Parasite Immunology, 2009, 31, 432–439 DOI: 10.1111/j.1365-3024.2009.01125.

Recruitment of CD8+ T cells expressing granzyme A is associated

with lesion progression in human cutaneous leishmaniasis

D. R. FARIA,1 P. E. A. SOUZA,2 F. V. DURffiES,1,3 E. M. CARVALHO,4 K. J. GOLLOB,3 P. R. MACHADO4 & W. O. DUTRA1

1
Department of Morphology, ICB, Universidade Federal de Minas Gerais, Minas Gerais, Brazil, 2Dentistry School, Pontif"cia Universidade
Cat#lica de Minas Gerais, Minas Gerais, Brazil, 3Department of Biochemistry and Immunology, ICB, Universidade Federal de Minas
Gerais, Minas Gerais, Brazil, 4Immunology Service, HUPES, Universidade Federal da Bahia, Bahia, Brazil

SUMMARY INTRODUCTION
Human infection with Leishmania braziliensis leads to the Leishmaniasis, caused by infection with parasites of the
establishment of cutaneous leishmaniasis (CL), character- Leishmania genus, affects millions of individuals worldwide
ized by the appearance of skin lesions that progress from causing serious morbidity and mortality. Individuals
nonulcerated to ulcerated forms. Our goal was to character- infected with Leishmania present with different clinical
ize the immunological kinetics associated with this progres- forms, depending on the species ⁄ strain of the parasite, as
sion, comparing the cellular composition, cytokines and well as characteristics of the host’s immune response (1).
granzyme expression between lesions of patients with early In Brazil, L. braziliensis is the most prevalent species, and
(E-CL) and late stages (L-CL) of CL. Histopathological can cause at least two major clinical forms: cutaneous
analysis showed that lesions from L-CL had more exuberant leishmaniasis (CL) and mucosal leishmaniasis (ML). CL is
inflammatory infiltrate as compared to E-CL. Although the most frequent form and is clinically characterized by
E-CL and L-CL lesions were predominantly mononuclear, the presence of a single skin ulcer with elevated borders
lesions from E-CL patients presented higher neutrophil and that can progress to spontaneous healing (2). While the
eosinophil counts than L-CL. While percentages of CD4+ self-healing of CL lesions appears to be associated with
and of CD68+ cells were slightly higher in L-CL, a fivefold natural resistance, the immunological mechanisms of resis-
increase of CD8+ cells was observed in L-CL, as compared tance have not been clearly defined. Nonself-healing
to E-CL. Moreover, CD8+ T-cells from L-CL expressed sig- lesions usually respond well to antimonial administration,
nificantly higher levels of granzyme A than E-CL. Interest- although this treatment is toxic to patients (3). We have
ingly, granzyme A expression was positively correlated with previously shown that early treatment fails to prevent
intensity of the inflammatory infiltrate in L-CL but not ulcer formation in CL (4). Thus, understanding the immu-
E-CL. Lastly, percentages of IFN-c+ and IL-10+ cells were nological kinetics of lesion progression in CL is critical for
higher in L-CL as compared to E-CL, with CD4+ T-cells better understanding the establishment of pathology and
and CD68+ monocytes as the main sources of these cyto- how it can be prevented.
kines, respectively. These results suggest that recruitment of Different patterns of cytokine expression by T cells are
CD8+ granzyme A+ T cells is involved in lesion progression related with severity of L. braziliensis infections in humans.
in human CL. It has been demonstrated that circulating CD4+ T cells of
CL patients, stimulated in vitro with soluble Leishmania
Keywords CD8+ T cells, granzyme, leishmaniasis, lesion, antigen, express IFN-c, but not IL-4 and IL-5, indicating
progression the presence of specific Th1 cells in this form of the illness
(5). Moreover, analysis of intralesional cytokine gene
expression showed that the Th1 cytokine mRNAs (IL-2,
Correspondence: Dra Walderez Ornelas Dutra, Departamento de IFN-c and lymphotoxin) were present in CL (6,7). Coutin-
Morfologia, ICB-Universidade Federal de Minas Gerais, Avenida ho et al. (8) have demonstrated a predominant production
Presidente Ant$nio Carlos 6627, Pampulha, Caixa Postal 486, of IFN-c by T cells from cured CL patients, suggesting a
CEP 31Æ270-901, Belo Horizonte, Minas Gerais, Brazil
(e-mail: waldutra@gmail.com).
protective role for this cytokine in CL (9). However, ML, a
Received: 24 November 2008 more aggressive clinical form that can also arise from
Accepted for publication: 19 March 2009 L. braziliensis infection, is associated with exuberant

