Key Event Title
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Key Event Components
Key Event Overview
AOPs Including This Key Event
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|Substance interaction with the cell membrane leading to lung fibrosis||KeyEvent|
Key Event Description
Proinflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Cell-derived proinflammatory mediators include cytokines, chemokines, and growth factors. Blood derived proinflammatory 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 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. Some of the proinflammatory 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 MD 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 proinflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas J et al., 2004). However, IL-1 family cytokines, IL-4, IL-5, IL-6, TNFa, IFNg are the commonly measured mediators in experimental animal models and in humans. Proinflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner.
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. 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 AK, 2002). Long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and ROS (Poland CA, 2008). Lung responses to long MWCNTs included high expression levels of MCP-1, TGF-β1, and TNF-α (Boyles MSP, 2015). In vitro, CNTs have been shown to induce increased secretion of cytokines and chemokines (He X, 2011; Herano S, 2010).
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.
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 proinflammatory mediators in several species including mouse, rat and humans, are readily available commercially.
ELISA assays – permit quantitative measurement of antigens in biological samples. For example, in a cytokine ELISA (sandwich ELISA), an antibody (capture antibody) specific to a cytokine is immobilised on microtitre wells (96-well, 386-well, etc.). Experimental samples or samples containing a known amount of the specific recombinant cytokine are then reacted with the immobilised antibody. Following removal of unbound antibody by thorough washing, plates are reacted with the secondary antibody (detection antibody) that is conjugated to an enzyme such as horseradish peroxidase, which when bound, will form a sandwich with the capture antibody and the cytokine (Amsen D et al., 2009). The secondary antibody can be conjugated to biotin, which is then detected by addition of streptavidin linked to horseradish peroxidase. A chromogenic substrate can also be added, which is the most commonly used method. Chromogenic substrate is chemically converted by the enzyme coupled to the detection antibody, resulting in colour change. The amount of colour detected is directly proportional to the amount of cytokine in the sample that is bound to the capture antibody. The results are read using a spectrophotometer and compared to the levels of cytokine in control samples where cytokine is not expected to be secreted or to the samples containing known recombinant cytokine levels.
Both ELISA and qRT-PCR assays 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 M 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 proinflammatory mediators and its downstream effectors in any given tissue (Costa PM 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 D et al., 2009).
Domain of Applicability
- Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochimica et Biophysica Acta 1843 (2014) 2563–2582.
- Javier Mestas and Christopher C. W. Hughes. Of mice and not men: differences between mouse and human immunology. J Immunol 2004; 172:2731-2738.
- Hubbard, Andrea K., Cynthia R. Timblin, Arti Shukla, Mercedes Rinco´n, and Brooke T. Mossman. Activation of NF-_B-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol 282: L968–L975, 2002; 10.1152
- Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, et al. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008; 3:423–428.
- Matthew S.P. Boyles, Lesley Young, David M. Brown, Laura MacCalman, Hilary Cowie, Anna Moisala, Fiona Smail, Paula J.W. Smith, Lorna Proudfoot, Alan H. Windle, Vicki Stone, Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos, In Toxicology in Vitro, Volume 29, Issue 7, 2015, Pages 1513-1528.
- He X, Young SH, Schwegler-Berry D, Chisholm WP, Fernback JE, Ma Q. Multiwalled carbon nanotubes induce a fibrogenic response by stimulating reactive oxygen species production, activating NF-kappaB signaling, and promoting fibroblast-to-myofibroblast transformation. Chem Res Toxicol. 2011;24:2237–2248.
- Seishiro Hirano, Yuji Fujitani, Akiko Furuyama, Sanae Kanno, Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells, In Toxicology and Applied Pharmacology, Volume 249, Issue 1, 2010, Pages 8-15.
- Tania Nolan, Rebecca E Hands & Stephen A Bustin. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82.
- Derk Amsen, Karin E. de Visser, and Terrence Town. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009; 511: 107–142.
- Mainul Husain, Dongmei Wu, Anne T. Saber, Nathalie Decan, Nicklas R. Jacobsen, Andrew Williams, Carole L. Yauk, Hakan Wallin, Ulla Vogel and Sabina Halappanavar. Intratracheally Instilled Titanium Dioxide Nanoparticles Translocate to Heart and Liver, and Activate Complement Cascade in the Heart of C57BL/6 Mice. Nanotoxicology. 2015;9(8):1013-22.
- Costa PM, Gosens I, Williams A, et al. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2017;1–13.