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Expression of cell adhesion molecules and their β1 and β2 integrin ligands in human liver peliosis
ARTICLE IN PRESS
Pathology – Research and Practice 201 (2005) 503–511 www.elsevier.de/prp
ORIGINAL ARTICLE
Expression of cell adhesion molecules and their b1 and b2 integrin ligands in human liver peliosis Maya Vladova Gulubova Department of General and Clinical Pathology, Medical Faculty, Thracian University, Stara Zagora, BG-6000, Bulgaria Received 13 August 2004; accepted 11 May 2005
Abstract The expression of the following cell adhesion molecules and their b1 and b2 integrin ligands was investigated in the liver tissue from 3 patients with non-bacillar peliosis using light and electron microscope immunohistochemistry: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), leukocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4). We found a parallel enhancement of the adhesion molecules expression in the dilated sinusoids and cavities in all 3 cases with peliosis. Mononuclear blood cells were detected in the sinusoids and sometimes perisinusoidally. These cells were mainly ICAM-1-, LFA-1-, and VLA-4-positive. At the ultrastructural level, ICAM-1-positive immune deposits were observed on the membrane of sinusoidal endothelial cells, Kupffer cells, and hepatocytes. The expression of cell adhesion molecules on liver sinusoids in peliosis is probably triggered by factors released from damaged endothelial cells and hepatocytes. The prevalence of the ICAM-1/LFA-1 and VCAM-1/VLA-4 patterns of mononuclear blood cell/sinusoidal cell interactions could support the macrophageinduced or lymphocyte-induced type of liver injury. PECAM-1 was also included in the non-specific immune response in peliosis. The presence of erythrostasis or thrombosis in liver sinusoids could participate in the induction of adhesion molecule expression in peliosis. r 2005 Elsevier GmbH. All rights reserved. Keywords: Adhesion molecules; Integrins; Peliosis; Liver; Sinusoidal endothelial cells
Introduction Peliosis hepatis is a condition affecting the human liver. Histologically, it is characterized by the presence of blood-filled cavities of variable size. It is found in association with sinusoidal dilation, and the distinction between the two conditions is not always sharp [8,36]. In humans, peliosis hepatis has been associated with the presence of malignant tumors [26], tuberculosis [36], anabolic steroid administration [11,29], selenium diet in animals [2], etc. Its pathogenesis remained unclear E-mail address: [email protected]. 0344-0338/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2005.05.006
although it may be triggered by more than 1 mechanism [8,36]. Theories regarding its origin include obstruction of the sinusoidal blood outflow at the junction of sinusoids and the centrilobular veins, liver cell necrosis [36], and endothelial cell damage [34]. Liver sinusoidal endothelial cell proliferation occurred in mice with melanoma, followed by peliosis [8]. Cell adhesion molecules are critical for inflammatory cells recruitment in liver inflammation and neoplasia [13,14,18]. Intercellular adhesion molecule-1 (ICAM-1) is expressed weakly, but constitutively, on normal liver sinusoidal endothelial cells [18]. Vascular cell adhesion molecule-1 (VCAM-1) and E-selectin are expressed by
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liver sinusoidal endothelial cells only after stimulation [33]. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is permanently found on large vessel endothelial cells [18] and in liver portal vessels [4]. Thrombosis is associated with inflammatory response in the vessel wall, and adhesion molecules also play a role in this process [32]. Erythrostasis and sometimes thrombosis were noted in the cavities in peliosis. In a previous study [15], we reported on an increased extracellular matrix occurrence in liver sinusoids in peliosis and on activation of hepatic stellate cells and Kupffer cells. Activated stellate cells also express ICAM-1 and VCAM-1 [20] and modulate the inflammatory cell transmigration through the sinusoidal wall [22]. A significant difference between inflammatory cells recruitment in the liver and most other organs lies in the fact that these events take place in liver sinusoids (capillaries) and not in postcapillary venules in other organs [18]. In the present study, we describe the adhesion molecule profile of liver sinusoids in human liver peliosis. We investigated immunohistochemically the expression of ICAM-1 (CD54); VCAM-1 (CD106), Eselectin (CD62E), PECAM-1 (CD31), and their ligands leukocyte function-associated antigen-1 (LFA-1 (a1); CD11a), macrophage antigen-1 (Mac-1 (aM; CD11b), and very late antigen 4 (VLA-a4; CD49d). We discuss the increased immunoreactivity of ICAM-1 on liver sinusoids and the de novo appearance of VCAM-1 and E-selectin in dilated sinusoids and cavities.
Table 1.
Sex
Materials and methods Tissue specimens We examined 3 patients (2 men and 1 woman between 40 and 43 years of age) for whom the diagnosis of peliosis hepatis had been rendered on sections of paraffin-embedded liver tissue. These patients had previously been examined for extracellular matrix disturbances in peliosis [15]. The main clinical and laboratory data are given in Table 1. Peliosis was attributed to tuberculosis in patient 1. Patient 2 had adrenal pheochromocytoma, detected 1 year after peliosis had been diagnosed. Patient 3 was treated with anabolic androgenic steroid hormones for endometriosis. Autopsy revealed asymptomatic peliosis. Ultrasonography and computed tomography showed enlarged livers without any specific lesions. Wedge-like liver surgical biopsies were obtained from patients with peliosis for diagnostic purposes. In addition, 3 patients (1 man and 2 women) between 39 and 61 years of age were used as controls. They underwent explorative laparotomy, and liver calcification (2 patients) was an occasional finding during operation. The controls had a histologically normal liver. None of the patients had a history of previous liver diseases, i.e., viral hepatitis, diabetes mellitus, hemochromatosis, hemotransfusions, or alcohol abuse (o20 g/per day). Informed consent was obtained from each patient.
