Kupffer cell iron overload induces intercellular adhesion molecule-1 expression on hepatocytes in genetic hemochromatosis

Kupffer Cell Iron Overload Induces Intercellular Adhesion Molecule-1 Expression on Hepatocytes in Genetic Hemochromatosis PER ST~L, ~'2 ULRIKA BROOMI~,1 ANNIKA SCHEYNIUS,3 RAGNAR BEFRITS,4 AND ROLF HULTCRANTZ4

The mechanisms underlying iron-induced liver fibrogenesis in patients with genetic hemochromatosis are poorly understood. We studied signs of Kupffer cell activation and inflammatory responses in liver biopsy specimens obtained from 15 patients with untreated and six patients with treated hemochromatosis. Immunohistochemistry was performed on 11 of the untreated and all treated patients. Three of the untreated patients (20%) had cirrhosis and eight (53%) had fibrosis. None had chronic active hepatitis (CAH). Immunohistochemistry indicated that 55% of the untreated patients had sparse intercellular adhesion molecule-1 (ICAM-1) expression by hepatocytes, and all of these had Kupffer cell iron overload. No ICAM-1 expression was seen by hepato. cytes in treated patients or healthy controls. ICAM-1 was strongly expressed by hepatocytes from control patients with inflammatory liver disease. HLA-DR reactivity was seen on sinnsoidal cells in all groups, but not on hepatocytes except for two of the control patients with CAH. Twenty-seven percent of the untreated hemochromatosis patients displayed moderate infiltration by CD3-positive lymphocytes. Electron microscopy of samples from untreated hemochromatosis patients showed hypertrophic Kupffer cells containing iron-rich remnants of phagocytosed hepatocytes. Fat-storing cells close to iron-laden hepatocytes contained multiple lipid droplets and adjacent collagen fibril bundles. Thus, in patients with untreated genetic hemochromatosis and Kupffer cell iron overload, hepatocytes occasionally express ICAM-1. In regions with heavy iron overload, Kupffer cell hypertrophy and transition of fat-storing cells are seen. Our findings indicate that release of factors from

Abbreviations: GH, genetic hemochromatosis; ICAM-1, intercellular adhesion molecule-I; CAH, chronic active hepatitis; ALD, alcohol-induced liver disease; PSC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis. From the 1Division of Gastroenterology and Hepatology and 2Clinical Research Center, Huddinge University Hospital; and the 3Department of Clinical Immunology and 4Internal Medicine, Karolinska Hospital, Karolinska Institutet, Stockholm, Sweden. Received May 10, 1994; accepted December 9, 1994. This study was supported by grants from the Swedish Medical Research Council (projects 7924 and 9127), the Swedish Work Environment Fund, the King Gustaf V Foundation, Funds from Nanna Swartz, and Rut and Richard Juhlin Foundation. Address reprint requests to: Per St~l, MD, Department of Gastroenterology and Hepatology, K 63, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2105-001353.00/0

iron-loaded, activated Kupffer cells is of importance for the transformation of fat-storing cells and increased collagen deposition seen in genetic hemochromatosis. (HEPATOLOGY 1995;21:1308-1316.)

Genetic hemochromatosis (GH) is an inherited disorder in which there is a progressive iron accumulation of the liver, initially in the periportal region of the liver lobule. 1'2 Excess iron catalyzes the formation of toxic oxyradicals, 3 which initiates lipid peroxidation of organelle membranes, 4 leading to impairment of cellular function. 5'8 The fibrogenic process is initiated at a critical threshold level of hepatic iron overload 7 and, if left untreated, the iron accumulation eventually results in hepatic cirrhosis. In many chronic liver diseases, development of fibrosis is preceded by an inflammatory process in the liver, s'9 Infiltrating lymphocytes release cytokines, which stimulate proliferation, activation, or transformation of fat-storing cells, lOTransformation of fat-storing cells to collagen-producing transitional cells or myofibroblasts is considered a crucial step in the series of events leading to fibrosis. 11 Thus, the inflammatory process is regarded to be of pathogenic importance in initiating the synthesis and deposition of collagen. GH, however, may progress to cirrhosis without signs of inflammation in the liver parenchyma. 1 The specific pathophysiologic mechanism by which excess iron stimulates hepatic fibrogenesis in GH is poorly understood. Several mechanisms have been suggested, including a direct-acting effect of iron or lipid peroxides on fat-storing cells, 12 release of profibrogenic factors from membrane-damaged hepatocytes, 13 or iron-induced activation of Kupffer cells, which in turn secrete cytokines transforming adjacent fat-storing cells to collagen-producing myofibroblasts. 14~ Inflammatory cells, such as lymphocytes or activated Kupffer cells, release proinflammatory cytokines that induce expression of intercellular adhesion molecules1 (ICAM-1) 17'~s or major histocompatibility complex class II antigens 19'2° on epithelial cells. Such an expression may be seen on hepatocyte cell surface membranes in chronic inflammatory liver diseases, as a sign of cytokine release from invading lymphocytes in these livers. 2~ Whether cytokine interaction exists in iron-overloaded GH livers is unknown. Our purpose in this study

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TABLE 1. C l i n i c a l D a t a o f G H P a t i e n t s B e f o r e a n d A f t e r T r e a t m e n t W i t h V e n e s e c t i o n T h e r a p y Patient No.

