Identification of the most active interleukin-32 isoform.
Cytokines are crucial in host defence against pathogens such as bacteria, viruses, fungi and parasites. A newly described cytokine, interleukin-32 (IL-32), induces various proinflammatory cytokines (tumour necrosis factor-alpha, IL-1beta, IL-6) and chemokines in both human and mouse cells through the nuclear factor-kappaB and p38 mitogen-activated protein kinase inflammatory signal pathway. The IL-32 primarily acts on monocytic cells rather than T cells. In an attempt to isolate the IL-32 soluble receptor, we used an IL-32 ligand-affinity column to purify neutrophil proteinase 3, which is a serine proteinase involved in many inflammatory diseases. IL-32 has biological activity associated with Mycobacterium tuberculosis and chronic proinflammatory diseases such as rheumatoid arthritis. IL-32 is transcribed as six alternative splice variants and the biological activity of each individual isoform remains unknown. Here, we cloned the complementary DNA of the four IL-32 isoforms (alpha, beta, gamma and delta) that are the most representative IL-32 transcripts. To produce recombinant protein with a high yield, the amino acids of two cysteine residues were mutated to serine residues, because serine residues are not conserved among different species. The multi-step purified recombinant IL-32 isoform proteins were assessed for their biological activities with different cytokine assays. The gamma isoform of IL-32 was the most active, although all isoforms were biologically active. The present study will provide a specific target to neutralize endogenous IL-32, which may contribute to basic and clinical immunology.
Choi
Ji-Da
JD
Laboratory of Cytokine Immunology, Institute of Biomedical Science and Technology, College of Medicine, Konkuk University, Seoul, Korea.
Bae
Su-Young
SY
Hong
Jae-Woo
JW
Azam
Tania
T
Dinarello
Charles A
CA
Her
Erk
E
Choi
Whan-Soo
WS
Kim
Bo-Kyung
BK
Lee
Chang-Kwon
CK
Yoon
Do-Young
DY
Kim
Sun-Jong
SJ
Kim
Soo-Hyun
SH
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics; D016207:Cytokines / Q000096:biosynthesis; D004847:Epithelial Cells / Q000276:immunology; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D007378:Interleukins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D017737:Macrophages, Peritoneal / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009154:Mutation; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D016207:Cytokines; C496971:IL32 protein, human; D007378:Interleukins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003545:Cysteine
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