Inhibition of Human Endothelial Cell Chemokine Production
by the Opportunistic Fungal Pathogen Cryptococcus
Neelufar Mozaffarian,* Arturo Casadevall,*†and Joan W. Berman2*‡
Cryptococcus neoformans is an encapsulated fungal pathogen commonly acquired by inhalation. Extrapulmonary dissemination
can lead to infection of the bloodstream and various organs, most commonly resulting in meningoencephalitis. However, infection
with C. neoformans is often characterized by a scant inflammatory response. The leukocyte response to infection depends in part
upon a gradient of chemotactic factors and adhesion molecules expressed by the host vascular endothelium, yet the inflammatory
response of human endothelial cells (EC) to C. neoformans has not been previously investigated. We found that incubation of
primary human EC with C. neoformans did not induce chemokine synthesis, and resulted in differential inhibition of cytokine-
induced IL-8, IFN-?-inducible protein-10, and monocyte chemoattractant protein-1. In contrast, C. neoformans had little effect on
EC surface expression of the leukocyte ligand, ICAM-1, as determined by flow cytometry. Modulation of chemokine production
was dependent on the chemokine under study, the inoculum of C. neoformans used, fungal viability, and cell-cell contact, but
independent of cryptococcal strain or encapsulation. These observations suggest a novel mechanism whereby C. neoformans can
affect EC function and interfere with the host inflammatory response. The Journal of Immunology, 2000, 165: 1541–1547.
lung, C. neoformans can disseminate and cause a fatal menin-
goencephalitis. Although the mechanisms of extrapulmonary
dissemination are poorly understood, there is considerable ev-
idence that C. neoformans invades the bloodstream. In one pro-
spective series, C. neoformans was the most common blood-
stream infection in febrile, HIV?adults (1). In animal models
of cryptococcosis, i.v. inoculation leads to dissemination and
intravascular granuloma formation (2), and C. neoformans is
detectable in the blood for prolonged periods of time (3). In
vitro studies have shown that C. neoformans adheres to and is
internalized by human endothelial cells (EC),3suggesting a po-
tential mechanism for entry into and exit from the vascular
compartment (4). Hence, there is both direct and circumstantial
evidence for contact between C. neoformans and endothelium
ryptococcus neoformans var. neoformans is a fungus
that is ubiquitous in the environment and infects hu-
mans via the respiratory tract. If not contained in the
in human infection, although the contribution of this interaction
to the pathogenesis of infection is not known.
It has been reported that cryptococcal infections often elicit little
or no inflammation (5). The phenomenon is not well understood,
but is generally assumed to be the result of fungal-induced immune
suppression. Vascular EC express chemotactic cytokines (chemo-
kines) and leukocyte adhesion molecules that mediate leukocyte
activation, migration, and diapedesis (6). Many infectious organ-
isms induce expression of chemokines and adhesion molecules in
human EC, including Candida albicans (7), Staphylococcus au-
reus (8, 9), Trypanosoma cruzi (10), Listeria monocytogenes (11),
dengue virus (12), Helicobacter pylori (13), CMV (14), Borrelia
burgdorferi (15), Rickettsia conorii (16), and Chlamydia pneu-
moniae (17, 18). Given the central role of EC in mediating inflam-
matory responses and the fact that C. neoformans is often found in
the vascular space, fungal-induced EC dysfunction may contribute
to the inadequate host response commonly associated with dissem-
Therefore, we investigated the effects of C. neoformans on the
expression of IL-8, IFN-?-inducible protein-10 (IP-10), monocyte
chemoattractant protein-1 (MCP-1), and the leukocyte ligand
ICAM-1, in primary HUVEC in the presence and absence of proin-
flammatory cytokines. IL-8 belongs to the CXC family of chemo-
kines and is the prototypic neutrophil chemoattractant (19, 20), but
also activates monocytes for firm adhesion to EC (21). IP-10
differs from other CXC chemokine family members in that it is
chemotactic for activated T cells (22, 23). MCP-1 is a well-char-
acterized member of the CC chemokine family, and is chemo-
tactic for monocytes and activated T cells (24). Remarkably,
we found that C. neoformans failed to induce chemokine or
adhesion molecule expression in resting EC, and differentially
inhibited chemokine production in cytokine-stimulated EC. Our
inflammatory signaling in human EC, and suggest that C. neo-
formans may alter leukocyte activation and trafficking in the
*Department of Microbiology and Immunology, Division of Infectious Diseases,
†Department of Medicine, and‡Department of Pathology, Albert Einstein College of
Medicine, Bronx, NY 10461
Received for publication January 11, 2000. Accepted for publication May 16, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1N.M. is supported in part by the Department of Pathology, Albert Einstein College
of Medicine, Yeshiva University. A.C. is supported in part by National Institutes of
Health Grants AI-33774, AI-13142, and HL-59842. J.W.B. is supported in part by
National Institutes of Health Grant PO1 NS 11920 and National Institute of Mental
Health Grant RO1 MH 52974. Data in this paper are from a thesis to be submitted by
N.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy,
Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Med-
icine, Yeshiva University.
