IL-32, a novel cytokine with a possible role in disease.

Dinarello, C A,Kim, S-H H. K. Lewis 2006 Annals of the rheumatic diseases Vol.65//SUP3 No.-

<P>IL-32 is the name given to the NK4 transcript first reported in IL-2 activated T lymphocytes and natural killer cells 13 years ago without known function. The novel cytokine has six isoforms. In an study to isolate a soluble form of the IL-32 receptor from human urine, IL-32alpha bound proteinase-3 with high affinity and was not affected by enzyme inhibition. IL-32alpha/IL-32gamma were expressed as recombinant molecules. The cytokine exhibits properties characteristic of proinflammatory cytokines and also induces the degradation of inhibitory kappaB and phosphorylation of mitogen activated protein p38. Monoclonal antibodies to IL-32 identify its presence in a variety of human tissues from diseases states. Epithelial cells from healthy subjects express low levels of the cytokine, but in disease conditions such as chronic obstructive pulmonary disease, Crohn's disease and psoriasis, the expression increases markedly. IL-32 is a major transcript in gene array studies in epithelial cells stimulated with IFNgamma in vitro. In rheumatoid arthritis, synovial tissues reveals increased content of IL-32, which correlates with severity of disease. A highly significant correlation has been observed between the number of synovial and macrophagic cells positive for IL-32 and the level of erythrocytes sedimentation, IL-1beta, tumour necrosis factor alpha, and IL-18. Thus, IL-32 exhibits many properties of proinflammatory cytokines and associations with disease severity.</P>

Differential Effects of Anti-IL-1R Accessory Protein Antibodies on IL-1α or IL-1β-induced Production of PGE₂ and IL-6 from 3T3-L1 Cells

Yoon, Do-Young,Dinarello, Charles A. Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.4

Soluble or cell-bound IL-1 receptor accessory protein (IL-1RAcP) does not bind IL-1 but rather forms a complex with IL-1 and IL-1 receptor type I (IL-1RI) resulting in signal transduction. Synthetic peptides to various regions in the Ig-like domains of IL-1RAcP were used to produce antibodies and these antibodies were affinity-purified using the respective antigens. An anti-peptide-4 antibody which targets domain III inhibited 70% of IL-$1\beta$-induced productions of IL-6 and PGE2 from 3T3-L1 cells. Anti-peptide-2 or 3 also inhibited IL-1-induced IL-6 production by 30%. However, antipeptide-1 which is directed against domain I had no effect. The antibody was more effective against IL-$1\beta$ compared to IL-$1\alpha$. IL-1-induced IL-6 production was augmented by coincubation with PGE2. The COX inhibitor ibuprofen blocked IL-1-induced IL-6 and PGE2 production. These results confirm that IL-1RAcP is essential for IL-1 signaling and that increased production of IL-6 by IL-1 needs the co-induction of PGE2. However, the effect of PGE2 is independent of expressions of IL-1RI and IL-1RAcP. Our data suggest that domain III of IL-1RAcP may be involved in the formation or stabilization of the IL-1RI/IL-1 complex by binding to epitopes on domain III of the IL-1RI created following IL-1 binding to the IL-1RI.

Interleukin-18 Binding Protein (IL-18BP): A Long Journey From Discovery to Clinical Application

Kim Soohyun,Yu Hyeon,Azam Tania,Dinarello Charles A. 대한면역학회 2024 Immune Network Vol.24 No.1

IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.