ALT* (IU/L)

Serum Ferritin* (pg/L)

Serum Transferrin Saturation (%)

69/M 63/M 61/M 42/M 40/M 38/M 31/M 31/M 26/F 25/F 24/M 66/F 65/F 64/M 54/M

65 123 97 285 115 101 94 71 46 28 63 45 86 34 ND

2,630 1,860 3,900 4,340 1,540 2,060 1,770 590 273 294 510 1,520 550 560 3,000

95 86 90 84 79 98 96 89 86 87 129 75 80 96 95

43/M 41/M 61/M 57/M 53/M 47/M

32 21 15 (112) 22 (87) 13 (108) 19 (68)

13 21 38 (2,180) 17 (2,690) 17 (1,510) 45 (9,340)

36 56 45 (90) 31 (82) 16 (80) 88 (92)

Age (yr)/Sex

Liver Histology

Patients before treatment 1 2 3 4 5 6t 7 8 9 10 11 12t 13? 14t 15t

Fibrosis Cirrhosis Cirrhosis Fibrosis Fibrosis Fibrosis Fibrosis Normal Normal Steatosis Normal Fibrosis Fibrosis Fibrosis Cirrhosis

Patients after treatment 4 5 16 17 18 19

Fibrosis Fibrosis Fibrosis Cirrhosis Cirrhosis, steatosis Cirrhosis

NOTE. Figures in parentheses denote values before treatment. Abbreviations: ALT, alanine transaminase; ND, not determined. * Normal values: ALT: males <40 IU/L, females <35 IU/L; serum ferritin: males 20-200 #g/L, females 10-130 #g/L. t Electron microscopic investigations performed.

was to a d d r e s s the h y p o t h e s i s t h a t iron-induced fibrogenesis involves cytokine release from a c t i v a t e d Kupffer cells. Hence, we a s k e d w h e t h e r iron overload induces expression of ICAM-1 a n d H L A - D R on h e p a t o c y t e s in livers of G H patients, a n d if such a n expression is a c c o m p a n i e d by Kupffer cell iron overload or presence of i n f l a m m a t o r y cells in the liver p a r e n c h y m a , a n d f u r t h e r r e l a t e d to u l t r a s t r u c t u r a l signs of K u p f f e r cell a c t i v a t i o n a n d lipocyte t r a n s f o r m a t i o n . PATIENTS

AND

METHODS

Patients a n d Controls. Fifteen patients with untreated GH were subjected to percutaneous liver biopsy as part of the investigation for the disease. The clinical data are outlined in Table 1. Liver biopsy specimens from 11 patients (nos. 1 to 11) were secured for immunohistochemical analysis, routine histological examination, and iron determination. Additional ultrastructural studies were made on liver tissue from patient no. 6. Four additional patients (nos. 12 to 15) were investigated with electron microscopic analysis, iron determination, and light microscopy, but not with immunohistochemical analysis because of limitations of liver tissue. Repeat biopsies were done on two of the patients (nos. 4 and 5) after completed venesection treatment, and liver tissue was secured for immunohistochemistry. An additional four patients (nos. 16 to 19), who had been diagnosed before the start of the study, also had repeat biopsies after treatment, and were included in the post-treatment group.

In all patients the diagnosis was based on the medical history, laboratory data, and liver histopathology. None of the patients had a history of oral iron supplementation or blood transfusions. Alcohol consumption was less than 30 g/ d in all patients. No patient was positive for hepatitis Bs antigen. All patients were negative for antibodies against hepatitis C tested with recombinant immunoblot assay II (Chiron Corporation, Emeryville, CA). The liver biopsy was made percutaneously with the Menghini needle (1.6 mm diameter) using the intercostal route, and approximately 3 cm of liver tissue was obtained from each patient. The tissue was immediately cut into three pieces; the first being fixed in formaldehyde for routine histology, the second being dry frozen in -70°C for determination of iron content, and the third being immediately snap frozen in chilled isopentane and stored at -70°C for immunohistochemical staining. In patients nos. 6 and 12 through 15, part of the biopsy specimen was cut into three to four pieces, each being approximately 1-mm thick, and these were immediately fixed for electron microscopic investigation. Five patients who underwent cholecystectomy served as healthy controls, and 11 patients with various liver diseases served as additional control patients. None of the healthy controls had any clinical or biochemical signs of iron overload, chronic liver disease, cholestasis, or cholecystitis. The liver specimens were taken preoperatively, immediately after opening of the abdomen, but before the cholecystectomy was performed. The specimens were cut into two pieces; one being fixed in formaldehyde for routine histopathology, and the

