Previous Article | Next Article 
Infection and Immunity, September 1999, p. 4620-4627, Vol. 67, No. 9
Department of Microbiology and Infectious
Diseases1 and Department of Internal
Medicine,3 University of Calgary, Calgary,
Alberta, Canada T2N 4N1, and Department of Microbiology,
University of Nevada, Reno, Nevada 595572
Received 28 December 1998/Returned for modification 27 January
1999/Accepted 3 June 1999
Cell-mediated immunity is critical for the host defense to
Cryptococcus neoformans, as demonstrated by numerous
animal studies and the prevalence of the infection in AIDS patients.
Previous studies have established that the polysaccharide capsule
contributes to the virulence of C. neoformans by
suppressing T-lymphocyte proliferation, which reflects the clonal
expansion of T lymphocytes that is a hallmark of cell-mediated
immunity. The present studies were performed to identify the major
mechanism by which polysaccharide impairs lymphocyte proliferation,
since capsular polysaccharide has the potential to affect the
development of T-lymphocyte responses by stimulating production
of interleukin-10 (IL-10), inhibiting phagocytosis, and inducing
shedding of cell surface receptors. We demonstrate that polysaccharide
inhibits lymphocyte proliferation predominantly by blocking uptake
of C. neoformans, which is crucial for subsequent
lymphocyte proliferation. In addition, we show that polysaccharide did
not suppress lymphocyte proliferation via an IL-10-dependent
mechanism, nor did it affect critical surface receptor interactions on
the T cell or antigen-presenting cell. Having established that
polysaccharide impairs phagocytosis, we performed studies to determine
whether opsonization with human serum or with anticapsular antibody
could reverse this effect. Impaired uptake and lymphocyte proliferation
that were induced by polysaccharide can be enhanced through
opsonization with monoclonal antibodies or human serum, suggesting that
antipolysaccharide antibodies might enhance the host defense by
restoring uptake of the organism and subsequent presentation to T
lymphocytes. These studies support the therapeutic potential of
stimulating cell-mediated immunity to C. neoformans with
anticapsular antibody.
Cryptococcus neoformans
is one of the leading fatal mycoses in AIDS (4, 13, 18).
Although cell-mediated immunity is of paramount importance in the host
defense to C. neoformans, the dominant mechanism by which
the major virulence factor, the polysaccharide capsule, might influence
the development of cell-mediated immunity and how this mechanism might
be overcome have not been determined.
We and others have established that capsular polysaccharide (CPS)
suppresses T-lymphocyte responses to both live and killed C. neoformans (5, 30, 33, 43). We have previously shown that strains of C. neoformans with a large capsule are less
able to stimulate proliferation of human lymphocytes than minimally encapsulated strains and that addition of purified CPS inhibits lymphocyte proliferation (33). CPS also impairs alveolar
macrophage-dependent T-cell responses to C. neoformans
(43). Since the clonal proliferation of T cells is a
hallmark of the cell-mediated immune response, we considered the
possibility that CPS-mediated suppression of lymphocyte proliferation
may be an important mechanism of virulence.
There are a number of mechanisms by which CPS could suppress the
development of the T-lymphocyte response. CPS can induce immunosuppressive cytokines such as interleukin-10 (IL-10)
(45), which suppresses lymphocyte proliferation by a number
of mechanisms (8, 12, 35, 40). CPS also causes shedding of
some cell surface receptors by an unknown mechanism (14).
Since antigen presentation is critically dependent on the expression of
costimulatory surface receptors, it is possible that CPS could
interfere with a critical receptor-ligand interaction between the
antigen-presenting cell and the T cell. CPS also inhibits uptake of the
organism by phagocytic cells (27, 32), which could inhibit
the antigen available for processing and presentation to the T cell.
Thus, there is the potential for CPS to suppress the antigen available for presentation by inhibiting the uptake of C. neoformans
by the antigen-presenting cell, by inhibiting cell-cell interactions necessary for costimulatory signals, or by stimulating production of
immunosuppressive IL-10.
By contrast to the role of cell-mediated immunity, the role of humoral
immunity has provided an arcanum in our understanding of cryptococcal
host defense. While administration of antibodies to CPS is protective
(37, 39), deficiencies of humoral immunity do not predispose
the host to cryptococcal infections. This suggests that natural humoral
mechanisms are unimportant in the host defense to C. neoformans but that administration of antibody, or vaccination with the development of antibodies, can augment mechanisms of host
defense. If CPS inhibits antigen presentation by inhibiting uptake of
C. neoformans, there is the potential to overcome this effect by opsonizing the organism with anticapsular antibody. In a
murine model, specific anticryptococcal antibodies that opsonize C. neoformans can augment cellular uptake (15, 36,
38), and antibodies to glucoronxylmannan conjugated to tetanus
toxoid promote phagocytosis of C. neoformans in the absence
of complement (51) and enhance survival via a CD4-dependent
mechanism (50). Thus, it is possible that specific
anticapsular antibody might enhance uptake and ultimately presentation
to T cells, resulting in activation, proliferation, and development of
cell-mediated immune responses that would provide an explanation for
the therapeutic efficacy of anticapsular antibodies.
To determine whether CPS suppresses lymphocyte proliferation by
production of IL-10, lymphocytes were stimulated with CPS-treated C. neoformans in the presence or absence of neutralizing
antibody to IL-10. To determine if CPS was affecting interactions
between antigen-presenting cells and T cells, CPS was added to the
peripheral blood mononuclear cells (PBMC) and the excess was removed
before stimulation with C. neoformans. To determine whether
the antiphagocytic properties of CPS contributed to a reduction in
lymphocyte proliferation, phagocytosis was correlated with
[3H]thymidine ([3H]TdR) incorporation.