432 ! 2009 Blackwell Publishing Ltd

Volume 31, Number 8, August 2009 Lesion progression in cutaneous leishmaniasis

production of IFN-c and TNF-a (10) and a deficient con- a positive skin Montenegro test, parasite isolation and ⁄ or
trol of inflammation due to low expression of IL-10 recep- histopathological analysis to confirm a diagnosis of CL.
tor (11). Moreover, we have shown that expression of Patients were classified as E-CL (approximately 15 days of
granzyme A is evident in lesions from CL and is even more illness, nonulcerated lesion) or L-CL (approximately
abundant in the severe ML form, suggesting a role for this 60 days of illness, ulcerated lesion), as previously estab-
molecule in tissue destruction (11). It is clear that a balance lished by us (4). For all cases, parasite species were typed
between immunoregulatory mechanisms that lead to para- to confirm that the disease was due to L. braziliensis infec-
site control vs. tissue destruction is critical for determining tion. E-CL patients enrolled in this study (n = 6) or L-CL
pathology or protection in human leishmaniasis. patients (n = 9) presented with a single nonulcerated or
With regard to the clinical evolution of CL, previous ulcerated lesion, respectively, and had not been previously
studies have shown that circulating T cells from patients diagnosed with or treated for leishmaniasis. At the time of
with early L. braziliensis infection, characterized by sample collection, the time of the active lesions were
approximately 15 days of illness and the presence of estimated among 15 (E-CL) or 30–60 days (L-CL), as
nonulcerated lesion, displayed a down-modulated Th1- reported by the patients themselves. Treatment was offered
type response as compared to cells from patients with late- to all patients as needed despite their enrolment in this pro-
stage CL (L-CL), characterized by approximately 60 days ject and was administered after sample collection. E-CL
of illness and the presence of ulcerated lesion (12). Under- and L-CL patients were not under treatment when samples
standing the kinetics of establishment of the cutaneous were collected. Lesions were collected at the Corte de Pedra
lesion, through the determination of which cells are healthcare facility. The Ethical Committees of Universidade
recruited to the lesion site, what is the level of expression Federal de Minas Gerais and Universidade Federal da
of immunomodulatory cytokines and cytotoxic molecules Bahia approved all procedures involved in this study.
will provide new insight towards the understanding of the
mechanism involved with the pathology associated with
Sample obtention
human CL. Thus, the aim of this study was to compare
the cellular composition, cytokine and granzyme expres- Skin biopsy specimens were taken from the borders of
sion in lesions of patients with early CL (E-CL) and active lesions using a 4-mm-diameter punch, as routinely
L-CL, to better understand the immunological mecha- done by us and others, as the central area of the lesion
nisms involved in the clinical evolution of CL. Our data comprises necrotic and haemorrhagic areas. In the case of
show that E-CL and L-CL are predominantly composed early lesions, biopsies were collected from the papule of
of mononuclear cells, although E-CL has a higher fre- the nonulcerated lesions, after the application of a local
quency of polymorphonuclear (PMN) cells. Moreover, we anaesthetic. Lesions were maintained in a 30% sucrose
demonstrated that the more exuberant inflammatory infil- solution for 30 min at 4"C and then transferred to OCT
trate observed in L-CL is consistent with lesion ulceration Tissue Tek freezing medium and immediately placed in
and is associated to higher IFN-c expression and the dry ice. The material was stored at )70"C until analysis.
recruitment of CD8+ T cells expressing granzyme A.
Importantly, an ongoing immunoregulation is present,
Histological and immunofluorescence staining
characterized by the increased frequency of IL-10 in
L-CL, consistent with further lesion resolution usually Individual 4–5 lm cryosections were placed in silane-pre-
observed in CL patients. coated slides and fixed for 10 min with acetone. Slides
were incubated with phosphate-buffered saline for 30 min
and subjected either to haematoxylin–eosin (HE) staining
MATERIALS AND METHODS
or to immunofluorescence staining using specific monoclo-
nal antibodies. Standard HE staining was performed to
Patients
ensure tissue integrity as well as for the evaluation of the
The patients analysed in this study were from Corte de intensity and composition of the inflammatory infiltrate.
Pedra, an endemic area for L. braziliensis, located 280 km Immunofluorescence reactions involved incubation with
south-west of Salvador, in the state of Bahia, Brazil. All fluorescein isothiocyanate (FITC)- and phycoerythrin
patients were volunteers, and informed consent was (PE)-labelled monoclonal antibodies directed to surface
obtained from all individuals prior to collection of lesion receptors (CD4 clone S3Æ5, CD8 clone 3B5 or CD68 clone
material. Diagnosis of leishmaniasis was performed based Ki-M7) and intracellular molecules (granzyme A clone
on clinical and laboratory criteria. Detection of suggestive CLB-GA28, IFN-c clone or B27, IL-10 clone 9D7) respec-
popular or ulcerated cutaneous lesions was associated with tively. Sections were incubated with antibodies mixture