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TABLE 2. I m m u n o h i s t o c h e m i e a l D a t a o f L i v e r B i o p s i e s F r o m C o n t r o l P a t i e n t s W i t h C h r o n i c L i v e r Diseases Other Than Genetic Hemoehromatosis Diagnosis

Liver Histology*

CD3+ Cells in Portal Tracts?~

CD3+ Cells in Lobuli?§

ICAM-1 Expression on Hepatocytest§

HLA-DR Expression on Hepatocytest§

CAH CAH CAH ALD ALD PSC PSC PSC PBC PBC PBC

Fibrosis, steatosis Cirrhosis, steatosis Cirrhosis, steatosis Fibrosis Cirrhosis, alcohol hepatitis Fibrosis Fibrosis Fibrosis Fibrosis Fibrosis Fibrosis

+++ +++ ++ + ++ ++ + ++ ++ +++ ++

+++ +++ ++ + +++ ++ + ++ + ++ ++

++ ++ +++ ++ ++++ + +++ ++ + ++ +++

+ + ----------

Abbreviations: CAH, chronic active hepatitis with serology positive for hepatitis C; ALD, alcohol-induced liver disease; PSC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis. * Staining with hematoxylin-eosin (paraffin-embedded sections). # Immunohistochemical staining (frozen sections). - - = Not significantly different from controls; + = slightly increased compared with controls; + + = moderate infiltrates, no piecemeal necrosis; ÷ + + = dense infiltrates, or with piecemeal necrosis. § - - = No positive cells; + = occasional positive cells; ÷+ = 1-3 clusters with >-3 positive cells; + + + = >4 clusters with ->3 positive cells; + + + + = 25-50% of cells are positive.

other frozen in chilled isopentane, stored a t -70°C, a n d used for i m m u n o h i s t o c h e m i s t r y . The 11 p a t i e n t s w i t h chronic liver disease were diagnosed as h a v i n g chronic active h e p a t i t i s (CAH), alcohol-induced liver disease (ALD), p r i m a r y sclerosing cholangitis (PSC), a n d p r i m a r y b i l i a r y cirrhosis (PBC), as is shown in Table 2. I n these patients, liver biopsies were performed p e r c u t a n e o u s l y as described above, and tissue specimens were fixed for routine histopathology a n d immunohistochemistry. The s t u d y was approved by t h e ethical committee a t the K a r o l i n s k a I n s t i t u t e t , a n d informed consent was given by p a t i e n t s a n d controls included in the study. Immunohistochemical Staining. Frozen, 6-#m thick sections of liver tissue were fixed in acetone for 5 m i n u t e s a t 4°C, a n d s t a i n e d by t h e a l k a l i n e p h o s p h a t a s e - m o u s e antialkaline p h o s p h a t a s e (APAAP) m e t h o d with Fast-Red. 22 To reduce nonspecific binding, n o r m a l r a b b i t s e r u m (1/10) was applied for 10 minutes. Sections were t h e n incubated with the following mouse monoclonal antibodies: a n t i - I C A M - 1 (CD54) diluted to 1:80 (hybridoma 84H10, Serotec, Oxford, U.K.), a n t i - H L A - D R diluted 1:128 (hybridoma L243, Becton Dickinson a n d Co., Oxnard, CA), a n d a n t i - L e u - 4 (anti-CD3) diluted 1:256 (Becton Dickinson). Rabbit anti-mouse i m m u noglobulin (diluted 1:40) obtained from D a k o p a t t s (Copenhagen, D e n m a r k ) served as a secondary antibody. The a l k a l i n e p h o s p h a t a s e - m o u s e a n t i - a l k a l i n e p h o s p h a t a s e complex in dilution 1:20 (Dakopatts) was t h e n applied to t h e sections for 30 minutes, followed by a n o t h e r 10 m i n u t e s ' incubation with r a b b i t anti-mouse immunoglobulin a n d a l k a l i n e phosphatase-mouse a n t i - a l k a l i n e p h o s p h a t a s e complex, respectively. The colored end product was developed with F a s t - R e d TR as a chromogen s u b s t r a t e , a n d t h e n sections were c o u n t e r s t a i n e d with Mayer's hematoxylin. Controls without the p r i m a r y antibody or with i r r e l e v a n t mouse monoclonal antibody gave no positive staining. The slides were e x a m i n e d by two independ e n t investigators u n a w a r e of t h e clinical s t a t u s of each patient. Hepatocytes were considered positive when the entire cell surface was outlined by a continuous positive staining.

