Summary
This paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens.
The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Iaantigens are not present on the LEC.
Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.
Similar content being viewed by others
Abbreviations
- KC :
-
Kupffer cells
- LEC :
-
Liver endothelial cells
- C :
-
Complement
- C3b :
-
Major fragment of C3 activation
- iC3b :
-
C3b that has been cleaved by factor I (C3b inactivator), present in serum
- meC3b :
-
C3b produced by treating purified human C3 with methyl amine
- trC3b :
-
C3b produced by treating purified human C3 with trypsin
- CR :
-
Complement receptors for C3b and iC3b
- IgG :
-
Immune globulin G
- IgM :
-
Immune globulin M
- E :
-
Erythrocytes
- E-IgG :
-
E covered with anti-E IgG
- E-IgM E :
-
covered with anti-E IgM
- E-C3b(h) :
-
E-IgM reacted with purified human C1, C4, oxidized C2 and C3 (E-IgMC14xyC2C3b)
- E-iC3b(m) :
-
E-IgM incubated with C5 deficient serum from AKR mice
- FcR :
-
Receptors for the Fc portion of IgG
- FITC :
-
Fluorescein isothiocyanate
- FITC-meC3b :
-
FITC conjugated to meC3b
- FITC-trC3b :
-
FITC conjugated to trC3b
- FA :
-
Fluorescein amine
- FA-OA :
-
Ovalbumin conjugated with FA
- FA-SA :
-
Serum albumin conjugated with FA
- FA-FSA :
-
Formaldehyde-treated serum albumin conjugated with FA
- Ia :
-
Immune response-associated AcE Acid unspecific esterase acting on alpha naphtyl acetate
- NASDAE :
-
Unspecific esterase acting on naphthol AS-D acetate
- NASDCAE :
-
Unspecific esterase acting on napthol AS-D chloroacetate
References
Bianco C, Griffin FM, Silverstein SC (1975) Studies of the macrophage complement receptor. Alteration of receptor function upon macrophage activation. J Exp Med 141:1278–1290
Blomhoff R, Eskild W, Berg T (1984a) Endocytosis of formaldehyde-treated serum albumin via scavenger pathway in liver endothelial cells. Biochem J 218:81–86
Blomhoff R, Smedrød B, Eskild W, Granum PE, Berg T (1984b) Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin. Exp Cell Res 150:194–203
Blouin A, Bolender RP, Weibel E (1977) Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. II. A stereological study. J Cell Biol 72:441–445
Bokisch VA, Müller-Eberhard HJ, Cochrane CG (1969) Isolation of fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum. J Exp Med 129:1109–1130
Borsos T, Rapp HJ (1967) Immune hemolysis: a simplified method for the preparation of EAC′4 with guinea pig or with human complement. J Immuno 199:263–268
Brouwer A, Barelds RJ, de Leeuw AM, Knook DL (1982) Maintenance cultures of Kupffer cells as a tool in experimental liver research. In: Knook DL, Wisse E (eds) Sinusoidal liver cells. Elsevier Biomedical Press, Amsterdam New York and Oxford, pp 327–334
Cohn ZA, Benson BJ (1965) The in vitro differentiation of mononuclear phagocytes. II. The influence of serum on granule formation, hydrolase production, and pinocytosis. J Exp Med 121:153–170
Cooper NR, Müller-Eberhard HJ (1968) A comparison of methods for the molecular quantitation of the fourth component of human complement. Immunochemistry 5:155–169
Emeis JJ, Planqué JJ (1976) Heterogeneity of cells isolated from rat liver by pronase digestion: ultrastructure, cytochemistry and cell culture. J Reticuloendothel Soc 20:11–29
Eriksson S, Fraser JRE, Laurent TC, Pertoft H, Smedsrød B (1983) Endothelial cells are a site of uptake and degradation of hyaluronic acid in the liver. Exp Cell Res 144:223–228
Graham RC, Karnovsky MJ (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney-ultrstructural cytochemistry by a new technique. J Histochem Cytochem 14:291–302
Hubbard AL, Wilson G, Ashwell G, Stukenbrok H (1979) An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125Iligands among the liver cell types. J Cell Biol 83:47–64
Hynes RO, Yamada KM (1982) Fibronectins: multifunctional modular glycoproteins. J Cell Biol 95:369–377
Jacob AI, Goldberg PK, Bloom N, Degensheim GA, Kozinn PS (1977) Endotoxin and bacteria in the portal blood. Gastroenterology 72:1268–1270
Johnson E, Bøgwald J, Eskeland T, Seljelid R (1983) Complement (C3) receptor-mediated attachment of agarose beads to mouse peritoneal macrophages and human monocytes. Scand J Immunol 17:403–410
Klareskog L, Forsum U, Wigzell H (1982) Murine synovial intima contains I-A-, I-E/C-positive bone-marrow-derived cells. Scand J Immunol 15:509–514
Kornfeld R, Kornfeld S (1980) Structure of glycoproteins and their oligosaccharide units. In: Lennartz JL (ed) The biochemistry of glycoproteins and proteoglycans. Plenum Press, New York, London, pp 1–34
Lagerlöf B, Sundström C, Öst Å (1979) Cytochemical stains of value in hematological disorders. Läkartidningen 76:4495–4498
Leeuw A-M de, Barelds RJ, de Zanger R, Knook DL (1982a) Primary cultures of endothelial cells of the rat liver. A model for ultrastructural and functional studies. Cell Tissue Res 223:201–215
Leeuw AM de, Martindale JE, Knook DL (1982b) Cultures and cocultures of rat liver Kupffer, endothelial and fat-storing cells. In: Knook DL, Wisse E (eds) Sinusoidal liver cells. Elsevier Biomedical Press, Amsterdam New York Oxford, pp 139–146
