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Distribution of the VLA family of integrins in normal and pathological human liver tissue
GASTROENTEROLOGY
1991;101:200-206
Distribution of the VIA Family of Integrins in Normal and Pathological Human Liver Tissue RICCARDO VOLPES, JOOST and VALEER J. DESMET
J. VAN DEN OORD,
Department of Pathology, Laboratory of Histochemistry and Cytochemistry, St. RafaBl, Catholic University of Leuven, Leuven, Belgium
The “very late activation” (VU) subgroup of tbe integrin superfamily of adhesion molecules plays a central role in cell-cell and cell-matrix interactions. The six different VLA dimers known so far consist of a common 8 subunit and a variable (Y (1 to 6) subunit. They serve as receptors for laminin, collab gen, and fibronectin or function as adhesion molecules for leukocytes and are therefore of great significance in embryogenesis, growth and repair, and in leukocyte recirculation. The distribution of tbe common f! and the variable (Ychains of the VLA were studied in normal, inflammatory, and cholestatic liver biopsy samples. In normal liver tissue, vascular endothelia express al, 2, 3, 5, and 6; bile duct epithelium 012, 3, 5, and 6; connective tissue stroma cul and 2; hepatocytes al and 5; sinusoidal lining cells al, 2, and 5; and mononuclear cells CK~. Whereas bile ducts and vascular endothelia do not show relevant changes in cxchain expression in liver diseases, hepatocytes de novo express membranous ~113 and 6 in inflammatory liver diseases. In view of the role of the VU-3 and VLA-6 as laminin recepfors, this finding is in line with the production of laminin in active liver disease. Moreover, de novo expression of “bile duct type” c112,3, and 6 on periportal hepatocytes in cholestatic liver disease likely illustrates a phenotypic switch of hepatocytes towards bile duct epithelium during cholestasis.
C importance in the development, organization, function, and repair of cells and tissues. This adheellular adhesion mechanisms
are of fundamental
sion occurs via soluble mediators and by direct contact. Three protein families of cell surface receptors are involved in an extensive network of cell-cell and cell-matrix interactions. The immunoglobulin (Z@ supe$amily (I) includes the antigen-specific T-cell receptor and B-cell immunoglobulins, as well as the
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Hospital
major histocompatibility complex antigens of class I and II, the antigen nonspecific intercellular adhesion molecule 1 (ICAM-l), vascular-cell adhesion molecule 1 (VCAM-l), lymphocyte function-associated antigen 3 (LFA-3), and CD2 antigen. The selectin family (2,3), e.g., the endothelial-leukocyte adhesion molecule 1 (ELM-l) and leukocyte adhesion molecule 1 (LAM-l), is involved in the binding of leukocytes to endothelial cells during their homing and extravasation. The receptors of the integrin family (a), which “integrate” signals from the extracellular environment with the intracellular cytoskeleton, consist of CX/~heterodimers divided into at least three subfamilies according to their p subunit. The fil integrins (also VLA subfamily, because the first two identified members appeared on T cells “very late after activation”) (5) contain six different dimers, composed of a common pl and six variable OLsubunits. These six members of the VLA subfamily are engaged in interactions with extracellular matrix components and with cell surface ligands. They serve as receptors for laminin (VIA- 1, 3, and 6)) collagen (VLA- 1, 2, and 3), fibronectin (VLA-4 and 5), or function as adhesion molecules for leukocytes (VLA-4) (Table 1) (6). Originally detected on T cells (7,8), the VLA integrins have been shown to be expressed by a variety of nonlymphoid cells as well (9,10). We have studied the
Abbreviations
used in this paper:
ELAM-1, endothelial-
leukocyte adhesion molecule 1;ICAM-1,intercellular adhesion molecule 1; LAM-l,leukocyte-adhesion molecule 1;LFA-1, lymphocyte function-associated antigen 1;LFA-3, lymphocyte function-associated antigen 3; Mah, monoclonal antibody; PAP, peroxidase-antiperoxidase technique; TCR, antigen-specific T-cell receptor; VCAM-1,vascular-cell adhesion molecule 1;VLA,very late activation antigen. o 1991 by the American Gastroenterological Association 0016~5065/91/$3.00
VLA INTEGRINS IN HUMAN LIVER TISSUE 201
July 1991
Table 1. Characteristics of the Six VLA Heterodimers Hetero-
Receptor VLA-1 VLA-2 VLA-3 VLA-4 VLA-5 VLA-6
dimers (mol wt) cdl (210) (Y2(165) (Y3 (135) cf4 (150) (u5 (130) (Y6 (140)
Known ligands Laminin, collagen Collagen, laminin 61 (136)
Laminin, collagen, fibronectin VCAM-1, fibronectin Fibronectin Laminin
distribution of the common pl and the variable cx chains of VLA integrins both in normal human liver biopsy specimens and in liver specimens with inflammatory and cholestatic disease.
All incubations were performed for 30 minutes at room temperature except for anti-VLA-cw5 Mabs 16 and BIIG2, which were applied overnight at 4°C. Each incubation step was followed by a wash in three changes of PBS, pH 7.2. The reaction product was developed by incubation for 15 minutes in 0.05 mol/L acetate buffer, pH 4.9, containing 0.05% 3-amino-9-ethylcarbazole and 0.01% H,O,, resulting in a bright red staining of immunoreactive sites. Only cells with membranous staining were considered; inflammatory cells with intense dotlike cytoplasmic immunoreactivity caused by endogenous peroxidase were ignored. Staining of the various components in the human liver specimens was graded semiquantitatively on a fivepoint scale [-, +/-, +, ++, and +++). Controls consisted of replacement of primary antibody by irrelevant Mabs of similar isotype or use of chromogen alone; these controls were consistently negative and revealed only cells with endogenous peroxidase activity.
