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

HEPATIC EOSINOPHIL GRANULOCYTOPOIESIS IN MURINEEXPERIMENTAL 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 livers 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 tothe presence of worms and their eggs, and Kupffer-cell stimulation and hyperplasiaare considered to be responsible for this extramedullary granulocytopoiesis.

AN INCREASE in the level of eosinophilgranulocytes in blood, bone marrow andinvolved tissues is characteristic of manyparasitic infections. It is particularlyprominent in those in which parasitesinhabit the circulatory system, or in whichtheir life cycles include the passage oflarvae through the host tissues (Basten,Boyer and Beeson, 1970; Colley, 1972).

Related to the presence and quantity ofparasite antigens in situ, eosinophilia isparticularly high in the acute phase ofhuman (Diaz-Rivera et al., 1956) and inexperimental (Colley, 1972; Byram,Imohiosen and von Lichtenberg, 1978)infection by Schistosoma mansoni.

In the acute phase of murine S. mansoniinfection eosinophil granulocyte infiltra-tion is 3-fold:

1. In periportal spaces eosinophils formpart of the inflammatory infiltrate.

2. In the inflammatory granulomatousreaction which forms around schistosomaleggs, the presence of eosinophils is relatedto the degree of maturation of the egg:they are particularly abundant in the"mixed inflammatory reaction" (vonLichtenberg, 1962) where they outnumberother types of cell. The presence ofimmature eosinophils in schistosomalgranulomas was described by Stenger,

Warren and Johnson (1967) and byBogitsh (1971), but the question of theirorigin was not solved.

3. In the hepatic parenchyma, inde-pendent of portal spaces and of periovulargranulomas, folliculi can be observed,composed of mature eosinophils, togetherwith immature forms and blastic cells. Inearlier works, Goennert (1955), Grimaudand Borojevic (1972) and Byram et al.(1978) have interpreted these hepaticfolliculi in murine schistosomiasis as sitesof local granulocytopoiesis.

Extramedullary eosinophil granulocyto-poiesis occurs in some eosinophil leuk-aemias (Gross, 1962; Anteunis et al., 1978),in young thymus (Bhatal and Campbell,1965; Sin and Saint-Marie, 1965) and, aspostulated by Dzury and Cohen (1958), ininflammatory reactions. Ultrastructuralfeatures of hepatic granulocytopoiesisduring the foetal period have been fol-lowed by Zamboni (1965), Fukuda (1974)and Enzan et al. (1978).The morphology of cells implicated in

ectopic extramedullary eosinophilicgranulocytopoiesis observed in experi-mental murine schistosomiasis, theirorigin and their relationship with thehepatic tissue, are the object of the presentreport.

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

HEPATIC GRANULOCYTOPOIESIS IN SCHISTOSOMIASIS

MATERIAL AND METHODS

Twenty inbred C3H rnice and 40 SwissAlbino TO mice were exposed as sucklings totranscutaneous penetration of 50 cercariae ofthe FS strain (Bahia, Brazil) of Schistosornamansoni. The animals were killed after 60 daysof infection. Small fragments of liver tissue were

fixed in 4%o buffered formalin for optical micros-copy, and treated by conventional histologicalmethods. For electron microscopy, 10% Os04,buffered in sodium cacodylate (015M) was used.Fixation was carried out at 4° for 60 inin.Embedding was carried out writh Epoxy resin.

Ultra-thin sections were made with a ReichertOM U2 mnicrotome, contrasted with uranyl-acetate/lead citrate solution and observed on thePlilips EM 300 microscope.

RESULTS

In acute mutine schistosomiasis eosino-philic granulocytes were found in hepaticparenchyma as a diffuse infiltrate, or

forming large distinct folliculi. Serialsections were studied in this work, toavoid the possible influence of neighbour-ing inflammatory portal spaces or peri-ovular granulomas. Only those fragmentsin which the vicinity of these inflam-matory structures was eliminated weretaken into consideration.