Clinical, laboratory, and histological data from 3 patients with peliosis hepatis and of control patients Patient 1, tuberculosis
Patient 2, pheochromocytoma
Control 1 Control 2 Control 3 Patient 3, Endometriosis/anabol steroids
Male
Male
Female
Male
Female
Female
43 No No
40 No No
40 No No
48 No No
61 No No
12.2
12.6
11
12
11.8
15/17
26/34
8/10
9/12
8/11
Normal S Ly, Mo Absent Absent Absent Absent
Normal S Ly, Mo Absent Absent Absent Absent
Normal S Ly, Mo Absent Absent Absent Absent
Absent Absent
Absent Absent
Absent Absent
Age (years) 40 Jaundice No Portal hypertension No Total serum Bilirubin (mmol/l) 15.2 GOT/GPT (U/L) 21/26 Histology PT II Moderate IC in PT Ly, Mo, Neu IC in sinusids Weak Types of IC in sinusoids Ly, Mo, S Neu Erythrostasis in sinusoids Present IC in Disse space Present IC attached to the wall of cavities Many Ly, Mo, Neu Ery aggreg in cavities Present Hydropic deg in h Absent Parenchym deg. in h Present
Moderate Weak Ly, Mo, Neu Ly, Mo, Neu Moderate Moderate Ly, Mo, S Pl Ly, Mo, Neu Present Present Present Present Moderate Ly, Mo, Neu Many Ly, Mo, Neu Present Present Absent Present Present Present
II – inflammatory infiltrate; PT – portal tract; IC – inflammatory cells; S – single; Ly – lymphocytes; Mo – monocytes; Neu – neutrophils; Pl – plasmocytes; Ery – erythrocytes; Aggreg – aggregation; Deg – degeneration; h – hepatocytes.
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Surgical biopsies of approximately 16 15 8 mm were taken from the lower border of the liver. Each of the biopsies was divided in 3 pieces and was processed as follows.
Routine histology Tissue samples (12 6 8 mm) were fixed in 10% neutral buffered formalin for routine histology. Paraffin sections were stained with hemalaun and eosin and Van Gieson staining. All 3 cases were investigated histologically.
Light and electron microscopical immunohistochemistry The floating section immunohistochemistry methodology was described previously by Gulubova and Vlaykova [17]. The two procedures were carried out simultaneously and according to the method of De Vos et al. [7]. Briefly, cryostat sections (5 mm thick for light microscope immunohistochemistry and 40–60 mm thick for electron microscope immunohistochemistry) were thawed in 0.1 M PB, pH 7.3, overnight. They were then incubated in 1.2% hydrogen peroxide in methanol for 30 min and rinsed in 0.1 M PB, pH 7.4, for 15 min. The sections were then blocked for 30 min with normal mouse serum (DAKO). After incubation with the primary mouse anti-human antibodies overnight, the cryostat sections were washed in 0.1 M PB, pH 7.4, incubated with a secondary anti-mouse biotinylated antibody (DAKO) for 4 h, and subsequently incubated with the streptavidin–HRP complex (DAKO) for 4 h. They were rinsed in 0.1 M PB, pH 7.4, and then in 0.05 M Tris-HCL buffer, pH 7.5, for 10 min. The reaction was made visible using a mixture of 3 mg 3,30 diaminobenzidine (DAB) (DAKO) in 15 ml 0.05 TrisHCL buffer, pH 7.5, and 36 ml 1% hydrogene peroxide for 10–20 min, rinsed in 0.1 M PB, pH 7.4. The cryostat sections (5 mm thick) were mounted on slides, dried overnight at room temperature, thawed in 951 and 1001 ethanol and xylene, and then mounted with entelan for light microscopy. The sections were not counterstained for better visualization of the DAB reaction product. The cryostat sections (40–60 mm thick) were postfixed in PB containing 2% osmium tetroxide for 30 min at 2 1C, followed by a rinse in 0.1 M PB, pH 7.4. Finally, the sections were dehydrated in graded concentrations of ethanol and propylene oxide and flat-embedded with Durcupan between celophane sheets. Ultrathin sections were cut from liver areas with dilated sinusoids. They were counterstained only with uranyl acetate and examined and photographed with an OPTON EM 109
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electron microscope at 50 kV. All procedures were started immediately after having taken the biopsies. Sections incubated with non-immune sera instead of the primary antibodies were used as negative controls. The optimal dilutions for the reagents were assessed in a series of stainings performed prior to this study: antiICAM-1 1:50, anti-VCAM-1 1:10, anti-E-selectin 1:100, anti-LFA-1 1:20, anti-Mac-1 1:10, and anti-VLA-4 1:10.
Immunochemicals The antibodies used were mouse anti-human ICAM-1 (CD54) (MCA 1135), mouse anti-human VCAM-1 (CD106) (MCA 981), mouse anti-human E-selectin (CD62E) (MCA 883), mouse anti-human Mac-1 (CD11b) (MCA 551), mouse anti-human LFA-1 (CD11a) (MCA 1149), and mouse anti-human VLA-a4 (CD49d) (MCA 699), all obtained from Serotec, Oxford, UK. The mouse anti-human PECAM-1 (CD31) (AM241-5M) was obtained from BioGenex Laboratories, San Ramon, CA, USA. The detection system used was DAKO LSABs2 System, HRP (K0675), and DAKOsDAB Chromogen tablets (S3000) (DAKO A/S Denmark).
