Interaction of antibodies with Entamoeba histolytica trophozoites from ...

Parasitol Res (2000) 86: 603±607

Ó Springer-Verlag 2000

SHORT COMMUNICATION

R. Campos-RodrõÂ guez á A. Jarillo-Luna J. Ventura-JuaÂrez á M. Shibayama J. Pacheco-YeÂpez á J. Serrano-Luna á V. Tsutsumi

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study Received: 26 September 1999 / Accepted: 24 November 1999

Abstract Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacri®ced at di€erent days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1±7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1±2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-speci®c during the ®rst 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host.

Introduction Although in vitro studies have shown that speci®c antibodies and complement can damage axenically cultured Entamoeba histolytica trophozoites, their in vivo role is still poorly understood. Most of the previous studies on human and experimental models of amebiasis have suggested that antibodies are harmless to amebas. The high systemic humoral response induced after an invasive amebiasis is unrelated to preventing subsequent reinfections or to curing the disease (Krupp 1970; Perches et al. 1970; Krupp and Powell 1971; Abioye et al. 1972; Juniper et al. 1972; Kagan 1973; ChacinBonilla and Bonpart 1981; Trissl 1982). In vivo, the trophozoites appear to endure the cytotoxic e€ect of the antibodies and complement; however, in vitro these molecules are very e€ective (Ortiz-Ortiz et al. 1974, 1978; Capin et al. 1978; Huldt et al. 1979; CalderoÂn and Schreiber 1985; Reed et al. 1986). In this study we analyzed the in vivo interaction between the IgG antibodies (non-speci®c and speci®c) and E. histolytica trophozoites present during the evolution of experimentally induced amebic liver abscess (ALA) in hamsters.

Materials and methods Experimental animals

R. Campos-RodrõÂ guez á A. Jarillo-Luna á J. Serrano-Luna Departamento de BioquõÂ mica, ESM-IPN, MeÂxico D.F., MeÂxico

Male adult hamsters (Mesocricetus auratus; 24 animals, each weighing 100 g) were used. Ameba culture

A. Jarillo-Luna á M. Shibayama á J. Pacheco-YeÂpez V. Tsutsumi (&) Departamento de PatologõÂ a Experimental, CINVESTAV-IPN, Av. IPN 2508, Col. Zacatenco, MeÂxico D.F. 07360, MeÂxico

Axenically cultured Entamoeba histolytica trophozoites (HM1IMSS strain; Diamond et al. 1978), cloned and passed three times through the hamster liver were used.

J. Ventura-JuaÂrez Centro Interdisciplinario de Ciencias de la Salud, IPN, MeÂxico D.F., MeÂxico

The anti-E. histolytica IgG antibodies present in the serum of the experimental and control groups were quanti®ed by the ELISA method (Tsutsumi et al. 1992).

Quanti®cation of hamster anti-ameba IgG antibodies

604

Amebic liver abscess production in hamsters Prior to surgery, animals were anesthetized by peritoneal injection of sodium pentobarbital (Anestesal) at doses of 4.72 mg/100 g body weight. Hamsters were intrahepatically inoculated with

1.25 ´ 105 trophozoites. At 24 h, 48 h, 72 h, 4 days, and 7 days post-inoculation, groups of four animals were sacri®ced. All animals were sacri®ced using overdoses of sodium pentobarbital (10 mg/100 g body weight), bled by cardiac puncture, and the whole liver and abscesses were dissected and weighed.