2Address correspondence and reprint requests to Dr. Joan W. Berman, Department of
Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
NY 10461. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: EC, endothelial cell; DAPI, 4?,6?-diamidino-2-
phenylindole; GXM, glucuronoxylomannan; IP-10, IFN-?-inducible protein-10;
LDH, lactate dehydrogenase; MCP, monocyte chemoattractant protein.
Copyright © 2000 by The American Association of Immunologists0022-1767/00/$02.00
Materials and Methods
Culture of EC
Primary human EC were isolated from umbilical cords, as previously
described (25). Briefly, umbilical veins were rinsed with sterile saline and
digested with 0.1% collagenase (Worthington Biochemical, Freehold, NJ).
EC were grown on gelatin-coated tissue culture plates (Falcon, Cock-
eysville, MD) at 37°C in a humidified chamber with 5% CO2. When con-
fluent, EC were passaged using trypsin (Life Technologies, Grand Island,
NY) digestion. For all experiments, EC were used at passages 3–4. EC
medium consisted of: M199 (Life Technologies) supplemented with 0.16%
bicarbonate, 11.1 mM HEPES (Calbiochem-Behring, La Jolla, CA), 1.6
mM L-glutamine (Life Technologies), 50 ?g/ml ascorbate (Fisher, Fair-
lawn, NJ), 25 ?g/ml heparin (Sigma, St. Louis, MO), 7.5 ?g/ml endothelial
cell growth factor (Sigma), 2.78 ?l/ml bovine brain extract (Clonetics, San
Diego, CA), 0.05 U/ml penicillin with 0.05 ?g/ml streptomycin (Life
Technologies), 20% newborn calf serum (Life Technologies), and 5% heat-
inactivated human serum (Biocell Laboratories, Rancho Dominguez, CA).
Culture of C. neoformans
Two encapsulated strains of C. neoformans (B-3501 and SB4) and one
acapsular strain (Cap 67) were used in this study. C. neoformans B-3501
(ATCC 34873; American Type Culture Collection, Manassas, VA) is a
serotype D strain that was chosen because it is the parent strain for the
acapsular mutant Cap 67 (ATCC 52817) (26), which has been shown to be
complemented to the encapsulated strain by a single gene (27). Serotype D
strains are pathogenic and are common clinical isolates in Europe (28–30).
