versión On-line ISSN 0717-9502
Int. J. Morphol. v.26 n.4 Temuco dic. 2008
Int. J. Morphol., 26(4):967-972, 2008.
Carbohydrates Detection in the Hepatic Egg - Granuloma System Using Lectin Histochemistry
Detección de Hidratos de Carbono en el Sistema Granuloma-Huevo Hepático Usando Lectina Histoquímica
*Mario Ribeiro Melo-Júnior; *Carmelita de Lima Bezerra Cavalcanti; *,**Nicodemos Teles de Pontes-Filho; *,***Luiz Bezerra de Carvalho Jr & *,***Eduardo Isidoro Carneiro Beltrão
* Laboratorio de Imunopatologia Keizo Asami (LIKA) - Universidade Federal de Pernambuco (UFPE), Brazil.
** Departamento de Patología - UFPE, Brazil.
*** Departamento de Bioquímica - Centro de Ciências Biológicas, CCB / UFPE, Brazil.
SUMMARY: This study aims to evalúate the egg-granuloma system in hepatic tissues using lectin histochemistry in experimental Schistosomiasis. Eight Swiss mice were infected with a local strain of Schistosoma mansoni, being submitted forty days later to a perfusión after which slices of liver (4|J.m) were prepared. The tissue samples were incubated with the following peroxidase conjugated lectins: Peanut agglutinin (PNA), Wheat Germ agglutinin (WGA), and Concanavalin A (Con A). All lectins recognized the glycoconjugates in the adult worm tegument. In the hepatic tissue, WGA presented the highest staining followed by PNA and Con A. The PNA presented the most intense staining of the egg-granuloma system while WGA stained the hepatic sinusoid cells and Con A bound preferentially the fibrosis rings of granuloma and the surrounding hepatic parenquima. WGA and PNA indicated the presence of residues of N-acetyl-glucosamine and galactose in the surface of Schistosoma mansoni eggs in the hepatic granulomas. In conclusión, using PNA, Con A and WGA our study presented different aspects of the egg-granuloma and Tegument of Schistosoma mansoni as well as indicated differences in the peri-ovular granulomas indicating alterations in the cellular mechanism of expression of surface carbohydrates during progression of the Schistosomiasis.
KEY WORDS: Lectins; Egg-granuloma system; Liver; Schistosoma mansoni.
RESUMEN: El objetivo del estudio fue evaluar el sistema de los huevos de los granulomas en los tejidos hepáticos, utilizando histoquímica de lectinas esquistosomiasis. Ocho ratones suizos experimentales fueron infectados con una cepa local de Schistosoma mansoni y luego a los cuarenta días fueron sometidos a la perfusión y se prepararon cortes de hígado (4 |J,m). Las muestras de los tejidos fueron incubadas con las siguientes peroxidasas lectinas conjugadas: aglutinina de maní (PNA), aglutinina de germenn de trigo (WGA), Concanavalin A (Con A). Todas las lectinas reconocieron las glicoconjugadas en el tegumento del gusano adulto. El tejido hepático con WGA presentó mayor coloración seguido de PNA y Con A. El PNA presentó la más intensa tinción de los huevos mientras el granuloma del sistema WGA tiñó las células hepáticas sinusoides y las Con A estuvieron siempre presentes en los anillos de la fibrosis y alrededor de los granulomas hepáticos del parénquima. WGA y PNA indicaron la presencia de residuos de N - acetil - glucosamina y galactosa en la superficie de los huevos de Schistosoma mansoni en los granulomas hepáticos de esquistosomiasis.
PALABRAS CLAVE: Lectinas; Sistema huevo-granuloma; Hígado; Schistosoma mansoni.
Lectins, carbohydrate-binding ubiquitous proteins, have found numerous applications in biological and medical áreas due to their broad recognition properties (Lehmann et al, 2006). They have been used as probes for prognosis and/or diagnosis in human and animal diseases (Mitchell & Schumacher, 1999). In histochemistry, lectins with different carbohydrate specificity can provide a sensitive detection system for changes in glycosylation and carbohydrate expression that may occur during embryogenesis, growth and disease and are sensitive, stable and easy-to-use tools to distinguish transformed from non-transformed cells (Ji et al, 2004).
