Vol.3, No.3, 172-178 (2011) Health
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Immunophenotyping of peripheral blood mononuclear
cells and intracellular detection of IL-2, IFNγ and IL-4 by
flow cytometry in patients with actinomycetoma by
Nocardia brasiliensis and Actinomadura madurae.
Findings in six patients
Heuze de Icaza Ivonne1,6, Castrillón Rivera Laura Estela1,7, Garibay-Escobar Adriana2,3,
Sandoval Trujillo Horacio1,7, Padilla Desgarennes Carmen4, Palma Ramos Alejandro1,
Santos-Argumedo Leopoldo2
1Universidad Autónoma Metropolitana, Campus Xochimilco. Calzada del Hueso 1100, Villa Quietud, Coyoacán, México D. F. CP
04960. México;
2Department of Molecular Biomedicine, Centro de Investigacion y Estudios Avanzados del IPN. Ave. Instituto Politécnico Nacional
No. 2508, Col. Zacatenco, CP 07360, México City, México;
3Universidad de Sonora. Hermosillo, Sonora, México;
4Laboratory of Mycology. Centro Dermatológico “Dr. Ladislao de la Pascua”, Servicios de Salud Pública of Distrito Federal. México
City, México;
5Departamento de Biolgía, Facultad de Química, UNAM. Ciudad Universitaria, México D.F., México;
6Heuze de Icaza Yvonne is in the Program of Doctorate in Biological Sciences. Universidad Autónoma Metropolitana, México City,
7Laura E. Castrillón R and Horacio Sandoval T. are part of Tutorial Committee of Yvonne Heuze in the Program of Doctorate in
Biological Sciences. Universidad Autónoma Metropolitana, México City, México.
Received 20 January 2011; revised 5 February 2011; accepted 18 February 2011.
Mycetoma is a chronic, granulomatous, progres-
sive inflammatory disease; it usually involves
the subcutaneous tissue after a traumatic
inoculation of the causative organism. Mycetoma
may be caused by true fungi or bacteria
(actinomycetes) and hence it is classified as
eumycetoma and actinomycetoma respectively.
Mycetoma immunological studies have fre-
quently addressed humoral aspects. Few reports
have addressed the role of cellular immunity in
humans, for this reason, we were interested in
finding differences in the circulating population
of mononuclear cells or in the ability of this
pathology to produce cytokines after mitogen
stimulation. In this study, immunophenotyping
of peripheral blood mononuclear cells (PBMN)
and intracellular detection by flow cytometry of
IL-2, IFNγ and IL-4 in patients with actino-
mycetoma by Nocardia brasiliensis and Actino-
madura madurae were evaluated. We investi-
gated the expression of T-cells (CD3, CD4 and
CD8), B-cells (CD19), monocytes (CD14), and
natural killer cells (CD16 and CD56) markers.
CD69 and CD25 were used to monitor individual
activated cell subsets. The percentage values of
the cells were calculated. Our results indicated
that PBMC from patients with mycetoma show
similar percentages of circulating cells when
compared with healthy donors. The expression
of IL-2 receptor (IL-2R) by mitogen activation
was similar in these two groups. These findings
suggest that circulating lymphocytes are not
affected by this pathology. Intracellular IL-4 was
increased only in patients with mycetoma by N.
brasiliensis, suggesting a TH2 profile; this ob-
servation has been reported by other authors.
Keywords: Actinomycetoma; Nocardia brasili ensis;
Actinomadura madurae; Cytokines; CD4+; CD8+;
Mycetoma is a chronic, granulomatous, progressive
inflammatory disease; it usually involves the subcuta-
neous tissue after a traumatic inoculation of the causa-
H. de I. Yvonne et al. / Health 3 (2011) 172-178
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
tive organism. Mycetoma may be caused by true fungi or
by bacteria (actinomycetes) and hence it is classified as
eumycetoma and actinomycetoma respectively [1,2].