The serotype A strain SB4 was chosen because it is a recent clinical isolate
that has been extensively studied (31–33), and represents the most common
serotype in clinical infection worldwide (34). Flasks of Sabouraud’s agar
broth (Difco Laboratories, Detroit, MI) were inoculated in sterile fashion
from frozen stocks (stored at ?70°C), and grown to late stationary phase
in a rotating shaker at 30°C for 3 days. To minimize endotoxin contami-
nation, all work was conducted in a biohazard hood. Fungal cells were
washed three times in sterile PBS, counted using a hemacytometer, and
diluted to the desired concentration in fresh EC medium. For experiments
requiring dead fungi, organisms were grown as above, pelleted, and resus-
pended in sterile PBS. One-half of this aliquot was subjected to autoclav-
ing, while the other half remained at room temperature. All samples were
then washed twice in PBS, counted, diluted in EC medium, and used in
parallel for treatment of EC monolayers. For other experiments, prepara-
tion of C. neoformans was conducted in an identical fashion, except that
fungal cultures were killed by exposure to 24 ?joules of UV light
(Stratalinker; Stratagene, La Jolla, CA) in place of autoclaving. Aliquots of
fungal cultures were streaked on plates of Sabouraud’s dextrose agar
(Difco Laboratories) to check for viability and to confirm colony
Treatment of EC for chemokine protein production
Confluent monolayers of EC were treated with cytokines and/or C. neo-
formans in a final volume of 0.6 ml/well in 24-well tissue culture plates
(Falcon). Human rTNF-? (R&D Systems, Minneapolis, MN) was used at
40 U/ml, human rIFN-? (Genzyme, Cambridge, MA) at 100 U/ml, and
human rIL-1? (National Cancer Institute, Frederick, MD) at 3 U/ml. In
some experiments, polyethylene cell culture inserts (Falcon) were used to
prevent contact between EC and fungi. These inserts allowed cell super-
natant to flow freely, but the 0.4 ?m pore size was too small to allow
passage of fungi. At the end of each time interval, EC supernatants were
separated from cells by centrifugation, transferred to new tubes, and stored
at ?20°C until assayed.
Determination of chemokine protein levels
Chemokine levels in EC supernatants were measured by ELISAs devel-
oped using flat-bottom 96-well plates (Costar, Corning, Corning, NY) and
paired anti-chemokine Abs, as directed by the manufacturer (R&D Sys-
tems). Plates were coated with capture Ab overnight and blocked with 1%
BSA/PBS. Samples were incubated overnight and followed by biotinylated
detection Ab and avidin-conjugated alkaline phosphatase. Wells were de-
veloped using tetramethylbenzidine peroxidase substrate (Kirkegaard &
Perry Laboratories, Gaithersburg, MD), and the reaction stopped with 1 M
phosphoric acid (Sigma). Absorbance was measured at 450 nm, and the
concentration of chemokine in each sample was calculated from a standard
curve generated using known amounts of recombinant human chemokine
(R&D Systems). Each sample was tested in duplicate, and results were
averaged to obtain the final concentration (ng/ml) of chemokine. The limit
of detection for these ELISAs is 15 pg/ml.
Flow-cytometric analysis of EC
EC cultures in six-well plates (Falcon) were treated with C. neoformans
and/or TNF-? for the times indicated. EC were rinsed, removed from plates
with 0.5 mM EDTA/PBS, and fixed using cold 2% formaldehyde/PBS.
Cells were stained with 2 ?g/ml of murine mAb to ICAM-1 (IgG1 anti-
human CD54; Dako, Carpenteria, CA) or nonspecific mouse IgG1 my-
eloma (ICN/Cappel, Aurora, OH) in 1% BSA/PBS. FITC-conjugated goat
anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL) was
used as the secondary Ab in 5% nonspecific goat serum/PBS. EC staining
was evaluated by flow cytometry using Lysis II software (FACScan; Bec-
ton Dickinson, Mountain View, CA).
Localization of endothelial NF-?B
For visualization of NF-?B translocation, EC were treated in 24-well plates
(Falcon) for 30 min, rinsed, and fixed in 100% cold methanol. Monolayers
were incubated with 1% BSA/PBS to block nonspecific Ab binding. EC
were incubated with rabbit Ab to NF-?B (polyclonal IgG anti-human Rel
A/p65; Santa Cruz Biotechnology, Santa Cruz, CA) or nonspecific rabbit
IgG (Southern Biotechnology Associates) in 1% BSA/PBS. Wells were
washed and incubated with biotinylated goat anti-rabbit Ig (Vector Labo-
ratories, Burlingame, CA) in 1% nonspecific goat serum/PBS (Vector Lab-
oratories), and followed by avidin-conjugated cy3 (Sigma). Nuclei were
counterstained with DAPI (5 ?g/ml in PBS; Molecular Probes, Eugene,
OR) before visualization under a fluorescence microscope (Olympus IX70,
At the end of some experiments, EC monolayers were gently rinsed to
remove unattached/extracellular fungi and examined by light microscopy
for confluence, morphology, and trypan blue exclusion. EC damage or
death after exposure to C. neoformans was measured by assessing the
release of lactate dehydrogenase (LDH). Cell-free EC supernatants were
incubated with a chromogenic substrate (Promega, Madison, WI) in du-
plicate wells of a 96-well plate (Falcon) for 30 min at room temperature.
