Br. J. exp. Path. (1981) 62, 480

HEPATIC GRANULOCYTOPOIESIS IN MURINE EXPERIMENTAL SCHISTOSOMIASIS MANSONI

R. BOROJEVIC*t, S. STOCKERt AND J. A. GRIMAUDt Fro0m the tinstitot Pasteur de Lyon, ERA 819, 77 Rue Pasteur, 69007 Lyon, France, and *Fundad4o Gon(alo M.1oniz, Sal(ador, Bahia, Brazil

Received fo)p)ublicationlMay 27, 1981

Summary.-In of mice with acute schistosomiasis eosinophil granulocyto- poietic folliculi are described. Exogen stem cells were observed in hepatic sinusoids. Their proliferation and progressive maturation are described. Specific reactions to the presence of worms and their eggs, and Kupffer-cell stimulation and hyperplasia are considered to be responsible for this extramedullary granulocytopoiesis.

AN INCREASE in the level of eosinophil Warren and Johnson (1967) and by in , and Bogitsh (1971), but the question of their involved tissues is characteristic of many origin was not solved. parasitic infections. It is particularly 3. In the hepatic parenchyma, inde- prominent in those in which parasites pendent of portal spaces and of periovular inhabit the circulatory system, or in which granulomas, folliculi can be observed, their life cycles include the passage of composed of mature , together larvae through the host tissues (Basten, with immature forms and blastic cells. In Boyer and Beeson, 1970; Colley, 1972). earlier works, Goennert (1955), Grimaud Related to the presence and quantity of and Borojevic (1972) and Byram et al. parasite antigens in situ, eosinophilia is (1978) have interpreted these hepatic particularly high in the acute phase of folliculi in murine schistosomiasis as sites human (Diaz-Rivera et al., 1956) and in of local granulocytopoiesis. experimental (Colley, 1972; Byram, Extramedullary eosinophil granulocyto- Imohiosen and von Lichtenberg, 1978) poiesis occurs in some eosinophil leuk- infection by Schistosoma mansoni. aemias (Gross, 1962; Anteunis et al., 1978), In the acute phase of murine S. mansoni in young thymus (Bhatal and Campbell, infection eosinophil infiltra- 1965; Sin and Saint-Marie, 1965) and, as tion is 3-fold: postulated by Dzury and Cohen (1958), in 1. In periportal spaces eosinophils form inflammatory reactions. Ultrastructural part of the inflammatory infiltrate. features of hepatic granulocytopoiesis 2. In the inflammatory granulomatous during the foetal period have been fol- reaction which forms around schistosomal lowed by Zamboni (1965), Fukuda (1974) eggs, the presence of eosinophils is related and Enzan et al. (1978). to the degree of maturation of the egg: The morphology of cells implicated in they are particularly abundant in the ectopic extramedullary eosinophilic "mixed inflammatory reaction" (von granulocytopoiesis observed in experi- Lichtenberg, 1962) where they outnumber mental murine schistosomiasis, their other types of cell. The presence of origin and their relationship with the immature eosinophils in schistosomal hepatic tissue, are the object of the present granulomas was described by Stenger, report.

Corriesponrden e to: Prof Radox an Borojexik, D)epartamento (le Bioquimica, Instittuto dle Qu'imica Universidade Fe(leral (lo Rio cle Janieiro, 21910 Ilha (lo Ftun(lio, Rio (1e Janeiro, Brasil. HEPATIC GRANULOCYTOPOIESIS IN SCHISTOSOMIASIS 481

