American Joumal of Pathology, Vol. 138, No. 5, May 1991Copyright © American Association ofPathologist

Modulation of Alpha Smooth Muscle Actinand Desmin Expression in PerisinusoidalCells of Normal and DiseasedHuman Livers

A. Schmitt-Graff,* S. Kruger,t F. Bochard,tG. Gabbiani,* and H. DenktFrom the Department ofPathology, University of Geneva,CMU, Geneva, Switzerland; the Department ofPathology,tUniversity ofDusseldorf, Dusseldorf, Federal Republic ofGermany; and the Department ofPathology,* University ofGraz, Graz, Austria

It has been suggested that perisinusoidal liver cells(PSC) play a pivotal role in the pathogenesis offi-brocontractive changes. Using light and electron mi-croscopic immunolocalization techniques; a series of207 normal and pathologic human liver specimenswere evaluated for the expression of alpha smoothmuscle (SM) actin and desmin in this and other non-parenchymal cell types, In normal adult liver tissue,PSCs were practically devoid of desmin and excep-tionally stained for a-SM actinr whereas this actinisoformfrequently was encountered in PSCsfrom theembryonic to the adolescent period A broad spec-trum ofpathologic conditions was accompanied bythe presence of a-SM actin containing PSCs; thesewere detected preferentially in periportal or perive-nular zones according to the predominant locationof the underlying hepatocellular damage. The occur-rence of this PSCphenotype generally was associatedwithfibrogenesis and was in some cases detected ear-lier than overt collagen accumulation. Fibrousbands subdividing liver tissue in cirrhosis andfocalnodular hyperplasig as well as desmoplastic reac-tion to malignant tumors, contained a-SM actin-richcells admixed with variable proportions of cells co-expressing desmin In end stages; thispopulation wasless numerous than in activefibrotic or cirrhoticpro-cesses Using immunogold electron microscopy, a-SMactin was localized in microfilament bundles of typ-ical PSCs Our results are compatible with the as-sumption that the appearance of a-SM actin anddesmin-expressing myofibroblasts results at least inpart from a phenotypic modulation ofPSCs (AmJPathol 1991, 138:1233-1242)

Perisinusoidal cells (PSC) are multifunctional mesenchy-mal cells residing beneath the endothelial cells in thespace of Disse. They also are called lipocytes, fat-storing,stellate, or Ito cells (for review see Aterman1). Althoughthey have already been mentioned by Boll in 18692 anddescribed as a specialized cell population by Ito andNemoto in 1952,3 their role in retinoid metabolism4 andcollagen production5-7 remains to be clarified.

Perisinusoidal cells initially were thought to be char-acterized by the presence of cytoplasmic lipid dropletsreacting intensely with gold chloride (for review seeWake8). However ultrastructural in vivo and in vitro stud-ies revealed that proliferation of PSCs may be associatedwith reduction of lipid droplets,9 development of roughendoplasmic reticulum,10 and of microfilament bun-dles.11 Conditions associated with liver fibrosis such astreatment of rats with carbon tetrachloride (CC14),12 feed-ing of baboons with ethanol,9 or alcoholism in humans13have been suggested to promote the transformation ofPSCs into transitional cells bearing ultrastructural featuresintermediate between this cell type and fibroblasts. Fur-thermore cells with a typical myofibroblastic morphologyhave been noted in human and experimental hepaticfibrosis1415 and cirrhosis.1617 Considerable interest re-cently was directed to the question of whether PSCs andmyofibroblasts in liver diseases are distinct populations18or represent the same cell type in various stages of dif-ferentiation and/or functional activity.1920

It is well accepted that in rats PSCs of the normal21and injured22 liver contain desmin as the major protein ofintermediate-sized filaments. However little information isavailable concerning the cytoskeletal components ofPSCs of the human liver.19 This led us to investigate theexpression of desmin and a-SM actin, the actin isoformtypical of smooth muscle cell differentiation,-324 which

Supported in part by the Swiss National Science Foundation grant 3.108-0.88.

Accepted for publication January 7, 1991.Address reprint requests to Giulio Gabbiani, MD, Department of Pa-

thology, University of Geneva, CMU, 1 rue Michel-Servet, 1211 Geneva 4,Switzerland.