Tumefaction and formation of sinus tracts characterize
mycetoma. The sinuses usually discharge purulent and
seropurulent exudate containing microcolonies called
grains. It may spread involving skin and the deep struc-
tures, resulting in destruction, deformity, and loss of
function. In rare occassions it may be fatal [3,4]. The
clinical presentation of mycetoma is almost identical,
regardless of the causal organism. However, the progress
is faster in actinomycetoma comparatively with eumy-
cetoma. In eumycetoma, the lesion grows slowly, with
clear defined margins and it remains encapsulated for a
long period, whereas, in actinomycetoma the lesion is
more inflammatory, more destructive, and it invades the
bone at an earlier period [1,4]. Mycetoma lesion has a
distinct appearance in a cytology smear which is charac-
terized by the presence of inflammatory cells consisting
of a mixture of neutrophils, lymphocytes, plasma cells,
histiocytes, macrophages, and foreign body giant cells in
grains [5]. In tissue sections, three types of tissue reac-
tions have been described (I, II and III). Type I is char-
acterized by actinomycetoma lesion [6,7] where the
grain is closely surrounded by polymorphonuclear leu-
kocytes that are the predominant cell type in the in-
flammatory infiltrate in the skin lesions and occasionally
infiltrated by neutrophils, causing its fragmentation.
Outside the neutrophil zone, monocytic cells and giant
cells are observed. The lymphocytes are scant where the
main population is TCD4+ [8].
Cenci et al. [9] demonstrated the cellular elements in
the inflammatory infiltrate in skin lesions of actinomy-
cetoma and eumycetoma and suggested that cellular me-
diated immunity may play a role in mycetoma patho-
genesis, in which a marked reduction of Langerhans
cells may reflect a depressed cell immune response, par-
tially explaining a chronic condition and unresponsive-
ness to the treatment.
Studies on the immunopathologic aspects of tissue
reaction are much less frequent, immunological studies
of mycetoma have frequently addressed humoral aspects
[10-13]. Few reports have addressed the role of cellular
immunity in humans. The main studies regarding cellu-
lar immune response by delayed hypersensitivity to an-
tigens from Nocardia was first describe in 1972 [14] and
protective effect after spleen cell transfer from guinea
pigs infected with Nocardia asteroids was demonstrated
five years later [15]. A defective T-cell mediated re-
sponse in eumycetoma patients was suggested as an im-
portant element in this pathology when the lymphocyte
proliferation stimulated with phytohemagglutinin af-
fected skin reactivity to dinitrochlorobenzene in these
patients [16]; in contrast, infections by Nocardia aster-
oides did not show these efects [17].
Cell-mediated immunity plays a major protection role
against intracellular microbes. Nocardia brasiliensis is a
facultative intracellular pathogen that grows in macro-
phages but not in PMN leukocytes [18]. The mecha-
nisms that allow explaining the evasion, resistance, or
neutralization of bactericidal action of macrophages and
neutrophils are: 1) production of high levels of catalase
and superoxide dismutase that reduce the oxygen toxic
products generated by phagocyte, 2) reduction of ly-
sosome enzymatic activity of some macrophage popula-
tions, 3) blockade of phagosome acidification, and 4)
inhibition of phagosome-lysosome fusion [19].
Regarding Actinomadura madurae infections, the
ability of this bacteria to persist inside murine macro-
phages in experimental in vivo and in vitro infections has
been reported [20]; as with nocardia infections, this
could explain the tendency to chronicity by actinomy-
cetales due their replicative capacity inside the phago-
cytes, probably this fact is related to blood dissemination
as it was demonstrated in experimental mycetoma by
Nocardia brasiliensis [21].
In mycetoma, the local host response is characterized
by neutrophil chemotaxis and small vessel congestion.