Results Histology In all 3 cases with peliosis, we observed accumulation of inflammatory cells consisting of lymphocytes, monocytes, and single neutrophils in the dilated sinusoids (Fig. 1a). Lymphocytes were noted in the perisinusoidal space attached to degenerative hepatocytes. In some cases, there was erythrocyte stasis in the sinusoids. In the cavities, mononuclear cells and erythrocytes aggregated to their wall (Fig. 1b). The results of the histological examination are given in Table 1.
Light microsopic immunohistochemistry Normally, the sinusoidal endothelial cells expressed ICAM-1 at a low level (Fig. 2a). In peliosis, sinusoidal endothelia of dilated sinusoids and cavities were ICAM1-positive (Fig. 2b), and so were the intrasinusoidal lymphocytes. VCAM-1 (Fig. 2c) and E-selectin (Fig. 2e) are usually not expressed by sinusoidal endothelia. In peliosis, expression of VCAM-1 (Fig. 2d) and E-selectin (Fig. 2f) was induced on sinusoidal endothelia in the dilated sinusoids and in cavities. PECAM-1 was found to be expressed in the normal liver on the endothelium of the portal vessels (Fig. 2g). By contrast, in peliosis, sinusoidal endothelia of the dilated sinusoids and cavities were PECAM-1-positive (Fig. 2h). Some of the
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Discussion
Fig. 1. (a) Part of a cavity (c) without endothelial cell lining and surrounded by sinusoids with mononuclear cells (lymphocytes and monocytes) (white circles). Patient 3. (b) Erythrostasis in the cavities. Mononuclear cells are attached to their wall. Patient 1. Magnifications: (a) 180, (b) 200.
cells in the sinusoids of peliotic liver were Mac-1positive (Fig. 3a). Most of the blood mononuclear cells in the sinusoids and portal tracts were LFA-1 (Fig. 3b, c)- and VLA-4 (Fig. 3d)-positive.
Electron microscopic immunohistochemistry The cavities were lined by endothelial cells with greatly enlarged fenestrae. Sinusoids were dilated and filled with many erythrocytes and cell debris (Fig. 4a). Intense ICAM-1 immune deposits were noted on the lateral hepatocyte membranes (Fig. 4b, c), on hepatocyte microvilli (Fig. 4c), and on sinusoidal endothelial cells (Fig. 4d). Some of the hepatocytes showed degenerative changes (Fig. 4b). Kupffer cells had many digitations (a sign of activation), which were also ICAM-1-positive (Fig. 4e). Sinusoidal endothelia in the dilated sinusoids were VCAM-1 (Fig. 4f)-, Eselectin-, and VLA-4-positive.
This report describes the upregulation of ICAM-1 and the de novo expression of endothelial VCAM-1, E-selectin, and PECAM-1 in the dilated sinusoids and cavities in human liver non-bacillar peliosis. In a previous study [15], we found that the sinusoids and cavities in peliosis were transformed into capillary vessels with collagen type IV and laminin-positive basement membrane, strengthened with collagen type III fibers. Hepatic stellate cells turned into transitional cells or myofibroblasts reminiscent of capillary pericytes. Sinusoidal endothelial cells showed signs of degeneration and enlarged fenestrae. A feature of sinusoidal endothelial cell transformation into endothelial cells of capillary vessels is their expression of PECAM-1 [5]. Therefore, in peliosis, the resemblance of the dilated sinusoids and cavities to capillary vessels could explain the adhesion molecule profile expressed on sinusoidal endothelial cells in that disease. In our cases, non-bacillar peliosis hepatis was associated with the presence of a malignant tumor, tuberculosis, and anabolic steroid administration. We observed hepatocyte and sinusoidal endothelial cell degeneration in all 3 cases. Endothelial cell deterioration such as presence of Ki-67-positive proliferating endothelial cells and intense lectin expression were detected in mouse livers with peliosis [8]. We also noted many lymphocytes, monocytes, and single neutrophils in the dilated sinusoids. Some of these cells were located in the perisinusoidal space, in close contact with other cells and with degenerative hepatocytes. In a previous study, we described sinusoidal dilation and lympho/monocyte aggregations in sinusoids in the livers of patients with malignant gastrointestinal tumors with or without liver metastases [16]. Probably, some tumor-derived factors such as vascular endothelial growth factor (VEGF) [34] and nitric oxide [33] are responsible for sinusoidal dilation through an effect on endothelial cell proliferation or act as a cytotoxic defence mechanism against metastatic spread. Therefore, one might conclude that the presence of inflammatory cells in the dilated sinusoids and cavities could be initiated by degenerative hepatocytes and sinusoidal endothelial cells that induce the release of interleukin-1 (IL-1), IL-12 and tumor necrosis factor a (TNF-a) by Kupffer cells [6,12]. These cytokines also induce the expression of ICAM-1, VCAM-1, and E-selectin on liver sinusoidal endothelial cells [23]. The degeneration of hepatocyte and endothelial cells noted in our cases could be initiated by drug administration or by some tumor-derived stimuli. We agree with Eising et al. [9], who suggest that peliosis hepatis is a non-specific immunologic response to a variety of noxious agents damaging liver parenchymal or non-parenchymal cells. Therefore, the expression of cell adhesion molecules is concomitant in all these changes.
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Fig. 2. (a) Low ICAM-1 positivity in the sinusoids (white arrow head) of a control liver. (b) ICAM-1-positivity in the cavities. Patient 3. (c) VCAM-1-positive endothelia in the portal vessels (white arrow head) in a control patient. (d) VCAM-1-positive sinusoidal cells in the dilated sinusoids and cavities. Patient 2. (e) E-selectin-positive structures in portal vessels (white arrow head) of a control patient. (f) E-selectin-positive endothelial cells in the dilated sinusoids. Patient 2. (g) PECAM-1-positive endothelial cells (white arrow head) in the portal blood vessels in a control liver. (h) PECAM-1-positive endothelial cells in the dilated sinusoids and cavities in peliosis. Patient 2. Magnifications: (a) 200, (b) 200, (c) 200, (d) 125, (e) 200, (f) 125, (g) 100, (h) 125.
