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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 91, Num. 3, 1996, pp. 381-384
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(1), May/June
1996
CD4^+ and CD8^+ T Cell Immune Responses of Immunocompetent and
Immunocompromised (AIDS) Patients with American Tegumentary
Leishmaniasis
Sergio G Coutinho^+, Alda Maria Da-Cruz, Marcia Pereira de
Oliveira, Sergio CF Mendonca, Alvaro L Bertho, Paula M De Luca
Laboratorio Imunidade Celular e Humoral, Departamento de
Protozoologia, Instituto Oswaldo Cruz, Av Brasil 4365, 21045-900
Rio de Janeiro, RJ, Brasil
Code Number: OC96075
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Key words: AIDS - tegumentary leishmaniasis -
Leishmaniabraziliensis - CD4^+/CD8^+ T cells -
cytokines
The majority of the Leishmania parasites which causes
American tegumentary leishmaniasis (ATL) in Brazil belongs to the
following species: L.braziliensis,
L.guyanensis and L.amazonensis.
However, in the area of Rio de Janeiro the only species that has
been detected infecting human and dogs is
L.braziliensis (H Momen et al. 1983 J Cell
Biochem 70 (Suppl): 28, G Grimaldi Jr et al. 1989
Am J Trop Med Hyg 41: 687). Transmission occurs
when the respective Phlebotominae vector inoculates the
promastigote forms of the parasite into the dermis of the
mammalian host during its blood meal. The promastigote forms are
then internalized by dermal phagocytic cells and transformed into
amastigote forms. Macrophages represent the preferential habitat
for the amastigote forms and the principal effector cells for
parasite destruction as well.
The host/parasite relationship can either be driven toward the
cure and destruction of the parasite or toward active disease
with production of tegumentary lesions, depending on the higher
or lower macrophage capacity for parasite destruction. Many
studies utilizing the mouse model (SG Coutinho et al. 1984
Parasite Immunol 6: 157, RG Titus et al. 1984 Clin Exp
Immunol 55: 157, P Heinzel et al. 1989 J Exp Med 169:
59, FY Liew et al. 1989 Eur J Immunol 19: 1227, RG Titus
et al. 1989 J Exp Med 170: 2097, RM Locksley et al. 1991
Res Immunol 142: 28, P Scott 1991 Exp Parasitol 75:
196, FP Heinzel et al. 1991 Proc Natl Acad Sci USA 88:
7011, SL Reiner et al. 1993 Science 259: 1457) have shown
that the T cell-mediated immune responses play a pivotal role in
these processes, either by activation of macrophages and killing
of the parasites or by inhibition of the macrophage functions.
In the first case, as observed in mouse strains resistant to
L.major infection, the Th1 CD4^+ T cell subsets are
preferentially activated with production of type 1 lymphokines
(e.g. interleukin 2 - IL-2, gamma interferon - IFN-gamma and
lymphotoxin) leading to activation of macrophages and destruction
of intracellular amastigotes. A delayed-type hypersensitivity
(DTH) to parasite antigens is elicited in resistant mice. In the
second case, as observed in mouse strains susceptible to
L.major infection, Th2 CD4^+ T cell subsets will be
preferentially activated leading to production of type 2
lymphokines (e.g. IL-4, IL-10) with inhibition of macrophage
activation allowing parasite multiplication into the
parasitophorus vacuole and aggravation of the disease. The cell
mediated immune response is depressed with negative DTH.
There is evidence that CD8^+ T cells may also play an important
role in the mechanisms of cure and resistance to
Leishmania infection, either by IFN- production and
activation of macrophages (I Muller et al. 1993 Infect
Immun 61: 33730, MMY Chan et al. 1993 Eur J
Immunol 23: 1181, I Muller et al. 1994 Infect
Immun 62: 2575, MM Stefani et al. 1994 Eur J
Immunol 24: 746) or by a cytolitic effect of CTL cells
upon parasitized macrophages (Coutinho et al. loc. cit.,
F Conceic o-Silva et al. 1994 Eur J Immunol 24:
2813) or by a combination of both effects.
