Angiogenin is upregulated during the alloreactive immune response and has no effect on the T-cell expansion phase, whereas it affects the contraction phase by inhibiting CD4+ T-cell apoptosis

Abstract

Under growth conditions, angiogenin is translocated into the nucleus, where it enhances ribosomal RNA transcription, facilitating increased protein synthesis and cellular proliferation. During stress conditions, angiogenin is sequestered in the cytoplasm, where it cleaves transfer RNA (tRNA) to produce tRNA-derived, stress-induced small RNAs (tiRNAs) that inhibit global protein synthesis, but increase the translation of anti-apoptotic factors. In the present study, the role of angiogenin in the human alloreactive immune response was evaluated using mixed lymphocyte reactions (MLRs) and neamine, an inhibitor of angiogenin nuclear translocation. In MLRs, angiogenin production was significantly (P<0.001) increased compared with resting peripheral blood mononuclear cells. The addition of neamine had no effect on cell proliferation, but did significantly (P<0.001) increase expression of Bcl-2-associated X protein and protein levels of activated caspase-3 in CD4⁺ T-cells isolated from the MLRs, indicating that angiogenin reduces apoptosis. In conclusion, angiogenin is upregulated during the alloreactive immune response, in which it does not affect the T-cell expansion phase, but inhibits the T-cell contraction phase by protecting against CD4⁺ T-cell apoptosis.

Keywords: angiogenin; apoptosis; cluster of differentiation 4 positive T-cells; proliferation.

Figure 1.

Effect of Nea on cell necrosis. Nea was not cytotoxic to resting PBMCs or MLRs. Ten different MLRs were performed. Results are presented as the mean ± standard deviation of triplicate readings of the percentage of cell death. PBMC, peripheral blood mononuclear cells; Nea, neamine; MLR, mixed lymphocyte reaction.

Figure 2.

Angiogenin expression in MLR in the presence or absence of Nea. Compared with resting PBMCs, expression of angiogenin in MLRs was significantly higher. Nea did not affect angiogenin expression in MLRs. Ten different MLRs were performed. Results are presented as the mean ± standard deviation. *P<0.001 vs. resting PMBCs. PBMC, peripheral blood mononuclear cells; MLR, mixed lymphocyte reaction; Nea, neamine.

Figure 3.

Effect of Nea on cell proliferation in MLRs. Nea did not affect proliferation index in MLRs. Ten different MLRs were performed. Results are presented as the mean ± standard deviation of triplicate readings. MLR, mixed lymphocyte reaction; Nea, neamine.

Figure 4.

The effect of Nea on the expression of Bax and Bcl-2, and protein levels of CC3 in MLR-derived CD4⁺ T-cells. (A) Western blot of expression levels in MLR-derived CD4⁺ T-cells. (B) Quantification of relative protein expression from western blots in MLR-derived CD4⁺ T-cells. Expression of pro-apoptotic Bax and protein levels of activated CC3 were significantly increased in CD4⁺ T-cells isolated from MLRs treated with Nea, whereas expression of anti-apoptotic Bcl-2 remained unaffected. Ten different MLRs were performed. Results are presented as the mean ± standard deviation. *P<0.001 vs. untreated MLR. MLR, mixed lymphocyte reaction; Nea, neamine; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; CC3, cleaved caspase-3.