obstructive and destructive portal vascular changes in- .... sinophilic infiltration near the sinusoids, in portal ... Thebright apple-green fluorescence observed.
Schisiosomal Hepatic Lesions Curative Chemotherapy
Evolution of the
in Mice
After
ZILTON A. ANDRADE, MD, and JEAN-ALEXIS GRIMAUD, MD
From Gonfalo Moniz Research Centre (Fiocruz), Bahia, Brazil, and Institut Pasteur, Lyon, France
Mice with 64-day-old Schistosoma mansoni infection (± 27 worms, 8-12 pairs) were treated simultaneously with oxamniquine and hycanthone. The cure rate was 100%, and changes occurring thereafter in the liver were sequentially followed by means of histologic, ultrastructural, and immunofluorescence methods. Soon after treatment, hepatitic changes cleared up and periovular granulomas diminished in size. The predominant Type III collagen in granulomas was reduced, and the Type I showed no apparent increase, whereas Type IV did not seem to participate in the process. Collagen fibrils in periovular granulomas changed in texture from dense and
more oriented to loose and disorganized. Fibroblasts, at first with marked signs of hyperfunction, became less so at a time when collagen fragments appeared within secondary lysosomes in macrophages and fibroblasts. Schistosomal ovular antigens remained sequestered inside the fibrotic granulomas up to the final, 39th day after treatment. Thus, specific treatment of schistosomiasis showed a beneficial effect upon the hepatic lesions from the very beginning and promoted changes in the periovular granulomas that indicated a rapid, although incomplete, resorption of fibrosis. (Am J Pathol 1986, 124:59-65)
THE ADVENT of highly effective, practical, safe, curative drugs against schistosomiasis represents a recent and significant advance in this field. Chemotherapy used on a large scale in Brazil has contributed to decreasing the number of hepatosplenic cases, acting either as a curative' or as a preventive agent.2 Experimentally, it has been shown that even the advanced and complex obstructive and destructive portal vascular changes induced by Schistosoma mansoni in mice can be reversed, almost completely, under specific chemotherapy.3 Also, the cure in the animals is followed by a period of residual immunity against reinfection.4 Therefore, the changes induced by curative chemotherapy in schistosomiasis offer new areas of study as well as pose many questions yet to be answered. This paper is an attempt to investigate with histologic, ultrastructural, and immunofluorescence methods the sequential changes occurring in the livers of infected mice following curative chemotherapy. Special interest was placed upon the alterations undergone by the collagen tissue as well as the fate of S mansoni antigens. Although these parameters have been extensively investigated both in vitro5s8 and in vivo,9'," the subsequent alterations induced by curative treatment have not been likewise studied.
Materials and Methods Albino Swiss mice of both sexes, weighing 20-22 g were submitted to infection by the tail method11 with 120 recently shed cercariae of a Puerto Rican strain of S mansoni. All infected mice presented viable parasite eggs in the stools 50-60 days after cercarial exposure. Infection was considered uniform and yielded approximately 27 worms, with 8-12 pairs, as seen after a perfusion technique."2 After 64 days of infection, the animals were divided into two groups: one group was treated simultaneously with 100 mg/kg body weight of oxamniquine (pure salt) administered by gavage, and 80 mg/kg body weight of hycanthone given intramuscularly (such combined treatment was observed to yield a 10007o cure rate.4) The other group was left untreated. A third group of normal, intact, age- and sex-matched control mice was added. After treatment, 2 animals, a male and a female, from each of the three groups were
Accepted for publication February 17, 1986. Address reprint requests to Zilton A. Andrade, Rua Valdemar Falcao 121 Brotas, 40.000 Bahia, Brazil.
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ANDRADE AND GRIMAUD
AJP * July 1986
Fragments of the liver from all the animals were fixed in aqueous Bouin's fluid and embedded in paraffin. The sections were stained with hematoxylin and eosin (H & E), Mallory's trichrome, and the Gordon-Sweet method for reticulum.
collagen of Types I and III (extracted from the skin and controlled by SDS-PAGE). The interspecies cross-reactivity of the anti-Type IV antiserum was also tested by ELISA with the use of microplates coated with mouse Type IV. Sera diluted 1:5 and 1:10 as well as undiluted sera were used. Control slides were observed after treatment with normal goat and rabbit sera and the fluorescent conjugate alone. An indirect technique was used. Slides were examined within 24 hours after preparation in a Leitz fluorescent microscope equipped with an HBO 200 mercury lamp.