The lectin-carbohydrate interactions have been used to survey the development of parasites since their antigenic glycoconjugates are responsible for immune response in animal infections (Hundt et al, 2000). Schistosoma mansoni antigens have been investigated using lectin histochemistry (Schottelius & Aisien, 1994; Okano et al, 1999; van Vliet et al., 2005). In different stages of development cell membrane glycoconjugates of Schistosoma have been found to be associated to the immune response level after infection (Nyame et al,,2000).
The tegument of Schistosoma eggs have complex structure where the cytoskeleton is rich in carbohydrates with extreme dynamic features that are used as defence barrier to the host immune response as well as to drug action (van Vliet et al).
Recent researches have characterized the surface carbohydrates of parasites during infection development (Restrepo et al, 2000; Hokke & Yazdanbakhsíi, 2005). Mapping of sugar residues of the surface of S. mansoni egg allowed identifying the strain of the infectious parasite and the host immune defence response against it, although many patho-biochemical changes in the cellular environment of the liver and intestine during infection by S. mansoni remain unknown (Muller et al, 2001).
Since little attention has been given to the physiology of the egg-granuloma system, this study aims to evalúate this system in hepatic tissues using lectin histochemistry in experimental schistosomiasis developed from a local strain of S. mansoni (SLM, Pernambuco - Brazil) isolated from Biomphalaria glabrata.
MATERIAL AND METHOD
Experimental Model. Eight Swiss mice were infected with a local strain of S. mansoni (SLM from Sao Lourengo da Mata - Zona da Mata of the State of Pernambuco - Brazil) isolated from B. glabrata. Infection was performed through tail immersion for 2h into an aqueous solution containing 50 cercariae. After forty days, the mice were submitted to perfusión and livers were preserved in 10% buffered-formalin solution until the inclusión of paraffin. Slices (4|im) were prepared and haematoxylin and eosin staining was developed.
Lectin Histochemistry. Concanavalin A (Con A), Wheat germ agglutinin (WGA) and Peanut agglutinin (PNA) lectins conjugated to peroxidase were supplied by Sigma Chemical Company (USA). Paraffin sections (4 |im) were deparaffinized in xylene and hydrated through graded alcohol. Tissue sections were treated with 0.1% (w/v) trypsin solution, at 37°C for 2 min, followed by 0.3% (v/v) methanolic hydrogen peroxide, for 20 min. and incubated with conjugated lectins (Con A, 25 |lg/mL; WGA and PNA, 50 |lg/mL), for 2h, at 4°C. Slides were washed (two 5-min) with 10 mM phosphate buffer, containing 0.15 M NaCl (PBS), pH 7.2, after each step. Peroxidase was visualized by incubation for 5 to 8 min in PBS containing diaminobenzidine (DAB) and hydrogen peroxide. The tissue sections were counterstained with haematoxylin and evaluated through optical microscopy. Inhibition assays were performed using the corresponding lectin specific sugar: methyl-a-D-mannoside for Con A, N-acetyl-glucosamine for WGA and D-galactose for PNA, at final concentration of 300 mM.
Morphometric analysis. Images of histological slices were captured using a digital video camera (Sony-USA) connected to a microscope and were processed using the OptimasTM 6.1 (Óptimas Corporation, USA) image analysis programme. Lectin-binding patterns were used to calcúlate the mean área of (mm2) and the mean number of cells (weak stain = 15-20%; modérate stain = 25-55%; intense stain = 60-95%). Semi-quantitative optical analysis was carried out taking account the intensity of staining pattern indicated as weak (+), modérate (++) or intense (+++), according to Ózer (2000).
In this study the lectin histochemistry was used to evalúate the egg-granuloma system in experimental schistosomiasis. All used lectins recognized the glycoconjugates in the adult worm tegument (AWT) in the hepatic tissue where WGA presented the highest staining followed by PNA and Con A.
WGA recognized carbohydrates residues in the parasites surface in a homogenous pattern presenting an intense staining at the hepatic sinusoid vessels, as well, during the chronic phase of the disease (Fig. 1).
