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Release in Response to Cryptococcus neoformans Polysaccharide Capsule1
The Evans Memorial Department of Clinical Research and Department of Medicine, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and a
proinflammatory response including TNF-
production. We investigated
whether TLRs participate in the host response to Cryptococcus
neoformans glucuronoxylomannan (GXM), the major capsular
polysaccharide of this fungus. Chinese hamster ovary fibroblasts
transfected with human TLR2, TLR4, and/or CD14 bound fluorescently
labeled GXM. The transfected Chinese hamster ovary cells were
challenged with GXM, and activation of an NF-B-dependent reporter
construct was evaluated. Activation was observed in cells transfected
with both CD14 and TLR4. GXM also stimulated nuclear NF-B
translocation in PBMC and RAW 264.7 cells. However, stimulation of
these cells with GXM resulted in neither TNF- secretion nor
activation of the extracellular signal-regulated kinase 1/2, p38, and
stress-activated protein kinase/c-Jun N-terminal kinase
mitogen-activated protein kinase pathways. These findings suggest that
TLRs, in conjunction with CD14, function as pattern recognition
receptors for GXM. Furthermore, whereas GXM stimulates cells to
translocate NF-B to the nucleus, it does not induce activation of
mitogen-activated protein kinase pathways or release of TNF-. Taken
together, these observations suggest a novel scenario whereby GXM
stimulates cells via CD14 and TLR4, resulting in an incomplete
activation of pathways necessary for TNF-
production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. neoformans is the only human pathogenic fungus with a polysaccharide capsule. The capsule is composed primarily of a high molecular mass glucuronoxylomannan (GXM)3 (4). Capsule-deficient mutants of C. neoformans have greatly reduced virulence compared with their parent strains. In addition to coating the surface of C. neoformans, GXM is shed from the fungus. In humans and experimental animals infected with C. neoformans, shed GXM can circulate in the blood and CSF at concentrations well into the micrograms per milliliter range, and it is likely that in infected tissues, local concentrations of GXM in the milligram per milliliter range are achieved (5, 6, 7).
A myriad of mechanisms has been postulated by which capsular polysaccharide, including GXM, helps the fungus to elude host defenses (1, 8). Capsular polysaccharide inhibits phagocytosis by presenting a surface that is not well recognized by phagocytes. In the absence of opsonins, most phagocytic cells will not bind to encapsulated strains of C. neoformans (9). GXM inhibits neutrophil migration by triggering those cells to shed the adhesion molecule L-selectin (10). GXM may further reduce neutrophil adherence by binding to CD18, thereby impairing complex formation with endothelial ICAM-1 (11). Other immune-suppressive effects attributed to the cryptococcal capsule include induction of suppressor T cells, inhibition of lymphoproliferation, and impairment of Ag presentation (8).
Prevention of cryptococcosis requires an immune response composed of
innate defenses and then, if unsuccessful, acquired immunity. The
innate immune system identifies infectious agents by means of pattern
recognition proteins. The host then attempts to dispose of the microbe
and to activate effector defense mechanisms (12).
Monocytes and macrophages are central to these processes. Whereas
disposal is attempted via phagocytosis and the generation and/or
release of antimicrobial substances, activation is accomplished by the
production of proinflammatory cytokines such as TNF-. Both processes
utilize a series of events initiated by cell surface binding and
mediated by intracellular signaling.
Toll-like receptors (TLR) are a recently described family of cell
surface receptors. Although no direct binding studies have been
reported describing TLR ligands, molecular genetic studies suggest that
the extracellular domain of TLRs recognizes conserved molecular
patterns associated with a range of microbial pathogens. The
cytoplasmic portion of all TLRs is homologous to the cytoplasmic domain
of the IL-1R, and is responsible for signal transduction
(13). The downstream pathways leading to the production
and release of TNF- and other proinflammatory cytokines are starting
to be defined. TLR engagement leads to binding of a cytoplasmic adapter
protein (MyD88), and activation of IL-1R-associated kinase, followed by
phosphorylation and activation of TNFR-associated factor 6 (TRAF6).