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1234 Schmitt-Graff et alAJP May 1991, Vol. 138, No. 5

appears temporarily in myofibroblasts during normalwound healing25 and more permanently in several fibro-contractive diseases,26 in nonparenchymal cells of nor-mal human fetal and adult hepatic tissue, as well as dur-ing a variety of pathologic settings. We show here thata-SM actin-expressing PSCs are rare in normal adultliver, while the appearance of an appreciable number ofthis cellular subset might be a hallmark of liver injury andfibrogenesis.

Materials and Methods

Specimens

Samples of liver tissue from 10- to 72-year-old patientswere obtained as percutaneous needle (n = 76) or sur-gical biopsies (n = 24). Furthermore 82 liver specimenswere collected from autopsies within 12 hours after death(age range, 1 day to 85 years). Four embryonic and 21fetal samples were collected from spontaneous miscar-riages or legal abortions. Liver tissue from newborn andadult Wistar rats (known to contain desmin-expressingPSCs) were used as controls.

Tissue samples were fixed in 4% phosphate-bufferedformaldehyde solution for light microscopy, in 2.5% cac-odylate buffered glutaraldehyde for electron microscopy,or in 0.1 moVI (molar) phosphate buffer containing 2%freshly prepared paraformaldehyde and 0.5% glutaral-dehyde for immunogold electron microscopy. Smallblocks of tissue were frozen in isopentane precooled inliquid nitrogen and stored at -80°C for immunofluores-cence staining.

Light Microscopy

Sections from paraffin-embedded material cut at 3 to 5 p.were stained with hematoxylin and eosin, Gomori's retic-ulin, Masson's trichrome, elastica van Gieson, and insome cases, Giemsa and periodic acid-Schiff solution.Parallel cryostat sections from samples studied by immu-nofluorescence microscopy also were processed forconventional light microscopy.

Antibodies, Immunofluorescence, andAvidin-Biotin-Complex Peroxidase (ABC-P)Staining

We used the following antibodies: 1) anti-aSM-1, amonoclonal IgG2a recognizing a-SM actin24; 2) affinitypurified polyclonal rabbit IgG against desmin27 and com-mercially available monoclonal desmin antibodies (DER-11, D33, Dakopatts, Hamburg, FRG); and 3) antibodies

to von Willebrand factor and macrophages (CD68, KPI)purchased from Dako AIS (Gloshun, Denmark). Second-ary antibodies for immunofluorescence microscopy wereFITC-labeled goat anti-rabbit IgG and TRITC-labeledgoat anti-mouse IgG (Nordic Immunological Laborato-ries, Tilburg, The Netherlands), and for immunogold elec-tron microscopy, goat anti-mouse Ig conjugated to 1-nmgold particles (Janssen Life Science Products, Olen, Bel-gium).

For avidin-biotin complex peroxidase staining, theVectastain kits antimouse IgG and anti-rabbit IgG (VectorLaboratories, Burlingame, CA) were used.

For immunofluorescence microscopy, cryostat sec-tions approximately 4 p.m thick, were fixed for 5 minutesin cold acetone (- 20°C), air dried for 2 hours, and incu-bated with the primary antibody diluted 1 :10 for 45 min-utes. After three rinsings in phosphate-buffered saline(PBS), the sections were incubated for 30 minutes withthe appropriate second antibody diluted 1:10 or 1:20,respectively. For double-labeled immunofluorescencemicroscopy, both primary antibodies were added simul-taneously, as were secondary antibodies. After three rins-ings in PBS, the sections were mounted in bufferedpolyvinylalcohol.28 Controls were performed using non-immune IgG in place of the primary antibody. Immuno-peroxidase staining for desmin was performed on cry-ostat sections. Sections were pretreated with normal goatserum and incubated for 1 hour with rabbit anti-desmindiluted 1:200.

