Pathogenesis of Acute Experimental Liver Amebiasis

Archives of Medical Research 37 (2006) 203–209

REVIEW ARTICLE

Pathogenesis of Acute Experimental Liver Amebiasis Ruy Pe´rez-Tamayo, Irmgard Montfort, Alfonso Olivos Garcı´a, Espiridio´n Ramos, and Carlos Barba Ostria Department of Experimental Medicine, National Autonomous University Medical School, Mexico City, Mexico Received for publication October 20, 2005; accepted October 21, 2005 (ARCMED-D-05-00434).

Classical descriptions of the pathology of amebiasis portray the parasite as the cause of tissue damage and destruction, and in recent years a number of amebic molecules have been identified as virulence factors. In this review we describe a series of experiments that suggest a more complex host–parasite relation, at least during the early stages of acute experimental amebic liver abscess in hamsters. The problems of extrapolating experiments in vitro to explain observations in vivo are discussed. The role of amebic cysteine proteases is examined and evidence presented to suggest that they are primarily related not to tissue damage but to amebic survival, which is required for the progression of the lesion. Inflammation is shown to be not only the major cause of tissue damage but also an absolute requirement for amebic survival in the liver, whereas complement and ischemia are not involved in the disappearance of the parasite in the absence of inflammation. Ó 2006 IMSS. Published by Elsevier Inc. Key Words: Experimental liver abscess, Hamster, Cysteine proteases, E-64, Inflammation, Complement, Ischemia.

Introduction Since the classic publication of Councilman and Lafleur (1) in 1891 entitled Amoebic Dysentery and based on the clinicopathological study of 14 autopsy cases, the pathology of the human disease has been periodically reviewed and adequately illustrated several times (2–8). A survey of the literature published on the subject since the last comprehensive review available failed to produce any new or significant information. Such a result is not something to celebrate, because many questions about the pathology of human amebiasis are yet to be answered. But it seems that their more likely explanations will not come from more and more careful and sophisticated morphological studies (although surprises are not excluded), but rather from cellular, biochemical, immunological and other molecular probings of the host–parasite relation, both in human and in experimental models of the disease. Instead of restating the well-known facts of the pathology of human

Address reprints requests to: Ruy Pe´rez-Tamayo, Departamento de Medicina Experimental, Facultad de Medicina de la UNAM, Hospital General de Me´xico, Dr. Balmis 148, Col. Doctores, Me´xico, D.F., 06814, Me´xico; E-mail: [email protected]

amebiasis, we have focused this review on some of the work performed in our laboratory exploring the mechanisms of tissue damage and destruction in early amebic lesions, using as a model the acute experimental liver abscess produced in hamsters (AEALAH) by the intraportal injection of axenically grown trophozoites of E. histolytica strain HM1-IMSS.

Background For many years (actually, since 1891), E. histolytica has been etiologically connected with the disease known as human amebiasis (9). It is interesting that some of the very first observations of the presence of the parasite in the affected patients (feces, pulmonary secretions) (10,11) were not considered as significant, but rather as opportunistic or coincidental infections. However, the pioneering human experiments of Walker and Sellards (12) in 1913 established the causal relation between the parasite and the disease, as reliably as was possible at that time. Further studies of the pathology of human amebiasis led to the definitive proof that E. histolytica was a true pathogen and strongly suggested that tissue damage in amebic disease was caused by the

0188-4409/06 $–see front matter. Copyright Ó 2006 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2005.10.007

