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Key Event Title
Suppression of IL-4 production
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Immune dysfunction induced by JAK3 inhibition||KeyEvent||Yasuhiro Yoshida (send email)||Under development: Not open for comment. Do not cite||Under Development|
|All life stages||High|
Key Event Description
IL-4 is a mammalian protein found in Homo sapiens. IL-4 is pivotal in shaping the nature of immune responses. Upon activation, naïve peripheral CD4+ T cells begin to synthesize and secrete cytokines. Type 2 helper cells (Th2 cells) produce IL-4, IL-5, IL-6, and IL-13. IL-4 is a 15-kD polypeptide with pleiotropic effects on many cell types. In T cells, binding of IL-4 to its receptor induces proliferation and differentiation into Th2 cells. Th2 cells assist B cells in promoting class switching from IgM to IgG1 and IgE (Choi and Reiser 1998).
STAT5 phosphorylation facilitates the dimerization of STAT5, transport to the nucleus, and gene regulation (Levy and Darnell 2002). DNaseI hypersensitivity sites II (HSⅡ) and III (HSⅢ) in intron 2 have been identified in several regions of the Il4/Il13 locus. STAT5A binding to sites near HSⅡ and HSⅢ could provide a mechanism through which STAT5A mediates IL-4 gene accessibility and participates in the induction of IL-4 production (Zhu, et al. 2003). The CD3 antibody-induced phosphorylation of STAT5 can be downregulated by tofacitinib, suggesting that JAK3 inhibition by tofacitinib can downregulate STAT5-dependent cytokine signaling. Tofacitinib was shown to abrogate anti-CD3-induced STAT5 activation in CD4+ T cells and inhibit IL-4 production from CD4+ T cells (Migita, et al. 2011).
How It Is Measured or Detected
In one study, CD4+ T cells were stimulated with CD3 monoclonal antibodies in the presence or absence of tofacitinib (CP-690550) for 48 h. Supernatants were collected and the levels of IL-4 production were measured by ELISA (Migita, et al. 2011). The authors also extract total RNA after 8 h or 24 h of stimulation and measured IL-4 mRNA expression was measured by real-time PCR (Migita, et al. 2011).
In another study, flow cytometry analysis involving intracellular staining was used to measure cytosolic IL-4 content in stimulated cells (Zhu, et al. 2001). Relative gene expression levels were determined by quantitative RT-PCR using Taqman Gene Expression primer probe sets and ABI PRISM 7700 or 7900 Taqman systems (Applied Biosystems). The comparative threshold cycle method and internal controls (cyclophillin or β-actin) were used to normalize the expression of target gene (IL-4) (Ghoreschi, et al. 2011).
Cytokine content was quantified in appropriately diluted samples in duplicate using ELISA kits to test matched antibody pairs with biotin-horseradish peroxidase-streptavidin detection and 3,3',5,5'-tetramethylbenzidine substrate. ELISA plates were scanned using the UVmax plate reader (Molecular Devices) using SOFT max software (Dumont, et al. 1998).
Domain of Applicability
In one study, only 1% of CD4 T cells from STAT5a-/- mice primed with soluble anti-CD3 and anti-CD28 with IL-2 produced IL-4, whereas 10.5% of control C57BL/6 CD4 T cells produced IL-4 (Cote-Sierra, et al. 2004).
Cells from STAT5A-deficient mice or cells treated with phospho-STAT5 peptide are defective in Th2 differentiation. STAT5A single-deficient mice showed impaired Th2 differentiation. Reconstituting STAT5A by retroviral infection restored the capacity of cells to induce IL-4 (Kagami, et al. 2001).
IL-2 directly activates STAT5A and STAT5B. T cells from mice deficient in either STAT5A or STAT5B did not show a dramatic change in T cell proliferation, but cells from mice in which both had been knocked out proliferated poorly in response to IL-4 (Moriggl, et al. 1999).
Choi P, Reiser H. 1998. IL-4: role in disease and regulation of production. Clin Exp Immunol 113:317-319. DOI: 10.1046/j.1365-2249.1998.00690.x.
Cote-Sierra J, Foucras G, Guo L, Chiodetti L, Young HA, Hu-Li J, Zhu J, Paul WE. 2004. Interleukin 2 plays a central role in Th2 differentiation. Proc Natl Acad Sci U S A 101:3880-3885. DOI: 10.1073/pnas.0400339101.
Dumont FJ, Staruch MJ, Fischer P, DaSilva C, Camacho R. 1998. Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK MAP kinase or calcineurin signaling pathways results in differential modulation of cytokine production. J Immunol 160:2579-2589.
Ghoreschi K, Jesson MI, Li X, Lee JL, Ghosh S, Alsup JW, Warner JD, Tanaka M, Steward-Tharp SM, Gadina M, Thomas CJ, Minnerly JC, Storer CE, LaBranche TP, Radi ZA, Dowty ME, Head RD, Meyer DM, Kishore N, O'Shea JJ. 2011. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol 186:4234-4243. DOI: 10.4049/jimmunol.1003668.
Kagami S, Nakajima H, Suto A, Hirose K, Suzuki K, Morita S, Kato I, Saito Y, Kitamura T, Iwamoto I. 2001. Stat5a regulates T helper cell differentiation by several distinct mechanisms. Blood 97:2358-2365. DOI: 10.1182/blood.v97.8.2358.
Levy DE, Darnell JE, Jr. 2002. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651-662. DOI: 10.1038/nrm909.
Migita K, Miyashita T, Izumi Y, Koga T, Komori A, Maeda Y, Jiuchi Y, Aiba Y, Yamasaki S, Kawakami A, Nakamura M, Ishibashi H. 2011. Inhibitory effects of the JAK inhibitor CP690,550 on human CD4(+) T lymphocyte cytokine production. BMC Immunol 12:51. DOI: 10.1186/1471-2172-12-51.
Moriggl R, Topham DJ, Teglund S, Sexl V, McKay C, Wang D, Hoffmeyer A, van Deursen J, Sangster MY, Bunting KD, Grosveld GC, Ihle JN. 1999. Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells. Immunity 10:249-259.
Zhu J, Cote-Sierra J, Guo L, Paul WE. 2003. Stat5 activation plays a critical role in Th2 differentiation. Immunity 19:739-748. DOI: 10.1016/s1074-7613(03)00292-9.
Zhu J, Guo L, Watson CJ, Hu-Li J, Paul WE. 2001. Stat6 is necessary and sufficient for IL-4's role in Th2 differentiation and cell expansion. J Immunol 166:7276-7281. DOI: 10.4049/jimmunol.166.12.7276.