Materials and Methods Thirty-four needle liver biopsy specimens obtained for diagnostic purposes were used for this study. Histologically, 5 specimens showed normal lobular architecture and complete absence of histological abnormalities and were considered normal. Five liver biopsies showed characteristics of acute hepatitis (2 HCV positive, 3 HBV positive); 3 of chronic persistent hepatitis (all 3 HBV positive); 9 of chronic active hepatitis with or without cirrhosis (3 HCV positive, 6 HBV positive]; 6 of alcoholic liver disease (3 with and 3 without cirrhosis); and 6 showed cholestatic features characterized by periportal ductular proliferation, 3 caused by primary biliary cirrhosis (stage 3-4), and 3 by longstanding extrahepatic bile duct obstruction. Each specimen was received freshly and divided into two parts. One part was fixed in B5 fixative, embedded in paraffin, and used for routine histology: the other part was snap-frozen in liquid nitrogen-cooled isopentane and used for immunohistochemistry. For conventional immunohistochemistry, 5-Frn serially cut frozen cryostat sections were dried overnight at room temperature and fixed in absolute acetone for 10 minutes. Cryostat sections were used immediately or wrapped in aluminum foil and stored in closed boxes at -20°C until further processing. In all cases, a three-step indirect immunoperoxidase procedure was performed. Rehydrated sections were incubated for 30 minutes with the monoclonal antibodies (Mabs) listed in Table 2. The secondary and tertiary antibodies consisted of peroxidase-conjugated rabbit anti-mouse and peroxidase-conjugated swine anti-rabbit (Igs), respectively (both obtained from Dakopatts a/s, Copenhagen, Denmark; working dilution I:50 and l:lOO, respectively). Anti-VLA-cy5 Mabs (16 and BIIG2) and anti-VLA-a6 Mab (GoH3) were demonstrated with a four-step unlabeled peroxidase-antiperoxidase (PAP) technique, using primary rat antibodies, followed by rabbit anti-rat Igs, swine antirabbit Igs, and rabbit-PAP complex. All primary antibodies were used in optimal dilutions, determined by titration on positive controls. All secondary and tertiary antisera as well as the PAP complex were diluted in phosphate-buffered saline (PBS), pH 7.2, containing 10% human AB-positive serum to reduce unwanted background staining.
Results Normal Liver Tissue
The distribution of the various different VLA chains in normal human liver tissue is shown in Table 3. VLA-al was expressed in portal tracts on vascular endothelium and in the connective tissue stroma but not on bile duct epithelium. In connective tissue it was not possible to specify by light microscopy the cellular or other localization of the reaction product. In the lobular parenchyma, sinusoidal lining cells stained positively and hepatocytes showed a moderate membranous VLA-al expression. Scattered mononuclear cells throughout the liver parenchyma were VLA-al negative. VLA-ol2 was found on portal vascular endothelium, bile duct epithelium, and connective tissue stroma. Sinusoidal lining cells were occasionally reactive with anti-VIA-o2 Mab. Hepatocytes and mononuclear cells were VLA-(~2 negative. VLA-11x3was exclusively expressed on the basolatera1 cell membrane of bile duct epithelium. VLA-a3 Table 2. Monoclonal Antibodies Used in This Study Mabs
Working dilution
TS2/7 lOGl1 J143
1:500 1:lO 1:lO
B5GlO B5E2 16 BIIGZ GoH3 A-lA5 4B4
1:20 1:20 1:lOO 1:50 1:50 1:2000 1:20
Source
Specificity
Dr. M. E. Hemler Dr. A. E. G. K. von dem Borne Dr. L. J. Old Dr. E. Klein Dr. M. E. Hemler Dr. M. E. Hemler Dr. K. M. Yamada Dr. C. Damsky Dr. A. E. G. K. von dem Borne Dr. M. E. Hemler Coulter Immunology (Hialeah,
VLA-(~1 chain VLA-u2 chain VLA-a3 chain
FL)
VLA-a4
chain
VLA-(~5 chain VLA-u6 chain VLA-J31 chain
202
VOLPES ET AL.
GASTROENTEROLOGY Vol. 101, No. 1
Table 3. Distribution ofthe Various Single VLA Chains in Normal Liver Tissue Vascular endothelium
Bile duct epithelium
Connective tissue stroma
+ +++
+
VLA-al
++
VLA-(uz VLA-ol3 VLA-a4 VLA-a5
+ + ++
++
VLA-ci6 VLA-l31
++ +++
+ +++
Sinusoidal lining cells
Mononuclear inflammatory cells
+
++
-
-
+I-
-
+
+
++ -
++
++
++
Hepatocytes
+
+++
-, No staining; +/-, variable staining; +, slight: + +, moderate; + + +, strong.
expression was also found on portal vascular endothelium but not on endothelium of the centrilobular vein. VLA-o14 was confined to few, scattered mononuclear cells, both in portal tracts and in the lobular parenchyma. Mabs B5E2 and B5GlO yielded the same results. VLA-a5 was found on all structures in portal tracts and lobular parenchyma, except for mononuclear inflammatory cells which were VLA-a5 negative. Hepatocytes and sinusoidal lining cells showed a weaker reactivity than bile duct epithelium and vascular endothelium. Mabs 16 and BIIG2 revealed identical patterns of immunoreactivity. VLA-~6 was restricted to portal vascular endothelium and bile duct epithelium, where it was selectively expressed on the basal cell membrane. The expression of VLA-pl chain corresponded to the sum of the reactivities of all six single VLA-ar chains. VLA-Pl was strongly expressed by vascular endothelium, bile duct epithelium, connective tissue stroma, and sinusoidal lining cells. Membranous staining of hepatocytes gave rise to a honeycomb pattern of immunoreactivity. In both portal tracts and in lobular parenchyma, mononuclear cells expressed VLA-pl. Inflammatory
and Cholestatic Liver Diseases
The distribution of VLA integrins in inflammatory and cholestatic liver disease is summarized in
Table 4 and illustrated in Figures 1 and 2, respectively. Compared with normal liver, hepatocytes showed increased VLA-al and VLA-cx5 membranous expression in liver biopsies with inflammation, without particular topographical correlation with the infiltrating inflammatory cells (Figure lA and B). De novo hepatocellular membranous VLA-ol3 and a6 positivity was found diffusely throughout the liver parenchyma in acute hepatitis and was restricted to periportal areas of inflammation in chronic active hepatitis (Figure 1C and D). Moreover, expression of VLA-(~2, (~3, and a6 was found on periportal hepatocytes in cases of cholestatic liver disease. Sinusoidal lining cells showed de novo VLA-(~4, as well as enhanced VLA-ol5 expression in periportal and intralobular areas of inflammation; this positivity was found both on sinusoidal endothelial cells and on Kupffer’s cells (Figure 1E’). Mononuclear inflammatory cells were numerous both in portal tracts and lobular parenchyma in inflammatory liver disease and showed strong VLA-a4 expression (Figure 1E). VLA-al expression by inflammatory cells was highly variable. Portal vascular endothelium and bile duct epithelium did not show relevant changes in VLA expression; proliferating bile ductules in cholestatic liver disease showed a strong VLA-(~2 and VLA-a6 but a weak VIA-(~3 expression (Figure 2). The connective
Table 4. Modifications in the Distribution of the Single VZA Chains in Inflammatory Bile duct epithelium
Vascular endothelium I
C
I
C
VIA-al
VLA-(r2 VLA-a3 VLA-(~4 VLA-(Y5 VLA-(Y6 VLA-I31
+++ ++
Connective tissue stroma C
I
+++
+++
+++
++
++ ++
++
NOTE. Only changes in VLA expression illustrated in Table 3. I, inflammation; C, cholestasis.
