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B Plays a Crucial Role for Organ Clearance of Candida albicans1
*
Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Kwangju, Korea;
Department of Immunology and Institute for Medical Sciences, Chonbuk National University Medical School, Chonju, Republic of Korea; and
Korean Research Institute of Chemical Technology, Daejon, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B in the organs resistant to C.
albicans, including the lung and spleen. In susceptible organs
such as the kidneys, early activation of NF-B was not observed. The
kinetics of TNF-
mRNA expression paralleled those of NF-B
activation in all organs examined. Blocking the effects of endogenous
platelet-activating factor (PAF) by pretreatment with the PAF
antagonist BN50739 or antioxidants significantly reduced the early
activity of NF-B and TNF- mRNA expression, and increased the
recovery of C. albicans in the lung and spleen.
Importantly, administration of PAF 5 min prior to the infection
resulted in the appearance of early activities of NF-B and TNF-
mRNA expression, followed by a nearly complete clearance of the
organisms in the kidneys. Pretreatment with anti-TNF- Ab
resulted in an enhanced susceptibility to C. albicans, and
the PAF-mediated resistance was abrogated by anti-TNF- in all
organs examined. These data indicated that endogenously produced PAF in
response to C. albicans is a key molecule involved in the
early activation of NF-B, which, in turn, renders the organ
resistant to the fungus by promoting the production of
anti-candidal proinflammatory cytokines such as TNF-.
Susceptible organs, including the kidneys, lack the capacity to
generate a sufficient PAF-induced early NF-B response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Platelet-activating factor (PAF),3 which
is produced by a variety of inflammatory cells, is a potent
lipid-derived first messenger involved in cellular activation,
fertilization, intracellular signaling, apoptosis, and diverse
inflammatory reactions (5, 6, 7, 8). We have recently reported
that PAF has a protective role in systemic murine candida infection and
that the effect of PAF appears to be mediated by TNF-
(9). PAF is released immediately in response to an
inflammatory stimulus and induces TNF- expression through the
activation of the inducible transcription factor NF-B
(10). NF-B plays a central role in the induction of
genes encoding proinflammatory cytokines and growth factors (e.g.,
TNF-, IL-1, and G-CSF) (11, 12), all of which can
confer protective activity against systemic C. albicans
infection (13, 14, 15). It is possible, therefore, that
PAF-induced NF-B activation plays a role in the pathogenesis of
organ-specific susceptibility to C. albicans infection.
In this study, we investigated the role of NF-B activation in the
organ resistance to systemic candida infection and the underlying
mechanism for NF-B activation. We found that clearance of C.
albicans in an organ is exclusively dependent upon the PAF-induced
early activation of NF-B in the organ. This may be a general
protective mechanism for infections in which cellular immunity plays a
major role.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. albicans NIH A-207 was kindly provided by Professor Hideoki Ogawa (University of Juntendo, Tokyo, Japan). C. albicans was grown to stationary phase at 28°C under slight agitation in Sabouraud dextrose broth (BBL Microbiology Systems, Cockeysville, MD). After a 24-h culture, cells were harvested by centrifugation (2000 x g), washed twice in PBS, diluted to the desired density, and injected i.v. via the tail vein in a volume of 0.1 ml.
Animals
Specific pathogen-free female BALB/c were provided by the Korean Institute of Chemistry Technology (Daejon, Korea) and were kept in our animal facility for 1–2 wk before use. All mice were used at 8–10 wk of age.
Reagents
Water-soluble PAF
(1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine),
(+)--tocopherol acid succinate (Vit. E), and
N-acetyl-L-cysteine (NAC) were purchased from
Sigma (St. Louis, MO). The PAF antagonist BN50739 (batch 51-884,
Mr 596.2, 50 mg/ml in DMSO), a
ginkoglide-derived synthetic PAF analogue, was a gift from Piere
Braquet (Institute Henri Beaufour, Le Plessis Robinson, France). The
PAF antagonist BN50739 is a synthetic PAF analogue counteracting the
effects of endogenous PAF by inhibiting PAF binding to its receptor and
the subsequent cellular responses. The efficacy of the BN50739 as a PAF
antagonist has been evaluated in previous studies (9, 10, 16, 17). Polyclonal anti-murine TNF- Ab was prepared from a
New Zealand White rabbit as previously described (9). The
IgG fraction from immune or preimmune serum was purified by protein G
chromatography (Pierce, Rockford, IL).
