temporary lymphocyte-endothelial adhesion in liver sinusoids. (Am J Pathol 1995, 146. 1406-1413). The presence of liver immune cells including Kupffer.
American Journal of Pathology, Vol. 146, No. 6, June 1995 Copyright ©) American Societyfor Investigative Pathology
Expression of Cell Adhesion Molecules on Liver-Associated Lymphocytes and Their Ligands on Sinusoidal Lining Cells in Patients with Benign or Malignant Liver Disease
Marfa Garcfa-Barcina,*t Barbara Lukomska,* Wanda Gawron,* Marfa Winnock,t Fernando Vidal-Vanaclocha,* Paulette Bioulac-Sage,t Charles Balabaud,t Waldemar Olszewskit From the Department of Cell Biology and Morphological Sciences,* Faculty of Medicine and Odontology, University of Basque Country, Leioa, Bizkaia, Spain; Surgical
Research and Transplantology Department,* Medical Research Center, Polish Academy of Sciences, Warsaw, Poland; and Laboratoire des Interactions Cellulaires,t Universite6 de Bordeaux II, Bordeaux, France
Liver sinusoids, in contrast with the capiUaries of other tissues, contain large numbers of sequestered lymphocytes. These blood-borne ceUs preferentialy home in the liver. The mechanism regulating the recruitment ofthese ceUs and molecular regulation ofthe recognition ofendothelial ceUs is as yet unclear. The present study sought to evaluate the ceU adhesion molecules on hunman liverassociated lymphocytes and their ligands on sinusoidal lining ceUs in 29 patients undergoing partial hepatectomy for liver tumors. Liver-
lymphocytes and peripheral blood lymphocytes were analyzed by flow cytometry associated
using monoclonal antibodies. Frozen sections of were stained according to alkaline
liver tissue
phosphatase anti-alkaline phosphatase method Cytometric analysis showed that virtualy aU liver-associated lymphocytes expressed on their surface the ceU adhesion molecules LFA-1 and VLA-4. This liver-associated lympbocyte population alsopresented a signiflcantly higherpercentage ofMac 1, ICAM-1, and LFA-3 and an increased surface expression of LFA-1, LFA-2, and NCAM in comparison with peripheral blood lympbocytes. It was likewise sbown that sinusoidal ceUs express ICAM-1, ICAM-2, ICAM-3, VCAM-1 and LFA-3 ligands. Liver-associated lymphocytes thus
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strongly express a number ofdifferent adhesion molecules. The corresponding ligands were also detected on sinusoidal lining ceUls. LFA-1 and VLA-4 would seem to be important pathways of temporary lymphocyte-endothelial adhesion in liver sinusoids. (Am J Pathol 1995, 146.
1406-1413) The presence of liver immune cells including Kupffer cells, endothelial cells, and marginating lymphocytes, and the reduced blood flow in the hepatic portal compartment, predispose this organ to fast recognition and trapping of foreign antigens and native cells. Lymphocyte recruitment by the liver is not the result of nonspecific trapping by sinusoids, but a physiologically significant phenomenon in immune defense. There is some evidence that lymphocytes marginating in liver sinusoids play a major role in the local destruction of tumor cells likely to form metastases in the liver.16 Human liver-associated lymphocytes (LAL) were identified by transmission electron microscopy in perfusion-fixed liver samples from control patients.7 The results of our experimental studies strongly suggest that these cells, of extrahepatic origin, are bloodborne cells preferentially homing in the liver.8 It is necessary to classify the mechanisms regulating the recruitment of these cells or their precursors in the liver, the recognition by and adherence to endothelial and tumoral cells, and their cytotoxic action on the latter, as well as to identify the stimulation of LAL. Supported by Le Conseil R6gional d'Aquitaine, FlSs 93/0714 and Gobierno Vasco. M. Garcia-Barcina has a grant from the Ministerio de Educaci6n y Ciencia (Spain) and B. Lukomska from Commission of the European Communities (ERBCiPACT 922139). Accepted for publication March 1, 1995. Address reprint requests to Maria Garcia-Barcina, Department of Cell Biology and Morphological Sciences (School of Medicine and Odontology), University of Basque Country, Sarriena s/n, Leioa, 48940 Bizkaia, Spain.