Immunoperoxidase staining for a-SM actin anddesmin was done on formalin-fixed and paraffin-embedded tissues. Sections were pretreated with H202/methanol and subsequently with 0.1 mol/l periodic acid,0.005 mol/A NaBH4, and normal horse serum. They wereincubated overnight at 40C with anti-aSM-1 or D33 at thedilutions of 1:500 and 1:50, respectively. The first incuba-tion was followed by ABC-P staining according to themanufacturers' instructions. The peroxidase activity wasrevealed with 30% 3,3'diaminobenzidine (Serva, Heidel-berg, FRG) in PBS containing 0.015% H202. Slides werecounterstained weakly with Mayer's hematoxylin, dehy-drated, and mounted in Eukitt. Controls were performedusing a rabbit or mouse IgG instead of the primary anti-body.

Estimation of the number of anti-aSM-1 and anti-desmin immunoreactive PSCs was done independentlyby two researchers on areas containing mainly longitudi-nally cut liver cell plates and analyzed separately in theperiportal zone, the intermediate zone, and the perivenu-lar zone of liver lobules. Immunoreactivity was catego-rized arbitrarily as follows: - = nothing; +/- = stainingof some PSCs occupying approximately less than 1% ofthe sinusoidal liver cell surface; + = staining of PSCsoccupying approximately 1% to 10% of the sinusoidal

Cytoskeleton of Perisinusoidal Cells 1235AJP May 1991, Vol. 138, No. 5

liver cell surface; + + = staining of PSCs occupyingapproximately 10% to 30% of the sinusoidal liver cell sur-face; + + + = staining of PSCs occupying more than30% of the sinusoidal liver cell surface. In conditions as-sociated with the formation of scars, fibrous septa, or des-moplasia, the number of anti-aSM-1 and anti-desmin im-munoreactive cells was evaluated using the followingscale: - = no positivity; +/- = positivity of less than10% of mesenchymal cells; + = positivity of 10% to 20%of mesenchymal cells; + + = positivity of 30% to 50% ofmesenchymal cells; + + + = positivity of more than50% of mesenchymal cells.

Photographs were taken with a Zeiss photomicro-scope (Carl Zeiss Inc., Oberkochen, FRG) equipped withepi-illumination using plan apo-chromate x 10/1.0 to 63/1.0 objectives and photographed on Kodak TMAX 400(Kodak Limited, Hemel, Hempstead, UK) or Ilford PANFblack and white films (Ilford, Basel, Switzerland).

Electron Microscopy

Tissue samples fixed with glutaraldehyde were postfixedin 1.33% osmium tetroxide, dehydrated, and embeddedin Epon. Semithin sections were stained with toluidineblue. Thin sections were stained with uranyl acetate andlead citrate and examined in a Philips 400 electron mi-croscope (Philips SA, ZOrich, Switzerland). For immu-nogold electron microscopy, fixed tissues were rinsed in0.1 moVI phosphate buffer, dehydrated, and embeddedin Lowicryl K4M resin (Chemische Werke Lowi, Wald-

kraiburg, FRG). Immunogold staining with anti-aSM-1was performed on sections collected on Formvar-coatednickel grids as previously described.2526 As control,grids were incubated with purified mouse IgG instead ofthe prmary antibody.

Results

In all cases examined, diagnoses were made on the ba-sis of histologic features correlated with clinical informa-tion. The patterns of immunofluorescence and immuno-peroxidase staining related to different kinds of liver dis-eases are given in Tables 1 to 3.

Normal Tissues and Pathologic LiverSpecimens with a Retained LobularStructure

The media of the portal vein, hepatic artery branches,and of intercalated and large hepatic vein branchesstained brightly for anti-aSM-1 (Figure 1a). Double im-munofluorescence showed that desmin-positive smoothmuscle cells (SMC) were less numerous than those pos-

itive for anti-aSM-1. Desmin-expressing SMCs usuallywere located in the external portion of the media (notshown). Rare mesenchymal cells embedded in the con-

nective tissue of portal tracts were positive for antiaSM-1and for desmin. Terminal hepatic venules of normal livergenerally were surrounded by a discontinuous singlelayer of SMCs staining for anti-aSM-1 (Figure 1 b). Inter-

Table 1. Expression of a-Smooth Muscle Actin in PSCs ofNormal and Diseased Livers with a Retained LobularStuctureCondition No of cases Periportal zone Intermediate zone Perivenular zone

