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Event: 1495

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Substance interaction with the lung resident cell membrane components

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help
Interaction with the lung cell membrane
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term
eukaryotic cell

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
pattern recognition receptor signaling pathway increased
toll-like receptor signaling pathway Toll-like receptor increased
toll-like receptor 4 signaling pathway Toll-like receptor 4 increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Substance interaction with the lung cell membrane leading to lung fibrosis MolecularInitiatingEvent Sabina Halappanavar (send email) Under development: Not open for comment. Do not cite EAGMST Under Review
Interaction with lung cells leads to lung cancer MolecularInitiatingEvent Penny Nymark (send email) Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI
human Homo sapiens High NCBI

Life Stages

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Life stage Evidence
Adults High

Sex Applicability

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Term Evidence
Male High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

The human lung consists of approximately 40 different resident cell types that play different roles during homeostasis, injury, repair and disease states (Franks et al., 2008). Of these, resident airway epithelial cells, alveolar/interstitial macrophages and dendritic cells are well characterised for their ability to sense the danger upon interaction with harmful substances and relay the message to mount the necessary immune/inflammatory response. The resident macrophages are present in all tissues, and in a steady state, macrophages contribute to epithelial integrity, survey the tissue for invading pathogens or chemicals and maintain an immunosuppressive environment. Their main function is to clear the incoming irritants and microbes. They are named differently based on the tissue type and their specific functions (Kierdorf et al., 2015).

Substance interactions:

The chemicals or pathogens interact with cellular membrane to gain access to the organisms’ interior. A predominant interaction mechanism involves the recognition of innate immune response agonists by pattern recognition receptors (PRRs) present on resident cells such as epithelial and alveolar macrophages. PRRs are also present on other immune and parenchymal cells. PRRs can be activated by two classes of ligands. Pathogen Associated Molecular Patterns (PAMPs) are microbial molecules derived from invading pathogens. PAMPs will not be discussed further as pathogens are not the focus for the AOP presented here. The other class of ligands are called Danger Associated Molecular Patterns (DAMPs) that include cellular fragments, nucleic acids, small molecules, proteins and even cytokines released from injured or dying cells. Most fibrogenic stressors discussed in this AOP act via DAMPs-driven PRR activation. High aspect ratio (HAR) materials such as asbestos or carbon nanotubes (CNTs) pierce the cellular membrane of epithelial cells or resident macrophages resulting in cell injury or non-programmed cellular death. Alveolar macrophages trying to engulf High Aspect Ratio (HARs) fibres that are long and stiff undergo frustrated phagocytosis because of their inability to engulf the piercing fibres and subsequently lead to cell injury (Mossman and Churg, 1998; Donaldson K et al., 2010). The cellular debris from injured or dying cell then serves as ligands for PRRs (Nakayama, 2018), leading to cell activation. In case of pro-fibrotic insoluble particles such as silica, coal dust and nanomaterials (NMs), the particle adsorbed opsonins such as, immunoglobulins, complement proteins, or serum proteins act as ligands to the receptors on the macrophage cell surface (Behzadi et al., 2017). The tissue response to these materials resembles that observed following foreign body invasion in lungs.

Toll-like receptors (TLRs) are highly conserved PRRs that are associated with fibrogenic stressors (Desai et al., 2018). Inhibition of TLR-4 is protective against bleomycin-induced fibrosis (Li et al., 2015). However, the exact role and mechanisms by which TLRs mediate lung fibrosis are yet to be uncovered and some studies have shown TLRs to be protective against lung fibrosis (Desai et al., 2018). Asbestos and silica crystals are suggested to engage scavenger receptors present on the macrophages. Mice deficient in class A scavenger receptor MARCO are shown to induce reduced fibrogenic response following chrysotile asbestos exposure; although, the direct binding of MARCO by asbestos is not investigated in the study (Murthy et al., 2015). In case of soluble substances such as bleomycin, paraquat (Dinis-Oliveira et al., 2008) (N, Ndimethyl-4, 4′-bipyridinium dichloride) and other soluble fibrogenic chemicals, direct damage of lung epithelial cells and resulting cellular debris or secreted cytokines (DAMPs) serve as triggers for downstream cascading pro-inflammatory events, tissue injury and fibrosis. Engagement of PRRs and consequent cell activation is observed in various organisms including flies and mammals (Matzinger, 2002).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

Detection of Danger Associated Molecular Patterns (DAMPs) or homeostasis-altering molecular processes (HAMPs):

Cellular interaction with substances or particles can be measured by assessing the release of DAMPs from stressed, injured or dying cells - indicative of binding of PRRs on the cell surface. Release of DAMPs is reflective of substance interaction with resident cells and their activation, a key step in the process of inflammation.