Differential Effects of Anti-IL-1R Accessory Protein Antibodies on IL-1α or IL-1β-induced Production of PGE2 and IL-6 from 3T3-L1 Cells

( Do Young Yoon ),( Charles A. Dinarello ) 생화학분자생물학회 2007 BMB Reports Vol.40 No.4

Identification of the most active interleukin-32 isoform

Choi, Ji-Da,Bae, Su-Young,Hong, Jae-Woo,Azam, Tania,Dinarello, Charles A.,Her, Erk,Choi, Whan-Soo,Kim, Bo-Kyung,Lee, Chang-Kwon,Yoon, Do-Young,Kim, Sun-Jong,Kim, Soo-Hyun Blackwell Publishing Ltd 2009 Immunology Vol.126 No.4

<P>Summary</P><P>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-&agr;, IL-1&bgr;, IL-6) and chemokines in both human and mouse cells through the nuclear factor-&kgr;B 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 <I>Mycobacterium tuberculosis</I> 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 (&agr;, &bgr;, &ggr; and &dgr;) 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 &ggr; 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.</P>

Individual LPS Responsiveness Depends on the Variation of Toll-Like Receptor(TLR) Expression Level

( Jaekal Jun ),( Edward Abraham ),( Tania Azam ),( Mihai G. Netea ),( Charles A. Dinarello ),( Jong Seok Lim ),( Young Yang ),( Do Young Yoon ),( Soo Hyun Kim ) 한국미생물생명공학회 2007 Journal of microbiology and biotechnology Vol.17 No.11

Dysregulation of IL-32 in myelodysplastic syndrome and chronic myelomonocytic leukemia modulates apoptosis and impairs NK function

Marcondes, A. M.,Mhyre, A. J.,Stirewalt, D. L.,Kim, S.-H.,Dinarello, C. A.,Deeg, H. J. Proceedings of the National Academy of Sciences 2008 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.105 No.8

<P>TNFalpha levels are elevated in the marrows of patients with myelodysplastic syndrome (MDS) and are associated with high rates of apoptosis, which contributes to hematopoietic failure. We observed that exposure of human marrow stroma cell lines HS5 and HS27a to TNFalpha increases levels of IL-32 mRNA. IL-32, in turn, induces TNFalpha. Marrow stroma from patients with MDS expressed 14- to 17-fold higher levels of IL-32 mRNA than healthy controls. In contrast, cells from patients with chronic myelomonocytic leukemia (CMML) expressed only one tenth the level of IL-32 measured in healthy controls. Human KG1a leukemia cells underwent apoptosis when cocultured with HS5 stromal cells, but knockdown of IL-32 in the stromal cells by using siRNA abrogated apoptosis in the leukemia cells. IL-32 knockdown cells also showed dysregulation of VEGF and other cytokines. Furthermore, CD56(+) natural killer cells from patients with MDS and CMML expressed IL-32 at lower levels than controls and exhibited reduced cytotoxic activity, which was unaffected by IL-2 treatment. We propose that IL-32 is a marrow stromal marker that distinguishes patients with MDS and CMML. Furthermore, IL-32 appears to contribute to the pathophysiology of MDS and may be a therapeutic target.</P>

Poster Session 2 : Oral Presentation ; Immunology : An antibody to the third domain of the IL-1 receptor accessory protein prevents IL-1-induced production of IL-6 from 3T3-L1 cells: PGE2 regulates the IL-1 responses via type 1 IL-1 receptor

( Do Young Yoon ),( Soo Hyun Kim ),( Charles. A. Dinarello ) 한국생화학분자생물학회 (구 한국생화학회) 2005 62회 KSBMB Annual Meeting in 2005 Vol.- No.-

IL-32-dependent effects of IL-1 on endothelial cell functions

Nold-Petry, C. A.,Nold, M. F.,Zepp, J. A.,Kim, S.-H.,Voelkel, N. F.,Dinarello, C. A. Proceedings of the National Academy of Sciences 2009 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.106 No.10