Activation of TRAF6 subsequently results in the activation of
NF-B-inducing kinase, followed by I-B (inhibitory protein that
dissociates from NF-B) kinase, phosphorylation of I-B, and its
dissociation from NF-B (14). These events ultimately
result in the translocation of NF-B to the nucleus and the
initiation of gene transcription. In addition to pathways leading to
NF-B activation, three mitogen-activated protein kinase (MAPK)
cascades, the extracellular signal-regulated kinase (ERK), p38 MAPK,
and stress-activated protein kinase (SAPK)/c-JUN N-terminal kinase
(JNK), appear necessary for optimal TNF- cytokine production
(15).
The diversity of microbial products to which TLR2 and 4, often in
association with CD14, mediate intracellular signaling has been
recently reviewed (16). Molecules derived from a range of
pathogens, including Gram-negative and Gram-positive bacteria,
mycobacteria, mycoplasmas, spirochetes, and the fungal product zymosan,
have been reported to activate cells via TLR2 (17, 18). To
date, TLR4 has only been reported to mediate intracellular signaling in
response to LPS (17) and Mycobacterium
tuberculosis (19). In the present study, we tested
the hypothesis that TLRs participate in mediating signaling responses
to C. neoformans. We found that whereas GXM, the major
cryptococcal polysaccharide capsule component, binds to and activates
cells to induce nuclear translocation of NF-B via an interaction
with TLR4 and CD14, it neither activates MAPK pathways nor induces
TNF- production. These findings suggest that the interaction between
GXM and TLR may represent a mechanism of immune dysregulation whereby
the organism incompletely activates cascades leading to TNF-
production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBS and trypsin/EDTA were obtained from BioWhittaker
(Walkersville, MD). Hams F12, RPMI 1640, and G418 were obtained from
Life Technologies (Gaithersburg, MD). FBS was from Life Technologies
(Grand Island, NY). Ciprofloxacin was obtained from Bayer
Pharmaceuticals (West Haven, CT). Hygromycin B was obtained from
Calbiochem (La Jolla, CA). Puromycin, streptomycin, penicillin,
Ficoll-Hypaque, and LPS (from Escherichia coli O111:B4) were
obtained from Sigma (St. Louis, MO). Where indicated, an LPS
preparation devoid of TLR2-stimulating activity (a kind gift of Dr.
Stephanie Vogel, Uniformed Services University of the Health Sciences,
Bethesda, MD) (20) was utilized in lieu of Sigma LPS.
Anti-CD25 mAb conjugated with FITC was obtained from Caltag
(Burlingame, CA). The 4-(4,6-dichlorotriazinyl)aminofluorescein (DTAF)
was obtained from Molecular Probes (Eugene, OR). rIL-1
was obtained
from Genzyme (Cambridge, MA).
All incubations were at 37°C in humidified air supplemented with 5% CO2, except where otherwise noted. All experiments were performed under conditions designed to minimize endotoxin contamination, as in our previous studies (21, 22, 23). Complete media is defined as RPMI 1640 with 10% FBS supplemented with penicillin and streptomycin. Media, FBS, and PBS contained less than 0.03 endotoxin U/ml, as certified by the manufacturer. Pooled human serum (PHS) was obtained by combining serum from >10 healthy donors under conditions designed to preserve complement activity. PHS was stored in aliquots at -70°C until use.
Purification of GXM
GXM was purified from culture supernatants of two serotype A strains of C. neoformans, J11a (a gift of Arturo Casadevall, Albert Einstein College of Medicine, New York, NY) and 6 (ATCC 62066), using a modification of the method of Cherniak (24). Serotype A is the most common serotype seen in clinical infections, and the structure of the GXM from both strains has been well characterized (24, 25). C. neoformans was grown in yeast nitrogen base media, supernatants collected, and polysaccharide precipitated with sodium acetate (taking care to keep the pH at 7 in order not to destroy acetyl groups), followed by 2.5 vol of ethanol. The precipitate (consisting of GXM and galactoxylomannan) was collected, quantitated by the phenol-sulfuric acid method (26), and solubilized in 0.2 M NaCl. GXM was separated from galactoxylomannan by combining 0.3% cetyltrimethylammonium bromide (CTAB) with the polysaccharide mixture, at a ratio of 3:1 by mass, to yield a precipitate containing CTAB bound to GXM. The CTAB-GXM precipitate was solubilized in 0.2 M NaCl, and 2 vol ethanol added to precipitate selectively the GXM. The precipitate was dissolved in 2 M NaCl and extensively dialyzed with a 10 kDa molecular mass cutoff against 1 M NaCl and then H2O. Quantitation was again by the phenol-sulfuric acid method.
