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Relationship: 1704


A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Recruitment of inflammatory cells leads to Loss of alveolar capillary membrane integrity

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
Substance interaction with the lung resident cell membrane components leading to lung fibrosis adjacent Moderate Moderate Sabina Halappanavar (send email) Under development: Not open for comment. Do not cite EAGMST Under Review

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 KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER.  More help

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER.  More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Acute lung injury followed by normal repair of the ACM results in rapid resolution of the tissue injury and restoration of tissue integrity and function. The irreversible loss of alveolar membrane integrity occurs when 1) acute inflammation is not able to get rid of the toxic substance or invading pathogen (this happens following exposure to a toxic substance that is persistent or when the host is repeatedly exposed to the substance over a long period of time, 2) acute inflammation, originally incited to protect the host from external stimuli and to maintain normal homeostasis, by itself damages the host, resulting in tissue injury, and 3) the host fails to initiate a resolution response, which is essential to override the self-perpetuating inflammation response (Nathan, 2002). Loss of type-1 epithelial cells and endothelial cells, the collapse of alveolar structures and fusion of basement membranes, and persistent proliferation of type II alveolar epithelial cells on a damaged ECM, mark this phase (Strieter and Mehrad, 2009). The lung tissues from patients diagnosed with idiopathic pulmonary fibrosis show ultrastructural damage to the ACM with type-1 pneumocyte and endothelial cell injury (Strieter and Mehrad, 2009). In rodents treated with bleomycin, the damaged ACM resembles that seen in the fibrotic human lung (Grande et al., 1998).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER.  For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings.  Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help
Biological Plausibility
Addresses the biological rationale for a connection between KEupstream and KEdownstream.  This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured.   More help

The biological plausibility of this KER is high. There is a mechanistic relationship between an increase in pro-inflammatory cells and mediators, and damage to the ACM (Bhalla et al., 2009; Ward 2003; Zemans et al., 2009).

Exposure to high doses of insoluble nanomaterials can impair the macrophage-mediated clearance process, initiating chronicity of inflammation characterized by cytokine release, ROS synthesis and the tissue damage cascade (Palecanda and Kobzik, 2001) and subsequently leading to tissue injury. For example, exposure to crystalline silica generates oxidative stress, increased release of pro-inflammatory cytokines (e.g. TNF-α, IL-1, IL-6), activation of transcription factors (e.g. NF-κB, AP-1), and other cell signalling pathways including MAP and ERK kinase (Hubbard et al., 2001; Hubbard et al., 2002; Fubini and Hubbard 2003). In silicosis, TNF-α is suggested to play a critical role in the observed pathogenicity (Castranova et al., 2004), which in turn, is dependent on activation of NF-κB and ROS synthesis (Shi et al.,1998; Cassel et al.,2008; Kawasaki et al., 2015). It has been proposed that IPF is a disorder of elevated oxidative stress, with the existence of an oxidant-antioxidant imbalance in distal alveolar air spaces (MacNee, 2001). Several studies have reported that anti-oxidant treatment attenuates the bleomycin-induced oxidative burden and subsequent pulmonary fibrosis (Wang et al., 2002; Serrano-Mollar et al., 2003; Punithavathi, et al., 2000).

Mice deficient in Nalp3 showed reduced inflammation, lower cytokine production and dampened fibrotic response following exposure to asbestos or silica (Dostert et al., 2008). SWCNT exposure induces alveolar macrophage activation, enhanced oxidative stress, increased and persistent expression of pro-inflammatory mediators associated with chronic inflammation and severe granuloma formation in mice (Chou et al., 2008). Bleomycin treatment induces increased lung weight, epithelial cell death, inflammation, increased hydroxyproline content, collagen accumulation and fibrotic lesions in mice, all of which were elevated in mice deficient in Nrf2 (Cho et al., 2004). MWCNT-induced fibrotic response is the result of interplay between oxidative stress and inflammation, which determines the severity of the fibrotic pathology. Mice lacking Nrf2 (the nuclear factor erythroid 2-related factor 2), that is associated with mounting anti-oxidant defense against oxidative stress, exhibit exuberant fibrotic responses to MWCNT (Dong and Ma, 2016).

Uncertainties and Inconsistencies
Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Although there is enough evidence to suggest a role for persistent inflammation and oxidative stress in ACM integrity loss, a direct relationship is hard to establish as studies involving inhibition of early pro-inflammatory cellular influx alter other immune cell types, thereby altering the end outcome.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references.  More help
Response-response Relationship
Provides sources of data that define the response-response relationships between the KEs.  More help
Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

One publication examined the timescale of KE induction with relation to this KER, in the context of AOP 173. Mo et al., 2019 found that KE2 (Event 1497) (1 and 3 days post-exposure) precedes KE3 (Event 1498) (3 and 7 days post-exposure) in mice exposed to 50 μg per mouse of nickel nanoparticles by intratracheal instillation.