+ +++ +++
Sinusoidal lining cells
Hepatocytes
I
+++ ++ +++
and Cholestatic Liver Biopsy Specimens
C
I
C
Mononuclear inflammatory cells I
C
+
+ ++ ++ ++
++t
++
compared with normal liver are reported. Empty spaces represent unmodified
VLA expression
as
July 1991
VLA INTEGRINS IN HUMAN LIVER TISSUE
Figure 1. Staining tory liver disease.
for VLA integrins
in inflamma-
A-C.
Chronic active hepatitis with cirrhosis. VLA-al is up-regulated on the surface of hepatocytes, as well as in the connective tissue stroma (A). Similarly, VLA-a5 shows enhanced expression on hepatocytes (B). VLA-(~6 is de novo expressed by hepatocytes in areas of inflammation (C).
D. Acute hepatitis.
VLA-a3 is de novo found on scattered periportal hepatocytes (arrowheads).
E. Chronic
active hepatitis. Numerous mononuclear inflammatory cells express VLA-a4, which is also found on sinusoidal lining cells (three-step indirect immunoperoxidase with Mab TS2/7 (A), Mab 16 (B), Mab GoH3 (C), Mab 1143 (D), and Mab B5E2 (E), counterstained with Harris’ hematoxylin; original magnification x250).
203
204 VOLPES ET AL.
Figure 2. Staining for VLA-a3 in cholestatic liver disease. VLA-(~3 is strongly expressed by bile ductules and is also present on periportal hepatocytes. The same expression pattern was found for VLA-a2 (three-step indirect immunoperoxidase with Mab J143,counterstained with Harris’ hematoxylin; original magnification X 400).
tissue stroma in inflamed portal tracts, as well as in fibrotic septa in cases of cirrhosis, showed enhanced VLA-~1, a2, a4, and a5 positivity. Discussion Cellular adhesion is mediated by a large number of cell surface molecules that serve as specific receptors for various ligands. The integrin family mediates both cell-cell and cell-matrix interactions. The VLA-pl subgroup of integrins serves as receptor for extracellular matrix components, such as laminin (VLA-1, 3, and 6), collagen, (VLA-1, 2, and 3), and fibronectin (VLA-4, and 5), as well as for molecules expressed by endothelial cells during leukocyte homing (VLA-4). In this study we have analyzed their distribution in normal human liver biopsy specimens to obtain data on adhesion events in normal conditions; their distribution in inflammatory and cholestatic liver diseases was studied to clarify whether these molecules play a role in liver diseases. The common p-1 subunit was expressed by virtually all structures in both portal tracts and lobular parenchyma, whereas staining for the various a-subunits revealed differential patterns of distribution on the various components of human liver tissue.
GASTROENTEROLOGY
Vol. 101, No. 1
VIA-1 is a receptor for laminin and collagen (6,11). In normal liver as well as in inflammatory and cholestatic liver diseases, VIA-1 was expressed on vascular endothelium, connective tissue stroma, sinusoidal lining cells, and hepatocytes but was constantly absent from bile duct epithelium. A diffuse increased hepatocellular membranous VLA-1 expression was found in both acute and chronic hepatitis; this is in agreement with the enhanced VLA-1 expression by the epithelium of inflamed intestinal mucosa (12) and suggests that VLA-1 up-regulation is a general adaptative response to prevent epithelial loss as a result of the ongoing tissue injury. VLA-2 is a receptor for collagen (13)~ although endothelial cells can use VLA-2 as laminin receptor (14). In normal liver tissue, VLA-2 was found on vascular endothelia, bile duct epithelium, connective tissue stroma, and occasionally on sinusoidal lining cells, whereas hepatocytes and mononuclear cells were VLA-2 negative. The enhanced VLA-2 expression in the connective tissue stroma in inflammatory and cholestatic liver diseases could reflect an increased collagen synthesis by various cell types (15,16). Moreover, de novo VLA-2 expression by periportal hepatocytes in cholestatic biopsy specimens and up-regulation of this integrin on proliferating bile ductules suggest that collagen plays a role in the “bile ductular reaction” and in the interaction of the ductules with surrounding liver tissue during cholestasis. VLA-3 is a multispecific receptor for laminin, collagen, and fibronectin (17). In normal liver, VLA-3 expression was confined to the basolateral membrane of bile duct epithelium and to portal vascular endothelium. Its de novo expression on periportal hepatocytes in inflammatory and cholestatic liver specimens is probably related to the function of VLA-3 as laminin receptor (see below). VLA-4 is expressed by the memory T-cell subset (18) and is involved in cell-cell interactions, because it participates in the binding of leukocytes to endothelial cells during lymphocyte homing (1920). In normal and inflamed liver biopsy specimens, VLA-4 expression was restricted to mononuclear inflammatory cells present in portal tracts and lobular parenchyma. Because VLA-4 is engaged in heterotypic interactions with the VCAM-1 expressed by activated endothelial cells (21), it is tempting to speculate that mononuclear inflammatory cells use VLA-4 as an adhesion molecule to bind to activated sinusoidal liver endothelium. This hypothesis is furthermore supported by our finding that VCAM-1 is de novo expressed by sinusoidal endothelial cells in areas of liver inflammation (R. Volpes, unpublished data). VLA-5 is expressed by adhering cells as receptor for fibronectin (22), a component of fibrillar extracellular
VLA INTEGRINS IN HUMAN LIVER TISSUE
July 1991