Size and purity of the GXM preparations were evaluated by SDS-PAGE,
followed by Periodic Acid-Schiff staining. Both 6 and J11a GXM migrated
approximately the same distance as a dextran standard of average
molecular mass 473,000. Low molecular mass bands were not seen, even
using overloaded gels, suggesting that both GXM solutions were free of
significant contamination with galactoxylomannan. The GXM was endotoxin
free to less than 0.03 endotoxin U/ml, as determined by the
Limulus amebocyte lysate assay (Associates of Cape Cod,
Woods Hole, MA). The negative Limulus amebocyte lysate assay
also suggests that the GXM was free of contamination with -glucan to
less than 1 ng (27). Finally, the GXM preparation used did
not contain detectable amounts of protein, as determined by the
bicinchoninic acid protein assay (Pierce, Rockford, IL). The
bicinchoninic acid assay was sensitive to 5 µg/ml.
Fluorescein-labeled GXM was prepared by covalent binding of the polysaccharide to DTAF per the method of Prigent (28), with modifications for GXM. In brief; DTAF and GXM were dissolved in a 0.05 M borate buffer solution at pH 9. The reaction was allowed to proceed for 24 h at 37oC, at which point 0.1 M glycine was added for 72 h to react with any unincorporated DTAF. DTAF-GXM was then purified by centrifuge filtration using a mini centrifuge filter (Nalgene, Rochester, NY) with a 10 kDa molecular mass cutoff.
Cells
The stably transfected cell lines used in the studies are listed
in Table I
. The cDNAs for TLR2 and TLR4
were cloned into the vector pFLAG, as described
(29). In addition to expressing the indicated human TLR
and/or CD14, all cell lines except Chinese hamster ovary (CHO)/TLR2
were transfected with the NF-B reporter plasmid, ELAM.tac. This
construct contains an NF-B-dependent portion of the endothelial
cell-leukocyte adhesion molecule (ELAM-1) promoter driving cell surface
expression of human CD25 (tac) (30, 31, 32). CHO cells were
grown as adherent monolayers in tissue culture flasks in F12 medium and
passed at least twice weekly to maintain logarithmic growth.
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Flow cytometric analysis of CHO cells
CHO cells were plated in 12-well tissue culture dishes at a concentration of 1 x 105 cells/ml, incubated for 24 h, and then stimulated for 18 h with GXM or LPS. Cells were detached from the surface using trypsin/EDTA and assessed by flow cytometry for the presence of surface CD25, as in our previous studies (32). Note that CD14, TLR2, TLR4, and CD4 are all resistant to standard trypsin treatment (data not shown). Cells analyzed for binding of GXM were likewise grown as adherent monolayers and detached from the surface using trypsin/EDTA. The cells were washed once with PBS, resuspended in PBS/10% FBS, incubated with fluorescein-labeled J11a GXM at 37°C for 30 min, washed twice with PBS, and evaluated for fluorescence by flow cytometry. Cell surface expression of heterologous receptors was verified by flow cytometry using anti-FLAG and anti-CD14 Abs to measure TLR and CD14 expression, respectively (19, 33).
Analysis of NF-B translocation
PBMC and RAW 264.7 cells were stimulated with 250 µg/ml of
J11a GXM and 100 ng/ml of LPS for 1 h. Cells were washed with PBS,
and nuclear extracts were prepared and analyzed using the EMSA, as
described (21, 23, 31). Briefly, nuclear extracts from
stimulated cells were prepared in the presence of protease inhibitors,
and protein concentration was determined using a commercial kit
(Bio-Rad Laboratories, Hercules, CA). An oligonuleotide containing the
NF-B-binding sequence (Promega) was end labeled with
[-32P]dATP and
[-32P]dCTP using Klenow DNA polymerase
(Promega, Madison, WI). Unincorporated nucleotides were removed with a
G-25 spin column (Pharmacia Biotech, Piscataway, NJ). Labeled probe
(0.2 ng) and nuclear extracts (4 µg) were incubated at room
temperature for 30 min in a 1x band shift buffer (10 mM Tris-HCl, pH
7.8, 1 mM EDTA, 40 mM KCl, 1 mM DTT) containing 50 mg/ml of poly(dI-dC)
and 5% glycerol. Reactions were then size fractionated by
electrophoresis in 4% native polyacrylamide gels, transferred to 3 MM
filter paper (Whatman Laboratory Products, Clifton, NJ), dried, and
visualized by autoradiography.