ATL IN IMMUNOCOMPETENT PATIENTS
Studies in humans have not shown a such well delimited poles
for the immune responses in ATL, in spite of some similarities
with the mouse model (J Convit 1974 Ethiopian Med J
12: 187, M Castes et al. 1983 Clin Immunol
Immunopathol 27: 176, EM Carvalho et al. 1985 J
Immunol 135: 4144, SCF Mendonca et al. 1986 Clin
Exp Immunol 64: 269, SG Coutinho et al. 1987 Mem
Inst Oswaldo Cruz 82 (Suppl I): 214, M Castes et al.
1988 J Clin Microbiol 26: 1207, F Conceic o-Silva
et al. 1990 Clin Exp Immunol 79: 221, C Pirmez et
al. 1990 J Immunol 145: 3100, A Barral et al. 1993
Proc Natl Acad Sci USA 90: 3442, J Convit
et al 1993 Trans R Soc Trop Med Hyg 87: 444).
Probably in the human cases of natural resistance to
dermatropic Leishmania species, without lesions, a typical
Th1 CD4^+ immune response may occurs with production of IFN-
(type 1 lymphokine) like in resistant mouse strains (M Kemp et
al. 1994 Clin Exp Immunol 96: 410). In endemic
areas of leishmaniasis in South America, cases of positive DTH
without lesion or typical scars have been found, pointing to the
occurrence of naturally resistant individuals (WJS Souza et al.
1992 Acta Tropica 52: 111, J Weigle et al. 1993
J Infect Dis 168: 699).
On the other hand, a small percentage of patients can develop
severe forms of ATL in endemic areas in Brazil: the diffuse
cutaneous leishmaniasis (DCL) and the mucocutaneous leishmaniasis
(MCL).
DCL is produced mainly by L.amazonensis. Nodular
disseminated lesions appear, full of parasitized macrophages.
A parasite specific cellular immune depression occurs, with
production of type 2 lymphokines (IL-4, IL-10), as well as
negative DTH, similarly to the L.major infection in
susceptible mouse strains (Castes et al. loc. cit., J
Convit et al. loc. cit., G Caceres-Dittmar et al. 1993
Clin Exp Immunol 91: 500).
MCL is produced mainly by L.braziliensis. Secondary
metastatic mucosal lesions occur in the face (nose, mouth) with
a extreme scarceness of parasites. The T cell-mediated immune
response is frequently enhanced with exacerbated hypersensitivity
(Carvalho et al. loc. cit., Coutinho et al. 1987 loc.
cit. Castes et al. loc. cit.). There are very few
experimental studies on this clinical form, because mice are
naturally resistant to experimental infection with
L.braziliensis.
The most common clinical form of ATL is the localized cutaneous
leishmaniasis (LCL) where a single or a few skin ulcers occur
with a tendency toward self healing or susceptibility to the
classical antimonial therapy. The scarceness of parasites in the
lesions and the presence of hypersensitivity to leishmanial
antigens (DTH) are the main immunoparasitological feature of
LCL.
Studies from our laboratory (AM Da-Cruz et al. 1994
Infect Immun 62: 2614, SG Coutinho et al.
manuscript in preparation) on LCL patients examined before
antimonial therapy (active disease) and at the end of the therapy
(cure) have shown that the lymphoproliferative responses (LPR)
of peripheral blood mononuclear cells (PBMC) after stimulation
in vitro with total L.braziliensis promastigotes
antigens, as measured by 3H thymidine incorporation, were not
significantly different before therapy (stimulation indices - SI
= 31.9 +/- 29.4) and at the end of the therapy (SI = 15.0 +/-
16.0) although a tendency to decline has been observed.
The phenotypes of leishmanial antigen-reactive T cell-
stimulated in vitro were also investigated in PBMC
cultures. After five days in culture, blast cells were separated
by centrifugation over a discontinuous Percoll (Sigma Chemical
Co., MO, USA) gradient incubated in the presence of monoclonal
antibodies for CD3^+ (T3-RD1, Coulter Immunology, FL, USA), CD4^+
(T4-FITC, Coulter Immunology) and CD8^+ (T8-RD1, Coulter
Immunology) and finally analyzed by flow cytometry. The
supernatant of each culture was also collected and stored at -70
C until use for determination of cytokine concentrations.