After the addition of 1 M acetic acid, absorbance was measured at 492 nm.
Results were compared with a standard curve generated using an LDH-
positive control made from lysed L929 fibroblasts (Promega) and are re-
ported as arbitrary units.
To examine the effect of varying cryptococcal inocula, statistical analyses
were conducted using one-way ANOVA, followed by Bonferroni correc-
tion (Primer of Biostatistics; McGraw-Hill, New York, NY). To examine
the effect of C. neoformans on EC chemokine production under various
conditions, data were analyzed using Wilcoxon Signed Rank (StatView;
Abacus Concepts, Berkeley, CA). For all other comparisons, paired, two-
tailed t test was used (Excel, Redmond, WA). For all tests, significance was
assigned in which p ? 0.05. To standardize for the different baseline levels
of chemokine production across EC obtained from different donors, values
in some experiments were normalized to the mean values obtained in the
absence of C. neoformans, and are represented as the pooled means ?
SEM. Normalization of protein concentrations revealed that relative
changes in EC chemokine production in response to C. neoformans re-
mained remarkably constant.
C. neoformans fails to induce chemokine production in human
EC and inhibits cytokine-induced chemokine expression
To determine whether C. neoformans affects EC chemokine pro-
duction, we measured protein levels of IL-8, IP-10, and MCP-1 in
EC supernatants at times ranging from 6 to 48 h. EC supernatants
were also assayed for LDH activity to monitor cell damage/death.
Treatment of EC with cytokines induced chemokine synthesis, as
evidenced by accumulation of IL-8, IP-10, and MCP-1 in super-
natants over time (Fig. 1). Surprisingly, C. neoformans strain
B-3501 (2.4 ? 107cells/well, an E:T ratio of 250:1) did not induce
EC chemokine production, either with or without cytokines. Be-
cause chemokine levels were followed for 48 h, it is unlikely that
there was merely a delay in induction of chemokines by C. neo-
formans. In fact, C. neoformans inhibited TNF-?- and IFN-?-in-
duced EC chemokine production (Fig. 1), without significantly in-
creasing LDH levels. C. neoformans inhibition of EC chemokine
C. neoformans INHIBITS ENDOTHELIAL CELL CHEMOKINE PRODUCTION
production was evident by 24 h, and persisted through the 48-h
study period (Fig. 1). Because inhibition of chemokine production
was optimally observed at the later time point, the following ex-
periments were conducted for 48 h, unless otherwise indicated.
Inhibition of chemokine expression is dependent upon the
inoculum of C. neoformans
To determine whether the fungal inhibition of chemokine produc-
tion was inoculum dependent, EC were treated with TNF-? and
various inocula of C. neoformans B-3501 (ranging from 9.6 ?
106/well to 9.6 ? 104/well, E:T ratios of 100:1 to 1:1). Significant
decreases in EC MCP-1 protein production were observed in the
presence of C. neoformans ?9.6 ? 105fungi/well (an E:T ratio of
10:1). In these cultures, LDH release was not significantly different
from control (Fig. 2), implying a noncytotoxic mechanism for re-
duction of MCP-1 levels. One inoculum of C. neoformans, which
was effective in significantly lowering MCP-1 expression (2.4 ?
106cells/well, an E:T ratio of 25:1), was selected for further study.