Finally, the ability of complement or anticapsular antibody to
ameliorate the effect on lymphocyte proliferation was tested with
pooled human sera and anticapsular monoclonal antibodies (MAb).
Isolation of PBMC and selection of lymphocyte populations.
Human peripheral blood was obtained from healthy adults by
venipuncture. The blood was anticoagulated with 10 U of heparin (Organon Teknika-Cappel, Scarborough, Ontario, Canada) per ml. PBMC
were purified by centrifugation (800 × g for 20 min)
on a Ficoll-Hypaque density gradient (Lymphoprep; Labquip, Woodbridge, Ontario, Canada). PBMC were washed three times in Hanks balanced salt
solution (Gibco, Burlington, Ontario, Canada), counted, and suspended
in medium containing RPMI 1640 (Gibco); 5% heat-inactivated pooled
human AB serum (lot 7M1809; BioWhittaker, Walkersville, Md.); and 2 mM
L-glutamine, 100 U of penicillin/ml, 100 µg of streptomycin/ml, 0.2 µg of amphotericin B/ml, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids (all from Gibco).
Preparation of C. neoformans and CPS.
C.
neoformans 67 (ATCC 52817; acapsular mutant) (21), 68 (ATCC 24064; lightly encapsulated, serotype A) (47), 3501 (ATCC 34873; lightly encapsulated, serotype D), 613 (ATCC 36556;
lightly encapsulated, serotype D) (24), T145 (ATCC 62070;
moderately encapsulated, serotype A) (41), and 6 (ATCC
62066; heavily encapsulated, serotype A) (41) were obtained
from the American Type Culture Collection (Rockville, Md.). The
organisms were maintained as previously described (34) on
Sabouraud slants (Difco, Detroit, Mich.) and passaged to fresh slants
monthly. The organisms were killed as previously described
(33) by autoclaving at 121°C for 15 min and were stored at
4°C. CPS was obtained from strain 68, serotype A (ATCC 24064), as
previously described (22). All reagents were prepared in
endotoxin-free water (Baxter, Mississauga, Ontario, Canada), and
glassware was baked prior to use.
Polysaccharide coating and staining of C. neoformans.
Acapsular C. neoformans (strain 67) was incubated in
purified polysaccharide for 1 h at 37°C. Unbound polysaccharide
was removed by washing in phosphate-buffered saline (PBS). The
polysaccharide-coated C. neoformans was then used in
proliferation and phagocytosis studies. Mucicarmine (Sigma, St. Louis,
Mo.) staining and microscopic examination were used to determine
whether CPS had bound to the surface of C. neoformans.
Treatment of C. neoformans with antibody or
sera.
For some experiments, heat-killed C. neoformans
was incubated for 1 h at 37°C with undiluted
non-heat-inactivated human AB serum (lots 5M1937 and 7M1809;
BioWhittaker), heat-inactivated (56°C for 60 min) serum, or an
anticapsular MAb (MAb 471) that was purified as previously described
(16, 42). This MAb is a murine immunoglobulin G1 antibody
that binds to serotype A and D polysaccharide. The organisms were then
washed three times in PBS and used in proliferation or phagocytosis assays.
Lymphocyte proliferation in response to C. neoformans.
To determine whether C. neoformans stimulated lymphocyte
proliferation, PBMC (2 × 105 cells/well) were
cultured in round-bottom wells of 96-well tissue culture plates
(Corning Glass Works, Corning, N.Y.). Whole C. neoformans
cells (2 × 105/well) were used to stimulate the
lymphocytes. Cultures were incubated for 7 days at 37°C with 5%
CO2. Sixteen hours before the end of incubation, 1 µCi of
[3H]TdR (ICN, Montreal, Quebec, Canada) was added. Cells
were harvested on glass filters, and counts per minute were determined
in a liquid scintillation counter. [3H]TdR incorporation
into cultures containing C. neoformans alone was routinely
less than 300 cpm. As a control, PBMC were stimulated with 10 µg of
concanavalin A (Sigma) per ml or 10 ELISA for IL-10.
The concentration of IL-10 in culture
supernatants was determined by an enzyme-linked immunosorbent assay
(ELISA). The capture antibody was monoclonal anti-IL-10 (1 µg/ml)
(18551 D; Pharmingen, San Diego, Calif.) or JES3-19F (American Type
Culture Collection). The secondary antibody was a biotinylated
anti-IL-10 MAb (1.5 µg/ml) (18562 D; Pharmingen), followed by
avidin-peroxidase (Sigma). The ELISA was developed by adding
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma; A-1888)
in 0.1 M citric acid buffer with 1 µl of 30% hydrogen peroxide per
ml. The ELISA was read spectrophotometrically at 405 nm. All results
were the means from duplicate samples, and the standard curve was
generated by using IL-10 from the Biological Response Modifiers
Program, National Institutes of Health, Bethesda, Md.
Phagocytosis of C. neoformans.
PBMC were cultured in
24-well plates containing plastic 13-mm-diameter coverslips (Nunc,
Naperville, Illinois) at 37°C in RPMI medium. After 1 h, the
nonadherent cells were removed by washing, and C. neoformans
(106 organisms/well) was added to the wells. At various
times, medium and unbound Cryptococcus were removed by
washing with PBS. Coverslips were removed, fixed in methanol, stained
with Giemsa stain (ICN), and then examined by light microscopy for the
number of cells that had bound or ingested C. neoformans
(3). Studies determined that Giemsa staining was as reliable
as fluorescein isothiocyanate labeling of C. neoformans and
quenching of extracellular fluorescence with trypan blue.