Comparing the proportions of CD4^+ and CD8^+ L.braziliensis-
reactive blast T cells before therapy (BT) and at the end of
therapy (ET) we observed an increase in the percentage of CD8^+
cells (BT=23.9 +/- 11.7; ET=42.6 +/- 21.7; p < 0.05), a
decline in the proportion of CD4^+ cells (BT=61.2 18.3; ET=40.9
+/- 21.7; p < 0.05) and a consequent reduction in the
CD4^+/CD8^+ ratio (BT=2.5; ET=0.9).
These results suggested that CD8^+ T cells could be implicated
in the mechanisms of cure of LCL. However it was not clear
whether the process of cure was associated only with the
increased percentages of CD8^+ Leishmania-reactive T cells
or whether it also depended on the balance between CD4^+ and
CD8^+ cells.
In this group of patients (and in another group of LCL patients
further studied) the levels of IFN-gamma and IL-4 production by
Leishmania-reactive T cells were determined, by testing
the supernatant of antigen-stimulated PBMC cultures. IFN-gamma
was measured by a solid-phase enzyme-linked immunosorbent assay
(ELISA test kit for quantification of human IFN-gamma; Holland
Biotechnology, Holland), and IL-4 was also measured by an ELISA
test (Intertest 4 - Genzyme Corporation, MA, USA). The mean
levels of IFN-gamma in supernatants from Leishmania-
stimulated cell cultures were 123.7 +/- 58.6 U/ml before therapy
(active disease) and 193.4 +/- 70 U/ml at the end of therapy
(cure). The mean levels of IL-4 at the same occasions were
respectively BT=415.8 +/- 633.1 pg/ml and ET= not detectable.
Hence in association with the CD4^+- CD8^+ switch in cured
patients it was also observed a slight, but not significant
increase of IFN-gamma production at the end of therapy, as well
as a striking significant decrease in the IL-4 production in
cured patients. Thus active LCL was characterized by
predominance of a CD4^+ T cell response with production of a
mixed type 1 (IFN-gamma) and type 2 (IL-4) cytokine profile. On
the other hand the process of cure was associated with a
predominance of a CD8^+ T cell response, with production of IFN-
gamma and absence of IL-4, characterizing an apparently
beneficial type 1 cytokine profile.
The lymphokine profiles determined in the skin lesions of
active cases of ATL (C Pirmez et al. 1993 J Clin Invest
91: 1390, Caceres-Dittmar et al. loc. cit.) have
also shown a mixture of type 1 and type 2 lymphokines with
relatively predominant of mRNA for type 1 lymphokines.
ATL IN IMMUNOCOMPROMISED PATIENTS (AIDS)
Patients in a state of immunosupression produced by the
acquired immunodeficience syndrome (AIDS) when also infected with
Leishmania parasites, display clinical signs and symptoms
that are either novel or more severe than usual. There are
several reports of AIDS patients who acquired visceral
leishmaniasis apparently as an opportunistic disease (R Badaro
et al. 1986 Lancet I: 647). Several cases of ATL and AIDS
have been described, all of them displaying unusual
immunopathological features (JR Coura et al. 1987 Mem Inst
Oswaldo Cruz 82: 581, M Scaglia 1989 Trans R Soc
Trop Med Hyg 83: 338, RMC Cunha et al. 1991 Rev
Soc Bras Med Trop 24 (Suppl 2): 109, E Machado et
al. 1992 Mem Inst Oswaldo Cruz 87: 487).
We have studied two cases of ATL/AIDS associated diseases: the
first one (AM Da-Cruz et al. 1992 Trans R Soc Trop Med Hyg
86: 511) displayed many lesions most of them with a
pustulonodular aspect, and high parasite load. A clear
depression of the T cell-mediated immune responses to
Leishmania-antigens occurred as detected by negative
intradermal test (DTH) and the absence of a lymphoproliferative
response to leishmanial antigens (SI=1.2). This picture is
similar to that observed in classical DCL. The HIV infection
provokes a decrease in the pool of circulating CD4^+ cells
leading to generalized immune depression (AIDS). Thus, the DCL-
like picture in our patient was probably related to the inability
of his T cell-mediated immune response to control the spread of
the Leishmania infection.