! 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 432–439 433

D. R. Faria et al. Parasite Immunology

overnight at 4"C. After staining, preparations were exten- each patient, and then the values were averaged for each
sively washed with phosphate-buffered saline, counter- group. The results are representative of two experiments per
stained with 4¢,6¢-diamidino-2-phenylindole (DAPI), and patient.
mounted using Antifade mounting medium (Molecular
Probes, Carlsbad, CA, USA). Slides were kept at 4"C,
Statistical analysis
protected from light, until acquisition in a laser scanning
confocal microscope (Zeiss, Thornwood, WV, USA). Statistical analysis of the data was performed using JMP
Isotype controls were analysed separately to confirm the statistical software from SAS (Cary, NC, USA) and
lack of nonspecific staining. Monoclonal antibodies BioEstat 3.0 statistical software. The comparisons of
were purchased from Caltag (Burlingame, CA, USA). percentage for a given parameter between the groups were
performed using the nonparametric t-test or Mann–
Whitney test. Spearmann correlation analysis test was also
Light microscopy and confocal analysis
performed. Results were considered statistically different
Haematoxylin–eosin stained sections were analysed using when the analysis returned a P < 0Æ05.
a light microscopy (Axiovert, Zeiss). We acquired the data
using a power magnification of 400·, and the frequencies
RESULTS
of neutrophils, eosinophils and mononuclear cells were
expressed as absolute numbers or percentage of the total
Distinct inflammatory profiles in E-CL and L-CL
cell count. A total of 16 fields ⁄ sample were acquired for
lesions
the histological analysis.
Confocal analysis were performed using a Meta-510 Zeiss The composition of the inflammatory infiltrate in lesions
laser scanning confocal system running LSMix software from E-CL and L-CL patients was determined using con-
coupled to a Zeiss microscope (Axiovert 100) with an oil ventional histological analysis as described in ‘Materials
immersion Plan-Apochromat objective (63·, 1Æ2 numerical and methods’. Sections stained with HE from E-CL
aperture) and Bio-Rad (Hercules, CA, USA) MRC 1024 patients were compared to L-CL patients. Histopathologi-
laser scanning confocal system running LaserSharp 3.0 soft- cal analysis of lesions from patients with both stages of
ware coupled to a Zeiss microscope (Axiovert 100) with a CL showed a keratinized stratified squamous epithelium,
water immersion objective (40·, 1Æ2 numerical aperture). A presenting hyperkeratosis, parakeratosis, acanthosis and a
water-cooled argon UV laser (488 nm) or a krypton ⁄ argon large number of cells with hydropic degeneration in the
laser was used to excite the preparation (through its 363, prickly layer (not shown). Diffuse chronic inflammation
488 or 568 nm line), and light emitted was selected with with predominantly mononuclear cell infiltration was
band-pass filters (522 ⁄ 35 for FITC or 598 ⁄ 40 for PE). For observed in the dense connective tissue (Table 1) classified
DAPI visualization a mercury lamp were used to excite the as productive and exudative type. Quantitative analysis
preparation (through its 20 ⁄ 80 nm line), and light emitted showed that lesions from patients with E-CL presented
was selected with band-pass filters (363 ⁄ 90 for DAPI). For higher percentage of PMN cells (P = 0Æ02), neutrophils
each section, the inflammatory infiltrate present in the (P = 0Æ04) and eosinophils (P = 0Æ01) when compared to
connective tissue adjacent to the epithelia was located and lesions from L-CL (Table 1). However, the frequency of
an area presenting with an uniform infiltrate was selected PMN cells in both groups was very low in relation to the
for analysis. Within this inflammatory area, a minimum of total inflammatory infiltrate (<2%). It was observed that
six images (fields) were collected. Image analysis and pro- the intensity of the inflammatory infiltrate (number of
cessing were performed with LSMix (Zeiss) or LaserSharp inflammatory cells per field) was higher in lesions from
(Bio-Rad), Confocal Assistant, Adobe Photoshop and patients with L-CL than E-CL (P = 0Æ004) (Table 1).
Image Tool software. Analyses were performed by counting
the total number of cells in six to nine fields acquired and
Lesions from L-CL display a higher proportion of CD8+
calculating the average of cells number per field for each
cells than E-CL lesions
patient. This procedure was performed for each parameter
analysed, allowing determination of the total number of Analysis of the frequencies of CD68+, CD4+ and CD8+
inflammatory cells (total number of DAPI+ cells within the cells was performed using confocal microscopy. The abso-
inflammatory infiltrate), the number of FITC or PE single- lute numbers of these cell populations increased in L-CL,
positive cells, and the number of double-positive cells. The as compared to E-CL, compatible with the more exuberant
counts were performed blindly, the results were expressed as inflammatory infiltrate observed in the former group. Thus,
the average of cells number per field for each parameter for we determined the percentages of these populations within

