Recombinant Cysteine Proteinase from Leishmania (Leishmania) chagasi Implicated in Human and Dog T-Cell Responses
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感染与免疫杂志 2005年第6期
Department of Microbiology, Immunology and Parasitology, Universidade Federal de So Paulo, Escola Paulista de Medicina, So Paulo, SP
Division of Parasitology, Faculdade NOVAFAPI, Teresina, PI
Division of Infectious and Parasitic Diseases, Universidade Federal do Piauí, Teresina, PI
Department of Clinical Veterinary, Universidade Federal do Piauí, Teresina, Piauí, PI, Brazil
ABSTRACT
High in vitro lymphoproliferative responses were induced in humans and dogs by a recombinant Leishmania (Leishmania) chagasi cysteine proteinase, with secretion of IFN- in asymptomatic subjects or of IFN-, interleukin 4 (IL-4), and IL-10 in oligosymptomatic subjects. In contrast, responses of symptomatic patients and dogs were lower, with production of IL-4 and IL-10.
TEXT
Visceral leishmaniasis (VL) is caused by Leishmania (Leishmania) chagasi in South America, and dogs are the main reservoir of the disease. Among the immunological changes found with VL, those involving T cells and interleukin 10 (IL-10) production have been correlated with pathology, whereas control of the infection has been associated with the production of gamma interferon (IFN-) (3, 4, 5, 8, 13). Leishmania (L.) infantum cysteine proteinases have been used for the evaluation of humoral and cellular immune responses in human and canine VL (9, 12). A recombinant cysteine proteinase from L. (L.) chagasi, rLdccys1, expressed by the Ldccys1 gene (10) in Escherichia coli, was shown to be a suitable tool for the diagnosis of American VL (6). The present study demonstrates that rLdccys1 elicits cellular immune responses in naturally infected humans and dogs in different stages of VL.
L. (L.) chagasi (MHOM/BR/1972/LD) amastigotes were isolated from spleens of infected hamsters, as previously described (2). The L. (L.) chagasi Ldccys1 was cloned and expressed as described elsewhere (6). The study population included human and canine blood samples obtained from subjects living in Teresina, Piauí State, Brazil, an area where VL is endemic, and were classified as shown in Table 1. The human and dog procedures were approved by the Ethical Committee for Human and Animal Care at the Universidade Federal de So Paulo, Escola Paulista de Medicina. The lymphoproliferative assays were performed with peripheral blood mononuclear cells (PBMC) purified by Ficoll-Hypaque density gradient centrifugation. For in vitro proliferation assays, the cells were cultured into 96-wells plates (1 x 106 cells/ml) in RPMI 1640 medium with 10% pooled human serum (R10) in the presence of rLdccys1 (2.5 μg/ml) or L. (L.) chagasi amastigote extracts (AM) (equivalent to 1 x 107 amastigotes). After 96 h at 37°C in 5% CO2, the cells were pulsed with 1 μCi of [3H]thymidine/well for 18 h, and [3H]thymidine uptake was determined after filtration on glass fiber filters. Lymphoproliferative responses were expressed as stimulation indexes (SI) determined by dividing the mean counts per minute for antigen-stimulated cells, in triplicate, by the mean counts for the control medium. Student's t test was used with SigmaStat software to evaluate the significance of the data (P < 0.05). After stimulation with rLdccys1, PBMC from asymptomatic, oligosymptomatic, symptomatic, and treated patients proliferated with mean stimulation indexes of 8.0, 6.25, 2.45, and 4.1, respectively, whereas healthy individuals showed basal levels of lymphoproliferation. Lower stimulation was obtained for PBMC from the same patients with AM (SI of 2.5, 5.0, 2.1, and 2.0 from asymptomatic, oligosymptomatic, symptomatic, and treated patients, respectively) (Fig. 1A). In PBMC from asymptomatic, oligosymptomatic, and symptomatic dogs, rLdccys1 induced SI means of 11, 3.3, and 1.8, respectively, whereas lower responses were obtained with AM (SI of 2.2, 1.2, and 0.6 from asymptomatic, oligosymptomatic, and symptomatic dogs, respectively). Corroborating our results, the lymphoproliferative responses