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Fig. 3. (a) Mac-1-positive cells in liver sinusoids in peliosis. Patient 3. (b) Many LFA-1-positive cells in a portal tract and around cavities. Patient 3. (c) Many LFA-1-positive cells in the liver in peliosis. Patient 1. (d) VLA-4-positive cells (white circle) in the sinusoids in peliosis. VLA-4-positive sinusoidal cells (white arrow head). Patient 2. Magnifications: (a) 200, (b) 200, (c) 125, (d) 125.
The lympho/monocytic cells in liver sinusoids and cavities were mainly LFA-1- and VLA-4-positive. Mac1-positive cells were fewer in number. A similar adhesion molecule profile of the inflammatory infiltrate was described in human chronic liver diseases [1,18,31,35] and in mouse models of liver injury [20,21]. For example, a great number of LFA-1-positive lymphocytes were observed in acute and chronic active hepatitis and cirrhosis [31], as well as in CCL4-induced liver injury [24]. Therefore, the adhesion molecule expression in dilated sinusoids and cavities supported the macrophage- or lymphocyte-induced pattern of liver injury, with a prevalence of the ICAM-1/LFA-1 and VCAM-1/VLA-4 patterns of inflammatory cell recruitment [18,30]. PECAM-1 is generally accepted to be constitutively and uniformly expressed on all types of endothelial cells and cannot be induced by cytokines [28]. PECAM-1 has been implicated in a number of important biologic processes, including leukocyte emigration at sites of inflammation [19], angiogenesis [3], and platelet aggre-
gation to injured endothelial cells [27]. Some authors suggest that it is not expressed on liver sinusoidal endothelial cells under normal conditions and after induction [4]. Others have demonstrated a constitutive expression of PECAM-1 on liver sinusoidal endothelial cells in normal and damaged rat and human livers [25]. It was reported recently that PECAM-1 contained a signaling sequence known as the immunoreceptor tyrosine-based inhibitory motif (ITIM), a motif originally described in receptors regulating immunity [3]. We think that PECAM-1 expression in the cavities in peliosis was directed towards maintenance of sinusoidal integrity, since altered sinusoidal endothelial cells and sinusoidal denudation were the main pathological problem in peliosis hepatis. PECAM-1 could also participate in the non-specific immune response in peliosis, inhibiting leukocyte and monocyte transmigration through the sinusoidal wall, and maintaining platelet aggregation in cavities. Our ultrastructural immunohistochemical finding of ICAM-1, and VCAM-1-positive sinusoidal endothelial
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Fig. 4. (a) Dilated sinusoid (cavity – C) filled with cell debris and erythrocytes (E). The endothelial lining covers enlarged Disse space. H – hepatocyte. Patient 1. (b) Intense ICAM-1 immune deposits on the lateral hepatocyte membrane. A hepatocyte with a dark cytoplasm is surrounded by two hepatocytes with bright and swollen cytoplasm. Patient 2. (c) ICAM-1 immune deposits on hepatocyte microvilli and on the lateral hepatocyte membrane. s – sinusoid. Patient 2. (d) Intense ICAM-1 immunoreactivity on the sinusoidal endothelial cell (E) membrane. s – sinusoid. Patient 1. (e) ICAM-1 immune deposits on the membrane of a Kupffer cell (K). s – sinusoid. Patient 1. (f) VCAM-1 immunoreactivity on the membrane of a sinusoidal endothelial cell. s – sinusoid. Patient 2. Magnifications: (a) 7000, (b) 12,000, (c) 12,000, (d) 12,000, (e) 7000, (f) 12,000.
cells in the dilated sinusoids confirmed the light microscopic finding. Degenerative hepatocytes with ICAM-1-positive hepatocyte membranes were well visible on electron microscopy. Therefore, degenerative hepatocytes, activated Kupffer cells and HSCs, already described by us [15], and the presence of
lymphocytes and monocytes in the dilated sinusoids and cavities, found in this report, could contribute to the altered cytokine and chemokine milieu in liver peliosis. This milieu could regulate the expression of the adhesion molecules on liver sinusoidal endothelial cells.
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We noted that erythrocyte stasis and mononuclear cells were attached to the wall of cavities. Somewhere, the endothelial lining was invisible as described by Zafrani et al. in 1984 [36]. Therefore, endothelial denudation exposed the subendothelial collagen to blood platelets and initiated their aggregation [30]. Expression of adhesion molecules on the endothelium of cavities and dilated sinusoids could also be initiated by the mediators, released from aggregated platelets, erythrocytes, and mononuclears [10]. In summary, we suggest a theory of the initiation of adhesion molecules expression in peliosis. The first step in this process could be the degeneration of some hepatocytes and of sinusoidal endothelial cells, triggered by toxic (steroids, tuberculostatics) or tumor-derived stimuli. The second step is the resultant sinusoidal dilation and cavity formation. This is accompanied by induction of the adhesion molecules expression in sinusoids, supported by cytokines and chemokines released in the first step. Simultaneously, there was recruitment of mononuclear cells in the sinusoids and sometimes in the space of Disse. Third, because of the impaired endothelial cell function, erythrostasis, platelet aggregation and activation, and thrombosis appeared somewhere in the cavities. Thrombogenesis also supports the process of activation of adhesion molecule expression. In conclusion, the data presented here support the hypothesis that the expression of liver sinusoidal adhesion molecules is susceptible to regulation by different stimuli, reflecting the different causes of human liver peliosis.