Histopathology. Liver tissue was fixed in formaldehyde, embedded in paraffin, and the sections were stained for hematoxylin-eosin, reticulin, van Gieson, a n d with Perls' blue, the l a t t e r to demonstrate iron deposits in histological sections. Electron Microscopy. Liver tissue for electron microscopic investigation was t r e a t e d as described previously. 23 In short, it was fixed in 3% g l u t a r a l d e h y d e in 0.1 mol/L sucrose a n d 0.1 mol/L cacodylate buffer. I t was postfixed in osmium a n d d e h y d r a t e d in g r a d e d ethanol, t h e n e m b e d d e d in Epon 812. Tissue was sectioned with a d i a m o n d knife a n d s t a i n e d in lead citrate. Sections were e x a m i n e d in a Philips 400 electron microscope. Biochemical Analyses. Iron content in liver tissue was m e a s u r e d in an atomic absorption spectrophotometer (Varian SpectrAA 400, Varian, Palo Alto, CA). Background correction was used. The s a m p l e s were hydrolyzed in 3.5% HNO3 overnight before analysis. 24 S e r u m m e a s u r e m e n t s of alanine t r a n s a m i n a s e , ferritin, a n d t r a n s f e r r i n s a t u r a t i o n were performed as routine l a b o r a t o r y investigations a t the Hospital's D e p a r t m e n t of Chemistry. RESULTS

Light Microscopy Untreated GH Patients (Nos. 1 Through 15). T h e h i s tological data and immunohistochemical results are s u m m a r i z e d i n T a b l e 3. A l l u n t r e a t e d p a t i e n t s h a d a typical histological appearance, with granular iron deposits in a periportal-to-centrilobular gradient. The patients with the highest liver iron content also had iron d e p o s i t s i n s i n u s o i d a l cells. K u p f f e r cell i r o n d e p o s i t s w e r e f o u n d i n 12 o f t h e u n t r e a t e d p a t i e n t s . A l l p a t i e n t s w i t h f i b r o s i s o r c i r r h o s i s h a d K u p f f e r cell i r o n o v e r l o a d , w h e r e a s o n l y o n e (no. 11) o f t h e f o u r y o u n g G H p a t i e n t s w i t h o u t f i b r o s i s d i s p l a y e d K u p f f e r cell i r o n d e p o s i t s . Occasional sideronecrotic hepatocytes were found in seven patients, all having fibrosis or cirrhosis. One pa-

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TABLE 3. H e p a t i c I r o n C o n t e n t , L i v e r H i s t o l o g y , a n d I m m u n o h i s t o c h e m i c a l D a t a o f L i v e r B i o p s i e s From GH Patients Before and After Venesection Therapy

Patient No.

Liver Iron (pg/g liver, wet weight)

Liver Histology*

Sideronecrosis of Hepatocytes*t

Kupffer Cell Iron~§

Biliary Iron~§

CD3+ Cells in Portal Tractslf¶

CD3+ Cells in Lobuliplt

ICAM-1 Expression on HepatocytesHt

++

+

÷÷

Patients before treatment 1

1,573

Fibrosis

+

++

+

2

1,948

Cirrhosis

+

+++

++

--

+

3

980

Cirrhosis

+

+ +

+

++

+

+

4 5

2,930 1,095

Fibrosis Fibrosis

+ +

+++ ++

+ ++

+ +

+ + +

+

6#

ND

Fibrosis

--

+ +

+

--

+

+

--

--

+

--

+

7

1,363

Fibrosis

--

8

758

Normal

.

9

578

Normal

--

10

530

Steatosis

.

++ .

.

.

-.

.

+

+

.

