| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy;
The Wistar Institute, Philadelphia, PA 19104; and
Roche Milano Ricerche, Milan, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. The failure to mount protective Th1
responses occurred despite the presence of an unimpaired innate
antifungal immune response, which correlated with unaltered IFN-
production, but defective production of, and responsiveness to,
inhibitory IL-10. IL-10 or IL-12 neutralization increased the innate
antifungal resistance in wild-type mice. However, in IL-12-deficient
mice, treatment with exogenous IL-12 or IL-10 impaired IL-4 production
and increased resistance to infection, through a negative effect on the
CTLA-4/B7-2 costimulatory pathway. These results confirm the obligatory
role of IL-12 in the induction of anticandidal Th1 responses, and
indicate the existence of a positive regulatory loop between IL-12 and
IL-10 that may adversely affect the innate antifungal response, but is
required for optimal costimulation of IL-12-dependent
CD4+Th1 cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(4) and requires the concerted action of
several cytokines (5, 6, 7, 8), including IL-12 (9, 10, 11). Susceptibility to
infection correlates with the presence of CD4+Th2 cells
producing IL-4 and IL-10 (4, 12, 13, 14) that have been implicated in
inhibiting the development of protective Th1 cells (15) and in
opposing the IFN--mediated activation of fungicidal macrophages
(16). Among factors contributing to CD4+Th development in mice with C. albicans infection, the initial handling of fungal pathogen by cells of the innate immune system appears to play a major role (17, 18, 19). The instructive role of the innate immune system in the adaptive immune response may operate at different levels (20, 21). In candidiasis, regulation of the early fungal burden (22), cytokine production including IL-10 and IL-12 (17, 18, 19), and expression of costimulatory molecules (8) are possible pathways through which the innate immune system may control CD4+Th immunity. Thus, qualitative or quantitative defects of antifungal effector and immunomodulatory functions of phagocytic cells result in the development of anticandidal Th2, rather than Th1, responses (17, 18, 19). Conversely, potentiation of innate antifungal immunity by neutralization of inhibitory cytokines, such as IL-10, leads to the development of protective Th1 responses in otherwise susceptible mice (13).
IL-12 is both required and prognostic for CD4+Th1
development in mice with candidiasis, being rapidly produced after
infection in both resistant and susceptible mice (9, 10, 11). In
genetically resistant mice, neutralization of IL-12 (9) or IFN- (5)
prevents development of protective Th1 responses, consistent with the
important role of IL-12-induced IFN- in activating host antifungal
effector cells (23) and CD4+Th1 lymphocytes (5). In
susceptible mice, IL-12 is negatively regulated by IL-4 and IL-10, and
IL-12-dependent activation of CD4+Th1 lymphocytes occurs
upon neutralization of these cytokines (9). However, exogenous IL-12
fails to increase resistance of susceptible mice to systemic or
gastrointestinal C. albicans infection and to effectively
oppose Th2 cell differentiation driven by IL-4/IL-10 (9, 11). These
data indicate the existence of complex immunoregulatory circuits
underlying the activity of IL-12 in candidiasis (11).
Production of IL-10 by T (24) and non-T (17, 18, 19) cells is induced by IL-12, IL-10 being a potent inhibitor of IL-12 (25). The importance of this negative regulatory loop between IL-10 and IL-12 is underlined by observations in IL-10-deficient mice infected with Toxoplasma gondii in which overproduction of IL-12 results in a toxic lethal syndrome (26). Thus, IL-10 appears to be a critical component of the host’s immune response to infectious agents (27). The finding that IL-12 induces production of IL-10 by T cells (9) as well as by neutrophils (18) in mice with candidiasis led us to investigate whether early induction of IL-10 by IL-12 could represent a major mechanism underlying the capacity of IL-12 to affect innate and adaptive antifungal immunity.
To test this hypothesis, IL-12 p40-deficient mice were assessed for pattern of cytokine production and for parameters of innate and acquired antifungal immunity during the course of systemic or gastrointestinal C. albicans infection. The results revealed a defective production of and responsiveness to IL-10, which correlated with the inability of IL-12 p40-deficient mice to mount protective anticandidal Th1 responses. This suggests the existence of an important, previously undefined, positive regulatory loop between IL-12 and IL-10, the latter being required for optimal costimulation of IL-12-induced CD4+Th1 cells in mice with C. albicans infection.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Breeding pairs of homozygous IL-12 p40-deficient (IL-12 p40-/-) and control IL-12 p40+/+ BALB/c mice (28) kindly provided by Dr. J. Magram (Hoffmann-La Roche, Nutley, NJ) were bred under specific pathogen-free conditions. Mice of both sexes, 8–10 wk old, were used. IL-12 p40-/- mice were devoided of IL-12 bioactivity, as assessed by the Ab capture assay for functional IL-12 (7). Procedures involving animals and their care were conducted in conformity with national and international laws and policies.