MATERIAL AND METHODS plasmic rim and flattened processes ex- Twenty inbred C3H rnice and 40 Swiss tending into the sinusoidal lumen, and Albino TO mice were exposed as sucklings to their cytoplasm was locally crowded with transcutaneous penetration of 50 cercariae of phagosomes, in which schistosomal pig- the FS strain (Bahia, Brazil) of Schistosorna ment and cell debris could be recognized. mansoni. The animals were killed after 60 days Cytoplasmic granules, corresponding to of infection. Small fragments of tissue were lysosomes and secretory granules, were fixed in 4%o buffered formalin for optical micros- copy, and treated by conventional histological abundant. methods. For electron microscopy, 10% Os04, Exogenous cells were mostly typical buffered in sodium cacodylate (015M) was used. mature blood leucocytes. Occasionally we Fixation was carried out at 4° for 60 inin. observed small cells with a very high Embedding was carried out writh Epoxy resin. Ultra-thin sections were made with a Reichert nucleo-cytoplasmic ratio, with subspheri- OM U2 mnicrotome, contrasted with uranyl- cal nuclei and cytoplasm rich in ribo- acetate/lead citrate solution and observed on the somes, but devoid of any particular in- Plilips EM 300 microscope. clusions or cytoplasmic differentiation. They had the characteristics of blastic cells, RESULTS but at this stage their precise identity was In acute mutine schistosomiasis eosino- difficult to tell. philic granulocytes were found in hepatic Small groups of these blastic cells were parenchyma as a diffuse infiltrate, or observed within the sinusoidal lumen, in forming large distinct folliculi. Serial close relation to the endothelial lining. In sections were studied in this work, to other places, the endothelial lining was avoid the possible influence of neighbour- interrupted, and blastic cells, together with ing inflammatory portal spaces or peri- occasional Kupffer cells, could be observed ovular granulomas. Only those fragments within Disse's space, in direct contact in which the vicinity of these inflam- with (Figs lb and 3). matory structures was eliminated were taken into consideration. Intraparenchymal folliculi These were observed most often in Hepatic parenchyma spaces corresponding to obliterated In our model of acute murine schisto- sinusoids. When small, folliculi did not somiasis the hepatic parenchyma pre- alter the trabecular organization of served its lobular architecture and the hepatic parenchyma, but when they were hepatocytes their trabecular organization. composed of several hundreds of cells they Hepatocellular irritation was indicated by could locally alter the normal hepatic an unusually high number of binucleate structure (Fig. 2a). cells, by the abnormal size and shape of Folliculi were composed of cells which the nucleoli and by dense, large mito- could be easily recognized as myeloid cells chondria, sometimes containing cristalloid of eosinophil granulocyte lineage. They bodies. Hepatocellular necrosis was not were remarkably closely synchronized in seen. their maturation and only exceptionally, in large folliculi, could very young cells be Hepatic sinusoids found together with mature eosinophils. The composite origin of these folliculi was These were loaded with cells. This was apparent. Three main categories of cells due simultaneously to an intense hyper- plasia of Kupffer cells and to infiltration could be recognized in folliculi: by exogenous cells. Kupffer cells had nuclei similar to those seen in endothelial Stage I cells cells, being irregular in shape and devoid Spherical or ovoid, these cells measured of nucleoli. They had a narrow cyto- 6-10 Htm in the largest diameter and had 482 R. BOROJEVIC, S. STOCKER AND J. A. GRIMAUD

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FIG. 1 Liver in acute murine schistosomiasis: intrasinusoidal formation of granulocytopoietic folliculi. (a) Semi-thin sections. x 333. (b) Electron micrograph showing Kupffer cells (K) and young eosinophil granulocytes. x 3078. HEPATIC GRANULOCYTOPOIESIS IN SCHISTOSOMIASIS 483

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FIG. 2.-Hepatic granulocytopoiesis in acute murine schistosomiasis. (a) Large mixed granulocytic follicle. Note the presence of Kupffer cells (K) and reticulin (R) in the location corresponding to Disse's space. x 2767. (b) Granulocytopoietic follicle composed of Stage I cells. x 7132.

33 484 R. BOROJEVIC, S. STOCKER AND J. A. GRIMAUD

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FIG. 3.-Hepatic granulocytopoiesis in acute murine schistosomiasis. (a) Granulocytopoietic follicle composed of Stage I and Stage II cells (x 5207). (b) Granulocytopoietic follicle in the intra- sinusoidal position, composed of Stage II cells. x 5180. (c) Eosinophil Stage II cell (): Golgi zone with formation of eosinophil granules. x 21,082. HEPATIC GRANULOCYTOPOIESIS IN SCHISTOSOMIASIS 485

