Parasitol Res (2004) 94: 207–212 DOI 10.1007/s00436-004-1193-6
O R I GI N A L P A P E R
Tatiani Uceli Maioli Æ Erica Takane Rosa Maria Esteves Arantes Juliana Lopes Rangel Fietto Luı´ s Carlos Crocco Afonso
Immune response induced by New World Leishmania species in C57BL/6 mice Received: 12 May 2004 / Accepted: 21 June 2004 / Published online: 1 September 2004 Springer-Verlag 2004
Abstract In the present study, C57BL/6 mice were inoculated with metacyclic Leishmania amazonensis or L. braziliensis promastigotes. While these animals were capable of controlling the infection by L. braziliensis, they developed chronic lesions with elevated numbers of parasites when infected by L. amazonensis. The differences in parasite control were associated with a decreased production of IFN-c and TNF by lymph node cells from L. amazonensis-infected mice. Furthermore, these animals presented decreased spleen cell proliferation and activation of germinal centers. In addition, we compared the ability of these parasites to hydrolyze extracellular ATP and AMP. While the ATPase activity of both parasite species was similar, L. amazonensis promastigotes presented higher AMP hydrolytic activity. This increased activity may lead to an increased production of adenosine, which has been shown to present anti-inflammatory activity and may thus be involved in the establishment of the immunosuppression observed in mice infected by L. amazonensis.
Introduction Studies on the immune response to Leishmania parasites often focus on a comparison of the outcome of the T. U. Maioli Æ E. Takane Æ J. L. R. Fietto Æ L. C. C. Afonso (&) Departamento de Cieˆncias Biolo´gicas, Instituto de Cieˆncias Exatas e Biolo´gicas/NUPEB, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto, Minas Gerais, Brazil E-mail:
[email protected] Tel.: +55-31-35591701 Fax: +55-31-35591680 R. M. E. Arantes Departamento de Patologia, Universidade Federal de Minas Gerais, CP 486, 30161-970 Belo Horizonte, Minas Gerais, Brazil
infection by one parasite species in genetically distinct hosts (for a review, see Sacks and Noben-Trauth 2002). While this approach undoubtedly contributes to the understanding of the immune mechanisms associated with resistance or susceptibility to the parasite (Heinzel et al. 1989; Scott 1991), it does not evaluate the role of parasite virulence factors in the establishment of the infection and its control. Infection by Leishmania parasites can cause, depending on the parasite species, a variety of disease outcomes in humans, ranging from single self-healing cutaneous lesions to visceral dissemination of the parasite, that may lead to death if not properly treated. Furthermore, New World Leishmania species may cause severe cutaneous forms, such as diffuse or mucocutaneous leishmaniasis, widening the range of possible outcomes. L. amazonensis is the causative agent of diffuse cutaneous leishmaniasis, a disease that is characterized by a decreased immune response in infected patients (Convit et al. 1972). In contrast, L. braziliensis, although responsible for one of the most severe forms of tegumentary leishmaniasis (mucocutaneous), is often associated with self-healing lesions. Regardless of the form, however, L. braziliensis infection induces a strong cellular response in the host (Carvalho et al. 1995; Coutinho et al. 1998; da Cruz et al. 1992; Lima et al. 1999). A similar outcome may be obtained in C57BL/6 mice when inoculated by these two parasite species. In order to look for parasite factors that might be involved in the establishment of this differentiated response, the present study compares the immune response to L. amazonensis and L. braziliensis in C57BL/6 mice and evaluates the activity of enzymes involved in the hydrolysis of extracellular nucleotides. Our data show that L. amazonensis induces a decreased immune response in infected mice, as measured by cytokine production, lymphoproliferation and histological evaluation. Furthermore, our data show that L. amazonensis presents an increased hydrolytic activity towards ATP and AMP when compared with L. braziliensis. The
208
Female C57BL/6 mice (4–8 weeks old) were obtained from CEBIO (Instituto de Cieˆncias Biolo´gicas, UFMG, Belo Horizonte, Brazil). Animals were given water and food ad libitum.
a glass tissue grinder. Tissue debris was removed by centrifugation at 50 g for 1 min and the supernatant was transferred to another tube and centrifuged at 1,540 g for 15 min. The pellet was resuspended in 0.5 ml Grace’s medium containing 20% FCS, 2 mML-glutamine and 10 lg/ml gentamycin. The parasite suspension was then serially diluted in 10-fold dilutions in duplicates to a final volume of 200 ll in 96-well plates. Pipette tips were replaced for each dilution. Plates were incubated for 10 days at 26 C and examined under an inverted microscope. Results were expressed as the negative logarithm of the last dilution in which parasites were detected, adjusted for the weight of the tissue sample.
