Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study

Heuff, Gijsbert; Steenbergen, Joke J. E.; Loosdrecht, Arjan A. van de; I, Sirovich; Dijkstra, C.D.; Meyer, S.; Beelen, Robert H.J.

doi:10.1016/0022-1759(93)90148-z

Cited by 22 publications

(15 citation statements)

References 19 publications

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“…(2007) should be only ≈ 7 × 10 3 KC g −1 , in strong contrast with our and other estimations (Emeis & Planque, 1976; Sleyster & Knook, 1982; Bouwens et al. 1986; Heuff et al. 1993).…”

Section: Discussioncontrasting

confidence: 99%

“…1995), leading, for example, to an oversampling of larger KC (Gundersen, 1986). Further, regarding quantitative data on KC, it is opportune to mention that cell isolation yields range between 3 and 12 × 10 6 KC g −1 of liver (Emeis & Planque, 1976; Sleyster & Knook, 1982; Heuff et al. 1993) or 80–120 × 10 6 KC per liver (Olynyk & Clarke, 1998; Valatas et al.…”

Section: Discussionmentioning

confidence: 99%

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An unbiased stereological study on subpopulations of rat liver macrophages and on their numerical relation with the hepatocytes and stellate cells

et al. 2009

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Studies on liver macrophages have elucidated their key roles in immunological, fibrotic and regenerative responses, and shown that macrophages are not a homogeneous population. In the rat, two sets of liver macrophages coexist, identified by ED1 and ED2 antibodies. Those sets have different quantitative responses in liver injuries and may have different tasks throughout the injury and recovery phases. Nevertheless, the total number (N), number per gram (N g -1 ) and proportion of those macrophages in relation to other liver cells has never been quantified using design-based stereology. Thus, we combined immunocytochemistry with those tools to produce an unbiased estimate of the N of ED1 + and of ED2 + cells. A smooth fractionator sampling scheme was applied to the liver of five male Wistar rats (3 months old), to obtain systematic uniform random sections (30 μ m thick); these were immunostained with the monoclonal antibodies: ED1, a pan-macrophagic marker; and ED2, which identifies the completely differentiated macrophages, i.e. Kupffer cells. The N of ED1 + cells was 340 × 10 6 , estimated with a coefficient of error (CE) of 0.04, and that of ED2 + cells was 283 × 10 6 , with a CE of 0.05. These figures correspond to 10.7% and 8.9%, respectively, of the total liver cells. The new data constitute reference values for correlative inferences. Also, the methodological strategy, by its accuracy and precision, is valuable for future investigations on the liver cell composition in various models of disease, and especially for studying the more subtle variations that occur during the injury and recovery phases.

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“…(2007) should be only ≈ 7 × 10 3 KC g −1 , in strong contrast with our and other estimations (Emeis & Planque, 1976; Sleyster & Knook, 1982; Bouwens et al. 1986; Heuff et al. 1993).…”

Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 99%

An unbiased stereological study on subpopulations of rat liver macrophages and on their numerical relation with the hepatocytes and stellate cells

et al. 2009

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“…12-085). After deep anesthesia by intravenous injection of sodium pentobarbital (20?mg/kg) and exsanguination, the lobes of the swine liver were dissected out and parenchymal hepatocytes were isolated by the perfusion of saline followed by collagenase into the portal vein [10,15,16]. Parenchymal hepatocytes were suspended in growth medium composed of DMEM (D6429, high-glucose type, Sigma-Aldrich, St. Louis, MO) containing 10% heat inactivated FCS (Sanko Junyaku Co. Ltd., Tokyo, Japan), supplemented with 100?µM ß-mercaptoethanol (M3148, Sigma-Aldrich), 10?µg/ml insulin (I5500, Sigma-Aldrich), 100?µg/ml streptomycin and 100?U/ml penicillin (15140-122, Life Technologies, Carlsbad, CA), and seeded into tissue culture flasks (surface area: 75?cm 2 , Sumitomo Bakelite Co., Ltd., Tokyo, Japan) at a density of 6.7 × 10 4?…”

Section: Methodsmentioning

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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“…Slides and cytospins were stained with Mayer's H&E (Klinipath, Duiven, The Netherlands), after which they were dehydrated in ethanol and embedded in Entallan (Merck, Haarlem, The Netherlands). The number of viable cells remaining in 96-well plates was determined with standard MTT assays as described by Heuff et al (28). The apoptosis inhibitor Z-VAD-fmk was purchased from Biocompare (San Francisco, CA).…”

Section: Adccmentioning

confidence: 99%

Targeting FcαRI on Polymorphonuclear Cells Induces Tumor Cell Killing through Autophagy

Bakema

Ganzevles

Fluitsma

et al. 2011

The Journal of Immunology

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Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B+ autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.

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Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study

Cited by 22 publications

References 19 publications

An unbiased stereological study on subpopulations of rat liver macrophages and on their numerical relation with the hepatocytes and stellate cells

An unbiased stereological study on subpopulations of rat liver macrophages and on their numerical relation with the hepatocytes and stellate cells

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

Targeting FcαRI on Polymorphonuclear Cells Induces Tumor Cell Killing through Autophagy

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