Cryptococci were incubated with confluent monolayers of EC
(with medium, TNF-?, IFN-?, or TNF-? plus IFN-?), and 48-h
supernatants were analyzed for chemokine expression by ELISA.
TNF-? induced IL-8 and MCP-1 and synergized with IFN-? for
IP-10 production. In EC treated with TNF-? ? IFN-?, C. neofor-
mans reduced protein levels ?30% for IL-8, 60% for IP-10, and
50% for MCP-1. Reduction of cytokine-induced chemokine pro-
tein by C. neoformans was statistically significant for all three
chemokines (Fig. 3). This inoculum was used for all of the fol-
lowing experiments, unless otherwise indicated.
Inhibition of EC chemokine production is independent of
To determine whether fungal inhibition of EC chemokine produc-
tion was strain dependent, we tested C. neoformans strain SB4
(serotype A). EC (n ? 2) were incubated with SB4 (at E:T ratios
of 25:1 and 0.25:1) with and without cytokines (TNF-?, IFN-?, or
TNF-? plus IFN-?). Supernatants were collected at 24 and 48 h
and analyzed for IL-8, IP-10, and MCP-1 by ELISA. We found
that SB4 inhibited EC chemokine production in a manner similar
to C. neoformans strain B-3501, and did not significantly augment
LDH release. As with B-3501, inhibition was optimally observed
at 48 h, and was dependent upon the inoculum of C. neoformans
used (data not shown).
and MCP-1. Primary human EC were treated with or without cytokines in
the absence (?) or in the presence (?) of C. neoformans (Cn), and su-
pernatants were analyzed for chemokine production at 6, 24, and 48 h.
Treatment of EC with C. neoformans did not induce chemokine produc-
tion, and inhibited cytokine-induced production of all three chemokines,
without significantly increasing the levels of LDH in these supernatants
(p ? 0.34 (TNF vs TNF ? Cn) and p ? 0.39 (IFN vs IFN ? Cn)).
Chemokine values were normalized to the mean protein values obtained in
the absence of C. neoformans, and represent the pooled means ? SEM
(n ? 3, except at 6 h, where n ? 1).
Effects of C. neoformans on EC production of IL-8, IP-10,
LDH release. EC were treated with TNF-? and varying inocula of C. neo-
formans (Cn) (ranging from 0 to 9.6 ? 106fungi/well), and supernatants
were analyzed for MCP-1 protein and LDH activity after 48 h. C. neofor-
mans inhibited production of MCP-1 from TNF-?-treated EC in an inoc-
ulum-dependent fashion, but did not significantly alter LDH release. Data
were analyzed using one-way ANOVA followed by Bonferroni correction.
Significant decreases in MCP-1 production were observed at Cn:EC ratios
?10:1 (?, represents p ? 0.05). LDH activity was not statistically different
between EC with and without C. neoformans. Values shown represent the
pooled means ? SEM, and were normalized to the mean values of protein
and LDH activity obtained in the absence of C. neoformans (n ? 2).
Influence of C. neoformans inoculum on EC MCP-1 and
duction by C. neoformans. EC were treated with or without cytokines
and/or C. neoformans (Cn) for 48 h, and EC supernatants were analyzed for
chemokine production. Cytokine treatment increased EC synthesis of IL-8,
IP-10, and MCP-1 proteins. Incubation of EC with C. neoformans resulted
in statistically significant reductions in the levels of all three chemokines.
In EC treated with TNF-? ? IFN-?, inhibition levels were ?30% for IL-8,
60% for IP-10, and 50% for MCP-1. Values were normalized to the mean
protein values (ng/ml) obtained in the absence of C. neoformans and rep-
resent the pooled means ? SEM (n ? 7–12). Statistical analysis was per-
formed using Wilcoxon Signed Rank on data values before normalization
(?, represents p ? 0.05; ??, represents p ? 0.005).
