Biological effects of a new IL-5 isoform in vitro

• Igor P. Shilovskiy
• Valeriia I. Kovchina
• Ekaterina D. Timotievich
• Tatyana E. Rusak
• Valery V. Smirnov
• Dmitry A. Kudlay
• Musa R. Khaitov

Abstract

Introduction. Recently, an alternative variant of IL-5 (IL-5d2) has been identified. Full-length IL-5 plays an important role in the maturation, activation and migration of proinflammatory eosinophil cells, while the biological role of IL-5d2 still unknown.

Therefore, the aim of this study was to investigate the biological effects of a new IL-5d2 isoform in vitro.

Material and methods. Full-length IL-5 and its novel isoform IL-5d2 were produced in HEK293T cells by transfection with plasmids carrying the corresponding genes. The concentration of IL-5 and IL-5d2 was determined by enzyme-linked immunosorbent assay (ELISA) using commercial monoclonal and polyclonal antibodies. Eosinophils were obtained by differentiation of BALB/c mouse bone marrow cells in the presence of growth factors GM-CSF, IL-3, and IL-5 or IL-5d2. Cytopreparations were prepared from the cells, followed by differential staining with azure and eosin and quantitative calculation of the proportion of eosinophils by light microscopy.

Results. The differentiation of eosinophils occurred best in the presence of all 3 factors (GM-CSF, IL-3 and IL-5). Replacing the full-size IL-5 with its splice variant led to a slowdown in the differentiation of eosinophils from mouse bone marrow cells. It should be noted that over the same period of time (5 days), the number of eosinophils in the presence of the splice variant IL-5d2 of the mouse decreased by 25 % compared with the full-size form of IL-5 of the mouse. Thus, the biological activity of the splice variant IL-5d2 of the mouse is lower compared to the full-size IL-5 of the mouse. Additional experiments have demonstrated that differentiation of eosinophils can occur only during incubation with IL-5 in the absence of GM-CSF and IL-3. The shortened isoform of IL-5d2 also contributed to the differentiation of eosinophils. Further, the ability of IL-5d2 to competitively inhibit IL-5-dependent differentiation of eosinophils was studied. As a result, it was shown that IL-5d2 doesn’t act as an inhibitor of the activity of full-sized mouse IL-5.

Conclusion. Thus, new isoform IL-5d2 exhibited own in vitro biological activity similar to the full-length IL-5. In addition, IL-5d2 does not inhibit IL-5-dependent eosinophil differentiation.

Keywords: bronchial asthma; Th2 cytokines; alternative splicing; novel splice variant IL-5; interleukin-5

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