Statistics.
Data are given as the mean ± standard
error of the mean (SEM) for the indicated number of experiments. Each
experiment was performed with different donors on different days.
[3H]TdR incorporation is expressed as the mean counts per
minute ± SEM for quadruplicate wells. To analyze the data
statistically, one-way analysis of variance was performed when allowed
by the F test (Statview 512+; Brainpower Inc., Calabasas,
Calif.). For experiments in which phagocytosis and lymphocyte
proliferation were determined, Wilcoxon-Mann-Whitney statistics were
used. In experiments comparing human serum to anti-CPS MAb, Friedman
two-way analysis of variance by ranks was performed. For these tests, a
P value of <0.05 was considered significant.
Inhibition of lymphocyte proliferation by CPS is independent of
IL-10.
Recombinant IL-10 can abrogate lymphocyte proliferation in
response to C. neoformans (35). Since CPS
stimulates production of IL-10 (8, 12), it was possible that
CPS could suppress lymphocyte proliferation via production of IL-10. To
determine whether the CPS induced sufficient IL-10 to influence
lymphocyte proliferation, PBMC were stimulated with C. neoformans in the presence or absence of purified polysaccharide
and anti-IL-10 or isotype-matched control antibody. The anti-IL-10 MAb
tended to augment lymphocyte proliferation but did not restore it to statistically significant levels (Fig.
1). To ensure that the CPS was capable of
stimulating IL-10 production, ELISA for IL-10 was performed on
CPS-stimulated supernatants. Modest concentrations of IL-10 were
detected (128 ± 40 pg/ml; n = 4), which were
greater than the concentration found in prior studies (45).
The anti-IL-10 antibody was active, since it enhanced C. neoformans-stimulated tumor necrosis factor alpha (TNF-
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Capsule of Cryptococcus neoformans Reduces
T-Lymphocyte Proliferation by Reducing Phagocytosis, Which Can Be
Restored with Anticapsular Antibody
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2 Leaf units of
tetanus toxoid (Connaught Laboratories, Mississauga, Ontario, Canada).
In some experiments, lidocaine (10 to 10,000 µM; Baxter) was added to
the culture wells. In other experiments, cells were incubated in the
presence of 100 to 1,000 ng of anti-IL-10 (Pharmingen) or
isotype-matched control antibody (Sigma) per ml.
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)
release. (The concentrations of TNF- were 4,742 pg/ml in
supernatants of PBMC that were stimulated with C. neoformans
plus polysaccharide plus control immunoglobulin G and 9,057 pg/ml in
supernatants of PBMC stimulated with C. neoformans plus
polysaccharide plus anti-IL-10.) This data suggests that IL-10 is not
the primary mechanism responsible for the CPS-induced lymphocyte
suppression.
View larger version (15K):
[in a new window]
FIG. 1.
Blocking of IL-10 does not restore lymphocyte
proliferation in response to acapsular C. neoformans 67 cultured in the presence of polysaccharide. PBMC and C. neoformans (Cn) were cultured in the presence or absence of
capsular polysaccharide (10 µg/ml) and anti-IL-10 or control antibody
(1 µg/ml). Lymphocyte proliferation was assessed 7 days later by
[3H]TdR incorporation. *, P < 0.05 by
analysis of variance. NS, not significantly different compared to
stimulated PBMC. 
, P < 0.05 compared to PBMC plus
C. neoformans. NS1, not significantly different
from PBMC plus C. neoformans plus CPS. The experiment was
repeated three times with similar results.
CPS suppresses lymphocyte proliferation by binding to C. neoformans rather than affecting interactions between antigen-presenting cells and T cells. CPS causes shedding of some cell surface receptors (14). Since receptor-ligand interactions are important in antigen presentation, the possibility that CPS interferes with a critical costimulatory signal was considered. PBMC were incubated with purified CPS, and the excess was removed by washing. The CPS-treated PBMC were stimulated with C. neoformans. Preincubation of PBMC with CPS had no effect on lymphocyte proliferation (Fig. 2). In parallel experiments, acapsular C. neoformans that had previously been incubated with purified polysaccharide and washed to remove the excess CPS was used to stimulate PBMC. Preincubation of C. neoformans with purified polysaccharide abrogated lymphocyte proliferation (Fig. 2). Mucicarmine staining confirmed that CPS was binding to C. neoformans (data not shown). Thus, the polysaccharide does not affect lymphocyte proliferation by affecting the interaction between antigen-presenting cells and T cells but rather exerts its effect by binding to the organism.
|
) were compared
to PBMC that were stimulated with C. neoformans (
); PBMC
that were stimulated with C. neoformans in the presence of
10 µg of CPS per ml
(
); PBMC
that were pretreated for 1 h at 37°C (Tx'd) with CPS, washed,
and stimulated with C. neoformans (
); or C. neoformans that was pretreated for 1 h at 37°C with CPS,
washed, and used to stimulate PBMC (
). After 7 days, lymphocyte
proliferation was determined by [3H]TdR incorporation.