Hepatic parenchymaIn our model of acute murine schisto-

somiasis the hepatic parenchyma pre-

served its lobular architecture and thehepatocytes their trabecular organization.Hepatocellular irritation was indicated byan unusually high number of binucleatecells, by the abnormal size and shape ofthe nucleoli and by dense, large mito-chondria, sometimes containing cristalloidbodies. Hepatocellular necrosis was notseen.

Hepatic sinusoidsThese were loaded with cells. This was

due simultaneously to an intense hyper-plasia of Kupffer cells and to infiltrationby exogenous cells. Kupffer cells hadnuclei similar to those seen in endothelialcells, being irregular in shape and devoidof nucleoli. They had a narrow cyto-

plasmic rim and flattened processes ex-tending into the sinusoidal lumen, andtheir cytoplasm was locally crowded withphagosomes, in which schistosomal pig-ment and cell debris could be recognized.Cytoplasmic granules, corresponding tolysosomes and secretory granules, wereabundant.Exogenous cells were mostly typical

mature blood leucocytes. Occasionally weobserved small cells with a very highnucleo-cytoplasmic ratio, with subspheri-cal nuclei and cytoplasm rich in ribo-somes, but devoid of any particular in-clusions or cytoplasmic differentiation.They had the characteristics of blastic cells,but at this stage their precise identity wasdifficult to tell.

Small groups of these blastic cells wereobserved within the sinusoidal lumen, inclose relation to the endothelial lining. Inother places, the endothelial lining wasinterrupted, and blastic cells, together withoccasional Kupffer cells, could be observedwithin Disse's space, in direct contactwith hepatocytes (Figs lb and 3).

Intraparenchymal folliculiThese were observed most often in

spaces corresponding to obliteratedsinusoids. When small, folliculi did notalter the trabecular organization ofhepatic parenchyma, but when they werecomposed of several hundreds of cells theycould locally alter the normal hepaticstructure (Fig. 2a).

Folliculi were composed of cells whichcould be easily recognized as myeloid cellsof eosinophil granulocyte lineage. Theywere remarkably closely synchronized intheir maturation and only exceptionally,in large folliculi, could very young cells befound together with mature eosinophils.The composite origin of these folliculi wasapparent. Three main categories of cellscould be recognized in folliculi:

Stage I cellsSpherical or ovoid, these cells measured

6-10 Htm in the largest diameter and had

481

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

b-

FIG. 1 Liver in acute murine schistosomiasis: intrasinusoidal formation of granulocytopoieticfolliculi. (a) Semi-thin sections. x 333. (b) Electron micrograph showing Kupffer cells (K) andyoung eosinophil granulocytes. x 3078.

482

W:

4.7

la

HEPATIC GRANULOCYTOPOIESIS IN SCHISTOSOMIASIS

tS .i *,4s ;<J=

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

33

483

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

Ntw

FIG. 3.-Hepatic granulocytopoiesis in acute murine schistosomiasis. (a) Granulocytopoietic folliclecomposed 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 (myelocyte):Golgi zone with formation of eosinophil granules. x 21,082.

484

HEPATIC GRANULOCYTOPOIESIS IN SCHISTOSOMIASIS 485

I~ ~~ #ltt 7

; ge 7|'.e *8*;8s r2s2.>t v% s eiFIG. 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 theStage III eosinophil granulocyte. x 7375.

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

nuclei with small indentations, slightlycondensed perichromation and an eccen-tric nucleolus. Numerous polyribosomes,scattered cisternae of rough endoplasmicreticulum and a few small mitochondriawere found in the narrow cytoplasmic rim(Fig. 2b).

Stage II cellsCharacterized by marked differentiation,

these were 10-13 ,um in diameter. Theyhad a decreased nucleo-cytoplasmic ratio,with an irregularly shaped nucleus andsparse chromatin clumped along thenuclear membrane. Their cytoplasm waselectron-clear, with many well developedmitochondria, polyribosomes and dilatedcisternae of rough endoplasmic reticulum.In the perinuclear region, 3-6 Golgisaccules could be observed: dense granuleswere found on the concave side of theGolgi complex, measuring up to 0 4 /tm intheir long axis (Fig. 3).

