Characterization of mutated transforming growth factor-β1 transformants and its modulatory effect on IgA isotype synthesis by murine B lymphocytes

Kijong Rhee, Sang Uk Nham, Jin Su Yoo, Se Won Yie, Gie Taek Chun, Eui Yul Choi, Joo Hung Park, Pyeung Hyeun Kim

Research output: Contribution to journalArticle

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Abstract

Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine having both inhibitory and stimulatory effects on the differentiation of a variety of cell types. In the context of B cell differentiation, it has been shown that purified TGF-β1 increases IgA isotype switching by murine and human B cells in vitro. However, it has not been formally accepted whether TGF-β1 is actually an important inducer of IgA synthesis in vivo. In addition, it is difficult to evaluate the autocrine and paracrine effect because endogenous TGF-β1 is secreted not only in small amounts but in an inactive form. As a first step in understanding the role of TGF-β1 in induction of IgA synthesis in vivo, CHO cells were stably transfected with mutated TGF-β1 cDNA under the control of a metallothionein promoter, and the biological function of the expressed proteins was characterized in a variety of ways. TGF-β1-transfected CHO cells (TT-CHO cells) induced by zinc sulfate without any artificial activation were found to secrete at least 5,000 pg/ml as measured by ELISA. In addition, the secreted TGF-β1 adhered to soluble type II TGF-β1 receptors. A bioassay using Mv1Lu cells showed that the supernatant from TT-CHO cells retained not less than 8,000 pg/ml of TGF-β1, confirmed by a blocking experiment with anti-TGF-β1 antibody. These results indicate that the mutated recombinant TGF-β1 expressed from TT-CHO cells is fully biologically active. We were then interested in the effect of the produced rTGF-β1 on IgA isotype synthesis by mouse spleen B cells. The TT-CHO cell supernatant, in combination with rIL-2, substantially increased IgA isotype production. Finally, LPS-activated murine spleen B cells were transfected transiently with mutated TGF-β1 cDNA, resulting in IgA isotype induction. Our results suggest that B cell transfectants produce rTGF-β1 in an active form that can modulate its own differentiation in an autocrine fashion.

Original languageEnglish
Pages (from-to)746-752
Number of pages7
JournalMolecules and cells
Volume6
Issue number6
Publication statusPublished - 1996 Dec 31

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Transforming Growth Factors
Immunoglobulin A
B-Lymphocytes
CHO Cells
Spleen
Complementary DNA
Immunoglobulin Class Switching
Zinc Sulfate
Growth Factor Receptors
Metallothionein
Biological Assay
Cell Differentiation
Enzyme-Linked Immunosorbent Assay

All Science Journal Classification (ASJC) codes

  • Medicine(all)

Cite this

Rhee, Kijong ; Nham, Sang Uk ; Yoo, Jin Su ; Yie, Se Won ; Chun, Gie Taek ; Choi, Eui Yul ; Park, Joo Hung ; Kim, Pyeung Hyeun. / Characterization of mutated transforming growth factor-β1 transformants and its modulatory effect on IgA isotype synthesis by murine B lymphocytes. In: Molecules and cells. 1996 ; Vol. 6, No. 6. pp. 746-752.
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abstract = "Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine having both inhibitory and stimulatory effects on the differentiation of a variety of cell types. In the context of B cell differentiation, it has been shown that purified TGF-β1 increases IgA isotype switching by murine and human B cells in vitro. However, it has not been formally accepted whether TGF-β1 is actually an important inducer of IgA synthesis in vivo. In addition, it is difficult to evaluate the autocrine and paracrine effect because endogenous TGF-β1 is secreted not only in small amounts but in an inactive form. As a first step in understanding the role of TGF-β1 in induction of IgA synthesis in vivo, CHO cells were stably transfected with mutated TGF-β1 cDNA under the control of a metallothionein promoter, and the biological function of the expressed proteins was characterized in a variety of ways. TGF-β1-transfected CHO cells (TT-CHO cells) induced by zinc sulfate without any artificial activation were found to secrete at least 5,000 pg/ml as measured by ELISA. In addition, the secreted TGF-β1 adhered to soluble type II TGF-β1 receptors. A bioassay using Mv1Lu cells showed that the supernatant from TT-CHO cells retained not less than 8,000 pg/ml of TGF-β1, confirmed by a blocking experiment with anti-TGF-β1 antibody. These results indicate that the mutated recombinant TGF-β1 expressed from TT-CHO cells is fully biologically active. We were then interested in the effect of the produced rTGF-β1 on IgA isotype synthesis by mouse spleen B cells. The TT-CHO cell supernatant, in combination with rIL-2, substantially increased IgA isotype production. Finally, LPS-activated murine spleen B cells were transfected transiently with mutated TGF-β1 cDNA, resulting in IgA isotype induction. Our results suggest that B cell transfectants produce rTGF-β1 in an active form that can modulate its own differentiation in an autocrine fashion.",
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Characterization of mutated transforming growth factor-β1 transformants and its modulatory effect on IgA isotype synthesis by murine B lymphocytes. / Rhee, Kijong; Nham, Sang Uk; Yoo, Jin Su; Yie, Se Won; Chun, Gie Taek; Choi, Eui Yul; Park, Joo Hung; Kim, Pyeung Hyeun.

In: Molecules and cells, Vol. 6, No. 6, 31.12.1996, p. 746-752.

Research output: Contribution to journalArticle

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AU - Rhee, Kijong

AU - Nham, Sang Uk

AU - Yoo, Jin Su

AU - Yie, Se Won

AU - Chun, Gie Taek

AU - Choi, Eui Yul

AU - Park, Joo Hung

AU - Kim, Pyeung Hyeun

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