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Pathology – Research and Practice 201 (2005) 503–511 www.elsevier.de/prp
ORIGINAL ARTICLE
Expression of cell adhesion molecules and their b1 and b2 integrin ligands in human liver peliosis Maya Vladova Gulubova Department of General and Clinical Pathology, Medical Faculty, Thracian University, Stara Zagora, BG-6000, Bulgaria Received 13 August 2004; accepted 11 May 2005
Abstract The expression of the following cell adhesion molecules and their b1 and b2 integrin ligands was investigated in the liver tissue from 3 patients with non-bacillar peliosis using light and electron microscope immunohistochemistry: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), leukocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4). We found a parallel enhancement of the adhesion molecules expression in the dilated sinusoids and cavities in all 3 cases with peliosis. Mononuclear blood cells were detected in the sinusoids and sometimes perisinusoidally. These cells were mainly ICAM-1-, LFA-1-, and VLA-4-positive. At the ultrastructural level, ICAM-1-positive immune deposits were observed on the membrane of sinusoidal endothelial cells, Kupffer cells, and hepatocytes. The expression of cell adhesion molecules on liver sinusoids in peliosis is probably triggered by factors released from damaged endothelial cells and hepatocytes. The prevalence of the ICAM-1/LFA-1 and VCAM-1/VLA-4 patterns of mononuclear blood cell/sinusoidal cell interactions could support the macrophageinduced or lymphocyte-induced type of liver injury. PECAM-1 was also included in the non-specific immune response in peliosis. The presence of erythrostasis or thrombosis in liver sinusoids could participate in the induction of adhesion molecule expression in peliosis. r 2005 Elsevier GmbH. All rights reserved. Keywords: Adhesion molecules; Integrins; Peliosis; Liver; Sinusoidal endothelial cells
Introduction Peliosis hepatis is a condition affecting the human liver. Histologically, it is characterized by the presence of blood-filled cavities of variable size. It is found in association with sinusoidal dilation, and the distinction between the two conditions is not always sharp [8,36]. In humans, peliosis hepatis has been associated with the presence of malignant tumors [26], tuberculosis [36], anabolic steroid administration [11,29], selenium diet in animals [2], etc. Its pathogenesis remained unclear E-mail address: [email protected]. 0344-0338/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2005.05.006
although it may be triggered by more than 1 mechanism [8,36]. Theories regarding its origin include obstruction of the sinusoidal blood outflow at the junction of sinusoids and the centrilobular veins, liver cell necrosis [36], and endothelial cell damage [34]. Liver sinusoidal endothelial cell proliferation occurred in mice with melanoma, followed by peliosis [8]. Cell adhesion molecules are critical for inflammatory cells recruitment in liver inflammation and neoplasia [13,14,18]. Intercellular adhesion molecule-1 (ICAM-1) is expressed weakly, but constitutively, on normal liver sinusoidal endothelial cells [18]. Vascular cell adhesion molecule-1 (VCAM-1) and E-selectin are expressed by
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liver sinusoidal endothelial cells only after stimulation [33]. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is permanently found on large vessel endothelial cells [18] and in liver portal vessels [4]. Thrombosis is associated with inflammatory response in the vessel wall, and adhesion molecules also play a role in this process [32]. Erythrostasis and sometimes thrombosis were noted in the cavities in peliosis. In a previous study [15], we reported on an increased extracellular matrix occurrence in liver sinusoids in peliosis and on activation of hepatic stellate cells and Kupffer cells. Activated stellate cells also express ICAM-1 and VCAM-1 [20] and modulate the inflammatory cell transmigration through the sinusoidal wall [22]. A significant difference between inflammatory cells recruitment in the liver and most other organs lies in the fact that these events take place in liver sinusoids (capillaries) and not in postcapillary venules in other organs [18]. In the present study, we describe the adhesion molecule profile of liver sinusoids in human liver peliosis. We investigated immunohistochemically the expression of ICAM-1 (CD54); VCAM-1 (CD106), Eselectin (CD62E), PECAM-1 (CD31), and their ligands leukocyte function-associated antigen-1 (LFA-1 (a1); CD11a), macrophage antigen-1 (Mac-1 (aM; CD11b), and very late antigen 4 (VLA-a4; CD49d). We discuss the increased immunoreactivity of ICAM-1 on liver sinusoids and the de novo appearance of VCAM-1 and E-selectin in dilated sinusoids and cavities.
Table 1.
Sex
Materials and methods Tissue specimens We examined 3 patients (2 men and 1 woman between 40 and 43 years of age) for whom the diagnosis of peliosis hepatis had been rendered on sections of paraffin-embedded liver tissue. These patients had previously been examined for extracellular matrix disturbances in peliosis [15]. The main clinical and laboratory data are given in Table 1. Peliosis was attributed to tuberculosis in patient 1. Patient 2 had adrenal pheochromocytoma, detected 1 year after peliosis had been diagnosed. Patient 3 was treated with anabolic androgenic steroid hormones for endometriosis. Autopsy revealed asymptomatic peliosis. Ultrasonography and computed tomography showed enlarged livers without any specific lesions. Wedge-like liver surgical biopsies were obtained from patients with peliosis for diagnostic purposes. In addition, 3 patients (1 man and 2 women) between 39 and 61 years of age were used as controls. They underwent explorative laparotomy, and liver calcification (2 patients) was an occasional finding during operation. The controls had a histologically normal liver. None of the patients had a history of previous liver diseases, i.e., viral hepatitis, diabetes mellitus, hemochromatosis, hemotransfusions, or alcohol abuse (o20 g/per day). Informed consent was obtained from each patient.