Yeasts, infections, in vivo analysis, and treatments
The origin and characteristics of the highly virulent C. albicans (CA-6) strain and the low virulence live vaccine strain, PCA-2, used in this study have been described previously (4, 10). For infection, cells were washed twice in saline, and diluted to the desired density to be injected i.v. via the lateral tail vein in a volume of 0.5 ml per mouse or intragastrically (i.g.;3 108 CA-6 cells), via an 18-gauge 4-cm-long plastic catheter, as described (29). Cell viability was >95% by trypan blue exclusion and quantitative cultures. Resistance to reinfection was assessed by i.v. injection of 106 CA-6 cells, 14 days after primary challenge, in mice surviving the primary infection. Quantification of yeast cells in organs of infected mice (6–8 mice per group) was performed by plating serial dilutions of homogenized organs in Sabouraud dextrose agar, and results (means ± SEM) were expressed as log10 CFU per organ. For histology, tissues were excised and immediately fixed in formalin. Sections (3–4 µm) of paraffin-embedded tissues were stained with periodic acid-Schiff reagent and examined for histology, as described (29). Mice succumbing to yeast challenge were routinely necropsied for histopathologic confirmation of disseminated candidiasis. Recombinant mouse IL-12 and polyclonal sheep anti-mouse IL-12 IgG Ab (Genetics Institute, Cambridge, MA) were given i.p. the day of and 1 and 2 days after challenge (100 ng/injection for rIL-12, and 500 µg/injection for anti-IL-12 Ab). rIL-10 (kind gift of Robert Coffman, DNAX, Palo Alto, CA) was given i.p. twice the day of and 1 and 2 days after challenge (1 µg/injection). Affinity-purified mAb anti-murine IL-10 (from the SXC-1 hybridoma; American Type Culture Collection, Manassas, VA) was given i.p., once the day before and the day of challenge (100 µg/injection). The murine CTLA4-Ig fusion protein (from the CTLA4-Ig-producing hybridoma, kind gift of Alan Sher, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD) or the control fusion protein was given i.v. via the lateral tail vein the day of and 1 day after challenge (500 µg/injection). Control groups were injected with saline or isotype-matched Ab (Zymed Laboratories, South San Francisco, CA). Endotoxin was removed from all solutions with Detoxi-gel (Pierce, Rockford, IL).
Purification and culture of cells
CD4+ T splenocytes (>90% pure on FACS analysis)
were positively selected by sequential adherence on anti-Ig-coated
plates and on anti-mouse CD4 (mAb GK1.5), and splenic macrophages
(>95% pure on esterase staining) were obtained by 2-h plastic
adherence, as described (6). CD4+ T cells were cultured
(5 x 106/ml) in the presence of accessory macrophages
(5 x 105/ml) and heat-inactivated yeast cells (5
x 105/ml). Total spleen cells (5 x
106/ml) were cultured in the presence of 10 µg/ml Con A
(Sigma, St. Louis, MO). Intragastric lymphocytes were isolated from the
whole stomach, after incubation in calcium and magnesium ion-free HBSS
containing DTT (0.145 µg/ml; Sigma) and EDTA (0.37 µg/ml), and
further digestion in RPMI 1640 containing collagenase VIII (100 U/ml;
Sigma) and DNase I (0.1 µg/ml; Sigma), as described (8).
Lymphocyte-enriched populations, containing cells of both CD8 (
75%)
and CD4 (7–10%) phenotypes, with CD4+ T cells expressing
the 
ß or 
TCR at approximately equal numbers on FACS
analysis, isolated at the 40–100% interface of a discontinuous
Percoll gradient (Pharmacia, Uppsala, Sweden), were cultured (5 x
105 cells/ml) in the presence of irradiated (2000 rad)
splenocytes and Con A (10 µg/ml). Cultures of splenic adherent
macrophages and purified peritoneal neutrophils, collected 18 h
after i.p. inoculation of aged, endotoxin-free 10% thioglycolate
solution (Difco, Detroit, MI), were conducted as described (17, 19), by
incubating 5 x 106 cells/ml in the presence of 400
U/ml IFN- and 40 ng/ml LPS (Sigma) or 5 x 105C. albicans cells/ml. Cytokines were quantitated in
supernatants collected after 48 h (for lymphocytes) or
24 h (for neutrophils), as described (17, 19).
Cytokine assays
The levels of IFN-, IL-4, and IL-10 in culture supernatants
were determined by cytokine-specific ELISAs, using pairs of
anti-cytokine mAbs, as described (6, 9). The Ab pairs used were as
follows, listed by capture/biotinylated detection: IFN-,
R4-6A2/XMG1.2; IL-4, BVD4-1D11/BVD6-24G2; IL-10, JES5-2A5/SXC-1
(PharMingen, San Diego, CA). Cytokine titers were calculated by
reference to standard curves constructed with known amounts of
recombinant cytokines (PharMingen).