Quantification of C. albicans
Lung, spleen, and kidneys from individual mice were removed aseptically and homogenized with 0.2 ml of PBS. The number of viable CFU in the specimens was determined by dilution plating on Sabouraud dextrose agar (BBL Microbiology Systems).
RT-PCR
RNA was prepared as previously described (10, 16).
Reverse transcription was performed using 1 µl of total RNA in a
10-µl reaction mixture (Promega, Madison, WI) containing oligo(dT)15
and avian myeloblastosis virus reverse transcriptase. cDNA (1 µl) was
amplified by PCR in a thermal cycler Perkin-Elmer System 2400 (Norwalk,
CT) (denaturation for 1 min at 94°C, annealing for 1 min at 60°C,
and elongation for 40 s at 72°C) using TNF- (30 cycles) or
-actin primers (23 cycles). The primers used in these analysis are
as follows: -actin, 5'-GGG TCA GAA CTC CTA TG-3' and 5'-GTA ACA ATG
CCA TGT TCA AT-3'; TNF-, 5'-CCT GTA GCC CAC GTC GTA GC-3' and 5'-TTG
ACC TCA GCG CTG AGT TG-3'.
Gel shift assay
The nuclear extracts were prepared from the lungs as described
previously (10, 16). To inhibit endogenous protease
activity, 1 mM PMSF was added. As a probe for the gel retardation
assay, an oligonucleotide containing the Ig-chain binding site
(B, 5'-CCG GTT AAC AGA GGG GGC TTT CCG AG-3') was synthesized. The
two complementary strands were annealed and labeled with
[-32P]dCTP. Labeled oligonucleotides (10,000 cpm), 10
µg of nuclear extracts, and binding buffer (10 mM Tris-HCl (pH 7.6),
500 mM KCl, 10 mM EDTA, 50% glycerol, 100 ng of poly(dI · dC),
and 1 mM DTT) were incubated for 30 min at room temperature in a final
volume of 20 µl. The reaction mixture was analyzed by electrophoresis
on a 4% polyacrylamide gel in 0.5x Tris-borate buffer. Specific
binding was controlled by competition with a 50-fold excess of cold
B or cAMP response element oligonucleotide.
Statistical analysis
The data are represented as the mean ± SE of multiple determinations from an experiment. Statistical significance was determined by Student’s t test when two data sets were analyzed or, alternatively, by ANOVA followed by the appropriate post hoc test for multiple data sets with the statistical software StatView (version 4.5; SAS Institute, Cary, NC). All experiments were conducted two or more times. Reproducible results were obtained and representative data are therefore shown in the figures.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is absent in the kidneys of
C. albicans-infected mice
The kinetics of NF-B activation in resistant (lung and spleen)
and susceptible (kidneys) organs to C. albicans was examined
for 5 days following infection. As shown in Fig. 1
, there were marked differences in the
kinetics of NF-B activation in these organs. The early (1–8 h) and
late (24–48 h) activation of NF-B was evident in the lung and
spleen (Fig. 1, A and B). In the kidneys,
however, the early activation of NF-B was absent. NF-B activation
in the kidney was seen after 24-h postcandida infection (Fig.
1C). TNF- is an important cytokine involved in resistance
to C. albicans infection (9, 13), and its
expression is tightly regulated by NF-B activity
(10, 11, 12). Time kinetics of TNF- mRNA expression
paralleled the kinetics of NF-B activation in all organs examined
(Fig. 1). Quantitative cultures revealed that the numbers of C.
albicans in the lung and spleen decreased from day 1. By contrast,
growth of the organisms in the kidneys increased from day 1. By days
3–5, the fungal burden in the kidneys was 10- to 1000-fold higher than
that of other organs (Fig. 2). These data
suggest that the early activation of NF-B is related to the early
clearance of C. albicans in an organ-specific manner.
