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Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

Liu Y, Sato H, Hamana M, Moonan NA, Yoneda M, Xia X, Kai C - J. Vet. Med. Sci. (2014)

Bottom Line: However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1.The expressed proIL-18 proteins were processed to their mature forms in the cells.These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

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Induction of IFN-γ by incubating canine or mouse immune cells with the supernatantfrom transfected cells. (A) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with canine PBMCs. After 48 hr, thesupernatant was examined for IFN-γ production with an ELISA. Three independentexperiments were performed (n=3). (B) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with mouse spleen cells in a polyvinylidenedifluoride (PVDF)-backed microplate coated with monoclonal antibody specific for mouseIFN-γ in a humidified 37°C incubator for 48 hr. The supernatant was examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).
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fig_002: Induction of IFN-γ by incubating canine or mouse immune cells with the supernatantfrom transfected cells. (A) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with canine PBMCs. After 48 hr, thesupernatant was examined for IFN-γ production with an ELISA. Three independentexperiments were performed (n=3). (B) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with mouse spleen cells in a polyvinylidenedifluoride (PVDF)-backed microplate coated with monoclonal antibody specific for mouseIFN-γ in a humidified 37°C incubator for 48 hr. The supernatant was examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).

Mentions: To examine the bioactivity of the cIL-18 secreted from the transfected cells, the culturesupernatant was cocultured with canine PBMCs, and the amount of canine IFN-γ produced by thePBMCs was measured. The supernatants of the cells expressing entire cIL-18 induced littleIFN-γ production compared with the control, whereas the supernatants of cells expressingcIL-12ss–cIL-18 slightly enhanced the induction efficiency, as in the previous report [37]. Interestingly, the supernatant of cells transfectedwith pFuse–hIL2ss–cIL18 induced significant IFN-γ production (Fig. 2AFig. 2.


Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

Liu Y, Sato H, Hamana M, Moonan NA, Yoneda M, Xia X, Kai C - J. Vet. Med. Sci. (2014)

Induction of IFN-γ by incubating canine or mouse immune cells with the supernatantfrom transfected cells. (A) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with canine PBMCs. After 48 hr, thesupernatant was examined for IFN-γ production with an ELISA. Three independentexperiments were performed (n=3). (B) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with mouse spleen cells in a polyvinylidenedifluoride (PVDF)-backed microplate coated with monoclonal antibody specific for mouseIFN-γ in a humidified 37°C incubator for 48 hr. The supernatant was examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197152&req=5

fig_002: Induction of IFN-γ by incubating canine or mouse immune cells with the supernatantfrom transfected cells. (A) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with canine PBMCs. After 48 hr, thesupernatant was examined for IFN-γ production with an ELISA. Three independentexperiments were performed (n=3). (B) The supernatant from transfected 293T cells ormock-transfected 293T cells was cocultured with mouse spleen cells in a polyvinylidenedifluoride (PVDF)-backed microplate coated with monoclonal antibody specific for mouseIFN-γ in a humidified 37°C incubator for 48 hr. The supernatant was examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).
Mentions: To examine the bioactivity of the cIL-18 secreted from the transfected cells, the culturesupernatant was cocultured with canine PBMCs, and the amount of canine IFN-γ produced by thePBMCs was measured. The supernatants of the cells expressing entire cIL-18 induced littleIFN-γ production compared with the control, whereas the supernatants of cells expressingcIL-12ss–cIL-18 slightly enhanced the induction efficiency, as in the previous report [37]. Interestingly, the supernatant of cells transfectedwith pFuse–hIL2ss–cIL18 induced significant IFN-γ production (Fig. 2AFig. 2.

Bottom Line: However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1.The expressed proIL-18 proteins were processed to their mature forms in the cells.These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

Show MeSH
Related in: MedlinePlus