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Volume 31, Number 8, August 2009 Lesion progression in cutaneous leishmaniasis

Table 1 Total number of inflammatory cells per area and frequencies of mononuclear and polymorphonuclear (neutrophil and eosinophil)
cells in lesions from patients with early cutaneous leishmaniasis and late cutaneous leishmaniasis

E-CL (n = 6) L-CL (n = 12)

Parameter Mean Median Minimum Maximum Mean Median Minimum Maximum P-value

Number of total cells ⁄ field 200Æ96 188Æ87 118Æ44 302Æ34 388Æ74 395Æ63 233Æ75 551Æ09 0Æ0037
% Mononuclear cells 97Æ92 97Æ97 97Æ13 98Æ45 98Æ66 98Æ95 96Æ83 99Æ45 0Æ0492
% Polymorphonuclear cells 2Æ73 2Æ83 2Æ17 3Æ25 1Æ61 1Æ17 0Æ66 3Æ80 0Æ0192
% Neutrophils 2Æ08 2Æ03 1Æ55 2Æ88 1Æ35 1Æ05 0Æ55 3Æ17 0Æ0492
% Eosinophils 0Æ65 0Æ58 0Æ34 1Æ22 0Æ26 0Æ18 0Æ07 0Æ62 0Æ0131

Haematoxilin–eosin stained sections were analysed using light microscopy. All connective tissue from each lesion was evaluated in a power
magnification of 400·. The comparisons of percentage for a given parameter between the groups were performed using the nonparametric
Mann–Whitney test.
E-CL, early cutaneous leishmaniasis; L-CL, late cutaneous leishmaniasis.