Measurement of TNF- production and gene expression
In 96-well flat-bottom plates, 105 PBMC
and RAW 264.7 cells were stimulated with 250 µg/ml of GXM or 100
ng/ml of LPS for 18 h in complete medium. PBMC were also
stimulated in RPMI with 10% PHS. Human and mouse TNF- were measured
in culture supernatants by sandwich ELISA using commercial kits
according to the manufacturer’s instructions (Duo Set ELISA
Development System, human and mouse TNF-; R&D Systems, Minneapolis,
MN). The ELISA were sensitive over a range of 10 to 3000 pg/ml.
Proinflammatory cytokine gene expression was measured by the RNase
protection assay (RPA), as in our previous studies (23).
Briefly, PBMC (5 x 106) were stimulated
with 100 ng/ml LPS or 250 µg/ml GXM in six-well polystyrene plates in
the presence of RPMI 1640 containing 10% FBS. Total cellular RNA was
extracted from PBMC using TRIzol reagent (Life Technologies, Grand
Island, NY) and analyzed for human TNF-, IL-1, and IL-6 mRNA by
RPA using a custom kit (PharMingen, San Diego, CA). Integrity of the
RNA and equal loading of lanes were verified using the housekeeping
genes L32 and GAPDH.
Analysis of activation of MAPK pathway
Phosphorylation of MAPK was evaluated in human PBMC and RAW 264.7 cells according to the manufacturer’s (New England Biolabs, Beverly, MA) protocol. Briefly, following stimulation with LPS or J11a GXM, cell lysates were prepared, separated by SDS-PAGE, and analyzed by Western immunoblotting using HRP-labeled Abs directed against the phosphorylated (activated) forms of p44/42 MAPK (ERK1/2), p38 MAPK, and SAPK/JNK. These Abs do not react with unphosphorylated forms of the protein kinases. Identity of the bands was confirmed using molecular mass markers and positive controls supplied by the manufacturer.
Statistics
Means and SE were compared by the two-tailed two-sample t test using a statistical software program (SigmaStat; Jandel Scientific Software, San Rafael, CA). Adjustments for significance of multiple comparisons were made using the Bonferroni correction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the contribution of TLR2, TLR4, and CD14 to binding
GXM, fluorescein-labeled J11a GXM was incubated with the six stably
transfected CHO cell lines listed in Table I
: CHO/ELAM.tac,
CHO/CD14/ELAM.tac, CHO/TLR2, CHO/TLR4/ELAM.tac,
CHO/CD14/TLR2/ELAM.tac, and CHO/CD14/TLR4/ELAM.tac. Compared with the
parent strain, an increase in fluorescence was observed in CHO cells
expressing CD14, TLR, or both (Fig. 1).
Preincubation of the cells with 250 µg/ml of unlabeled J11a GXM
nearly completely competed out the increase in fluorescence observed
following incubation with GXM-DTAF (Fig. 2). These data suggest CD14, TLR2, and
TLR4 participate in binding to GXM.
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We next sought to determine whether binding of GXM to transfected
CHO cells led to cellular activation as measured by nuclear
translocation of NF-B. To accomplish this, we used CHO reporter
cells stably transfected with an inducible promoter-driving surface
expression of CD25 in response to NF-B activation. Activation of
cells was measured by flow cytometry using FITC-labeled Ab to
CD25.
