Immune Regulation Of Chicken Interleukin-2 Receptor During RNA-associated Virus Infections

Using a pair of specific primers designed from chicken interleukin-2 receptor a chain (chCD25),βchain (chCD122) and y chain (chCD132), the full cDNAs for chCD25, chCD132 and partial cDNA for chCD122 were amplified from a Chinese local breed Sanhuang broilers. The lengths of chCD25 and chCD132 cDNAs were 1027 bp and 1047 bp, respectively, whose precursor proteins comprised of 211 and 348 amino acids (aa), respectively. The predicted analysis of bioinformaitics revealed that the predicted mature chCD25 protein was composed of an extracellular domain (172 aa), a membrane-spanning region (15 aa) and a cytoplasmic domain (4 aa). The predicted mature chCD132 protein was composed of an extracellular domain (210 aa), a membrane-spanning region (23 aa) and a cytoplasmic domain (94 aa). The partial fragment of chCD122 was 660 bp in length, which encoded partial extracellular domain. In a further analysis, chCD25 gene was predicted to locus on the chromosome 1, which had five exons and four introns. chCD122 gene was also predicted to locus on the chromosome 1, which had 12 exons and 11 introns. chCD132 gene was predicted to locus on the chromosome 4, which had 8 exons and 7 introns. The gene structures of chCD25 and chCD122 were quite different from those of human and mouse, but not for chCD132. It suggests that chicken interleukin-2 receptor (chIL-2R) gene has obvious species specificity. In addition, the sequences encoding entire extracellular domains of chCD25 and chCD132 and partial extracellular domain of chCD122, were inserted into the plasmid pET32a, or pET28a vectors, respectively. Then, these recombinant plasmids were transformed E.coli BL21(DE3) and induced by 1 mM IPTG. The analysises of SDS-PAGE and Western blot showed that chCD25, chCD122 and chCD132 proteins were successfully expressed in the prokaryotic system, with molecular weights of 38 KD a,30 KD a and 28 KD a, respectively. The highly pure chCD25, chCD122 and chCD132 prokaryotic proteins were acquired.Using chCD25, chCD122 and chCD132 prokaryotic proteins as immunogen to immune BALB/c mosue, the stable cell lines secreting seven monoclonal antibodies (mAbs)to chCD25(4H5,6C9,6A1,5E1,1G1,6C2,1E1), four mAbs to chCD122 (1G11,5A5,5B12,2C11) and six mAbs to chCD132 (1A1,1G11,1B9,3G11,F8,C10) were acquired. The result of Western blot showed that all these antibodies could react to corresponding antigens. By immunocytochemistry or indirect immunofluorescence assay, the result demonstrated that all mAbs could react to chIL-2R eukaryotic proteins except of 5B12 mAb to chCD122 and 1A1 mAb to chCD132. The vitro proliferation experiments of Con A-stimulated T lymphocytes demonstrated that chCD25 mAbs (5E1 and 6C9), chCD122 mAbs (5A5,1G11,2C11) and chCD132 mAb (C10) had abilities to binding with proteins expressed on the surface of lymphocytes. The study on the inhibiton of lymphocytes proliferation in vitro indicated that mAb 6C9 to chCD25 could effectively inhibited chicken interleukin-2 (chIL-2) to stimulating the proliferation of T lymphocytes, which was dose-dependent. This hints that chCD25 gene here was specific to chIL-2.Chicken embryonic fibroblast (CEF) was inoculated with IBDV, and then the expression of chIL-2R and chCD132 cytokines mRNAs or proteins at different time point post-infection (p.i) was analyzed. The result demonstrated that the transcriptions of chCD25, chCD122, chCD132, chIL-2, chicken interleukin-4 (chIL-4), chicken interleukin-7 (chIL-7), chicken interleukin-9 (chIL-9), chicken interleukin-15 (chIL-15) up-regulated obviously in CEF, but chCD132 mRNA was inhibited. At 48h p.i, the expression of chCD25 and chCD122 proteins could be detected in IBDV-inoculated CEF, but not for chCD132, which was the same to that at mRNA level. Pathogenic IBDV was inoculated with SPF chicken, and then the expression of chIL-2R and chCD132 cytokines was analyzed at mRNA and amino acid levels. The result demonstrated that the transcriptions of chCD25, chCD122, chCD132, chIL-2, chIL-4, chIL-7, chIL-9, chIL-15 were inhibited obviously in thymus; In contrast, the expression of chCD25 