Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Homo sapiens||Homo sapiens||High||NCBI|
|Mus musculus||Mus musculus||High||NCBI|
|cynomolgus monkey||Macaca fascicularis||High||NCBI|
|All life stages||High|
Key Event Description
Production of T cell cytokines including Interleukin (IL)-2 and IL-4 is regulated by nuclear factor of activated T cells (NFAT)/ activator protein-1 (AP-1) complexes. Activated NFAT/AP-1 complex that bind at the site of the IL-2 and IL-4 promoters, thereby induces transcription of IL-2 (Jain et al. 1993). For IL-2, NFAT proteins are necessary for IL-2 gene expression and cooperation of NFAT with AP-1 is required for IL-2 gene transcription. For IL-4, At least five different NFAT sites have been described in the IL-4 promoter with at least three of them being composite sites binding NFAT and AP-1 (Macián et al. 2001).
IL-2 binds to IL-2 receptor (IL-2R) and acts on T cell. CD25 is one of IL-2R. Basiliximab (Simulect) is known as anti-CD25 antibody. Basiliximab binds to IL-2R and blocks IL-2 signaling. Clinical transplantation study of basiliximab reveals decreases in rejections. On the other hand, basiliximab inhibits the activation of antigen specific T cells (Novartis Pharma 2016).
Calcineurin inhibitors (CNIs) such as FK506 and cyclosporin A (CsA) hinder the formation of the functional NFAT/AP-1 complexes by interfering with NFAT nuclear localization (Flanagan et al. 1991). Reduced binding of NFAT/AP-1 complexes at the promoter site of the IL-2 gene lowers the transcription of the mRNA of IL-2 and the following cytokine production.
Transcription of IL-4 is also inhibited by CNIs in the same manner as IL-2 (Dumont et al. 1998).
In CD3/ phorbol 12-myristate-13-acetate (PMA)-activated human T cells, FK506 suppressed production of IL-2, IL-4, and Interferon (IFN)-γ at the concentrations of 1.2 to 12.5 nM after 22 to 24 hours culture as well as inhibited expression of IL-2, IL-4, and IFN-γ mRNA in a dose-dependent (10 nM) manner (Dumont et al. 1998).
Treatment with CsA completely eliminated detectable IL-2 release from 3A9 T cells co-cultured with antigen-bearing Ch27 B cells with an IC25 and IC50 for IL-2 production of 1.19 nM and 1.99 nM. Treatment with other immunosuppressant compounds (dexamethasone, azathioprine, methotrexate, benzo(a)pyrene and urethane) also resulted in decreased IL-2 release from stimulated 3A9 T cells at non-cytotoxic concentrations. Urethane, a weakly immunosuppressive chemical, was least potent in the assay, with an IC25 and IC50 for IL-2 secretion of 4.24 mM and 13.26 mM (D.M. Lehmann. et al. 2018).
In male CD-1 mice, chronic psychosocial stress (types of social outcome occurred: residents becoming subordinates) reduced IL-2 release in response to keyhole limpet hemocyanine (KLH) (Alessandro, B. et al. 2003).
In female B6C3F1 mice, 1,2:5,6-dibenzanthracene exposure reduced production of IL-2 in spleen cell culture supernatants after in vitro stimulation with Concanavalin A or lipopolysaccharide (Donna, C. et al. 2010).
Treatment with CsA at 50 mg/kg BID via oral gavage or 2C1.1 (a fully human anti-ORAI1 monoclonal antibody) at 25 mg/kg single IV resulted in reduction of IL-2, IL-4, IL-5, and IL-17 cytokine production from PMA/ionomycin stimulation of whole blood in the cynomolgus monkey (Kevin, G. et al. 2014).
CNIs is considered to increase carcinogenicity through the suppression of IL-2 and IL-4 production.
- Renal transplant patients on immunosuppressive therapy were found to develop cancer within 10 years after surgery (Luster, M.I. et al. 1993).
In experimental animal studies, carcinogenicity of FK506 was reported as follows.
- In mice subjected to topical application testing, in which 100 μL of FK506 ointment was applied once daily for two years to roughly 40% of the total body area, an increased incidence of lymphoma was found in mice of the 0.1% ointment group showing high blood concentrations of the drug (Maruho Co., Ltd 2014).
- In hairless albino mice, virtually all of which developed skin tumors after a 40-week exposure to ultraviolet light, application of a 1% FK506 ointment reduced the time to outbreak of the skin tumors. (Maruho Co., Ltd 2014).
How It Is Measured or Detected
Quantitation of cytokine content was done on appropriately diluted samples, run in duplicate, using Sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) kits to test matched Antibody pairs with biotin-horseradish peroxidase-streptavidin detection and 3,3',5,5'-tetramethylbenzidine substrate. ELISA plates were scanned in a Molecular Devices UVmax plate reader (Menlo Park, CA), using SOFT max software (Molecular Devices) (Dumont et al. 1998).