*, P < 0.05 compared to unstimulated PBMC. NS, not
significantly different compared to C. neoformans-stimulated
PBMC. Blocking of phagocytosis inhibits lymphocyte proliferation in response to C. neoformans. One of the major effects of the polysaccharide capsule is to inhibit phagocytosis (25). To determine whether impaired phagocytosis might explain the reduced lymphocyte proliferation, the uptake of untreated acapsular C. neoformans was compared to that of CPS-coated C. neoformans and correlated with lymphocyte proliferation. Preincubation of C. neoformans with purified polysaccharide reduced the number of cells that had taken up by C. neoformans by 60 to 70% across a broad range of numbers of organisms (Fig. 3). The number of organisms that had been internalized was proportional to the number that had bound to cells (data not shown), and the number of cells that had internalized C. neoformans correlated with a reduction in lymphocyte proliferation (Fig. 3).
|
|
Opsonization of polysaccharide-treated C. neoformans overcomes suppression of lymphocyte proliferation. To determine if opsonization with human serum could neutralize the inhibition of lymphocyte proliferation in response to acapsular C. neoformans that had been coated with CPS, treated C. neoformans was incubated for 1 h in non-heat-inactivated human serum and then used to stimulate lymphocytes to proliferate. Human serum augmented lymphocyte proliferation in response to polysaccharide-coated C. neoformans (Fig. 5A). This correlated with an increase in the percentage of cells that had taken up CPS-treated C. neoformans, which went from 7.0% ± 3.5% of the cells in the absence of serum to 26.9% ± 7.5% of the cells in the presence of serum (n = 4 experiments).
|
).
CPS-treated organisms were incubated in human serum (HS) (A) or with 10 µg of MAb to polysaccharide per ml (B) (
). In panel A the
percentage of cells that had taken up CPS-treated C. neoformans increased from 8% of cells without serum to 29% of
cells with serum opsonization. In panel B the percentage of cells that
had taken up CPS-treated C. neoformans increased from 10%
of cells in the absence of antibody to 45% of cells in the presence of
antibody. *, P < 0.05 compared to PBMC plus C. neoformans. Opsonization of encapsulated C. neoformans enhances lymphocyte proliferation. Having established the effects of an opsonic antibody on acapsular organisms that had been coated with capsular polysaccharide, we performed experiments to compare the abilities of anti-CPS MAb and human serum to augment lymphocyte proliferation in response to four different encapsulated strains of C. neoformans. Preincubation in normal human serum or with anticryptococcal antibody increased lymphocyte proliferative responses to encapsulated strains of C. neoformans regardless of the serotype (Fig. 6), which correlated with increased association of C. neoformans with adherent PBMC (data not shown). For all but the most highly encapsulated strains tested, lymphocyte proliferation was greater when organisms were treated with anticapsular antibody than when they were treated with human serum. Thus, treatment with MAb to CPS was an effective way to augment lymphocyte proliferation.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
We have made three observations: (i) CPS did not inhibit lymphocyte proliferation by an IL-10-dependent mechanism or by directly affecting antigen-presenting or accessory cells; (ii) CPS suppressed phagocytosis, and this correlated with lymphocyte proliferation; and (iii) opsonization with an anticapsular MAb increased lymphocyte proliferation and phagocytosis and was more effective than opsonization with human serum.
One of the most important virulence factors of C. neoformans
is its polysaccharide capsule. CPS has a number of important effects on the immune response. It inhibits phagocytosis
(25) and induces the release of immunosuppressive cytokines
such as IL-10 (45). CPS can also inhibit production of
TNF- and IL-1
(44), which are important in the host
defense to C. neoformans (1, 2, 20). Further, CPS
can affect leukocyte infiltration in inflammatory responses by
causing shedding of L-selectin and TNF receptors (14). We
and others have previously shown that there is an inverse correlation
between the size of the capsule and lymphocyte proliferation (5,
33). Further, the addition of exogenous capsule inhibits
lymphocyte proliferation in response to an acapsular strain
(33). In this study, we found that the major mechanism by
which CPS suppressed lymphocyte proliferation was by inhibiting uptake
of the organisms that was necessary for presentation to lymphocytes,
rather than by inducing IL-10 or by directly suppressing the function
of antigen-presenting cells or T cells.
A previous study suggested that CPS-induced IL-10 production was an important mechanism in the CPS-mediated suppression of lymphocyte proliferation (40). However, three pieces of evidence indicate that CPS-induced IL-10 production was not responsible for suppression of lymphocyte proliferation in our studies. First, preincubation of responding cells with CPS failed to affect subsequent lymphocyte proliferation. If incubation of PBMC with CPS had stimulated the production of inhibitory concentrations of IL-10, it should have affected lymphocyte proliferation. We found no suppression when PBMC were incubated with CPS, suggesting that the effect was not due to IL-10. Second, previous studies have demonstrated that an encapsulated strain of C. neoformans does not suppress the response to an acapsular strain (33). If the encapsulated strain had been inducing IL-10 production that suppressed lymphocyte proliferation, the IL-10 produced in response to the encapsulated strain should have suppressed the proliferation in response to the acapsular strain. The fact that the encapsulated strain did not suppress lymphocyte proliferation in response to the acapsular strain suggests that IL-10 was not responsible for the effect. Finally, an anti-IL-10 antibody did not restore lymphocyte proliferation, suggesting that IL-10 is not the primary reason that CPS reduces proliferative responses to C. neoformans.
The reason for the discrepancy between our studies and the previous
studies is not apparent; however, the previous studies evaluated the
contribution of IL-10 in response to two nonisogenic strains of
C. neoformans (40), while in our studies, the
same strain was used to compare the responses with and without CPS. Thus, it may be that the effect is related to phenotypic differences of
the strains. We considered the possibility that the amount of
polysaccharide in our cultures overwhelmed the ability of anti-IL-10 to
neutralize the cytokine. This seems unlikely, since low levels of IL-10
are secreted in response to CPS, and anti-IL-10 antibody was able to
enhance CPS-induced TNF- release. Our data does agree with the
finding by Retini et al. (40) that CPS impairs phagocytosis and extends this observation to demonstrate that this is an important mechanism responsible for reduced lymphocyte proliferation.