the infiltrates of both forms, to access the contribution of quency of cells expressing granzyme A, a cytolytic mole-
each population to the overall inflammatory infiltrate, com- cule, in E-CL and L-CL lesions. Our results showed that
paring between E-CL and L-CL. Our analysis showed that the expression of granzyme A by cells within the inflam-
the percentages of CD4+ cells and CD68+ cells were higher matory infiltrate was over five times higher in L-CL that
in L-CL as compared to E-CL (Figure 1). Strikingly, a five- E-CL (Figures 2 and 3b). Moreover, while approximately
fold increase in the percentage of CD8+ cells was observed 20% of the CD8+ cells from E-CL expressed granzyme A,
in L-CL when compared to E-CL group (Figure 1). approximately 50% of the CD8+ cells from L-CL
expressed this cytolytic molecule. Interestingly, a positive
correlation was observed between the intensity of the
Recruitment of CD8+ T cells expressing a cytolytic
inflammatory infiltrate and the number of CD8+ gran-
molecule, granzyme A, is associated with lesion
zyme A+ cells in lesions from L-CL but not E-CL (Fig-
ulceration in CL
ure 2). These results suggest that the expression of
To evaluate whether the establishment of CL lesions was granzyme A by CD8+ T cells is involved in tissue destruc-
associated to cytotoxic responses, we determined the fre- tion during the progression of CL to the ulcerated stage.

E-CL
E Ongoing immunoregulatory mechanism is observed dur-
L-CL ing the evolution of CL lesions
40 *
35 * The frequencies of cells expressing the pro-inflammatory
cytokine IFN-c or the anti-inflammatory cytokine IL-10
Positive cells (%)

30
were determined using confocal analysis. The results
25
showed that the percentages of cells expressing IFN-c and
20 IL-10 were higher in L-CL as compared to E-CL (Fig-
15 ure 3a and b). However, no differences were observed as
10 for the sources of these cytokines between the groups, with
CD4+ and CD68+ cells as the main sources of IFN-c and
5
IL-10 respectively.
0
CD4 CD8 CD68
Figure 1 Percentages of T CD4+, T CD8+ and CD68+ cells in the DISCUSSION
inflammatory infiltrate from early cutaneous leishmaniasis (E-CL)
(n = 6) and late cutaneous leishmaniasis (L-CL) (n = 9) lesions. The most common clinical form of the disease caused by
Frozen tissue sections were stained with FITC-labelled anti-CD4, L. braziliensis is localized CL, which is characterized by
anti-CD8 or anti-CD68 monoclonal antibodies and were counter- single or multiple ulcerated dermal lesions that usually
stained with DAPI as described in ‘Materials and methods’. heal spontaneously. Patients with a short period of illness
Results are expressed as bars of the mean percentages for each
group. Standard deviation indicated by above line bars. Asterisks
(<2 weeks) have acneiform lesions or small bleeding ulcers
indicate statistically significant differences between groups at that clearly differ from the classical CL ulcer observed in
P < 0Æ05. patients with L-CL. We have previously shown that early

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 D. R. Faria et al. Parasite Immunology

(a) 80
70 * *
60
Cells (%)

50
40 E -CL
L -CL
30
20
10
0
Gr A+ cells Granzyme A expression
within CD8+ cells
(b)
Figure 2 (a) Percentages of cells express-
CD8+Granzyme A+ cells

15 60 ing granzyme A (Gr A+) and % gran-

R2 = 0·08 R2 = 0·74
12·5 p = 0·62 50 p = 0·0026 zyme A expression within CD8+ T cells in
early cutaneous leishmaniasis (E-CL)
10 40
(n = 6) and late cutaneous leishmaniasis
7·5 30 (n = 9) lesions. Asterisks indicate statisti-
5 20 cally significant differences between
groups at P < 0.05. (b) Correlation
2·5 10
E-CL L-CL analysis between the frequency of CD8+
0 0 granzyme A+ cells and intensity of
50 100 150 200 250 300 0 500 1000 1500 2000 2500
inflammatory infiltrate in lesions from
Number of inflammatory cells E-CL and L-CL.

