Purification of murine Kupffer cells by centrifugal elutriation

Janousek, Jaromir; Strmen, E.; Gervais, Francine

doi:10.1016/0022-1759(93)90281-b

Cited by 15 publications

(8 citation statements)

References 19 publications

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“…scribed in 1977 by Knook and Sleyster (1977). More recently the purification of KC from mice, using the same technique has been reported by other workers (Janousek et al. 1993).…”

Section: Introductionmentioning

confidence: 59%

Isolation and characterization of murine Kupffer cells and splenic macrophages

Hagen

Vianen

Bakker-Woudenberg

1996

Journal of Immunological Methods

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A method is described using counterflow centrifugation elutriation to isolate macrophages from murine liver and spleen. In this study three, size fractionated, macrophage populations were collected. Isolation resulted in a high yield of pure Kupffer cells (total of 10 x 10(6) /g liver) and enrichment of splenic macrophages to 20%. In addition to standard methods such as non-specific esterase staining, the isolated macrophages were also characterized by flow cytometry using specific monoclonal antibodies. In addition, a rapid flow cytometry method was introduced to determine the percentage of macrophages based on autofluorescence. A strong correlation was found between the percentages of macrophages found by non-specific esterase staining and autofluorescence. Functional tests revealed differences between the isolated macrophages in terms of tumor necrosis factor-alpha (TNF-alpha) production, oxygen metabolism and the production of nitric oxide. However, no significant differences in phagocytic activity was observed between the fractions. After two weeks of culture without the addition of antibiotics the cells still exhibited the above mentioned functions.

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“…scribed in 1977 by Knook and Sleyster (1977). More recently the purification of KC from mice, using the same technique has been reported by other workers (Janousek et al. 1993).…”

Section: Introductionmentioning

confidence: 59%

Isolation and characterization of murine Kupffer cells and splenic macrophages

Hagen

Vianen

Bakker-Woudenberg

1996

Journal of Immunological Methods

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“…The conventional method of liver-macrophages starts with dissociation by collagenase perfusion, pronase treatment to remove parenchymal hepatocytes and finally counterflow centrifugal elutriation to separate the liver-macrophages from other nonparenchymal cells [6,7]. In addition, various modifications of this method for specific animal species have been reported, including human [8,9] and bovine [10,11] applications.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the liver-macrophages isolated from a mixed primary culture of neonatal swine hepatocytes

Kitani

Yoshioka

Takenouchi

et al. 2014

Results in Immunology

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We recently developed a novel procedure to obtain liver-macrophages in sufficient number and purity using a mixed primary culture of rat and bovine hepatocytes. In this study, we aim to apply this method to the neonatal swine liver. Swine parenchymal hepatocytes were isolated by a two-step collagenase perfusion method and cultured in T75 culture flasks. Similar to the rat and bovine cells, the swine hepatocytes retained an epithelial cell morphology for only a few days and progressively changed into fibroblastic cells. After 5–13 days of culture, macrophage-like cells actively proliferated on the mixed fibroblastic cell sheet. Gentle shaking of the culture flask followed by the transfer and brief incubation of the culture supernatant resulted in a quick and selective adhesion of macrophage-like cells to a plastic dish surface. After rinsing dishes with saline, the attached macrophage-like cells were collected at a yield of 106 cells per T75 culture flask at 2–3 day intervals for more than 3 weeks. The isolated cells displayed a typical macrophage morphology and were strongly positive for macrophage markers, such as CD172a, Iba-1 and KT022, but negative for cytokeratin, desmin and a-smooth muscle actin, indicating a highly purified macrophage population. The isolated cells exhibited phagocytosis of polystyrene microbeads and a release of inflammatory cytokines upon lipopolysaccharide stimulation. This shaking and attachment method is applicable to the swine liver and provides a sufficient number of macrophages without any need of complex laboratory equipments.

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“…Notably, Kupffer cells retain their structural and functional characteristics in vitro. Cultivated Kupffer cells are known to synthesize cytokines such as tumor necrosis factor ␣, interleukin 1, interleukin 6, and transforming growth factor ␤, [27][28][29] to express major histocompatibility complex class I molecules as well as complement and Fc receptors, and are fully capable of phagocytosis and lysosomal digestion. [30][31][32] Using our in vitro model, we show that P. berghei and P. yoelii sporozoites actively invade and safely passage through Kupffer cells before hepatocyte invasion.…”

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confidence: 99%

Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion

2001

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Malaria sporozoites have to cross the layer of sinusoidal liver cells to reach their initial site of multiplication in the mammalian host, the hepatocytes. To determine the sinusoidal cell type sporozoites use for extravasation, endothelia or Kupffer cells, we quantified sporozoite adhesion to and invasion of sinusoidal cells isolated from rat liver. In vitro invasion assays reveal that Plasmodium berghei and P. yoelii sporozoites attach to and enter Kupffer cells, but not sinusoidal endothelia. Unlike hepatocytes and other nonphagocytic cells, which are invaded in vitro only within the first hour of parasite exposure, the number of intracellular sporozoites in Kupffer cells increases for up to 12 hours. By confocal and electron microscopy, sporozoites are enclosed in a vacuole that does not colocalize with lysosomal markers. Inhibition of phagocytosis with gadolinium chloride has no effect on Kupffer cell invasion, but abolishes phagocytosis of inactivated sporozoites. Malaria-infected anopheline mosquitoes inoculate Plasmodium sporozoites into the avascular dermis and subcutaneous connective tissue of the mammalian host. 1 Within minutes after entering a blood capillary at the mosquito bite site, the sporozoites travel in the bloodstream to the liver where they invade their initial target cells, the hepatocytes. 2 The arrest of the sporozoites in the liver sinusoid is thought to be mediated by the specific binding of the major Plasmodium sporozoite surface proteins, the circumsporozoite (CS) protein and the thrombospondin-related adhesive protein, to the unique heparan sulfate proteoglycans (HSPGs) of the liver. 3-5 For example, recombinant CS protein binds to the HSPGs on the basolateral cell surface of hepatocytes in the space of Disse. This interaction occurs between the conserved regions I and II-plus of the CS protein and highly sulfated, heparin-like oligosaccharides in heparan sulfate. 6 Furthermore, recombinant CS protein is selectively removed from the bloodstream by binding to liver HSPGs, 3,6,7 a clearance mechanism also used by lipoprotein remnants. [8][9][10][11] Since P. berghei sporozoites compete with ␤ very low density lipoprotein for binding to HSPGs, 12 it appears that malaria sporozoites utilize an existing clearance mechanism of the host for targeting the liver sinusoid.After the initial arrest in the liver sinusoid, Plasmodium sporozoites have to cross the sinusoidal cell layer prior to invasion of hepatocytes. The sinusoid is composed of fenestrated endothelial cells and interspersed with Kupffer cells. Kupffer cells are resident macrophages of the liver that are strategically positioned in the sinusoidal lumen and exhibit numerous pseudopodia. To date, the route that sporozoites use for extravasation has remained uncertain. [13][14][15] have proposed that the parasites infect hepatocytes directly by squeezing through the endothelial fenestration, although they exceed the maximum pore size of the sieve plates (0.1 m 16 ) by an order of magnitude. However, this event has never been observ...

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Purification of murine Kupffer cells by centrifugal elutriation

Cited by 15 publications

References 19 publications

Isolation and characterization of murine Kupffer cells and splenic macrophages

Isolation and characterization of murine Kupffer cells and splenic macrophages

Characterization of the liver-macrophages isolated from a mixed primary culture of neonatal swine hepatocytes

Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion

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