Immunofluorescence Study
Electron-Microscopic Study
One lobe of the liver was snap-frozen in liquid nitro(-196 C) and stored in a tightly closed plastic box at - 70 C until sectioned in an automatic cryotome at 20 C. The sections were treated with fluoresceinated anti-soluble egg antigen (SEA) serum and several anticollagen sera. The anti-SEA serum was obtained through the courtesy of Dr. Deelder (Leiden, Holland) and was prepared in goats given repeated injections of purified fractions obtained from isolated S mansoni eggs. Anti-collagen antibodies were raised in rabbits through several injections of Type I, III, and IV human collagen and of Type III human procollagen. Type I and III collagen was extracted from human cirrhotic liver. 13 Type IV collagen and Type III procollagen were extracted from human placenta, and the antibodies raised against Type III procollagen were reacted with the aminopropetide (pN) portion of the molecule. The absence of detectable contaminant (another type of collagen or glycoprotein) in each preparation was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Monospecificity of the antibody against a specific type of collagen was ensured by adsorption of the antisera with the other types and purification by affinity chromatography. 13 The monospecificity of each purified antiserum was then controlled by ELISA, with the use of microplates coated with the different types of pure collagens. With the same assay, no cross-reaction was detected between the anti-Type IV and laminin. The cross-reactivity of the anti-Type I and anti-Type III antisera with mouse collagen was demonstrated by ELISA, with the use of microplates coated with purified
Small pieces of the liver were rapidly fixed in 1% osmium tetroxyde in 0.3 M sodium cacodylate buffer, pH 7.4, 400 mOsm, and embedded in epoxy resin. Sections were obtained by using an automatic LKB ultramicrotome with a diamond knife, contrasted by the uranyl acetate-lead citrate method, and observed with a Philips EM 300 microscope at 80 mv.
sacrificed after 1, 3, 5, 10, 20, and 39 days. The animals killed by neck dislocation, the abdomen was opened, and the liver was quickly removed and submitted to the following technical procedures. were
Histologic Study
gen -
mouse
Results In untreated animals, periovular granulomas around mature eggs in the liver were numerous and large, with a mixture of macrophages, fibroblasts, and eosinophils, and showed an irregular peripheral delimitation and a small area of central necrosis. Besides the granulomas, the liver showed focal areas of mononuclear and eosinophilic infiltration near the sinusoids, in portal spaces, and around central veins (Figure la). Focal areas of coagulative necrosis were sometimes seen. In the treated group, only a few adult dead worms were found from the first to the third day after treatment. By the fifth day, all worms were found dead and disintegrating. The first histologic changes in the liver which could be attributed to treatment were evident from Day 3 on. The focal inflammatory infiltration in the parenchyma, in portal spaces, and around central veins was the first change to disappear. The eosinophilic infiltration within and around the periovular granulomas (Figure lb) gradually diminished. The granulomas without the exudative component began to appear smaller with fibroblasts and macrophages found at random within the collagenous matrix. Later, the
Figure 1a-The predominant type of S mansoni periovular granuloma in the liver of an untreated mouse with a 10-week-old infection. Eosinophils are b-Hepatic periovular granulonumerous, and the fibrous component of the lesion is marked by the predominantly exudative reaction. (H&E, x 100) ma as observed 10 days after treatment. There can be seen fibroblasts, macrophages, and a few scattered eosinophils. The periphery of the granuloma in scattered all seen throughout the liver. (H&E, were granulomas uniformly shows a distinct delimitation from the hepatic parenchyma. Such changes x 100) c-There is a rich pattern of fibers forming a mesh around the schistosome egg and showing specific fluorescence for procollagen Type d-Bright fluorescence for the soluble egg antigen obIll. The eggshell exhibits autofluorescence. Untreated mouse, 12-week-old infection. (x 250) served within the egg in the liver of a mouse that had been treated 39 days ago. The amorphous material seen inside the eggshell was observed in control sections to be free of cells or eosinophil granules. Cryostat section treated with anti-SEA serum followed by fluoresceinated anti-goat IgG. ( x 450)
Fop, AML.