Stage III cellsThese had a typical small and seg-

mented nucleus, without nucleoli. Thecytoplasmic rim was large, containingnumerous small mitochondria, many lyso-somes, a well developed Golgi apparatus,few elongated rough endoplasmic cisternaeand 2 types of granules: small (0.2-0.5 ,min diameter), round or ovoid, homo-geneously electron-dense, and large (meas-uring up to 0-5-0-8 ,tm in the long axis)containing most often an electron-clearcristalloid inclusion typical of eosinophilgranulocytes. At the cellular peripherynumerous pinocytic and coated vesicleswere observed (Fig. 4).

Granulocytopoietic folliculi were re-markably well delimited and homo-geneous. With the exception of Kupffercells, no cell line other than myeloid typeswere observed within the folliculi. Kupffercells were scattered at the periphery andthroughout the folliculi, forming longextensions and pseudopodia between myel-oid cells, but without any cell-contactdifferentiations.

At their periphery folliculi were in directcontact with hepatocytes. At the interface,hepatocytes were flattened, without anysign of irritation or reaction to the pre-sence of myeloid cells. Collagen bundles,thin and resembling the perisinusoidal"reticulin", were sometimes observed atthe periphery of folliculi. These bundleswere topographically related to pre-exist-ing sinusoids, and no fibrotic reactionaround folliculi was detected. The Disse'sspace of neighbouring sinusoids was incontinuity with the follicular space.

Cell divisions were frequent in folliculi.They were not restricted to blastic cells ofthe Stage I type, but were also found inmore differentiated cells including matureeosinophils with typical eosinophilic gran-ules in the cytoplasm (Fig. 4b).The diffuse eosinophilic infiltrate of the

hepatic parenchyma in acute murineschistosomiasis is composed of matureindividual eosinophil granulocytes. Theywere found mostly in perisinusoidal spacesand were more frequent in the vicinity ofportal spaces.

DISCUSSION

In earlier works, Goennert (1955),Grimaud and Borojevic (1972) and Byramet al. (1978) have indicated hepatic eosino-phil granulocytopoiesis in the acute phaseof murine infection with Schistosomamansoni. By electron microscopy Stengeret al. (1967) and Bogitsh (1971) have indi-cated the presence of immature granulo-cytes in hepatic periovular granulomas inthe same experimental model. In the pre-sent work, we describe the morphology ofhepatic granulocytopoiesis and its rela-tionship with the hepatic tissues.

Hepatic granulocytopoiesis in acuteschistosomiasis follows the same patternobserved in the bone marrow and inhepatic tissues during the prenatal period.The 3 morphological stages of myeloidcells observed in hepatic folliculi corres-pond exactly to promyelocytes, myelo-cytes and young mature eosinophil granu-locytes observed in normal granulocyto-

486

HE1PATlC GRANULO('YTOPOIESIS IN SCHISTOSOMIASIS

poiesis, as described in foetal liver byFukuda (1974) and Enzan et al. (1978).The origin of these myeloid cell lines andthe reasons which lead to this extra-medullary eosinophilopoiesis in acute in-flammation should be emphasized.

