium only (Control) or in the presence
of heat-inactivated Y or GT cells of C. albicans strain BP
(monocyte/Candida ratio = 1:2). Where indicated,
IFN- (100 U/ml) was added to the cultures as an IL-12 costimulant.
IL-10 and IL-12 p70 production was evaluated by ELISA. (B) IL-10 gene
expression in Candida-stimulated monocytes. Unstimulated
control (C), Y cell-stimulated (Y), or GT cell-stimulated (GT)
monocytes were analyzed, at the indicated times, for IL-10 mRNA
expression by semiquantitative RT-PCR, as described in the text. Lane
M, molecular weight markers. Data are from one representative
experiment out of two performed with similar results.
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Phagocytosis and killing of C. albicans by monocytes
and relationship with IL-12 induction.
Since Y cells of C. albicans are easily phagocytized by human monocytes while GT cells
are not (31), and knowing the importance of phagocytosis for
non-T-cell-dependent, early IL-12 production in other host-parasite
interactions (12), we also asked whether differences in
phagocytosis might indeed account for differential cytokine production.
In addition, we investigated whether there was any relationship between
IL-12 production and the phagocytes' ability to kill the two forms of
growth of Candida.
As shown in Fig.
3A and
4, the
human monocytes were much less able to phagocytize the GT cells than
the Y cells of
C. albicans.
CFU enumeration showed, however,
that the monocytes were equally
capable of killing the two forms of
growth (Fig.
3C). The addition
of cytochalasin D to the monocyte
cultures dose dependently inhibited
phagocytosis of Y cells (from 70 to
0% in the representative experiment
shown in Fig.
3B [also Fig.
4B]). This drug also inhibited the
killing of Y cells but not that of
the GT cells (Fig.
3C), which
thus predominantly occurred by
phagocytosis-independent, extracellular
mechanisms (Fig.
3C). No
difference was found in the extent of
phagocytosis of heat-inactivated
Y cells of the germinative and
the agerminative strains (data not
shown).
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FIG. 3.
Phagocytosis and killing of Y  and GT  cells of
C. albicans by human monocytes: effect of cytochalasin D. (A) Monocytes (5 × 106/ml) were cocultured with live
Y or GT cells of C. albicans at the indicated
monocyte/Candida (E:T) ratios. Phagocytosis was evaluated
after 2 h by a 3H-glucose uptake inhibition assay, as
described in Materials and Methods. *, P < 0.01; **,
P < 0.05 (both determined by Student's t test,
two tailed [comparing percent phagocytosis of Y and GT cells]). (B)
Effect of cytochalasin D on the phagocytosis of Y cells by monocytes.
Cytochalasin D (ctcl D), at the indicated doses, was added to monocyte
cultures 30 min before Candida cells (E:T ratio = 1:1),
and phagocytosis was measured as described in the legend to Fig. 3A. No
ctclD, control culture (not treated with the drug). *, P < 0.01; **, P < 0.05 (both determined by Student's
t test [comparing values measured in the absence of
cytochalasin D]). (C) Killing of Y cells and GT cells by monocytes and
effect of phagocytosis blockade with cytochalasin D. Monocytes (2 × 106/ml) were cultured for 4 h with Y cells or GT of
C. albicans, at the indicated monocyte/Candida
(E:T) ratios, in the presence or absence of cytochalasin D (1 µg/ml).
Killing activity was evaluated by Candida CFU counts, as
reported in Materials and Methods. *, P < 0.01
(Student's t test) as compared to killing values measured
in the absence of cytochalasin D. Differences of killing activity
against Y and GT cells were not significant. Values are means ± standard deviations (error bars) of three independent determinations.
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FIG. 4.
Microscopic evaluation of phagocytosis of Y and GT
cells of C. albicans by human monocytes. Monocytes (5 × 106/ml) were cultured for 4 h at 37°C under 5%
CO2, with heat-inactivated Y or GT cells of C. albicans strain BP (5 × 106/ml). Samples of the
cocultures were smeared onto silanized microscope slides, fixed, and
periodic acid-Schiff stained. (A) Y cells ingested by monocytes; (B) Y
cells uningested by monocytes in the presence of cytochalasin D (1 mg/ml); (C) GT cells uningested by monocytes. Magnification for all
panels, ×400.
