Event: 1495

Key Event Title


Increased, interaction with the resident cell membrane components

Short name


Interaction with the cell membrane

Biological Context


Level of Biological Organization

Cell term


Organ term


Key Event Components


Process Object Action

Key Event Overview

AOPs Including This Key Event


AOP Name Role of event in AOP
Substance interaction with the cell membrane leading to lung fibrosis MolecularInitiatingEvent



Taxonomic Applicability


Life Stages


Sex Applicability


Key Event Description



The human lung with the surface area of ~100 m2 is ventilated by 10-20,000 litres of air per day. Thus, it is the largest organ of the human body that is exposed to the environment contaminated with pathogens, organic and inorganic materials. The gas exchange between blood and the inhaled air takes place across a membrane that is only 1-2 µm thick, making the lung an easy target for foreign invasion and toxic chemicals in the environment. To combat the constant attack by the pathogens and chemicals, the lung is equipped with several defence mechanisms; a layer of epithelial cells acting as physical barrier to limit entry of pathogens and specialised cells of the immune system with defence functions (reviewed in Weitnauer M et al., 2016). The human lung consists of approximately 40 different resident cell types that play different roles during homeostasis, injury, repair and disease states (Franks TJ et al., 2008). Of these, resident airway epithelial cells, alveolar 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.

Receptor-mediated interactions

The chemicals or pathogens interact with cellular membrane to gain access to the organisms’ interior. This interaction between the pathogens/substances and lung cells can occur via binding of the pathogens or pathogen associated molecular patterns (PAMPs, microbial molecules) to the receptors present on the surfaces of the resident cells. For example, the airway epithelium and the mucosal layer form a physical barrier and contribute to the first line of defence. The resident epithelial cells, by default, under the normal homeostatic conditions, are adjusted to the local microbial burden and therefore, are less responsive to microbial stimulation. The resident epithelial cell surfaces express receptors that recognise and sense the presence of pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) resulting in epithelial cell activation. PRRs are also present on other resident cells including alveolar macrophages. PRRs can be activated by interaction with debris from dying cells (cellular fragments) as a result of interaction with toxic substances. Engagement of PRRs and consequent cell activation is observed in various organisms including flies and mammals (Matzinger, 2002).

Opsonisation driven interactions

In case of non-pathogens such as insoluble particles or engineered nanomaterials (ENMs), interaction with cellular membrane can occur via the process of opsonisation. For example, the surfaces of ENMs can be adsorbed with opsonins (protein corona) such as immunoglobulins, complement proteins, or serum proteins that are then recognised by the phagocytes (macrophages). Opsonised ENMs attach to the cellular membrane of phagocytes via ligand-receptor interactions. Some of the receptors on the cell surface that are engaged by the opsonins include Fc receptors and complement protein receptors (reviewed in Behzadi S et al., 2017). The endocytosis processes such as phagocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis and micropinocytosis define the interactions between cell membrane and the particles (airborne or ENMs).

Physical or mechanical interactions

The other type of interaction can involve physical damage to cells. High aspect ratio (HAR) materials such as asbestos or carbon nanotubes (CNTs) can pierce the cellular membrane of epithelial cells or resident macrophages resulting in cell injury or non-programmed cellular death. 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. Resident macrophages in the lung, bone, brain, liver, spleen, skin, and in the intestine are known as alveolar macrophages, osteoclasts, microglia, Kupffer cells, splenic macrophages, Langerhans cells and intestinal macrophages, respectively (Kierdorf K et al., 2015). It has been shown that alveolar macrophages trying to engulf HARs including asbestos and CNTs that are long and stiff undergo frustrated phagocytosis because of their inability to engulf the piercing fibres and subsequently to cell injury (Mossman BT et al., 1998; Donaldson K et al., 2010).


How It Is Measured or Detected


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 or activation of TLR receptors - indicative of binding of PAMPs to 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. DAMPs, also called as alarmins are endogenous molecules that are released by stressed cells. A few of the putative alarmins that can be effectively measured in biological samples including cultured cells are High Mobility Group Binding 1 (HMGB1) protein, Heat Shock proteins (HSPs), uric acid, annexins, S100 proteins and IL-1α (Bianchi ME, 2007).

Of all, IL-1a is the most commonly measured alarmin. IL-1a is the principal pro-inflammatory moiety and is a designated ‘alarmin’ in the cell that alerts the host to injury or damage (Di Paolo NC, 2016). It is shown that administration of necrotic cells to mice results in neutrophilic inflammation that was entirely mediated by IL-1a released from the dying or necrosed cells and consequent activation of IL-1 Receptor 1 (IL-1R1) signalling (Suwaraa MI et al., 2014). IL-1a is released following exposure to MWCNTs (Nikota J et al., 2017) and silica (Rabolli V et al., 2014). IL-1a can be cleaved from its precursor form to the active form; however, it is not a prerequisite since both precursor and cleaved forms of IL-1a are active that bind and activate IL-1R1 signaling.

The release of DAMPs can be measured by the techniques listed below.

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 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.

IL-1a is activated or secreted into the cytosol following stimulus. Targeted ELISA for IL-1a can be used to quantify IL-1a 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.