Clinical, laboratory, and histological data from 3 patients with peliosis hepatis and of control patients Patient 1, tuberculosis
Patient 2, pheochromocytoma
Control 1 Control 2 Control 3 Patient 3, Endometriosis/anabol steroids
Male
Male
Female
Male
Female
Female
43 No No
40 No No
40 No No
48 No No
61 No No
12.2
12.6
11
12
11.8
15/17
26/34
8/10
9/12
8/11
Normal S Ly, Mo Absent Absent Absent Absent
Normal S Ly, Mo Absent Absent Absent Absent
Normal S Ly, Mo Absent Absent Absent Absent
Absent Absent
Absent Absent
Absent Absent
Age (years) 40 Jaundice No Portal hypertension No Total serum Bilirubin (mmol/l) 15.2 GOT/GPT (U/L) 21/26 Histology PT II Moderate IC in PT Ly, Mo, Neu IC in sinusids Weak Types of IC in sinusoids Ly, Mo, S Neu Erythrostasis in sinusoids Present IC in Disse space Present IC attached to the wall of cavities Many Ly, Mo, Neu Ery aggreg in cavities Present Hydropic deg in h Absent Parenchym deg. in h Present
Moderate Weak Ly, Mo, Neu Ly, Mo, Neu Moderate Moderate Ly, Mo, S Pl Ly, Mo, Neu Present Present Present Present Moderate Ly, Mo, Neu Many Ly, Mo, Neu Present Present Absent Present Present Present
II – inflammatory infiltrate; PT – portal tract; IC – inflammatory cells; S – single; Ly – lymphocytes; Mo – monocytes; Neu – neutrophils; Pl – plasmocytes; Ery – erythrocytes; Aggreg – aggregation; Deg – degeneration; h – hepatocytes.
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Surgical biopsies of approximately 16 15 8 mm were taken from the lower border of the liver. Each of the biopsies was divided in 3 pieces and was processed as follows.
Routine histology Tissue samples (12 6 8 mm) were fixed in 10% neutral buffered formalin for routine histology. Paraffin sections were stained with hemalaun and eosin and Van Gieson staining. All 3 cases were investigated histologically.
Light and electron microscopical immunohistochemistry The floating section immunohistochemistry methodology was described previously by Gulubova and Vlaykova [17]. The two procedures were carried out simultaneously and according to the method of De Vos et al. [7]. Briefly, cryostat sections (5 mm thick for light microscope immunohistochemistry and 40–60 mm thick for electron microscope immunohistochemistry) were thawed in 0.1 M PB, pH 7.3, overnight. They were then incubated in 1.2% hydrogen peroxide in methanol for 30 min and rinsed in 0.1 M PB, pH 7.4, for 15 min. The sections were then blocked for 30 min with normal mouse serum (DAKO). After incubation with the primary mouse anti-human antibodies overnight, the cryostat sections were washed in 0.1 M PB, pH 7.4, incubated with a secondary anti-mouse biotinylated antibody (DAKO) for 4 h, and subsequently incubated with the streptavidin–HRP complex (DAKO) for 4 h. They were rinsed in 0.1 M PB, pH 7.4, and then in 0.05 M Tris-HCL buffer, pH 7.5, for 10 min. The reaction was made visible using a mixture of 3 mg 3,30 diaminobenzidine (DAB) (DAKO) in 15 ml 0.05 TrisHCL buffer, pH 7.5, and 36 ml 1% hydrogene peroxide for 10–20 min, rinsed in 0.1 M PB, pH 7.4. The cryostat sections (5 mm thick) were mounted on slides, dried overnight at room temperature, thawed in 951 and 1001 ethanol and xylene, and then mounted with entelan for light microscopy. The sections were not counterstained for better visualization of the DAB reaction product. The cryostat sections (40–60 mm thick) were postfixed in PB containing 2% osmium tetroxide for 30 min at 2 1C, followed by a rinse in 0.1 M PB, pH 7.4. Finally, the sections were dehydrated in graded concentrations of ethanol and propylene oxide and flat-embedded with Durcupan between celophane sheets. Ultrathin sections were cut from liver areas with dilated sinusoids. They were counterstained only with uranyl acetate and examined and photographed with an OPTON EM 109
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electron microscope at 50 kV. All procedures were started immediately after having taken the biopsies. Sections incubated with non-immune sera instead of the primary antibodies were used as negative controls. The optimal dilutions for the reagents were assessed in a series of stainings performed prior to this study: antiICAM-1 1:50, anti-VCAM-1 1:10, anti-E-selectin 1:100, anti-LFA-1 1:20, anti-Mac-1 1:10, and anti-VLA-4 1:10.
Immunochemicals The antibodies used were mouse anti-human ICAM-1 (CD54) (MCA 1135), mouse anti-human VCAM-1 (CD106) (MCA 981), mouse anti-human E-selectin (CD62E) (MCA 883), mouse anti-human Mac-1 (CD11b) (MCA 551), mouse anti-human LFA-1 (CD11a) (MCA 1149), and mouse anti-human VLA-a4 (CD49d) (MCA 699), all obtained from Serotec, Oxford, UK. The mouse anti-human PECAM-1 (CD31) (AM241-5M) was obtained from BioGenex Laboratories, San Ramon, CA, USA. The detection system used was DAKO LSABs2 System, HRP (K0675), and DAKOsDAB Chromogen tablets (S3000) (DAKO A/S Denmark).
Results Histology In all 3 cases with peliosis, we observed accumulation of inflammatory cells consisting of lymphocytes, monocytes, and single neutrophils in the dilated sinusoids (Fig. 1a). Lymphocytes were noted in the perisinusoidal space attached to degenerative hepatocytes. In some cases, there was erythrocyte stasis in the sinusoids. In the cavities, mononuclear cells and erythrocytes aggregated to their wall (Fig. 1b). The results of the histological examination are given in Table 1.