Differential inhibition of cytokine-induced chemokine pro-
1543The Journal of Immunology
Inhibition of EC chemokine production is independent of fungal
encapsulation, but is dependent upon cell-cell contact and
Encapsulation of C. neoformans is important for virulence in ro-
dent models of infection. The cryptococcal capsule is composed
predominantly (80–90%) of glucuronoxylomannan (GXM), and
several acapsular mutants lack the ability to produce this polysac-
charide (26). To determine whether capsular GXM is required for
modulation of chemokine production, C. neoformans strain
B-3501 was compared with its acapsular variant, Cap 67.
C. neoformans B-3501 was used to treat confluent monolayers
of EC (in the presence of medium, TNF-?, IFN-?, or TNF-? plus
IFN-?), and 48-h supernatants were analyzed for chemokine ex-
pression by ELISA. C. neoformans did not induce MCP-1, but did
diminish cytokine-induced chemokine production. The effects of
Cap 67 on EC chemokine production were similar to those of
B-3501 (Fig. 4A), suggesting that a fungal component other than
GXM was responsible for inhibition of proinflammatory signaling
in EC. Acapsular or encapsulated C. neoformans that were phys-
ically separated from EC (Fig. 4B), heat killed (Fig. 4C), or UV
irradiated (Fig. 4D) did not inhibit MCP-1 production. These data
suggest that cell-cell contact between EC and metabolically active
fungi or a heat/UV-labile surface molecule were required.
Inhibition of cytokine-induced chemokine production by C.
neoformans is not specific to TNF-?
To establish whether the inhibitory effect of C. neoformans was
cytokine specific, EC cultures were treated with IL-1?, with and
without IFN-? and/or C. neoformans strain B-3501. Like TNF-?,
IL-1? induced IL-8 and MCP-1 protein synthesis and synergized
with IFN-? for IP-10 production (data not shown). C. neoformans
down-modulated IL-1?-induced EC chemokine expression in a
fashion comparable with that observed in the presence of TNF-?,
and this inhibition was reversed by the use of cell culture inserts or
by prior UV killing of the yeasts (data not shown). Baseline ex-
pression of MCP-1 by untreated EC was also inhibited by C. neo-
formans (Figs. 1, 3, and 4).
EC expression of ICAM-1 is not altered in the presence of C.
To determine whether the inhibitory effect of C. neoformans was
specific for chemokine production, we examined EC expression of
the leukocyte adhesion molecule ICAM-1 (CD54). ICAM-1 is a
member of the Ig gene superfamily that mediates firm adhesion of
activated leukocytes to EC before extravasation (35). Untreated
EC constitutively express low levels of surface ICAM-1, which is
up-regulated by proinflammatory cytokines such as TNF-?, and
peaks after ?12 h of treatment (36). EC were treated with C. neo-
formans B-3501 in the presence and absence of TNF-? for 12 h,
and examined for ICAM-1 staining by flow cytometry. Untreated
EC expressed ICAM-1, which was increased by treatment with
TNF-? and was not altered by C. neoformans (Fig. 5). Comparison
of the geometric mean fluorescence intensities using paired, two-
tailed, t tests confirmed that there were no significant differences
between EC with and without C. neoformans (n ? 3, data not
Treatment of EC with C. neoformans does not cause nuclear
translocation of NF-?B
Nuclear translocation of NF-?B is involved in the transcription of
many proinflammatory genes in EC, including adhesion molecules
and chemokines (37, 38). Activation of this pathway is rapid, and
NF-?B movement to the nucleus can often be detected within min-
utes after treatment of EC. To determine whether C. neoformans
activated translocation of NF-?B, EC were incubated with me-
dium, TNF-?, or C. neoformans for 30 min, and stained for NF-?B
p65. Immunofluorescence revealed that cells treated with C. neo-
formans or medium had prominent cytoplasmic staining, with little
nuclear staining, suggesting a lack of NF-?B translocation (Fig. 6).
In contrast, TNF-?-treated EC exhibited intense nuclear staining
for p65 (Fig. 6). EC treated with TNF-? plus C. neoformans also
showed nuclear staining. However, we were not able to determine
whether this nuclear staining was significantly different from that
seen in EC treated with TNF-? alone due to the qualitative nature
of this assay (data not shown).