Leeuw AM de, Brouwer A, Barelds RJ, Knook DL (1983) Maintenance cultures of Kupffer cells isolated from rats of various ages: ultrastructure, enzyme cytochemistry, and endocytosis. Hepatology 3:497–506
Lundwall Å, Hellman U, Eggertsen G, Sjöquist J (1982) Isolation of tryptic fragments of human C4 expressing Chido and Rodgers antigens. Mol Immunol 19:1655–1665
Lundwall Å, Hellman U, Eggertsen G, Sjöquist J (1984) Chemical characterization of cyanogen bromide fragments from the ß- chain of human complement factor C3. FEBS 169:57–62
Mego JL, Bertini F, McQueen JD (1967) The use of formaldehydetreated 131I-albumin in the study of digestive vacuoles and some properties of these particles from mouse liver. J Cell Biol 32:699–707
Müller-Eberhard HJ (1977) Preparation of hemolytic intermediates from human complement. In: Williams CA, Chase MW (eds) Methods in immunology and immunochemistry. Academic Press, Inc. New York, San Francisco, London, pp 176–180
Mørland B, Kaplan G (1977) Macrophage activation in vivo and in vitro. Exp Cell Res 108:279–288
Munthe-Kaas AC, Berg T, Seglen PO, Seljelid R (1975) Mass isolation and culture of rat Kupffer cells. J Exp Med 141:1–10
Munthe-Kaas AC, Kaplan G, Seljelid R (1976) On the mechanism of internalization of opsonized particles by rat Kupffer cells in vitro. Exp Cell Res 103:201–212
Neufeld EF, Ashwell G (1980) Carbohydrate recognition systems for receptor-mediated pinocytosis. In: Lennarz WJ (ed) The biochemistry of glycoproteins and proteoglycans. Plenum Press, New York, London, pp 241–266
Niesen TE, Alpers DH, Stahl PD, Rosenblum JL (1984) Metabolism of glycosylated human salivary amylase: in vivo plasma clearance by rat hepatic endothelial cells and in vitro receptor mediated pinocytosis by rat macrophages. J Leukocyte Biol 36:307–320
Polley MJ, Müller-Eberhard HJ (1968) The second component of human complement: its isolation, fragmentation by C′1 esterase, and incorporation into C′3 convertase. J Exp Med 128:531–551
Pommier CG, Inada S, Fies LF, Tokahashi T, Frank MM, Brown EJ (1983) Plasma fibronectin enhances phagocytosis of opsonized particles by human peripheral blood monocytes. J Exp Med 157:1844–1854
Praaning-van Dalen DP, de Leeuw AM, Brouwer A, de Ruiter GCF, Knook DL (1982) Ultrastructural and biochemical characterization of endocytic mechanisms in rat liver Kupffer and endothelial cells. In: Knook DL, Wisse E (eds) Sinusoidal liver cells. Elsevier Biomedical Press, Amsterdam, New York, Oxford, pp 271–278
Pulford K, Souhami RL (1981) The surface properties and antigenpresenting function of hepatic non-parenchymal cells. Clin Exp Immunol 46:581–588
Rabinovitch M (1967) Attachment of modified erythrocytes to phagocytic cells in the absence of serum. Proc Soc Exp Biol Med 124:396–399
Rabinovitch M, De Stefano MJ (1973 a) Macrophage spreading in vitro. Exp Cell Res 77:323–334
Rabinovitch M, De Stefano MJ (1973b) Particle recognition by cultivated macrophages. J Immunol 110:695–701
Reynolds HY, Atkinson JP, Newball HH, Frank MM (1975) Receptors for immunoglobulin and complement on human alveolar macrophages. J Immunol 114:1813–1819
Seljelid R (1980) Properties of Kupffer cells. In: van Furth R (ed) Mononuclear phagocytes. Functional aspects. Martinus Nijhoff Publishers The Hague, Boston London, pp 157–199
Smedsrød B, Pertoft H (1985) Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence. J Leukocyte Biol 38, no 1
Smedsrød B, Eriksson S, Fraser JRE, Laurent TC, Pertoft H (1982) Properties of liver endothelial cells in primary monolayer cultures. In: Knook DL, Wisse E (eds) Sinusoidal liver cells. Elsevier Biomedical Press, Amsterdam New York Oxford, pp 263–270
Smedsrød B, Pertoft H, Eriksson S, Fraser JRE, Laurent TC (1984) Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells. Biochem J 223:617–626
Steffan A-M, Lecerf F, Keller F, Cinqualbre J, Kirn A (1981) Isolement et culture de cellules endothéliales de foies humain et murin. CR Acad Sc Paris, t292, pp 809–815
Summerfïeld JA, Vergalla J, Jones EA (1982) Modulation of a glycoprotein recognition system on rat hepatic endothelial cells by glucose and diabetes mellitus. J Clin Invest 69:1337–1347
Sundström C, Nilsson K (1977) Cytochemical profile of human haematopoietic biopsy cells and derived cell lines. Br J Haematol 37:489–501
Vuento M, Vaheri A (1979) Purification of fibronectin from human placenta by affinity chromatography under non-denaturing conditions. Biochem J 183:331–337
Wisse E (1972) An ultrastructural characterization of the endothelial cell in the rat liver sinusoidal under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells. J Ultrastruct Res 38:528–562
Wisse E, van't Noordende JM, van der Meulen J, Daens WTh (1976) The pit cell: description of a new type of cell occurring in rat liver sinusoids and peripheral blood. Cell Tissue Res 173:423–435
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Smedsrød, B., Pertoft, H., Eggertsen, G. et al. Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll, and selective substrate adherence. Cell Tissue Res. 241, 639–649 (1985). https://doi.org/10.1007/BF00214586
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00214586