Candidacidal assay and nitrite determination
For the candidacidal assay, splenic-adherent macrophages or elicited peritoneal neutrophils (5 x 105 cells/ml) were incubated with 105 C. albicans cells/ml for 4 or 1 h, respectively, and the number of CFU was determined as described (16). The percentage of CFU inhibition (means ± SE) was determined as percentage of colony formation inhibition = 100 - (CFU experimental group/CFU control cultures) x 100. Nitrite concentration, a measure of nitric oxide (NO) synthesis, was assayed in culture supernatants by a standard Griess reaction adapted to microplates, as described (16). The data represent the means ± SE of quadruplicate determinations and are expressed as µM NO2-/107 cells.
Flow cytometry
Spleen cells were harvested, and 1 x 106 cells were sequentially reacted with saturating amounts of FITC-conjugated anti-CD80 (B7-1, hamster IgGmAb, IG-10A1) and anti-CD86 (B7-2, rat IgG2a, mAb GL1) mAbs and phycoerythrin-conjugated anti-CD11b (rat IgG2b, mAb M1/70) (PharMingen). Cells were analyzed with a FACSort flow cytometer (Becton Dickinson, Mountain View, CA) using Cell Quest software for Macintosh, as described (7, 30). Nonviable cells were excluded from analysis by accepted procedures involving propidium iodide staining and narrow forward-angle light scatter gating.
Reverse-transcriptase PCR (RT-PCR)
RNA extraction and amplification of synthesized cDNA from macrophages, purified CD4+ splenocytes, and elicited peritoneal neutrophils were performed as described (6, 19). For hypoxanthine-guanine phosphoribosyltransferase (HPRT) and IL-10, the cycles and temperatures were as described (8). For IL-10R, the sequences of 5'-sense primers and 3'-antisense primers were as follows: 5'-AGGCAGAGGCAGCAGGCCCAGCAGAATGCT; 3'-TGGAGCCTGGCTAGCTGGTCACAGTAGGTCT.
For IL-10R, each cycle consisted of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. The HPRT primers were used as a control for both reverse transcription and the PCR reaction itself, and also for comparing the amount of products from samples obtained with the same primer. The PCR fragments were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. PCR-assisted mRNA amplification was repeated at least twice for at least two separately prepared cDNA samples for each experiment. Data are representative of at least three different experiments.
Competitive RT-PCR
The semiquantitative competitive PCR developed by Reiner et al.
(31) was performed using the competitor construct containing sequences
for multiple cytokines, the primers for HPRT, IFN-, and IL-4 and the
PCR conditions described by the authors. Briefly, aliquots of cDNA were
assayed for levels of HPRT by placing serial dilutions from 1/1 to 1/40
of the experimental cDNA against a fixed concentration of the
competitor construct and examining the ratio of competitor-to-wild-type
band intensity after amplification with HPRT-specific primers.
Adjustments were made in the amount of cDNAs needed to standardize the
HPRT levels to comparable levels among all groups. Serial dilutions of
these adjusted volumes of cDNA were then used to quantitate cytokine
levels using a fixed concentration of competitor (3 pg/ml for HPRT and
1.5 pg/ml for IFN- and IL-4, respectively) in the presence of
cytokine-specific primers. The PCR products were separated by
electrophoresis in 2% agarose gels containing ethidium bromide. The
point of equivalence in intensity between the competitor (upper band)
and the cDNA (lower band) indicates the relative concentration of mRNA.
Statistical analysis
Survival data from each group of wild-type mice were compared with those from IL-12-deficient mice using the Mann-Whitney U test; significance was defined as p < 0.05. Student’s t test was used to determine statistical significance between cytokine production and organ clearance by the two groups. In vivo groups consisted of four to six mice. The data reported were pooled from three experiments.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-12 p40-/- and IL-12 p40+/+ mice
were injected i.v. with 106 highly virulent C.
albicans (CA-6) or low virulence PCA-2, or i.g. with
108 CA-6. Mice were monitored for resistance to primary and
secondary infections in terms of survival (Table I
) and fungal growth in the kidneys and
stomach (Fig. 1). Survival to acute
systemic infection with CA-6 was increased in IL-12
p40-/- mice as compared with wild-type mice, whereas
survival to primary systemic infection with PCA-2 or to i.g. infection
with CA-6 did not differ between IL-12 p40-/- and IL-12
p40+/+ mice. However, upon a lethal CA-6 challenge of
survivors, IL-12 p40+/+ mice either survived or showed a
remarkable resistance to reinfection, while IL-12-deficient mice did
not. Resistance to acute systemic infection with CA-6 correlated with a
significant decrease of Candida growth in the target organ,
the kidney (Fig. 1A). In the gastrointestinal infection,
local fungal growth in the stomach did not significantly differ between
IL-12 p40-/- and IL-12 p40+/+ mice in the
early stage of infection. However, 4 wk after infection, when this was
almost cleared in wild-type mice, a remarkable fungal growth was still
present in the stomachs of IL-12-deficient mice (Fig. 1B).
Susceptibility to reinfection of IL-12-deficient mice surviving primary
systemic or i.g. infection was associated with an increased fungal
growth in the kidneys, as compared with resistant wild-type mice (Fig. 1C). Histopathologic examination of the kidneys of
IL-12-deficient mice with primary or secondary systemic infection
revealed lesions similar to those observed in resistant or susceptible
strains of mice upon i.v. Candida infection (4, 14).