Because some stimuli require longer incubation times to effect
nuclear translocation of NF-?B (12), EC were also examined after
24 h of treatment. These EC had NF-?B staining patterns similar
to those obtained after 30 min of treatment (data not shown).
EC viability is not reduced by C. neoformans
At the end of some experiments, we examined EC for viability
after the prolonged contact with C. neoformans. EC were rinsed to
viability on EC MCP-1 production. EC were treated with encapsulated
(B-3501) or acapsular (Cap 67) C. neoformans (Cn) for 48 h in the pres-
ence and absence of cytokines. The effect of live fungal cultures was com-
pared with that using C. neoformans separated from EC by a 0.4-?m mem-
brane, and heat- or UV-killed C. neoformans. EC production of MCP-1
was reduced by live C. neoformans (A), but not by C. neoformans that had
been physically separated from EC (B), autoclaved (C), or UV irradiated
(D). Values were normalized to the mean protein values obtained in the
absence of C. neoformans, and represent the pooled means ? SEM (A, n ?
7–9; B, n ? 2–6; C, n ? 2–4; and D, n ? 4 in the absence of TNF, n ?
2 in the presence of TNF).
Effects of cryptococcal capsule, cell-cell contact, and fungal
C. neoformans INHIBITS ENDOTHELIAL CELL CHEMOKINE PRODUCTION
remove unattached/extracellular fungi and examined by light mi-
croscopy. EC cultures maintained confluence and retained a nor-
mal cobblestone appearance. Several EC in C. neoformans-treated
wells were in contact with the yeasts and/or contained intracellular
organisms. Trypan blue was excluded from ?99% of EC in every
well, regardless of treatment. DAPI staining revealed nonapoptotic
nuclei (Fig. 6), and analysis of EC supernatants revealed no in-
crease in LDH release over controls. In agreement with our find-
ings, EC cultured in 24-well plates and exposed to encapsulated
C. neoformans (107fungi/well) showed no signs of cell damage or
death after 8 h, as measured by51Cr release (4).
C. neoformans is a fungus that can cause life-threatening disease in
immunocompromised hosts. The mechanisms of fungal dissemi-
nation are not well understood, but dissemination is believed to be
inversely correlated with the host leukocyte response (32, 39, 40).
Incubation of cryptococci with EC in our experiments did not lead
to expression of IL-8, IP-10, MCP-1, lymphotactin, RANTES,
MIP-1?, MIP-1?, or I-309. In fact, C. neoformans inhibited cyto-
kine-induced chemokine production from human EC. Inhibition of
chemokine production occurred whether EC were treated with
TNF-?, IL-1?, IFN-?, or combinations of these cytokines, indi-
cating a general down-modulatory effect on cytokine signaling.
C. neoformans caused greater reductions in EC expression of the
mononuclear cell chemoattractants, IP-10 and MCP-1, than in the
neutrophil chemoattractant IL-8. These data demonstrate that
C. neoformans is capable of differential inhibition of CXC and CC
chemokine synthesis from human EC, consistent with different sig-
naling pathways for induction of these chemokines. The fact that
C. neoformans only minimally reduces EC production of IL-8 is
intriguing, as elevated IL-8 levels have been documented in the
CSF of HIV-1?patients with cryptococcal meningitis (41),
and pulmonary cryptococcal infection in mice induces expres-
sion of neutrophil chemoattractants, but fails to induce MCP-1
or IP-10 (42).
