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|
|Substance interaction with the lung cell membrane leading to lung fibrosis||KeyEvent|
Key Event Description
How this KE works
Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-3, IL-5 and GM-CSF are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (IL-1a, IL-1b, IL-1ra, IL-18, IL-36a, IL-36b, IL-36g, IL-36Ra, IL-37), IL-6 family, TNF family, IL-17, and IFNg (Turner et al., 2014). While IL-4 and IL-5 are considered T helper (Th) cell type 2 response, IFNg is suggested to be Th1 type response.
Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, IL-1 family cytokines, IL-4, IL-5, IL-6, TNFa, IFNg are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002).
Evidence for its perturbation
Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of CNT types and other fibrogenic materials. Poland et al., 2008) showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and ROS (reactive oxygen species). Exposure to crystalline silica induces release of inflammatory cytokines (TNFa, IL-1, IL-6), transcription factors (NF-kb, AP-1) and kinase signalling pathways in mice that contain NFkB luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long MWCNTs included high expression levels of pro-inflammatory mediators MCP-1, TGF-β1, and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of NMs induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2010; Husain et al., 2015). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).
How It Is Measured or Detected
The selection of proinflammatory mediators for investigation varies based on the expertise of the lab, cell type studied and availability of the specific antibodies.
How it is measured or detected
The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.
qRT-PCR – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan T et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding flourophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.
ELISA assays – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE.
Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample.
Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen et al., 2009).
Domain of Applicability
Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of lietrature is avaiable to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.
- Alberts, D., Chen, H., Woolfenden, J., Moon, T., Chang, S., Hall, J., Himmelstein, K., Gross, J. and Salmon, S. (1979). Pharmacokinetics of bleomycin in man. Cancer Chemotherapy and Pharmacology, 3(1).
- Brömme, D., Rossi, A., Smeekens, S., Anderson, D. and Payan, D. (1996). Human Bleomycin Hydrolase: Molecular Cloning, Sequencing, Functional Expression, and Enzymatic Characterization. Biochemistry, 35(21), pp.6706-6714.
- Canellos, G., Anderson, J., Propert, K., Nissen, N., Cooper, M., Henderson, E., Green, M., Gottlieb, A. and Peterson, B. (1992). Chemotherapy of Advanced Hodgkin's Disease with MOPP, ABVD, or MOPP Alternating with ABVD. New England Journal of Medicine, 327(21), pp.1478-1484.
- Claussen, C. and Long, E. (1999). Nucleic Acid Recognition by Metal Complexes of Bleomycin. Chemical Reviews, 99(9), pp.2797-2816.
- Forn-Cuní, G., Varela, M., Pereiro, P., Novoa, B. and Figueras, A. (2017). Conserved gene regulation during acute inflammation between zebrafish and mammals. Scientific Reports, 7(1).
- Froudarakis, M., Hatzimichael, E., Kyriazopoulou, L., Lagos, K., Pappas, P., Tzakos, A., Karavasilis, V., Daliani, D., Papandreou, C. and Briasoulis, E. (2013). Revisiting bleomycin from pathophysiology to safe clinical use. Critical Reviews in Oncology/Hematology, 87(1), pp.90-100.
- Hay, J., Shahzeidi, S. and Laurent, G. (1991). Mechanisms of bleomycin-induced lung damage. Archives of Toxicology, 65(2), pp.81-94.
- Hubbard, A., Timblin, C., Shukla, A., Rincón, M. and Mossman, B. (2002). Activation of NF-κB-dependent gene expression by silica in lungs of luciferase reporter mice. American Journal of Physiology-Lung Cellular and Molecular Physiology, 282(5), pp.L968-L975.
- Ohnuma, T., Holland, J., Masuda, H., Waligunda, J. and Goldberg, G. (1974). Microbiological assay of bleomycin: Inactivation, tissue distribution, and clearance. Cancer, 33(5), pp.1230-1238.
- Sleijfer, S. (2001). Bleomycin-Induced Pneumonitis. Chest, 120(2), pp.617-624.
- Tashiro, J., Rubio, G., Limper, A., Williams, K., Elliot, S., Ninou, I., Aidinis, V., Tzouvelekis, A. and Glassberg, M. (2017). Exploring Animal Models That Resemble Idiopathic Pulmonary Fibrosis. Frontiers in Medicine, 4.
- Twentyman, P. (1983). Bleomycin—mode of action with particular reference to the cell cycle. Pharmacology & Therapeutics, 23(3), pp.417-441.
- Umezawa, H., Maeda, K., Takeuchi, T. and Okami, Y. (1966). NEW ANTIBIOTICS, BLEOMYCIN A AND B. The Journal of Antibiotics, XIX(5), pp.200-209.
- Umezawa, H., Suhara, Y., Takita, T. and Maeda, K. (2019). PURIFICATION OF BLEOMYCINS. Journal of Antibiotics, XIX(5), pp.210-215.
- Yu, Z., Schmaltz, R., Bozeman, T., Paul, R., Rishel, M., Tsosie, K. and Hecht, S. (2013). Selective Tumor Cell Targeting by the Disaccharide Moiety of Bleomycin. Journal of the American Chemical Society, 135(8), pp.2883-2886.