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LAL are sufficiently strong attached to endothelial and Kupffer cells in sinusoids to resist the physiological pressure of portal blood flow. Anchoring is regulated by membrane glycoproteins mediating cell-tocell contact. Lymphocyte/endothelial cell adhesion depends on multiple interactions between complementary adhesion molecules present on both cell surfaces. The cell surface expression of intercellular adhesion molecules is a characteristic on lymphocytes and endothelial cells but could also be the result of activation in response to a variety of cytokines.915 Three main molecular families of adhesion receptor/ ligand molecules have been identified: the integrin, immunoglobulin, and selectin families. The integrin family is composed of non-covalently associated a and ,B subunits classified into subfamilies according to the respective subunits as f31 (CD29): VLA-1 (CD49a), VLA-2 (CD49b), VLA-3 (CD49c), VLA-4 (CD49d), VLA-5 (CD49e), VLA-6 (CD49f); 12 (CD1 8): LFA-1 (CD11a), Mac 1 (CD11b), p150,95 (CD11c), and f33 (CD61): CD41 and CD51. The immunoglobulin superfamily is characterized by immunoglobulin domains and comprises LFA-2 (CD2), LFA-3 (CD58), ICAM-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD50), VCAM-1 (CD106), NCAM (CD56), and PECAM-1 (CD31). The selectin or leukocyte endothelial adhesion molecules (LECAM) are glycoproteins involved in fast intravascular adhesion and homing: L-selectin
(CD62L), E-selectin (CD62E), and P-selectin (CD62P). Studies on cell binding have shown that lymphocytes express (31 and 32 integrins. The endothelial ligands corresponding to these three families are respectively vascular cell adhesion molecules (VCAM-1), intercellular adhesion molecules (ICAM-1, ICAM-2, and ICAM-3) and selectin molecules. Although many adhesion molecules have been identified, it is not clear what type of receptors and what kind of regulation are involved in the interaction between sinusoidal endothelial cells and LAL. It is thought that this interaction plays an important role in the sequestration of lymphocytes in hepatic sinusoids.
The aim of the study was to characterize the surface molecules on human LAL as well as their ligands on sinusoidal lining cells (SLC) in patients with benign and malignant disease.
Materials and Methods Patients Twenty-nine patients aged 28-75 years were included in this study. All were undergoing partial hepatectomy for malignant (19 patients) or benign (10 patients) liver diseases (Table 1). All patients gave their
Table 1. Patients Grouped According to Liver Disease Patient
Sex
Age
Liver pathology
Tumor size
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
M
54 68 75 54 70 53 44 60 73 59 28 59 69 56 54 57 59 69 39 61 50 21 37 31 29 41 34 48 50
Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (testis) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (urothelial) Metastasis (colorectal) Metastasis (colorectal) Metastasis (colorectal) Metastasis (duodenum) Focal nodular hyperplasia Focal nodular hyperplasia Focal nodular hyperplasia Focal nodular hyperplasia Focal nodular hyperplasia Focal nodular hyperplasia Adenoma Adenoma Angioma Angioma (ruptured)
2x2 8 x 5, 8 x 6, 3 x 3 8 x 8, 10 x 10 3 x 2.5 15 x 10 6.5 x 6.5 3.5 x 3.5 7.5 x 5 6x6 3x2 7.5 x 7.5 4x4 5x5 2x2 11 x 8,6 x 7 3x3 3 x 3, 1 x 1 3x2 7 x 3.5 3x3 8x8 10 x 10 6.5 x 6.5 6x5 5 5 x 2.5 6x6 7.5 x 7.5 ND
ND: not etermined.
F
M M M M F F
M M M F
M F
M M M F
M F F
M F F F F F
M F
1408 Garcia-Barcina et al AJP June 1995, Vol. 146, No. 6
informed consent, and the trial was approved by the Bordeaux University Ethics Committee.
Peripheral Blood Fifty ml peripheral blood was collected at the start of the operation from each patient.
Liver Lavage After partial hepatectomy, non-tumoral tissue was washed by high pressure perfusion (50-cm water column) using a peristaltic pump, with warm (37 C) Ca2+-free and Mg2+-free Dulbecco's solution supplemented with 0.1 % EDTA by catheterization of the portal or hepatic vein. The eluate evacuated was collected in sterile conditions.