Light microsopic immunohistochemistry Normally, the sinusoidal endothelial cells expressed ICAM-1 at a low level (Fig. 2a). In peliosis, sinusoidal endothelia of dilated sinusoids and cavities were ICAM1-positive (Fig. 2b), and so were the intrasinusoidal lymphocytes. VCAM-1 (Fig. 2c) and E-selectin (Fig. 2e) are usually not expressed by sinusoidal endothelia. In peliosis, expression of VCAM-1 (Fig. 2d) and E-selectin (Fig. 2f) was induced on sinusoidal endothelia in the dilated sinusoids and in cavities. PECAM-1 was found to be expressed in the normal liver on the endothelium of the portal vessels (Fig. 2g). By contrast, in peliosis, sinusoidal endothelia of the dilated sinusoids and cavities were PECAM-1-positive (Fig. 2h). Some of the
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Discussion
Fig. 1. (a) Part of a cavity (c) without endothelial cell lining and surrounded by sinusoids with mononuclear cells (lymphocytes and monocytes) (white circles). Patient 3. (b) Erythrostasis in the cavities. Mononuclear cells are attached to their wall. Patient 1. Magnifications: (a) 180, (b) 200.
cells in the sinusoids of peliotic liver were Mac-1positive (Fig. 3a). Most of the blood mononuclear cells in the sinusoids and portal tracts were LFA-1 (Fig. 3b, c)- and VLA-4 (Fig. 3d)-positive.
Electron microscopic immunohistochemistry The cavities were lined by endothelial cells with greatly enlarged fenestrae. Sinusoids were dilated and filled with many erythrocytes and cell debris (Fig. 4a). Intense ICAM-1 immune deposits were noted on the lateral hepatocyte membranes (Fig. 4b, c), on hepatocyte microvilli (Fig. 4c), and on sinusoidal endothelial cells (Fig. 4d). Some of the hepatocytes showed degenerative changes (Fig. 4b). Kupffer cells had many digitations (a sign of activation), which were also ICAM-1-positive (Fig. 4e). Sinusoidal endothelia in the dilated sinusoids were VCAM-1 (Fig. 4f)-, Eselectin-, and VLA-4-positive.
This report describes the upregulation of ICAM-1 and the de novo expression of endothelial VCAM-1, E-selectin, and PECAM-1 in the dilated sinusoids and cavities in human liver non-bacillar peliosis. In a previous study [15], we found that the sinusoids and cavities in peliosis were transformed into capillary vessels with collagen type IV and laminin-positive basement membrane, strengthened with collagen type III fibers. Hepatic stellate cells turned into transitional cells or myofibroblasts reminiscent of capillary pericytes. Sinusoidal endothelial cells showed signs of degeneration and enlarged fenestrae. A feature of sinusoidal endothelial cell transformation into endothelial cells of capillary vessels is their expression of PECAM-1 [5]. Therefore, in peliosis, the resemblance of the dilated sinusoids and cavities to capillary vessels could explain the adhesion molecule profile expressed on sinusoidal endothelial cells in that disease. In our cases, non-bacillar peliosis hepatis was associated with the presence of a malignant tumor, tuberculosis, and anabolic steroid administration. We observed hepatocyte and sinusoidal endothelial cell degeneration in all 3 cases. Endothelial cell deterioration such as presence of Ki-67-positive proliferating endothelial cells and intense lectin expression were detected in mouse livers with peliosis [8]. We also noted many lymphocytes, monocytes, and single neutrophils in the dilated sinusoids. Some of these cells were located in the perisinusoidal space, in close contact with other cells and with degenerative hepatocytes. In a previous study, we described sinusoidal dilation and lympho/monocyte aggregations in sinusoids in the livers of patients with malignant gastrointestinal tumors with or without liver metastases [16]. Probably, some tumor-derived factors such as vascular endothelial growth factor (VEGF) [34] and nitric oxide [33] are responsible for sinusoidal dilation through an effect on endothelial cell proliferation or act as a cytotoxic defence mechanism against metastatic spread. Therefore, one might conclude that the presence of inflammatory cells in the dilated sinusoids and cavities could be initiated by degenerative hepatocytes and sinusoidal endothelial cells that induce the release of interleukin-1 (IL-1), IL-12 and tumor necrosis factor a (TNF-a) by Kupffer cells [6,12]. These cytokines also induce the expression of ICAM-1, VCAM-1, and E-selectin on liver sinusoidal endothelial cells [23]. The degeneration of hepatocyte and endothelial cells noted in our cases could be initiated by drug administration or by some tumor-derived stimuli. We agree with Eising et al. [9], who suggest that peliosis hepatis is a non-specific immunologic response to a variety of noxious agents damaging liver parenchymal or non-parenchymal cells. Therefore, the expression of cell adhesion molecules is concomitant in all these changes.
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Fig. 2. (a) Low ICAM-1 positivity in the sinusoids (white arrow head) of a control liver. (b) ICAM-1-positivity in the cavities. Patient 3. (c) VCAM-1-positive endothelia in the portal vessels (white arrow head) in a control patient. (d) VCAM-1-positive sinusoidal cells in the dilated sinusoids and cavities. Patient 2. (e) E-selectin-positive structures in portal vessels (white arrow head) of a control patient. (f) E-selectin-positive endothelial cells in the dilated sinusoids. Patient 2. (g) PECAM-1-positive endothelial cells (white arrow head) in the portal blood vessels in a control liver. (h) PECAM-1-positive endothelial cells in the dilated sinusoids and cavities in peliosis. Patient 2. Magnifications: (a) 200, (b) 200, (c) 200, (d) 125, (e) 200, (f) 125, (g) 100, (h) 125.
