Cellular and humoral immune responses of a patient with American cutaneous leishmaniasis and AIDS

511

TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICALMEDICIKE AND HYGIENE (1992) 86, 511-512

Cellular and humoral immune leishmaniasis and AIDS

responses

of a patient

with American

cutaneous

Alda M. Da-Cruz’, Elizabeth S. Machado2, Jacqueline A. Menezes 2, Marcio S. Rutowitsch2 and Sergio G. Coutinho’ IInstituto Oswald0 Cruz, Rio deJaneiro, Brazil; 2Hospital dos Servidores do Estado do Rio deJaneiro, Brazil Abstract

The lymphocyte responsivenessto leishmanial antigens and its influence on the course of cutaneous leishmaniasis was studied in a patient with AIDS-associatedAmerican cutaneous leishmaniasis causedby Leishmania braziliensis. The patient had cutaneous disseminated erythematous papules or nodules and mucosal lesions as well as moniliasis and weight loss. The patient had a poor delayed-type hypersensitivity to leishmanial antigens, showing 3 mm of induration. The cellular immune responseswere studied in vitro by lymphocyte proliferative assaysinduced by leishmanial antigens and concanavalin A. The T cell phenotypes were analysed by flow cytometry. The peripheral blood mononuclear cells before proliferation showed an inversion of the CD4iCD8 ratio (0.28: 1). The lymphoproliferative responsesto antigen and mitogen were very low (indices <2.5). The blast-like cell phenotypes after antigen stimulation in culture were: CD3+ 44.8%, CD4+ 7.53% and CD8+ 17.45%. In AIDS patients the decreasein the pool of CD4+ cells, and consequent diminution of the CD4iCD8 ratio, produced by HIV infection provokes a generalized immune depression. The patient’s disseminated clinical picture was probably related to the inability of his T cell-mediated immune responsesto control the spreadof Leishmania infection. Introduction

New immunopathological aspectsof parasitic diseases have emerged in studies of patients who are in a state of immunosuppression. In particular, patients with the acquired immune deficiency syndrome (AIDS) who are also infected with parasitic diseasesdisplay clinical symptoms that are either novel or more severe than usual (CANN-

rose gel electrophoresis), according to GRIMALDI et al. (1990. In orde; to induce the delayed-type hypersensitivitv (DTH’, reactions to leishmanial antigens. 0.1 ml of leisl&anin i40 pg protein/ml) was injected intradermally. The DTH response (3 mm) was considered negative since, 48 h later, the induration had a diameter of less than 5 mm.

ING, 1990).

The influence of immunodepression induced by human immunodeficiency virus (HIV) on the outcome of leishmaniasis has not been studied in depth. There are several reports of immunocompromised individuals who acquired visceral leishmaniasis apparently as an opportunistic disease(BADAR~) et al., 1986;PETERSet al., 1990). Four casesof cutaneous leishmaniasis plus HIV infection have been described as far as we know (COURAet al., 1987; SCAGLIA et al., 1989; PIALOUX et al., 1990; CUNHA et al., 1991). The T cell-mediated immune response in

leishmaniasis is critical both in the processesof cure and in immunopathological mechanisms responsible for aggravating the disease(COUTINHO et al., 1987). In American cutaneous leishmaniasis (ACL), a wellmodulated cellular immune response can lead to selfhealing; on the other hand, an enhanced or depressedT cell-mediated immune response can lead to serious illness, in which mucosal or diffuse lesions may occur. KOUTINHO et al., 1987; CONCEICAO-SILVA et al., 1990). As far as we know, the present report is the f&t study both of the T cell-mediated immune responseto leishmanial antigens in a Datient with AIDS ulus ACL and of its influence on the cdurse of cutaneous ieishmaniasis. Case report

A 36 years old male, living on the outskirts of Rio de Janeiro, Brazil (registration no. 786225, HSE-RJ), presented in July 1990 with laryngeal inflammatory lesions that extended to the nasal septum and skin of the nose. Subsequently, in addition to oral moniliasis and loss of weight, he displayed disseminated erythematous papular or nodular lesions on his skin. In spite of the un;s;al clinical picture, the suspicion of ACL was confirmed when the Darasitewas detected in a biopsy of the lesions (by examination of histological preparations stained with haematoxylin and eosin and of a culture in NNN medium). The parasite was characterized asLeishmania braziliensis using species-specificmonoclonal antibodies (serodeme analysis) and isoenzyme analysis (agaAddress for correspondence: Sergio G. Coutinho, Laboratbrio de Imunidade Celular e Humoral, Departamento de Protozoologia, Pav. Carlos Chagas, 5” andar, IOC-FIOCR~Z,Av. k&4365, Caixa Postal 926, CEP 21 040, Rio de Janeiro, RJ,