Mononuclear Cell Isolation Mononuclear cells were isolated from peripheral blood (PBL) and liver lavage (LAL) by centrifugation on a Ficoll-Paque (Pharmacia LKB Biotechnologies, Inc., Piscataway, NJ) density gradient. The cells collected from the interphase were washed three times and resuspended in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin, which we shall refer to as complete medium.
Flow Cytometry Twenty-two patients (13 metastasis and 9 benign liver tumors) were included in flow cytometric analysis. Isolated PBL and LAL (0.5 x 106) were incubated with 20 p1 of fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled murine monoclonal antibodies (MAb) in 100 pl PBS for 30 minutes at 4 C, washed twice, and fixed with 1% paraformaldehyde in PBS. All the MAb used for immunofluorescence (CD2, CD3, CD8, CD11a, CD11b, CD11c, CD18, CD29, CD49b, CD49d, CD49f, CD54, and CD58) were purchased from Immunotech (Marseille, France), with the exception of CD56 from Becton Dickinson (Mountain View, CA). One-color (argon laser, 488 nm) or two-color (green fluorescence at 535 nm and red fluorescence at 580 nm) analysis was performed on a FACScan (Becton Dickinson). IgGl and IgG2a mouse isotypes were used as controls.
Immunohistochemical Staining Immunohistochemical studies were performed on liver fragments taken as far away as possible from the neoplastic lesion, in macroscopically nonaffected areas. Liver fragments from 10 patients (eight metastasis and two focal nodular hyperplasia) were snap frozen in liquid nitrogen and transferred into the cryostat. Five-p thick sections of frozen tissue mounted individually on glass slides were dried for at least 2 hours in front of an electric fan. For immunohistochemical staining indirect alkaline phosphatase anti-alkaline phosphatase (APAAP) technique was applied. Liver tissue sections fixed in cold acetone were dried and incubated with normal rabbit serum, followed by application of primary mouse monoclonal antibodies: CD54 (ICAM-1) from Immunotech; CD102 (ICAM-2) and CD50 (ICAM-3), a gift from Dr. T. Springer; CD106 (VCAM-1) (2G7 and E1/6), donated by Dr. W. Newman; CD62E (E-selectin) (H4/18 and H18/7), a gift from Dr. M. Bevilacqua; and CD58 (LFA-3), obtained from Becton Dickinson. Secondary and tertiary antibodies in the form of rabbit anti-mouse immunoglobulins and APAAP complex were then also applied. All incubations were carried out at room temperature for 30 minutes followed by a wash in two changes of TBS for 10 minutes. The reaction product was developed by incubation with chromogenic alkaline phosphatase substrate for 15 minutes. Tissue sections were then counterstained with hematoxylin and mounted in glycergel.
Statistics Results are reported as mean percentages ±SEM. For statistical analysis, the nonparametric Wilcoxon's rank sum test and Mann-Whitney test were used.
