Long-term stimulation of trout head kidney cells with the cytokines MCSF, IL-2 and IL-6

gene expression dynamics

Yolanda Corripio-Miyar, Christopher J. Secombes, Jun Zou

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The production of salmonid leukocyte cell lines from primary cell cultures has been attempted on several occasions, however, to date only monocyte/macrophage like cell lines exist (e.g. RTS-11 and SHK-1 cells). With the increasing number of cytokines discovered in fish in recent years, many of which are growth factors for leukocytes, we now have the possibility of using these molecules to promote leukocyte development and differentiation in culture.

We have generated stable cell lines transfected with a variety of plasmids expressing cytokines (Interleukin (IL)-2, IL-6 and Macrophage Colony Stimulating Factor (MCSF)), in order to produce conditioned media rich in these cytokines. The cytokine-conditioned media were used to assess their activity and ability to support the growth of primary head kidney (HK) leukocyte cultures. Here, we describe a series of experiments aimed to determine which cell population(s) of primary HK cultures is supported and will grow in conditioned media containing MCSF, IL-2 or IL-6. For a period of 5 weeks, cells were incubated at 22 C and media were changed every 3-4 days. Samples were taken at different time points, from freshly isolated HK cells (TO), one week post-stimulation (1-WPS), 3-WPS and 5-WPS for RNA extraction. A variety of cell lineage markers (MCSF Receptor 2 (MCSFR2) for macrophages, CD4 and CD8a for T cells and IgM heavy chain for B cells) were then analysed by real-time qPCR to study the cell population dynamics as influenced by the different recombinant cytokines in the cultures. We show here that whilst MCSF appears to drive macrophage differentiation and maintenance, IL-2 and IL-6 seem to preferentially drive lymphocyte differentiation. (C) 2011 Elsevier Ltd. All rights reserved.

Original languageEnglish
Pages (from-to)35-44
Number of pages10
JournalFish & Shellfish Immunology
Volume32
Issue number1
Early online date25 Oct 2011
DOIs
Publication statusPublished - Jan 2012

Keywords

  • interleukin
  • cell line
  • long-term stimulation
  • teleost
  • conditioned media
  • chronic lymphocytic-leukemia
  • oncorhynchus-mykiss
  • In-Vitro
  • T-cells
  • macrophage growth
  • B-cells
  • lines
  • generation
  • identification
  • establishment

Cite this

Long-term stimulation of trout head kidney cells with the cytokines MCSF, IL-2 and IL-6 : gene expression dynamics. / Corripio-Miyar, Yolanda; Secombes, Christopher J.; Zou, Jun.

In: Fish & Shellfish Immunology, Vol. 32, No. 1, 01.2012, p. 35-44.

Research output: Contribution to journalArticle

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AB - The production of salmonid leukocyte cell lines from primary cell cultures has been attempted on several occasions, however, to date only monocyte/macrophage like cell lines exist (e.g. RTS-11 and SHK-1 cells). With the increasing number of cytokines discovered in fish in recent years, many of which are growth factors for leukocytes, we now have the possibility of using these molecules to promote leukocyte development and differentiation in culture.We have generated stable cell lines transfected with a variety of plasmids expressing cytokines (Interleukin (IL)-2, IL-6 and Macrophage Colony Stimulating Factor (MCSF)), in order to produce conditioned media rich in these cytokines. The cytokine-conditioned media were used to assess their activity and ability to support the growth of primary head kidney (HK) leukocyte cultures. Here, we describe a series of experiments aimed to determine which cell population(s) of primary HK cultures is supported and will grow in conditioned media containing MCSF, IL-2 or IL-6. For a period of 5 weeks, cells were incubated at 22 C and media were changed every 3-4 days. Samples were taken at different time points, from freshly isolated HK cells (TO), one week post-stimulation (1-WPS), 3-WPS and 5-WPS for RNA extraction. A variety of cell lineage markers (MCSF Receptor 2 (MCSFR2) for macrophages, CD4 and CD8a for T cells and IgM heavy chain for B cells) were then analysed by real-time qPCR to study the cell population dynamics as influenced by the different recombinant cytokines in the cultures. We show here that whilst MCSF appears to drive macrophage differentiation and maintenance, IL-2 and IL-6 seem to preferentially drive lymphocyte differentiation. (C) 2011 Elsevier Ltd. All rights reserved.

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KW - oncorhynchus-mykiss

KW - In-Vitro

KW - T-cells

KW - macrophage growth

KW - B-cells

KW - lines

KW - generation

KW - identification

KW - establishment

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