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parasite (13,14). The pathogenesis of the lesions was believed to be directly secondary to the histolytic properties of amebas that, upon arrival in the intestine or other tissues, induced extensive and often progressive necrosis with little reactive inflammation (15). Early suggestions that amebic proteases were responsible for tissue destruction were inconclusive, and although gelatinase (16,17), glutaminase (18) and casease (19) activities were demonstrated in extracts of pathogenic amebas, the substrates used were denatured and no proteases capable of degrading proteins in their native state were observed. In addition, the same proteases were present in nonpathogenic amebas. A hyaluronidase was also described in amebas (20,21), but careful studies failed to reveal any correlation between such enzyme and parasite virulence (22). In 1970, in a monograph on human amebiasis one of the authors (5) wrote: ‘‘Therefore, it must be concluded that, although the more probable mechanism of amebic tissue penetration and cellular destruction is enzymatic lysis, there still is no evident proof of it, and the name ‘‘histolytica’’ is yet to be justified on basis firmer than pure morphologic inference.’’ Since that time a vast literature has accumulated incriminating several amebic molecules, some with hydrolytic and other biologic activities, as directly responsible for cell and tissue damage in amebiasis. Those more frequently mentioned are amebapore (23,24), an 18-kDa protein with no enzyme activity but with the ability to create ion channels in cell membranes; a galactose and N-acetyl-D-galactosamine (Gal/GalNAc)-specific lectin, also non-enzymatic but that mediates adhesion of the parasite to colonic mucins (25,26); phospholipase A (27) and collagenase (28), proteolytic enzymes with better-defined substrates and at least 20 genetically different cysteine proteinases (EhCPs) (29,30) with a broad protein-substrate spectrum (31). A critical analysis of the relevant information supporting the claim that each of the amebic molecules mentioned plays a significant role in cell and tissue destruction in both experimental and human amebiasis reveals the following problems. In Vitro Experiments Purified amebapore has a cytotoxic effect on several cell lines, provided it is tested in acid pH (5.6-6.0 u) (32). Purified EhCPs have a cytopathic effect on monolayers of HeLa cells (33), BHK cells (31), and human fibroblasts (34). A 30-kDa EhCP is effectively cytolytic on dead rat and hamster hepatocytes and this activity is blocked by E-64, a specific inhibitor of such enzymes (35). Decrease of EhCP5 expression induced in E. histolytica by antisense mRNA correlates with decreased phagocytosis but cytopathic effect and hemolytic activity remain unchanged (36). Overexpression of EhCP2 in both E. histolytica and E. dispar

effectively increases the cytopathic activity of those two amebic species (37). Experiments using purified amebic molecules tested against different cell lines under in vitro conditions adequate for cell culture provide interesting results, but they are far removed from the in vivo situations in amebic disease. Major differences in pH, O2 concentration, and the presence of a very different environment with many additional components may completely change the nature of the results obtained in vitro. In Vivo Experiments E. histolytica trophozoites grown axenically and in the presence of E-64 (38) or laminin (39) have a decreased capacity to produce liver abscesses in immunodeficient mice (SCID). Lysates of virulent E. histolytica lower the transepithelial electric resistance (TER) in the cecum of gerbils, and this effect is inhibited by E-64 (40). Decrease in EhCP5 expression, induced in E. histolytica by means of antisense mRNA, correlates with a decreased capacity of the parasite to induce liver abscesses in hamsters (41) and to cause inflammation, to secrete Il-2 and to convert proIl-1 to Il-1 in human intestine transplanted to SCID mice (42). The experiments mentioned above and others similar in design and results certainly suggest that some amebic molecules, especially EhCPs, may play a role in tissue damage in amebiasis. But the evidence is far from conclusive because in many of those experiments the effect of the amebic manipulation on the viability and other functions of the parasite were not tested, and in those few that included such observations it was clear that both growth and viability of amebas were compromised (43). CPs are present in many other species of parasites (44), and when their enzymatic activity is blocked with inhibitors their survival and nutritional metabolism are severely damaged (45). Thus, the results of in vivo experiments with interference of EhCPs could be equally interpreted as indicating an important role of such enzymes in the survival of the parasite, which is necessary for the initiation and progression of tissue damage, probably related to other molecular mechanisms of the parasite and/or of the host. That the host is involved in tissue damage and destruction in amebiasis has been suspected for a long time, since the existence of ‘‘healthy carriers’’ was experimentally established in humans by Walker and Sellards in 1913 (12). That virulent E. histolytica isolated from the feces of a patient suffering from clinical amebiasis fails to produce the disease in healthy subjects when they are infected with such parasite and become cyst passers, and these cysts are further fed to other healthy subjects and some of them develop the full disease while others remain asymptomatic, was considered proof that some hosts either lack something that the parasite needs to cause the disease or have something that actively prevents them from doing it. Whatever the