Results Expression of Cell Adhesion Molecules on Isolated LAL The flow cytometric analysis, after incubation with labeled monoclonal antibodies against cell adhesion molecules, showed that LAL presented a high proportion of positive cells for CD1 1 a (96 ± 1) and CD1 8 (94 ± 2) a and a chains of LFA-1, CD49d (98 ± 2) and CD29 (99 ± 1) a and f3 of VLA-4, and CD2 or LFA-2 (72 ± 5). Moreover, quantitative differences in the expression of surface antigens between LAL and PBL were observed. In liver lavage and peripheral blood, the percentages of CD3 (61 ± 3 versus 71 + 3) (Wilcoxon, P = 0.002), CD8 (54 ± 3 versus 28 ± 2)
Cell Adhesion Molecules on Hepatic Sinusoidal Cells 1409 AJP June 1995, Vol. 146, No. 6
(Wilcoxon, P = 0.001), CD11b (57 3 versus 41 ± 5) (Wilcoxon, P = 0.01), CD54 (27 5 versus 5 ± 1) (Wilcoxon, P = 0.0003), CD56 (51 3 versus 28 ± 4) (Wilcoxon, P = 0.006), and CD58 (90 ± 3 versus 69 ± 4) (Wilcoxon, P = 0.005) positive cells were significantly different (Figure 1). The strong expression of CD49d and CD29 observed in LAL was found as well as in PBL (91 ± 2 and 98 ± 1, respectively). Major differences were noted in the composition of liver lavage and peripheral blood with regard to CD49b (24 ± 6 versus 9 ± 2) (Wilcoxon, P = 0.02) and CD49f (22 ± 4 versus 38 ± 8) (Wilcoxon, P = 0.2249) (Figure 2). The observation of fluorescence intensity values revealed a selective expansion of cell subsets expressing high surface levels (bright population) of cell adhesion molecules among LAL, in comparison with PBL. LAL showed a significantly higher percentage of cells with a bright phenotype for CD2 (44 ± 4), CD8 (34 ± 2), CD1 1a (81 ± 2), CD18 (66 ± 5), and CD56 (22 ± 3)thanPBL(25 ±3,16 ±1,41 ± 4,41 ± 5and 5 ± 2, respectively) (Table 2). There were no apparent differences (MannWhitney) in the expression of cell adhesion molecules by PBL and LAL isolated from non-tumoral liver tissue between patients with benign or malignant liver dis-
100
E80,
~40 03 POL.
20
U LAL 0 CD49d
CD49b
CD49f
CD29
Figure 2. Percentage of(1 integrins presented by LAL and PBL. Results are expressed as mean ± SEM. Differences were statistically sig-
nificant for *P < 0.05.
Table 2. Percentages of Bright and Dim Populations of CD2, CD8, CD1la, CD18, and CD56 Positive Cells Among LAL and PBL
Cell adhesion molecules CD2 CD8 CD11a CD18 CD56
LAL
Bright
Dim
4*** 33 ± 13*** 2** 20 ± 2** 2*** 15 ± 2*** 5** 30 ± 5** 3*** 27 ± 3 Differences were statistically significant 44 ± 34 ± 81 ± 66 + 22 ±
PBL Bright Dim
25 ± 3 16 ± 1 41 ± 4 41 ± 5 5±2
53 ± 3 12 ± 1 53 ± 3 55 ± 5 21 ± 4
between PBL and LAL for *P < 0.05, **P < 0.01 and ***P < 0.001. Discrimination between dim and bright population was established for every monoclonal antibody: channels 20 to 80 and 80 to 250 for CD2, 10 to 450 and 450 to 2600 for CD8, 10 to 96 and 96 to 500 for CD1 la and CD18, and 25 to 170 and 170 to 1600 for CD56, respectively.
eases.
Expression of Adhesion Molecules on SLC Analysis of the expression of lymphocyte adhesion ligand molecules on the different endothelial cell types of the liver revealed a specific pattern. Significant differences between sinusoidal endothelial cells and other liver cell endothelia were observed. A summary of the expression of the different adhesion molecules is given in Table 3. A strong presence of ICAM-1 antigen (CD54) was observed on SLC (endothelial and Kupffer cells). On the endothelium of the central vein, portal vein, and hepatic artery expression was faint (Figure 3). Weak 100-.*
" 80: 60-
~ ~~~~~~~~~0POL
20
ULAL
0
CD3
CD8
CD1ib
CD54
CD56
CDS$
Figure 1. Expression of cell adbesion molecules on LAL and PBL. Results are expressed as mean ± SEM. Differences were statistically signifcantfor P < 0.05, #P < 0.01 and P < 0.001.
ICAM-2 (CD102) reactivity was found on SLC and on hepatic artery endothelia. No staining was detected on portal and central venular endothelial cells. Expression of ICAM-3 (CD50) molecules, a third ligand for LFA-1, was restricted to Kupffer cells and to mononuclear cells present in the sinusoidal lumen. In all cases, VCAM-1-positive SLC were observed, but VCAM-1 membrane expression was not detected on any of the other liver vessel endothelia (Figure 4). Two monoclonal antibodies (2G7 and E 1/6) recognized two different epitopes of VCAM-1 molecule, showed the same distribution pattern. SLC, like other endothelial cell types in the liver, do not display detectable levels of E-selectin (CD62E). Monoclonal antibodies H4/18 and H18/7 yielded the same results. LFA-3 (CD58), a ligand of the LFA-2 molecule, was present on SLC but this molecule was not expressed on central venous, portal venous and hepatic arterial endothelial cells. There were no variations in the expression and distribution of the adhesion molecules between the non-tumoral liver tissue from patients with malignant or benign diseases. Immunohistochemical staining performed in this study revealed that hepatocytes were negative for all tested adhesion molecules.
