FEMS Immunology and Medical Microbiology 42 (2004) 219224

www.fems-microbiology.org

Apoptosis of phagocytic cells induced by Candida albicans

and production of IL-10
Tha
s Helena Gasparoto a, Luis Carlos Jabur Gaziri b, Eva Burger c,
Ricardo S
ergio Couto de Almeida a, Ionice Felipe a,*
a
b

Departamento de Ci^encias Patol
ogicas, CCB, Universidade Estadual de Londrina, Cx P. 6001, 86051-990 Londrina, Brazil
Departamento de Ci^encias Fisiol
ogicas, CCB, Universidade Estadual de Londrina, Cx P. 6001, 86051-990 Londrina, Brazil
c
Departamento de Imunologia, Universidade de S~ao Paulo, S~ao Paulo, Brazil
Received 12 January 2004; received in revised form 30 April 2004; accepted 14 May 2004
First published online 9 June 2004

Abstract
Macrophages co-incubated with Candida albicans strain CR1 in vitro showed early signs of apoptosis, but evolved to necrosis
after 2 h. In this study, we investigated whether strain CR1 caused apoptosis or necrosis of macrophages after its inoculation into
mice peritoneal cavity, and whether this correlated with the secretion of IL-10. Peritoneal macrophages from mice that received an
inoculum of C. albicans CR1 showed signs of apoptosis and necrosis from 30 min to 2 h afterwards, whereas heat-killed C. albicans
did not cause those eects. IL-10 production was low during the rst 6 h post-infection, when macrophages predominated in the
peritoneal exudate, whereas its higher production after 24 h correlated with an increase of neutrophils in the exudate. Treatment of
CR1 with pepstatin (an inhibitor of proteinases) prevented the process of apoptosis and signicantly reduced IL-10 production,
suggesting that the increased production of IL-10 was caused by processes occurring during the initial phase of infection, such as
apoptosis, necrosis and uptake of death cells.

2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Candida albicans; Apoptosis; Necrosis; IL-10

1. Introduction
Candida albicans causes several diseases, ranging
from mucocutaneous infection to systemic infection.
Moreover, C. albicans may cause peritonitis when it
reaches the peritoneal cavity by its iatrogenic inoculation through contaminated plastic devices and uids
during continuous ambulatory peritoneal dialysis [1,2].
Several putative virulence factors of C. albicans were
identied, such as ability to form germ tubes, adherence
to host cells, and secretion of proteinases [36]. Candida
proteinases decreased the opsonic and bactericidal activities of human serum by degradation of the Fc portion of immunoglobulin G and proteolysis of C3 of
*

Corresponding author. Tel.: +55-43-371-4267; fax: +55-43-33714267.

E-mail address: ionice@uel.br (I. Felipe).

complement [7]. Furthermore, we observed destruction

of macrophages co-incubated with C. albicans, which
was prevented by the proteinase inhibitor pepstatin [8];
it was also demonstrated that there is expression of secreted aspartyl proteinases Sap4 to Sap6 from C. albicans in murine macrophages [9]. A triple mutant in
Sap46 induced a signicantly reduced activity of alanine aminotransferase, which was used as parameter for
damage of the liver in comparison to the reference strain
[10]. Induction of programmed cell death or apoptosis
by microrganisms may constitute an important factor in
pathogenesis, since cell death, particularly death of
phagocytes, might favour invasion by pathogens [11
13].
Murine peritoneal macrophages co-incubated with a
strain of C. albicans isolated from a HIV-infected individual showed early signs of apoptosis, such as membrane exposure of phosphatidylserine, but ultimately

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2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsim.2004.05.006

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T.H. Gasparoto et al. / FEMS Immunology and Medical Microbiology 42 (2004) 219224

progressed to necrosis after 2 h of incubation [14]. Since

these latter observations were carried out in vitro, they
raised questions as to whether that strain of C. albicans
would induce apoptosis of phagocytes in vivo and
whether immune response mediator IL-10 would show
changes corresponding to that presumed apoptosis;
these two questions were addressed in the present study.