The second case (AM Da-Cruz et al., manuscript in preparation)
displayed most of the ATL lesions in the face and presented
resistance to the classical antimonial therapy. Surprisingly,
when a combined Leishmania antigen-immunotherapy
associated with antimonial was tried (W Mayrink et al. 1992
Parassitologia 34: 159), the patient had clinical cure of
the ATL lesions, in spite of no apparent clinical improvement of
the HIV infection. The lymphocyte proliferative response induced
by leishmanial antigens which was negative before the combined
therapy (active ATL lesions) became positive after that therapy
(healed ATL lesions). The majority of the antigen-responding
cells after therapy belonged to the CD8^+ phenotype as measured
by flow cytometry. The levels of IFN-gamma in the supernatants
of the antigen stimulated PBMC cultures were also not detectable
before therapy and positive after therapy.
These results suggest that activation of CD8^+ T cells and
production of IFN-gamma may have a beneficial effect in ATL,
although we can not reject the possibility that the IFN-gamma
detected in the culture supernatants would be produced by other
cell types (e.g.: natural killer cells).
FINAL COMMENTS
We have shown that cure in LCL either in immunocompetent
individuals or in immunocompro-mised patients (AIDS), can be
associated with predominant CD8^+ T cell activation and
production of IFN-gamma in vitro. This does not mean that
CD4^+ Leishmania-reactive T-cells are not important in the
mechanisms for healing of lesions, because the observed decrease
in the CD4^+ subpopulations in AIDS patients aggravates the
parasitic disease.
Three hypothesis at least could arise to explain the
immunological changes observed after therapy: (a) the effect of
therapy and the CD4^+ T cells functions (production of mixed type
1 and type 2 lymphokines) led to decreased parasite load and
healing of lesions. In this case CD8^+ T cells would represent
an epiphenomenon, just replacing the actually effective CD4^+ T
cells, which would suffer apoptosis after their activation.
However, the second AIDS patient mentioned above had a tendence
to cure associated with CD8^+ and not CD4^+ T cell responses.
Moreover, results from our laboratory (SCF Mendonca et al. 1995
Am J Trop Med Hyg 53: 195) on vaccination
of human volunteers with a crude promastigote leishmanial vaccine
(W Mayrink et al. 1979 Trans R Soc Trop Med Hyg 73: 385)
have shown that the majority of the Leishmania-responding
T cells in assays of lymphopro-liferative response to the
parasite antigen belonged to the CD8^+ phenotype. In this case
the CD8^+ T cell response seems to be involved in the mechanisms
of protective immunity since there is evidence that the vaccine
is able to induce protection in approximately 50% of cases (CMF
Antunes et al. 1986 Int J Epidemiol 15: 5732); (b) CD8^+
T cell would play an important role for cure of leishmaniasis by
production of type 1 lymphokines leading to immunomodulation of
hypersensitivity and/or activation of macrophages for parasite
destruction; (c) there is also evidence that CD8^+ T cells can
have a cytotoxic effect (CTL) on parasitized macrophages, with
a beneficial effect on the follow-up of the disease (Conceic o-
Silva et al. loc. cit.). However, this effect when
exacerbated, could be detrimental for the patient (A Barral et
al. 1993 Mem Inst Oswaldo Cruz 88 (Suppl): 29).
Transmission of ATL is mainly restricted to silvatic or
periurban areas. However, because of the expansion of HIV
infection in Brazil, a rise in the frequency of ATL/AIDS
associated infections could occur and consequently higher number
of severe forms of the disease.
Supported by grants from the UNDP/World Bank/WHO Special Program
for Research and Training in Tropical Diseases, The European
Community, Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico (CNPq) and Coordenadoria de Apoio a Pesquisa e Ensino
Superior (CAPES).
^+Corresponding author. Fax: 55-21-280.1589
Received 7 December 1995
Accepted 10 January 1996
Copyright 1996 Fundacao Oswaldo Cruz
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