We found that CPS affected lymphocyte proliferation by binding to C. neoformans. Binding of free polysaccharide to cryptococcal organisms has been well described (22). Incubation of nonencapsulated Cryptococcus with purified cryptococcal polysaccharide renders nonencapsulated cells resistant to phagocytosis (23, 25, 26). We found that CPS bound to the organisms, which limited uptake and resulted in a significant reduction in lymphocyte proliferation.
Knowing that diminished lymphocyte proliferation in the presence of CPS was due to decreased uptake of the organism, we were interested in determining whether opsonizing the organism might increase uptake and restore lymphocyte proliferation. Initially, we used human serum as a source of opsonins. Human serum with active complement can opsonize C. neoformans, while heat-inactivated serum does not (7). Cryptococcus is opsonized by C3 fragments, which bind to the capsule and opsonize cryptococci, increasing uptake (28, 29, 31). Highly encapsulated strains are more potent activators of complement than acapsular strains (48). We found that preincubation in complement-sufficient human serum enhanced uptake of polysaccharide-coated organisms and that this was associated with improved lymphocyte proliferation.
The role of natural antibody in the host defense to C. neoformans is controversial. Patients who are predisposed to cryptococcal infections have defects in cell-mediated immunity. By contrast, patients with isolated defects in antibody production do not have a meaningful increase in the incidence of cryptococcal infections. This has led to the assumption that T-cell-mediated immunity is important, while humoral immunity is not. However, there are numerous studies demonstrating that specific anticryptococcal antibody enhances granuloma formation (17) and is protective in murine models (9, 15, 19, 50). Since T cells are clearly important in cryptococcal host defense, we considered the possibility that protective antibody might somehow influence T-cell responses and enhance the host defense by this mechanism. This is supported by recent studies demonstrating that T cells can cooperate with administered antibody to induce protective responses and increase survival in a murine model (50) and by other studies where anticryptococcal antibody augmented lymphocyte proliferation (46). We were interested in determining whether an anticapsular MAb was more effective at restoring lymphocyte proliferation than human serum, reasoning that following vaccination (passive or active therapy with specific anti-CPS antibody), both would exert their effect in vivo.
Anticryptococcal antibodies can have protective, nonprotective, or disease-enhancing effects on the host defense (36, 38, 49), suggesting that they have multiple effects on the immune response. For our studies, we selected antibodies that had been demonstrated to be protective in a murine system. We found that lymphocyte proliferation in the presence of a specific anticapsular antibody was significantly better than that in the presence of human serum for all but the most highly encapsulated strains of C. neoformans.
Recently, the presence of anticapsular antibody has been found to
increase production of IL-1, TNF-, and IL-2 (46). Our studies indicate that one mechanism by which anticapsular antibody enhances cell-mediated immunity is by promoting the uptake of organisms
by the antigen-presenting cells, which facilitates presentation to T
cells. Additionally, increased uptake of the organisms could enhance
activation of antigen-presenting cells, which would in turn enhance
production of IL-1 and TNF- (46). The increased presentation of antigen that resulted from enhanced antigen uptake might also enhance IL-2 production (46) and hence lymphocyte proliferation. Thus, the effects of anti-CPS antibody on cytokine levels can be explained by increased antigen being presented to T cells
as well as increased stimulation and production of favorable cytokines.
Our studies support the rationale for vaccine therapy for cryptococcosis. A vaccine has been prepared by conjugating glucoronxylmannan to tetanus toxoid (11). This vaccine elicits murine and human antibody responses (10, 11, 39). Antibodies to glucoronxylmannan conjugated to tetanus toxoid promote phagocytosis of C. neoformans in the absence of complement (51), and the vaccine is protective in a murine model (10). Our data suggests that one mechanism by which these antibodies may be effective is by enhancing the uptake of organisms by antigen-presenting cells and thus enhancing presentation to T cells, augmenting cell-mediated immunity.
In summary, we have shown that exogenous polysaccharide can inhibit cell-mediated immune responses by binding to organisms and reducing their uptake by antigen-presenting cells. Further, opsonization by normal human serum and by anticapsular antibody can augment the responses to C. neoformans. Our studies support the therapeutic potential of stimulating cell-mediated immunity with anticapsular antibody.
| |
ACKNOWLEDGMENTS |
|---|
We thank Jason Spurrell and Peter Warren for technical assistance.
This work is supported by a grants from the Medical Research Council and The Canadian Foundation for AIDS Research and by Public Health Service grant AI14209. R.M.S. was supported by a National Health Research and Development Program Studentship. C.H.M. is a Scholar of the Alberta Heritage Foundation for Medical Research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Division of Pulmonary Medicine, Room 273, Heritage Medical Research Building, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1. Phone: (403) 220-5979. Fax: (403) 270-2772. E-mail: cmody{at}ucalgary.ca.