2. Materials and methods

2.1. C. albicans culture
Candida albicans CR1 was isolated from the oral
mucosa of a HIV-infected individual at the Dentistry
School, Universidade Estadual de Londrina, Brazil. The
fungi were maintained at room temperature on Sabouraud dextrose agar. The blastoconidia were grown in
Sabouraud dextrose broth for 24 h at 28 C, and then
were harvested by centrifugation (2000g 6 min), washed with PBS and resuspended at 107 cells/0.5 ml in
RPMI medium (Sigma) containing 1% added human
albumin. As a control, C. albicans CR1 was autoclaved
for 15 min, washed and resuspended as before. The
viable yeasts were incubated with pepstatin (30 lg)
(Sigma) for 15 min at 37 C before infection to analyse
the eect of proteinases on the apoptosis and IL-10
production.
2.2. Infection of mice
Four groups of Swiss male mice weighing 2832 g and
6070 days old were infected intraperitoneally with either viable or autoclaved C. albicans CR1 (107 yeast/ml
RPMI). One group of ve mice was used as control.
2.3. Ex-vivo assays
Animals that received an inoculum of either strain of
viable C. albicans or autoclaved controls (ve mice per
group) were sacriced after 30 min, 2, 6 and 24 h, and
their peritoneal exudate cells were collected. The animals
infected with C. albicans pre-treated with pepstatin were
sacriced after 30 min and 24 h and peritoneal exudate
cells and IL-10 analysed. Aliquots (100 ll) of peritoneal
exudate were plated on Sabouraud agar at all time
points studied and the number of CFU was determined
after incubation for 48 h at 37 C.
2.4. Evaluation of phagocytic cells
Exudate cells were obtained by rinsing the peritoneal
cavity with 2 ml of RPMI-albumin medium. After centrifugation (3000 rpm 4 min, at 4 C), the pellet was
resuspended in 1 ml RPMI-albumin medium. The cells
were distributed on coverslips (0.2 ml/coverslip) and

incubated at 37 C, under 5% CO2 , for 30 min. The

following tests were applied to the cells: (1) staining by
May GrumwaldGiemsa (Merck) and analysis by light
microscopy to evaluate the populations of phagocytes,
mean number of blastoconidia cells per phagocyte and
phagocytising cells, by counting 10 elds; (2) acridine
orange (140 lg in 20 ll PBS; Sigma) for observation of
macrophages and neutrophils; (3) annexin V-FITC
(R&D System) for detection of apoptosis; (4) staining
with propidium iodide and propidium iodide plus 6carboxyuorescein diacetate (6-CFDA) (Sigma) to
evaluate the presence of necrotic and viable cells. All
tests using uorescent stains were observed in a
uorescence microscope (Zeiss) and photographed
(Blue-violet irradiation: heat lter, BG-38 and BG-12
excitation lters and 530-barrier lter).
2.5. Assays of IL-10
Supernatants were collected after centrifugation of
peritoneal exudates obtained from each mouse from all
the experimental groups and submitted to capture
ELISA using monoclonal antibodies (mAb) against
murine cytokines Jess 2A5 (2 lg/ml) and biotinylated
SXC-1 (2 lg/ml) for IL-10 (PharMingen, San Diego,
CA). The ELISA procedure was performed according to
the manufacturers protocol. The concentrations of cytokines were determined with reference to a standard
curve for serial twofold dilutions of murine recombinant
cytokines; optical absorption was measured at 492 nm.
The lowest limit of each recombinant standard curve
detection was 7.8 pg/ml for IL-10.
2.6. Statistical analysis
Dierences between the control and experimental
groups were analysed by the Tukeys test. A p-value of
<0.01 was considered signicant.

3. Results
3.1. Phagocytic cells
Mice that received an inoculum of viable C. albicans
CR1 showed a signicant reduction in the population of
peritoneal macrophages 6 h post-infection, which returned to control levels after 24 h (Fig. 1(a)). The population of neutrophils signicantly increased in the
group infected with viable CR1 strain after 6 h, and was
greatly increased at 24 h of infection (Fig. 1(b)). A large
percent of macrophages showed at least one phagocytosed blastoconidia 30 min after infection, and this
percent was greatly reduced thereafter for both viable
and autoclaved C. albicans (Table 1). The results obtained by plating of the peritoneal exudates (Table 2)

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221

ulum of autoclaved CR1 resulted in lower phagocytosis

by macrophages and neutrophils compared with viable
CR1 (Table 1).

