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|
|All life stages||High|
Key Event Description
Production of T cell cytokines including Interleukin-2 (IL-2) and interleukin-4 (IL-4) is regulated by NFAT/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).
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/PMA-activated human T cells, FK506 suppressed production of IL-2, IL-4, and 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).
CN inhibition 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 ELISA kits to test matched Ab pairs with biotin-horseradish peroxidase (HRP)-streptavidin detection and 3,3',5,5'-tetramethylbenzidine (TMB) 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).
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, 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, TNF-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).
These facts indicate that CN-NFAT system-mediated suppression of cytokines is commonly found in humans 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.
- 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