Initially, the response is nonspecific, later, macrophages
and monocytes present at the infection site are activated
by interferon gamma and tumor necrosis factor-alpha;
these cells have enhanced microbicidal activity [22]. For
these reasons, the production of pro-inflammatory and
anti-inflammatory cytokines and the expression of their
receptors, after stimulation are important indicators of
mononuclear cell activation in this pathology.
2.1. Patients
Mononuclear cells from four patients with mycetoma
by Nocardia brasiliensis, two patients with mycetoma
by Actinomadura madurae, and five healthy subjects
were evaluated. These patients were diagnosed and
treated by the Mycology Service of the Dermatologic
Center “Ladislao de la Pascua” in Mexico City. This
study was carried out with the written consent of all pa-
tients and it was approved by the Ethics Committees of
each participating institution.
2.2. Samples
Peripheral blood samples from healthy volunteers and
patients were collected in sodium heparin VACU-
TAINERR tubes (Becton Dickinson, San Jose, CA). Per-
centages and absolute counts of mature human lym-
H. de I. Yvonne et al. / Health 3 (2011) 172-178
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phocytes subsets were evaluated from PBMC as follows:
T lymphocytes (CD3+), B lymphocytes (CD19+), helper
/inducer T lymphocytes (CD3+CD4+), cytotoxic T lym-
phocytes (CD3+CD8+), and natural killer (NK) lym-
phocytes (CD3-CD16 + CD56+). CD69 is expressed on
all activated lymphocytes, thus it represents a generic
marker to monitor individual subset responses to differ-
ent stimuli [23].
2.3. CD25 (IL-2αR) Expression
PBMC were stimulated with 20 µg/mL phytohae-
maglutinin (PHA Sigma) in RPMI, in 24 well flat bot-
tom plates (Corning Glass Works, Corning, NY) for 24 h
at 37˚C and 5% CO2. After incubation, PBMC were
harvested by centrifugation at 500 × g for 5 min and then
stained with a three-color combination using the follow-
ing mAbs: anti-CD25PE (IL-2R) (Phar Mingen), anti-
CD4FITC (Phar Mingen) and anti-CD3PerCP (Immmu-
notech, Marseille, France). Percenteges of T lympho-
cytes (gated on CD3+) expressing CD25 were measured
by flow cytometry.
2.4. Intracellular Cytokine Detection: IFN-γ,
IL-2 and IL-4 [24]
The production of cytokines was measured through
intracellular staining. Briefly, capped polystyrene Falcon
tubes (Becton Dickinson) were used to incubate whole
blood samples with 25 ng/ml phorbol myristate acetate
(PMA) (Sigma), 1 µg/ml ionomycin (Sigma), and 10
µg/ml brefeldin A (BFA) (Sigma) or BFA only (control),
for 5 h at 37˚C and 5% CO2. Anti-CD3 peridinin chlo-
rophyll protein (PerCP) (Becton Dickinson) was added
to aliquots of the stimulated and non-stimulated blood
and then incubated for 15 min at room temperature in the
dark. After incubation, erythrocytes were lysed with
FACS lysing solution (Becton Dickinson) and the sam-
ples were incubated for another 10 min at room tem-
perature in the dark. The cells were then centrifuged for
5 min at 500 × g and supernatants were aspirated with-
out disturbing the pellets. FACS permeabilizing solution
(Becton Dickinson) was added to the pellets and incu-
bated for 10 min at room temperature in the dark; cell
suspensions were then washed with phosphate buffered
saline (PBS) containing 0.1% bovine serum albumin
(BSA) (Research Organics, Cleveland, OH) and 0.01%
sodium azide (PBA), and centrifuged for 5 min at 500 ×
G, the supernatants were removed. For intracellular stain-
ing, anti-cytokine monoclonal antibodies (mAbs) were
added to the pellets and cell suspensions were incubated
at room temperature for 30 min in the dark, ac- cording
to the following protocol: FastImmuneR INF-γ fluo-
rescein isothiocyanate (FITC)/IL-4 phycoerythrin (PE)
(Becton Dickinson) or FastImmuneR anti-HuIL-2 FITC
(Becton Dickinson), and anti-CD69PE (Becton Dickin-
son) were added to the pellets previously stained with
anti-CD3PerCP. Simultest γ2a FITC/γ1PE with anti-CD3
PerCP was used as isotype control.