Pathogenesis of Acute Experimental Liver Amebiasis

Because standard purification procedures of EhCPs have very low yields, we developed a new scheme based on ethyl ether extracts of axenically grown trophozoites of E. histolytica strain HM1-IMSS, which includes molecular filtration and electroelution and yields milligrams instead of micrograms of active protein. Sequence analysis of the purified product revealed EhCP2 plus ubiquitin(s). Electrophoretic migration patterns, isoelectric point determination and Western blot failed to reveal other EhCP molecules. Polyclonal antibodies produced in rabbits against the purified EhCP2 either stabilized or enhanced the enzyme activity in a dose response fashion. Purified EhCP2 was enclosed within inert resin microspheres (22–44 mm in diameter) and injected into the portal vein of normal hamsters. In the liver, control microspheres (without EhCP2) caused no reaction, whereas those carrying EhCP2 caused mild acute inflammation as early as 3 h after injection, accompanied by minimal necrosis of surrounding liver cells present 24 h after injection. Such changes disappeared in a short time. Liver sections were immunohistochemically stained with the antiEhCP antibody and the microspheres were positive for only a very short period (1 h) after injection. These ‘‘synthetic amebas’’ are admittedly a very crude model of the real parasites, but they represent the first attempt to examine the role of a single molecular component of E. histolytica in the causation of tissue damage in an in vivo model of amebiasis, in the absence of all other possible participating amebic elements. Immunohistochemical studies of liver sections using the anti-EhCP2 antibody of AEALAH (1 day, 5 days) routinely revealed intense staining (with both peroxidase or Au-silver techniques) of EhCP2 within amebas, but there was never positive staining of amebic EhCP2 in the extra-amebic space, not even in the immediate vicinity of the parasite. Such negative results could be explained by either no EhCP2 secretion or by a very low concentration of the enzyme in the extra-amebic spaces, below the sensitivity of the techniques used to detect it (46). E. histolytica and E. dispar made by transfection to overexpress EhCP2 (2.7 fold) were injected into gerbil livers and 7 days later

conclusion, it became established that the host plays a significant role in the nature of the host–parasite relation that is established when pathogenic cysts of E. histolytica enter the human organism. Epidemiological studies established that only about 10% of those infected with E. histolytica develop amebiasis, and efforts to account for the lack of susceptibility of those that remain as ‘‘healthy carriers’’ on the basis of acquired immunity or genetic features have so far been unsuccessful.

Current Work in Our Laboratory In our laboratory we have conducted a series of experiments aimed to explore with more detail the pathogenic role of both amebas and the host in the development of one specific type of early amebic tissue damage, namely, AEALAH. On the side of E. histolytica and because of their prominence in the literature as probable virulence factors, so far we have closely examined the role of EhCPs. On the side of the host and because of their alleged contribution to the early stages in the development of tissue lesions, we have studied the participation of polymorphonuclear leukocytes (PMNLs) and other inflammatory cells, of complement, and of the ischemia that accompanies the liver lesions since their inception. We are fully aware that this is just the beginning of a survey that will eventually include many other elements but are encouraged by the results obtained so far. We are also conscious that our data are only valid for the very specific model under study, and that things may be more or less different in later stages of the development of the lesions, or in other tissues of the same host, or in other susceptible species. On the other hand, we know that virulent amebic trophozoites preserved in vitro are in a medium and under conditions very different from those present in vivo, not only because their physical and chemical environment is completely new but also because of their sudden exposure to the cells and other host factors. Figure 1 illustrates the general hypothesis used to design the first series of experimental observations.

EhCPs

?

?

host

inflammation

?

amoebic survival

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tissue damage

Ischemia

? x

complement and high O2 tension

?

Figure 1. Role of EhCPs in tissue damage and destruction in AEALAH.