stimulated by the recombinant L. (L.) infantum cysteine proteinase B (rCPB) in Iranian dogs with active VL were weaker in symptomatic than in asymptomatic animals. However, in contrast to our results, asymptomatic dogs showed higher lymphoproliferative responses to the total L. (L.) infantum lysate than to rCPB. In addition, for asymptomatic dogs, we found that higher lymphoproliferative responses were elicited by rLdccys1 (SI = 11) than by rCPB (SI 4.0) (9, 12). For evaluation of lymphokine production, 1 x 107 PBMC from VL patients and dogs were cultured in 1 ml of R10 and maintained for 72 h in the presence of 2.5 μg/ml rLdccys1. Culture supernatants were tested for IFN-, IL-4, and IL-10 using a specific enzyme-linked immunosorbent assay. PBMC from asymptomatic patients secreted high levels of IFN- when stimulated with rLdccys1 (1.722 ng/ml). Lower levels of IFN- (0.871 ng/ml) were produced by PBMC from oligosymptomatic patients, whereas this lymphokine was not detected in symptomatic individuals. IL-4 and IL-10 were secreted by PBMC from oligosymptomatic patients (0.277 ng/ml and 0.317 ng/ml, respectively), and a significant increase in the secretion of these two lymphokines was detected in symptomatic individuals (0.848 ng/ml and 0.725 ng/ml for IL-4 and IL-10, respectively). No lymphokine secretion was found in the PBMC supernatants from normal and treated patients (Table 2). These results corroborate literature data which show that VL patients with a controlled form of the disease display Th1-type lymphokines, whereas immunosuppression and a Th2 profile predominate in individuals with exacerbated disease (1, 8, 14). Secretion of significant levels of IFN-, IL-4, and IL-10 by lymphocytes from oligosymptomatic patients is in agreement with data which showed both resistant (IFN-) and susceptible (IL-10) profiles in this form of VL (7). PBMC from asymptomatic and oligosymptomatic dogs secreted IFN-, whereas IL-4 and IL-10 were released by PBMC from symptomatic animals (Table 3). Lymphocytes from oligosymptomatic dogs did not secrete IL-4; however, IL-10 values were comparable in the supernatants of PBMC from humans and dogs presenting symptomatic and oligosymptomatic signs of VL. Few studies have reported cytokine dosage in dogs with VL (15), impairing comparison with our results. The production of nitric oxide (NO) was also evaluated for the supernatants from human and dog lymphocytes stimulated by rLdccys1, by use of the Griess reagent. PBMC from asymptomatic and oligosymptomatic patients secreted significant levels of NO (108.42 μM ± 19.22 μM and 58.71 μM ± 7.78 μM, respectively), whereas symptomatic and treated patients secreted lower NO concentrations (19.22 μM ± 0.8 μM and 5.02 μM ± 0.2 μM, respectively) (Fig. 2A). A similar relationship between NO production and the clinical signs of VL was observed with PBMC cultures from dogs stimulated with rLdccys1 (82.16 μM ± 8.33 μM, 25.21 μM ± 7.44 μM, and 7.79 μM ± 0.34 μM from asymptomatic, oligosymptomatic, and symptomatic dogs, respectively) (Fig. 2B). The observed direct correlation between the secretion of IFN- and NO during the development of VL in humans and dogs has been previously reported (11, 16).
Overall, our results showed that the rLdccys1 antigen is a suitable immunological marker for several stages of VL in humans and dogs.
ACKNOWLEDGMENTS
We thank Adriana Nunes Pinheiro and Joo Batista Teles from the Instituto de Doenas Tropicais Natan Portella, Teresina, Piauí State, Brazil, for excellent assistance.
This study was supported by FAPESP (Fundao de Amparo a Pesquisa do Estado de So Paulo), CAPES (Conselho Nacional de Desenvolvimento Científico e Tecnologico), and FACULDADE NOVAFAPI.
REFERENCES
1. Bacellar, O., M. Barral-Neto, R. Badaro, and E. M. Carvalho. 1991. Gamma interferon production by lymphocytes from children infected with L. chagasi (L.) chagasi. Braz. J. Med. Biol. Res. 24:791-795.
2. Barbieri, C. L., A. I. Doine, and E. Freymüller. 1990. Lysosomal depletion in macrophages from spleen and foot lesions of Leishmania-infected hamster. Exp. Parasitol. 71:218-228.