We exposed CHO/CD14/ELAM.tac, CHO/CD14/TLR2/ELAM.tac, and
CHO/CD14/TLR4.tac cells to J11a GXM. As a positive control, cells were
incubated with LPS, a known stimulant of CHO/CD14/ELAM.tac,
CHO/CD14/TLR2/ELAM.tac, and CHO/CD14/TLR4/ELAM.tac cells (29, 34). Both GXM and LPS stimulated CHO/CD14/TLR4/ELAM.tac cells to
translocate NF-B to the nucleus, as indicated by increased surface
expression of CD25 (Fig. 3). Surface
expression of CD25 increased in a concentration-dependent manner in
response to escalating concentrations of GXM from 62.5 to 250 µg/ml.
LPS, but not GXM, increased surface expression of CD25 among CHO cells
expressing either CD14 alone or CD14 and TLR2. Incubation of CHO cells
with 25 µg/ml of polymixin B inhibited responses to LPS, but not GXM
(data not shown). This result in combination with our finding that our
GXM preparations were free of LPS, as determined by Limulus
amebocyte lysates, made it unlikely that the results with GXM
stimulation were secondary to LPS contamination. Similar results were
obtained comparing GXM preparations from strains J11a and 6 (data not
shown).
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B to the nucleus. Next, we sought to determine whether TLR4
alone, in the absence of CD14, could activate NF-B. We exposed
CHO/TLR4/ELAM.tac to 250 µg/ml of J11a GXM, 1 µg/ml of LPS, and 3
ng/ml of IL-1. CHO/TLR4/ELAM.tac cells were observed to increase
surface expression of CD25 only minimally in response to GXM or LPS
(Fig. 3). Consistent with previous observations, only IL-1, but not
LPS, activated the cells to translocate NF-B in the absence of
membrane-bound CD14 (35, 36). Thus, GXM stimulated a
vigorous NF-B response only in the presence of coexpression of both
human TLR4 and CD14.
GXM induces nuclear translocation of NF-B among PBMC and RAW
264.7 cells
The next set of experiments assessed whether GXM stimulates
NF-B nuclear translocation in professional phagocytic cells, as it
does in the CHO/CD14/TLR4/ELAM.tac cell line. Human PBMC and murine RAW
264.7 cells were stimulated with 250 µg/ml of J11a GXM or 100
ng/ml of LPS for 1 h. Both cell types were noted to increase
translocation of NF-B to the nucleus in response to J11a GXM and LPS
when compared with controls (Fig. 4). Unopsonized C.
neoformans strain 145 (21), which presents a
surface coated with serotype A GXM, also stimulated NF-B
translocation.
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gene expression and release
Having found that GXM stimulates NF-B nuclear translocation, we
next sought to determine whether TNF- release was likewise
stimulated. Mononuclear phagocytes from two sources were studied: human
PBMC and RAW 264.7 cells. In both cell types, levels of TNF-
following an 18-h stimulation with 250 µg/ml of GXM were not
significantly different from levels seen in unstimulated cells (Fig. 5A). As expected, an 18-h
incubation with 100 ng/ml of LPS stimulated large amounts of TNF-.
Substitution of FBS with PHS in the media did not alter TNF-
responses (data not shown). Moreover, in experiments using three
different donors each tested in triplicate, preincubation of PBMC with
250 µg/ml of GXM did not significantly alter TNF- release induced
by 10 ng/ml of an LPS preparation that is devoid of TLR2-stimulating
activity (mean ± SEM TNF release 1295 ± 214 and 1273
± 216 ng/ml in the absence and presence of GXM, respectively).
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release was not secondary to
posttranscriptional mechanisms as GXM also failed to stimulate TNF-
mRNA in PBMC (Fig. 5B). In addition, cellular levels of
IL-1 and IL-6 mRNA did not change following GXM stimulation. In
contrast, LPS stimulated gene expression of all three proinflammatory
cytokines. GXM fails to activate MAPK pathways among PBMC and RAW 264.7 cells
The MAPK are a group of signaling kinases that, by modulating the
phosphorylation status of transcription factors, link transmembrane
signaling with gene induction. Three major groups of MAPKs have been
identified in mammalian cells: ERK1/2, p38 MAPK, and SAPK/JNK. All
three MAPKs are thought to be necessary for optimal TNF- induction
in LPS-stimulated macrophages (37, 38). To assess whether
the above MAPK pathways were activated by GXM, PBMC (data not shown)
and RAW 264.7 cells were exposed to 250 µg/ml of strain 6 GXM for 10,
20, 40, and 60 min. As a positive control, the cells were likewise
stimulated with 100 ng/ml of LPS. Activation of the MAPK pathways was
measured by Western blot using Abs reactive to phosphorylated ERK1/2,
p38, and SAPK/JNK. LPS activated all three MAPK pathways, peaking by 20
min and falling off at 60 min (Fig. 6).