Ex vivo whole blood stimulated cytokine (IL-2, IL-4, IL-5, and IL-17) production assay in the supernatants were determined using an electrochemiluminescent immunoassay from Meso Scale Discovery (MSD; Gaithersburg, MD) (Kevin, G. et al. 2014).
Total RNA was extracted using RNeasy mini kit (Qiagen, Chatsworth, CA) and quantitated by absorbance at 260 nm. Cytokine mRNAs were detected using a RiboQuant MultiProbe RPA system (PharMingen, San Diego, CA). Riboprobes were 32P-labeled and hybridized overnight with 10 to 30 mg of the RNA samples. The hybridized RNA was treated with RNase and purified according to the RiboQuant protocol. The samples were then electrophoresed in 6% polyacrylamide-Tris-borate-EDTA-urea gels using the Seqi -Gen GT Nucleic Acid Electrophoresis Cell (Bio-Rad, Hercules, CA), or minigels (Novex, San Diego, CA). The gels were dried, exposed and quantitated in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using the ImageQuant software (Dumont et al. 1998).
Domain of Applicability
CNIs suppress production of IL-2, IL-3, IL-4, IL-5, IFN-γ, Granulocyte Macrophage colony-stimulating Factor (GM-CSF), and other cytokines, as induced by CD2/CD3 or CD3/CD26 stimulation, in human peripheral blood mononuclear cells (PBMC) (Sakuma et al. 2001a). Also, CNIs (FK506 and CsA) suppress production of IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, Tumor Necrosis Factor-α, IFN-γ, and GM-CSF, as induced by CD3/PMA stimulation, in human PBMC (Dumont et al. 1998).
CNIs (FK506 and CsA) exhibit suppression of IL-2 production induced from mixed lymphocyte reactions in mice and humans (Kino, T et al. 1987a).
Treatment with CsA or 2C1.1 resulted in reduction of IL-2, IL-4, IL-5, and IL-17 cytokine production from PMA/ionomycin stimulation of whole blood in the cynomolgus monkey (Kevin, G. et al. 2014).
These facts indicate that Calcineurin-NFAT system-mediated suppression of cytokines is commonly found in humans, monkey and mice.
Evidence for Perturbation by Stressor
- Dumont, F.J., Staruch, M.J., Fischer, P., DaSilva, C. and 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. Journal of immunology 160 (6): 2579-89.
- Flanagan, W.M., Corthésy, B., Bram, R.J. and Crabtree, G.R. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352 (6338): 803-7.
- Jain, J., McCaffrey, P. G., Valge-Archer, V. E. and Rao, A. (1992). Nuclear factor of activated T cells contains Fos and Jun. Nature. 356(6372): 801-804.
- Jain, J., Miner, Z. and Rao, A. (1993). Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. Journal of immunology. 151(2): 837-848.
- Kino, T., Hatanaka, H., Miyata, S., Inamura, N., Nishiyama, M., Yajima, T., Goto, T., Okuhara, M., Kohsaka, M. and Aoki, H. (1987a). FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. Journal of antibiotics. 40(9): 1256-1265.
- Macián, F., López-Rodríguez, C. and Rao, A. (2001). Partners in transcription: NFAT and AP-1. Oncogene. 20(19): 2476-89.
- Novartis Pharma K.K. (2016). Drug interview form Simulect i.v. injection 20 mg. 10th edition.
- Sakuma, S., Higashi, Y., Sato, N., Sasakawa, T., Sengoku, T., Ohkubo, Y., Amaya, T., and Goto, T. (2001a). Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system. (Comparison with steroids). International Immunopharmacology 1(6): 1219-26.
- Schreiber, SL., and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42.
- Luster, M.I., and Rosenthal, G.J. (1993). Environmental Health Perspectives. 100: 219-36.
- Maruho Co.,Ltd. (2014) Drug interview form Protopic ointment 0.1% Revised 16th edition.
- Alessandro B, Paola S, Alberto E. Paneraic, Tiziana P,Paola Palanzaa and Stefano P(2003). Chronic psychosocial stress-induced down-regulation of immunity depends upon individual factors Journal of Neuroimmunology 141: 58–64
- Donna C. S, Matthew J. S and Kimber L. W Jr. (2010) Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1 mice, Journal of Immunotoxicology, 7:3, 219-231
- Kevin G, Hossein S, Raju S, Valerie A, Anna K, Ming Z, Fen-Fen L, Hung Q. N, Lei Z, John K. S, Min W and Helen J. M(2015) Inhibition of CRAC with a human anti-ORAI1 monoclonal antibody inhibits T-cell-derived cytokine production but fails to inhibit a T-cell-dependent antibody response in the cynomolgus monkey, Journal of Immunotoxicology, 12:2, 164-173,
- D.M. Lehmann, W.C. Williams.(2018) Development and Utilization of a Unique In Vitro Antigen Presentation Co-culture Model for Detection of Immunomodulating Substances. Toxicol In Vitro.53: 20–28.