The egg-granuloma system was uniform and intensively stained by PNA, being this lectin the best one to characterize disease features. PNA presented also apositive staining to the AWT (Fig. 2).
Con A recognized preferentially the fibrosis rings of the granulomas and their adjacent hepatic parenquima. Con A presented the most unspecific binding pattern among the three lectins used. WGA and PNA indicated the presence of N-acetyl-glucosamine and galactose, respectively, in the surface of S. mansoni eggs in the hepatic granuloma environment (Table I).
Lectin binding was completely abolished when the lectins were previously incubated with their respective carbohydrate at final concentration of 300 mM. This result confirms the staining pattern observed after lectin histochemistry of the schistosomiasis experimental model.
Lectin carbohydrate recognition ability has been used to explore different aspects of saccharide content and distribution on cell surface or tissues (Haseley et al, 1994). Schsitosomosa mansoni have been studied in the Brazil for almost hundred years. Epidemiology and histopaphology are among the most studied aspects of schistosomiasis since trien (Rutitzky et al, 2005).
Molecular, immunohistochemical and histochemical techniques are used to investigate the development of Schistosoma eggs and the role of their secretions in the initiation, development and activation of immune response in the host tissues (Ashton et al, 2001; Lehmann et al).
The distinct localization of the staining patterns observed in our study using Con A, WGA and PNA, which have different carbohydrate specificity, indicated the expression of certain sort of saccharides in the egg-granuloma system developed experimentally.
Experimental evidences suggest that sugars of the cell surface are involved in the processes of recognition between parasite and host and that changes in this array of surface carbohydrates induce structural and functional modifications in the host cells (Hartgers, 2006).
Here, positive staining was observed to S. mansoni parasite tegument for the used lectins, although PNA, Con A and WGA failed to distinguish between the female and male parasites via carbohydrate array present in their tegument as reported by Gobert et al. (1998) for S. japonicum and S. mansoni. Similar results were fond with other lectins (PHA and UEA-I) used for the characterization of the AWT of S. japonicum (Gazzinelli et al, 1991).
The interest in mapping the glycoconjugates in S. mansoni tegument aróse from the fact that during its development, the worm incorporates in its membrane glycolipids, glycoproteins and imunoglobulins from the host. Since tríese structures are not recognized as non-self by the host, the worms become safe from the attack of the host antibodies (Faveeuw, 2002).
Our results showed that the egg-granuloma system was evidenced by the PNA and Con A binding. One possible explanation for this lectin binding pattern is that the lectins recognized glycoconjugates present in the egg surface (Jacobson & Doyle, 1996) or egg secreted glycoproteins diffused through and anchored in the adjacent granulomas tissue characterizing glycoconjugates expression by the granuloma cells (Ashton et al).
In this work, PNA binding pattern was diffuse and non-specific for the cellular environment present in the granulomas. Studies have observed that carbohydrates of eosinofils surface are involved in the degranulation process (Ohmori & Nawa, 2000), fact which raised attention for the importance of expression and liberation of producís from cells in the hepatic granulomas acting as fibrosis inhibitors (van de Vrjver et al, 2006).
Lectin binding intensity can be influenced by the stage of development of the Schistosoma egg, reflecting the content of carbohydrates in the egg tegument. Ashton and co-workers observed that mature eggs present a higher quantity of (glyco) proteins trian the former egg stage. This difference was observed in our study since Con A presented a different binding pattern to different stages of the egg-granuloma system. D-glucose and D-mannose were previously indicated as sugars involved in the parasite-host interaction during infection (Jacobson & Doyle; Lee et al, 1999; Opanasopit et al, 2001).
Recently, many studies involved were developed in our laboratories to investigate different pathologic granuloma system process (Meló-Júnior et al., 2004; Melo-Junior et al., 2006a; 2006b; Araújo-Filho et al, 2006) indicating an important contribution of lectins in those biomedical analysis.
Lectin histochemistry has shown to be an interesting tool in the investigation of the structure and function of glycoconjugates in the parasites and host-cells besides helping to light the biochemical alterations during the evolution of parasitosis (Nyame et al.; Eichinger, 2001).
