Identification and expression analysis of IL-2 receptors in large yellow croaker (Larimichthys crocea)

Abstract

Interleukin-2 (IL-2) signals influence various lymphocyte subsets during differentiation, immune responses and homeostasis. IL-2 acts on different cells by binding to its receptors (IL-2R), which consists of three subunits, IL-2Rα (CD25), IL-2Rβ (CD122), and the common gamma chain or γc (CD132). In the present study, three IL-2 receptor subunits, designated as LcCD25-like (LcCD25L), LcIL-2Rβ and Lcγc, were characterized in large yellow croaker (Larimichthys crocea). The LcCD25L, like other teleost CD25L or IL-2/IL-15Rα, contains only one sushi domain at N-terminus. The synteny of CD25L from different teleost are conserved. The deduced protein of LcIL-2Rβ and Lcγc exhibits a typical class I cytokine receptors architecture, including a cytokine-binding homology domain (CHD) consisting of two fibronectin type-III (FNIII) domains (D1 and D2) and a conserved WSXWS motif in D2 domain. These three IL-2 receptor subunits were constitutively expressed in all tissues and primary immune-related cells examined. The LcCD25L was highly expressed in blood, while LcIL-2Rβ and Lcγc were highly expressed in spleen and gill. For immune-related cells, LcCD25L and LcIL-2Rβ were highly expressed in PKLs, while the Lcγc exhibited the highest expression in PKMs. These three IL-2 receptor subunits could be dramatically induced by T cell mitogen PHA in PKLs, which mainly composed of T and B lymphocytes. The results presented indicated that large yellow croaker IL-2R might exercise function on lymphocytes, especially on activated T cells.

Keywords: Expression modulation; Interleukin 2 receptor; Large yellow croaker; Molecular evolution.

Fig. 1

Phylogenetic tree based on amino acids of IL-2/IL-15R subunits without signal peptide from different species. Deduced amino acid sequences of IL-2Rs and IL-15Rs family members without signal peptide were aligned with mafft software (v7.453) and phylogenetic tree was constructed with maybayes method using phylosuite software (v1.2.1) based on the multiple alignment result. The percentage of bootstrap values is shown next to the branches based on 20,000 bootstrap replications. The LcIL-2Rs are marked with black triangles.

Fig. 2

Expression analysis of LcIL-2Rs gene in different tissues and immune-related cells. Expression levels of LcIL-2Rs gene in different tissues and immune-related cells including PKMs, PKGs and PKLs were investigated. The expression of Lcβ-actin was used as an internal control. The lowest expression of each LcIL-2Rs in a specific tissue or cells was designated as 1, and the other expression was expressed as the ratio to it. Deviation bars represent the standard errors of the mean (± SEM).

Fig. 3

Expression analysis of LcIL-2Rs genes in vivo after immune stimulation. The expression of LcIL-2Rs genes in head kidney and spleen during 48 h of stimulation with Poly(I:C) or V. alginolyticus was examined by real-time PCR. Total RNA was extracted from head kidney and spleen of large yellow croaker collected at 3, 6, 12, 24 and 48 hps, and used for real-time PCR detection. Expression levels of Lcβ-actin were set as internal control. The expression levels of each gene was expressed as fold change relative to that in PBS-injected fish. All experiments were performed at least in triplicate. Deviation bars represent the standard errors of the mean (± SEM) at each time point. ^∗P < 0.05, ^∗∗P < 0.01.

Fig. 4

Expression analysis of LcIL-2Rs genes in PKLs after immune stimulation. The PKLs were stimulated with PHA, Con-A, Poly(I:C), inactivated V. alginolyticus or with PBS as control for 4, 8 and 24 hps. The expression level was normalized to that of the Lcβ-actin from the same sample, and expressed as fold change relative to the expression levels in control PKLs. Deviation bars represent the standard errors of the mean (± SEM) at each time point. ^∗P < 0.05, ^∗∗P < 0.01.