and chCD122 proteins up-regulated markly in bursa of Fabricius and spleen, but chCD132 mRNA only weakly increased. The expression of chCD25 and chCD122 proteins could be detected in IBDV-inoculated bursa of Fabricius and spleen, but not for chCD132 protein, which was in accordance with that at mRNA level. These results suggest that the infection of IBDV obviously inhibited chCD25, chCD122 and chCD132 to express in thymus, enhanced chCD25 and chCD122 to express in cells from bursa of Fabricius, but nearly not affected chCD132 in bursa of Fabricius. Thus, we made sure that the immunoregulation mechanisms of IL-2R were different between cellular central immune and humoral centran immune during IBDV infection. In IBDV-inoculated non-immnue tissues, chCD25, chCD122 and chCD132 mRNAs down-regulated in brain, duodenum and jejunum, gradually up-regulated in glandular stomach, cecum, kidney, lung and pancreas; chCD25, chCD122 and chCD132 mRNAs expressed in gizard down-regulated in the initial stage, up-regulated in the intermediate stage and down-regulated in the end stage; in the liver, the expression of chCD122 and chCD132 mRNAs upregulated at the whole time of infection, but the expression of chCD25 mRNA initially up-regulated, then down-regulated; in heart, chCD132 mRNA up-regulated, differently, chCD25 mRNA up-regulated in the initial stage, down-regulated in the intermediate stage and up-regulated in the end stage, and chCD122 mRNA down-regulated in the initial stage and up-regulated finally. The transcriptional expression of chIL-4 was inhibited in the brain, and up-regulated in the kidney; The transcriptional expression of chIL-7 up-regulated in the gizard and was inhibited in the proventriculus; The transcriptional expression of chIL-2, chIL-7, chIL-9 was inhibited in the duodenum and up-regulated in the cecum; chIL-15 mRNA increased in the duodenum and was inhibited in the jejunum and cecum. ChIL-2, chIL-7, chIL-9, chIL-15 mRNAs were inhibited in the jejunum and up-regulated in the cecum and heart. These results confirmed that the immunoregulation mechanisms of IL-2R were different in each non-immune tissue.CEF was inoculated with H5N1, and then the expression of chIL-2R and chCD132 cytokines mRNAs or proteins at different time point post-infection (p.i) was analyzed. The result demonstrated that the infection of H5N1 vigorously increase the expression of chIL-2R and chCD132 cytokines mRNAs in CEF. Meanwhile, the chCD25, chCD122 and chCD132 proteins could be detected by the indirect immunofluorescence assay. This result suggests that immunoregulation of chCD132 happened in the infection of H5N1 was quite different from that of IBDV. On this basis, SPF chicken was inoculated with H5N1 in order to vivo analysis. The result demonstrated that the expression of chCD25, chCD122 and chCD132 mRNAs up-regulated obviously in the spleen. In bursa of Fabricius, chCD25 and chCD132 mRNAs increased. In contrast, chCD25, chCD122 and chCD132 mRNAs were obviously down-regulated in the thymus. The result of immunohistochemistry also confirmed that the quantities of chCD25+, chCD122+ and chCD132+cells were more than those of bursa of Fabricius, but no positive signals appeared in the thymus. ChIL-2 and chIL-4 mRNAs increased obviously in the spleen and thymus, but down-regulated in the bursa of Fabricius. This suggests that the infection of H5N1 causes that chIL-2R+cells migrated from central immune organ (thymus) to peripheral immune organ, as well as activation of TH1 and TH2 cellular immune responses. To other chCD132 cytokines, chIL-15 mRNA did not change obviously in the spleen, thymus and bursa of Fabricius. It hints that chIL-15 does not play a positive role in the process of H5N1 infection. ChIL-7 mRNA obviously increased in the spleen and bursa of Fabricius, but did not change in the thymus. This phenomenon indicated that chIL-7 played an important role in the maintaining balance of peripheral T cells. In conclusion, the infection of H5N1 caused that chCD25, chCD122 and chCD132 down-regulated in the central immune, and up-regulated in the peripheral immune organs, and chIL-15 demaged the immunoregulation functions of chIL-2 and chIL-4.