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Septal fibrosis of the liver in C. hepatica-infected ratsBrazilian Journal of Medical and Biological Research (2003) 36: 1201-1207ISSN 0100-879X

Immunological basis of septalfibrosis of the liver in Capillariahepatica-infected rats

Laboratório de Patologia Experimental, Centro de Pesquisa Gonçalo Moniz,Fundação Oswaldo Cruz, Salvador, BA, Brasil

Q.T. Lemos,I.F. Magalhães-Santos

and Z.A. Andrade

Abstract

Rats infected with the helminth Capillaria hepatica regularly developseptal fibrosis of the liver similar to that induced by repeated ipinjections of pig serum. Fibrosis starts when the focal parasitic lesionsbegin to show signs of resorption, thus suggesting an immunologicallymediated pathogenesis of this fibrosis. To explore this possibility, thedevelopment of C. hepatica-related hepatic fibrosis was observed inrats exposed to worm antigens from the first neonatal day onward.Wistar rats (150 g) were either injected ip with an extract of C.hepatica eggs (protein concentration: 1 mg/ml) or received immatureeggs by gavage from the first neonatal day until adult life and werethen infected with 500 embryonated eggs. Changes were monitored onthe basis of serum levels of anti-worm antibodies and hepatic histopa-thology. Rats submitted to immunological oral tolerance markedlysuppressed C. hepatica-related serum antibodies and septal fibrosis ofthe liver when infected with the helminth later on. Tolerance trialswith ip injections of worm antigens gave essentially negative results.The partial suppression of septal fibrosis of the liver after the induc-tion of immunological tolerance to C. hepatica antigens in rats indi-cates an immunological basis for the fibrosis and emphasizes theimportance of immunological factors in the pathogenesis of hepaticfibrosis.

CorrespondenceZ.A. Andrade

Rua Valdemar Falcão, 121

41295-001 Salvador, BA

Brasil

E-mail: zilton@cpqgm.fiocruz.br

Research supported by PAPES III

(FIOCRUZ) and PRONEX.

Received August 20, 2002

Accepted June 3, 2003

Key words• Capillaria hepatica model• Hepatic fibrosis• Immunological tolerance

Introduction

Recent in vitro studies have demonstratedthat soluble mediators are released by stimu-lated Kupffer cells (1-4), including severalcytokines, such as transforming growth fac-tor-ß (4). Mediators induce Ito’s stellate cellsto proliferate, differentiate and secrete extra-cellular matrix components (5). It is to beexpected that this hepatic cellular axis, com-posed of two nonparenchymal cell types lo-cated within and around the hepatic sinuso-

ids, would play a decisive role in the patho-genesis of the types of hepatic fibrosis desig-nated as perisinusoidal and septal fibrosis.

Variable degrees of hepatic fibrosis wereobtained in rats submitted to repeated andprolonged injections of foreign proteins (6-8). A peculiar model emerged from theseexperimental trials carried out to induce fi-brosis by immunological means when wholepig serum or its albumin fraction was used(9). In the pig serum model, fibrosis gradu-ally and insidiously developed, without be-

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ing preceded by hepatocellular necrosis orchronic inflammation (10), later progressingto a morphologic picture of cirrhosis (11).Bhunchet et al. (12) demonstrated that pigserum acts as an antigen, with the immuneresponse to it being the decisive factor con-tributing to the pathogenesis of this peculiartype of fibrosis, which therefore proved torest on an immunological basis. These inves-tigators made rats tolerant to pig serum albu-min by inoculating them with this proteinfrom the first neonatal day to adult life.Then, at the end of the tolerance inductionperiod, the animals developed extremely lowantibody levels and no evidence of hepaticfibrosis when submitted to a further periodof repeated injections with pig serum albu-min. This procedure resulted in high serumantibody levels and widespread hepatic sep-tal fibrosis in nontolerant controls.

In 1993, Ferreira and Andrade (13) de-scribed the regular appearance of septal fi-brosis of the liver similar to that observed inthe pig serum model in rats infected with thehelminth Capillaria hepatica. In this host,C. hepatica usually dies and disintegrates byabout 20-30 days of infection, after deposi-tion of a considerable number of eggs withinthe liver. Septal fibrosis was seen to startafter the focal parasitic necroinflammatorylesions were showing signs of encapsulationand resorption. It was then speculated thatthe slow and continuous release of solubleproducts from the encapsulated parasitic le-sions was stimulating cells of the Kupffer-Ito axis to produce fibrosis. The morphologi-cal and pathogenetic similarities betweenthe pig serum model and the C. hepaticamodel were evident, probably sharing animmunological basis. To further understandthe pathogenesis of fibrosis associated with C.hepatica infection, a trial on antigenic toler-ance along the same lines as those used for thepig serum model seemed to be appropriate.

