Preparation Of IL-33 Transgenic Mice And IL-33 Promotes Pulmonary Inflammation

Interleukin-33 is a recent found cytokine which belongs to the cytokine family of Interleukin-1. The orphan receptor ST2 which was found in 1989, is ragarded as IL-33 receptor on target cell surface. ST2 was mainly expressed on the surface of Th2 helper lymphocytes and mast cells. IL-33 was regarded as a pro-inflammatory factor to promote Th2 cells functions. IL-33 and ST2 play an important role in inflammatory diseases including hypersensitive diseases like asthma, autoimmune diseases like rheumatoid arthritis, cardiovascular diseases like heart failure and neurodegenerative diseases like Alzheimer's disease. The exogenous administration of recombinant IL33 induced splenomegaly changes and severe airway and digestive trace inflammation in mice. Currently there were no report on endogenous up-regulation of IL-33 in animals. IL-33 contains an IL-1-like cytokine fold in its carboxyl-terminus and chromatin binding domain in amino-terminus. Without a signal peptide for secretion, it is necessary to understand the functional structure of IL-33, that is the cleavage and secretion of this cytokine. A lot of researchers had tried to solve this question, but there are conflicts now.Here we reported the generation of an IL-33 transgenic mouse, in which mouse IL-33 full length cDNA was cloned out of neonate mouse tissue, inserted into euocaryotic expression vector pCDNA3.1, controlled under the CMV promoter. Immuno-blot results showed that the transgenic IL-33 was released as a cleaved form with molecular weight of 18 kDa in pulmonary, nephritic, cardiac and pancreatic tissues in transgenic mice and the pâ… of 18 kDa peptide was about pH3-5 on the 2-D PAGE, which was similar with the activated IL-33 peptide of 109-266 aa.The birth-rates, body weights, and sex ratios of ltransgenic mice were recorded and there were no difference compared with that of C57BL/6 wild-type mice. The gross anatomic and histological analyses were performed on the tissues of heart, liver, spleen, kidney, pancreas, intestine and brain of IL-33 transgenic mice at 2-month-old. No obvious changes were observed. Histological analysis showed massive pulmonary inflammation with infiltration of eosinophils around bronchi and small blood vessels, hyperplasia of goblet cells and accumulation of mucus-like material in pulmonary tissue of transgenic mice. An increase of IL-5, IL-8, IL-13 and IgE was detected in bronchoalveolar lavage fluid (BALF) of transgenic mice, which are inflammatory factors. sST2 and the anti-inflammatory factor IL-10 were also up-regulated to conterbalance the inlfamatroy damage of IL-33. These findings suggest transgenic IL-33 could be cleaved and secreted in an activated form and play an important role in the pathogenesis of pulmonary inflammation. Through our IL-33 transgenic research, the endogenous over-expression of mouse IL-33 was achived in mouse pulmonary tissue. And we detected the endogenous cleavage of IL-33 in mouse, and the character of the cleaved IL-33 was similar to the peptide with cytokine activity. The endogenous over-expression of IL-33 leads to spontaneous pulmonary inflammation, which proved the pro-inflammatory character of IL-33. The evolution of species is the result of nature selection. The genetic material would also changes with the evolution of species. In fact the evolution of species is also the evolution of the genomes. There were different kinds of changes of the gene, including the appearance of new gene.120 million years ago, the primate like human being had the same ancestor with the rodent. With the evolution of the two kinds of animals, there were significant changes in their genomes. Based on the genomic sequence analysis of these two kinds of animals, there were about 1%genes, which only exist in one kind, not in the other. These genes were regarded as species specific genes.We've found some species specific genes through the sequence comparison of these two kinds of animals. XAGE-3 is one of the primate species specific gene. In this study, we firstly confirm the species specific character of XAGE-3 by bio-information method. Subsequently we cloned this gene out of human placenta, and inserted this gene into eukaryotic expression vector pCDNA3.1. The XAGE-3 transgenic mice were got by micro-injection. We analyzed the XAGE-3 expression in transcripts from different tissues. Over-expression of XAGE-3 can be detected in intestine, thymus and testis in transgenic mice.3-week-old male mice were labeled with Brdu. More spermatocytes were labeled inXAGE-3 transgenic mice compared with negative control. The results suggests the human XAGE-3 transgene may affect the proliferation and development of spermatocytes. MAN2C1 is an a-mannosidase, which regulates protein glycosylation. Previous studies have showed its effect on tumor proliferation. In this study, the enzyme activity of a-mannosidase in liver and spleen of MAN2C1 transgenic mice were analysed. A rabbit polyclonal antibody against hMAN2Cl was raised to detect the transgenic MAN2C1 expression in 2 lines of hMAN2Cl transgenic mice. The transgenic model was used to prove the effect of MAN2C1 on proliferation and migration of grafted tumors. For better understanding the biological function of this gene, a transgenic gene silence mouse model was made. The expression of the targeted gene had been inhibited in transcripts, but there no no changes in the enzyme activity in the tissue from gene silenced mice.