In contrast, GXM failed to stimulate phosphorylation of any of the
MAPKs. Simultaneous costimulation of PBMC and RAW 264.7 cells with 250
µg/ml of strain 6 GXM and 100 ng/ml of LPS did not abrogate
LPS-induced MAPK pathway activation, suggesting that GXM does not
deactivate stimulated pathways. These data suggest failure to stimulate
MAPK pathways as a possible mechanism for the lack of TNF- release
by GXM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation provides
precedence for fungal stimulation of cells via TLR (18).
However, our studies are the first to demonstrate a role for mammalian
TLR in interactions with products derived from a fungus of
unquestionable pathogenicity. Interestingly, in the fruit fly
Drosophila melanogaster, the TLR homologue Toll
mediates antifungal responses by inducing genes that encode for the
antifungal peptide drosomycin. This interaction occurs by way of
Rel-Cactus complexes, which are functionally and structurally
equivalent to vertebrate NF-B/I-B complexes
(40). The conjugation of fluorescein to GXM was performed at an alkaline pH. It is possible that these conditions resulted in loss of O-acetyl groups on the polysaccharide. However, it is unlikely that this substantially altered the binding characteristics of the labeled GXM as unconjugated native GXM was able to displace its binding to transfected CHO cells.
Nuclear translocation of NF-B, as measured by reporter gene
expression, was stimulated by GXM only in CHO cells heterologously
expressing both CD14 and TLR4. NF-B activation in response to GXM
did not occur in CHO cells expressing CD14 and TLR2 despite our finding
that those cells effectively bound GXM. Thus, receptor occupancy by GXM
does not necessarily lead to a signaling response. In addition to
stimulating nuclear translocation of NF-B in CHO/CD14/TLR4/ELAM.tac,
GXM activated NF-B in human PBMC and the murine macrophage cell line
RAW 264.7. This is consistent with the finding that mononuclear
phagocytes express both CD14 and TLR4 (16). The failure of
CHO/CD14 cells, which express endogenous TLR4, to react to stimulation
with GXM may be due to the inability of hamster TLR4 to respond to this
ligand. Recently, it has been reported that human and hamster TLR4
mediate disparate, species-specific responses to identical ligands
(34). Furthermore, it is possible that GXM stimulation of
transfected CHO cells, but not mononuclear phagocytes, is dependent
upon overexpression of TLR4.
Our data demonstrating the capacity of GXM to stimulate nuclear
translocation of NF-B provide a putative mechanism for the
observation that capsular polysaccharide enhances HIV-1 infection
(41, 42). NF-B binding sites are present in the HIV
long terminal repeat promoter, and as a consequence, stimuli that
induce nuclear translocation of NF-B generally also induce HIV
replication (43, 44).
Cytokine dysregulation may contribute to the capacity of GXM to act as
a virulence factor. GXM has been reported to stimulate IL-6 and IL-10
production by monocytes and to inhibit LPS-induced secretion of
cytokines (45, 46). Vecchiarelli et al. reported that GXM
induces IL-8 secretion by human PMN and that the effect was due to
complement activation with generation of C3a and C5a (47).
Whereas opsonized C. neoformans stimulates cells to produce
TNF- (22, 48), this response was not seen with either
GXM or unopsonized organisms. In contrast to our finding, two
laboratories have reported that GXM induced small amounts of TNF-
release from human PBMC, but only in the presence of
complement-sufficient PHS (49, 50). The reasons for this
disparity are unclear, although the possibility remains that some of
the GXM preparations used might have been degraded and/or contaminated
by galactoxylomannan, mannoprotein, -glucans, and/or bacterial
endotoxin. Using sensitive techniques, we demonstrated that the GXM
preparations utilized in our studies were free of significant amounts
of these contaminants and migrated as a single high molecular mass
band.
