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Entamoeba histolytica Induces Cyclooxygenase-2 Expression in Macrophages During Amebic Liver Abscess Formation
Archives of Medical Research 31 (2000) S122–S123
Entamoeba histolytica Induces Cyclooxygenase-2 Expression in Macrophages During Amebic Liver Abscess Formation Blanca E. Sánchez-Ramírez,* Marcela Ramírez-Gil,* Ernesto Ramos-Martínez** and Patricia Talamás Rohana*** *Facultad de Ciencias Químicas, **Departamento de Anatomía Patológica del Hospital Central Universitario, Facultad de Medicina, Universidad Autónoma de Chihuahua (UACH), Chihuahua, Mexico ***Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
Key Words: Entamoeba histolytica, Cyclooxygenase, Prostaglandin, Inflammation.
Introduction During amebic liver abscess (ALA) formation, macrophage activity is probably suppressed by inflammation-derived mediators, such as prostaglandins (1). Recently, we have demonstrated that development of experimental ALA courses with an increase in both systemic levels of PGE2 and local COX activity in liver microsomes (2). In addition, in vivo treatment of infected hamsters with indomethacin (Indo) reduced ALA formation by 30%. Conversion of arachidonic acid to PGE2 is catalyzed by COX, which exist in at least two isoforms: COX-1, a constitutive enzyme, and COX-2, an inducible isoform associated with inflammation (3). Nonsteroidal, anti-inflammatory drugs such as Indo inhibit both active COX enzymes. We recently reported that COX2 protein is expressed in ALA-derived macrophages (AMAs) (4). To demonstrate that COX-2 protein is expressed in vivo during liver infection with E. histolytica, we examined the localization of COX-2 by immunohistochemical analysis.
Materials and Methods Male in-bred hamsters (Mesocricetus auratus), weighing approximately 100 g, were infected intrahepatically (i.h.) with 1 ⫻ 106 trophozoites, as described previously (2). Noninfected hamsters were used as controls. At 2, 4, and 7 days postinfection the animals were anesthetized and killed
Address reprint requests to: Dra. Blanca E. Sánchez-Ramírez, Facultad de Ciencias Químicas, UACH, Apdo. Postal 1542-C, 31000 Chihuahua, Chihuahua, México. Telefax: (⫹52) (14) 144-492; E-mail: [email protected] Presenting author: Blanca E. Sánchez-Ramírez.
by exsanguination, and livers and abscesses were removed to calculate the percentage of liver damage. Liver samples obtained from normal and infected hamsters and from areas near to and far from the abscess were fixed with 10% phosphate-buffered formalin. Kidneys from normal hamsters were used as COX-2 controls. After embedding in paraffin, 6–8 m-thick sections were tested by immunohistochemistry following the protocol enclosed with the Histostain-Plus Kit (Zymed Lab., Inc.). The primary antibody, either polyclonal rabbit anti-COX-2 antiserum (1:250 dilution, Cayman Chem. Co.) or polyclonal rabbit antiameba antiserum (1:1000 dilution) was detected using secondary biotinylated affinity-purified antirabbit IgG antibody, avidin–peroxidase, and freshly prepared substrate. Controls with unspecific or preimmune antiserum were done. Samples were stained with hematoxylin and eosin (H&E).
Results Biologically active prostanoids such as PGE2 have several housekeeping functions in the kidney. Immunohistochemistry revealed COX-2 protein expression in the cortex and the medulla of the normal kidney (Figure 1A); the normal liver showed dotted signals of COX-2 protein, particularly on portal areas (Figure 1C). Amebic antigens were detected in livers from infected hamsters, showing a strong reaction on trophozoites and distributed throughout necrotic areas (Figure 1B). On the other hand, COX-2 expression in infected livers appeared mainly inside the trophozoites at 2, 4, and 7 days postinfection (Figure 1D). However, some dotted signals were observed in the parenchyma near the trophozoites. To determine whether the signal was inside the cells, we did a contrast stain with hematoxylin (1:10 dilution) after immunohistochemistry. COX-2 expression was detected in
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 1 2 2 - 3
XIV Seminar on Amebiasis, Mexico City / Archives of Medical Research 31 (2000) S122–S123
S123
Discussion Our results confirm that E. histolytica induces COX-2 expression in macrophages at the site of infection, which is consistent with the high levels of PGE2 produced by AMAs (1). Additionally, COX-2 protein expression in invasive trophozoites is consistent with the observation that E. histolytica produces PGE2 (5). Furthermore, PGE2 itself may feed back to increase the expression of COX-2; COX-2 induction in macrophages probably depends on continuous interaction with trophozoites or amebic excretion products. Studies are in progress to elucidate the modulatory role of COX-2 on the cellular immune response and the participation of E. histolytica antigens in COX-2 induction. Acknowledgments Figure 1. COX-2 expression in liver from hamsters infected i.h. with 1 ⫻ 106 trophozoites of E. histolytica at 4 days postinfection. (A) COX-2 expression in cells from normal kidney (arrowheads); (B) immunohistochemistry with a polyclonal antiameba antiserum in liver from infected hamster. Trophozoites showed a strong peroxidase reaction (arrowheads) and amebic antigens in the area (arrows); C) COX-2 expression in cells from normal liver (arrowheads), and (D) COX-2 expression in liver from infected hamster. Staining was detected inside the trophozoites (arrowheads) and in areas near the trophozoites. Original magnification ⫻40.