Measuring cellular uptake of ENMs

Lysosomal localisation – In vitro, interaction of ENMs with the cellular membrane can be investigated by assessing their uptake by the lysosomes (Varela JA 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 N et al., 2016) or confocal microscopy to visualise co-localisation of fluorescence labelled nanoparticles with lysosomal markers have been used.

Domain of Applicability


Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event


Not many studies investigate the exact mode of interaction between the toxic substance and the cellular membrane components. Rather, the consequences of such interaction such as, the release of intracellular contents (alarmins), increases in genes or protein synthesis downstream of receptor binding or cellular uptake are measured as indicative of occurrence of such interactions (Nel AE et al., 2009; Cheng L-C et al., 2013). Because of the physical –chemical properties such as surface charge, ENMs and asbestos like materials can bind to cellular macromolecules and cell surface/membrane components, which in turn, facilitate their uptake and intracellular sequestration by the cells (NIOSH 2011 a; Pascolo L et al., 2013). Intratracheally instilled crystalline silica interacts with lung epithelial cells and alveolar macrophages and induces proinflammatory cascade via NF-kb signaling pathway (Hubbard AK, 2002).

Exposure to MWCNTs results in increased IL-1a protein levels in the bronchoalveolar lavage fluid (BALF) of lungs in mice (Nikota J et al., 2017) and lung epithelial cells exposed to silica show lysosomal accumulation of silca in a size dependent manner (Decan N et al., 2016).



  1. Weitnauer M, Mijosek V, Dalpke AH. Control of local immunity by airway epithelial cells. Mucosal Immunology (2016) 9, 287–29.
  2. Franks TJ, Colby TV, Travis WD, Tuder RM, ....Williams MC. Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function.  Proc Am Thorac SocBeh.. 2008 Sep 15;5(7):763-6.
  3. Polly Matzinger. The Danger Model: A Renewed Sense of Self Science 2002 Apr 12;296(5566):301-5.
  4. Behzadi S, Serpooshan V, Tao W, Hamaly MA, ....Mahmoudi M. Cellular uptake of nanoparticles: journey inside the cell. Chem. Soc. Rev., 2017 Jul 17;46(14):4218-4244
  5. Kierdorf K, Prinz M, Geissmann F, Perdiguero EG. Development and function of tissue resident macrophages in mice. Seminars in immunology. 2015;27(6):369-378.
  6. Ken Donaldson, Fiona A Murphy, Rodger Duffin, Craig A Poland. Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. Particle and Fibre Toxicology 2010, 7:5
  7. Brooke T. Mossman and Andrew Churg. Mechanisms in the Pathogenesis of Asbestosis and Silicosis. Am J Respir Crit Care Med. 1998 Vol 157. pp 1666–1680.
  8. Marco E. Bianchi. DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of Leukocyte Biology 2007 Jan;81(1):1-5.

  9. Nelson C Di Paolo & Dmitry M Shayakhmetov. Interleukin 1α and the inflammatory process. Nat Immunol. 2016 Jul 19;17(8):906-13.

  10. Suwaraa MI, Greena NJ, Borthwicka LA, ..... and DA mann. IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunology (2014) 7, 684–693.

  11. Nikota J, Banville A, .......and Halappanavar S. 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. Part Fibre Toxicol. 2017; 14: 37.

  12. Rabolli V, Badissi AA, Devosse R, .......and Huaux F. The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Part Fibre Toxicol. 2014; 11: 69.

  13. Amsen D, de Visser KE, Town T. Approaches to Determine Expression of Inflammatory Cytokines. Methods in molecular biology (Clifton, NJ). 2009;511:107-142.

  14. Juan A Varela, Mariana G Bexiga, Christoffer Åberg, Jeremy C Simpson and Kenneth A Dawson. Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells. Journal of Nanobiotechnology 2012, 10:39.

  15. Nathalie Decan, Dongmei Wu, Andrew Williams, Stéphane Bernatchez, Michael Johnston, Myriam Hill, Sabina Halappanavar, Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Volume 796, 2016, Pages 8-22.

  16. Nel AE, …Klaessiga F, Castranova V and Thompson M. Understanding biophysicochemical interactions at the nano–bio interface. Nature Materials 8, 543–557 (2009).

  17. Liang-Chien Cheng, Xiumei Jiang, Jing Wang, Chunying Chen and Ru-Shi Liu. Nano–bio effects: interaction of nanomaterials with cells. Nanoscale, 2013, 5, 3547-3569.

  18. National Institute of Occupational Safety and Health. 2011a. Current Intelligence Bulletin 62. Asbestos fibers and other elongate mineral particles: state of the science and roadmap for research. Department of Health and Human Services, Centers for Disease Control and Prevention, DHHS (NIOSH) Publication Number 2011-159. 

  19. Lorella Pascolo, Alessandra Gianoncelli, Giulia Schneider, Murielle Salome, Manuela Schneider,Carla Calligaro, Maya Kiskinova, Mauro Melato & Clara Rizzardi. The interaction of asbestos and iron in lung tissue revealed by synchrotron basedscanning X-ray microscopy. Scientific Reports 3, 2013. Article number: 1123.

  20. 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.

  21. Nikota J, Banville A, .......and Halappanavar S. 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. Part Fibre Toxicol. 2017; 14: 37.

  22. Rabolli V, Badissi A, Devosse R, .......and Huaux F. The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Part Fibre Toxicol. 2014; 11: 69.