605 b Fig. 1a±f Immunocytochemical assay of the interaction of hamster IgG with Entamoeba histolytica trophozoites. The ®rst antibody used in a±c (1, 4, and 7 days respectively) was rabbit anti-ameba, and in d±f (1, 4, and 7 days) we used rabbit anti-hamster IgG. In both cases, the secondary antibody was goat anti-rabbit IgG labeled with peroxidase. The trophozoites (arrows) stained with anti-ameba serum showed the same intensity of labeling at all post-inoculation times (a±c). In the samples of hepatic tissue obtained on day 1 post-inoculation (d) and reacted with anti-hamster IgG, the trophozoites displayed high staining over the complete surface. At later stages, the staining decreased progressively as shown in samples from 4 days (e) and 7 days (f) post-inoculation. ´1,200

Immunocytochemistry of experimental ALA Selected liver fragments containing amebic lesions were ®xed with 10% formaldehyde in a phosphate-bu€ered saline (PBS) and processed for paran embedding. Sections (6 lm thick) were mounted on glass slides. After dewaxing with xylene and rehydration, endogenous peroxidase activity was blocked by incubation in 1% H2O2 in methanol for 30 min. The sections were washed three times with PBS, and incubated with 10% serum bovine albumin in PBS. The slides of each fragment were incubated with the primary rabbit antibodies (anti-ameba or antihamster IgG) and the secondary antibody (a goat anti-rabbit IgG labeled with peroxidase). The controls were incubated only with PBS instead of the primary rabbit antibody. The reaction was developed using immunopure metal-enhanced diaminobenzidine (DAB: Pierce). Finally the slides were counterstained with hematoxylin, dehydrated with alcohol, cleared in xylene and mounted with synthetic resin and examined with a light microscope. Determination of the number of trophozoites in hepatic lesions The number of trophozoites present in liver lesions were counted in an area 0.25 mm2 and the intensity of cytochemical staining was graded from + to ++++. Reactions were performed in three di€erent samples from each animal, and the mean of the number of positive amebas was obtained. Trophozoite isolation from liver abscesses The abscesses were dissected and isolated. A fragment of liver was cut into multiple small pieces, forming a turbid cell suspension, then washed in PBS and centrifuged twice at 1,500 rpm at 4 °C. Finally, the cells were ®xed with 4% paraformaldehyde for 30 min at room temperature and then washed three times in PBS for 5 min. Detection of the IgG on the trophozoite membranes A sample of cell suspension (100 ll) containing amebas obtained from the abscess were incubated for 1 h at room temperature with 100 ll serum (diluted 1:100) obtained from hamsters with ALA. After incubation, the preparations were washed in PBS three times for 5 min and then incubated with peroxidase-labeled rabbit anti-hamster IgG (Dako) diluted 1:100 in PBS for 1 h at room temperature. The reaction was developed using immunopure metal-enhanced DAB for 10 min and washed with PBS. Statistical analysis The results were analyzed by one way analysis of variance. A difference was considered signi®cant with P < 0.05.

Results The trophozoites obtained from hamster hepatic lesions at all post-inoculation times reacted positively to rabbit IgG anti-Entamoeba histolytica (Fig. 1a±c). Twenty-four hours after intrahepatic inoculation with virulent trophozoites, approximately 80% of the trophozoites were seen labeling cell surfaces and inside the cells when anti-hamster IgG antibodies were used (Fig. 1d). This happened in spite of the absence of speci®c anti-ameba antibodies in the blood serum (Table 1) and suggests a non-speci®c interaction. The number of cells stained and the intensity of labeling lessened progressively, and the results at day 4 post-inoculation are shown in Fig. 1e. At day 7 post-inoculation, only 30% of trophozoites were lightly stained (Fig. 1f), although at that time speci®c anti-ameba antibodies were detected in the sera using the ELISA method (Table 1). Amebic antigens were detected on the surface of amebas isolated from abscesses; however, hamster immunoglobulins were scarce (Fig. 2a, b). Only 10% of the trophozoites isolated from the abscesses obtained day 7 post-inoculation showed IgG on the plasma membranes (Table 2). However, the percentage of trophozoites interacting with speci®c antibodies increased notably (78%: Table 2, Fig. 2) when the amebas were incubated with serum from the same animal which had developed an abscess and produced IgG anti-ameba antibodies.