Editor: J. R. McGhee
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Aguirre, K., E. A. Havell, G. W. Gibson, and L. L. Johnson. 1995. Role of tumor necrosis factor and gamma interferon in acquired resistance to Cryptococcus neoformans in the central nervous system of mice. Infect. Immun. 63:1725-1731[Abstract]. |
| 2. | Blasi, E., R. Barluzzi, R. Mazzolla, L. Pitzurra, M. Puliti, S. Saleppico, and F. Bistoni. 1995. Biomolecular events involved in anticryptococcal resistance in the brain. Infect. Immun. 63:1218-1222[Abstract]. |
| 3. | Chaka, W., J. Scharringa, A. F. Verheul, J. Verhoef, A. G. Van Strijp, and I. M. Hoepelman. 1995. Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry. Clin. Lab. Immunol. 2:753-759. |
| 4. | Coker, R. J. 1992. Cryptococcal infection in AIDS. Int. J. Sex. Transm. Dis. AIDS 3:168-172. |
| 5. |
Collins, H. L., and G. J. Bancroft.
1991.
Encapsulation of Cryptococcus neoformans impairs antigen-specific T-cell responses.
Infect. Immun.
59:3883-3888 |
| 6. | Das, K. C., and H. P. Misra. 1994. Impairment of RAW 264.7 macrophage function by antiarrhythmic drugs. Mol. Cell. Biochem. 132:151-162[Medline]. |
| 7. | Davies, S. R., D. P. Clifford, J. R. Hoidal, and J. E. Repine. 1982. Opsonic requirements for the uptake of Cryptococcus neoformans by human polymorphonuclear leukocytes and monocytes. J. Infect. Dis. 145:870-874[Medline]. |
| 8. | Del Prete, G., M. De Carli, F. Almerigogna, M. G. Giudizi, R. Biagiotti, and S. Romagnani. 1993. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J. Immunol. 150:353-360[Abstract]. |
| 9. |
Deshaw, M., and L. A. Pirofski.
1995.
Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan are ubiquitous in serum from HIV+ and HIV individuals.
Clin. Exp. Immunol.
99:425-432[Medline].
|
| 10. | Devi, S. J. 1996. Preclinical efficacy of a glucuronoxylomannan-tetanus toxoid conjugate vaccine of Cryptococcus neoformans in a murine model. Vaccine 14:841-844[Medline]. |
| 11. |
Devi, S. G. N.,
R. Schneerson,
W. Egan,
T. J. Ulrich,
D. Bryla,
J. B. Robbins, and J. E. Bennett.
1991.
Cryptococcus neoformans serotype A glucuronoxylomannan-protein conjugate vaccines: synthesis, characterization, and immunogenicity.
Infect. Immun.
59:3700-3707 |
| 12. |
de Waal Malefyt, R.,
J. Abrams,
B. Bennett,
C. G. Figdor, and J. E. de Vries.
1991.
Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocyte: an autoregulatory role of IL-10 produced by monocytes.
J. Exp. Med.
174:1209-1220 |
| 13. | Dismukes, W. E. 1988. Cryptococcal meningitis in patients with AIDS. J. Infect. Dis. 157:624-628[Medline]. |
| 14. | Dong, Z. M., and J. W. Murphy. 1996. Cryptococcal polysaccharides induce L-selectin shedding and tumor necrosis factor receptor loss from the surface of human neutrophils. J. Clin. Invest. 97:689-698[Medline]. |
| 15. | Dromer, J., C. Perronne, J. Barge, J. L. Vilde, and P. Yeni. 1989. Role of IgG and the complement component C5 in the initial course of experimental cryptococcosis. Clin. Exp. Immunol. 78:412-417[Medline]. |
| 16. |
Eckert, T. F., and T. R. Kozel.
1987.
Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide.
Infect. Immun.
55:1895-1899 |
| 17. | Feldmesser, M., and A. Casadevall. 1997. Effect of serum IgG1 to Cryptococcus neoformans glucuronoxylomannan on murine pulmonary infection. J. Immunol. 158:790-799[Abstract]. |
| 18. | Grant, I. H., and D. Armstrong. 1988. Fungal infections in AIDS. Cryptococcosis. Infect. Dis. Clin. N. Am. 2:457-464[Medline]. |
| 19. |
Houpt, D. C.,
G. S. Pfrommer,
B. J. Young,
T. A. Larson, and T. R. Kozel.
1994.
Occurrences, immunoglobulin classes, and biological activities of antibodies in normal human serum that are reactive with Cryptococcus neoformans glucuronoxylomannan.
Infect. Immun.
62:2857-2864 |
| 20. |
Huffnagle, G. B.,
G. B. Toews,
M. D. Burdick,
M. B. Boyd,
K. S. McAllister,
R. A. McDonald,
S. L. Kunkel, and R. M. Strieter.
1996.
Afferent phase production of TNF- is required for development of protective T cell immunity to Cryptococcus neoformans.
J. Immunol.
157:4529-4536[Abstract].
|
| 21. |
Jacobson, E. S.,
D. J. Ayers,
A. C. Harrell, and C. C. Nicholas.
1982.
Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans.
J. Bacteriol.
150:1292-1296 |
| 22. |
Kozel, T. R., and C. A. Hermerath.
1984.
Binding of cryptococcal polysaccharide to Cryptococcus neoformans.
Infect. Immun.
43:879-886 |
| 23. |
Kozel, T. R.
1977.
Nonencapsulated variant of Cryptococcus neoformans. II. Surface receptors for cryptococcal polysaccharide and their role in inhibition of phagocytosis by polysaccharide.
Infect. Immun.
16:99-106 |
| 24. |
Kozel, T. R., and J. Cazin.
1971.
Nonencapsulated variant of Cryptococcus neoformans. I. Virulence studies of characterization of soluble polysaccharide.
Infect. Immun.
3:287-294 |
| 25. |
Kozel, T. R., and R. P. Mastroianni.