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B and
efficient clearance of C. albicans in the lung and spleen
Based on the facts that PAF is released immediately in response to
an inflammatory stimulus (10) and is a potent inducer of
NF-B activation (10, 18, 19), we investigated whether
PAF is involved in the early activation of NF-B seen in the lung and
spleen. Blocking of endogenous PAF by pretreatment with the PAF
antagonist BN50739 resulted in the inhibition of most of the early
NF-B activation and TNF- mRNA expression in the lung and spleen.
In parallel, pretreatment with the PAF antagonist resulted in a
progressive increase (2- to 5-fold) in the recovery of C.
albicans over the 3 days after infection in the lung and spleen
(Fig. 3, A and B).
NF-B activation and TNF- mRNA expression were not significantly
altered by pretreatment with BN50739 in the kidneys (Fig.
3C), but a slight increase in the recovery of C.
albicans was observed. We further clarified the role of early
NF-B activation by blocking NF-B activity using the antioxidants
Vit. E and NAC. Reactive oxygen intermediates are strong inducers of
NF-B (20, 21). We have recently demonstrated that
antioxidants selectively inhibited PAF-induced activation of NF-B
(22). The efficacy of antioxidants in blocking LPS-induced
NF-B activation was determined in preliminary experiments (data not
shown). Pretreatment of the mice with antioxidants before infection
exerted similar effects as did BN50739 pretreatment (Fig. 4). These data indicated that PAF is
involved in the early activation of NF-B, which is critical for
organ-specific clearance of C. albicans.
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B and
efficient clearance of C. albicans in the kidneys by
exogenous PAF
Administration of exogenous PAF has been reported to protect mice
from death and reduce the growth of C. albicans in the
kidneys (9). Thus, we examined how exogenous PAF regulates
NF-B activation and fungal burden in the kidneys. Administration of
PAF 5 min before the infection resulted in the early activation of
NF-B and early expression of TNF- mRNA, followed by efficient
clearance of the organisms in the kidneys (Fig.
5A). The fungal burden in the
kidneys after PAF treatment was <1% of control (230 x
102 vs 2 x 102). Exogenous PAF exerted
protective effects in the lung and spleen, albeit to a much lesser
degree than that of exogenous PAF in the kidneys; it increased NF-B
activation and TNF- mRNA expression at the earlier time point and
slightly decreased the recovery of C. albicans in the lung
and spleen (Fig. 5, B and C). The PAF-induced
early activation of NF-B, TNF- mRNA expression, and efficient
clearance of the fungus were completely blocked by either BN50739 or
Vit. E (Fig. 6) in the kidney, indicating
that early activation of NF-B and efficient clearance of the fungus
in the kidneys are an exclusively PAF-dependent process. These data
further support the potential involvement of PAF in the early
activation of NF-B following C. albicans infection and
support the hypothesis that failure to efficiently clear C.
albicans in the kidneys is due to the absence of PAF-induced early
activation of NF-B.
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inhibition on the resistance to
C. albicans and PAF-induced enhanced resistance
To elucidate the efficacy of NF-B-dependent cytokine, we
examined the effect of TNF- inhibition on clearances of C.
albicans in the various organs by using anti-TNF-
polyclonal Ab. Administration of anti-TNF- 10 min before
C. albicans infection resulted in an enhanced susceptibility
to C. albicans (Fig.
7A). The anti-TNF-, but
not control Ab, pretreatment increased the number of the organism in
the kidneys from (7.8 ± 1.2) x 104 to
(14.8 ± 2.7) x 104 CFU (*, p <
0.05). Furthermore, it also increased the fungal recovery in the
lung from (4.9 ± 1.1) x 103 to (9.2 ±
1.7) x 103 CFU (*, p < 0.05) and
in the spleen from (6.4 ± 1.3) x 103 to
(10.8 ± 2.0) x 103 CFU (*, p <
0.05), supporting that a protective effect in candidiasis is
mediated, at least in part, by TNF-. We next examined the effect of
TNF- on PAF-induced enhanced resistance. As shown in Fig.