Embryo 4 + + + +Fetus 21 + + ++Infant/child 7 +/- + ++Adolescent 3 +/- + +Adult 6 +I- +l- +/-Steatosis 3 +/- +/- +/-Vitamin A intoxication 2 + + + + + +Circulatory shock 6 +/-+I- ++Septic shock 4 +/- + +Chronic congestion 12 + + + + + + +Veno-occlusive disease 1 + + + + +Alcoholic hepatitis 15 + + + + + +Chronic active hepatitis 4 + + + +/-Chronic persistent hepatitis 3 + +/- +1Chronic large bile duct obstruction 7 + ++ + +/-Inactive secondary biliary fibrosis 3 + +I- +I-Primary biliary cirrhosis, stages

and 11 2 ++ + +Acute liver rejection 1 + + + +Acute myeloid leukemia 5 + + +Chronic myeloid leukemia 4 + + +Chronic myelomonocytic leukemia 3 + + + + + +

PSC, perisinusoidal cells.

1236 Schmitt-Graff et alAJP May 1991, Vol. 138, No. 5

Table 2. Expression of a-Smooth Muscle Actin and Desmin in Mesenchymal Cells of CirrhoticLivers

Perisinusoidal cells Fibrous septa

Condition No of cases asm-1 Desmin axsm-1 Desmin

Cirrhosis with alcoholic hepatitis 11 + + +- + + + +Inactive alcoholic cirrhosis 9 + - + + +/-Chronic active hepatitis with cirrhosis 9 + +- +++ +Primary biliary cirrhosis, stage IV 2 + + ND + + + NDChronic graft-versus-host disease 2 + + ND + + + NDSecondary biliary fibrosis with

developing cirrhosis 9 + ND + + + NDInactive portal fibrosis with

partial cirrhosis 3 + ND + + NDCirrhosis in metabolic disorders 4 + ND + + NDInactive cirrhosis of unknown etiology 5 + ND + + ND

ND, not done.

estingly on several occasions, no anti-aSM-1 -positivecells were seen in this location. Scattered star-shapedcells within the lobules of embryonic, fetal, childhood, andadolescent livers were decorated with antiaSM-1 (Fig-ure 1 b). These cells were consistently negative for von

Willebrand factor and the macrophage marker KPI, andhence they represented PSCs. They were located in theperisinusoidal space adjacent to hepatocytes and were

more numerous in perivenular than in intermediate or

periportal areas. In fetal tissues, some staining for desminwas noted in these PSCs. In all other specimens, PSCslacked desmin immunoreactivity, contrary to what we

(and previous workers) have observed in rat liver (datanot shown). In normal adult liver, some a-SM actin-expressing PSCs were noted, which is contrary to whatwas observed in rat liver (data not shown). The vast ma-jority of liver lobules of adult controls, however, was de-void of this PSC phenotype (Figure 1 a). A pattern similarto controls was observed in nonalcoholic steatosis with-out inflammation or necrosis (Figure 2a). An increase ina-SM actin-expressing PSCs was encountered in differ-ent pathologic situations showing hepatocellular degen-eration, inflammation and fibrosis (Table 1). Two patientswith chronic hypervitaminosis A showed perivenular fi-brosis and prominent anti-aSM-1 immunoreactive PSCscontaining cytoplasmic vacuoles indicating fat droplets(Figures 2b, c). This cell type was abundant, especially

around the terminal hepatic venule and in the perivenularand intermediate zones of hepatic lobules. Postmortemliver specimens from patients with acute heart failure andshock surviving longer than 48 hours showed anti-aSM-1 -positive PSCs around areas of perivenular necro-

sis and collapse. In patients with prolonged septic shockand hyperbilirubinaemia, canalicular cholestasis and fociof hepatocellular necrosis were associated with a slightincrease in ant[-cSM-1 -positive PSCs. Biopsy and au-

topsy specimens obtained in chronic central congestiondue to right-sided heart failure were characterized by a

striking accumulation of anti-aSM-1-positive PSCs sur-

rounding terminal venules and cords of atrophic hepato-cytes adjacent to engorged sinusoids (Figure 2d). A sim-ilar pattern was observed in veno-occlusive diseasecomplicating allogenic bone marrow transplantation.These findings were most prominent in perivenular andintermediate zones. Alcoholic hepatitis with fattychanges, ballooning of hepatocytes, and Mallory bodiesshowed increased anti-aSM-1 positivity in the perivenu-lar zone; this change was clear even in cases in whichpericellular fibrosis was not detected by means of Go-mori's reticulin or Masson's trichrome stain. Alcoholichepatitis associated with central sclerosis showed con-

sistently perivenular accumulation of anti-aSM-1 -positivePSCs (Figure 2e). In the majority of cases with pericellularfibrosis of the 'chicken wire' type, we observed

