Immunomodulatory activity of recombinant Ricin toxin binding Subunit B (RTB)

Abstract

Ricin toxin binding subunit B (RTB) is one of the subunits of the ricin protein. RTB has been used as adjuvant, but little is known about its mechanism. In this study, we found that RTB increased not only nitric oxide (NO) release, but also tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in mouse macrophage cell line RAW264.7 cells. They subsequently exhibited enhanced ConA-induced T-cell and LPS-induced B-cell proliferative responses. We also examined the cytokines that were produced from splenocytes following in vitro RTB administration. Increased levels of IL-2, interferon (IFN)-γ and TNF-α were observed, while IL-4 and IL-5 were unaffected. These results demonstrate that recombinant RTB can act on the immune system and activate T-cells by introducing a Th1 immune response. Th1 cells might be the primary cellular target affected by RTB. Our results suggest that the recombinant RTB can promote the activation of macrophages and has a beneficial effect on immunomodulatory activity.

Figure 1

Expression and purification of recombinant ricin toxin B subunit (RTB). (A) Lane M contains molecular weight markers (Invitrogen); lane 1, bacterial strain BL21(DE3) pLys extract; lane 2, cell extract containing expressed RTB protein; (B) Lane M contains molecular weight markers (Invitrogen); lane 1, RTB protein after purification by Ni2⁺ affinity column chromatography.

Figure 2

Effects of RTB on cell viability. RAW264.7 cells were treated with various concentrations of RTB for 24, 36 or 48 h, respectively. Cell viability was determined by alamarBlue assay, as described in Materials and Methods. Cell viability in absence of RTB treatment was taken as 100%. The results were expressed as the mean ± SD of three independent experiments.

Figure 3

NO production in RAW264.7 cells treated with RTB. RAW264.7 cells were added with RTB (1, 10 and 100 μg/mL) or LPS (50 ng/mL) for 24, 36 and 48 h, respectively. NO production was measured by Griess reagent. Data are presented as the means ± SD of three replicates. *p < 0.01 compared to the control group.

Figure 4

IL-6 and TNF-α production in RAW264.7 cells treated with RTB. (A) IL-6 production in RAW264.7 cells treated with RTB; (B) TNF-α production in RAW264.7 cells treated with RTB. RAW264.7 cells were added with RTB (1, 10 and 100 μg/mL) or LPS (50 ng/mL) for 24 h. TNF-α and IL-6 production was measured by ELISA. Data are presented as the means ± SD of three replicates. *p < 0.05, **p < 0.01 compared to the control group.

Figure 5

Effects of RTB on proliferation of mouse splenocytes in vitro. (A) Splenocytes were measured with treated RTB (from 0 to 100 μg/mL) for 48 h; (B) Splenocytes were measured with RTB (1, 10 and 100 μg/mL) in the presence of 2.5 μg/mL ConA or 5 μg/mL LPS for 48 h. The absorbance at 570 nm was measured. SI (RTB) = mean A570 of RTB/mean A570 of media control, SI (mitogen) = mean A570 of RTB + ConA or LPS/mean A570 of ConA or LPS. Values are the mean ± SD of three replicates. *p < 0.01 compared to the control group.

Figure 6

Expression of TNF-α, IFN-γ, IL-2, IL-4 and IL-5 in spleen cells from mice with different concentration of RTB through cytometric bead array immunoassay (CBA) analysis. Splenocytes were stimulated with RTB (1, 10 and 100 μg/mL) in the presence of 2.5 μg/mL ConA for 48 h. The culture supernatants were collected and assessed for cytokine production (TNF-α, IFN-γ, IL-2, IL-5, IL-4) by CBA analysis. Data are presented as the means ± SD of three replicates. *p < 0.01 compared to the control group.