For all conditions, the expression of CD69 was meas-
ured as an activation control. The samples were washed
with PBA and fixed with PBS containing 1% parafor-
maldehyde (PFA). A fluorescent activated cell sorter
FACSCaliburR (Becton Dickinson) equipped with a 15
mW argon ion laser and filter settings for FITC (530 nm),
PE (585 nm) and PerCP(Becton Dickinson), emitting in
the deep red (> 650 nm) was used. Cells (10,000) were
computed in list mode and analyzed gating on CD3
(lymphocytes) using the CellQuestR software.
3. Results
3.1. Mononuclear Cell Populations
The analysis of mononuclear cell populations in nor-
mal subjects and from patients with mycetoma by No-
cardia brasiliensis and Actinomadura madurae is pre-
sented in Table 1. The results from healthy volunteers
showed: CD3+T cells (84%), in which the percentages
for the subpopulations were TCD4+ (56.6%), TCD8+
(28.2%); B cells (13.2%); NK cells and monocytes both
were at 1%. In mycetoma patients, the percentages were
as follows: T lymphocytes (CD3+) were 84.7%, in which
58.1% and 26.6% corresponded to CD4+ and CD8+
lymphocytes, respectively. B lymphocytes were 12.3%,
NK cells 1% and monocytes 0.6%.
3.2. IL-2ΑR Expression Analysis
A kinetic analysis for the expression of CD25+
(IL-2αR) was made. PBMC from healthy subjects were
stimulated with 20 µg/mL PHA for 0, 24, 48 and 72 h
and stained for CD25 (IL-2αR). Percentages of lympho-
cytes positive for CD25 were mesured at each time by
Table 1. Percentage of monocytes (CD14+), NK (CD16 +
CD56+), TCD8 + (CD3+) and B (CD19+) lymphocytes from
total PBMC in healthy and actinomycetoma patients.
Patients with mycetoma by
brasiliensis 0.15 ± 0.0858.4 ± 13.3 25 ± 12.0 2.0 ± 1.2 14.5 ± 10.4
madurae 1.1 ± 1.657.8 ± 11.9 28.3 ± 14.9 2.4 ± 1.810.2 ± 10.1
donors 1.0 ± 1.956.6 ± 13.7 28.2 ± 1.9 1.0 ± 1.213.2 ± 6.7
H. de I. Yvonne et al. / Health 3 (2011) 172-178
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Figure 1. Percent of expression of IL-2αR at different hours of
flow cytometry. As it can be seen in Figure 1, maximum
expression of IL-2αR (58.66% CD25+) was detected after
72 h of activation; however, at this time, the percentage
of cell death was also high (60% of cells were positive
for propidium iodide staining, data no shown). For this
reason, all determinations were done after 24 h of
3.3. Percentages of TCD4+ Lymphocytes
from Healthy Donors, and Mycetoma
Patients Expressing IL-2αR (CD25+)
The percentages of CD4+ T cells expressing IL-2 re-
ceptor (CD25+) showed that 6.3% of healthy donors ex-
pressed CD25 upon activation; in contrast, 15.3% and
13.3% CD4+ lymphocytes from patients suffering my-
cetoma by Nocardia brasiliensis or by Actinomadura
madurae respectively were positive for this molecule.
Table 2.
3.4. Detection of the Intracellular Cytokines:
IL-2, IFN-γ and IL-4
Lymphocytes from healthy donors, (gated on CD3+ cells)
expressed CD69 (12.81%), IL-2 (3.19%), IFN-γ (17.34
%) and IL-4 (0.43%), only when they were activated
with PMA and ionomycin (Table 3). The absence or low
Table 2. Percentage of TCD4+ lymphocytes from healthy do-
nors and actinomycetoma patients, expressing IL-2αR (CD25+).