<P>Increasing evidence demonstrates that interleukin (IL)-32 is a pro-inflammatory cytokine, inducing IL-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and chemokines via nuclear factor (NF)-kappaB, p38 mitogen-activated protein kinase (MAPK), and activating protein (AP)-1 activation. Here we report that IL-32 is expressed and is also functional in human vascular endothelial cells (EC) of various origins. Compared with primary blood monocytes, high levels of IL-32 are constitutively produced in human umbilical vein EC (HUVEC), aortic macrovascular EC, and cardiac as well as pulmonary microvascular EC. At concentrations as low as 0.1 ng/ml, IL-1beta stimulated IL-32 up to 15-fold over constitutive levels, whereas 10 ng/ml of TNFalpha or 100 ng/ml of lipopolysaccharide (LPS) were required to induce similar quantities of IL-32. IL-1beta-induced IL-32 was reduced by inhibition of the IkappaB kinase-beta/NF-kappaB and ERK pathways. In addition to IL-1beta, pro-coagulant concentrations of thrombin or fresh platelets increased IL-32 protein up to 6-fold. IL-1beta and thrombin induced an isoform-switch in steady-state mRNA levels from IL-32alpha/gamma to beta/epsilon. Adult EC responded in a similar fashion. To prove functionality, we silenced endogenous IL-32 with siRNA, decreasing intracellular IL-32 protein levels by 86%. The knockdown of IL-32 resulted in reduction of constitutive as well as IL-1beta-induced intercellular adhesion molecule-1 (ICAM-1) (of 55% and 54%, respectively), IL-1alpha (of 62% and 43%), IL-6 (of 53% and 43%), and IL-8 (of 46% and 42%). In contrast, the anti-inflammatory/anti-coagulant CD141/thrombomodulin increased markedly when IL-32 was silenced. This study introduces IL-32 as a critical regulator of endothelial function, expanding the properties of this cytokine relevant to coagulation, endothelial inflammation, and atherosclerosis.</P>

Endogenous IL-32 controls cytokine and HIV-1 production.

Nold, Marcel F,Nold-Petry, Claudia A,Pott, Gregory B,Zepp, Jarod A,Saavedra, Milene T,Kim, Soo-Hyun,Dinarello, Charles A Williams Wilkins 2008 JOURNAL OF IMMUNOLOGY Vol.181 No.1

<P>IL-32, a proinflammatory cytokine that activates the p38MAPK and NF-kappaB pathways, induces other cytokines, for example, IL-1beta, IL-6, and TNF-alpha. This study investigated the role of endogenous IL-32 in HIV-1 infection by reducing IL-32 with small interfering (si)RNA in freshly infected PBMC and in the latently infected U1 macrophage cell line. When PBMC were pretreated with siRNA to IL-32 (siIL-32), IL-6, IFN-gamma, and TNF-alpha were reduced by 57, 51, and 36%, respectively, compared with scrambled siRNA. Cotransfection of NF-kappaB and AP-1 reporter constructs with siIL-32 decreased DNA binding of these transcription factors by 42 and 46%, respectively. Cytokine protein array analysis revealed that the inhibitory activity of siIL-32 primarily targeted Th1 and proinflammatory cytokines and chemokines, e.g., MIP-1alpha/beta. Unexpectedly, HIV-1 production (as measured by p24) increased 4-fold in these same PBMC when endogenous IL-32 was reduced. Because IFN-gamma was lower in siIL-32-treated PBMC, we blocked IFN-gamma bioactivity, which enhanced the augmentation of p24 by siIL-32. Furthermore, siIL-32 reduced the natural ligands of the HIV-1 coreceptors CCR5 (MIP-1alpha/beta and RANTES) and CXCR4 (SDF-1). Inhibition of endogenous IL-32 in U1 macrophages also increased HIV-1. When rhIL-32gamma was added to these cells, p24 levels fell by 72%; however, in the same cultures IFN-alpha increased 4-fold. Blockade of IFN-alpha/beta bioactivity in IL-32gamma-stimulated U1 cells revealed that IFN-alpha conveys the anti-HIV-1 effect of rhIL-32gamma. In summary, depletion of endogenous IL-32 reduced the levels of Th1 and proinflammatory cytokines but paradoxically increased p24, proposing IL-32 as a natural inhibitor of HIV-1.</P>