Table 3. Expression of a-Smooth Muscle Actin and Desmin in Mesenchymal Cells ofNeoplastic and Tumorlike Lesionsuwth Alteration ofLobular Structure

Tissue surrounding Cellslesions within lesions

Condition No. of cases asm-1 Desmin asm-1 Desmin

Focal nodular hyperplasia 6 + + + + + + +Adenoma 5 + + ND + NDNodular transformation 4 + ND +/- NDHepatocellular carcinoma 4 + + + ND + + NDMetastatic carcinoma 12 + + + + + + + +Lymphoma 7 + + ND + ND

ND, not done.

Cytoskeleton of Perisinusoidal Cells 1237AJP May 1991, Vol. 138, No. 5

V

JFigure 1. Anti-asm-1-positive cells are seen only in the blood vessel wall of normal adult liver (a, X50), while a specimen obtainedfroma 9-year-old child shows staining ofboth vascular SMCs and PSCs (b, X200). Double-immunofluorescence staining shows abundant a-SMactin-expressing cells infibrous tissue ofposthepaitic cir*osis (C, x 100),focal nodal hyperplasia (e, x 100), and ofdesmnoplasia associatedwith metastatic breast cancer (g, x200). Note coexpression of desmin by a small (d, x1C00) to moderate (f, x100; g, x200) number ofstromal cells. In focal nodal byperplasia (e), anti-asn-1-immunoreactive cells are seen also within hepatocyte nodules (arouieads). Bymeans ofimmunoelectron microscopy, a-SM actin appears localized in stromal cells offocal nodal byperpIasia, wbich contain lipid droplets(arrowheads; i, x 10,000; j, x13,000). Intense labeling of microfilament bundles and of attenuated cytoplasmic processes is noted. 7heextracellular matrix is ricb in collagen ftbers.

anti-xSM-1 -positive PSCs surrounding hepatocytes (Fig-ure 2f). Active alcoholic hepatitis contained many immu-noreactive cells in fibrous scars linking perivenular areasto portal tracts. Findings in acute viral hepatitis were in-consistent and therefore not included in Table 1. Inchronic active hepatitis with piecemeal necrosis,

anti-aSM-1 -positive PSCs were observed adjacent to theinflammatory infiltrate trapping clusters of hepatocytes(Figure 2g). In chronic persistent hepatitis, an increase inanti-aSM-1 -positive PSCs was not evident. Chronic largebile duct obstruction yielded many anti-aSM-1 -positivePSCs adjacent to portal areas presenting cholestasis,

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1238 Schmitt-Graff et alAJP May 1991, Vol. 138, No. 5

bile ductular proliferation, inflammation, and fibrosis.Anti-aSM-1 immunoreactive cells also were present infibrous septa of chronic biliary obstruction. Secondary

biliary fibrosis, without signs of activity, ie, inflammation orcholestasis, showed only a small number of antiaSM-1 -positive PSCs. In primary biliary cirrhosis with florid duct

--g--

Cytoskeleton of Perisinusoidal Cells 1239AJP May 1991, Vol. 138, No. 5

lesions, ductular proliferation and expanding portal tracts(stages and II), anti-aSM-1 -positive PSCs were encoun-tered predominantly in the periportal zone. Furthermorescattered immunoreactive cells were present in interme-diate and perivenular zones. In acute liver rejection withmixed portal infiltrate, bile duct damage and central veinendothelialitis, scattered anti-aSM-1 -positive cells wereseen, predominantly in the perivenular zone. Portal tractand sinusoidal infiltration by acute and chronic leukemiaswas associated with a slight increase in anti-aSM-1 -

positive SMCs. A more pronounced proliferation of thesecells was observed in chronic myelomonocytic leukemia.