3. Carvalho, E. M., O. Bacellar, C. Brownell, T. Regis, R. L. Coffman, and S. G. Reed. 1994. Restoration of IFN- production and lymphocyte proliferation in visceral leishmaniasis. J. Immunol. 152:5949-5956.
4. Carvalho, E. M., A. Barral, D. Pedral-Sampaio, M. Barral-Netto, R. Badaro, H. Rocha, and W. D. Johnson. 1992. Immunologic markers of clinical evolution in children recently infected with Leishmania donovani chagasi. J. Infect. Dis. 165:535-540.
5. Carvalho, E. M., R. S. Teixeira, and W. D. Johnson, Jr. 1981. Cell-mediated immunity in American visceral leishmaniasis: reversible immunosuppression during acute infection. Infect. Immun. 22:649-656.
6. Dias, S. S., P. H. C. Pinheiro, S. Katz, M. R. M. dos Santos, and C. L. Barbieri. 2005. A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi suitable for serodiagnosis of American visceral leishmaniasis. Am. J. Med. Trop. Hyg. 72:126-132.
7. Gama, M. E. A., J. M. L. Costa, J. C. R. Pereira, C. M. C. Gomes, and C. E. P. Corbett. 2004. Serum cytokine profile in the subclinical form of visceral leishmaniasis. Braz. J. Med. Biol. Res. 37:129-136.
8. Ghalib, H. W., M. R. Piuvezam, Y. A. Sheiky, M. Siddig, F. A. Hashim, A. M. el-Hassan, D. M. Russo, and S. G. Reed. 1993. Interleukin 10 production correlates with pathology in human Leishmania donovani infections. J. Clin. Investig. 92:324-329.
9. Nakhaee, A., T. Taheri, M. Taghikhani, M. Mohebali, A.-H. Salmanian, N. Fasel, and S. Rafati. 2004. Humoral and cellular immune responses against Type I cysteine proteinase of Leishmania infantum are higher in asymptomatic than symptomatic dogs selected from a naturally infected population. Vet. Parasitol. 119:107-123.
10. Omara-Opyene, A. L., and L. Gedamu. 1997. Molecular cloning, characterization and overexpression of two distinct cysteine protease cDNAs from Leishmania donovani chagasi. Mol. Biochem. Parasitol. 90:247-267.
11. Panaro, M. A., A. Acquafredda, S. Lisi, D. D. Lofrumento, T. Trotta, R. Satalino, M. Saccia, V. Mitolo, and O. Brandonisio. 1999. Inducible nitric oxide synthase and nitric oxide production in Leishmania infantum-infected human macrophages stimulated with interferon-gamma and bacterial lipopolysaccharide. Int. J. Clin. Lab. Res. 29:122-127.
12. Rafati, S., A. Nakhaee, T. Taheri, A. H. Ghashghaii, A. H. Salmanian, M. Jimenez, M. Mohebali, S. Masina, and N. Fasel. 2003. Expression of cysteine proteinase type I and II of Leishmania infantum and their recognition by sera during canine and human visceral leishmaniasis. Exp. Parasitol. 103:143-151.
13. Rezai, H. R., S. M. Ardehali, G. Amirhakimi, and A. Kharazmi. 1978. Immunological features of kala-azar. Am. J. Trop. Med. Hyg. 27:1079-1083.
14. Sang, D. K., J. H. Ouma, C. C. John, C. C. Whalen, C. L. King, A. A. F. Mahmoud, and F. P. Heinzel. 1999. Increased levels of soluble interleukin-4 receptor in the sera of patients with visceral leishmaniasis. J. Infect. Dis. 179:743-746.
15. Santos-Gomes, G. M., R. Rosa, C. Leandro, S. Cortes, P. Romo, and H. Silveira. 2002. Cytokine expression during the outcome of canine experimental infection by Leishmania infantum. Vet. Immunol. Immunopathol. 88:21-30.