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FIG. 4.-Hepatic granulocytopoiesis in acute murine schistosomiasis. (a) Stage III follicle: note the continuity between the follicular space and the adjacent Disse's space. x 7375. (b) Mitosis in the Stage III eosinophil granulocyte. x 7375. 486 R. BOROJEVIC, S. STOCKER AND J. A. GRIMAUD nuclei with small indentations, slightly At their periphery folliculi were in direct condensed perichromation and an eccen- contact with hepatocytes. At the interface, tric nucleolus. Numerous polyribosomes, hepatocytes were flattened, without any scattered cisternae of rough endoplasmic sign of irritation or reaction to the pre- reticulum and a few small mitochondria sence of myeloid cells. bundles, were found in the narrow cytoplasmic rim thin and resembling the perisinusoidal (Fig. 2b). "reticulin", were sometimes observed at the periphery of folliculi. These bundles Stage II cells were topographically related to pre-exist- ing sinusoids, and no fibrotic reaction Characterized by marked differentiation, around folliculi was detected. The Disse's these were 10-13 ,um in diameter. They space of neighbouring sinusoids was in had a decreased nucleo-cytoplasmic ratio, continuity with the follicular space. with an irregularly shaped nucleus and Cell divisions were frequent in folliculi. sparse chromatin clumped along the They were not restricted to blastic cells of nuclear membrane. Their cytoplasm was the Stage I type, but were also found in electron-clear, with many well developed more differentiated cells including mature mitochondria, polyribosomes and dilated eosinophils with typical eosinophilic gran- cisternae of rough endoplasmic reticulum. ules in the cytoplasm (Fig. 4b). In the perinuclear region, 3-6 Golgi The diffuse eosinophilic infiltrate of the saccules could be observed: dense granules hepatic parenchyma in acute murine were found on the concave side of the schistosomiasis is composed of mature Golgi complex, measuring up to 0 4 /tm in individual eosinophil granulocytes. They their long axis (Fig. 3). were found mostly in perisinusoidal spaces and were more frequent in the vicinity of Stage III cells portal spaces. These had a typical small and seg- mented nucleus, without nucleoli. The DISCUSSION cytoplasmic rim was large, containing numerous small mitochondria, many lyso- In earlier works, Goennert (1955), somes, a well developed Golgi apparatus, Grimaud and Borojevic (1972) and Byram few elongated rough endoplasmic cisternae et al. (1978) have indicated hepatic eosino- and 2 types of granules: small (0.2-0.5 ,m phil granulocytopoiesis in the acute phase in diameter), round or ovoid, homo- of murine infection with Schistosoma geneously electron-dense, and large (meas- mansoni. By electron microscopy Stenger uring up to 0-5-0-8 ,tm in the long axis) et al. (1967) and Bogitsh (1971) have indi- containing most often an electron-clear cated the presence of immature granulo- cristalloid inclusion typical of eosinophil cytes in hepatic periovular granulomas in granulocytes. At the cellular periphery the same experimental model. In the pre- numerous pinocytic and coated vesicles sent work, we describe the morphology of were observed (Fig. 4). hepatic granulocytopoiesis and its rela- Granulocytopoietic folliculi were re- tionship with the hepatic tissues. markably well delimited and homo- Hepatic granulocytopoiesis in acute geneous. With the exception of Kupffer schistosomiasis follows the same pattern cells, no cell line other than myeloid types observed in the bone marrow and in were observed within the folliculi. Kupffer hepatic tissues during the prenatal period. cells were scattered at the periphery and The 3 morphological stages of myeloid throughout the folliculi, forming long cells observed in hepatic folliculi corres- extensions and pseudopodia between myel- pond exactly to , myelo- oid cells, but without any cell-contact cytes and young mature eosinophil granu- differentiations. locytes observed in normal granulocyto- HE1PATlC GRANULO('YTOPOIESIS IN SCHISTOSOMIASIS 487 poiesis, as described in foetal liver by the environment which stimulates local Fukuda (1974) and Enzan et al. (1978). haemopoiesis (Cline and Golde, 1979). In The origin of these myeloid cell lines and our model, there was no topographical the reasons which lead to this extra- relationship between the connective tissue medullary eosinophilopoiesis in acute in- and myeloid foci, the first groups of flammation should be emphasized. myeloid cells being observed in the In the liver of acute murine schisto- sinusoidal lumen. A constant association somiasis, several factors influencing was, on the other hand, observed between eosinophilia are locally produced. Schisto- Kupffer