Establishment of a long-term culture system for rat colon epithelial cells

The regulation of the synthesis by the cytokines interleukin-1 (IL-1) and IL-6 of the positive acute-phase protein alpha 1-acid glycoprotein (AGP) and of the negative acute-phase protein alpha 2-HS glycoprotein (AHSG) has been studied in a long-term culture system of the human hepatoma cell line Hep3B. The culture system contained 30 nM-sodium selenite as the only supplement. This allowed maintenance of the synthesis of the proteins under study at a near steady state for over 3 months. An increase in AGP mRNA and a decrease in AHSG mRNA were observed when cells were treated for two successive 48 h-periods with monocyte-conditioned medium. A return to basal levels was obtained after cessation of the cytokine addition. Two further additions of cytokines led to alterations in mRNA levels similar to those observed following the first cytokine treatment. The amounts of AGP and AHSG secreted were altered in accordance with the mRNA modifications. These results suggest that new cytokine receptors were being constantly synthesized during cell culture. When cytokines were present in the culture medium for 10 days, maximum alterations in AGP and AHSG synthesis were obtained following 2 and 4 days of treatment respectively, but further alterations in protein levels could not be observed afterwards. Expression of IL-6 receptor mRNA was not up-regulated by cytokines, but only by 1 microM-dexamethasone. Our results show that, in this long-term culture system, cytokines induce a response in hepatoma cells similar to that observed in vivo during human inflammatory states. This model could be used to evaluate the effects of agonists or antagonists of cytokines responsible for the hepatic acute-phase protein response.

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Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

Virologica Sinica ◽

10.1007/s12250-020-00253-y ◽

2020 ◽

Vol 35 (3) ◽

pp. 311-320 ◽

Cited By ~ 3

Author(s):

Siyu Xia ◽

Ming Wu ◽

Si Chen ◽

Tao Zhang ◽

Lina Ye ◽

...

Keyword(s):

Epithelial Cells ◽

Proximal Tubule ◽

Ex Vivo ◽

Kidney Injury ◽

Human Kidney ◽

Ex Vivo Model ◽

Long Term Culture ◽

Kidney Proximal Tubule

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Secretion of cytokines by rat alveolar epithelial cells: possible regulatory role for SP-A

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.2.l148 ◽

1994 ◽

Vol 266 (2) ◽

pp. L148-L155 ◽

Cited By ~ 9

Author(s):

H. Blau ◽

S. Riklis ◽

V. Kravtsov ◽

M. Kalina

Keyword(s):

Epithelial Cells ◽

Alveolar Epithelial Cells ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Type Ii Cells ◽

Long Term Culture ◽

Alveolar Epithelial ◽

Gm Csf

Cultured alveolar type II cells and pulmonary epithelial (PE) cells in long-term culture were found to secrete colony-stimulating factors (CSF) into the medium in similar fashion to alveolar macrophages. CSF activity was determined by using the in vitro assay for myeloid progenitor cells [colony-forming units in culture (CFU-C)]. Both lipopolisaccharide (LPS) and interleukin-1 alpha (IL-1 alpha) were found to upregulate the secretion 6.5- to 8-fold from alveolar type II cells and macrophages. However, no stimulatory effect of these factors was observed in PE cells that release CSF into the medium constitutively, possibly due to the conditions of long-term culture. The CSF activity was partially neutralized (70% inhibition) by antibodies against murine granulocyte/macrophage (GM)-CSF and IL-3, thus indicating the presence of both GM-CSF and IL-3-like factors in the CSF. However, the presence of other cytokines in the CSF is highly probable. Surfactant-associated protein A (SP-A), which is known to play a central role in surfactant homeostasis and function, was also found to upregulate secretion of CSF (at concentrations of 0.1-5 micrograms/ml) from alveolar type II cells and macrophages. Control cells such as rat peritoneal macrophages, alveolar fibroblasts, and 3T3/NIH cell line could not be elicited by SP-A to release CSF. The results are discussed in relation to the possible participation of the alveolar epithelial cells in various intercellular signaling networks. Our studies suggest that alveolar type II cells and SP-A may play an important regulatory role in the modulation of immune and inflammatory effector cells within the alveolar space.

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Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor

Blood ◽

10.1182/blood.v83.9.2594.2594 ◽

1994 ◽

Vol 83 (9) ◽

pp. 2594-2601 ◽

Cited By ~ 146

Author(s):

JS Miller ◽

KA Alley ◽

P McGlave

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Culture System ◽

Interleukin 2 ◽

Early Event ◽

Hematopoietic Progenitors ◽

Long Term Culture ◽

Lineage Differentiation ◽

Marrow Stroma

Abstract We have recently described a marrow stroma-dependent long-term culture system that supports differentiation of CD34+ human marrow primitive progenitors into natural killer (NK) cells. We postulate that CD7 expression may be an early event in commitment of hematopoietic progenitors to the NK lineage. Here we compare the characteristics of CD34+7- and CD34+7+ marrow cells cultivated in the stroma-based NK culture system. These CD34+ populations were further compared with a marrow derived, more committed, CD34–7+ progenitor to emphasize the continuum of NK development and to highlight differences between progenitors in our assays. No progenitor proliferated when plated in media without stroma, underscoring the importance of stroma in NK differentiation. Plating progenitor populations in interleukin-2 containing media directly on preestablished, allogeneic, irradiated marrow stroma for 5 weeks resulted in CD56+CD3- NK cells; however, characteristics of the cultured populations differed. Fold expansion and cloning efficiency of the CD34+7+ population, determined by a functional limiting dilution assay was significantly higher than of the CD34+7- or CD34+7+ populations. This suggests that the CD34+7+ population is highly enriched for an NK progenitor and a possible intermediate in NK lineage differentiation. Further dividing the CD34+7+ population by the relative fluorescence of CD7 into CD34+7+dim and CD34+7+bright populations showed that the CD34+7+bright population exhibited a significantly higher cloning frequency than parallel experiments with CD34+7+dim cells (11.8% +/- 2.4% v 2.4% +/- 0.7%, n = 6; P = .005). Plating of the more primitive CD34+7- population in a transwell system (which separates progenitors from stroma by a microporous membrane) prevents differentiation into NK cells. In contrast, plating of CD34+7+ progenitors in transwells resulted in generation of NK cells. These data suggest that primitive, but not more mature NK progenitors may require direct contact with stroma for the initial differentiation steps. Finally, differentiation of the NK progenitors in this stroma-dependent model results in expression of CD2 not present on any of the starting populations. This observation suggests that marrow stroma can stimulate CD2 expression on NK progenitors in a previously undescribed fashion that may be analogous to the thymic effect on CD2 expression in immature T lymphocytes. These observations identify early steps in the commitment of primitive marrow CD34+ hematopoietic progenitors to a lymphoid lineage and underscore the importance of coexpression of CD7 with CD34 as an early lymphoid commitment characteristic and direct progenitor-stroma interactions in this process.

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