Similarly to what was observed in resistant wild-type mice (Fig. 2, B and D),
reduced fungal growth and absence of pathologic lesions characterized
the kidneys of IL-12-deficient mice that were resistant to acute
systemic infection with PCA-2 (Fig. 2A). In contrast, an
extensive fungal growth, associated with the presence of numerous foci
of inflammatory reaction (consisting mainly of polymorphonuclear cells)
throughout the kidney parenchyma, was observed in IL-12
p40-/- (Fig. 2C), but not IL-12
p40+/+ (Fig. 2D) mice upon reinfection. Four
weeks after gastrointestinal infection, stomach sections from IL-12
p40-/- mice (Fig. 2E) revealed numerous
intraepithelial abscesses consisting of a thickened keratinized outer
layer that enveloped a dense aggregate of hyphae and a few infiltrating
cells. Unexpectedly, erosions were present through the thickness of the
gastric mucosa, characterized by epithelial necrosis, presence of
numerous fungal cells, and a prominent inflammatory cell infiltrate;
conspicuous signs of acanthosis and hyperkeratosis were also
visible. Importantly, abscesses and mucosal infiltration were observed
in the glandular region of the gastric mucosa rather than in the
cardial-atrium fold, which is a major site of colonization by C.
albicans in the murine gastrointestinal tract (29). In wild-type
mice (Fig. 2F), fewer yeast cells were present in the
keratinized layer, with a limited inflammatory reaction observed only
at the level of the cardial-atrium fold. Altogether, these data clearly
indicate a two-stage control of C. albicans infection in
IL-12-deficient mice, which efficiently oppose infectivity in the
initial stage of infection, but fail to acquire resistance to it.
|
|
|
Protective acquired resistance to C. albicans
correlates with the induction of CD4+Th1 responses (1, 2, 3).
To assess the pattern of Th1 (IFN-) and Th2 (IL-4) cytokine
production, IL-12 p40-/- and IL-12 p40+/+
mice were infected under conditions that induce protective
CD4+Th1 responses, such as i.v. infection with PCA-2 or
i.g. infection with CA-6, followed by reinfection with CA-6 14 days
later. We found that production of IFN- was not significantly
decreased in IL-12-deficient as compared with wild-type mice, early in
infection. Comparable levels of IFN- were detected in culture
supernatants of mitogen-stimulated splenocytes or i.g. lymphocytes from
both wild-type and IL-12-deficient mice upon primary i.v. (Fig. 3A) or i.g. (Fig. 3B) infection, respectively. In contrast, IFN- production
was severely impaired in CD4+ T splenocytes from
IL-12-deficient mice upon reinfection (Fig. 3C). In these
mice, defective production of IL-2 was also observed (data not shown).
IL-4 production was always present in IL-12 p40-/- mice
(Fig. 3, A, B, and C), whereas it was
considerably reduced in IL-12 p40+/+ mice upon primary i.v.
(Fig. 3A) or secondary infection (Fig. 3C). Local
production of IL-4 was comparable in IL-12 p40-/- and
IL-12 p40+/+ mice with gastrointestinal infection (Fig. 3B). Analysis of IFN- and IL-4 gene expression in
CD4+ T cells by quantitative RT-PCR confirmed the results
of cytokine secretion. IFN- gene expression was not different in
wild-type and IL-12-deficient mice early in the course of primary i.v.
infection, as opposed to the twofold decrease observed in
CD4+ T splenocytes from IL-12-deficient mice upon
reinfection (Fig. 3D). In contrast, a twofold increase of
IL-4 gene expression was observed in IL-12 p40-/-, but
not IL-12 p40+/+ mice upon both primary and secondary i.v.
infection. Therefore, IL-12 deficiency does not impair the early
production of IFN- in mice with C. albicans infection,
but rather prevents the development of CD4+ cells producing
IFN- and IL-2. In contrast, an elevated production of IL-4 was
observed early in infection as well as in reinfection. Production of
IL-4 paralleled that of circulating Candida-specific IgE,
which were detected at high levels during the course of primary or
secondary infection (data not shown). These data indicate that impaired
development of protective CD4+Th1 responses in
IL-12-deficient mice is associated with differentiation of
nonprotective IL-4-producing CD4+Th2 cells, which occurs in
spite of IFN- production.
|
To evaluate the contribution of innate immunity in the ability of
IL-12-deficient mice to efficiently oppose infectivity in the early
stage of infection, we assessed the antifungal activities of
macrophages and neutrophils under conditions of vigorous innate system
activation (17), that is mice infected i.v. with virulent C.