m
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ANDRADE AND GRIMAUD
AJP * July 1986
Figure 2-Fibrotic periovular granulomas in the liver of a mouse with a 10-week S mansoni infection, 39 days after curative treatment. There is a synchronous involution of all granulomas. (Masson's trichrome, x 50)
granulomas appeared shrunken and dense, assuming the appearance of a focal scar by the end of 39 days (Figure 2), when a progressive accumulation of a dark brown pigment appeared within and around them. Reticulum stain showed that the thin prolongations of collagen tracts radiating from the granuloma into the parenchyma disappeared soon after treatment. Immunotyping of collagen disclosed the constant presence of both Types I and III in variable amount in the periovular granulomas. Usually the Type III was predominant and tended to appear at the periphery. A bright fluorescence which stained fibers all over the granulomas was observed when antiserum for Protocollagen III was used (Figure lc). Staining for Type I collagen was more frequently seen in the central part of the granuloma, and its intensity showed a progressive increase after treatment. The fluorescence determined by Type III antiserum did not seem to be likewise influenced by treatment, and anti-Procollagen III continued to give bright fluorescence even when the granulomas were shrunken. Type IV collagen was detected near the granulomas in basement membranes of blood vessels and biliary structures but not within the granulomas. Egg antigens were detected inside the miracidium
present within the eggshell by means of the anti-SEA serum. The bright apple-green fluorescence observed remained positive even 39 days after the cure of schistosomiasis (Figure Id). At the ultrastructural level, the periovular granulomas in untreated mice appeared composed of several cellular elements, with a predominance of macrophages, eosinophils, and fibroblasts. Fibroblasts presented with a rich endoplasmic reticulum and prominent Golgi cisternae filled with fine granular material. The abundant collagen fibers oriented in various directions formed a loose structure with cells inside its meshes in some granulomas, while in others these fibers showed a progressive accumulation and a concentric orientation. Eosinophils usually presented a few typical granules and exhibited a rich endoplasmic reticulum. Macrophages contained many lysosomes and ring-shaped dark schistosomal pigments. A few typical myofibroblasts could also be observed. The main change observed in these granulomas after treatment was a breakdown of the collagen fibers, which appeared fragmented and dispersed into a loose amorphous and abundant matrix (Figure 3a). Some macrophages and fibroblasts showed fragments of collagen fibers inside secondary lysosomes (Figure 3b). The fibroblasts and occasion-
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CHEMOTHERAPY OF SCHISTOSOMIASIS
Vol. 124 * No. 1
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Figure 3-Treated animals. a-Schistosomal periovular granuloma 10 days after treatment. Collagen fibrils appear disorganized and fragmented, with variable thickness, dispersed in an amorphous, loose matrix. (x 29,750) b-Same as above. In the center of the picture a fibroblast discloses several secondary lysosomes, within which there are fragments of collagen (arrow). (x 52,000)
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ANDRADE AND GRIMAUD
ally some myofibroblasts continued to exhibit dilated and hyperplastic endoplasmic reticulum, whereas the numbers of eosinophils and other polymorphonuclear cells decreased. The changes described above for the treated animals could also, although rarely, be observed in those untreated. The differences were quantitative, rather than qualitative.
Discussion A striking early amelioration of the hepatic lesions provoked by S mansoni in mice was observed after specific chemotherapy. For some time there was the impression that treatment of schistosomiasis could at first increase the intensity of the lesions by causing the worms to disintegrate and to liberate "toxic" and antigenic products. The elevation of the levels of transaminases, eosinophils, and antibodies in peripheral blood observed soon after treatment would favor such interpretation.14 However, the present findings would suggest that such assumptions may be exaggerated. Patients invariably show general improvement after treatment, even those with the acute "toxemic" form of schistosomiasis. I At the tissue level, both in the granulomas and in the hepatic parenchyma and portal spaces, the inflammatory changes decreased considerably after treatment. This effect was noted a few days after the administration of the drugs. It could not be attributed to the death of the miracidia, because we have seen (unpublished data) that they can remain alive 12-15 days following a proven cure by oxamniquine-hycanthone. It is probably related to worm death, through some mechanism as yet unknown. In favor of such an interpretation is the fact that the in vitro periovular granuloma forms better in the presence of live worms.16 A strong fluorescence for soluble egg antigen was found inside the egg, regardless the viability of the miracidium. Thirty-nine days after treatment, when the miracidium was already disintegrated and the egg looked almost empty, a strong apple-green fluorescent reaction could still be detected. It has been said that the schistosomal egg granuloma is a focal inflammatory reaction aimed at sequestering antigens of low diffusibility. 7 Such sequestration may be very effective indeed. It may provide a means for a continuous and slow antigenic release that may keep the host immune system stimulated for some time after the cure of schistosomiasis. It has been said that curative treatment of schistosomiasis causes the periovular granulomas to shrink and be transformed into small focal scars. 18-20 Immunotyping of collagen and electron-microscopic observations permitted some details of these processes to be analyzed.