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revealed no differences with the results of control experiments with wild E. histolytica and E. dispar, i.e., the former parasitic strain did not increase in virulence and the latter remained non-pathogenic (37). Together these results are complementary and could be explained if overexpression of EhCP2 is not accompanied by secretion of the enzyme. We are satisfied that our antibody does not cross-react with EhCP5, but it has not been tested against other EhCPs. Thus it remains possible that EhCPs other than EhCP2 are secreted and contribute to the extensive tissue destruction that rapidly develops surrounding the parasite. Our work and that of others support the suggestion that EhCP2 is not secreted and is not directly involved in tissue damage and that other amebic molecules, either other EhCPs or other different amebic components, may be responsible for tissue lesions during amebiasis. The role of EhCPs in tissue damage in AEALAH has been further explored in a series of in vitro and in vivo experiments using E-64 as a specific and irreversible inhibitor. The effect of EhCPs inhibition on the parasite viability, replication, complement resistance, cytotoxicity, TER, erythrophagocytosis, hemolytic capacity, virulence and survival was established. Complete inhibition of total EhCP activity of axenic trophozoites of E. histolytica grown in vitro in the presence of E-64 and for as long as 72 h had minor or no effects on viability, growth curve, cytotoxicity, hemolytic capacity and complement resistance. On the other hand, erythrocyte phagocytosis and TER of MDCK monolayers were somewhat decreased. Preservation of viability and other properties of amebas in vitro despite the complete and prolonged inhibition of total EhCP activity suggest that the culture medium must contain all the elements required for their normal metabolism. Use of E. histolytica trophozoites grown for 72 h in the presence of E-64 to produce AEALAH in hamsters treated with daily intraperitoneal injections of E-64 revealed no lesions 5 days after injection, when controls displayed both gross and microscopic multiple abscesses. In animals specially prepared to detect lesions at earlier periods, there were small groups of PMNLs with poorly preserved amebas still present 8 h after injection. Twenty h after injection no amebas were present and there were only small foci of PMNLs leucop. Thus, EhCP activity appears to be an absolute requirement for survival of amebas in in vivo conditions, at least in the experimental situation described in this work. The possibility that EhCP activity is directly related to the initiation of inflammation and of tissue damage ignores the fact that full inhibition of EhCP activity has no influence on amebic cytotoxicity in vitro and on initial inflammation in vivo and also fails to explain the rapid disappearance of amebas when there is no cell and tissue destruction. An alternative explanation would be that EhCP activity is primarily necessary for survival of amebas in in vivo conditions, and that amebic survival is required

for the development of tissue damage caused by various mechanisms, which may very well include other EhCP activity (47).

Role of Inflammation, Complement, Ischemia and Amebic Survival in AEALAH The pioneering morphologic studies of Tsutsumi et al. (48,49) published 20 years ago of the earlier stages in the development of AEALAH were the first to suggest rather strongly that tissue damage and destruction were not caused directly by E. histolytica but rather by the lysosomal enzymes of disintegrating inflammatory cells that accumulate rapidly around the parasites and are killed by them. Their observations were confirmed in several laboratories and the experimentum leucop, i.e., the attempt to produce AEALAH in leukopenic animals yielded negative results. In such experiments viable amebas were microscopically present within liver sinusoids up to 6 h after their intraportal injection; there were no inflammatory cells surrounding them and no liver cell and tissue damage in their vicinity. An unexpected finding was that amebas were no longer detectable after that period, suggesting that the tissue lesion was necessary for their survival (50). Following such observations, we examined the role of inflammatory cells, serum complement and ischemia on the development of AEALAH (51). In hamsters made leukopenic by whole body radiation (800 rads), it takes 48 h for the peripheral PMNLs to drop from slightly over 10,000 cells/mm3 to !300 cells/mm3; the cell count remains at this level until the animal dies, usually after 7 days. Hamsters injected intraportally with 1 3 106 axenic trophozoites 2 h after whole body radiation and sacrificed 24 h after the injection revealed liver lesions differing from controls in size and degree of inflammation but still characterized by dense infiltration with PMNLs and macrophages (Mos) surrounding well-preserved amebas and minimal hepatocyte damage, restricted to the immediately surrounding liver tissue. Similarly treated animals sacrificed 6 days after the injection of amebas showed normal livers with no lesions and no amebas. The same experiment performed 24 h after total body radiation gave the same results. When the intraportal injection of amebas was given 48 h after total body radiation the results were different from the previous experiments: in animals sacrificed 6 h after amebic injection the liver revealed lesions and parasites identical to those found in controls; in those sacrificed 24 h after the injection of trophozoites there were very few and small lesions with few inflammatory cells, minimal necrosis and few or no amebas, whereas radiated animals sacrificed 6 days after the injection of trophozoites revealed neither lesions nor amebas. Thus, when virulent amebic trophozoites reach the liver soon after radiation, they are still able to stimulate inflammation and initiate an early lesion, which subsides