7B, anti-TNF- abrogated the majority of PAF-induced
enhanced resistance, suggesting that the PAF-induced protection is
mediated by TNF-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(9). The data of the present study demonstrated
that the organ-specific protective effect of PAF against systemic
candidiasis was due to its ability to induce the early activation of
NF-B. This process, in turn, inhibited the growth of C.
albicans by promoting the production of proinflammatory cytokines
such as TNF-. The important difference between organs resistant to
C. albicans such as lung and spleen and a susceptible organ
such as the kidney was the kinetics of NF-B activation as well as
TNF- mRNA expression. NF-B activation and TNF- mRNA expression
occurred 1–2 h after C. albicans infection only in
resistant, but not in susceptible organs. These results suggest that
the early activation of NF-B may determine the resistance to
C. albicans within a specific organ.
A key element necessary for the early activation of NF-B was
determined to be the lipid-derived mediator PAF. The presence of PAF
and subsequent early activation of NF-B is necessary for limiting or
suppressing C. albicans growth. This conclusion came from
the observations that 1) the PAF antagonist BN50739 significantly
abrogated the early activation of NF-B as well as the early
expression of TNF- mRNA, followed by a 2- to 5-fold increase in the
recovery of C. albicans in the lung and spleen; and 2)
blocking of early activity of NF-B using the antioxidants, Vit. E
and NAC, showed comparable effects to those of BN50739. In this study,
we used Vit. E and NAC as inhibitors of NF-B activation.
Antioxidants have been previously reported to selectively inhibit the
ability of PAF to activate NF-B-activating activity
(22). Thus, the inhibition of PAF-induced early activation
of NF-B as well as efficient clearance of C. albicans by
Vit. E and NAC indicated that PAF exerted its effect through the
generation of reactive oxygen intermediates.
The administration of exogenous PAF resulted in the appearance of the
early NF-B activation as well as early TNF- mRNA expression and
efficient clearance of C. albicans in the kidneys. These
effects were blocked by the PAF antagonist BN50739 or Vit. E. These
data clearly indicated that the protective effect of exogenous PAF
could be attributed to its ability to induce early NF-B activation
within the susceptible kidneys. However, much fewer alterations of
these end points were seen in the lung and spleen by the administration
of exogenous PAF. Thus, it seems likely that the PAF-mediated NF-B
activation process is particularly important in the kidneys, whereas
other protective mechanism rather than PAF-mediated NF-B-dependent
protection may exist in the lung and spleen.
The mechanism underlying organ differences in PAF-mediated early
activation of NF-B is unclear. Since PAF is released or produced
largely from inflammatory cells, it seems likely that organ-specific
tissue macrophages represent the source of endogenous PAF in response
to C. albicans. It is possible that the amount of PAF
produced from tissue macrophages is not sufficient in susceptible
organs to induce the early NF-B activation. An impaired NF-B
response in the kidneys cannot, however, be responsible for the lack of
C. albicans clearance since exogenous PAF administration
resulted in NF-B activation similar to what was observed in the lung
and spleen. In contrast, much fewer changes in the level of NF-B
activation and fungal recovery in the lung and spleen occurred
following exogenous PAF administration. Such findings suggests that
endogenously produced PAF in the lung and spleen following infection
contributes to the almost maximum protective effect. It is likely,
therefore, that the amount of PAF produced endogenously in response to
C. albicans in an organ may be the most important factor
determining the level of resistance against the organisms. In systemic
candidasis, the heart shows only minor lesions in normal mice. We
examined the fungal recovery and NF-B activation in the heart. The
CFU recovered from the heart was much less (comprising 0.1–1%) than
those from the other organs, and kinetic study revealed that very weak
NF-B activation was seen 24 h postinfection (data not shown).