In conclusión, using PNA, Con A and WGA our study presented different aspects of the egg-granuloma and AWT of S. mansoni, as well as indicated differences in the peri-ovular granulomas showing alterations in the cellular mechanism of expression of surface carbohydrates during the progression of schistosomiasis.
We thank Raquel A.L. Coélho, Ph.D. for scientific assistance. This work was supported by CNPq (CTPETRO no 463655/001). 2008.
Araújo-Filho, J. L. S.; Melo-Junior, M. R.; Carvalho Jr, L. B. & Pontes-Filho, N. T. Galectin-3 in prostatic tumours: immunohistochemistry and digital image analysis. J. Bras. Patol. Med. Lab., 42:469-75, 2006. [ Links ]
Ashton, P D.; Harrop, R.; Shah, B. & Wilson, R. A. The schistosome egg: development and secretions. Parasitol, 722:329-33,2001. [ Links ]
Eichinger, D. A role for a galactose lectin and ligands during encystment of Entamoeba. Histol. Histopathol, 14(1): 217-26,2001. [ Links ]
Faveeuw, C. Antigen Presentation by CDld Contributes to the Amplification of Th2 Responses to Schistosoma mansoni Glycoconjugates in mice. .J. Immunol, 7 (59:906-12,2002. [ Links ]
Gazzinelli, R. T.; Romanha, A. J.; Fontes, G.; Egler, C; Gazzinelli, G. & Brener, Z. Distribution of carbohydrates recognized by the lectins EVE and Con A in monoxemic and heteroxemic trypanosomatids. J. Protozool, 38:320-5,1991. [ Links ]
Gobert, G. N.; Jones, M. K. & Stenzel, D. J. Ultrastrutural analysis of the adult Schistosoma japonicum by lectin cytochemistry. Int. J. Parasitol, 28(9): 1445-52,1998. [ Links ]
Hartgers, F. Adaptive and Regulatory Responses in Schistosomiasis: Relationship to Allergy. Capron, M. & Trottein, F (eds): Parasites and Allergy. Chem. Immunol. Allergy. Basel, Karger, 90:157-75, 2006. [ Links ]
Haseley, S. R.; Talaga, P; Kamerling, J. P & Vliegenthart, J. F G. Characterization of the carbohydrate binding specificity and kinetic parameters of lectins by using surface plasmon resonance. Anal. Biochem., 74:203-10,1994. [ Links ]
Hokke, C. H. & Yazdanbakhsh, M. Schistosome glycans and innate immunity. Parasite Immunol, 27(8):257-64, 2005. [ Links ]
Hundt, M.; Heiken, H. & Schmidt, R. E. Association of low mannose-binding lectin serum concentrations and bacterial pneumonia in HIV infection. AIDS, 14(12): 1853-4, 2000. [ Links ]
Jacobson, R. L. & Doyle, R. J. Lectin-parasite interactions (review). Parásitol. Today, 12(2):55-61, 1996. [ Links ]
Ji, H.; House, M.; Long, V.; McMillan, P. & Chien, E. Comparison of proteoglycan expression in rat and human cervix using lectin histochemistry. Am. J. Obst. Gynecol, 193(6):44, 2004. [ Links ]
Lee, R. T.; Shinohara, Y; Hasegawa, Y. & Lee, Y. C. Lectin-carbohydrate interactions: fine specificity difference between two mannose-binding proteins. Biosci. Reports., 19(4):283-92,1999. [ Links ]
Lehmann, E; Tiralongo, E. & Tiralongo, J. Sialic Acid-Specific Lectins: Occurrence, Specificity and Function. Cell. Mol. Life Sel, 63(72)1331-54, 2006. [ Links ]
Melo-Júnior, M. R.; Araújo-Filho, J. L. S.; Patú, V. J. R. M.; Beltrao, E. I. C. & Carvalho Jr, L. B. Digital image analysis of skin neoplasms evaluated by lectin histochemistry: potential marker to biochemical alterations and tumour differential diagnosis. J.Bras. Patol. Med. Lab., 42:455-60, 2006a. [ Links ]