C. hepatica is a nematode that parasitizesthe liver of a wide variety of mammalianhosts, especially rodents. It presents world-

wide distribution and may cause human in-fection (14). After being deposited, the eggsare metabolically active within the liver for aprolonged period of time, but remain imma-ture. They need to reach the outer environ-ment (after the death of the host) and, after aperiod of 28-30 days, under favorable condi-tions of moisture and temperature, becomeembryonated (15). Host ingestion of imma-ture eggs results in a “spurious” infection. Inthis case, the immature eggs pass through thedigestive tract and are eliminated in the stools.Ingestion of embryonated eggs results in theproduction of larvae that invade the intestineand reach the liver to form adult worms, thuscausing a “true” infection.

Material and Methods

Animals

Young healthy Wistar rats of both sexes,weighing approximately 150 g, maintainedon a commercial balanced diet and water adlibitum were used. When manipulating theanimals during the neonatal period, specialcare was taken to determine that the motherswere not going to refuse suckling by theirlitters.

Experimental groups

Group I. Twenty animals intraperitone-ally (ip) injected with a saline extract ofimmature C. hepatica eggs (from 0.1 to 0.5ml). Injections were started on the very firstday after birth and continued twice a weekfor 30 weeks until adult life. After comple-tion of the tolerance-inducing proceduresthe animals were submitted to a surgicalliver biopsy. Under sterile conditions andether anesthesia, the abdomen was openedand a small piece of the liver was ligated andremoved. No complications were recorded,and all animals recovered from surgery. Twodays later they received by gavage 500 em-bryonated C. hepatica eggs and a new liver

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biopsy was obtained 40 days later. The ani-mals were then sacrificed 20 days later, 60days after infection.

Group II. Twenty animals receiving bygavage a suspension of immature C. he-patica eggs (500 up to 2500) from the firstneonatal day on instead of the injections ofsoluble antigen preparations. All other pro-cedures were exactly the same as for theprevious group.

Group III. Control of C. hepatica “true”infection. Ten rats were ip injected withsaline from the first neonatal day to adult lifeunder the same conditions as the animals ofgroup I. After the 30th injection, they under-went a liver biopsy and were submitted toinfection with 500 embryonated eggs ad-ministered by gavage. The animals were sub-mitted to a surgical liver biopsy 40 days afterinoculation and sacrificed 20 days later (60days after infection).

Group IV. Control of C. hepatica “spuri-ous” infection. Five healthy adult rats re-ceived by gavage 2500 immature eggs twicea week for 3 weeks. The animals underwenta liver biopsy 40 days after the last inocula-tion and were sacrificed 20 days later.

Capillaria hepatica eggs and antigens

Immature eggs were recovered from in-fected rats around the 40th day of infection.The livers were removed, washed and cleanedof debris and homogenized in a blender at1300 rpm for 5 min in the presence of a smallamount of distilled water.

The material was washed with distilledwater, the sediment was decanted, severaltimes, and finally centrifuged. The sediment,containing the eggs, was suspended in a 3:2mixture of saline and Percoll (Sigma, St.Louis, MO, USA) and again washed in dis-tilled water. The clean eggs were placed on aPetri dish, humidified with 0.5% formalinand kept at room temperature (around 26ºC)for 28-30 days to embryonate. For antigenpreparation, clean immature eggs were sus-

pended in sterilized PBS and smashed in amanual homogenizer after several cycles offreezing and thawing. The supernatant wascollected after centrifugation at 14,000 rpmfor 40 min in a refrigerated Eppendorf cen-trifuge at 4ºC. Protein concentration wasmeasured with a BCA protein assay reagentkit (Pierce, Rockford, IL, USA).