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When cytochalasin D was used in experiments of IL-12 production upon
stimulation by Y cells, at the concentration totally
inhibiting Y cell
phagocytosis, IL-12 heterodimer production was
also practically
abolished. This was seen to occur through inhibition
of p40 monomer
production (Table
2). Cytochalasin D did
not cause
any inhibition of IL-12 p40 or p70 production by
LPS-stimulated
monocytes, which is known to be independent of
phagocytosis, attesting
to the specificity of the action exerted by the
phagocytosis inhibitor
for the Y
Candida cell-induced IL-12
production (Table
2).
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TABLE 2.
Effect of cytochalasin D on IL-12 p70 and IL-12 p40
production by monocyte cultures stimulated with Candida
cells or LPSa
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DISCUSSION |
In this study we have shown that the GT forms of C. albicans are significantly less efficient than the Y forms in
stimulating human monocytes to release IL-12, a key cytokine in the
development of protective anti-Candida immunity. We measured
the in vitro IL-12 production by human monocytes following stimulation
with predifferentiated and heat-inactivated Y or GT cells of the fungus or with live cells from a virulent, GT-forming strain and compared the
results to those for live cells of an avirulent, nongerminative strain.
The results of these experiments demonstrated that the virulent
germinative strain, in its viable state, was substantially unable to
induce IL-12 production by the monocytes and that this inability could
be only partially reversed by overstimulation with IFN-. The
experiments also indicated that transition to the GT form was the cause
of the lower efficiency of the virulent strain in stimulating IL-12
production, as heat-inactivated Y cells, but not the inactivated GT
cells, of this virulent strain stimulated the monocytes to produce
quantities of IL-12 comparable to those detected upon stimulation with
the corresponding Y form of the avirulent, non-GT-forming strain.
Our observations appear to be of some importance for the supposed role
of GT cells in C. albicans pathogenicity and immune response. IL-12 is a key promoter of protective immunity against this
pathogen. Endogenous IL-12 production is a critical requirement for the
development of a Th1, protective response in mice with candidiasis, and
even its suboptimal production has been clearly linked to a nonhealing
disease (22, 24, 25). On the other hand, GT formation is a
key feature of the interaction of this fungus with its host. During
this process, most of the host-impacting cell surface molecules of
C. albicans are modified and consistent rearrangements of
the fungal antigenic makeup, inclusive of antigens with protective
value, are observed (3, 4, 16, 17). Mutation of genes
involved in or regulating GT transition have been shown to obviously
affect fungal virulence (8). Data presented in this paper
suggest that the greater virulence attributed to the Y-GT transition of
C. albicans might also be due, at least in part, to
deficient induction, by GT cells, of protection-associated cytokine
responses by the host. Importantly, GT cells proved to be suboptimal
stimulators of IL-12 production even in the presence of IFN-, a Th1
cytokine that appears to be important not only in stimulating IL-12
production but also in maintaining responsiveness to this cytokine
(6). We have recently observed that GT cells were also much
less efficient than Y cells in stimulating chemokine (in particular
MCP-1) production by human monocytes and neutrophils (27a).
The two processes could evidently synergize in vivo, as the low IL-12
production by the monocytes encountering the GT cells could match the
suboptimal influx of those natural immunoeffectors which are devoted,
among other things, to an early production of IL-12 in the absence of
activated T cells (23, 30).
The lower IL-12 response generated by GT cells might be due, in
principle, to selective stimulation of IL-10, a major IL-12-regulating cytokine with anti-inflammatory and Th2 differentiation-promoting activity. This was not the case, since GT cells did not significantly differ from Y cells in their IL-10 stimulation ability. Since IL-10 has
been recognized as a potent downregulator of the
anti-Candida functions of human phagocytes (21),
this observation is also in line with the preserved killing activity
exerted by monocytes against the GT forms of C. albicans.