1976.
Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis.
Infect. Immun.
14:62-67 |
| 26. |
Kozel, T. R., and T. G. McGaw.
1979.
Opsonization of Cryptococcus neoformans by human immunoglobulin G: role of immunoglobulin G in phagocytosis by macrophages.
Infect. Immun.
25:255-261 |
| 27. |
Kozel, T. R.,
G. S. T. Pfrommer,
A. S. Guerlain,
B. A. Highison, and G. J. Highison.
1988.
Strain variation in phagocytosis of Cryptococcus neoformans: dissociation of susceptibility to phagocytosis from activation and binding of opsonic fragments of C3.
Infect. Immun.
56:2794-2800 |
| 28. |
Levitz, S. M., and D. J. DiBenedetto.
1988.
Differential stimulation of murine resident peritoneal cells by selectively opsonized encapsulated and acapsular Cryptococcus neoformans.
Infect. Immun.
56:2544-2551 |
| 29. |
Levitz, S. M., and T. P. Farrell.
1990.
Growth inhibition of Cryptococcus neoformans by cultured human monocytes: role of the capsule opsonins, the culture surface, and cytokines.
Infect. Immun.
58:1201-1209 |
| 30. | Levitz, S. M., T. P. Farrell, and R. T. Maziarz. 1991. Killing of Cryptococcus neoformans by human peripheral blood mononuclear cells stimulated in culture. J. Infect. Dis. 163:1108-1113[Medline]. |
| 31. | Levitz, S. M., and A. Tabuni. 1991. Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin. J. Clin. Invest. 87:528-535. |
| 32. |
Mitchell, T. G., and L. Friedman.
1972.
In vitro phagocytosis and intracellular fate of variously encapsulated strains of Cryptococcus neoformans.
Infect. Immun.
5:491-498 |
| 33. |
Mody, C. H., and R. M. Syme.
1993.
Effect of polysaccharide capsule and methods of preparation on human lymphocyte proliferation in response to Cryptococcus neoformans.
Infect. Immun.
61:464-469 |
| 34. |
Mody, C. H.,
G. B. Toews, and M. F. Lipscomb.
1988.
Cyclosporin A inhibits the growth of Cryptococcus neoformans in a murine model.
Infect. Immun.
56:7-12 |
| 35. | Monari, C., C. Retini, B. Palazzetti, F. Bistoni, and A. Vecchiarelli. 1997. Regulatory role of exogenous IL-10 in the development of immune response versus Cryptococcus neoformans. Clin. Exp. Immunol. 109:242-247[Medline]. |
| 36. |
Mukherjee, J.,
G. Nussbaum,
M. D. Scharff, and A. Casadevall.
1995.
Protective and nonprotective monoclonal antibodies to Cryptococcus neoformans originating from one B cell.
J. Exp. Med.
181:405-409 |
| 37. |
Mukherjee, J.,
L. S. Zuckier,
M. D. Scharff, and A. Casadevall.
1994.
Therapeutic efficacy of monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan alone and in combination with amphotericin B.
Antimicrob. Agents Chemother.
38:580-587 |
| 38. | Mukherjee, S., S. C. Lee, and A. Casadevall. 1994. Antibodies to Cryptococcus neoformans glucuronoxylomannan enhance antifungal activity of murine macrophages. Infect. Immun. 63:573-579[Abstract]. |
| 39. | Pirofski, L., R. Lui, M. DeShaw, A. B. Kressel, and Z. Zhong. 1995. Analysis of human monoclonal antibodies elicited by vaccination with a Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide vaccine. Infect. Immun. 63:3005-3014[Abstract]. |
| 40. |
Retini, C.,
A. Vecchiarelli,
C. Monari,
F. Bistoni, and T. R. Kozel.
1998.
Encapsulation of Cryptococcus neoformans with glucuronoxylomannan inhibits the antigen-presenting capacity of monocytes.
Infect. Immun.
66:664-669 |
| 41. |
Small, J. M.,
T. G. Mitchell, and R. W. Wheat.
1986.
Strain variation in composition and molecular size of the capsular polysaccharide of Cryptococcus neoformans serotype A.
Infect. Immun.
54:735-741 |
| 42. |
Spiropulu, C.,
R. A. Eppard,
E. Otteson, and T. R. Kozel.
1989.
Antigenic variation within serotypes of Cryptococcus neoformans detected by monoclonal antibodies specific for the capsular polysaccharide.
Infect. Immun.
57:3240-3242 |
| 43. | Vecchiarelli, A., D. Pietrella, M. Dottorini, C. Monari, C. Retini, T. Todisco, and F. Bistoni. 1994. Encapsulation of Cryptococcus neoformans regulates fungicidal activity and the antigen presentation process in human alveolar macrophages. Clin. Exp. Immunol. 98:217-223[Medline]. |
| 44. |
Vecchiarelli, A.,
C. Retini,
D. Pietrella,
C. Monari,
C. Tascini,
T. Beccari, and T. R. Kozel.
1995.
Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 secretion from human monocytes.
Infect. Immun.
63:2919-2923[Abstract].
|
| 45. | Vecchiarelli, A., C. Retini, C. Monari, C. Tascini, F. Bistoni, and T. R. Kozel. 1996. Purified capsular polysaccharide of Cryptococcus neoformans induces interleukin-10 secretion by human monocytes. Infect. Immun. 64:2846-2849[Abstract]. |
| 46. |
Vecchiarelli, A.,
C. Retini,
C. Monari, and A. Casadevall.
1998.