16. Voldoukis, I., J. C. Drapier, A. K. Nüssler, Y. Tselentis, O. A. Silva, M. Gentilini, D. M. Mossalayi, L. Monjour, and B. Dugas. 1996. Canine visceral leishmaniasis: successful chemotherapy induces macrophage antileishmanial activity via the L-arginine nitric oxide pathway. Antimicrob. Agents Chemother. 40:253-256.(Paulo Henrique da Costa P)
Division of Parasitology, Faculdade NOVAFAPI, Teresina, PI
Division of Infectious and Parasitic Diseases, Universidade Federal do Piauí, Teresina, PI
Department of Clinical Veterinary, Universidade Federal do Piauí, Teresina, Piauí, PI, Brazil
ABSTRACT
High in vitro lymphoproliferative responses were induced in humans and dogs by a recombinant Leishmania (Leishmania) chagasi cysteine proteinase, with secretion of IFN- in asymptomatic subjects or of IFN-, interleukin 4 (IL-4), and IL-10 in oligosymptomatic subjects. In contrast, responses of symptomatic patients and dogs were lower, with production of IL-4 and IL-10.
TEXT
Visceral leishmaniasis (VL) is caused by Leishmania (Leishmania) chagasi in South America, and dogs are the main reservoir of the disease. Among the immunological changes found with VL, those involving T cells and interleukin 10 (IL-10) production have been correlated with pathology, whereas control of the infection has been associated with the production of gamma interferon (IFN-) (3, 4, 5, 8, 13). Leishmania (L.) infantum cysteine proteinases have been used for the evaluation of humoral and cellular immune responses in human and canine VL (9, 12). A recombinant cysteine proteinase from L. (L.) chagasi, rLdccys1, expressed by the Ldccys1 gene (10) in Escherichia coli, was shown to be a suitable tool for the diagnosis of American VL (6). The present study demonstrates that rLdccys1 elicits cellular immune responses in naturally infected humans and dogs in different stages of VL.
L. (L.) chagasi (MHOM/BR/1972/LD) amastigotes were isolated from spleens of infected hamsters, as previously described (2). The L. (L.) chagasi Ldccys1 was cloned and expressed as described elsewhere (6). The study population included human and canine blood samples obtained from subjects living in Teresina, Piauí State, Brazil, an area where VL is endemic, and were classified as shown in Table 1. The human and dog procedures were approved by the Ethical Committee for Human and Animal Care at the Universidade Federal de So Paulo, Escola Paulista de Medicina. The lymphoproliferative assays were performed with peripheral blood mononuclear cells (PBMC) purified by Ficoll-Hypaque density gradient centrifugation. For in vitro proliferation assays, the cells were cultured into 96-wells plates (1 x 106 cells/ml) in RPMI 1640 medium with 10% pooled human serum (R10) in the presence of rLdccys1 (2.5 μg/ml) or L. (L.) chagasi amastigote extracts (AM) (equivalent to 1 x 107 amastigotes). After 96 h at 37°C in 5% CO2, the cells were pulsed with 1 μCi of [3H]thymidine/well for 18 h, and [3H]thymidine uptake was determined after filtration on glass fiber filters. Lymphoproliferative responses were expressed as stimulation indexes (SI) determined by dividing the mean counts per minute for antigen-stimulated cells, in triplicate, by the mean counts for the control medium. Student's t test was used with SigmaStat software to evaluate the significance of the data (P < 0.05). After stimulation with rLdccys1, PBMC from asymptomatic, oligosymptomatic, symptomatic, and treated patients proliferated with mean stimulation indexes of 8.0, 6.25, 2.45, and 4.1, respectively, whereas healthy individuals showed basal levels of lymphoproliferation. Lower stimulation was obtained for PBMC from the same patients with AM (SI of 2.5, 5.0, 2.1, and 2.0 from asymptomatic, oligosymptomatic, symptomatic, and treated patients, respectively) (Fig. 1A). In PBMC from asymptomatic, oligosymptomatic, and symptomatic dogs, rLdccys1 induced SI means of 11, 3.3, and 1.8, respectively, whereas lower responses were obtained with AM (SI of 2.2, 1.2, and 0.6 from asymptomatic, oligosymptomatic, and symptomatic dogs, respectively). Corroborating our results, the lymphoproliferative