cells and myeloid foci. Stimulated some egg antigens and schistosome meta- macrophagic cells are known to induce bolic antigens are known to induce an haemopoiesis (Dimitrov et al., 1975; intense reaginic response (Sadun, 1972; Buhles and Shifrine, 1978; Foster, 1978), Dessaint et al., 1975). Anaphylactic reac- and focal haemopoiesis was induced in rat tions and immune complexes formed with liver by a activator, glucan reaginic antibodies are chemotactic for (Deimann and Fahimi, 1980). Endothelial eosinophils (Goetzel, 1976). Simultaneous- and Kupifer-cell hyperplasia are character- ly, specifically stimulated lymphocytes istic of schistosomal liver (Grimaud, and entire granulomas elicited by schisto- Borojevic and Santos, 1976), and Kupffer some eggs are known to produce an cells are known to be one of the sources of Eosinophil-Stimulating Promoter (ESP) the Colony Stimulating Factor (CSF) and to induce eosinophilopoiesis (Colley, which induces the proliferation of myeloid 1973; James and Colley, 1975; Miller, lines (Joyce & Chervenick, 1975). Colley and McGarry, 1976). Byram et al. The cells which are the origin of (1978) have thus proposed that eosino- granulocytopoietic-cell proliferation in philic islands in schistosome-infected liver are apparently exogenous, as the organs might be elicited by lymphokines least differentiated forms were observed secreted by sensitized T lymphocytes in within the hepatic sinusoids. The syn- the granulomas themselves. Immune com- chronous differentiation in each folliculum plexes, ESP and lymphokines are probably suggests that each corresponds to the directly responsible for eosinophil infiltra- proliferation of a single stem cell. Cells tion in periovular granulomas and in which may, under appropriate conditions, inflammatory-tissue infiltrates in acute initiate the production of granulocytic schistosomiasis. As indicated by Bogitsh colonies are known to be present, although (1971), Byram et al. (1978) and by our in small number, in the circulating blood own observations (unpublished), immature (Chervenick and Boggs, 1971). We may eosinophils are found in liver, spleen and thus assume that, while circulating in the mesenteric tissues, but never in granul- liver sinusoids, these cells are subjected to omas formed in lungs and in the intestinal local conditions created by thWe local wall, tissues where granulomas are par- immune reaction to the presence of worms ticularly abundant. Extramedullary gran- and their eggs and by the secretory ulocytopoiesis thus occurs only in tissues activity of stimulated Kupffer cells, which with recognized haemopoietic potential, could induce them to initiate the produc- and corresponds to true myeloid meta- tion in situ of an eosinophilic granulocyte plasia, as specified earlier by Grimaud and colony. Borojevic (1972). The presence of ESP, It is remarkable that glucan stimulation lymphokines and reaginic immune com- of induces complete haemo- plexes does not seem to be sufficient per se poiesis in rat liver (Deimann and Fahimi, to induce ectopic granulocytopoiesis in 1980), while in our model only eosinophil this model. granulocytopoiesis was observed. It is Connective-tissue cells and extracellular known that granulocyte progenitor cells stroma are considered to be responsible for are heterogeneous, and specific eosinophil 488 R. BOROJEVIC, S. STOCKER AND J. A. GRIMAUD precursors were recognized in bone mar- (ESP): A Lymphokine Induced by Specific Anti- gen or Phytohemagglutinin. J. Immunol., 110, row, spleen and peripheral blood of mice 1419. (Johnson, Dresch and Metcalf, 1977; DEIMANN, W. & FAHIMI, H. D. (1980) Induction of Johnson and the Focal Hemapoiesis in Adult Rat Liver by Glucan, Metcalf, 1980). On other a Macrophage Activator. Lab. Invest., 42, 217. hand, specific CSF, which stimulates only DESSAINT, J. P., CAPRON, M., BOUT, D. & CAPRON, A. eosinophil granulocyte proliferation, has (1975) Quantitative Determination of Specific IgE Antibodies to Schistosome Antigens and been identified (Nicola et al., 1979). The Serum IgE Levels in Patients with Schisto- selective stimulation of eosinophilopoiesis somiasis (S. mansoni or S. haematobium). Clin. could be achieved either by progenitor exp. Immunol., 20, 427. DIAZ-RIVERA, R. S., RAMOS-MORALES, F., KOPPISCK, selection or through specific stimulation E., PALMEIRA, G. M. R., RIVEIRA, C. A., MARCH- of less differentiated stem cells. The AND, E. J., GONZALES, 0. & TORREGROSA, M. V. (1956) Acute Manson's Schistosomiasis. Am. J. mechanisms of the control of granulocyte Med., 21, 918. proliferation in human and in experi- DIMITROV, V., ANDRE, S., ELIPOULOS, G. & mental schistosomiasis are currently under HALPERN, B. 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