albicans 3 days earlier. Splenic macrophages and peritoneal
neutrophils were assessed for candidacidal activity and production of
NO. Candidacidal activity and NO production from both cell types were
higher in uninfected IL-12 p40-/- than IL-12
p40+/+ mice (Fig. 4). Upon
infection, the candidacidal activity continued to be elevated in
IL-12-deficient mice, as opposed to the inhibition observed in
wild-type mice, whereas NO production decreased in IL-12
p40-/- mice, although to a lesser extent than in IL-12
p40+/+ mice. Because the number of peripheral neutrophils
increased similarly in IL-12 p40-/- and IL-12
p40+/+ mice upon infection (from 1168–8520
cells/mm3 in IL-12 p40-/- mice, and from
1116–9680 cells/mm3 in IL-12 p40+/+ mice),
these data indicate that an activated innate antifungal immune response
can successfully control early infectivity in IL-12-deficient mice.
|
The finding that IL-10 is a potent deactivating signal for
fungicidal effector phagocytes (16) and is induced by IL-12 (27)
prompted us to investigate whether defective IL-10 production occurred
in IL-12-deficient mice. Macrophages and neutrophils, obtained as
above, were assessed for IL-10 gene expression and ability to secrete
IL-10 upon exposure to IFN- plus LPS in vitro. In addition, the
capacity of these cells to respond to IL-10 was evaluated by assessing
their levels of IL-10R gene expression. We found that both IL-10
production (Fig. 5A) and IL-10
gene expression (Fig. 5B) were decreased significantly in
macrophages and neutrophils from infected IL-12 p40-/-
compared with wild-type mice. The expression of the IL-10R was also
down-regulated upon infection in macrophages and neutrophils from IL-12
p40-/-, but not IL-12 p40+/+ mice (Fig. 5B). In vitro, exposure of macrophages and neutrophils to
IL-12 greatly increased their ability to release IL-10 upon stimulation
with Candida cells (data not shown). These results would
predict that an exaggerated production of IL-12-induced IL-10 may be
involved in the higher susceptibility of IL-12 p40+/+ mice
to C. albicans infection. This was indeed the case, as
either IL-10 or IL-12 neutralization decreased fungal burden and
increased survival in mice infected with virulent C.
albicans (Fig. 6). These results
indicate that a defective production of and responsiveness to IL-10 by
phagocytic cells may represent a mechanism underlying the unimpaired
innate antifungal response occurring in IL-12-deficient mice.
|
|
In mice with C. albicans infection, early production of
IL-4 is associated with the expansion of a CD4+ population
expressing the activated phenotype, i.e., low levels of L-selectin
(Mel-14) and high levels of CD44, through the CTLA-4/B7-2 costimulatory
pathway (30). To define whether blockade of this costimulatory pathway
also affects IL-4 production in IL-12-deficient mice, mice were
systemically infected with PCA-2, treated with CTLA4-Ig, a soluble
ligand for B7 (32), and assessed 3 days later for IL-4 and IFN-
production by CD4+ T cells. We also measured cytokine
production upon short low dose treatment with either recombinant IL-10
or IL-12. Each treatment significantly reduced IL-4 and increased
IFN- production (Table II), and this
correlated with a reduced expansion of activated CD4+ T
cells (data not shown). Each treatment also increased resistance of
IL-12-deficient mice to infection, as assessed by the decreased fungal
growth in their kidneys. Therefore, these results indicate that early
IL-4 production in IL-12-deficient mice relies on the expansion of a
CD4+ population through a CTLA-4/B7-dependent pathway that
is sensitive to IL-10 and IL-12.
|
The dependency of IL-4 production on the CTLA-4/B7 costimulatory
pathway, together with the finding that IL-10 production, which is
known to affect expression of B7 molecules on accessory cells (33), was
reduced in IL-12-deficient mice, led us to investigate whether B7-1 and
B7-2 costimulatory molecules were differentially expressed in vivo in
C. albicans-infected IL-12 p40-/- and IL-12
p40+/+ mice, and were modified upon IL-10 or IL-12
administration or neutralization, respectively. Three days after
infection, the expression of costimulatory molecules on splenic
macrophages was assessed by flow cytometry (Fig. 7). The expression of B7-2, and to a
lesser extent of B7-1, significantly increased upon infection in IL-12
p40-/- as compared with wild-type mice. However,
administration of IL-10 or IL-12 inhibited the up-regulated expression
of B7-2 and, interestingly, increased that of B7-1. Likewise, IL-10,
and in part IL-12, neutralization resulted in an increased expression
of B7-2 in IL-12 p40+/+ mice. These results and those of
Table II
indicate that the expansion of IL-4-producing CD4+
T cells in mice with C. albicans infection is associated
with up-regulated expression of B7-2 on macrophages. IL-10, and partly
IL-12, inhibit the expression of B7-2, while slightly increasing that
of B7-1. Interestingly, the expression of the CD40 molecule is
increased upon IL-10 administration, while that of MHC class II Ag is
decreased (unpublished data). These results thus suggest that IL-10,
through its ability to modulate costimulatory molecules on accessory
cells, may play a key role in IL-12-dependent Th1 cell development in
mice with candidiasis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and type 1 cytokine responses (28, 36) and to mount a
polarized Th2 cell response upon infection with Leishmania
major (37).