Laboratory studies Indirect immunofluorescence antibody testfor leishmaniasis

Promastigote forms were used as antigens with goat anti-human immunoglobulin (Ig) G or M conjugate (Sigma). Reciprocal antibody titres 245 were considered nositive. The patients’s serum showed a positive IgG titre of 180. whereas IZM was not reactive. The susnicion of HI+ infection “arosefrom the unusual clinic;1 picture that resembled that of disseminated cutaneous leishmaniasis. This suspicion was confirmed when an enzyme immunoassav (Abbott. recombinant HIV-l and HIV-2 EIA@) and ‘&direct &munofluorescence assay using KE37-3 cells (H9 subclonal) infected with human lymphotropic virus 3 (produced by Biomanguinhos-FIOCRUZ) revealed the presenceof anti-HIV antibodies. 1

Cellular immune response

The cellular immune response to Leishmania was studied by determining the proliferative responseof peripheral blood mononuclear cells (PBMCs), induced in vitro by Leishmania antigens. PBMCs were separatedby centrifugation on a gradient of Ficoll-Hypaque (Histopaque 1077@,Sigma). The viable PBMCs were resuspended in RPM1 1640 medium (Sigma) supplemented with 10% heat-inactivated human AB Rh+ serum, 10 mM HEPES, 1.5 mM L-glutamine, 0.04 mM 2-mercaptoethanol and antibiotics (200 iuiml penicilin and 200 pg/ml streptomycin). The cells (3 x 105/well) were cultured in triplicate in round-bottomed 96-well microtitre plates (Nunc) for 5 d at 37°Cin a humidified atmosphere of 5% CO2 in air, in the presence of 4 pg concanavalin A (Pharmacia) per well or lo6 sonicated L. braziZiensis promastigotes per well. Sixteen hours before harvesting, 1 @i [3H]thymidine (Amersham) was added to each well, and the radioactivity uptake was measured in a scintillation p counter (Beckman LS 8100). Cell proliferation was expressed as stimulation indexes (SI) and was considered positive if SI22.5 ~MENDONCA et al., 1986). In parallel, the PBMCs (3 x 10 /well) were cultured in 24-well plates (Nunc) in the presenceof the sameproliferative stimuli. After 5 d of culture the blast-like cells were separatedon a Percoll@(Sigma) discontinuous gradient. Phenotypes of the PBMCs and the blast-like cells stimulated in vitro by

512 L. bruziliensis were characterized using the following monoclonal antibodies: T3-RDI, T4-FITC and T8-RDl

casesof leishmaniasis.

(Coulter). Briefly, 0.5~ lo6 washed cells were incubated

Acknowledgements

with 5 ~1 of each monoclonal antibody in 0.05% sodium

azide and 0.1% formalin (4%) in phosphate-buffered saline for 30 min at 4°C. The cells were analysed by flow cytometry (Coulter, EPICS 751@). There was a marked decreasein the number of CD4+ cells and an inversion of the CD4ICD8 ratio before stimulation with L. braziZiensis (Table). The lymphoproliferative indices of the T cell microcultures containina L. braziliensis antigens as well as concanavalin A were very low, showing that the T lymphocytes were not able to respond properly to the antigens or to the mitogen. The T cell phenotypes, after 5 d in culture with L. bruziliensis antigens, were quite different from those of HIVnegative leishmaniasis patients. Moreover, these cells could not be considered specific to L. braziliensis since stimulation indices were very the lymphoproliferative

We aregratefulto Mr P. R. Z. Antas for assistance in the la-

boratory, to Mr A. Bertho and MS M. Santiago for cell cytometry analysis, and to MS R Pelegrino for excellent secretarial assistance. This work was supported by the UNDPiWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases.bv Coordenadoria de Aooio a Pesauisa e Ensino Suuerior (CAP&) and by Conselho NaGonal de Dksenvolvimento dientifico e Tecnologico (CNPq). Badaro, R., Carvalho, E. M., Rocha, H., Queiroz, A. C. & Jones, T. C. (1986). Leishmania donovani: an opportunistic microbe associatedwith progressive diseasein three immunocompromised patients. Lancet, i, 647-648. Cannina. E. U. (1990). Protozoan infections. Transactions of the Royay‘Society of Tropical Medicine and Hygiene, 84, supplement 1,19-24.

Table. Lymphocyte surface phenotypes of peripheral blood mononuclear cells in patients with cutaneous leishmaniasis and AIDS before and after stimulation with Leishmania brazillien&

Delayed-type Lymphoproliferative hypersensitivity response* Lb CD3 response Con. A

Lymphocytesurfacephenotypes(%) Beforestimulation After stimulation CD4 CD8 Ratio CD3 CD4 CD8 Ratio

Patients Leishmaniasisplus *ms 3mm 2.0 1.2 83.49 18.60 64.84 Leishmaniasiswithout AID@ 11 mm 33.3 57.4 66.87 44.60 25.50 AIDS without leishmaniasis Not done 2.9 0.8 63.47 1.03 60.48 2.0 52.63 29.63 10.32 Healthy controls Negative 40.1 “Stimulationindices in responseto concanavatinA (Con. A) and L. braziliensis(Lb). b3 caseswith active American cutaneousleishmaniasis.