AJP * July 1986
Apparently, there was a preponderance of Type III collagen in the granulomas, and this could be related to the rapid, although partial, reversibility of these lesions after treatment. However, 39 days after treatment both Type I and Type III were present in similar amounts in the shrunken granuloma. A more prolonged time of observation will be necessary for us to see whether Type III collagen would completely disappear. Wu et als observed that in mice with an 8-week-old infection with 50 S mansoni cercariae the Type I collagen increased 11 times in the liver, and the Type III, 22 times. They performed a biochemical study, but one must assume that most of the collagen was present in the periovular granulomas. Another point of agreement between the present immunofluorescent findings and the biochemical data of Wu et al is the observation that collagen Type IV is not a component of the fibrous changes occurring in hepatic murine schistosomiasis. The modification of the collagen structure is also a prominent feature brought about by chemotherapy. The periovular granuloma is an essentially fibrosing lesion. It shows signs of increased collagen synthesis, as well as resorption8'0; and the collagen fibers at first deposited with a loose texture soon become more compact and concentrically oriented. Treatment brings about a fragmentation of the collagen fibers with the disappearance of many of them. All indications point to an enzymatic process with a possible cellular (phagocytic) participation. Our findings are in keeping with the suggestion that after cessation of the stimulus for fibrogenesis, the associated fibrolytic process takes over.2" Curative treatment of schistosomiasis does not induce something qualitatively new to the periovular granuloma. During the spontaneous evolution of the granuloma in the liver, it passes through phases of inflammation, increased fibrogenesis, death of the miracidium, and total or partial resorption of fibrosis. Treatment only provokes the appearance of the latter change in all granulomas at the same time. Phagocytosis of collagen has been observed in both macrophages and fibroblasts and interpreted as a mechanism of collagen resorption.22 During the postpartum involution of the rat uterus, Henell et a123 have pointed out, three mechanisms can be accounted for in the reduction in size of the organ: autophagia, heterophagia (phagocytose), and crinophagia. The latter means that the secreting cells can destroy part of their own secretion when that is in excess. This mechanism would explain the presence of collagen fragments in the digestive vacuoles of fibroblasts. The rapid and intense collagen synthesis which occurs in the periovular granulomas in schistosomiasis and the profound modifications in collagen content and structural rear-
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rangement after chemotherapy indicate that one can take advantage of the murine model of schistosomiasis for the studies concerning collagen metabolism.
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12. Duvall RH, De Witt W: An improved perfusion technique for recovering adult schistosomes from laboratory animals. Am J Trop Med Hyg 1967, 16:483-486 13. Grimaud JA, Druguet M, Peyrol S, Chevalier 0, Herbage D, Badrawy N: Collagen immunotyping in human liver: Light and electron microscopic study. J Histochem Cytochem 1980, 28:1145-1156 14. Silva LC, Hoshino-Shimizu S, Kanamura H, Strassman PC, Camargo ME, Sette H Jr, Lopes D, Chamone DAF, Raia S, Silva GR: Serum antibodies changes after repeated chemotherapic series in "parasitologically cured" patients with schistosomiasis mansoni. Rev Inst Med Tlop S Paulo 1976, 18:206-210 15. Neves J: Diagnostico e tratamento das doencas infectuo sas e parasitarias. Edited by Guanabara-Koogan. Rio de Janeiro, 1978 16. Doughty BL, Phillips SM: Delayed hypersensitivity granuloma formation around Schistosoma mansoni eggs in vitro: I. Definition of the model. J Immunol 1982, 128:30-36 17. Lichtenberg F: Host response to eggs of S mansoni: I. Granuloma formation in the unsensitized mouse. Am J Pathol 1963, 41:711-723 18. Gonnert R: Schistosomiasis Studien: IV. Zur Pathologie der Schistosomiasis der Maus. Ztschr Tropenmed Parasitol 1965, 6:279-336 19. Cameron GR, Ganguly NC: An experimental study of the pathogenesis and reversibility of schistosomal hepatic fibrosis. J Pathol Bacteriol 1964, 87:217-237 20. Warren KS: The influence of treatment on the development and course of murine hepatosplenic schistosomiasis mansoni. Trans R Soc RTop Med Hyg 1972, 56:510-519 21. Montfort J, Perez Tamayo R: Collagenase in experimental carbon tetrachloride cirrhosis of the liver. Am J Pathol 1978, 92:411-420 22. Svoboda ELA, Shiga A, Deporter DA: A sterologic analysis of collagen phagocytosis by fibroblasts in three soft connective tissues with differing rates of collagen turnover. Anat Rec 1981, 199:473-480 23. Henell F, Bricsson JLE, Glaumann H: An electron microscopic study of the post-partum involution of the rat uterus: With a note on apparent crinophagy of collagen. Virchows Arch [Cell Pathol] 1983, 42:271-287