In vitro/in vivo/population study


KE1 (Event 1496)

  KE2 (Event 1497)

KE3 (Event 1498)

KE6 (Event 1501)

Mo Y et al., 2019

In vivo

Mice C57BL/6, 50 mg per mouse intratracheal instillation


1- and 3-days post-exposure

Neutrophil content

1 and 3 days


LDH activity, oxidative stress protein content

3- and 7-days post-exposure

Hydroxyproline content

42 days post-exposure

Known Feedforward/Feedback loops influencing this KER
Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help


List of the literature that was cited for this KER description. More help
  1. Arras M et al. Interleukine-9 reduces lung fibrosis and type 2 immune polarization induced by silica particles in a murine model. Am J Respir Cell Mol Biol, 2001, 24, 368-375.
  2. Barosova H et al. Use of epiAlveolar lung model to predict fibrotic potential of multiwalled carbon nanotubes. ACSNANO, 2020, 14(4): 3941-3956.
  3. Bhalla, D. K., Hirata, F., Rishi, A. K., & Gairola, C. G. (2009). Cigarette smoke, inflammation, and lung injury: a mechanistic perspective. Journal of toxicology and environmental health. Part B, Critical reviews, 12(1), 45–64.
  4. Blum et al. Short-term inhalation of cadmium oxide nanparticles alters pulmonary dynamics associated with lung injury, inflammation, and repair in a mouse model. Inhalation toxicology, 2014, 26(1): 48-58.
  5. Caielli, S., Banchereau, J. and Pascual, V. (2012). Neutrophils come of age in chronic inflammation. Current Opinion in Immunology, 24(6), pp.671-677.
  6. Cassel, S., Eisenbarth, S., Iyer, S., Sadler, J., Colegio, O., Tephly, L., Carter, A., Rothman, P., Flavell, R. and Sutterwala, F. (2008). The Nalp3 inflammasome is essential for the development of silicosis. Proceedings of the National Academy of Sciences, 105(26), pp.9035- 9040.
  7. Castranova, V. (2004). Signaling Pathways Controlling The Production Of Inflammatory Mediators in Response To Crystalline Silica Exposure: Role Of Reactive Oxygen/Nitrogen Species. Free Radical Biology and Medicine, 37(7), pp.916-925.
  8. Chaudhary, N., Schnapp, A. and Park, J. (2006). Pharmacologic Differentiation of Inflammation and Fibrosis in the Rat Bleomycin Model. American Journal of Respiratory and Critical Care Medicine, 173(7), pp.769-776.
  9. Cho, H., Reddy, S., Yamamoto, M. and Kleeberger, S. (2004). The transcription factor NRF2 protects against pulmonary fibrosis. The FASEB Journal, 18(11), pp.1258-1260.
  10. Chou, C., Hsiao, H., Hong, Q., Chen, C., Peng, Y., Chen, H. and Yang, P. (2008). Single-Walled Carbon Nanotubes Can Induce Pulmonary Injury in Mouse Model. Nano Letters, 8(2), pp.437-445.
  11. Cui A et al. VCAM-1 mediated neutrophil infiltration exacerbates ambient fine particle-induced lung injury. Toxicology Letters, 2018, 1;302: 60-74.
  12. Dong, J. and Ma, Q. (2016). In vivo activation of a T helper 2-driven innate immune response in lung fibrosis induced by multi-walled carbon nanotubes. Archives of Toxicology, 90(9), pp.2231-2248.
  13. Dostert, C., Petrilli, V., Van Bruggen, R., Steele, C., Mossman, B. and Tschopp, J. (2008). Innate Immune Activation Through Nalp3 Inflammasome Sensing of Asbestos and Silica. Science, 320(5876), pp.674-677.
  14. Fubini, B. and Hubbard, A. (2003). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radical Biology and Medicine, 34(12), pp.1507-1516.
  15. Gautam N et al. Kinetics of leukocyte-induced changes in endothelial barrier function. British Journal of Pharmacology, 1998, 125, 1109-1114.
  16. Grande, N. R., Peao, M. N. ., de Sa, C. M., & Aguas, A. P. (1998). Lung Fibrosis Induced by Bleomycin: Structural Changes and Overview of Recent Advances. Scanning Microsc, 12(3), 487–494.
  17. Hubbard, A., Timblin, C., Rincon, M. and Mossman, B. (2001). Use of Transgenic Luciferase Reporter Mice To Determine Activation of Transcription Factors and Gene Expression by Fibrogenic Particles. Chest, 120(1), pp.S24-S25.