structures and of basal lamina in all tissues, which plays a central role in cell adhesion and migration (23). In normal liver tissue VLA-5 was found on vascular endothelia, bile duct epithelium, sinusoidal lining cells, and hepatocytes. In line with the active production of fibronectin by various cell types during active liver disease (15), we observed a strong expression of VLA-5 in the connective tissue stroma of portal tracts in both acute and chronic hepatitis, in particular in the inflamed fibrotic septa in active cirrhosis. Moreover, up-regulation of VLA-5 expression was found on hepatocytes and sinusoidal lining cells in areas of inflammation in both acute and chronic inflammatory liver disease. This finding is in line with the enhanced hepatocellular synthesis and secretion of fibronectin (24) and its deposition in areas of inflammation during chronic active liver disease (25). This suggests a role for VIA-5-positive hepatocytes in liver regeneration after tissue injury (26). Moreover, because sinusoidal lining cells show enhanced expression of VLA-5 in areas of inflammation and because VLA-5 and VLA-4 each bind to different fragments of fibronectin (27), a fibronectin-mediated binding might be one of the mechanisms by which VLA-&positive lymphocytes adhere to VLA-5-positive sinusoidal liver endothelial cells. VLA-6 is the classical receptor for laminin (28). Its restricted presence on portal vascular endothelium and on the basal membrane of bile duct epithelium parallels the occurrence of laminin in basement membranes (29). The increased VLA-6 expression on proliferating bile ductules in cholestatic liver biopsy specimens suggests a role for VLA-6 in the anchorage of the neoformed bile ductules in the stroma. In contrast to normal conditions, VLA-6 was found on hepatocytes in focal areas of inflammation, as well as in periportal areas in cholestatic liver disease. De novo VLA-6 and, to a lesser extent, VLA-3 expression by hepatocytes in inflammatory and cholestatic liver diseases is in line with the hepatocellular production of basement membrane laminin in active liver disease (25) and might contribute to the reconstitution of the original architecture by allowing regenerating hepatocytes to attach to the pericellular environment (26). Finally, the de novo expression by periportal hepatocytes in cholestatic biopsy specimens of those VLA molecules (VLA-2, VLA-3, and VIA-6) normally expressed on bile duct epithelium illustrates a phenotypic switch of hepatocytes towards bile duct epithelium during cholestasis. Together, these data provide insights into the role of VLA integrins in cell-cell and cell-matrix interactions in the liver under normal and pathological conditions. The modification in VLA expression observed in inflammatory and cholestatic liver diseases suggests that the modulating signals generated through
205
the various VLA receptors are important steps in the regulation of interactions in conditions of liver cell damage, repair, and architectural rearrangement as well as of leukocyte migration during inflammatory responses. References 1. Williams AF, Barclay AN. The immunoglobulin
domains
for cell surface
recognition.
Annu
superfamilyRev Immunol
1988;6:381-405. 2. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone
MA. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Nat1 Acad Sci USA 1987;84:9238-9242. 3. Tedder TF, Isaacs CM, Ernst TJ, Demetri GD, Adler DA, Disteche CM. Isolation and chromosomal localization of CDNAs encoding a novel human lymphocyte cell surface molecule, LAM-l. Homology with the mouse lymphocyte homing receptor and other humans adhesion proteins. J Exp Med 1989;170:123-133, 4. Hynes RO. Integrins: a family of cell surface receptors. Cell 1987;48:549-554. 5. Hemler ME, Huang C, Schwarz L. The VLA protein family.
Characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight B subunit. J Biol Chem 1987;262:3300-3309. 6. Larson RS, Springer TA. Structure and function of leukocyte integrins. ImmunolRev 1990;114:181-217. 7. Hemler ME, Jacobson JG, Brenner MB, Mann D, Strominger JL. VLA-I: a T cell surface antigen which defines a novel Iate stage of human T cell activation. Eur J Immunol1985;15:502-508. 8. Hemler ME, Jacobson JG, Strominger JL. Biochemical characterization of VLA-1 and VLA-2. Cell surface heterodimers on activated T cells. J Biol Chem 1985;260:15246-15252. 9. Konter U, Kellner I, Klein E, Kaufmann R, Mielke V, Sterry W. Adhesion molecule mapping in normal human skin. Arch DermatolRes 1989;281:454-462. 10. Choy M-Y, Richman PI, Horton MA, MacDonald TT. Expression of the VLA family of in&grins in human intestine. J Path01 1990;160:35-40. 11. Shimizu Y, van Seventer
12.
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15.
GA, Horgan KJ, Shaw S. Roles of adhesion molecules in T-cell recognition: fundamental similarities between four integrins on resting human T cells (LFA-1, U-4, VLA-5, VLA-6) in expression, binding, and costimulation.ImmunolRev 1990;114:109-143. MacDonald TT, Horton MA, Choy MY, Richman PI. Increased expression of laminin/collagen receptor (VLA-1) on epithelium of inflamed human intestine. J Clin Path01 1990;43:313-315. Takada Y, Hemler ME. The primary structure of the VLA-Z/ collagen receptor a2 subunit (platelet GPIa): homology to other integrins and presence of a possible collagen-binding domain. J Cell Biol 1989;109:397-407. Languino LR, Gehlsen KR, Wayner E, Carter WG, Engvall E, Ruoslahti E. Endothelial cells use (~261 integrin as a laminin receptor. J Cell Biol 1989;109:2455-2462. Clement B, Grimaud J-A, Campion J-P, Deugnier Y, Guillouzo A. Cell types involved in collagen and fibronectin production in normal and fibrotic human liver. Hepatology 1986;6:225-
234. 16. Scharffetter
K, Heckmann M, Hatamochi A, Mauch C, Stein B, Riethmiiller G, Ziegler-Heitbrock H-WL, Krieg T. Synergistic effect of tumor necrosis factor-o and interferon-y on collagen synthesis of human skin fibroblasts in vitro. Exp Cell Res 1989;181:409-419. 17. Carter WG, Wayner EA, Bouchard TS, Kaur P. The role of integrins a281 and (~3B1 in cell-cell and cell-substrate adhe-