Specific antibody to Cryptococcus neoformans alters human leukocyte cytokine synthesis and promotes T-cell proliferation.
Infect. Immun.
66:1244-1247 |
| 47. | Wilson, D. E., J. E. Bennett, and J. W. Bailey. 1968. Serologic grouping of Cryptococcus neoformans. Proc. Soc. Exp. Biol. Med. 127:820-823[Medline]. |
| 48. |
Young, B. J., and T. R. Kozel.
1993.
Effects of strain variation, serotype, and structural modification on kinetics for activation and binding of C3 to Cryptococcus neoformans.
Infect. Immun.
61:2966-2972 |
| 49. | Yuan, R., A. Casadevall, G. Spira, and M. D. Scharff. 1995. Isotype switching from IgG3 to IgG1 converts a nonprotective murine antibody to Cryptococcus neoformans into a protective antibody. J. Immunol. 154:1810-1816[Abstract]. |
| 50. |
Yuan, R.,
A. Casadevall, and M. D. Scharff.
1997.
T cells cooperate with passive antibody to modify Cryptococcus neoformans infection in mice.
Proc. Natl. Acad. Sci. USA
94:2483-2488 |
| 51. | Zhong, Z., and L. Pirofski. 1996. Opsonization of Cryptococcus neoformans by human anticryptococcal glucuronoxylomannan antibodies. Infect. Immun. 64:3446-3450[Abstract]. |
Infection and Immunity, September 1999, p. 4620-4627, Vol. 67, No. 9
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Geunes-Boyer, S., Oliver, T. N., Janbon, G., Lodge, J. K., Heitman, J., Perfect, J. R., Wright, J. R.
(2009). Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival. Infect. Immun.
77: 2783-2794
[Abstract] [Full Text] -
Marr, K. J., Jones, G. J., Zheng, C., Huston, S. M., Timm-McCann, M., Islam, A., Berenger, B. M., Ma, L. L., Wiseman, J. C. D., Mody, C. H.
(2009). Cryptococcus neoformans Directly Stimulates Perforin Production and Rearms NK Cells for Enhanced Anticryptococcal Microbicidal Activity. Infect. Immun.
77: 2436-2446
[Abstract] [Full Text] -
Jain, N., Li, L., Hsueh, Y.-P., Guerrero, A., Heitman, J., Goldman, D. L., Fries, B. C.
(2009). Loss of Allergen 1 Confers a Hypervirulent Phenotype That Resembles Mucoid Switch Variants of Cryptococcus neoformans. Infect. Immun.
77: 128-140
[Abstract] [Full Text] -
Guerrero, A., Fries, B. C.
(2008). Phenotypic Switching in Cryptococcus neoformans Contributes to Virulence by Changing the Immunological Host Response. Infect. Immun.
76: 4322-4331
[Abstract] [Full Text] -
Monari, C., Kozel, T. R., Paganelli, F., Pericolini, E., Perito, S., Bistoni, F., Casadevall, A., Vecchiarelli, A.
(2006). Microbial Immune Suppression Mediated by Direct Engagement of Inhibitory Fc Receptor. J. Immunol.
177: 6842-6851
[Abstract] [Full Text] -
Martinez, L. R., Casadevall, A.
(2005). Specific Antibody Can Prevent Fungal Biofilm Formation and This Effect Correlates with Protective Efficacy. Infect. Immun.
73: 6350-6362
[Abstract] [Full Text] -
Monari, C., Pericolini, E., Bistoni, G., Casadevall, A., Kozel, T. R., Vecchiarelli, A.
(2005). Cryptococcus neoformans Capsular Glucuronoxylomannan Induces Expression of Fas Ligand in Macrophages. J. Immunol.
174: 3461-3468
[Abstract] [Full Text] -
Syme, R. M., Spurrell, J. C. L., Amankwah, E. K., Green, F. H. Y., Mody, C. H.
(2002). Primary Dendritic Cells Phagocytose Cryptococcus neoformans via Mannose Receptors and Fc{gamma} Receptor II for Presentation to T Lymphocytes. Infect. Immun.
70: 5972-5981
[Abstract] [Full Text] -
Shapiro, S., Beenhouwer, D. O., Feldmesser, M., Taborda, C., Carroll, M. C., Casadevall, A., Scharff, M. D.
(2002). Immunoglobulin G Monoclonal Antibodies to Cryptococcus neoformans Protect Mice Deficient in Complement Component C3. Infect. Immun.
70: 2598-2604
[Abstract] [Full Text] -
Feldmesser, M., Mednick, A., Casadevall, A.
(2002). Antibody-Mediated Protection in Murine Cryptococcus neoformans Infection Is Associated with Pleotrophic Effects on Cytokine and Leukocyte Responses. Infect. Immun.
70: 1571-1580
[Abstract] [Full Text] -
Ma, L. L., Mody, C. H.
(2002). When There Is a Fungus Among Us, What Makes it Virulent?. Am. J. Respir. Cell Mol. Bio.
26: 273-276
[Full Text] -
Syme, R. M., Spurrell, J. C. L., Ma, L. L., Green, F. H. Y., Mody, C. H.
(2000). Phagocytosis and Protein Processing Are Required for Presentation of Cryptococcus neoformans Mitogen to T Lymphocytes. Infect. Immun.
68: 6147-6153
[Abstract] [Full Text] -
Feldmesser, M., Rivera, J., Kress, Y., Kozel, T. R., Casadevall, A.
(2000). Antibody Interactions with the Capsule of Cryptococcus neoformans. Infect. Immun.
68: 3642-3650
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»