Parasites and infection protocol
Analysis of cytokine production
Leishmania (Viannia) braziliensis promastigotes, strain M2903 (MHOM/BR/75/M2903) and L. (L.) amazonensis, PH8 strain (IFLA/BR/67/PH8) were cultured in Grace’s insect medium (GIBCO BRL, Grand Island, N.Y., USA) supplemented with 20% heat-inactivated fetal calf serum (FCS; Nutricell, Campinas, Brazil), 2 mML-glutamine (GIBCO BRL) and 20 lg/ml gentamycin sulfate (Schering Plough, Rio de Janeiro, Brazil), at 26 C. Mice were inoculated in the left hind footpad with 1·105 metacyclic promastigotes obtained by gradient centrifugation (Spa¨th and Beverley 2001) of parasites at the late log phase of culture (day 5) over Ficoll 400 (Amersham Biosciences do Brasil, Sa˜o Paulo, Brazil). Lesion development was followed weekly with a dial micrometer (model 1015MA; L.S. Starret Co., Itu, Brazil) and expressed as the difference in size between the infected footpad and the contralateral uninfected footpad (Afonso and Scott 1993).
Single-cell suspensions were prepared from the popliteal lymph nodes of the infected footpad harvested at 6 weeks after infection. Cells were adjusted to a concentration of 5·106 cells/ml in Dulbecco’s minimal essential medium (GIBCO BRL) containing 10% FCS, 2 mML-glutamine and 10 lg/ml gentamycin, 25 mM N-2-hydroxiethylpiperazine-N’-2-ethanosulfonic acid (HEPES), 50 lM 2-mercaptoethanol and were plated at 0.5 ml/well in 48-well tissue culture plates. Cells were stimulated with parasite extract at 50 lg/ml. Supernatants were harvested after 24 h for tumor necrosis factor (TNF) measurement and at 72 h for IFN-c and IL-4 determination, as described by Afonso and Scott (1993). R46A2 and 11B11 hybridomas were obtained from the Rio de Janeiro Cell Bank. All monoclonal antibodies were produced in our laboratory. TNF concentration was measured in a bioassay as described by Lattime et al. (1988).
Antigen preparation
Lymphocyte proliferation
Leishmania antigen was obtained from logarithmic phase cultures of L. braziliensis or L. amazonensis promastigotes. Promastigotes were washed twice in PBS and pellets were stored frozen at 20 C until enough material accumulated for preparation of antigen. Pellets were submitted to seven cycles of freezing in liquid nitrogen followed by thawing at 37 C. The preparations were visually inspected for the presence of intact parasites (Afonso and Scott 1993). Protein content of preparations was assayed by the Lowry method (Lowry et al. 1951) and adjusted to 1 mg/ml protein. Antigen preparation was aliquoted and stored frozen at 70 C. Antigens were thawed immediately before use in cell cultures.
Lymph and spleen cells from mice infected for 6 weeks were plated at 5·106 cells/ml as described above and incubated with 50 lg/ml autologous antigen for 5 days or with 8 lg/ml ConA for 3 days. Proliferation was assessed by [3H-methyl]-thymidine incorporation for the last 16 h of culture (Toledo et al. 2001).
possible involvement of these enzymes in the determination of the immune response in the infected host is discussed.
Materials and methods Animals
Estimation of parasite load The number of parasites in the footpad was estimated by a limiting dilution assay (Afonso and Scott 1993). Mice were sacrificed and the whole lesion was removed and weighed. A fragment of known weight was obtained and ground in cold PBS containing 10 lg/ml gentamycin, in
Histology Histological analysis of footpad lesions and spleen of infected mice was done in paraffin-embedded sections stained by hematoxylin–eosin. Sections were inspected for the presence of the parasite and evaluation of the inflammatory infiltrate was performed under an optical microscope (original magnification 80·) Ectonucleotidase assays Intact live parasite cells (108 metacyclic promastigotes obtained at the fifth day of culture) were washed twice
209
with 0.8% NaCl and suspended in 250 ll of 50 mM Hepes-Tris buffer, pH 7.4, containing 116 mM KCl (5.4 mM) and 5.5 mM glucose. Ectonucleotidase activity was started by addition of 5 mmol of the indicated nucleotide substrate (Fietto et al. 2004). Enzymatic activity was measured for 1 h at 37 C and the amount of inorganic phosphate released was measured (Taussky and Shorr 1953). Statistical analysis All experiments used groups of four mice. Results are represented as mean +SEM. Differences between groups were compared using the Student’s t-test. P