3.2. Apoptosis and necrosis

Fig. 1. Populations of phagocytic cells in the peritoneal exudate on the

course of infection with either viable (shaded bars) or autoclaved (open
bars) C. albicans CR1. (a) Macrophages and (b) neutrophils. Results
represent the mean values SEM for ve mice. *p < 0:001 compared
with values from control mice (closed bars).

show that there was a close correlation between reduction of C. albicans in the peritoneal cavity from 30 min
to 24 h post-infection and the percentage of macrophages and neutrophils phagocytosing them. The inoc-

Macrophages collected 30 min or 2 h after infection

with strain CR1 bound annexin V-FITC (Fig. 2(a) and
(b)); macrophages from those same samples were permeable to propidium iodide 30 min or 2 h after infection
(Fig. 2(c) and (d)). Pre-incubation of C. albicans with
pepstatin abolished the process of apoptosis that
otherwise was detected 30 min after infection (micrographs not shown). Apoptotic cells were not detected 24
h after infection, and few necrotic cells were then detected (Fig. 2(f)). The population of phagocytic cells
presented a predominance of neutrophils after staining
with acridine orange 24 h post-infection (micrographs
not shown). No apoptotic cells were found in the peritoneal exudates of mice that received an inoculum of
autoclaved C. albicans CR1 for all time points studied
up to 24 h. Propidium iodide, which stains only nuclei of
cells with disrupted plasma membranes, which is a
hallmark of necrosis [15], showed only death cells, and
for this reason we used 6-CFDA to detect viable phagocytic cells and death cells simultaneously. Fig. 2(e)
shows a higher number of necrotic cells after 2 h, as
compared to 24 h of infection (Fig. 2(f)). Since 6-CFDA
stains viable cells, it was possible to observe viable yeast
and germ tubes cells inside of macrophages (Fig. 2(e)),
besides macrophages and neutrophils, which could not
be seen when only propidium was used (Fig. 2(c) and
(d)). A macrophage containing a phagocytosed necrotic
cell can be observed in Fig. 2(e).

Table 1
Phagocytosis by macrophages and neutrophils

Conditions
CR1 V
CR1 A

Macrophages

Neutrophils

Time post-infection (h)

Time post-infection (h)

1/2
42.0 1.4
23.5 2.1

2
24.0 2.70
8.20 1.65

6
2.7 0.3
6.6 0.9

24
1.2 0.6
6.9 2.0

1/2
0
0

2
33.5 4.8
29.6 3.8

6
7.14 1.9
9.3 1.5

24
0.2 0.44
2.0 0.50

Percentages of macrophages and neutrophils phagocytosing C. albicans CR1 viable (V) autoclaved (A) at dierent times. Data are reported as
means SEM for ve experiments.

Table 2
Recovery of C. albicans from peritoneal exudates (CFU)
Time post-infection (h)
Conditions
CR1

1/2
5 105 1.6 102

2
3.3 104 1 102

6
3.5 103 1 102

24
1.8 102 7.3 101

Number of C. albicans CR1 (colony forming units) in the peritoneal exudate after inoculation of 107 yeast cells. Data are reported as means 
SEM for ve experiments.

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T.H. Gasparoto et al. / FEMS Immunology and Medical Microbiology 42 (2004) 219224

Fig. 2. Fluorescence micrograph of phagocytic cells stained with (a) annexin V-FITC 30 min and (b) 2 h post-infection with viable C. albicans CR1;
propidium iodide on the same preparation (c) 30 min and (d) 2 h; or propidium iodide plus 6-CFDA (e) 2 h. Arrow indicates macrophage
phagocytosing dead cell and (f) 24 h post-infection with viable CR1.