206 VOLPES ET AL.
sion of human epidermal cells. J Cell Biol 1990;110:13871404. 18.Volpes R, van den Oord JJ, Desmet VJ. “Memory” T-cells represent the predominant lymphocyte subset in acute and chronic liver inflammation. Hepatology 1991 (in press). 19. Takada Y, Elites MJ, Crouse C, Hemler ME. The primary structure of the a4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function. EMBO J 1989;8:1361-1368. 20. Hemler ME, Elites MJ, Parker C, Takada Y. Structure of the integrin VLA-4 and its cell-cell and cell-matrix adhesion functions. Immunol Rev 1990;114:45-65. 21. Elites MJ, Osborn L, TakadaY, Crouse C, Luhowskyi S, Hemler ME, Lobb RR. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 1990;60:577-584. 22. Takada Y, Huang C, Hemler ME. Fibronectin receptor structures in the VLA family of heterodimers. Nature 1987;326:607609. 23. Hynes R. Molecular biology of fibronectin. Annu Rev Cell Biol 1985;1:67-90. 24. Tamkun JW, Hynes RO. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem 1983;258:4641-4647. 25. Bianchi FB, Biagini G, Ballardini G, Cenacchi G, Faccani A, Pisi E, Laschi R, Liotta L, Garbisa S. Basement membrane production by hepatocytes in chronic liver disease. Hepatology 1984;4:1167-1172. 26. Carlsson R, Engvall E, Freeman A, Ruoslahti E. Laminin and fibronectin in cell adhesion: enhanced adhesion of cells from
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regenerating liver to laminin. Prnc Nat1 Acad Sci USA 1981;78: 2403-2406. 27. Guan J-L, Hynes RO. Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor (~461. Cell 1990;60:53-61. 28. Sonnenberg A, Modderman PW, Hogervorst F. Laminin receptor on platelets is the integrin VLA-6.Nature 1988;336:487489. 29. Clement B, Rescan P-Y, Baffet G, Loreal 0, Lehry D, Campion J-P, Guillouzo A. Hepatocytes may produce laminin in fibrotic liver and in primary culture. Hepatology 1988;8:794-803.
Received July 12,1999. Accepted December 1,199O. Address requests for reprints to: Riccardo Volpes, M.D., Department of Pathology, Laboratory of Histochemistry and Cytochemistry, University Hospital St. Rafael, Catholic University of Leuven, Minderbroedersstraat 12, B-3600 Leuven, Belgium. This study was supported by a grant from the Fonds voor Geneeskundig Wetenschappelijk Onderzoek (FGWO), Belgium. Dr. M. E. Hemler (Harvard Medical School, Boston, MA), Dr. A. E. G. K. von dem Borne (University of Amsterdam, The Netherlands), Dr. L. J. Old (Memorial Sloan-Kettering Cancer Center, New York, NY), Dr. E. Klein (University of Ulm, Germany), Dr. K. M. Yamada (National Institutes of Health, Bethesda, MD), and Dr. C. Damsky (University of San Francisco, CA) are greatly acknowledged for their generous gifts of monoclonal antibodies. The authors thank C. Van den Broeck for excellent technical assistance and M. Rooseleers for preparation of the photographs.
1991;101:200-206
Distribution of the VIA Family of Integrins in Normal and Pathological Human Liver Tissue RICCARDO VOLPES, JOOST and VALEER J. DESMET
J. VAN DEN OORD,
Department of Pathology, Laboratory of Histochemistry and Cytochemistry, St. RafaBl, Catholic University of Leuven, Leuven, Belgium
The “very late activation” (VU) subgroup of tbe integrin superfamily of adhesion molecules plays a central role in cell-cell and cell-matrix interactions. The six different VLA dimers known so far consist of a common 8 subunit and a variable (Y (1 to 6) subunit. They serve as receptors for laminin, collab gen, and fibronectin or function as adhesion molecules for leukocytes and are therefore of great significance in embryogenesis, growth and repair, and in leukocyte recirculation. The distribution of tbe common f! and the variable (Ychains of the VLA were studied in normal, inflammatory, and cholestatic liver biopsy samples. In normal liver tissue, vascular endothelia express al, 2, 3, 5, and 6; bile duct epithelium 012, 3, 5, and 6; connective tissue stroma cul and 2; hepatocytes al and 5; sinusoidal lining cells al, 2, and 5; and mononuclear cells CK~. Whereas bile ducts and vascular endothelia do not show relevant changes in cxchain expression in liver diseases, hepatocytes de novo express membranous ~113 and 6 in inflammatory liver diseases. In view of the role of the VU-3 and VLA-6 as laminin recepfors, this finding is in line with the production of laminin in active liver disease. Moreover, de novo expression of “bile duct type” c112,3, and 6 on periportal hepatocytes in cholestatic liver disease likely illustrates a phenotypic switch of hepatocytes towards bile duct epithelium during cholestasis.
C importance in the development, organization, function, and repair of cells and tissues. This adheellular adhesion mechanisms
are of fundamental
sion occurs via soluble mediators and by direct contact. Three protein families of cell surface receptors are involved in an extensive network of cell-cell and cell-matrix interactions. The immunoglobulin (Z@ supe$amily (I) includes the antigen-specific T-cell receptor and B-cell immunoglobulins, as well as the
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Hospital
major histocompatibility complex antigens of class I and II, the antigen nonspecific intercellular adhesion molecule 1 (ICAM-l), vascular-cell adhesion molecule 1 (VCAM-l), lymphocyte function-associated antigen 3 (LFA-3), and CD2 antigen. The selectin family (2,3), e.g., the endothelial-leukocyte adhesion molecule 1 (ELM-l) and leukocyte adhesion molecule 1 (LAM-l), is involved in the binding of leukocytes to endothelial cells during their homing and extravasation. The receptors of the integrin family (a), which “integrate” signals from the extracellular environment with the intracellular cytoskeleton, consist of CX/~heterodimers divided into at least three subfamilies according to their p subunit. The fil integrins (also VLA subfamily, because the first two identified members appeared on T cells “very late after activation”) (5) contain six different dimers, composed of a common pl and six variable OLsubunits. These six members of the VLA subfamily are engaged in interactions with extracellular matrix components and with cell surface ligands. They serve as receptors for laminin (VIA- 1, 3, and 6)) collagen (VLA- 1, 2, and 3), fibronectin (VLA-4 and 5), or function as adhesion molecules for leukocytes (VLA-4) (Table 1) (6). Originally detected on T cells (7,8), the VLA integrins have been shown to be expressed by a variety of nonlymphoid cells as well (9,10). We have studied the
Abbreviations
used in this paper:
ELAM-1, endothelial-
leukocyte adhesion molecule 1;ICAM-1,intercellular adhesion molecule 1; LAM-l,leukocyte-adhesion molecule 1;LFA-1, lymphocyte function-associated antigen 1;LFA-3, lymphocyte function-associated antigen 3; Mah, monoclonal antibody; PAP, peroxidase-antiperoxidase technique; TCR, antigen-specific T-cell receptor; VCAM-1,vascular-cell adhesion molecule 1;VLA,very late activation antigen. o 1991 by the American Gastroenterological Association 0016~5065/91/$3.00
VLA INTEGRINS IN HUMAN LIVER TISSUE 201
July 1991
Table 1. Characteristics of the Six VLA Heterodimers Hetero-
Receptor VLA-1 VLA-2 VLA-3 VLA-4 VLA-5 VLA-6
dimers (mol wt) cdl (210) (Y2(165) (Y3 (135) cf4 (150) (u5 (130) (Y6 (140)
Known ligands Laminin, collagen Collagen, laminin 61 (136)
Laminin, collagen, fibronectin VCAM-1, fibronectin Fibronectin Laminin
distribution of the common pl and the variable cx chains of VLA integrins both in normal human liver biopsy specimens and in liver specimens with inflammatory and cholestatic disease.