+ + ++

11 12#

ND 5,325

Normal Fibrosis

-+

++ + ++

-+

++ ND

+ ND

ND

13#

2,825

Fibrosis

--

++

--

ND

ND

ND

14#

ND

Fibrosis

--

+

+

ND

ND

ND

15#

6,650

Cirrhosis

+

++ +

+

ND

ND

ND

Patients after treatment 4

53

Fibrosis

--

+

--

--

+

5

55

Fibrosis

--

--

--

+

+

16

113

Fibrosis

--

--

--

+

+

17

33

Cirrhosis

--

--

--

+

+

18

13

Cirrhosis, Steatosis

--

+

--

+

++

19

332

Cirrhosis

--

--

--

+

+

N O T E . N o r m a l v a l u e for l i v e r i r o n : < 2 7 5 # g / g l i v e r ( w e t w e i g h t ) . Abbreviation: ND, not determined. * Staining with hematoxylin-eosin (paraffin-embedded sections). t --

N o p o s i t i v e cells; + = o c c a s i o n a l p o s i t i v e cells; + +

= 1-3 c l u s t e r s w i t h -~3 p o s i t i v e ceils; + + +

= > 3 c l u s t e r s w i t h ->3 p o s i t i v e

cells. $ Staining with Perls' blue (paraffin-embedded sections). § --

- N o i r o n - p o s i t i v e cells; + = o c c a s i o n a l i r o n - p o s i t i v e cells; + +

= < 5 0 % i r o n - p o s i t i v e cells; + + + = > 5 0 % i r o n - p o s i t i v e cells.

III m m u n o h i s t o c h e m i c a l s t a i n i n g ( f r o z e n s e c t i o n s ) . ¶ - - = N o t significantly different f r o m controls; + = slightly i n c r e a s e d c o m p a r e d w i t h controls; + + = m o d e r a t e infiltrates, no p i e c e m e a l necrosis. # Electron microscopic investigations performed.

tient had a slight hepatic steatosis. None of the patients had signs of CAH. T r e a t e d GH P a t i e n t s (Nos. 4, 5, 16 T h r o u g h 19). After iron mobilization with phlebotomy treatment, no patients displayed positive granular iron deposits in hepatocytes with Perls' blue staining. Two patients (nos. 4 and 18) had occasional Kupffer cells that were positive with Perls' blue. Three patients had cirrhosis, and three had fibrosis. There were no signs of sideronecrosis or biliary iron in any of the posttreatment biopsies. Healthy Controls ( n = 5 ) . None had any signs of increased collagen content or inflammatory infiltrates in portal tracts or lobuli. Perls' blue staining was negative in all biopsy specimens. Controls With Chronic L i v e r Disease (n = 11). The two patients with CAH both had signs of piecemeal necrosis, occasional inflammatory cells in lobuli, and fi-

brosis. The ALD patients (n = 2) both had cirrhosis, with slight or moderate infiltration of inflammatory cells in portal tracts, but without piecemeal necrosis. The patients with PSC (n = 3) and PBC (n = 3) did all have portal inflammation, bile duct proliferation, and fibrosis. None of the PBC or PSC patients had cirrhosis. Staining with Perls' blue was negative in all control patients. l m m u n o h i s t o c h e m i e a l Investigation U n t r e a t e d GH P a t i e n t s (Nos. 1 Through 11).

The

re-

of the immunohistochemical investigations are shown in Table 3. In three patients (nos. 1, 3, and 11), there was a moderate infiltration of CD3-positive lymphocytes in the portal tracts, but without signs of piecemeal necrosis. An additional three patients (nos. 4, 5, and 9) had a slightly increased number of CD3-positive cells in the portal tracts compared with controls. Occasional CD3-positive cells were found in hepatic lobules sults

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FIG. 1. (A) A periportal area of a liver biopsy specimen'from patient no. 1 before treatment. A cluster of 4 to 5 ICAM-l-positive hepatocytes is seen in an area with marked iron overload (arrow). The iron deposits are seen as brownish pigment located intracellularly in the hepatocytes (double arrows). (B) HLA-DRreactivity from a liver biopsy specimen in an area with similar iron overloadand from the same patient. The sinusoidal cells, but not hepatocytes, are HLA-DRpositive. The expression of ICAM-1 and HLA-DRis visualized with the APAAP method. (Original magnification x750.)

in all biopsies. In one patient (no. 4), three adjacent CD3-positive cells were seen in the liver lobule, but no patients had lobular infiltrates of more t h a n three CD3positive lymphocytes. I C A M - l - p o s i t i v e hepatocytes were found in six untreated patients. A cluster of I C A M - l - p o s i t i v e hepatocytes in an area of iron loading (patient no. 1) is demonstrated in Fig. 1A. Also, patient no. 11 had a group with three to four hepatocytes expressing ICAM-1, whereas in the other four patients occasional single hepatocytes were positive. The I C A M - l - p o s i t i v e hepatocytes were all located in iron-loaded areas, but there was no clear-cut correlation between the degree of iron loading in hepatocytes and ICAM-1 positivity. Thus, in periportal areas with a marked iron load, ICAM-1expressing hepatocytes were not always found. However, ICAM-1 was never expressed on hepatocytes in areas without visible iron deposits. We found ICAM1-positive cells in five of seven patients with fibrosis/ cirrhosis, and in one of four patients without fibrosis. All six patients positive for ICAM-1 displayed Kupffer cell iron overload, whereas the three patients without iron deposits in Kupffer cells were all negative for ICAM-1 on hepatocytes. There was no correlation to the number of CD3-positive lymphocytes in portal tracts or lobuli. Sideronecrosis was found in five of the six pa-