The release of DAMPs can be measured by the techniques listed in the published literature (Suwara et al., 2014; Nikota et al., 2017; Rabolli et al., 2014).

Targeted enzyme-linked immunosorbent assays (ELISA) (routinely used and recommended):

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 and De Visser, 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.

IL-1a and IL-1b is activated or secreted into the cytosol following stimulus. Targeted ELISA can be used to quantify IL-1a or IL1b that is released in the culture supernatant of the cells exposed to toxicants, in bronchoalveolar lavage fluid and serum of exposed animals. The assay is also applicable to human serum, cerebrospinal fluid, and peritoneal fluids.

Similarly, other alarmins can also be quantified by ELISA. Westernblot is another method that can be used to quantify the release of various alarmins using specific antibodies. qRT-PCR or ELISA assays can also be used to quantify expression of genes or proteins that are regulated by the receptor binding – e.g. downstream of TLR binding.

Frustrated phagocytosis and cellular uptake of NMs:

In vitro, interaction of NMs with the cellular membrane is investigated by assessing their uptake by lysosomes (Varela et al., 2012). Immunohistochemistry methods targeting lysosome specific proteins are regularly employed for this purpose. In co-localisation experiments, lysosomal marker LAMP1 antibody is used to detect particle co-localisation with lysosomes. A combination of Cytoviva hyperspectral microscope and immunolocalisation (Decan et al., 2016) or confocal microscopy to visualise co-localisation of fluorescence labelled nanoparticles with lysosomal markers have been used. Frustrated phagocytosis is assessed using microscopic techniques (Donaldson et al., 2010).

Cellular co-culture models of the pulmonary epithelium:

Complex co-culture systems, such as those containing epithelial cells and immune cells, better model the environment of the lung epithelium and can be used to study the interaction of potentially pro-fibrotic fibres and particles with resident lung cells. This type of model has been used, alongside electron microscopy, to study lung cell interactions with CNTs following 24 Hr in vitro exposure (Clift et al., 2014). More recently, the EpiAlveolar model, which contains primary human alveolar epithelial cells, endothelial cells, as well as fibroblasts was assessed for its ability to predict fibrosis induced by CNTs (Barasova et al., 2020). Using laser scanning, fluorescence, and enhanced darkfield microscopy, CNT interaction with the resident cells of the model was shown, and this interaction induced the formation of holes in the epithelial model (Barasova et al., 2020). While new co-culture models are a better recapitulation of the native lung environment as compared to traditional mono-cultures, the increased complexity necessitates enhanced expertise in tissue culture techniques, and can make them less practical as compared to submerged mono culture methods. 

Ex vivo model of the lung – Precision Cut Lung Slices:

Even closer to the in vivo condition than co-culture models, precision cut lung slice (PCLS) techniques capture the native lung architecture, cell-cell communication and cellularity of the lung. Advancement in culturing and cryopreservation techniques has increased accessibility and use of PCLS for longer term studies (Bai et al., 2016, Neuhaus et al., 2017). These slices can be cultured ex vivo for up to a week with minimal reduction in viability, and the technique has recently been assessed for its applicability to assess nanomaterial induced fibrosis ex vivo (Rahman et al., 2020). Using MWCNT and darkfield microscopy, interaction between the nanofibers and the lung epithelium could be determined. The main downside of this technique is the animal requirement, which precludes their use in a first-pass screening context for the MIE.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

Human, mouse, rat.

Although the expression of DAMPs following exposure to pro-fibrotic substances is not assessed across species, it is known that alarmins are released after trauma or injury, and their release is important for initiating the inflammatory response in all species including humans. The immediate acute inflammatory response involving DAMP signalling is also observed in human IPF; however, anti-inflammatory drugs have proven ineffective for treating IPF. Danger signalling axis including uric acid, ATP and IL-33/ST2 has been proven to promote lung fibrosis in animals.


List of the literature that was cited for this KE description. More help

1. Amsen, D. and De Visser, K. (2009). Approaches to Determine Expression of Inflammatory Cytokines. Methods in molecular biology.. 511th ed. (Clifton, NJ), pp.107-142.