All incubations were performed for 30 minutes at room temperature except for anti-VLA-cw5 Mabs 16 and BIIG2, which were applied overnight at 4°C. Each incubation step was followed by a wash in three changes of PBS, pH 7.2. The reaction product was developed by incubation for 15 minutes in 0.05 mol/L acetate buffer, pH 4.9, containing 0.05% 3-amino-9-ethylcarbazole and 0.01% H,O,, resulting in a bright red staining of immunoreactive sites. Only cells with membranous staining were considered; inflammatory cells with intense dotlike cytoplasmic immunoreactivity caused by endogenous peroxidase were ignored. Staining of the various components in the human liver specimens was graded semiquantitatively on a fivepoint scale [-, +/-, +, ++, and +++). Controls consisted of replacement of primary antibody by irrelevant Mabs of similar isotype or use of chromogen alone; these controls were consistently negative and revealed only cells with endogenous peroxidase activity.
Materials and Methods Thirty-four needle liver biopsy specimens obtained for diagnostic purposes were used for this study. Histologically, 5 specimens showed normal lobular architecture and complete absence of histological abnormalities and were considered normal. Five liver biopsies showed characteristics of acute hepatitis (2 HCV positive, 3 HBV positive); 3 of chronic persistent hepatitis (all 3 HBV positive); 9 of chronic active hepatitis with or without cirrhosis (3 HCV positive, 6 HBV positive]; 6 of alcoholic liver disease (3 with and 3 without cirrhosis); and 6 showed cholestatic features characterized by periportal ductular proliferation, 3 caused by primary biliary cirrhosis (stage 3-4), and 3 by longstanding extrahepatic bile duct obstruction. Each specimen was received freshly and divided into two parts. One part was fixed in B5 fixative, embedded in paraffin, and used for routine histology: the other part was snap-frozen in liquid nitrogen-cooled isopentane and used for immunohistochemistry. For conventional immunohistochemistry, 5-Frn serially cut frozen cryostat sections were dried overnight at room temperature and fixed in absolute acetone for 10 minutes. Cryostat sections were used immediately or wrapped in aluminum foil and stored in closed boxes at -20°C until further processing. In all cases, a three-step indirect immunoperoxidase procedure was performed. Rehydrated sections were incubated for 30 minutes with the monoclonal antibodies (Mabs) listed in Table 2. The secondary and tertiary antibodies consisted of peroxidase-conjugated rabbit anti-mouse and peroxidase-conjugated swine anti-rabbit (Igs), respectively (both obtained from Dakopatts a/s, Copenhagen, Denmark; working dilution I:50 and l:lOO, respectively). Anti-VLA-cy5 Mabs (16 and BIIG2) and anti-VLA-a6 Mab (GoH3) were demonstrated with a four-step unlabeled peroxidase-antiperoxidase (PAP) technique, using primary rat antibodies, followed by rabbit anti-rat Igs, swine antirabbit Igs, and rabbit-PAP complex. All primary antibodies were used in optimal dilutions, determined by titration on positive controls. All secondary and tertiary antisera as well as the PAP complex were diluted in phosphate-buffered saline (PBS), pH 7.2, containing 10% human AB-positive serum to reduce unwanted background staining.
Results Normal Liver Tissue
The distribution of the various different VLA chains in normal human liver tissue is shown in Table 3. VLA-al was expressed in portal tracts on vascular endothelium and in the connective tissue stroma but not on bile duct epithelium. In connective tissue it was not possible to specify by light microscopy the cellular or other localization of the reaction product. In the lobular parenchyma, sinusoidal lining cells stained positively and hepatocytes showed a moderate membranous VLA-al expression. Scattered mononuclear cells throughout the liver parenchyma were VLA-al negative. VLA-ol2 was found on portal vascular endothelium, bile duct epithelium, and connective tissue stroma. Sinusoidal lining cells were occasionally reactive with anti-VIA-o2 Mab. Hepatocytes and mononuclear cells were VLA-(~2 negative. VLA-11x3was exclusively expressed on the basolatera1 cell membrane of bile duct epithelium. VLA-a3 Table 2. Monoclonal Antibodies Used in This Study Mabs
Working dilution
TS2/7 lOGl1 J143
1:500 1:lO 1:lO
B5GlO B5E2 16 BIIGZ GoH3 A-lA5 4B4
1:20 1:20 1:lOO 1:50 1:50 1:2000 1:20
Source
Specificity
Dr. M. E. Hemler Dr. A. E. G. K. von dem Borne Dr. L. J. Old Dr. E. Klein Dr. M. E. Hemler Dr. M. E. Hemler Dr. K. M. Yamada Dr. C. Damsky Dr. A. E. G. K. von dem Borne Dr. M. E. Hemler Coulter Immunology (Hialeah,
VLA-(~1 chain VLA-u2 chain VLA-a3 chain
FL)
VLA-a4
chain
VLA-(~5 chain VLA-u6 chain VLA-J31 chain
202
VOLPES ET AL.