Antibody detection

Sera from the animals of the variousgroups were collected a) on the last day ofthe tolerance-inducing procedure, b) 40 daysafter challenge with a “true” or “spurious”infection with C. hepatica, and c) at the timeof sacrifice. Sera were submitted to an ELISAtest for detection of total immunoglobulinantibodies using a peroxidase-conjugatedgoat anti-rat IgG (Sigma). The plates weresensitized with 10 µg/ml C. hepatica eggantigen diluted in carbonate buffer, pH 9.6,and readings were taken with a microplatereader (Thermomax spectrophotometer,Molecular Devices, Sunnyvale, CA, USA)at 450 nm connected to a computer withMDS-Soft Max.

Histopathology

Liver fragments obtained from surgicalbiopsies or at the time of sacrifice wereimmediately placed in buffered neutral for-malin for fixation. The tissue was dehy-drated in graded alcohol, cleared in xyleneand embedded in paraffin. Five micrometer-thick sections were stained with hematoxy-lin and eosin and Sirius-red for collagenvisualization.

Statistical analysis

The Kruskal-Wallis and the Dunn mul-tiple comparison nonparametric tests wereused to compare serological data obtainedfrom different experimental groups, consid-ering P < 0.05 as significant.

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C. hepatica antibodies by the end of thetolerance induction period (Figure 1A). Theconcentration of these antibodies was in-creased 30 and 40 days after infection withembryonated eggs. Hepatic histopathologicexamination 40 days after infection withembryonated eggs revealed encapsulated fociof chronic inflammation and necrosis con-taining dead worm debris and immature eggs.These results were similar to those obtainedfor group III, which was the control of “true”C. hepatica infection. In addition, through-out the hepatic parenchyma, fine fibroussepta were seen connecting portal spaces,central canals, portal spaces to central canalsor vice-versa, in several combinations thatresulted in a mosaic pattern (Figure 2A andB).

Animals of group II, rendered tolerant byoral administration of immature C. hepaticaeggs, exhibited either low titers or no C.hepatica antibodies after the tolerance in-

Figure 1. A, Levels of serum anti-Capillaria hepaticaantibody (ELISA) in rats submitted to trials of immuno-logical tolerance. Control, normal rat serum; ip (groupI), animals repeatedly injected with egg antigen by theip route since the neonatal period; gastric (group II),animals receiving by gavage immature C. hepaticaeggs since the neonatal period. P < 0.001 for controlvs ip; P < 0.05 for control vs gastric; P < 0.01 for ip vsgastric (Kruskal-Wallis and the Dunn multiple compari-son nonparametric tests). B, Anti-C. hepatica antibodylevels (ELISA) in the sera of rats 40 days after oraladministration of either immature (“spurious” infec-tion) or embryonated (“true” infection) of C. hepaticaeggs. PI, pre-infection sera; TI (group III), “true” infec-tion (embryonated eggs); SI (group IV), “spurius” in-fection (immature eggs). P < 0.01 for PI vs TI; P > 0.05for PI vs SI (Kruskal-Wallis and the Dunn multiplecomparison nonparametric tests). C, Anti-C. hepaticaantibodies in the sera of rats submitted to a tolerancetest by gastric administration of immature C. hepaticaeggs, as compared to pre-infection levels and to “true”infection in previously normal rats. PI, pre-infectionsera (normal rats); TI (group III), “true” infection; gas-tric + TI (group II), animals made tolerant by gastricadministration of immature eggs and challenged witha “true” infection. P < 0.001 for PI vs TI; P < 0.01 for PIvs gastric + TI; P > 0.05 for TI vs gastric + TI (Kruskal-Wallis and the Dunn multiple comparison nonparamet-ric tests).

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Results

Animals from group I, which were in-jected with egg antigen by the ip route, al-ready exhibited high levels of serum anti-

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duction period (Figure 1A). Antibodies werepresent in all animals from this group afterthey were inoculated with embryonated eggs,but still at low levels (Figure 1C). Forty daysafter a true infection with C. hepatica, histo-pathologic alterations in the liver markedlydiffered from those seen in group I and incontrols (group III). The focal parasitic le-sions were present in the liver of tolerantanimals, with encapsulation, necrosis andinflammation, but the eggs in the lesionsseemed to be much less numerous than incontrols, usually exhibiting a thin and trans-parent shell (Figure 2C). Septal fibrosis waslimited to a few spurs radiating from thecapsule of the focal parasitic lesions (Figure3A), while the rest of the liver parenchymamaintained its normal structure. Occasion-ally, septal fibrosis extended further intosmall areas of the hepatic parenchyma, awayfrom the parasitic lesions. In this case, thesepta were fewer and thinner, sometimesincomplete when compared to controls. Someof the C. hepatica lesions still contained liveworms surrounded by a scanty inflammatoryreaction and no eggs, even by 60 days afterinoculation with embryonated eggs (Figure3B). Live worms are usually not found afterthe 40th day of infection in rats. Animalssolely receiving immature eggs by gavagedid not develop infection, but did presentlow titers of anti-C. hepatica antibodies inserum (Figure 1B). Microscopic examina-tion of hepatic tissue did not show abnor-malities in this group of animals.