In the liver of acute murine schisto-somiasis, several factors influencingeosinophilia are locally produced. Schisto-some egg antigens and schistosome meta-bolic antigens are known to induce anintense reaginic response (Sadun, 1972;Dessaint et al., 1975). Anaphylactic reac-tions and immune complexes formed withreaginic antibodies are chemotactic foreosinophils (Goetzel, 1976). Simultaneous-ly, specifically stimulated lymphocytesand entire granulomas elicited by schisto-some eggs are known to produce anEosinophil-Stimulating Promoter (ESP)and to induce eosinophilopoiesis (Colley,1973; James and Colley, 1975; Miller,Colley and McGarry, 1976). Byram et al.(1978) have thus proposed that eosino-philic islands in schistosome-infectedorgans might be elicited by lymphokinessecreted by sensitized T lymphocytes inthe granulomas themselves. Immune com-plexes, ESP and lymphokines are probablydirectly responsible for eosinophil infiltra-tion in periovular granulomas and ininflammatory-tissue infiltrates in acuteschistosomiasis. As indicated by Bogitsh(1971), Byram et al. (1978) and by ourown observations (unpublished), immatureeosinophils are found in liver, spleen andmesenteric tissues, but never in granul-omas formed in lungs and in the intestinalwall, tissues where granulomas are par-ticularly abundant. Extramedullary gran-ulocytopoiesis thus occurs only in tissueswith recognized haemopoietic potential,and corresponds to true myeloid meta-plasia, as specified earlier by Grimaud andBorojevic (1972). The presence of ESP,lymphokines and reaginic immune com-plexes does not seem to be sufficient per seto induce ectopic granulocytopoiesis inthis model.

Connective-tissue cells and extracellularstroma are considered to be responsible for

the environment which stimulates localhaemopoiesis (Cline and Golde, 1979). Inour model, there was no topographicalrelationship between the connective tissueand myeloid foci, the first groups ofmyeloid cells being observed in thesinusoidal lumen. A constant associationwas, on the other hand, observed betweenKupffer cells and myeloid foci. Stimulatedmacrophagic cells are known to inducehaemopoiesis (Dimitrov et al., 1975;Buhles and Shifrine, 1978; Foster, 1978),and focal haemopoiesis was induced in ratliver by a macrophage activator, glucan(Deimann and Fahimi, 1980). Endothelialand Kupifer-cell hyperplasia are character-istic of schistosomal liver (Grimaud,Borojevic and Santos, 1976), and Kupffercells are known to be one of the sources ofthe Colony Stimulating Factor (CSF)which induces the proliferation of myeloidlines (Joyce & Chervenick, 1975).The cells which are the origin of

granulocytopoietic-cell proliferation inliver are apparently exogenous, as theleast differentiated forms were observedwithin the hepatic sinusoids. The syn-chronous differentiation in each folliculumsuggests that each corresponds to theproliferation of a single stem cell. Cellswhich may, under appropriate conditions,initiate the production of granulocyticcolonies are known to be present, althoughin small number, in the circulating blood(Chervenick and Boggs, 1971). We maythus assume that, while circulating in theliver sinusoids, these cells are subjected tolocal conditions created by thWe localimmune reaction to the presence of wormsand their eggs and by the secretoryactivity of stimulated Kupffer cells, whichcould induce them to initiate the produc-tion in situ of an eosinophilic granulocytecolony.

It is remarkable that glucan stimulationof macrophages induces complete haemo-poiesis in rat liver (Deimann and Fahimi,1980), while in our model only eosinophilgranulocytopoiesis was observed. It isknown that granulocyte progenitor cellsare heterogeneous, and specific eosinophil

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488 R. BOROJEVIC, S. STOCKER AND J. A. GRIMAUD

precursors were recognized in bone mar-row, spleen and peripheral blood of mice(Johnson, Dresch and Metcalf, 1977;Johnson and Metcalf, 1980). On the otherhand, specific CSF, which stimulates onlyeosinophil granulocyte proliferation, hasbeen identified (Nicola et al., 1979). Theselective stimulation of eosinophilopoiesiscould be achieved either by progenitorselection or through specific stimulationof less differentiated stem cells. Themechanisms of the control of granulocyteproliferation in human and in experi-mental schistosomiasis are currently understudy.

This work was supported by theConselho Nacional de DesenvolvimentoCientlfico e Tecnologico (CNPq), Braziland by the Societe d'Hepatologie Experi-mentale, Lyon, France.

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