macrophages near trophozoites and in areas far from necrosis; COX-2 signal was observed in some polymorphonuclear cells (data not shown). Controls with preimmune serum did not show crossreaction with E. histolytica antigens; Western blot analysis of protein extracts from cultured trophozoites incubated with anti-COX-2 antiserum (1:100 dilution) showed no signal for this protein.
The authors thank A. Ríos for critical review of the manuscript. This work was supported by a grant from the Francisco Villa Research System of Conacyt (Mexico) (SIVILLA-Conacyt Reg. No. 9502-121).
References 1. Wang W, Chadee K. Entamoeba histolytica alters arachidonic acid metabolism in macrophages in vitro and in vivo. Immunology 1992;76:242. 2. Sánchez-Ramírez B, Escalante B, Rosales-Encina JL, Talamás-Rohana P. Role of prostaglandin E2 on amoebic liver abscess formation in hamsters. Prostaglandins 1997;53:411. 3. Holtzman MJ, Turk J, Shornick LP. Identification of a pharmacologically distinct prostaglandin h synthase in cultured epithelial cells. J Biol Chem 1992;267:21438. 4. Sánchez-Ramírez B, Escalante B, Rosales-Encina JL, Talamás-Rohana P. Entamoeba histolytica induces cyclooxygenase-2 expression in macrophages isolated from liver abscess. Proceedings, 9th Int. Congress of Parasitology. In: Tada I, Kojima S, Tsuji M, editors. Japan: Monduzzi Edit.;1998. p. 691. 5. Belley A, Chadee K. Eicosanoid production by parasites: from pathogenesis to immunomodulation? Parasitol Today 1995;11:227.
Entamoeba histolytica Induces Cyclooxygenase-2 Expression in Macrophages During Amebic Liver Abscess Formation Blanca E. Sánchez-Ramírez,* Marcela Ramírez-Gil,* Ernesto Ramos-Martínez** and Patricia Talamás Rohana*** *Facultad de Ciencias Químicas, **Departamento de Anatomía Patológica del Hospital Central Universitario, Facultad de Medicina, Universidad Autónoma de Chihuahua (UACH), Chihuahua, Mexico ***Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
Key Words: Entamoeba histolytica, Cyclooxygenase, Prostaglandin, Inflammation.
Introduction During amebic liver abscess (ALA) formation, macrophage activity is probably suppressed by inflammation-derived mediators, such as prostaglandins (1). Recently, we have demonstrated that development of experimental ALA courses with an increase in both systemic levels of PGE2 and local COX activity in liver microsomes (2). In addition, in vivo treatment of infected hamsters with indomethacin (Indo) reduced ALA formation by 30%. Conversion of arachidonic acid to PGE2 is catalyzed by COX, which exist in at least two isoforms: COX-1, a constitutive enzyme, and COX-2, an inducible isoform associated with inflammation (3). Nonsteroidal, anti-inflammatory drugs such as Indo inhibit both active COX enzymes. We recently reported that COX2 protein is expressed in ALA-derived macrophages (AMAs) (4). To demonstrate that COX-2 protein is expressed in vivo during liver infection with E. histolytica, we examined the localization of COX-2 by immunohistochemical analysis.
Materials and Methods Male in-bred hamsters (Mesocricetus auratus), weighing approximately 100 g, were infected intrahepatically (i.h.) with 1 ⫻ 106 trophozoites, as described previously (2). Noninfected hamsters were used as controls. At 2, 4, and 7 days postinfection the animals were anesthetized and killed
Address reprint requests to: Dra. Blanca E. Sánchez-Ramírez, Facultad de Ciencias Químicas, UACH, Apdo. Postal 1542-C, 31000 Chihuahua, Chihuahua, México. Telefax: (⫹52) (14) 144-492; E-mail: [email protected] Presenting author: Blanca E. Sánchez-Ramírez.