responses stimulated by the recombinant L. (L.) infantum cysteine proteinase B (rCPB) in Iranian dogs with active VL were weaker in symptomatic than in asymptomatic animals. However, in contrast to our results, asymptomatic dogs showed higher lymphoproliferative responses to the total L. (L.) infantum lysate than to rCPB. In addition, for asymptomatic dogs, we found that higher lymphoproliferative responses were elicited by rLdccys1 (SI = 11) than by rCPB (SI 4.0) (9, 12). For evaluation of lymphokine production, 1 x 107 PBMC from VL patients and dogs were cultured in 1 ml of R10 and maintained for 72 h in the presence of 2.5 μg/ml rLdccys1. Culture supernatants were tested for IFN-, IL-4, and IL-10 using a specific enzyme-linked immunosorbent assay. PBMC from asymptomatic patients secreted high levels of IFN- when stimulated with rLdccys1 (1.722 ng/ml). Lower levels of IFN- (0.871 ng/ml) were produced by PBMC from oligosymptomatic patients, whereas this lymphokine was not detected in symptomatic individuals. IL-4 and IL-10 were secreted by PBMC from oligosymptomatic patients (0.277 ng/ml and 0.317 ng/ml, respectively), and a significant increase in the secretion of these two lymphokines was detected in symptomatic individuals (0.848 ng/ml and 0.725 ng/ml for IL-4 and IL-10, respectively). No lymphokine secretion was found in the PBMC supernatants from normal and treated patients (Table 2). These results corroborate literature data which show that VL patients with a controlled form of the disease display Th1-type lymphokines, whereas immunosuppression and a Th2 profile predominate in individuals with exacerbated disease (1, 8, 14). Secretion of significant levels of IFN-, IL-4, and IL-10 by lymphocytes from oligosymptomatic patients is in agreement with data which showed both resistant (IFN-) and susceptible (IL-10) profiles in this form of VL (7). PBMC from asymptomatic and oligosymptomatic dogs secreted IFN-, whereas IL-4 and IL-10 were released by PBMC from symptomatic animals (Table 3). Lymphocytes from oligosymptomatic dogs did not secrete IL-4; however, IL-10 values were comparable in the supernatants of PBMC from humans and dogs presenting symptomatic and oligosymptomatic signs of VL. Few studies have reported cytokine dosage in dogs with VL (15), impairing comparison with our results. The production of nitric oxide (NO) was also evaluated for the supernatants from human and dog lymphocytes stimulated by rLdccys1, by use of the Griess reagent. PBMC from asymptomatic and oligosymptomatic patients secreted significant levels of NO (108.42 μM ± 19.22 μM and 58.71 μM ± 7.78 μM, respectively), whereas symptomatic and treated patients secreted lower NO concentrations (19.22 μM ± 0.8 μM and 5.02 μM ± 0.2 μM, respectively) (Fig. 2A). A similar relationship between NO production and the clinical signs of VL was observed with PBMC cultures from dogs stimulated with rLdccys1 (82.16 μM ± 8.33 μM, 25.21 μM ± 7.44 μM, and 7.79 μM ± 0.34 μM from asymptomatic, oligosymptomatic, and symptomatic dogs, respectively) (Fig. 2B). The observed direct correlation between the secretion of IFN- and NO during the development of VL in humans and dogs has been previously reported (11, 16).
Overall, our results showed that the rLdccys1 antigen is a suitable immunological marker for several stages of VL in humans and dogs.
ACKNOWLEDGMENTS
We thank Adriana Nunes Pinheiro and Joo Batista Teles from the Instituto de Doenas Tropicais Natan Portella, Teresina, Piauí State, Brazil, for excellent assistance.
This study was supported by FAPESP (Fundao de Amparo a Pesquisa do Estado de So Paulo), CAPES (Conselho Nacional de Desenvolvimento Científico e Tecnologico), and FACULDADE NOVAFAPI.
REFERENCES
1. Bacellar, O., M. Barral-Neto, R. Badaro, and E. M. Carvalho. 1991. Gamma interferon production by lymphocytes from children infected with L. chagasi (L.) chagasi. Braz. J. Med. Biol. Res. 24:791-795.
2. Barbieri, C. L., A. I. Doine, and E. Freymüller. 1990. Lysosomal depletion in macrophages from spleen and foot lesions of Leishmania-infected hamster. Exp. Parasitol. 71:218-228.