tients with the highest liver iron content, and four of these patients also expressed ICAM-1. Endothelial and Kupffer cells were positive for ICAM-1 and HLA-DR in all patients. No HLA-DR reactivity was found on hepatocytes in any of the patients. Figure 1B demonstrates HLA-DR reactivity on sinusoidal cells in patient no. 1 with u n t r e a t e d GH. Treated GH Patients (Nos. 4, 5, 16 Through 19). Five treated patients had a slightly increased n u m b e r of CD3-positive cells in the portal tracts, as demonstrated in Table 3. As before treatment, occasional CD3-positive lymphocytes were found in hepatic lobules of all biopsy specimens, and one of the treated patients (no. 18) had a cluster of three CD3-positive cells in the lobule. In patient no. 5, no significant change in the number of CD3-positive cells in portal tracts was seen before or after treatment, whereas patient no. 4 had fewer lymphocytes in portal tracts in the p o s t t r e a t m e n t liver specimen t h a n in the biopsy t a k e n before iron depletion. None of the phlebotomized patients displayed any I C A M - l - p o s i t i v e hepatocytes in the biopsies. Patients no. 4 and 5, who were ICAM-1 positive before treatment, were negative after completed venesection therapy. Endothelial and Kupffer cells were positive for ICAM-1 and HLA-DR in all t r e a t e d patients. No HLADR reactivity was found on hepatocytes.

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Healthy Controls (n = 5). I n c o n t r o l p a t i e n t s , occas i o n a l CD3-positive cells were found in the liver lob-

ules, and occasional cells or scarce infiltration in portal tracts. All control patients were negative for ICAM-1 and HLA-DR on hepatocytes. As in untreated and treated patients, endothelial and Kupffer cells were positive for ICAM-1 and HLA-DR in all controls. The expression of ICAM-1 and HLA-DR on Kupffer and endothelial cells did not differ between healthy controls, treated, or untreated GH patients. Controls With Chronic Liver Disease (n = 11). T h e

results of the immunohistochemical investigation are demonstrated in Table 2. All patients except two (having ALD and PSC) had moderate or marked increase of CD3-positive cells in portal tracts or lobuli. All patients were positive for ICAM-1 on hepatocytes, ranging from occasional hepatocytes expressing ICAM-1 in two patients having PSC and PBC, to over 25% positive hepatocytes in one patient with ALD. Hepatocytes expressing ICAM-1 were located in proximity to clusters of mononuclear cells in lobuli, or adjacent to the portal tracts. HLA-DR expression on hepatocytes was only seen on occasional hepatocytes in livers from patients with CAH, and not found in the other control patients with chronic liver diseases. The expression of HLADR and ICAM-1 on Kupffer and endothelial cells was similar to that of healthy controls and GH patients. Some of the control patients with cholestatic liver disease have been included in a previous study. 2° Electron Microscopy Untreated Patients (Nos. 6, 12 T h r o u g h 15). T h e electron microscopical investigation showed iron overload in hepatocytes, with ferritin granules, hemosiderin deposits, and lysosomal hypertrophy as described previously. 23 In areas of heavily iron-overloaded hepatocytes, hypertrophic ferritin-containing Kupffer cells were abundant. In such hypertrophic Kupffer cells, glycogen was present in large heterophagic vacuoles, suggesting that phagocytosed hepatocytes gave rise to these vacuoles. Kupffer cells also contained considerable amounts of ferritin and hemosiderin (Fig. 2). Fat-storing cells were found throughout the liver lobule. In some of these, numerous small lipid droplets and adjacent bundles of collagen fibrils were encountered, as has been described for transitional cells or myofibroblast-like cells in other liver diseases 25 (Fig. 3). High-density granules with the appearance of ferritin particles could be seen in occasional fat-storing cells in areas with a heavy iron overload, as demonstrated in Fig. 4. However, ferritin particles were always less abundant in fat-storing cells than in adjacent hepatocytes. DISCUSSION

In the current study, we found expression of ICAMI on hepatocytes in iron-overloaded livers from patients with GH, a finding we interpret as an indirect sign of cytokine release in these livers. The expression was only seen in livers with simultaneous Kupffer cell iron