Nevertheless, if the balance of IL-10 and IL-12 production is
considered, it follows that while Y cell forms induce both cytokines,
GT cells predominantly induce IL-10, a cytokine which has been
associated with nonhealing disease in mice with candidosis
(23). Although preliminary, these observations suggest that
unbalanced IL-12 versus IL-10 induction might be an additional feature
of GT forms of C. albicans, leading to increased pathogenicity and evasion from host protective immunity. They are also
in keeping with the different IL-12 and IL-10 responses associated with
spontaneously healing or nonhealing disseminated murine infections by
avirulent, agerminative or virulent, germinative C. albicans
strains, respectively (23). Additional in vivo studies are,
however, necessary to assess the precise role of the different cytokine
response induced by the two forms of growth of C. albicans in modifying fungal pathogenicity and the final outcome of the infection.
A clear relationship has emerged from our study between the lower
efficiency of GT cells in IL-12 induction and the monocyte inability to
ingest this form of growth of the fungus. This apparent association has
been reinforced by the observation that Y cell-stimulated IL-12
production is totally suppressed by the phagocytosis inhibitor cytochalasin D. In this context, the process of phagocytosis seems to
be a major signal for Candida-mediated IL-12 induction, as also suggested for other microorganisms by some authors
(12).
Recently, Pitzurra et al. (19) have shown that an early,
non-T-cell-dependent IL-12 production by human monocytes is stimulated by either secreted or cell wall-associated mannoproteins of C. neoformans, acting, at least in part, through an internalization process in the endocytic pathway. We have previously shown intense IL-12 production by human phagocytes from both human immunodeficiency virus-positive and -negative subjects stimulated with a mannoprotein fraction of C. albicans (5). Since the other main
cell wall constituent of this fungus, i.e., glucan, does not stimulate
IL-12 production (A. Torosantucci, unpublished observation), cell
wall-expressed mannoproteins of the Y form of C. albicans
are probably involved in the interaction between the fungal cell and
the phagocyte, leading to the subsequent activation of endocytosis.
Mannoproteins are cell wall components of C. albicans which
are mostly secreted and rearranged during GT formation, with remarkable
losses of Y cell-expressed molecules (3, 17). Further
studies of specific fungal molecules mediating adhesion to the
phagocyte, thus fostering fungal internalization, and how these
molecules are differentially expressed on Y and GT forms of C. albicans, could greatly contribute to a further understanding of
the mechanisms whereby GT and Y cells differ in IL-12 induction by monocytes.
Finally, of interest is the observation that the lower production of
IL-12 by the GT-stimulated monocytes did not affect their candidacidal
capacity. Extracellular killing mechanisms have been shown to be
possessed by most professional phagocytes, and GT cells were
demonstrated to be highly sensitive to these nonphagocytic fungicidal
mechanisms, particularly in the presence of opsonins and cytokine
activators (31). It is also known that IL-12 does not, per
se, enhance the candidacidal activity of both mononuclear and
polymorphonucleate phagocytes (6). In the present
investigation, low-level IL-12 stimulator GT cells appeared to be
killed by human monocytes by nonphagocytic, extracellular mechanisms to
exactly the same extent, in vitro, as the high-level IL-12 stimulator Y
cells. Thus, the effect of GT cells on IL-12 production is uncoupled to
monocyte candidacidal activity, suggesting that the process of GT
formation and filamentation in vivo may critically impair the
development of a protective response against the fungus rather than the
immediate outcome of the local Candida-host encounter.
| |
ACKNOWLEDGMENTS |
Thanks are due to Antonio Cassone for helpful discussion during
the course of this work and for critical reading of the manuscript. We
also express our gratitude to A. Botzios for help in the preparation of
the manuscript.
This work was partly supported by the National AIDS Program, contract
50C/B.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Bacteriology and Medical Mycology, Istituto Superiore di Sanità,
Viale Regina Elena, 299 00161 Rome, Italy. Phone: 39-0649902824. Fax: 39-06-49387112. E-mail: torosan{at}iss.it.
Editor:
R. N. Moore
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Infection and Immunity, October 2000, p. 5628-5634, Vol. 68, No. 10
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