Percentage of positive cells
Patients with
mycetoma by
CD4+ CD25+ CD4+ - CD25+
Nocardia brasiliensis 58.4 ± 13.321.5 ± 13.2 15.3 ± 7.2
madurae 57.8 ± 11.916.4 ± 11.6 13.3 ± 2.7
Healthy donors 56.6 ± 13.712.5 ± 5.1 6.3 ± 3.6
Table 3. Percentage of lymphocytes expressing CD69 and
producing cytokines upon stimulation.
CD3 + lymphocytes expressing
Patients with
by CD69+ IL-2 (%) IFN-γ (%) IL4 (%)
brasiliensis 13.3 ± 3.25.7 ± 3.5 17.3 ± 18.43.7 ± 2.6
madurae 13.3 ± 2.34.9 ± 0.9 15.9 ± 13.00.4 ± 0.4
donors 12.8 ± 6.33.2 ± 2.1 17.3 ± 10.70.4 ± 0.4
levels of IL-4 production, confirms the difficulty of de-
tecting this Th2-type cytokine by flow cytometry [25].
Table 3 also shows the expression of these cytokines in
six patients. An average of 14.2% of T cells expressed
CD 69, while 5.9% of these produced IL-2, 15.7% pro-
duced IFN-γ and 0.68% produced IL-4.
4. Discussion
Although mycetoma was first described more than
3,000 years ago, the studies on physiopathology began
only some decades ago. Many aspects about the immune
response during the evolution of this disease are still
unknown. A basic question related to the nature of the
immunological response in this pathology could be re-
lated to the existence of differences in the circulating
mononuclear cells in these patients. In our analysis, pe-
ripheral blood monocytes, CD4+ and CD8+ T lympho-
cytes and natural killer cells CD56+ were present with
similar values in patients as well as control subjects (Ta-
ble 1). This fact suggests that the immune response is
developed locally and the patients that suffer this
chronical disease do not present changes in circulating
mononuclear cells (Figure 2).
Host immune response is present in both, human
(natural infection) and experimental infections (animal
models or in vitro) with Nocardia brasiliensis and Ac-
tinomadura madurae. The response is characterized by
an intense infiltration of polymorphonuclear cells, being
this a constant finding during the entire active infection
[26]. Infections by Noca rdia have been associated with:
a) inhibition of the bacteria by monocytes and polymer-
phonuclear leukocytes, b) activation of macrophages and
3) specific cytotoxicity from a T cell subset [27]. How-
ever,in the long run, Nocardia depends on the meta-
bolic state of macrophage and neutrophil, the concentra-
tion and type of lysosomal enzymes inside the cell and
the type of strain of Nocardia [17] and also, probably,
the presence of L forms of bacteria inside cells involved
in tissue process [28]. A deficiency of one of these fac-
tors could augment the host’s subseptibility to acquire
the illness produced by this bacterium. The ideal candi-
dates to modulate these functions are the cytokines which
H. de I. Yvonne et al. / Health 3 (2011) 172-178
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Figure 2. Percentage from PBMC evaluated by flow cytometry from mononuclear peripheral blood from healthy
volunteers (a) and (d); with mycetoma by Nocardia brasiliensis patients (b) and (e); and Actinomadura madurae
patients (c) and (f). PBMC were stimulated with 20 µg/mL phytohaemaglutinin in RPMI, for 24 h at 37˚C and 5 %
CO2, then the cells were stained with: anti-CD25PE, anti-CD4FITC, anti-CD8PE and anti-CD3PerCP. Percentages of
T lymphocytes (gated on CD3+) expressing CD25 were measured by flow cytometry.
are proteins that regulate the origin, proliferation and
activation of the immune cellular response.