FIG. 2. Electron micrograph of a hypertrophic Kupffer cell (K) from 1 of the cirrhotic patients (no. 15). The cell contains heterophagic vacuoles with phagocytosed material containing hemosiderin, ferritin deposits, and glycogen, indicating remnants from necrotic and iron-loaded hepatocytes. Adjacent to the large Kupffer cell surrounded by hepatocytes (H) is a fat-storing cell (F) with numerous lipid droplets and bundles of collagen located between the 2 cell types (arrow). (Original magnification ×5,200.)

overload. Ultrastructurally, we also found Kupffer cell iron deposits and collagen bundles adjacent to fat-storing cells displaying signs of transition, in livers with marked iron overload. ICAM-1 was neither expressed on hepatocytes in early stages of the disease, when sinusoidal iron deposits were scarce, nor after iron depletion with phlebotomies, nor in healthy controls. The ICAM-1 expression in iron-overloaded livers was sparse, as compared with that seen in livers from patients having chronic liver diseases with an inflammatory component. In GH, only occasional hepatocytes in iron-loaded areas expressed ICAM-1, whereas in CAH, ALD, PSC, or PBC clusters of ICAM-l-positive cells, in some cases more than 25% of all hepatocytes seen in the biopsy were positive and located in proximity to inflammatory cells. In these chronic liver diseases, intense inflammatory cell infiltrates with subsequent destruction of parenchymal cells are commonly seen, s'9 and release of soluble inflammatory mediators from invading lymphocytes has been implicated to trigger the onset of fibrogenesis. In contrast, inflammation has never been emphasized as a major histopathological feature in the classical description of liver histopathology in GH. 1'2 However, investigators have recently focused attention on the presence of scarce or moderate inflammatory cell infiltrates in GH. 2~ In our study, three of the untreated GH patients displayed a moderate portal infiltration of CD3-positive lymphocytes that could be classified as a chronic persistent hepatitis. However, no patient had signs of CAH such as piecemeal necrosis or lobular infiltrates. Chronic viral hepatitis was excluded in all patients. Thus, the scarce or moderate portal mononuclear infiltration found in a fraction of GH patients contrasts to the intense inflammatory reaction seen in other chronic liver dis-

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from damaged, iron-overloaded parenchymal cells possibly could enhance proliferation of collagen-producing cells. The finding that aldehydic products from lipid peroxidation may enhance collagen messenger RNA levels in cultured fibroblasts is in line with this hypothesis. 35 However, m a n y authors emphasize the activated Kupffer cell as being the major determinant in fat-storing cell transformation. 14~6'3s In liver biopsies from GH patients, Kupffer cell iron deposits are seen adjacent to areas with massive hepatocyte iron loading and sideronecrosisy and ultrastructurally, they appear to originate from phagocytosis of sideronecrotic hepatoc y t e s 9 Kupffer cell iron overload may be a strong inducer of fibrosis in animal models. ~ In culture, Kupffer cells secrete factors that may activate fat-storing

FIG. 3. A fat-storing cell (F) adjacent to hepatocytes (H) from patient no. 14, with fibrosis. The cell displays smaller lipid vacuoles and adjacent bundles of collagen fibrils (arrow). (Original magnification ×10,800.)

eases, and it has been interpreted as a phenomenon related to sideronecrosis. 2s Therefore, other factors t h a n cytokine-producing lymphocytes have been suggested to trigger the fibrogenic process in GH. Experimental evidence indicates that initiation of the fibrotic process in iron overload has the same lowest common denominator as in other chronic liver diseases, namely, transformation of fat-storing cells to transitional cells, and further to myofibroblasts producing collagen and proteoglycans. 1°'11'27'2s However, the mechanisms by which iron transforms fat-storing cells and induces fibrogenesis still remain poorly understood, although several studies are being carried out. 29'3° Different factors responsible for activation of fat-storing cells have been suggested. '2-14'3°'31 Hypothetically, iron or aldehydic products from ironinduced lipid peroxidation (such as malondialdehyde) released from degenerated or sideronecrotic ironloaded hepatocytes could have a direct-acting effect on the fat-storing cell. 32 We found electron-dense granules with the appearance of ferritin particles in fat-storing cells, although never as abundant as in hepatocytes or Kupffer cells. Others have demonstrated ferritin receptors at the cell surface of fat-storing cells. ~2 Thus, a direct-acting effect of iron or ferritin on fat-storing cells cannot be ruled out, although results are conflicting as to whether collagen gene expression is increased in experimental iron overload without sideronecrosis or fibrosis. 33'34 Furthermore, hepatocytes contain a cytosolic factor with mitostimulatory effects on cultured fat-storing cells.13 Consequently, release of this protein

FIG. 4. A fat-storing cell (F) with lipid vacuoles (V) from patient no. 15. The fat-storing cell is located between a sinusoid (S) and a hepatocyte (H) containing abundant electron-dense granules with the appearance of ferritin particles (double arrow). Electron-dense granules are also encountered in the cytoplasm of the fat-storing cell (arrow), but are less numerous. (Original magnification )<68,450.)