Discussion The trophozoites of Entamoeba histolytica present during the ®rst 2 days of ALA evolution in hamsters probably capture a high concentration of serum immunoglobulins from the host, which are non-speci®c to E. histolytica. This assumption is based on the fact that speci®c antibodies to this protozoan cannot be detected Table 1 In vivo interaction of hamster antibodies with Entamoeba histolytica trophozoites. Data were obtained from three di€erent assays and represent the mean of the number of positive cells with the antibodies anti-ameba and anti-hamster IgG. The intensity of the reaction is graduated from + (weak) to ++++ (intense). The amount of IgG antibodies anti-E. histolytica was measured by ELISA and values in the table represent the mean and standard error (for each group, n = 6) of absorbance at 492 nm. Signi®cant di€erences appear on day 4 (P = 0.043) and day 7 (P < 0.001), compared with the 1st day (*). These di€erences were calculated by one-way analysis of variance Time (days)

Positive amebas (%) and intensity (+) Anti-ameba

Anti-IgG hamster

ELISA Absorbance at 490 nm

1 2 3 4 7

100 100 100 100 100

77 (++/+++) 65 (++) 63 (++) 42* (+) 30* (+)

0.54 0.85 0.123 0.245* 0.565*

(++++) (++++) (++++) (++++) (++++)

‹ ‹ ‹ ‹ ‹

0.01 0.02 0.01 0.04 0.05

606

Fig. 2a, b Detection of IgG and surface antigens in trophozoites isolated from amebic liver abscesses on day 7 post-inoculation. Isolated amebas from the abscess were either not incubated (a) or incubated (b) with hamster serum from animals with amebic liver abscess and containing anti-E histolytica IgG antibodies. The secondary antibody was rabbit anti-hamster IgG labeled with peroxidase. ´1,200

in the blood serum before the 4th day after intrahepatic inoculation. The process in which the amebas capture antibodies can be due to a normal course of endocytosis or to a phenomenon mediated by a molecule similar to a receptor for the Fc fragment of IgG (Galatiuc et al. 1981). In the chronic stages of ALA evolution in hamsters, the expression of immunoglobulins by the trophozoites contained in the abscess was weaker. We suggest that this lack of antigen expression on the membrane of the parasite is due either to a failure in the circulatory system (ischemia) in the area surrounding the liver abscess (Tsutsumi et al. 1984; PeÂrez-Tamayo et al. 1992), or to an in vivo destruction of the antibodies by amebas, as has been demonstrated in vitro by Tran et al. (1998). We suggest that all these factors are participating at least in part in hindering the contact between the antibodies and the trophozoites. Nevertheless, it was established by immunohistochemical procedures that the amebas isolated from the abscess had membrane proteins which were recognized in vivo and in vitro by the anti-ameba antibodies. However, the percentage of trophozoites with hamster Table 2 In vitro interaction of hamster antibodies with trophozoites of E. histolytica obtained from amebic liver abscess. Amebas obtained from the axenic culture or from the hepatic abscess were ®xed with paraformaldehyde and incubated with or without hamster serum containing IgG anti-E. histolytica antibodies. Cell suspensions were incubated and washed, and then IgG of rabbit anti-IgG of hamster (labeled with peroxidase) was added and revealed with diaminobenzidine. Results are shown as mean and standard error of three di€erent experiments Amebas from

Axenic culture Hepatic abscess

Amebas with label (%) Without Abs

With Abs

0 10 ‹ 6

93 ‹ 3 78 ‹ 4

IgG on the surface was lower. This supports the hypothesis that the antibodies have diculty interacting in vivo with the trophozoites located in the liver lesions, probably due to circulatory problems around the hepatic necrosis. This could in part explain why the immunoglobulins do not have a relevant role against amebic invasive infection. Acknowledgements This work was supported in part by grants from CONACYT (MeÂxico) and COFAA-IPN (MeÂxico).

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