Cirrhosis

Bands of fibrous tissue surrounding parenchymal nod-ules in all types of cirrhosis examined contained mesen-

chymal cells immunoreactive for ant-aSM-1 (Table 2,Figure 1c). Double immunofluorescence showed that a

portion of them coexpressed desmin (Figure 1 d). No dif-ferences were noted between micro- and macronodularpatterns. The frequency of ant-aSM-1 -positive cells en-countered in fibrous septa and scars was related to theactivity of the cirrhotic process. In active cirrhosis with ablurred interface between fibrous tissue and hepato-cytes, inflammatory infiltrates and cell damage, manya-SM actin-expressing cells were present in expandingsepta as well as in the perisinusoidal space of residualhepatic parenchyma (Figure 2i). However, in areas dem-onstrating regenerative activity, the number ofanti-aSM-1 -positive PSCs was restricted to the peripheryof the regenerative cell plates. End stages with sharplyoutlined nodules showed a diminished number of immu-noreactive cells in fibrous septa and absence ofanti-aSM-1 -positive cells in a perisinusoidal localization.Chronic graft-versus-host disease with piecemeal necro-sis and developing cirrhosis was characterized by thepresence of a-SM actin-expressing cells in the periportalzone and within fibrous septa (Figure 2h).

Neoplastic or Tumorlike Lesions withAlteration of Lobular Structure

Focal nodular hyperplasia was characterized by thepresence of abundant anti-aSM-1 -positive cells, some of

which coexpressed desmin (Table 3, Figures 1 e, f). Thisphenotype was encountered mainly in the central stellatescar and the fibrous septa with chronic inflammation andproliferating bile ductules. Venules revealed a thickenedlayer of anti-aSM-1 - and anti-desmin-positive SMCsconsistent with fibromuscular hyperplasia. The nodularparenchyma bore many a-SM actin-expressing cells ad-jacent to hepatocytes. This pattern was quite differentfrom that seen in inactive cirrhosis, despite the histologicresemblance that sometimes occurred between the twolesions. The hepatocellular adenoma containedanti-aSM-1l-positive cells mainly in the fibrous capsuleand foci of hemorrhage. In nodular transformation,anti-aSM-1 -positive PSCs were present in zones of atro-phic internodular hepatocytes; within the nodules, a pro-liferation of PSCs expressing a-SM actin was exceptional.All specimens obtained from hepatocellular carcinomas,metastatic carcinoma, and lymphomas of high-grademalignancy producing bulky masses resulted in a strik-ing proliferation of ant-aISM-1 -positive cells in bands offibrous tissue surrounding tumor masses as well as withinthe lesions (Figure 1 g). In these desmoplastic reactions,a small number of cells coexpressing a-SM actin anddesmin were present (Figure 1 h). No differences in thestaining pattern for antiaSM-1 were noted in hepatocel-lular carcinomas compared to metastatic tumors. Gener-ally the non-neoplastic liver tissue adjacent to a benign ormalignant lesion contained an increased number ofanti-aSM-1 -positive cells associated with congestionand inflammation.

Immunogold Electron Microscopy

Immunogold electron microscopy was done on surgicalspecimens from focal nodular hyperplasia and primarybiliary cirrhosis; it showed antiaSM-1 labeling of micro-filament bundles present in stellate-shaped nonparen-

chymal cells embedded in a collagenous matrix (Figures1i, j). The cells displayed indented nuclei, a well-developed endoplasmic reticulum, and microfilamentbundles with dense bodies scattered within, predomi-nantly in the peripheral cytoplasm. Furthermore conspic-uous lipid droplets, as seen in typical fat-storing PSCs,were noted in several of them. These morphologic fea-tures, which correlated well with those observed in con-