2. Barosova, H., Maione, A. G., Septiadi, D., Sharma, M., Haeni, L., Balog, S., O'Connell, O., Jackson, G. R., Brown, D., Clippinger, A. J., Hayden, P., Petri-Fink, A., Stone, V., & Rothen-Rutishauser, B. (2020). Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS nano, 14(4), 3941–3956.

3. Bai Y, Krishnamoorthy N, Patel KR, Rosas I, Sanderson MJ, Ai X. (2016). Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists. Am J Respir Cell Mol Biol. 2016 May;54(5):656-63.

4. Behzadi, S., Serpooshan, V., Tao, W., Hamaly, M., Alkawareek, M., Dreaden, E., Brown, D., Alkilany, A., Farokhzad, O. and Mahmoudi, M. (2017). Cellular uptake of nanoparticles: journey inside the cell. Chemical Society Reviews, 46(14), pp.4218-4244.

5. Bianchi, M. (2006). DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of Leukocyte Biology, 81(1), pp.1-5.

6. Boyles, M., Young, L., Brown, D., MacCalman, L., Cowie, H., Moisala, A., Smail, F., Smith, P., Proudfoot, L., Windle, A. and Stone, V. (2015). Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicology in Vitro, 29(7), pp.1513-1528.

7. Brown, D., Kinloch, I., Bangert, U., Windle, A., Walter, D., Walker, G., Scotchford, C., Donaldson, K. and Stone, V. (2007). An in vitro study of the potential of carbon nanotubes and nanofibres to induce inflammatory mediators and frustrated phagocytosis. Carbon, 45(9), pp.1743-1756.

8. Cheng, L., Jiang, X., Wang, J., Chen, C. and Liu, R. (2013). Nano–bio effects: interaction of nanomaterials with cells. Nanoscale, 5(9), p.3547.

9. Clift, M. J., Endes, C., Vanhecke, D., Wick, P., Gehr, P., Schins, R. P., Petri-Fink, A., & Rothen-Rutishauser, B. (2014). A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes. Toxicological sciences : an official journal of the Society of Toxicology, 137(1), 55–64.

10. Decan, N., Wu, D., Williams, A., Bernatchez, S., Johnston, M., Hill, M., & Halappanavar, S. (2016). Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes. Mutation research. Genetic toxicology and environmental mutagenesis, 796, 8–22.

11. Denholm, E. and Phan, S. (1990). Bleomycin Binding Sites on Alveolar Macrophages. Journal of Leukocyte Biology, 48(6), pp.519-523.

12. Desai, O., Winkler, J., Minasyan, M. and Herzog, E. (2018). The Role of Immune and Inflammatory Cells in Idiopathic Pulmonary Fibrosis. Frontiers in Medicine, 5.

13. Di Paolo, N. and Shayakhmetov, D. (2016). Interleukin 1α and the inflammatory process. Nature Immunology, 17(8), pp.906-913.

14. Dinis-Oliveira, R., Duarte, J., Sánchez-Navarro, A., Remião, F., Bastos, M. and Carvalho, F. (2008). Paraquat Poisonings: Mechanisms of Lung Toxicity, Clinical Features, and Treatment. Critical Reviews in Toxicology, 38(1), pp.13-71.

15. Donaldson, K., Murphy, F., Duffin, R. and Poland, C. (2010). Asbestos, carbon nanotubes and the pleural mesothelium: a review and the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. Particle and Fibre Toxicology, 7(1), p.5.

16. Dörger, M., Münzing, S., Allmeling, A., Messmer, K. and Krombach, F. (2001). Differential Responses of Rat Alveolar and Peritoneal Macrophages to Man-Made Vitreous Fibers in Vitro. Environmental Research, 85(3), pp.207-214.

17. Franks, T., Colby, T., Travis, W., Tuder, R., Reynolds, H., Brody, A., Cardoso, W., Crystal, R., Drake, C., Engelhardt, J., Frid, M., Herzog, E., Mason, R., Phan, S., Randell, S., Rose, M., Stevens, T., Serge, J., Sunday, M., Voynow, J., Weinstein, B., Whitsett, J. and Williams, M. (2008). Resident Cellular Components of the Human Lung: Current Knowledge and Goals for Research on Cell Phenotyping and Function. Proceedings of the American Thoracic Society, 5(7), pp.763-766.

