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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

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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.

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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

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Authors and Affiliations

  1. Department of Medical and Physiological Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden

    Bård Smedsrød

  2. Department of Medical and Physiological Chemistry, University of Uppsala, Uppsala, Sweden

    Håkan Pertoft & Gösta Eggertsen

  3. Department of Pathology, University of Uppsala, Uppsala, Sweden

    Christer Sundström

Authors
  1. Bård Smedsrød
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  2. Håkan Pertoft
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  3. Gösta Eggertsen
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  4. Christer Sundström
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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

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