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Fig. 3. (a) Mac-1-positive cells in liver sinusoids in peliosis. Patient 3. (b) Many LFA-1-positive cells in a portal tract and around cavities. Patient 3. (c) Many LFA-1-positive cells in the liver in peliosis. Patient 1. (d) VLA-4-positive cells (white circle) in the sinusoids in peliosis. VLA-4-positive sinusoidal cells (white arrow head). Patient 2. Magnifications: (a) 200, (b) 200, (c) 125, (d) 125.
The lympho/monocytic cells in liver sinusoids and cavities were mainly LFA-1- and VLA-4-positive. Mac1-positive cells were fewer in number. A similar adhesion molecule profile of the inflammatory infiltrate was described in human chronic liver diseases [1,18,31,35] and in mouse models of liver injury [20,21]. For example, a great number of LFA-1-positive lymphocytes were observed in acute and chronic active hepatitis and cirrhosis [31], as well as in CCL4-induced liver injury [24]. Therefore, the adhesion molecule expression in dilated sinusoids and cavities supported the macrophage- or lymphocyte-induced pattern of liver injury, with a prevalence of the ICAM-1/LFA-1 and VCAM-1/VLA-4 patterns of inflammatory cell recruitment [18,30]. PECAM-1 is generally accepted to be constitutively and uniformly expressed on all types of endothelial cells and cannot be induced by cytokines [28]. PECAM-1 has been implicated in a number of important biologic processes, including leukocyte emigration at sites of inflammation [19], angiogenesis [3], and platelet aggre-
gation to injured endothelial cells [27]. Some authors suggest that it is not expressed on liver sinusoidal endothelial cells under normal conditions and after induction [4]. Others have demonstrated a constitutive expression of PECAM-1 on liver sinusoidal endothelial cells in normal and damaged rat and human livers [25]. It was reported recently that PECAM-1 contained a signaling sequence known as the immunoreceptor tyrosine-based inhibitory motif (ITIM), a motif originally described in receptors regulating immunity [3]. We think that PECAM-1 expression in the cavities in peliosis was directed towards maintenance of sinusoidal integrity, since altered sinusoidal endothelial cells and sinusoidal denudation were the main pathological problem in peliosis hepatis. PECAM-1 could also participate in the non-specific immune response in peliosis, inhibiting leukocyte and monocyte transmigration through the sinusoidal wall, and maintaining platelet aggregation in cavities. Our ultrastructural immunohistochemical finding of ICAM-1, and VCAM-1-positive sinusoidal endothelial
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Fig. 4. (a) Dilated sinusoid (cavity – C) filled with cell debris and erythrocytes (E). The endothelial lining covers enlarged Disse space. H – hepatocyte. Patient 1. (b) Intense ICAM-1 immune deposits on the lateral hepatocyte membrane. A hepatocyte with a dark cytoplasm is surrounded by two hepatocytes with bright and swollen cytoplasm. Patient 2. (c) ICAM-1 immune deposits on hepatocyte microvilli and on the lateral hepatocyte membrane. s – sinusoid. Patient 2. (d) Intense ICAM-1 immunoreactivity on the sinusoidal endothelial cell (E) membrane. s – sinusoid. Patient 1. (e) ICAM-1 immune deposits on the membrane of a Kupffer cell (K). s – sinusoid. Patient 1. (f) VCAM-1 immunoreactivity on the membrane of a sinusoidal endothelial cell. s – sinusoid. Patient 2. Magnifications: (a) 7000, (b) 12,000, (c) 12,000, (d) 12,000, (e) 7000, (f) 12,000.
cells in the dilated sinusoids confirmed the light microscopic finding. Degenerative hepatocytes with ICAM-1-positive hepatocyte membranes were well visible on electron microscopy. Therefore, degenerative hepatocytes, activated Kupffer cells and HSCs, already described by us [15], and the presence of
lymphocytes and monocytes in the dilated sinusoids and cavities, found in this report, could contribute to the altered cytokine and chemokine milieu in liver peliosis. This milieu could regulate the expression of the adhesion molecules on liver sinusoidal endothelial cells.
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We noted that erythrocyte stasis and mononuclear cells were attached to the wall of cavities. Somewhere, the endothelial lining was invisible as described by Zafrani et al. in 1984 [36]. Therefore, endothelial denudation exposed the subendothelial collagen to blood platelets and initiated their aggregation [30]. Expression of adhesion molecules on the endothelium of cavities and dilated sinusoids could also be initiated by the mediators, released from aggregated platelets, erythrocytes, and mononuclears [10]. In summary, we suggest a theory of the initiation of adhesion molecules expression in peliosis. The first step in this process could be the degeneration of some hepatocytes and of sinusoidal endothelial cells, triggered by toxic (steroids, tuberculostatics) or tumor-derived stimuli. The second step is the resultant sinusoidal dilation and cavity formation. This is accompanied by induction of the adhesion molecules expression in sinusoids, supported by cytokines and chemokines released in the first step. Simultaneously, there was recruitment of mononuclear cells in the sinusoids and sometimes in the space of Disse. Third, because of the impaired endothelial cell function, erythrostasis, platelet aggregation and activation, and thrombosis appeared somewhere in the cavities. Thrombogenesis also supports the process of activation of adhesion molecule expression. In conclusion, the data presented here support the hypothesis that the expression of liver sinusoidal adhesion molecules is susceptible to regulation by different stimuli, reflecting the different causes of human liver peliosis.
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