Although we do not know exactly the reason of low fungal recovery
despite very weak NF-B activation in the heart, this may be due to
the fact that, in contrast to other organs which contain many
mononuclear phagocytic cells such as lung, liver, spleen, and kidneys,
there are very few tissue macrophages in the heart. Given the entry of
the fungus into an organ is achieved by uptake of the fungus by tissue
macrophages and intracellular killing activity of the cells plays an
important role in host defenses against fungal diseases, it is possible
that the heart is infected by much fewer C. albicans as
compared to other organs. Alternatively proinflammatory cytokines such
as IL-6 and IL-10 produced by cardiomyocytes (23, 24) may
influence the growth of the organism.
NF-B is normally present in the cytosol as part of an inactive
complex that includes inhibitory proteins known as IBs.
Phosphorylation of IBs by a variety of inducers triggers their
degradation and induced dissociation of NF-B from IB followed by
translocation of NF-B is then free to translocate to the nucleus
where it transactivates the expression of various proinflammatory
cytokine genes (11, 12). Thus, the critical role for
NF-B activity in the resistance to C. albicans implies
that anti-candidal cytokines under the transcriptional regulation
by NF-B are involved in the process. Cytokines under
NF-B-dependent regulation includes TNF-, IL-1, and G-CSF
(11, 12), all of which can confer protective activity
against systemic C. albicans infection
(13, 14, 15). TNF- plays an important role in host
resistance to infections caused by various microorganisms including
C. albicans (25, 26), and TNF- is a key
molecule in PAF-induced protection against C. albicans
(9). In the present study, we observed that there was
always a complete correlation between the early activation of NF-B
and the early expression of the TNF- gene, strongly suggesting that
the potential protective effect of NF-B against the organisms is
attributed to the early production of NF-B-dependent cytokines such
as TNF-.
It is presently unclear by what specific mechanism the early NF-B
activation and subsequent TNF- expression exert an
anti-candidacidal effect. Given that TNF- and G-CSF have been
reported to enhance host resistance to disseminated candidiasis through
recruitment of neutrophils to the site of infection (15, 27), we determined whether exogenous PAF-induced enhanced
clearance of C. albicans in the kidneys was mediated through
neutrophil recruitment. This possibility was unlikely, however, because
no evidence of cellular infiltration was found in these organs up to 3
days after infection (data not shown). Alternatively, exogenous PAF may
exert its protective effect through enhancing candidacidal activity of
tissue macrophages in the kidneys. This issue is currently under
investigation.
In summary, our data suggest that endogenously produced PAF in response
to C. albicans in an organ is a key mediator in the early
activation of NF-B. The activation of NF-B, in turn, renders the
organ resistant to the fungus by promoting the production of
anti-candidal proinflammatory cytokines such as TNF-.
Susceptible organs, such as the kidney, to C. albicans is
attributed to the inability of the organs to undergo this process. To
our knowledge, this is the first report revealing the mechanism by
which organ susceptibility in candida infection occurs and may provide
a fundamental protective mechanism against infectious diseases caused
by intracellular pathogens in which cellular immunity plays a
major role.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Suhn-Young Im, Department of Biological Sciences, College of Natural Sciences, Chonnam National University Kwangju 500-757, Republic of Korea.
3 Abbreviations used in this paper: PAF, platelet-activating factor; Vit. E, (+)--tocopherol acid succinate; NAC, N-acetyl-L-cysteine.
Received for publication August 20, 2000. Accepted for publication February 14, 2001.
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B in platelet-activating factor-mediated tumor necrosis factor- expression. Eur. J. Immunol. 27:2800.[Medline]
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B and Rel proteins in innate immunity. Adv. Immunol. 58:1.
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B activation through a G protein-coupled pathway. J. Biol. Chem. 270:14928.B and immunoglobulin expression in response to platelet-activating factor in a human B cell line. Cell. Immunol. 155:292.[Medline]
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lymphotoxin- double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans. J. Immunol. 163:1498.This article has been cited by other articles:
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