treatment of CL fails to prevent ulcer development (4). In the early phases of L. braziliensis infection. Moreover, they
the present study, we compared the cellular composition, suggested that this phenomenon would allow the parasite
cytokine and granzyme A expression in lesions between survival and growth, leading to the development of disease.
individuals with E-CL and L-CL to better understand the We observed that the intensity of inflammation was
progression of this disease towards lesion formation. higher in lesions from patients with L-CL than E-CL,
The composition of the inflammatory infiltrate in compatible with lesion progression. Furthermore, this
lesions from E-CL and L-CL patients was determined increased inflammatory infiltrate was associated with a
using conventional histological analysis, and it was verified striking recruitment of CD8+ T cells to L-CL lesions. We
that both clinical forms showed diffuse chronic inflamma- hypothesized that this increased inflammatory infiltrate
tion, with the presence of dense fibrous connective tissue was associated with the expression of the inflammatory
classified as productive and exudative type with a predom- cytokine IFN-c. IFN-c has been considered a critical cyto-
inantly mononuclear cellular infiltrate. Moreover, lesions kine involved in the pathogenesis of CL, due to its parasiti-
from patients with E-CL presented higher frequencies of cidal ability and also due to its inflammatory activity
PMN neutrophils and eosinophils when compared to (11,15). Our data showed an increase in IFN-c expression
lesions from L-CL. Previous studies had documented that in L-CL as compared to E-CL lesions, which is consistent
ulcerated lesions from CL patients are mainly composed with our hypothesis. We have previously shown that CD4+
of mononuclear cells (11,13). Although the frequency of Th1 cells are responsible for the majority of IFN-c pro-
PMN cells is low in relation to mononuclear cells (<2%), duction in peripheral blood mononuclear cells (PBMC)
the presence of PMN cells in E-CL suggests a role for from CL patients (5). Recently, our group demonstrated
these cells during early infection. A recent paper using the that CD4+ cells represented the majority of IFN-c-produc-
murine model of Leishmania infection has demonstrated a ing cells, followed by CD8+ cells and CD4)CD8) cells in
role for PMN cells, specifically neutrophils, in hosting the PBMC and lesions from CL patients (11,16). The analyses
parasite during early infection, favouring parasite survival performed here showed that CD4+ T cells are the main
(14). Although we did not evaluate the frequency of Leish- source of IFN-c in L-CL and also E-CL, accounting for
mania-infected PMN cells in E-CL lesions due to material over 50% of the IFN-c expression in E-CL and 75% in
limitation and preservation, it is possible that these cells L-CL. Thus, with progression of the disease, a higher fre-
also host the parasite in early stages of human infection. quency of CD4+ T cells become engaged with expression
Further analysis to evaluate parasite burden in E-CL and of this cytokine, although statistical analysis of these data
L-CL will be performed. Rocha et al. (12) showed that a did not show significance (P = 0Æ06). Our suggestion that
down regulation of the Th1-type response occurs during IFN-c is associated with disease progression is in accor-

436 ! 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 432–439

Volume 31, Number 8, August 2009 Lesion progression in cutaneous leishmaniasis

(a) 120 100

*
*

IFN-γ + cells (%)

100

IL-10+ cells (%)

80
80
60
60
40
40
20 20
0 0
E-CL L-CL E-CL L-CL
80
120

IL-10+ expression (%)