Inhibition of cellular inflammatory mediators has been previ-
ously reported for C. neoformans. In vitro, C. neoformans down-
modulated production of IL-12 by a murine macrophage cell line
(43), NO by murine peritoneal macrophages (44), and TNF-? and
GM-CSF by human NK cells (45). Cryptococcal inhibition of
these factors was mediated by reduced transcription of these genes,
was dependent on direct contact between the fungus and the leu-
kocytes, and was not due to fungal killing of these leukocytes
(43–45). In addition, reduction of macrophage NO was not medi-
ated by cryptococcal capsular GXM, as two acapsular strains of
C. neoformans also inhibited nitrite production (44).
ICAM-1 is a leukocyte adhesion molecule constitutively ex-
pressed by resting EC, and is up-regulated within hours after treat-
ment with inflammatory stimuli (9, 11, 18, 46, 47). Binding to
ICAM-1 allows for firm adhesion of leukocytes to the vessel wall,
three donors were treated for 12 h with TNF-? and/or C. neoformans and
analyzed for ICAM-1 staining by flow cytometry. Representative histo-
grams from one set of EC are shown. A, Untreated EC expressed low levels
of ICAM-1 (anti-ICAM-1, shaded; control IgG1, open). B, Treatment of
EC with C. neoformans had no effect on ICAM-1 staining (untreated, shad-
ed; C. neoformans treated, open). C, TNF-? up-regulated the expression of
ICAM-1 as compared with untreated EC (untreated, shaded; TNF-?
treated, open). D, Treatment of EC with C. neoformans had no effect on
cytokine-induced ICAM-1 staining (TNF-? treated, shaded; C. neoformans
Effect of C. neoformans on ICAM-1 expression. EC from
immunofluorescence. Untreated and C. neoformans-treated EC showed NF-?B reactivity located mainly in the cytoplasm, while in TNF-?-treated EC,
NF-?B staining was predominantly nuclear. No reactivity was observed in EC stained with control rabbit IgG. Nuclei were counterstained with DAPI and
exhibit normal morphology (original magnification, ?200).
Visualization of NF-?B. EC were treated for 30 min with medium, TNF-?, or C. neoformans, and NF-?B localization was determined by
1545 The Journal of Immunology
and is an important step before leukocyte extravasation (36, 48).
We found that incubation of EC with C. neoformans did not induce
ICAM-1 expression, and did not significantly alter baseline or
TNF-?-induced ICAM-1 levels. However, C. neoformans-induced
down-modulation of chemokine synthesis may affect the ability of
leukocytes to adhere to adhesion molecules, as binding is triggered
by chemoattractant-dependent activation of integrin counterrecep-
tors on the leukocyte surface (49–51).
Activation of NF-?B signaling in EC and subsequent adhesion
molecule and chemokine expression has been documented for
S. aureus (our unpublished observations), C. albicans (7), T. cruzi
(10), L. monocytogenes (11), B. burgdorferi (15), dengue virus
(12), and respiratory syncytial virus (38), and may represent a
common final pathway in the EC response to infectious agents.
The failure of C. neoformans to induce nuclear translocation of
NF-?B is consistent with the finding that C. neoformans does not
induce expression of ICAM-1 or chemokines in human EC.
The conditions under which C. neoformans inhibited EC che-
mokine synthesis suggest that cell-bound and/or highly labile cryp-
tococcal/EC products are involved in this process, such as those
documented for other pathogens with viability- and contact-depen-
dent effects on mammalian cells (52). Schistosomula of Schisto-
soma mansoni produce a lipophilic substance that interferes with
NF-?B activity in EC, most likely via the activation of the cAMP/
protein kinase A pathway (53). Treatment of human EC with
cAMP-elevating agents did not affect TNF-?-induced expression
of ICAM-1 (54), but did reduce EC chemokine synthesis (55).
Therefore, the study of lipids and/or cAMP signaling in EC may be
a promising avenue of investigation for understanding the immu-
nomodulatory effects of C. neoformans.
In summary, our results indicate that C. neoformans fails to
activate human EC for chemokine and ICAM-1 production and
suppresses cytokine-induced chemokine synthesis, with greater in-
hibition of the mononuclear cell chemoattractants IP-10 and
MCP-1, than the neutrophil chemoattractant, IL-8. These effects
require fungal viability and cell-cell contact, and suggest a general
down-modulatory effect of C. neoformans on EC activation. Re-
duced chemokine production could interfere with the ability of the
host to mount an adequate inflammatory response at sites of cryp-
tococcal infection. Future work will focus on the investigation of
the signaling pathways responsible for the effects that we have
We thank Dr. Tina Calderon for critical reading of this manuscript.
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1547The Journal of Immunology