low (Table). In control patients with the active form of ACL, but without AIDS, all had high L. braziliensis stimulation indices with a predominance of CD4+ blastlike cells after proliferation (Table). Discussion The most common clinical picture of ACL produced by L. braziliensis is a single open lesion. Sometimes multiple ulcers may appear. Our patient presented an unusual clinical feature: he displayed many lesions, most of them with a pustulonodular aspect, and a high parasite load, similar to those found in other reported casesof ACL plus HIV infection (COURA et al., 1987; CUNHA et al., 1991). This clinical nicture is similar to that presented’ in classical diffuse cutaneous leishmaniasis fDCL). which. in Brazil. is usuallv caused bv L. ama,&en& In this clinical form, a clear and siecific depression of T cell-mediated immune responsesto Leishmania antigens occurs. This immunological condition leads to a high parasite load in the diffuse nodular lesions and to negative intradermal delayed-type hypersensitivity (DTH) tests, in the absenceof a lymnhoproliferative responseto leishmanial antigens andwiihout any alteration in the CD4/CD8 ratio in ueriuheral blood (CASTESet al.. 1988). In AIDS patients, on the other hand,‘HIV infection provokes a decreasein the pool of circulating CD4+ cells and a conseauent decreasein the CD4iCD8 ratio. leading: to generalized immune depression. Thus, the DCL-liki cliGica1 picture in our patient was probably related to the

inabilitv of his T cell-mediated immune resnonseto control the-spreadof the Leishmania infection. ’ The high anti-Leishmaniu IgG antibody titre observed in this patient corroborates the hypothesis that humoral immunity per seis not able to bring about a cure for leishmaniasis. In Brazil, HIV infection occurs mainly in big cities, whereas ACL transmission is mainly confined to silvatic or periurban areas. However, given the current expansion of HIV infection, it is possible that there will be a rise in the number of AIDS caseswith this kind of disease association and, consequently, in the number of severe

0.28 1.74 0.01 2.87

44.87 84.19

7.53 83.27

17.45 9.91

0.43 14.0

No proliferation No proliferation

Castes, M., Cabrera, M., Trujillo, D. & Convit, J. (1988). T cell sub-populations, expression of interleukin-2 receptor and production of interleukin-2 and gamma interferon in human American cutaneous leishmaniasis. 7ournal of Clinical Microbiology, 24, 1207-1213. Conceiclo-Stlva, F., Dbrea, R. C. C., Pirmez, C., Schubach, A. & Coutinho, S. G. (1990). Quantitative studv of Leishmania braziliensis braziliensis reactive T cells in peripheral blood and in the lesions of patients with American cutaneous leishmaniasis. Clinical and Experimental Immunology, 79,221-226. Coma, J. R., Galvao-Castro, B. & Grimaldi, G:, jr (1987). Disseminated American cutaneous leishmaniasis m a patient with AIDS. Membrias

do Instituto Oswald0 Cruz, 82,581-582.

Coutinho, S. G., Pirmez, C., Mendonca, S. C. F,., ConceicaoSilva, F. & Dorea, R. C. C. (1987). Pathogenesrsand immunopathology of letshmaniasis. Memdrias do Instituto Oswald0 Cruz, 82, supplement 1,214-228. Cunha, R. M. C., Hallack, K. A. & Castanon, M. C. M. N. (1991). Leishmaniose tegumentar difusa em paciente infectado pelo HIV: relato de urn case. Revistu da Socidedade Brasileira de Medicina Tropical, 24, supplement0 2,109-l 10. Grimaldi, G., jr, Momen, H., Naiff, R. D., McMahon-Pratt, D. & Barrett, T. V. (1991). Characterization and classitication of leishmanial parasites from humans, wild animals, and sandflies in the Amazon region of Brazil. AmericanJournal of Tropical Medicine and Hygiene, 44,645661.

Mendonca, S. C. F., Coutinho, S. G., Amendoeira, M. R. R., Marzochi, M. C. A. & Pirmez, C. (1986). Human American cutaneous leishmaniasis (Leishmania b. bruziliensis) in Brazil: lymphoproliferative responsesand influence of therapy. Clinical and Experimental Immunology, 64,269-276.

Peters, B. S., Fish, D., Golden, R., Evans, D. A., Bryceson, A. D. M. & Pinching, A. J. (1990). Visceral leishmaniasis in HIV infection and AIDS: clinical features and responseto therapy. QuarterlyJournal of Medicine, 283, 1101-l 111. Pialoux, G., Christophe, H,., DuPont, B. & Ravisse, P. (1990). Cutaneousleishmaniasis m an AIDS patients: cure with itraconazole.Journal of Infectious Diseases, 162, 1221-1222. Scaglia, M.? Villa, M., Gatti, S. & Fabio, F. (1989). Cutaneous leishmamasis in acquired immunodeficiency syndrome. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83,33&339.

Received 17 September 1991; revised 2 January acceptedfor publication 6 February 1992

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