GASTROENTEROLOGY Vol. 101, No. 1
Table 3. Distribution ofthe Various Single VLA Chains in Normal Liver Tissue Vascular endothelium
Bile duct epithelium
Connective tissue stroma
+ +++
+
VLA-al
++
VLA-(uz VLA-ol3 VLA-a4 VLA-a5
+ + ++
++
VLA-ci6 VLA-l31
++ +++
+ +++
Sinusoidal lining cells
Mononuclear inflammatory cells
+
++
-
-
+I-
-
+
+
++ -
++
++
++
Hepatocytes
+
+++
-, No staining; +/-, variable staining; +, slight: + +, moderate; + + +, strong.
expression was also found on portal vascular endothelium but not on endothelium of the centrilobular vein. VLA-o14 was confined to few, scattered mononuclear cells, both in portal tracts and in the lobular parenchyma. Mabs B5E2 and B5GlO yielded the same results. VLA-a5 was found on all structures in portal tracts and lobular parenchyma, except for mononuclear inflammatory cells which were VLA-a5 negative. Hepatocytes and sinusoidal lining cells showed a weaker reactivity than bile duct epithelium and vascular endothelium. Mabs 16 and BIIG2 revealed identical patterns of immunoreactivity. VLA-~6 was restricted to portal vascular endothelium and bile duct epithelium, where it was selectively expressed on the basal cell membrane. The expression of VLA-pl chain corresponded to the sum of the reactivities of all six single VLA-ar chains. VLA-Pl was strongly expressed by vascular endothelium, bile duct epithelium, connective tissue stroma, and sinusoidal lining cells. Membranous staining of hepatocytes gave rise to a honeycomb pattern of immunoreactivity. In both portal tracts and in lobular parenchyma, mononuclear cells expressed VLA-pl. Inflammatory
and Cholestatic Liver Diseases
The distribution of VLA integrins in inflammatory and cholestatic liver disease is summarized in
Table 4 and illustrated in Figures 1 and 2, respectively. Compared with normal liver, hepatocytes showed increased VLA-al and VLA-cx5 membranous expression in liver biopsies with inflammation, without particular topographical correlation with the infiltrating inflammatory cells (Figure lA and B). De novo hepatocellular membranous VLA-ol3 and a6 positivity was found diffusely throughout the liver parenchyma in acute hepatitis and was restricted to periportal areas of inflammation in chronic active hepatitis (Figure 1C and D). Moreover, expression of VLA-(~2, (~3, and a6 was found on periportal hepatocytes in cases of cholestatic liver disease. Sinusoidal lining cells showed de novo VLA-(~4, as well as enhanced VLA-ol5 expression in periportal and intralobular areas of inflammation; this positivity was found both on sinusoidal endothelial cells and on Kupffer’s cells (Figure 1E’). Mononuclear inflammatory cells were numerous both in portal tracts and lobular parenchyma in inflammatory liver disease and showed strong VLA-a4 expression (Figure 1E). VLA-al expression by inflammatory cells was highly variable. Portal vascular endothelium and bile duct epithelium did not show relevant changes in VLA expression; proliferating bile ductules in cholestatic liver disease showed a strong VLA-(~2 and VLA-a6 but a weak VIA-(~3 expression (Figure 2). The connective
Table 4. Modifications in the Distribution of the Single VZA Chains in Inflammatory Bile duct epithelium
Vascular endothelium I
C
I
C
VIA-al
VLA-(r2 VLA-a3 VLA-(~4 VLA-(Y5 VLA-(Y6 VLA-I31
+++ ++
Connective tissue stroma C
I
+++
+++
+++
++
++ ++
++
NOTE. Only changes in VLA expression illustrated in Table 3. I, inflammation; C, cholestasis.
+ +++ +++
Sinusoidal lining cells
Hepatocytes
I
+++ ++ +++
and Cholestatic Liver Biopsy Specimens
C
I
C
Mononuclear inflammatory cells I
C
+
+ ++ ++ ++
++t
++
compared with normal liver are reported. Empty spaces represent unmodified
VLA expression
as
July 1991
VLA INTEGRINS IN HUMAN LIVER TISSUE
Figure 1. Staining tory liver disease.
for VLA integrins
in inflamma-
A-C.
Chronic active hepatitis with cirrhosis. VLA-al is up-regulated on the surface of hepatocytes, as well as in the connective tissue stroma (A). Similarly, VLA-a5 shows enhanced expression on hepatocytes (B). VLA-(~6 is de novo expressed by hepatocytes in areas of inflammation (C).
D. Acute hepatitis.
VLA-a3 is de novo found on scattered periportal hepatocytes (arrowheads).
E. Chronic
active hepatitis. Numerous mononuclear inflammatory cells express VLA-a4, which is also found on sinusoidal lining cells (three-step indirect immunoperoxidase with Mab TS2/7 (A), Mab 16 (B), Mab GoH3 (C), Mab 1143 (D), and Mab B5E2 (E), counterstained with Harris’ hematoxylin; original magnification x250).
203
204 VOLPES ET AL.
Figure 2. Staining for VLA-a3 in cholestatic liver disease. VLA-(~3 is strongly expressed by bile ductules and is also present on periportal hepatocytes. The same expression pattern was found for VLA-a2 (three-step indirect immunoperoxidase with Mab J143,counterstained with Harris’ hematoxylin; original magnification X 400).
tissue stroma in inflamed portal tracts, as well as in fibrotic septa in cases of cirrhosis, showed enhanced VLA-~1, a2, a4, and a5 positivity. Discussion Cellular adhesion is mediated by a large number of cell surface molecules that serve as specific receptors for various ligands. The integrin family mediates both cell-cell and cell-matrix interactions. The VLA-pl subgroup of integrins serves as receptor for extracellular matrix components, such as laminin (VLA-1, 3, and 6), collagen, (VLA-1, 2, and 3), and fibronectin (VLA-4, and 5), as well as for molecules expressed by endothelial cells during leukocyte homing (VLA-4). In this study we have analyzed their distribution in normal human liver biopsy specimens to obtain data on adhesion events in normal conditions; their distribution in inflammatory and cholestatic liver diseases was studied to clarify whether these molecules play a role in liver diseases. The common p-1 subunit was expressed by virtually all structures in both portal tracts and lobular parenchyma, whereas staining for the various a-subunits revealed differential patterns of distribution on the various components of human liver tissue.