by exsanguination, and livers and abscesses were removed to calculate the percentage of liver damage. Liver samples obtained from normal and infected hamsters and from areas near to and far from the abscess were fixed with 10% phosphate-buffered formalin. Kidneys from normal hamsters were used as COX-2 controls. After embedding in paraffin, 6–8 m-thick sections were tested by immunohistochemistry following the protocol enclosed with the Histostain-Plus Kit (Zymed Lab., Inc.). The primary antibody, either polyclonal rabbit anti-COX-2 antiserum (1:250 dilution, Cayman Chem. Co.) or polyclonal rabbit antiameba antiserum (1:1000 dilution) was detected using secondary biotinylated affinity-purified antirabbit IgG antibody, avidin–peroxidase, and freshly prepared substrate. Controls with unspecific or preimmune antiserum were done. Samples were stained with hematoxylin and eosin (H&E).
Results Biologically active prostanoids such as PGE2 have several housekeeping functions in the kidney. Immunohistochemistry revealed COX-2 protein expression in the cortex and the medulla of the normal kidney (Figure 1A); the normal liver showed dotted signals of COX-2 protein, particularly on portal areas (Figure 1C). Amebic antigens were detected in livers from infected hamsters, showing a strong reaction on trophozoites and distributed throughout necrotic areas (Figure 1B). On the other hand, COX-2 expression in infected livers appeared mainly inside the trophozoites at 2, 4, and 7 days postinfection (Figure 1D). However, some dotted signals were observed in the parenchyma near the trophozoites. To determine whether the signal was inside the cells, we did a contrast stain with hematoxylin (1:10 dilution) after immunohistochemistry. COX-2 expression was detected in
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 1 2 2 - 3
XIV Seminar on Amebiasis, Mexico City / Archives of Medical Research 31 (2000) S122–S123
S123
Discussion Our results confirm that E. histolytica induces COX-2 expression in macrophages at the site of infection, which is consistent with the high levels of PGE2 produced by AMAs (1). Additionally, COX-2 protein expression in invasive trophozoites is consistent with the observation that E. histolytica produces PGE2 (5). Furthermore, PGE2 itself may feed back to increase the expression of COX-2; COX-2 induction in macrophages probably depends on continuous interaction with trophozoites or amebic excretion products. Studies are in progress to elucidate the modulatory role of COX-2 on the cellular immune response and the participation of E. histolytica antigens in COX-2 induction. Acknowledgments Figure 1. COX-2 expression in liver from hamsters infected i.h. with 1 ⫻ 106 trophozoites of E. histolytica at 4 days postinfection. (A) COX-2 expression in cells from normal kidney (arrowheads); (B) immunohistochemistry with a polyclonal antiameba antiserum in liver from infected hamster. Trophozoites showed a strong peroxidase reaction (arrowheads) and amebic antigens in the area (arrows); C) COX-2 expression in cells from normal liver (arrowheads), and (D) COX-2 expression in liver from infected hamster. Staining was detected inside the trophozoites (arrowheads) and in areas near the trophozoites. Original magnification ⫻40.
macrophages near trophozoites and in areas far from necrosis; COX-2 signal was observed in some polymorphonuclear cells (data not shown). Controls with preimmune serum did not show crossreaction with E. histolytica antigens; Western blot analysis of protein extracts from cultured trophozoites incubated with anti-COX-2 antiserum (1:100 dilution) showed no signal for this protein.
The authors thank A. Ríos for critical review of the manuscript. This work was supported by a grant from the Francisco Villa Research System of Conacyt (Mexico) (SIVILLA-Conacyt Reg. No. 9502-121).
References 1. Wang W, Chadee K. Entamoeba histolytica alters arachidonic acid metabolism in macrophages in vitro and in vivo. Immunology 1992;76:242. 2. Sánchez-Ramírez B, Escalante B, Rosales-Encina JL, Talamás-Rohana P. Role of prostaglandin E2 on amoebic liver abscess formation in hamsters. Prostaglandins 1997;53:411. 3. Holtzman MJ, Turk J, Shornick LP. Identification of a pharmacologically distinct prostaglandin h synthase in cultured epithelial cells. J Biol Chem 1992;267:21438. 4. Sánchez-Ramírez B, Escalante B, Rosales-Encina JL, Talamás-Rohana P. Entamoeba histolytica induces cyclooxygenase-2 expression in macrophages isolated from liver abscess. Proceedings, 9th Int. Congress of Parasitology. In: Tada I, Kojima S, Tsuji M, editors. Japan: Monduzzi Edit.;1998. p. 691. 5. Belley A, Chadee K. Eicosanoid production by parasites: from pathogenesis to immunomodulation? Parasitol Today 1995;11:227.