CD68+ contribution to
IFN-γ + expression (%)
70
CD4+contribution to

100 60
80 50
50
60 40
40 30
20
20 10
0 0
E-CL L-CL E-CL L-CL

(b) CD8-FITC CD4-FITC CD68-FITC

Granzyme A -PE IFN-γ-PE IL-10 -PE
% 7 +/–5 % 25 +/–9 % 21 +/–11

E-CL

% 39 +/–16* % 60 +/–27* % 50 +/–12*

L-CL

Figure 3 (a) Percentages, in early cutaneous leishmaniasis (E-CL) (n = 6) and late cutaneous leishmaniasis (n = 9) lesions, of cells express-
ing IFN-c and IL-10 and the percentage contribution of CD4+ and CD68+ cells for the expression of IFN-c and IL-10 respectively. Frozen
tissue sections were stained with FITC-labelled anti-CD4 or anti-CD68 monoclonal antibodies and with PE-labelled anti-IFN-c or anti-IL-
10 antibody, and were counterstained with DAPI as described in ‘Materials and methods’. Standard deviation indicated by above line bars.
Asterisks indicate statistically significant differences between groups at P < 0Æ05 (t-test). (b) Representative images from confocal micros-
copy analyses for determination of the frequencies of CD8+ granzyme A, CD4+ IFN-c+ and CD68+ IL-10+ cells in E-CL and L-CL
lesions. Frozen tissue sections were co-stained with FITC-labelled anti-CD8 or anti-CD4 or anti-CD68 monoclonal antibody and PE-
labelled anti-granzyme A or anti-IFN-c or anti-IL-10, and were counterstained with DAPI as described in ‘Materials and methods’. The
three optical sections for each patient were obtained simultaneously with 363, 488 and 568 nm line of the argon ⁄ krypton laser and the
proper set of filters. The overlay for CD4 or CD8 or CD68 (green), granzyme A or IFN-c or IL-10 (red) and DAPI (blue) in E-CL and
L-CL lesions is shown. The cells that are double positive for each pair of staining (CD8+ granzyme A+ or CD4+ IFN-c+ or CD8+ IL-10+)
appear in yellow. These images are representative of each group. Values represent the average € standard deviation of each group following
numeric determination of the percentage of positive cells for the indicated molecules. *Indicates statistically significant differences at
P < 0Æ05. The bar = 10 lm.

dance with previous studies showing that the higher the demonstrated a higher frequency of granzyme A expres-
frequencies of IFN-c or TNF-a producing circulating T sion in lesions from ML patients, as compared to CL (11)
lymphocytes, the larger the ulcerated lesion of CL patients and that CL lesions display a high number of TIA-1+ cells
(17). (21). In this work, we observed that the percentages of
Another important biological function of IFN-c is the granzyme A+ cells and CD8+ granzyme A+ cells were sig-
induction of cytotoxic activity, which is directly correlated nificantly higher in L-CL than in E-CL lesions. These data
with granzyme A and TIA-1 expression (18–20). Previous suggest that the higher frequency of cells expressing gran-
studies using murine infection with Leishmania major have zyme A is consistent with the extensive tissue destruction
shown that the frequency of T cells expressing granzyme observed in L-CL and the main cell subpopulation
A was significantly higher in susceptible BALB ⁄ c than in involved in this process is the CD8+, which were preferen-
resistant C57BL ⁄ 6 mice (20). Also, we have previously tially recruited to L-CL. Thus, execution of cytolytic func-

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D. R. Faria et al. Parasite Immunology

tion by CD8+ T cells may be an important mechanism of Noronha e Annamaria Vago for critical review of the
tissue destruction in CL. Moreover, this cytolytic function manuscript and valuable suggestions and Dr Jos' Barbosa
could also be important for parasite killing. While elimi- Jfflnior for valuable help with the confocal figures.
nating the parasite, cell death would occur due to cytotox-
icity and thus, tissue destruction and parasite elimination
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by the fact that ML patients do not respond to IL-10, 10 Bacellar O, Lessa H, Schriefer A, et al. Up-regulation of Th1-
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Taken together, our data indicate that the progression of observed in mucosal leishmaniasis. Infect Immun 2005; 73:
CL lesions from nonulcerated to ulcerated states is associ- 7853–7859.
ated with the presence of an intense inflammatory infil- 12 Rocha PN, Almeida RP, Bacellar O, et al. Down-regulation of
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13 Ribeiro-de-Jesus A, Almeida RP, Lessa H, Bacellar O & Carv-
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ACKNOWLEDGEMENTS into macrophages. J Immunol 2004; 173: 6521–6525.
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The authors are grateful to the financing agencies CNPq, gizes with IFN-gamma in mediating killing of Leishmania
FAPEMIG, CAPES and NIH ⁄ TMRC. We also would like major through the induction of nitric oxide. J Immunol 1990;
to thank Drs Alda Cruz, Cristina Guatimosim, F&tima 145: 4306–4310.

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Volume 31, Number 8, August 2009 Lesion progression in cutaneous leishmaniasis

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20 Moll H, M*ller C, Gillitzer R, et al. Expression of T-cell-asso- KJ. Antigen specific correlations of cellular immune responses
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