3.3. Cytokine analysis

The amount of IL-10 in supernatants of peritoneal
exudates from animals that received an inoculum of
viable C. albicans CR1 was low from 30 min to 6 h, and
signicantly increased 24 h post-infection (Fig. 3). The
amount of IL-10 in animals which received an inoculum
of autoclaved C. albicans CR1 did not show such an
increase at 24 h, although at 2 and 6 h post-infection it
showed levels similar to those detected after inoculation
of viable strain CR1 (Fig. 3). IL-10 was not detected in
the peritoneal exudate of non-infected mice.

isolated from skin lesions of an individual not infected

with HIV [14]. In this work,we veried that macrophages from the peritoneal exudate of mice that had
received an intraperitoneal inoculum of strain CR1

4. Discussion
We previously reported that C. albicans CR1, which
was isolated from a HIV-infected individual, induced
early apoptotic changes in macrophages that phagocytosed them in vitro, but that those macrophages
progressed to necrosis and lysis 2 h thereafter; similar
pro-apoptotic changes were not observed in macrophages co-incubated with C. albicans 577, which was

Fig. 3. Dosage of IL-10 from supernatants of peritoneal exudate by

ELISA after infection with C. albicans. Infection with viable (shaded
bar) and autoclaved (open bars) C. albicans CR1. Data are reported as
means SEM for three experiments.

T.H. Gasparoto et al. / FEMS Immunology and Medical Microbiology 42 (2004) 219224

bound annexin V-FITC and became permeable to propidium iodide, and that these changes occurred from 30
min to 2 h after in vivo infection, in agreement with the
prior in vitro observations. Thus, the results obtained in
this in vivo study parallel the ones previously observed
in vitro. Moreover, heat-killed strain CR1 did not induce similar changes in peritoneal exudate macrophages
for up to 24 h of observation, which suggests that induction of macrophage apoptosis by C. albicans CR1
depends on a factor released by the viable pathogen.
Of the numerous cells reported to recognize and remove apoptotic cells, the macrophages are the most
prominent [15,16], and we detected macrophages
phagocytosing dead cells 2 h post-infection. Macrophages appear to recognize apoptotic cells via dierent
mechanisms, including integrins, phosphatidylserine
recognition, scavanger receptors, and lectins [17,18]. In
this study, we observed that both apoptosis and necrosis
increased from 30 min to 2 h of infection. Phagocytosis
of apoptotic cells, due to the exposure of phosphatidylserine for instance, could explain the signicant reduction of macrophages detected 6 h after infection.
Considering that incubation of CR1 with pepstatin before infection prevented the process of apoptosis, it
seems that proteinases released by the pathogen inside
of macrophages participate in this process. As expected,
since apoptosis and necrosis occurred in an early phase
of the infection, and those cells were therefore removed,
after 24 h the cells present in exudate were mostly viable,
and the incoming neutrophils were the most prominent
cells.
A small amount of IL-10 was detected in the peritoneal exudates of mice infected with either viable or autoclaved C. albicans CR1, 6 h post-infection. This
observation is in agreement with that of Romani et al.
[19], who found that macrophages have a poor ability to
release IL-10. On the other hand, those authors detected
a high production of IL-10 by neutrophils from CA-6
infected non-healer mice, although those cells were unable to produce IL-12. In this study, we observed that
the increased amount of IL-10 in the peritoneal exudates
24 h after infection with viable C. albicans CR1 correlated with an increase in both the amount and the proportion of neutrophils, suggesting that the increased
production of IL-10 depends on processes taking place
during the initial phase of infection, such as induction of
apoptosis or necrosis by viable C. albicans CR1 and
uptake of death cells by incoming phagocytes. Further
corroborating this conclusion, we observed that pepstatin reduced the apoptosis and necrosis of macrophages during the rst 2 h after infection and abolished
the increase in IL-10 otherwise observed 24 h after infection with viable strain CR1. An apoptotic cell exposes
phosphatidylserine externally, thus engaging the phosphatidylserine receptor and providing a dominant antiinammatory signal [20,21], in agreement with this

223

study. These results suggest that the ability of C. albicans CR1 to induce early apoptosis of macrophages
might induce or modulate an anti-inammatory pattern
of immune response afterwards.
Acknowledgements
We gratefully acknowledge Renata Scavone and
V^ania Darc for technical assistance. CAPES, CPG/
UEL, CNPq and Araucaria Fundation funded this
study.
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