There is a wide range of disease severeness for many
human pathologic conditions; it is likely to be a spec-
trum of cytokine production, imbalance, or cytokine
dysregulation, at different stages of the disease. In infec-
tions like mycetoma, IFN-γ may induce or activate bac-
tericidal mechanisms of infected macrophages and clear
the infection. It may induce TH1 lymphocyte prolifera-
tion, activate macrophages and reduce the production of
other interleukins such as IL-4 and IL-10. In an experi-
mental actinomycetoma model in mice, it was demon-
strated that circulating IFN-γ levels increased 10 times
the basal levels during the 4 days of infection and de-
creased with the progression of the disease [29].
The identification of a specific cell surface marker for
type 1 or type 2 cytokine-producing cells or the ability to
stain intracellulary such cytokines would greatly facili-
tate the study of these cells and their role in human dis-
eases. Direct intracellular detection of cytokines by flow
cytometry has been accomplished, in some cases in
combination with cell surface marker expression [30]. In
this work, intracellular cytokines produced by stimulated
T lymphocytes from patients with mycetoma showed an
adequate response to activation. Similar percentages of
IFN-γ producing cells were detected in both healthy do-
nors and patients. These results demonstrate that cellular
immune response of patients is not affected by the pa-
thology, at least in these cytokines, as shown in Figure
Instead the cytokine quantification, the study of re-
ceptor activity may be a more suitable indicator of cel-
lular activation ability. The measurement of functional
receptors by the binding of labeled cytokine has been
reported [25]; in mycetoma patients, the expression of
IL-2 receptor (CD25) by PHA stimulation showed an
increase in patients, this finding demonstrates that pa-
tient’s CD3+CD4 +CD2 5+ cells are able to bind to IL-2
and consequently to be stimulated for proliferation and
activation (Table 2).
There is an inverse relationship between cell-mediated
(CMI) and humoral immunity in response to antigenic
stimuli. TH1 cells are recongnized to provide a better
helper activity for CMI whereas TH2 cells are more im-
portant for B-cell development and antibody production.
Human illnesses regarding type 1 and type 2 cytokine
imbalances may explain part of the pathology in this
disease; thus restoring the balance may ameliorate the
disease [31]. The profile of TH1-TH2 responses in my-
cetoma is unknown.
TH2 cytokines also induce IgE production and eosi-
nophilia. These responses are characteristic of helminth
infections, atopic disorders, and uncontrolled infections
with intracellular pathogens (e.g. visceral leishmaniasis
and lepromatous leprosy) [32].
In this study, T-lymphocytes producing IL-4 were
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Figure 3. Intracelular cytokines produced by stimulated T lymphocytes of healthy donor (a) and (d), Nocardia
brasiliensis (b) and (e), and Actinomadura madurae patients (c) and (f). Whole blood was incubated with 25 ng/mL
PMA, 1 µg/mL ionomycin and 10 µg/mL BFA for 5H, then linage was determined by surface staining (CD3). Panels
(a), (b) and (c) are non-stimulated cells, and (d),(e) and (f), are stimulated cells.
higher only in the patients with N. brasiliensis infection; in
contrast, the cells from A. madurae patients were normal.
This fact probably indicates that TH2 profile is present
in actinomycetoma Nocardia patients. This type of im-
mune response may favor the disease progression. These
observations are in agreement with the study of El Has-
san [33] who demonstrated the TH2 cytokine pattern in
lymph nodes from patients suffering mycetoma by
Streptomyces somaliensis, as well as the cytokine pro-
duction analysis in patients with actinomycetoma caused
by Nocardia brasiliensis [34] However, further studies
in actinomycetoma are needed to determine the produc-
tion of other cytokines (TNFα, IL-10).
The authors thank M. Sc. Hector Romero Ramirez, DVM Jorge
Fernández Hernández, and QFB Victor Hugo Rosales Garcia for their
help at different stages of this work.
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