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Table 3. Expression of Adhesion Ligand Molecules in the Non-Tumoral Liver of Patients Undergoing Partial Hepatectomy
Cell adhesion molecules
ICAM-1 ICAM-2 ICAM-3 VCAM-1 (2G7) VCAM-1(E1/6) LFA-3 ELAM-1 (H 4/18) ELAM-1 (H 18/7)
Sinusoidal lining cells Endothelial Kupffer cells cells +++
++ + ++
+++ + + ++ + ++
-
-
+
Endothelial cells in other liver vascular compartments Portal Central Hepatic vein vein artery +
+
-
-
+ +
-, no staining; +, slight; ++, moderate; +++, strong.
Figure 3. Immunostaining of a normal human liver section uith a monoclonal antibody against ICAM-1. The micrograph shows the strong expression of ICAM-1 by SLC, drawing the outline of the sinusoid (S). Centrolobular vein endothelia (CLV) was weakly stained. (X 600).
Discussion Liver SLC display a specific pattern of adhesion molecules expression. It can be assumed that this specific distribution of endothelial ligand molecules in liver sinusoids may be a functional prerequisite for the interaction with inflowing blood lymphocytes. It is also possible that there may be a relation between this specific pattern and the intravascular control of lymphocytes homing in the liver.
Various adhesion pathways essential for lymphocyte-to-endothelial adhesion have been characterized. The first includes LFA-1/ICAM-1 molecules. The results of our study reveal that most LAL that accumulate in liver sinusoids express a higher
surface intensity of LFA-1 (CD1 la + CD18) than that seen on PBL (bright versus dim population). This high expression corresponds to the presence of ICAM-1, ICAM-2, and ICAM-3 ligands on SLC that have found
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Figure 4. Immunostaining of a normal human liver section with a monoclonal antibody 2G7 against VCAM-1. Tbe micrograph shows the strong but heterogeneous expression of VCAM-1 by SLC. S, sinusoid. (X 600).
to be ligands for LFA-1 and Mac 1. We have shown strong expression of ICAM-1 on sinusoidal endothelium in non-tumoral liver. This finding confirms and extends certain previously published results that showed the presence of ICAM-1 on normal human liver.1619 Moreover, in common with other capillary endothelial cells, SLC also express ICAM-2. The presence of both ICAM-1 and ICAM-2 on the hepatic sinusoidal endothelium could promote the binding of particular lymphocyte subsets. Constitutive expression of ICAM-2 in SLC, which takes part in lymphocyte binding to unstimulated endothelium, may argue for it. LFA-1 and Mac 1 expressed by LAL and ICAM-3 molecules expressed by Kupffer cells may also contribute to the adhesion of these two cell types. Although ICAM-1 is a vascular adhesion molecule, it is widely expressed on other cell types, including activated lymphocytes. Our studies show that a high percentage of LAL express ICAM-1. Two known ligands of ICAM-1, LFA-1, and Mac 1 have been shown to be present on Kupffer cells.20 The expression of ICAM-1 molecules on LAL allow them to interact with
(32 integrins on Kupffer cells. This interaction may reinforce lymphocyte adhesion in the sinusoids. The second adhesion pathway involves the interaction of VLA-4/VCAM-1. Several in vitro studies have demonstrated that, under normal conditions, endothelial cells do not express VCAM-1.1821,22 Volpes18 found VCAM-1 expression on the sinusoidal endothelium in inflammatory liver disease but not in normal liver tissue. Steinhoff observed VCAM-1-positive sinusoidal endothelial cells in the tissue with chronic rejection of liver transplant.21 We observed faint staining of VCAM-1 on SLC of non-tumoral tissue in patients with benign and malignant liver disease. VCAM-1-positive staining was not the result of tumor presence, since our previous studies have shown VCAM-1 molecules present on SLC of normal liver tissue obtained from non-cancer patients undergoing elective surgery.23 Similar observations have previously been reported by Adams.19 VLA-4, the natural receptor for VCAM-1, is expressed by nearly all LAL. The interaction between VLA-4 present on LAL and