Melo-Júnior, M. R.; Araújo-Filho, J. L. S.; Patú, V. J. R. M.; Mello, L. A. & Carvalho Jr, L. B. Langerhans cells in cutaneous tumours: immunohistochemistry study using a computer image analysis system. J.Mol. Histol, 37:321-5, 2006b. [ Links ]
Melo-Junior, M. R.; lelles, A. M. S. & Albuquerque, F E. B. Altered lectin-binding sites in normal colon and ulcerative colitis. J.Bras. Patol. Med. Lab., 40(2):123-5, 2004. [ Links ]
Mitchell, B. S. & Schumacher, U. Ihe use of the lectin Helix pomatia agglutinin (HPA) as a prognostic indicator and as a tool in cáncer research. Histol. Histopathol, 14(1): 217-26, 1999. [ Links ]
Muller, E.; Brunet, L. R.; Fried, B. & Sherma, J. Effects on the neutral lipid contents of liver, ileum and serum during experimental schistosomiasis. Int. J. Parasitol., 31(3): 285-7, 2001. [ Links ]
Nyame, A. K.; Leppanen, A. M.; Bogitsh, B. J. & Cummings, R. D. Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-míecieá mice and expression of the glycan among schistosomes. Exp. Parasitol, 96(4):202-12, 2000. [ Links ]
Ohmori, J. & Nawa, Y. Localization of sialyl glycoconjugates in eosinophil-specific granules after degranulation stimuli. Int. Arch. Allergy Immunol, 122(1):2-5, 2000. [ Links ]
Okano, M.; Satoskar, A. R.; Nishizaki, K.; Abe, M. & Harn, D. A. Induction of the responses and IgE is largely due to carbohydrates functioning as adjuvants on Schistosoma mansoni egg antigens. J.Immunol, 163(12):612-1, 1999. [ Links ]
Opanasopit, P; Shirashi, K.; Nishikawa, M.; Yamashita, F; Takakura, Y. & Hashida, M. In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein. Am. J. Physiol, 280(5):879-89, 2001. [ Links ]
Ózer, E. Effects of prenatal exposure on neuronal migration, neurogenesis and brain myelinization in the mice brain. Clin. Neuropathol, 19(1):21-5, 2000. [ Links ]
Restrepo, B. I.; Obregón-Henao, A.; Mesa, M.; Gil, D. L.; Ortiz, B. L.; Mejía, J. S.; Villota, G. E.; Sanzón, F & Teale, J. M. Characterization of the carbohydrate components of Taenia solium metacestode glycoprotein antigens. Int. J. Parasitol, 30(6):689-96, 2000. [ Links ]
Rutitzky, L. I.; Hernández, H. J.; Yim, Y. S.; Ricklan, D. E.; Finger, E.; Mohán, C; Peter, I.; Wakeland, E. K. & Stadecker, M. J. Enhanced egg-induced immunopathology correlates with high IFN-g in murine schistosomiasis: Identification of two epistatic genetic intervals. J.Immunol, 174:435-40, 2005. [ Links ]
Schottelius, J. & Aisien, M. S. O. In: Doyle, R. J. & Slifklin, M. (eds) Lectin-microorganisms interactions. Marcel, Dekker, 1994. pp. 225-48. [ Links ]
van Vliet, S. J.; van Liempt, E.; Saeland, E.; Aamoudse, C. A.; Appelmelk, B.; Irimura, 1.; Geijtenbeek, 1. B. H.; Blixt, O.; Alvarez, R.; van Die, I. & van Kooyk, Y. Carbohydrate profiling reveáis a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells. Int. Immunol, 17(5):661-9, 2005. [ Links ]
Van de Vijver, K. K.; Deeider, A. M.; Jacobs, W.; Van Marck, E. A. & Hokke, C. H. LacNAc-containing glycans induce granulomas in an in vivo model for schistosome egg-induced hepatic granuloma formation. GlycobioL, 16(3):231-43, 2006. [ Links ]
Correspondence to: Mario Ribeiro de Melo-Júnior.
Laboratorio de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco. Cidade Universitaria. EP 50670-420. Recife. Pernambuco, BRAZIL. Phone: 55-81-3271-8486 FAX : 55-81-3271-8484. E-mail: email@example.com
Received: 08-05-2008, Accepted: 22-09-2008.