The statistical analysis of the serologicalresults is presented in Figure 1.

Although the median values did show

Figure 2. A and B, Aspect of the septal fibrosis ob-served in the liver of rats infected with the helminthCapillaria hepatica (group III - “true” infection), 40 daysafter infection. Foci of egg deposition can be seen atthe top of panel B. Sirius-red method for collagen,200X. C, Encapsulated necroinflammatory lesionformed around dead worm debris and a few transpar-ent eggs. Hematoxylin and eosin, 200X.

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Discussion

The results obtained from the presentinvestigation suggested that the task of mak-ing rats completely tolerant to C. hepatica ismore difficult than making rats tolerant towhole pig serum or to pure pig serum albu-min. This could be expected, since in thecase of C. hepatica much more complexantigens are probable involved. Serum albu-min is a single protein molecule, although itpresents multiple antigenic determinants.However, in regard to C. hepatica, not onlyprotein and somatic and metabolic polysac-charide antigens derived from the wormsand their eggs, but also possibly neo-anti-gens derived from host-parasite interactions,can be expected to play a rather complex rolein the process. The considerable reductionof septal fibrosis of the liver, which wasobserved in the animals receiving immatureeggs by gavage from the neonatal periodonward, can be interpreted as immunologi-cal tolerance, albeit partial. It can thus beassumed that septal fibrosis occurring in ratcapillariasis has an immunological basis.Although tolerance has not been achieved bythe parenteral administration of antigen, aswas the case with pig serum-treated rats, asdemonstrated by Bhunchet et al. (12), thesimilarity of the two models can also beassumed by their responses regarding theprocess of induced tolerance.

The behavior of serum antibodies is alsoin keeping with this conclusion. The up-surges of antibodies after the “tolerant” ratsbecame infected with C. hepatica may indi-cate a derepression phenomenon. Higginsand Weiner (16) have called attention to thefact that disease manifestations can be par-tially or completely suppressed in toleranthosts in a dose-dependent manner.

The use of ip injections of soluble anti-gens to make rats tolerant to C. hepatica wasattempted because of the excellent resultsobtained by Bhunchet et al. (12) in theirstudies with the pig serum model. As men-

Figure 3. A, Large collection of immature Capillaria hepatica eggs in the liver of a rat madeimmunologically tolerant by oral administration of immature egg antigens from the firstneonatal day until adult life (group II). Only a scanty amount of septal fibrosis radiates fromthe capsule of the parasitic lesion 40 days after a “true” infection. Sirius-red method forcollagen, 200X. B, Young adult worms surrounded by a mild inflammatory reaction arepresent in the liver of a C. hepatica-tolerant rat from group II, 60 days after infection withembryonated eggs. Hematoxylin and eosin, 200X.

differences between the two experimentalgroups, these differences were not statisti-cally significant, indicating only a partialstate of immunosuppression.

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tioned above, the ip route, under the presentexperimental circumstances, proved to betotally inadequate for the rat C. hepaticamodel. It is well known that not only thedoses but also the route of administration ofantigens have considerable effects on theimmune response (17-20). Also, for the pro-cess of immunological tolerance it is impor-tant that the antigens remain for a prolongedperiod of time within the organism, other-wise only a transient tolerance would beinduced (21). To emphasize the importanceof the route of administration, Andrade andGodoy (22) demonstrated that rats treated

with whole pig serum by subcutaneous in-jections failed to develop hepatic fibrosis.Under the same experimental protocol, sep-tal hepatic fibrosis developed in 65% of therats injected ip.

In attempts to induce hepatic fibrosis byimmunological means, the C. hepatica mo-del can be considered an alternative to thepig serum model. Although immunologicallymore complex, the C. hepatica model hasthe advantage of coursing with almost nomortality and with fibrosis occurring in 100%of the infected animals (13).

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