Immune Netw.  2015 Dec;15(6):313-318. 10.4110/in.2015.15.6.313.

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

• Jung Y

• KMID: 2132705
• DOI: http://doi.org/10.4110/in.2015.15.6.313

Abstract

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 microM dbcAMP or 0.5 microM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 microM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 microM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 microM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

Keyword

EoL-1 cells; DbcAMP; Butyric acid; In vitro differentiation; Eosinophils

MeSH Terms

Bucladesine
Butyric Acid*
Cytokines
Eosinophils*
Humans*
Hypereosinophilic Syndrome*
Leukocytes
Bucladesine
Butyric Acid
Cytokines

Figure

• 

  Figure 1 Effects of dbcAMP and butyric acid on the morphologic features and proliferation capacity of EoL-1 cells. (A) EoL-1 cells were incubated for the indicated periods in the absence or presence of 100 µM dbcAMP or 0.5 µM butyric acid. Cell number was adjusted to 5×105/ml every 3 days. Diff-Quik staining of unstimulated EoL-1 cells and EoL-1 cells stimulated with dbcAMP or butyric acid. Arrows denote cells showing nuclear lobulation and arrow heads indicate cells showing shrinkage and chromatin condensation. Original magnification, ×40. (B, C) EoL-1 cells were incubated for 8 or 9 days in medium containing indicated concentration of dbcAMP or butyric acid. The cells were then harvested and enumerated (B) and the viability of the cells was determined by flow cytometry analysis of 7-amino-actinomycin D (7-AAD) (C). All data are representative of two or more independent experiments. Data are mean±s.e.m. values. *p<0.05, **p<0.01, and ***p<0.001 (Student's t-test) vs. the control.

• 

  Figure 2 Effects of dbcAMP and butyric acid on the differentiation of EoL-1 cells. (A) cDNA was prepared from total RNA obtained from undifferentiated EoL-1 cells (day 0) and EoL-1 cells stimulated with indicated concentrations dbcAMP or butyric acid for 3 or 6 days. mRNA expressions of PRG2, EPX, CCR3, Il5RA, and GATA1 were analyzed by real-time PCR. Data are mean±s.e.m. values. *p<0.05, **p<0.01, and ***p<0.001 (Student's t-test) of 0.5 µM butyric acid vs. 100 µM dbcAMP stimulation. (B) mRNA expressions of PRG2, EPX, CCR3, Il5RA, and GATA1 of undifferentiated EoL-1 cells (control) and EoL-1 cells stimulated with 100 µM dbcAMP or 0.5 µM butyric acid for 3 or 6 days. Data are mean±s.e.m. values. *p<0.05, **p<0.01, and ***p<0.001 (Student's t-test) vs. the control.

• 

  Figure 3 Effects of dbcAMP and butyric acid on the expression of CCR3 and IL-5Rα in EoL-1 cells. The expression of CCR3 or IL-5Rα in undifferentiated EoL-1 cells and EoL-1 cells stimulated with 100 µM dbcAMP or 0.5 µM butyric acid for 6 days was determined by flow cytometry. Flow cytometric expression of CCR3 or IL-5Rα in indicated cell groups was shown as mean fluorescence intensity (MFI). Data are representative of two or more independent experiments. Data are mean±s.e.m. values. **p<0.01 (Student's t-test) vs. the control.

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