18. Kierdorf, K., Prinz, M., Geissmann, F. and Gomez Perdiguero, E. (2015). Development and function of tissue resident macrophages in mice. Seminars in Immunology, 27(6), pp.369-378.

19. Kim, J., Lim, H., Minai-Tehrani, A., Kwon, J., Shin, J., Woo, C., Choi, M., Baek, J., Jeong, D., Ha, Y., Chae, C., Song, K., Ahn, K., Lee, J., Sung, H., Yu, I., Beck, G. and Cho, M. (2010). Toxicity and Clearance of Intratracheally Administered Multiwalled Carbon Nanotubes from Murine Lung. Journal of Toxicology and Environmental Health, Part A, 73(21-22), pp.1530-1543.

20. Li, X., Jiang, D., Huang, X., Guo, S., Yuan, W. and Dai, H. (2015). Toll-like receptor 4 promotes fibrosis in bleomycin-induced lung injury in mice. Genetics and Molecular Research, 14(4), pp.17391-17398.

21. Matzinger, P. (2002). The Danger Model: A Renewed Sense of Self. Science, 296(5566), pp.301-305.

22. MOSSMAN, B. and CHURG, A. (1998). Mechanisms in the Pathogenesis of Asbestosis and Silicosis. American Journal of Respiratory and Critical Care Medicine, 157(5), pp.1666-1680.

23. Murthy, S., Larson-Casey, J., Ryan, A., He, C., Kobzik, L. and Carter, A. (2015). Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure. The FASEB Journal, 29(8), pp.3527-3536.

24. Nakayama, M. (2018). Macrophage Recognition of Crystals and Nanoparticles. Frontiers in Immunology, 9.

25. National Institute of Occupational Safety and Health (NIOSH) (2011). Asbestos fibers and other elongate mineral particles: state of the science and roadmap for research.. pp.Current Intelligence Bulletin 62. Publication Number 2011-159.

26. Nel, A., Mädler, L., Velegol, D., Xia, T., Hoek, E., Somasundaran, P., Klaessig, F., Castranova, V. and Thompson, M. (2009). Understanding biophysicochemical interactions at the nano–bio interface. Nature Materials, 8(7), pp.543-557.

27. Neuhaus V, Schaudien D, Golovina T, Temann UA, Thompson C, Lippmann T, Bersch C, Pfennig O, Jonigk D, Braubach P, Fieguth HG, Warnecke G, Yusibov V, Sewald K, Braun A. Assessment of long-term cultivated human precision-cut lung slices as an ex vivo system for evaluation of chronic cytotoxicity and functionality. J Occup Med Toxicol. 2017 May 26;12:13.

28. Nikota, J., Banville, A., Goodwin, L., Wu, D., Williams, A., Yauk, C., Wallin, H., Vogel, U. and Halappanavar, S. (2017). Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Particle and Fibre Toxicology, 14(1).

29. Pascolo, L., Gianoncelli, A., Schneider, G., Salomé, M., Schneider, M., Calligaro, C., Kiskinova, M., Melato, M. and Rizzardi, C. (2013). The interaction of asbestos and iron in lung tissue revealed by synchrotron-based scanning X-ray microscopy. Scientific Reports, 3(1).

30. Poland, C., Duffin, R., Kinloch, I., Maynard, A., Wallace, W., Seaton, A., Stone, V., Brown, S., MacNee, W. and Donaldson, K. (2008). Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nature Nanotechnology, 3(7), pp.423-428.

31. Rabolli, V., Badissi, A., Devosse, R., Uwambayinema, F., Yakoub, Y., Palmai-Pallag, M., Lebrun, A., De Gussem, V., Couillin, I., Ryffel, B., Marbaix, E., Lison, D. and Huaux, F. (2014). The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica microand nanoparticles. Particle and Fibre Toxicology, 11(1).

32. Rahman, L., Williams, A., Gelda, K., Nikota, J., Wu, D., Vogel, U., & Halappanavar, S. (2020). 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small, 16(36), e2000272.

33. Suwara, M., Green, N., Borthwick, L., Mann, J., Mayer-Barber, K., Barron, L., Corris, P., Farrow, S., Wynn, T., Fisher, A. and Mann, D. (2013). IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunology, 7(3), pp.684-693.

34. Varela, J., Bexiga, M., Åberg, C., Simpson, J. and Dawson, K. (2012). Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells. Journal of Nanobiotechnology, 10(1), p.39.