Isoflurane decreases interleukin-2 production by increasing c-Cbl and Cbl-b expression in rat peripheral blood mononuclear cells

Abstract

Objective To evaluate how isoflurane affects T-cell function by assaying interleukin (IL)-2 production and the expression of two Casitas B-lineage lymphoma (Cbl) family proto-oncogenes (c-Cbl and Cbl-b) in rat peripheral blood mononuclear cells (PBMCs). Methods Adult male Sprague-Dawley rats were randomly allocated to those that underwent blood collection after brief isoflurane anesthesia (control group), immediately after 4 hours of isoflurane general anesthesia (4I group), and 1 day after 4 hours of isoflurane general anesthesia (1D 4I group). IL-1, IL-2, and IL-6 mRNA levels and c-Cbl and Cbl-b levels in PBMCs were determined by polymerase chain reaction. Ubiquitination of protein kinase Cθ (PKCθ) and phospholipase C-γ1 (PLC-γ1) in PBMCs was assessed by immunoprecipitation. Results The IL-2 mRNA level in rat PBMCs was significantly lower in the 4I and 1D 4I groups than in the control group. c-Cbl, Cbl-b, and ubiquitin expression was significantly increased and zeta-chain-associated protein kinase 70, PLC-γ1, and PKCθ protein levels were significantly decreased in the 4I group. Ubiquitination of PLC-γ1 and PKCθ was significantly increased in the 4I group. Conclusion Isoflurane influences ubiquitin, c-Cbl, and Cbl-b expression in rat PBMCs, indicating suppression of receptor tyrosine kinase signaling pathways. These results suggest that isoflurane suppresses T-cell function.

Keywords: Cbl family; PKCθ; PLC-γ1; interleukin-2; isoflurane; ubiquitin.

Figure 1.

Interleukin (IL)-1β, IL-6, and IL-2 mRNA levels in peripheral blood mononuclear cells (PBMCs) of the control, 4I, and 1D 4I groups. The IL-2 mRNA level was significantly decreased (0.40-fold) in the 4I group and non-significantly decreased (0.57-fold) in the 1D 4I group compared with in the control group. ∗P < 0.05.

Figure 2.

Effects of isoflurane on c-Cbl, Cbl-b, ubiquitin, ZAP70, and PLC-γ1 levels in rat peripheral blood mononuclear cells. (a) Casitas B-lineage lymphoma proto-oncogene (c-Cbl) and Cbl-b protein levels were increased after exposure to isoflurane. The levels of phosphorylated c-Cbl and Cbl-b were increased and decreased, respectively, in the 4I group. (b) The level of conjugated ubiquitin was increased, and that of monoubiquitin was decreased, in the 4I group. (c) The levels of the phosphorylated and non-phosphorylated forms of zeta-chain-associated protein kinase 70 (ZAP70) and phospholipase C-γ1 (PLC-γ1) were decreased in the 4I and 1D 4I groups compared with in the control group.

Figure 3.

Ubiquitination of PLC-γ1 and PKCθ after 4 hours of exposure to isoflurane. (a) Ubiquitination of PLC-γ1 was increased in the 4I and 1D 4I groups compared with in the control group. (b) Ubiquitination of protein kinase C (PKC)θ was increased in the 4I and 1D 4I groups.

Figure 4.

c-Cbl and Cbl-b expression in CD4⁺ and CD28⁺ T cells after 4 hours of exposure to isoflurane. c-Cbl and Cbl-b expression by (a and b) CD4⁺ T cells and (c and d) CD28⁺ T cells was significantly increased in the 4I group and non-significantly increased in the 1D 4I group compared with in the control group.