ventional transmission electron microscopy, were com-

Figure 2. ABC-P staining of liver tissue for a-SM actin. In a fatty liver ofa diabetic patient that does not show inflammatory infiltration,immunoreactive PSCs are absent (a, X200). a-SM actin containing PSCs are visble in a case ofvitamin A intoxiation charactenzed by aconspicuousproliferation ofPSCs containing lipid vacuoles (b, X 100; C, X550); in chronic congestion (d, x200); in alcoholic liver diseasewith central sclerosis associated with many PSCs around terminal hepatic venule (e, x200); in alcoholic hepatits withprominentpercellularfibrosis (f, x450); in chronic active hepatitis with stellate-shaped PSCs at the margin ofpiecemeal necrosis (9, x400); in chronic graft-versus-bost disease withpiecemeal necrosis containing PSCs in ibrous tissue near theportal tract (h, x 100); in active cirrosis with alcoholichepatitis a conspicuous immunoreactivity for anti-sm-1 is visible in stromal cells ofthe scars, fibrous septa expanding in theparenchyma,and in PSCs scattered within the nodules (i, x60).

1240 Schmitt-Graff et alAJP May 1991, Vol. 138, No. 5

patible with the assumption that a-SM actin develops inhepatic PSCs.

Discussion

It appears more and more clear that stromal fibroblasticcells are heterogeneous in several respects, includingthe expression of cytoskeletal markers.29 Thus they mayexpress markers of myogenic differentiation such asa-SM actin and/or desmin in several locations such as thelymph nodes and spleen,3'31 tonsil and thymus,32 bonemarrow,33 and uterine mucosa.34 In addition, stromalcells can acquire, temporarily25 or more permanently,26these markers during pathologic situations such aswound healing or fibrocontractive diseases. We considerthat a high proportion of the a-SM actin-expressing cellsthat we have identified in liver parenchyma belong to thePSC population because of their characteristic location,their stellate morphology with the presence of long cyto-plasmic processes, and the electron microscopic immu-nolocalization showing lipid droplets and microfilamentslabeled by gold particles in the same cell. Furthermorelipid droplets were clearly visible even by light micros-copy in cells expressing a-SM actin in vitamin A intoxica-tion. However we cannot exclude that some a-SM actin-expressing cells, especially those of fibrous septa, origi-nate from pre-existing fibroblastic cells, as well as fromSMCs or pericytes. It was documented previously thatmyofibroblasts of cirrhotic rat liver produced by CCd4possess a contractile potential.17 Connective tissuesepta of cirrhosis and focal nodular hyperplasia or des-moplastic stroma of tumor metastasis contain not onlya-SM actin-positive cells but also a smaller proportion ofcells also expressing desmin. Desmin antibodies did notstain PSCs of normal adults livers, while rat PSCs werehighly labeled. It was mentioned previously that humanPSCs may lack desmin intermediate filaments.19 In hu-man fetal specimens of our series, however, some PSCswere indeed stained by desmin antibodies. Furthermorea-SM actin-expressing PSCs were numerous in the liversfrom the embryonic period to the age of adolescence,whereas this phenotype was rare in normal adult livertissue. These observations suggest that the cytoskeletalcomposition of PSCs may be modulated during develop-ment and in response to various stimuli in the adult, as itwas shown previously for stromal cells of other organssuch as bone marrow33 or intestine.35

Nonparenchymal liver cells are now generally con-sidered to be the primary source of collagen in normaland fibrotic liver, 7101236 although hepatocytes alsohave been suggested to contribute to collagen produc-tion.379 However, in a rat model of CCI4-induced liverfibrosis6 and in experimental biliary fibrosis,7 increasing

amounts of a2 (1), a1 (111), and a1 (IV) procollagen tran-scripts were demonstrated in cells of expanding fibroussepta and in PSCs, but not in hepatocytes. As shown byMilani et al,7 procollagen-expressing PSCs were colocal-ized with desmin-immunoreactive cells suggesting thatPSCs and modified PSCs (transitional cells) were impli-cated in collagen deposition. This correlates with an invitro study indicating that collagen derives from PSCs inprimary hepatocyte cultures.20