3. Carvalho, E. M., O. Bacellar, C. Brownell, T. Regis, R. L. Coffman, and S. G. Reed. 1994. Restoration of IFN- production and lymphocyte proliferation in visceral leishmaniasis. J. Immunol. 152:5949-5956.
4. Carvalho, E. M., A. Barral, D. Pedral-Sampaio, M. Barral-Netto, R. Badaro, H. Rocha, and W. D. Johnson. 1992. Immunologic markers of clinical evolution in children recently infected with Leishmania donovani chagasi. J. Infect. Dis. 165:535-540.
5. Carvalho, E. M., R. S. Teixeira, and W. D. Johnson, Jr. 1981. Cell-mediated immunity in American visceral leishmaniasis: reversible immunosuppression during acute infection. Infect. Immun. 22:649-656.
6. Dias, S. S., P. H. C. Pinheiro, S. Katz, M. R. M. dos Santos, and C. L. Barbieri. 2005. A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi suitable for serodiagnosis of American visceral leishmaniasis. Am. J. Med. Trop. Hyg. 72:126-132.
7. Gama, M. E. A., J. M. L. Costa, J. C. R. Pereira, C. M. C. Gomes, and C. E. P. Corbett. 2004. Serum cytokine profile in the subclinical form of visceral leishmaniasis. Braz. J. Med. Biol. Res. 37:129-136.
8. Ghalib, H. W., M. R. Piuvezam, Y. A. Sheiky, M. Siddig, F. A. Hashim, A. M. el-Hassan, D. M. Russo, and S. G. Reed. 1993. Interleukin 10 production correlates with pathology in human Leishmania donovani infections. J. Clin. Investig. 92:324-329.
9. Nakhaee, A., T. Taheri, M. Taghikhani, M. Mohebali, A.-H. Salmanian, N. Fasel, and S. Rafati. 2004. Humoral and cellular immune responses against Type I cysteine proteinase of Leishmania infantum are higher in asymptomatic than symptomatic dogs selected from a naturally infected population. Vet. Parasitol. 119:107-123.
10. Omara-Opyene, A. L., and L. Gedamu. 1997. Molecular cloning, characterization and overexpression of two distinct cysteine protease cDNAs from Leishmania donovani chagasi. Mol. Biochem. Parasitol. 90:247-267.
11. Panaro, M. A., A. Acquafredda, S. Lisi, D. D. Lofrumento, T. Trotta, R. Satalino, M. Saccia, V. Mitolo, and O. Brandonisio. 1999. Inducible nitric oxide synthase and nitric oxide production in Leishmania infantum-infected human macrophages stimulated with interferon-gamma and bacterial lipopolysaccharide. Int. J. Clin. Lab. Res. 29:122-127.
12. Rafati, S., A. Nakhaee, T. Taheri, A. H. Ghashghaii, A. H. Salmanian, M. Jimenez, M. Mohebali, S. Masina, and N. Fasel. 2003. Expression of cysteine proteinase type I and II of Leishmania infantum and their recognition by sera during canine and human visceral leishmaniasis. Exp. Parasitol. 103:143-151.
13. Rezai, H. R., S. M. Ardehali, G. Amirhakimi, and A. Kharazmi. 1978. Immunological features of kala-azar. Am. J. Trop. Med. Hyg. 27:1079-1083.
14. Sang, D. K., J. H. Ouma, C. C. John, C. C. Whalen, C. L. King, A. A. F. Mahmoud, and F. P. Heinzel. 1999. Increased levels of soluble interleukin-4 receptor in the sera of patients with visceral leishmaniasis. J. Infect. Dis. 179:743-746.
15. Santos-Gomes, G. M., R. Rosa, C. Leandro, S. Cortes, P. Romo, and H. Silveira. 2002. Cytokine expression during the outcome of canine experimental infection by Leishmania infantum. Vet. Immunol. Immunopathol. 88:21-30.
16. Voldoukis, I., J. C. Drapier, A. K. Nüssler, Y. Tselentis, O. A. Silva, M. Gentilini, D. M. Mossalayi, L. Monjour, and B. Dugas. 1996. Canine visceral leishmaniasis: successful chemotherapy induces macrophage antileishmanial activity via the L-arginine nitric oxide pathway. Antimicrob. Agents Chemother. 40:253-256.(Paulo Henrique da Costa P)