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cells. 14 We found signs of Kupffer cell activation in liv- terial. However, ICAM-1 expression was never found ers with heavy or massive iron overload. Ultrastruc- in livers from iron-depleted GH patients, healthy contural signs of activation included Kupffer cell hypertro- trols, or early diagnosed cases of GH without sinusoidal phy and phagocytosis of iron-rich cellular remnants in iron loading, but only seen in livers with marked parenmainly periportal areas. We also had ultrastructural chymal iron loading and Kupffer cell iron deposits. Thus, we may propose the following series of events. indications of the presence of transitional cells with lipid droplets and bundles of fibrils adjacent to iron- Iron overload leads to enhanced lipid peroxidation, overloaded Kupffer cells; similar changes of fat-storing caused by an increased oxidative stress through the production of toxic free oxyradicals.3 Peroxidation of cells have been described in other liver diseases. 11 The expression of ICAM-1 on hepatocytes in livers membrane lipids 4 causes a variety of organelle funcwith Kupffer cell iron overload indicates cytokine re- tional abnormalities, 5'6 and eventually impairment of lease in these livers. In inflammatory liver diseases, cellular function results in sideronecrosis. The ironconcomitant expression of ICAM-1 and HLA-DR is usu- rich cellular remnants are phagocytosed by Kupffer ally seen in areas of lymphocytic infiltration, sug- cells leading to hypertrophy and activation. Finally, gesting that a common regulation existsY '3s The lack activated Kupffer cells release cytokines, possibly of of HLA-DR coexpression on iron-loaded hepatocytes, as pathogenic importance for the transformation of fatwell as the low number of lymphocytes in GH, leads to storing cells and initiation of the fibrogenic process our proposal that the induced ICAM-1 expression is a in GH. secondary sign of cytokine release from adjacent actiAcknowledgment: We thank Kristina Eckes for vated Kupffer cells. Taken together, our ultrastructural findings and hepatocyte ICAM-1 expression skillful technical assistance, Anne Svensson for immustrongly suggest a role of the iron-loaded, activated nohistochemical preparation, and Ingrid Jusinski for Kupffer cell in releasing factors of importance for the preparation of electron microscopical specimens. transformation of fat-storing cells to collagen-producREFERENCES ing myofibroblasts. Our results indicate that cytokine release in GH livers is of a low degree compared with 1. Powell LW, Kerr JFR. The pathology of the liver in hemochromathat found in other chronic liver diseases. Clinically, tosis. In: Joachim H, ed. Pathobiology annual. New York: Applethe fibrotic process has a slower course in GH than in ton Century Crofts, 1975:317-337. 2. MacSween, Jackson J. Haemochromatosis: a clinico-pathological inflammatory liver diseases, which is consistent with review of 37 cases. Scot Med J 1966; 11:395-400. this hypothesis. 3. Bacon BR, Britton RS. The pathology of hepatic iron overload: We only investigated patients with iron overload due a free radical mediated process? HEPATOLOGY1990; 11:127-137. to genetic hemochromatosis, and not patients with iron 4. Bacon BR, Tavill AS, Brittenham GM, Park CH, Recknagel RO. Hepatic lipid peroxidation in vivo in rats with chronic iron overoverload due to repeated transfusions. In secondary load. J Clin Invest 1983;71:429-439. hemochromatosis, iron deposition of reticuloendothe5. Myers BM, Prendergast FG, Holman R, Kuntz SM, LaRusso NF. lial cells probably precedes that of hepatocytes. Thus, Alterations in the structure, physicochemical properties, and pH it is unknown if Kupffer cell iron overload due to phagoof hepatocyte lysosomes in experimental iron overload. J Clin cytosis of transfused erythrocytes also will lead to actiInvest 1991;88:1207-1215. 6. Bacon BR, Park CH, Brittenham GM, O'Neill R, Tavill AS. Hevation and cytokine release. Furthermore, hepatocyte patic mitochondrial oxidative metabolism in rats with chronic iron overload with sideronecrosis may be a feature of dietary iron overload. HEPATOLOGY1985;5:789-797. advanced secondary, as well as primary hemochroma7. Bassett ML, Halliday JW, Powell LW. Value of hepatic iron meatosis. 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