However, we found that production of IFN- was not decreased early in
the course of systemic or gastrointestinal infection. This indicates
that IFN- production in the early phase of C. albicans
infection is relatively IL-12 independent, and that cytokines other
than IL-12 induce the release of IFN- (38). Conversely, production
of IFN- was greatly impaired in IL-12-deficient mice upon
reinfection, and correlated with the failure to activate
CD4+Th1 cells producing IFN- and IL-2 and with their
inability to resist reinfection upon systemic or mucosal immunization.
Both resistance to reinfection and resolution of the mucosal infection
in murine candidiasis depend on the activation of Th1 responses (4, 29). Thus, regulation of IFN- production early in the course of
infection may not represent a primary mechanism of IL-12 activity in
C. albicans infection.
IL-12 induces IL-10 production by T (24) and non-T (17, 19) cells. In
the present study, we demonstrated the occurrence of this positive
regulatory loop between IL-12 and IL-10 in mice with C.
albicans infection. Both production of and responsiveness to IL-10
were decreased in phagocytic cells from infected IL-12-deficient mice,
very likely accounting for the unimpaired innate antifungal immunity
and the increased resistance of these mice to acute systemic infection.
IL-10 is a potent inhibitor of NO production and candidacidal activity
of IFN--activated phagocytic cells (16). Both activities were indeed
up-regulated in IL-12-deficient mice, although IL-12 appeared to be
required for optimal production of NO by IFN--activated phagocytic
cells.
IL-10, but also IL-12, neutralization increased the innate antifungal resistance of wild-type mice, a finding in line with previous data showing that unphysiologic administration of either IL-10 (15) or IL-12 (9) exacerbated candidiasis in mice. However, defective IL-10 production was associated with up-regulated expression of B7 costimulatory molecules, particularly B7-2, on macrophages. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting up-regulation of B7 (33) and MHC (39) expression. It has been suggested that IL-10, by down-regulating costimulatory molecules on accessory cells, could play an indirect role in T cell activation (33, 40). We have shown previously that the effects of exogenous IL-10 in mice with candidiasis were critically dependent on the dose administered, the highest doses of IL-10 exacerbating the infection (15). In contrast, at lower doses, IL-10 paradoxically ameliorated the infection, and this was associated with a decreased production of IL-4 (15, 30). In this study, we have shown that treatment with low dose IL-10 prevents up-regulation of B7-2 expression on macrophages and concomitantly decreases IL-4 production. Interestingly, B7-1 expression was slightly increased, indicating a differential effect of IL-10 on B7-1 and B7-2 expression by accessory cells, as already suggested (40).
The engagement of appropriate costimulatory molecules is required
for Ag-specific T cell activation (41) and for optimal IL-12-induced
proliferation and cytokine production (42, 43). Triggering of the
CD28/CTLA-4 receptors on T cells by their natural ligands, B7-1 and
B7-2, provides an effective costimulus for T cell activation and
cytokine production (44, 45). Whether B7-1 and B7-2 mediate distinct or
overlapping costimulatory functions remains controversial (46).
However, when coexpressed with B7-1 on accessory cells, B7-2 provides a
predominant costimulatory signal (47) and primes for production of IL-4
and IFN- (46).
Although the extent to which B7-1 and B7-2 may participate in Th selection in response to C. albicans awaits further clarification, the results of the present study indicate that B7-2, rather than B7-1, is required for the production of IL-4 by activated CD4+ T cells. In this respect, it is interesting to note that Candida-induced proliferation of human memory T cells is highly dependent on B7-2 expression on monocytes (48). In murine C. albicans infection, production of IL-4 by CD4+ T cells is critically dependent upon the CTLA-4/B7 interaction (30). Interference with this costimulatory pathway during infection results in a decreased IL-2-dependent IL-4 production by CD4+ T cells expressing an activated phenotype, i.e., Mel-14lowCD44high, and in the activation of CD4+Th1 cells (30). In the present study, we found that blockade of this costimulatory pathway by CTLA4-Ig, IL-10, or IL-12 administration prevents the expansion of CD4+Mel-14lowCD44high cells and results in decreased IL-4 production by infected IL-12-deficient mice. Thus, rather than opposing the Th1-promoting activity of IL-12, endogenous IL-10 appears to be required for optimal costimulation of IL-12-induced CD4+Th1 cells.
IL-10 has been implicated in regulation of inflammation and of a wide variety of immune responses (49, 50), but many of the studies have relied upon the unphysiologic administration of exogenous IL-10. Although studies on IL-10-deficient or IL-10-overexpressing mice will definitely clarify the issue, the experimental models described in the present study would suggest that an overproduction of IL-10 may adversely affect the innate control of C. albicans infection; yet, endogenously produced IL-10 is required for development of antifungal Th1 responses. This is in line with recent findings showing that Th1-mediated resistance to L. major is unaffected in mice overexpressing IL-10 (51). The use of C. albicans strains of different virulence, thus having a different impact on the innate immune system (18), allows us to dissect the opposite effects of IL-10 in anticandidal immunity. It is conceivable that the ultimate effect of IL-10 in C. albicans infections may depend on the relative contribution of innate vs specific immune responses in the control of the infection and its pathology.