NF-B nuclear translocation is thought to be necessary, but not
sufficient, for TNF- production. Our finding that GXM stimulated
NF-B without release of TNF- prompted us to examine whether GXM
stimulated other pathways thought necessary for optimal TNF-
release, notably the MAPK cascades (15, 38, 51). None of
three MAPK pathways were activated by GXM, suggesting a mechanism to
explain the lack of TNF-. At present, significant gaps remain in our
understanding of how signaling via TLR results in activation of MAPK
and NF-B. The adapter protein MyD88 acts to transduce signals in
response to activation of CD14/TLR complexes. The TLR signaling pathway
that leads to SAP/JNK and NF-B activation appears to diverge
downstream of MyD88 and upstream of TRAF6 (52).
Nevertheless, MyD88 knockout mice activate MAPK and NF-B in response
to LPS, although activation does not lead to TNF- production
(53). Our experiments indicate that GXM activates NF-B,
but not the MAPK cascades in mononuclear phagocytes. Moreover, we found
that GXM does not inhibit MAPK pathways or TNF- production induced
by LPS. The mechanisms responsible for this are unclear at the present
time. It could be postulated that GXM and LPS interact with various
components of the TLR/CD14 complex in a fashion dissimilar enough to
yield divergent downstream signals. Alternatively, internalization of
GXM may influence the activity of proteins distal to the site at which
NF-B and MAPK pathways separate.
Our studies utilized concentrations of GXM up to 250 µg/ml. The
molecular mass of GXM and LPS have been estimated to be approximately
1 x 106 and 1 x
104, respectively (4, 54). Due to
the very high molecular mass of GXM, on a molar basis, LPS and GXM did
not vary greatly in their ability to activate NF-B. From a clinical
standpoint, in patients with AIDS and cryptococcosis, the fungal burden
tends to be very high, and GXM concentrations approaching 1 mg/ml may
be attained in the serum and cerebrospinal fluid (5). More
importantly though, in infected tissues such as the brain, even larger
concentrations of GXM are presumably found (6). Consistent
with our data, accumulation of GXM in vivo preferentially occurs in
macrophages (5), cells known to express CD14 and TLR4
(55, 56). In addition to the effects of shed GXM on cell
function, our data demonstrate that whole C. neoformans,
which presents a surface predominately consisting of GXM, also
stimulates NF-B. Like purified GXM, unopsonized encapsulated
C. neoformans does not stimulate PBMC to make TNF-
(22).
Taken together, our data suggest a novel scenario whereby GXM interacts
with CD14 and TLR4 to stimulate NF-B nuclear translocation without
MAPK activation or TNF- production. The clinical consequences of
these findings remain speculative. As discussed above, GXM is present
in high concentrations in patients with cryptococcosis and is the major
virulence factor of C. neoformans. Thus, the interaction
between GXM and CD14/TLR4 could exert an immunodysregulatory effect
deleterious for the host.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stuart M. Levitz, Room X626, Section of Infectious Diseases, Boston University School of Medicine, 650 Albany Street, Boston, MA 02118-2393.
3 Abbreviations used in this paper: GXM, glucuronoxylomannan; CHO, Chinese hamster ovary; CTAB, cetyltrimethylammonium bromide; DTAF, 4-(4,6-dichlorotriazinyl)aminofluorescein; ELAM, endothelial cell-leukocyte adhesion molecule; ERK, extracellular signal-regulated kinase; I-B, inhibitory protein that dissociates from NF-B; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PHS, pooled human serum; RPA, RNase protection assay; SAPK, stress-activated protein kinase; TLR, Toll-like receptor; TRAF6, TNFR-associated factor 6.
Received for publication September 26, 2000. Accepted for publication January 22, 2001.
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References |
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  L. Guillot, S. F. Carroll, R. Homer, and S. T. Qureshi Enhanced Innate Immune Responsiveness to Pulmonary Cryptococcus neoformans Infection Is Associated with Resistance to Progressive Infection Infect. Immun., October 1, 2008; 76(10): 4745 - 4756. [Abstract] [Full Text] [PDF]
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