Pathogenesis of Acute Experimental Liver Amebiasis

and disappears as leucopenia becomes more pronounced. Once leucopenia is nearly complete, little or no tissue damage is elicited by amebas, which disappear in less than 24 h. Thus, in the absence of inflammatory cells, virulent E. histolytica causes no liver damage in hamsters and disappears in a very short time period (6 h). Susceptibility of axenic amebic trophozoites to serum complement has been repeatedly reported, although there is a wide variety in the percent of amebas killed depending on the animal species tested as serum donor. No amebas survive after exposure for 24 h in vitro to hamster serum, whereas exposure for the same time period to serum obtained from hamsters previously treated with cobra venom factor (CVF) results not only in full amebic survival but in a normal increase in numbers through replication. Leukopenic hamsters were used to examine the role of complement in the disappearance of amebas when their presence in the liver elicits no inflammation and tissue damage. Hamsters made leukopenic by whole body radiation were injected with CVF and their serum shown to have lost completely its amebic-killing capacity in vitro. Such animals were then injected intraportally with 1 3 106 axenically grown amebic trophozoites and their livers examined after different time periods (6, 24, 48 and 72 h). Amebas were no longer present after 48 and 72 h. Therefore, complement seems to play no role in the disappearance of amebas in the leukopenic hamster. To examine the effect of focal ischemia induced in radiated animals that show no inflammation surrounding the injected amebas, and therefore develop no lesion-related ischemia, 1 3 106 axenic trophozoites were mixed with 2 3 106 Superdex 75 microspheres and injected intraportally in hamsters 48 h after whole-body radiation with 800 rads and 3 h after the first intraperitoneal injection of 16 mg of glycogen, which was repeated evey 24 h. The animals were sacrificed at 6, 24, 48 and 72 h after the intraportal injection of amebas mixed with Sephadex 75 microspheres. Microscopically well-preserved amebas were observed without surrounding inflammatory cells lying in the midst of well-

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outlined ischemic areas only 6 h after injection. After this period, no amebas were visible, although ischemic areas remained detectable for as long as 72 h.

Conclusions The diagram used to specify the different possible interactions in the complex host–parasite relation in AEALAH may be modified as follows (see Figure 2). Thus, early AEALAH inflammation is necessary for both tissue damage and amebic survival, which in turn is required for the progression of the lesion. It is clear that the major contribution to liver cell and tissue destruction is made by inflammatory cells, although some participation of amebas cannot be excluded. On the other hand, it is not known why amebas fail to survive in the absence of inflammation, because neither complement nor ischemia seems to be involved in their disappearance in the absence of inflammation. The current notion that amebas survive because they resist the potent killing mechanisms of PMNLs and Mos that work so well with other virulent microorganisms is inadequate to explain the results of our experiments. Perhaps the opposite may be true, that inflammatory cells provide amebas with some kind of stimulus necessary for their survival in the liver. This sounds counterintuitive; nevertheless, it may still be true. Analysis of the more complex pathology of AEALAH characterized by extensive tissue necrosis, granulomatous metaplasia of Mos, fibrous capsule formation, massive proliferation of amebas, conspicuous scarcity of inflammatory cells and coexistence of lesions of very different ages, among many other elements not present in AEALAH, suggests that it may be a more complex biological problem in which both host and parasite play different and probably changing roles. Before entering such an elaborate and difficult field, we would do well to concentrate on the (apparently) simpler problems presented by AEALAH, namely, the basic requirements for amebic trophozoite survival once they enter the portal vein.

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complement and high O2 tension X

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Figure 2.

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Acknowledgments We gratefully acknowledge the excellent technical help of Pedro Balderas Flores and Marco E. Gudin˜o Salas, as well as the professional animal care of Ricardo Vargas Orozco and Daniel Sa´nchez Almaraz. This work was partially supported by CONACYT Grants 30831-M and 25119-M, and by DGAPA Grant No. IN-225998.

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