IL-4 may antagonize IL-12 in Th development (11, 52). However, recent
studies have revealed a more complex relationship between IL-12 and
IL-4 in CD4+Th differentiation and phenotype stability.
IL-4 appears to be a component of IL-12-dependent activation of
CD4+Th1 lymphocytes (53, 54, 55): it is produced by
CD4+ T lymphocytes in response to IL-12 (54), and the IL-4R
is expressed on Th1 cells (54, 56). We have recently observed that
endogenous IL-4 is indeed physiologically required for development and
maintenance of CD4+Th1 responses in mice with candidiasis
(22, 57). Therefore, a finely regulated balance of directive cytokines,
such as IL-4, IL-10, and IL-12, rather than the relative absence of
opposing cytokines, appears to be required for optimal development and
maintenance of Th1 responses in mice with candidiasis. In particular,
IL-12-dependent Th1 differentiation in vivo appears to occur in the
presence of appropriate levels of IL-4 (22) and IL-10 (this study) in
addition to other cytokines, including IFN- (5), TGF-ß (30), IL-6
(6), and TNF- (8). It is conceivable that IL-12 acts at different
levels to favor anticandidal Th1 responses. By inducing IL-10 (9, 19)
and TGF-ß (unpublished observation), IL-12 may limit the consequences
of an exaggerated proinflammatory response, including aberrant
expression of costimulatory molecules on accessory cells. At the level
of CD4+ T cell priming, IL-12, rather than IFN-, acts as
a limiting factor for optimal Th1 development (11). The occurrence of
early and sustained levels of IFN- in Candida-driven Th2
development makes it unlikely that IL-12 plays only an indirect role in
Th1 differentiation, by inducing soon after infection IFN- as an
essential requirement for CD4+Th1 differentiation (10). It
has recently been demonstrated that IFN- is required for expression
of the IL-12Rß2 message in activated CD4+ Th1 cells (58).
The finding that IL-12Rß2 transcripts are similarly expressed in
IL-12-deficient and wild-type mice (unpublished data) could suggest
that IFN- is required for the induction of IL-12 responsiveness in
naive CD4+ T cells, but that IL-12 is required to induce
IFN- production by CD4+ Th1 cells.
Overall, the data in this study stress the obligatory role of IL-12 in the induction of anticandidal Th1 responses. They also highlight a positive regulatory loop between IL-12 and IL-10 that negatively affects the innate antifungal response, but is required for the induction of optimal adaptive immune response to C. albicans.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Luigina Romani, 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: i.g., intragastrically; HPRT, hypoxanthine-guanine phosphoribosyltransferase; NO, nitric oxide.
Received for publication April 27, 1998. Accepted for publication August 8, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-double deficient mice infected with Candida albicans. Int. Immunol. 10:37. production correlates with induction of T helper type-1 phenotype in murine candidiasis. Eur. J. Immunol. 24:909.[Medline]
and interleukin-10. J. Exp. Med. 183:2559. production by suppressing natural killer cell stimulatory factor/interleukin-12 synthesis in accessory cells. J. Exp. Med. 178:1041. and TNF-. J. Immunol. 157:798.[Abstract]
). Curr. Opin. Immunol. 9:17.[Medline]
production and type 1 cytokine responses. Immunity 4:471.[Medline]
. J. Immunol. 155:785.[Abstract]
production by T cells. Nature 378:88.[Medline]
production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223. production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.-producing T cells studied by lineage ablation of IL-4-producing cells. Cell 7:985.