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Vol. 101, No. 1
VIA-1 is a receptor for laminin and collagen (6,11). In normal liver as well as in inflammatory and cholestatic liver diseases, VIA-1 was expressed on vascular endothelium, connective tissue stroma, sinusoidal lining cells, and hepatocytes but was constantly absent from bile duct epithelium. A diffuse increased hepatocellular membranous VLA-1 expression was found in both acute and chronic hepatitis; this is in agreement with the enhanced VLA-1 expression by the epithelium of inflamed intestinal mucosa (12) and suggests that VLA-1 up-regulation is a general adaptative response to prevent epithelial loss as a result of the ongoing tissue injury. VLA-2 is a receptor for collagen (13)~ although endothelial cells can use VLA-2 as laminin receptor (14). In normal liver tissue, VLA-2 was found on vascular endothelia, bile duct epithelium, connective tissue stroma, and occasionally on sinusoidal lining cells, whereas hepatocytes and mononuclear cells were VLA-2 negative. The enhanced VLA-2 expression in the connective tissue stroma in inflammatory and cholestatic liver diseases could reflect an increased collagen synthesis by various cell types (15,16). Moreover, de novo VLA-2 expression by periportal hepatocytes in cholestatic biopsy specimens and up-regulation of this integrin on proliferating bile ductules suggest that collagen plays a role in the “bile ductular reaction” and in the interaction of the ductules with surrounding liver tissue during cholestasis. VLA-3 is a multispecific receptor for laminin, collagen, and fibronectin (17). In normal liver, VLA-3 expression was confined to the basolateral membrane of bile duct epithelium and to portal vascular endothelium. Its de novo expression on periportal hepatocytes in inflammatory and cholestatic liver specimens is probably related to the function of VLA-3 as laminin receptor (see below). VLA-4 is expressed by the memory T-cell subset (18) and is involved in cell-cell interactions, because it participates in the binding of leukocytes to endothelial cells during lymphocyte homing (1920). In normal and inflamed liver biopsy specimens, VLA-4 expression was restricted to mononuclear inflammatory cells present in portal tracts and lobular parenchyma. Because VLA-4 is engaged in heterotypic interactions with the VCAM-1 expressed by activated endothelial cells (21), it is tempting to speculate that mononuclear inflammatory cells use VLA-4 as an adhesion molecule to bind to activated sinusoidal liver endothelium. This hypothesis is furthermore supported by our finding that VCAM-1 is de novo expressed by sinusoidal endothelial cells in areas of liver inflammation (R. Volpes, unpublished data). VLA-5 is expressed by adhering cells as receptor for fibronectin (22), a component of fibrillar extracellular
VLA INTEGRINS IN HUMAN LIVER TISSUE
July 1991
structures and of basal lamina in all tissues, which plays a central role in cell adhesion and migration (23). In normal liver tissue VLA-5 was found on vascular endothelia, bile duct epithelium, sinusoidal lining cells, and hepatocytes. In line with the active production of fibronectin by various cell types during active liver disease (15), we observed a strong expression of VLA-5 in the connective tissue stroma of portal tracts in both acute and chronic hepatitis, in particular in the inflamed fibrotic septa in active cirrhosis. Moreover, up-regulation of VLA-5 expression was found on hepatocytes and sinusoidal lining cells in areas of inflammation in both acute and chronic inflammatory liver disease. This finding is in line with the enhanced hepatocellular synthesis and secretion of fibronectin (24) and its deposition in areas of inflammation during chronic active liver disease (25). This suggests a role for VIA-5-positive hepatocytes in liver regeneration after tissue injury (26). Moreover, because sinusoidal lining cells show enhanced expression of VLA-5 in areas of inflammation and because VLA-5 and VLA-4 each bind to different fragments of fibronectin (27), a fibronectin-mediated binding might be one of the mechanisms by which VLA-&positive lymphocytes adhere to VLA-5-positive sinusoidal liver endothelial cells. VLA-6 is the classical receptor for laminin (28). Its restricted presence on portal vascular endothelium and on the basal membrane of bile duct epithelium parallels the occurrence of laminin in basement membranes (29). The increased VLA-6 expression on proliferating bile ductules in cholestatic liver biopsy specimens suggests a role for VLA-6 in the anchorage of the neoformed bile ductules in the stroma. In contrast to normal conditions, VLA-6 was found on hepatocytes in focal areas of inflammation, as well as in periportal areas in cholestatic liver disease. De novo VLA-6 and, to a lesser extent, VLA-3 expression by hepatocytes in inflammatory and cholestatic liver diseases is in line with the hepatocellular production of basement membrane laminin in active liver disease (25) and might contribute to the reconstitution of the original architecture by allowing regenerating hepatocytes to attach to the pericellular environment (26). Finally, the de novo expression by periportal hepatocytes in cholestatic biopsy specimens of those VLA molecules (VLA-2, VLA-3, and VIA-6) normally expressed on bile duct epithelium illustrates a phenotypic switch of hepatocytes towards bile duct epithelium during cholestasis. Together, these data provide insights into the role of VLA integrins in cell-cell and cell-matrix interactions in the liver under normal and pathological conditions. The modification in VLA expression observed in inflammatory and cholestatic liver diseases suggests that the modulating signals generated through
205
the various VLA receptors are important steps in the regulation of interactions in conditions of liver cell damage, repair, and architectural rearrangement as well as of leukocyte migration during inflammatory responses. References 1. Williams AF, Barclay AN. The immunoglobulin
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Received July 12,1999. Accepted December 1,199O. Address requests for reprints to: Riccardo Volpes, M.D., Department of Pathology, Laboratory of Histochemistry and Cytochemistry, University Hospital St. Rafael, Catholic University of Leuven, Minderbroedersstraat 12, B-3600 Leuven, Belgium. This study was supported by a grant from the Fonds voor Geneeskundig Wetenschappelijk Onderzoek (FGWO), Belgium. Dr. M. E. Hemler (Harvard Medical School, Boston, MA), Dr. A. E. G. K. von dem Borne (University of Amsterdam, The Netherlands), Dr. L. J. Old (Memorial Sloan-Kettering Cancer Center, New York, NY), Dr. E. Klein (University of Ulm, Germany), Dr. K. M. Yamada (National Institutes of Health, Bethesda, MD), and Dr. C. Damsky (University of San Francisco, CA) are greatly acknowledged for their generous gifts of monoclonal antibodies. The authors thank C. Van den Broeck for excellent technical assistance and M. Rooseleers for preparation of the photographs.