Our findings indicate that a large variety of hepaticpathologic conditions is accompanied by an increase inPSCs expressing a-SM actin. Most of these pathologicsettings are characterized by fibrosis. Appearance ofanti-aSM-1 -positive PSCs was more prominent in activestages of fibrosis and cirrhosis accompanied by celldeath than in advanced or inactive stages. However it isworth noting that the occurrence of a-SM actin-expressing PSCs is already clear at a stage of diseaseearlier than overt hepatic fibrosis. Examples include earlystages of alcoholic hepatitis and acute liver allograft re-jection. Furthermore a high proportion of anti-aSM-1-positive PSCs in the absence of fibrosis was detectedin shock necrosis and leukemic infiltration. In the contextof our study, it is of special interest that the response of ratliver to acute CC14 injury has been found to result in anincrease in desmin-positive PSCs in areas of necrosis asearly as 48 hours after intoxication,' while in other ex-periments increased procollagen expression has beendemonstrated after 2 weeks of CC14 treatment.6 In hu-mans the anti-aSM-1-positive population may corre-spond to PSCs with morphologic traits of transitional cellsor myofibroblasts described in experimental rat liver in-jury. This phenotypic change of PSCs has becomeknown as activation of PSCs.9,4142 Feeding of rats withalcohol and a high-fat low-protein diet resulted in perisi-nusoidal cell activation characterized by a significantlyincreased area of rough endoplasmic reticulum both inPSCs located near sinusoids and within scars.42 Mat-suoka et a143 have reported that PSCs isolated from ratliver with alcoholic fibrogenesis are activated to producemore collagen and are stimulated more efficiently byKupffer cell-derived factors compared to PSCs from nor-mal livers. It has been suggested that cytokines such astransforming growth factor 1 (TGF-,B1), interleukin-1 (IL-1), and tumor necrosis factor (TNF) may be released frommonocytes/macrophages in conditions such as CC14 oralcohol intoxication and schistosomiasis.44 Recent evi-dence indicates an important role of TGF-P1 in hepaticfibrogenesis.44 In a study of the effect of cytokines on cellproliferation and extracellular matrix formation by culturedPSCs, TGF-11 inhibited fat-storing cell proliferation whileenhancing collagen accumulation in the medium.45Interleukin-1a and TNFa had a mitogenic effect but in-duced an inhibition of collagen formation. It is tempting to

Cytoskeleton of Perisinusoidal Cells 1241AJP May 1991, Vol. 138, No. 5

speculate that cytokines may modulate the expression ofa-SM actin by PSCs in human liver. Indeeda-SM-positive cells frequently are visible around focalhepatocellular necrosis during septic shock. This condi-tion is characterized by high levels of TNF.46 Increasedserum levels of TNF also have been described in patientswith chronic congestive heart failure.47 In our study,chronic venous congestion was associated with a strikingaccumulation of PSCs expressing a-SM actin. Thus thispattern may not merely be a consequence of the me-chanical hydrostatic pressure and hypoxic cell damage,but also may be related to the effects of TNF. Tumornecrosis factor a has been incriminated in the pathogen-esis of pulmonary fibrosis.' However we do not knowwhether cytokines influence PSCs directly or indirectly,eg, via Kupffer cells. Furthermore infiltration of sinusoidsby monocytes in chronic and acute myelomonocytic leu-kemia is accompanied by the presence of many a-SMactin-positive cells. This finding is in agreement with theprevious observation that bone marrow stromal cells aremodulated to express a-SM actin in these types ofleukemia.'

Little is presently known on the features influencing theappearance of a-SM actin and desmin in stromal fibro-blastic cells. It has been reported that y-interferon de-creases the expression of a-SM actin in SMCs andfibroblasts,49 whereas local application of GM-CSF in-creases the expression of this marker in fibroblasts sur-rounding the application site.50 Our findings correlatewith the assumption that the appearance of a-SM actinand desmin-positive fibroblasts coincides with the devel-opment of fibrotic changes in several organs andtissues' (for review Sappino et ala9). Further informationon the factors regulating the expression of these proteinsin stromal cells will help the understanding of the patho-genetic mechanism leading to the development of fibro-contractive diseases, including those of the liver.

Acknowledgments

The authors thank Dr. 0. Fryc for fumishing specimens of normalliver; G. Chakroun, A. Lockenhoff, and M. Schroder for technicalhelp; J. C. Rumbeli and E. Denkinger for photographic work; andM. -M. Rossire and M. Giovanola for typing the manuscript.

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