-producing CD4+ T cells following activation of naive CD4+ T cells. J. Immunol. 158:1085.[Abstract]
in stabilizing the T helper cell type 1 and 2 phenotype. J. Immunol. 158:2648.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  A. Pina, S. Bernardino, and V. L. G. Calich Alveolar macrophages from susceptible mice are more competent than those of resistant mice to control initial Paracoccidioides brasiliensis infection J. Leukoc. Biol., May 1, 2008; 83(5): 1088 - 1099. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Fradin, A. L. Mavor, G. Weindl, M. Schaller, K. Hanke, S. H. E. Kaufmann, H. Mollenkopf, and B. Hube The Early Transcriptional Response of Human Granulocytes to Infection with Candida albicans Is Not Essential for Killing but Reflects Cellular Communications Infect. Immun., March 1, 2007; 75(3): 1493 - 1501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. De Bernardis, R. Lucciarini, M. Boccanera, C. Amantini, S. Arancia, S. Morrone, M. Mosca, A. Cassone, and G. Santoni Phenotypic and Functional Characterization of Vaginal Dendritic Cells in a Rat Model of Candida albicans Vaginitis Infect. Immun., July 1, 2006; 74(7): 4282 - 4294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bozza, F. Fallarino, L. Pitzurra, T. Zelante, C. Montagnoli, S. Bellocchio, P. Mosci, C. Vacca, P. Puccetti, and L. Romani A Crucial Role for Tryptophan Catabolism at the Host/Candida albicans Interface J. Immunol., March 1, 2005; 174(5): 2910 - 2918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zafirova, M. Yordanov, and R. Kalfin Antiarthritic effect of VIP in relation to the host resistance against Candida albicans infection Int. Immunol., August 1, 2004; 16(8): 1125 - 1131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Deepe Jr. and R. S. Gibbons Protective and Memory Immunity to Histoplasma capsulatum in the Absence of IL-10 J. Immunol., November 15, 2003; 171(10): 5353 - 5362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Fieschi, S. Dupuis, E. Catherinot, J. Feinberg, J. Bustamante, A. Breiman, F. Altare, R. Baretto, F. Le Deist, S. Kayal, et al. Low Penetrance, Broad Resistance, and Favorable Outcome of Interleukin 12 Receptor {beta}1 Deficiency: Medical and Immunological Implications J. Exp. Med., February 17, 2003; 197(4): 527 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Montagnoli, A. Bacci, S. Bozza, R. Gaziano, P. Mosci, A. H. Sharpe, and L. Romani B7/CD28-Dependent CD4+CD25+ Regulatory T Cells Are Essential Components of the Memory-Protective Immunity to Candida albicans J. Immunol., December 1, 2002; 169(11): 6298 - 6308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mencacci, C. Montagnoli, A. Bacci, E. Cenci, L. Pitzurra, A. Spreca, M. Kopf, A. H. Sharpe, and L. Romani CD80+Gr-1+ Myeloid Cells Inhibit Development of Antifungal Th1 Immunity in Mice with Candidiasis J. Immunol., September 15, 2002; 169(6): 3180 - 3190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bacci, C. Montagnoli, K. Perruccio, S. Bozza, R. Gaziano, L. Pitzurra, A. Velardi, C. F. d'Ostiani, J. E. Cutler, and L. Romani Dendritic Cells Pulsed with Fungal RNA Induce Protective Immunity to Candida albicans in Hematopoietic Transplantation J. Immunol., March 15, 2002; 168(6): 2904 - 2913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Newman and A. Holly Candida albicans Is Phagocytosed, Killed, and Processed for Antigen Presentation by Human Dendritic Cells Infect. Immun., November 1, 2001; 69(11): 6813 - 6822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. O. Beenhouwer, S. Shapiro, M. Feldmesser, A. Casadevall, and M. D. Scharff Both Th1 and Th2 Cytokines Affect the Ability of Monoclonal Antibodies To Protect Mice against Cryptococcus neoformans Infect. Immun., October 1, 2001; 69(10): 6445 - 6455. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Bliss, L. C. Gavrilescu, A. Alcaraz, and E. Y. Denkers Neutrophil Depletion during Toxoplasma gondii Infection Leads to Impaired Immunity and Lethal Systemic Pathology Infect. Immun., August 1, 2001; 69(8): 4898 - 4905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Elahi, G. Pang, R. Clancy, and R. B. Ashman Cellular and Cytokine Correlates of Mucosal Protection in Murine Model of Oral Candidiasis Infect. Immun., October 1, 2000; 68(10): 5771 - 5777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mencacci, A. Bacci, E. Cenci, C. Montagnoli, S. Fiorucci, A. Casagrande, R. A. Flavell, F. Bistoni, and L. Romani Interleukin 18 Restores Defective Th1 Immunity to Candida albicans in Caspase 1-Deficient Mice Infect. Immun., September 1, 2000; 68(9): 5126 - 5131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Romani Innate and adaptive immunity in Candida albicans infections and saprophytism J. Leukoc. Biol., August 1, 2000; 68(2): 175 - 179. [Abstract] [Full Text]
|
|
|
|
 |
|
 K. E. Balashov, M. Comabella, T. Ohashi, S. J. Khoury, and H. L. Weiner Defective regulation of IFN{gamma} and IL-12 by endogenous IL-10 in progressive MS Neurology, July 25, 2000; 55(2): 192 - 198. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Nagayama, K. Sato, H. Kawasaki, M. Enomoto, C. Morimoto, K. Tadokoro, T. Juji, S. Asano, and T. A. Takahashi IL-12 Responsiveness and Expression of IL-12 Receptor in Human Peripheral Blood Monocyte-Derived Dendritic Cells J. Immunol., July 1, 2000; 165(1): 59 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Cenci, A. Mencacci, A. Bacci, F. Bistoni, V. P. Kurup, and L. Romani T Cell Vaccination in Mice with Invasive Pulmonary Aspergillosis J. Immunol., July 1, 2000; 165(1): 381 - 388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Cain and G. S. Deepe Jr. Interleukin-12 Neutralization Alters Lung Inflammation and Leukocyte Expression of CD80, CD86, and Major Histocompatibility Complex Class II in Mice Infected with Histoplasma capsulatum Infect. Immun., April 1, 2000; 68(4): 2069 - 2076. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