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VCAM-1 expressed at high levels by SLC may contribute to the temporary adhesion of lymphocytes to the sinusoidal wall. The non-expression of E-selectin (CD62E) on SLC would infer that this molecule is not induced in liver sinusoids. It thought that E-selectin is the main regulatory molecule of the initial phase of neutrophil adherence to activated endothelial cells. It would not seem to play a structural role in the adhesion of LAL to SLC. To summarize, the results of our studies suggest that LFA-1/lCAMs and VLA-4N/CAM-1 are the main pathways of the lymphocyte/endothelial adhesion process in liver sinusoids. This interaction could be further modulated and potentiated by the presence of other cell surface molecules. LAL strongly express LFA-2 (CD2), for which specific ligand LFA-3 (CD58) is constitutively present on SLC. The action of these molecules may represent an additional factor in the adhesion of LAL to endothelial and Kupffer cells in the sinusoidal lumen. There remains the following question: is the presence of identified adhesion molecules sufficient to ensure lymphocyte margination in sinusoids or are additional signals needed for the firm attachment of these cells? The mechanism of cell-to-cell adhesion found in hepatic sinusoids appears to be different from that in other capillary vessels, and is no doubt adapted to the specific liver microenvironment. The existence of unique environmental factors and organ specific receptors in the liver may contribute to the temporary margination of a certain population of lymphocytes in hepatic sinusoids.
Acknowledgments The authors wish to thank Prof. J. Saric (Service de Chirurgie Digestive, Hopital Saint-Andr6, Bordeaux) for the provision of liver tissue and Ms. C. Bedin, Ms. L. Boussarie, and Ms. L. Latapie for their excellent technical assistance.
References 1. Wiltrout RH, Herberman RB, Zhang SR, Chirigos MA, Ortaldo JR, Green KM, Talmadge JE: Role of organ associated NK cells in decreased formation of experimental metastasis in lung and liver. J Immunol 1985, 134:4267-4275 2. Cohen SA, Tzung SP, Doerr RJ, Goldrosen MH: Role
of asialo GM-1 positive liver cells from athymic nude or polyinosinic-polycytidilic acid-treated mice in suppressing colon derived experimental hepatic metastasis. Cancer Res 1990, 50:1834-1840
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17. Steinhoff G, Schrader B, Duijvestijn AM, Wonigeit K: Expression patterns of leukocyte adhesion ligand molecules on human liver endothelia. Am J Pathol 1993, 142:481-488 18. Volpes R, Van den Oord JJ, Desmet VJ: Vascular adhesion molecules in acute and chronic inflammation. Hepatology 1992, 15:269-275 19. Adams DH, Burra P, Hubscher SG, Elias E, Newman W: Endothelial activation and circulating vascular adhesion molecules in alcoholic liver disease. Hepatology 1994, 19:588-594 20. Scoazec JY, Feldman G: The cell adhesion molecules of hepatic sinusoidal endothelial cells. J Hepatol 1994, 20:296-300 21. Steinhoff G, Berhrend M, Schrader B, Pilchmar R:
Intercellular adhesion molecules in human liver transplants. Overview on expression patterns of leukocyte receptor and ligand molecules. Hepatology 1993, 18:440-453 22. Scoazec JY, Racine, Couvelard A, Flejou JF, Feldman G: Endothelial cell heterogeneity in the normal human liver acinus: in situ immunohistochemical demonstration. Liver 1994, 14:113-123 23. Lukomska B, Garcia-Barcina M, Gawron W, Winnock M, Bioulac-Sage P, Balabaud C, Olszewski WL: Adhesion molecules on liver-associated lymphocytes and sinusoidal lining cells of human livers. Cells of the Hepatic Sinusoid. Edited by DL Knook, E Wise, and K Wake. Leiden, The Netherlands, Kupffer Cell Foundation, 1995 (in press)