  18. 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.
  19. Inoue H et al. Ultrastructural changes of the air-blood barrier in mice after intratracheal instillation of lipopolysaccharide and ultrafine carbon black particles. Experimental and toxicology pathology, 2009, 61: 51-58.
  20. Janga H et al. Site-specific and endothelial-mediated dysfunction of the alveolar-capillary barrier in response to lipopolysaccharides. J Cell Mol Med, 2018, 22(2): 982-998.
  21. Kasai T et al. Lung carcinogenicity of inhaled multi-walled carbon nanotube in rats. Particle and Fibre Toxicology, 2016, 13:53.
  22. Kasper J et al. Inflammatory and cytotoxic responses of an alveolar-capillary co-culture model to silica nanoparticles: Comparison with conventional monocultures. Particle and Fibre Toxicology, 2011,8:6.
  23. Kawasaki, H. (2015). A mechanistic review of silica-induced inhalation toxicity. Inhalation Toxicology, 27(8), pp.363-377.
  24. Kim H et al. Polyhexamethylene guanidine phosphate aerosol particles induce pulmonary inflammatory and fibrotic responses. Arch Toxicol. 2015, 90(3): 617-32.
  25. Kim, S., Lee, J., Yang, H., Cho, J., Kwon, S., Kim, Y., Her, J., Cho, K., Song, C. and Lee, K. (2010). Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice. Toxicological Research, 26(3), pp.217-222.
  26. Koli, K., Myllärniemi, M., Keski-Oja, J. and Kinnula, V. (2008). Transforming Growth Factor-β Activation in the Lung: Focus on Fibrosis and Reactive Oxygen Species. Antioxidants & Redox Signaling, 10(2), pp.333-342.
  27. Ma, B., Whiteford, J., Nourshargh, S. and Woodfin, A. (2016). Underlying chronic inflammation alters the profile and mechanisms of acute neutrophil recruitment. The Journal of Pathology, 240(3), pp.291-303.
  28. MacNee, W. (2001). Oxidative stress and lung inflammation in airways disease. European Journal of Pharmacology, 429(1-3), pp.195-207.
  29. Marcus B et al. Loss of endothelial barrier function requires neutrophil adhesion. Surgery, 1997: 420-427
  30. Mo Y et al. Comparative mouse lung injury by nickel nanoparticles with differential surface modification. Journal of Nanobiotechnology, 2019, 17:2.
  31. Morimoto Y et al. Pulmonary toxicity of well-dispersed cerium oxide nanoparticles following intratracheal instillation and inhalation. J Nanopart Res, 2015, 17:442.
  32. Nathan, C. (2002). Points of control in inflammation. Nature, 420(6917), pp.846-852.
  33. Nemmar A et al. Chronic exposure to water-pipe smoke induces alveolar enlargement, DNA damage and impairment of lung function. Cell Physiol Biochem, 2016, 38:382-992.
  34. Pacheco Y et al. Granulomatous lung inflammation is nanoparticle type-dependent. Experimental lung research, 2018, 44(1): 25-39.
  35. Palecanda, A. and Kobzik, L (2001). Receptors for Unopsonized Particles: The Role of Alveolar Macrophage Scavenger Receptors. Current Molecular Medicine, 1(5), pp.589-595.
  36. Park K et al. Bronchoalveolar lavage findings of radiation induced lung damage in rats. J. Radiat. Res., 2009, 50: 177-182.
  37. Porter D et al. Acute pulmonary dose-responses to inhaled multi-walled carbon nanotubes. Nanotoxicology, 2013, 7(7): 1179-1194.
  38. Punithavathi, D., Venkatesan, N. and Babu, M. (2000). Curcumin inhibition of bleomycin-induced pulmonary fibrosis in rats. British Journal of Pharmacology, 131(2), pp.169-172.
  39. Sapoznikov A et al. Early disruption of the alveolar-capillary barrier in a ricin-induced ARDS mouse model: neutrophil-dependent and -independent impairment of junction proteins. Am J Physiol Lung Cell Mol Physiol, 2019, 316(1): L255-L268.
  40. Sellamuthu R et al. Molecular mechanisms of pulmonary response progression in crystalline silica exposed rats. Inhalation toxicology, 2017, 29(2): 53-64.
  41. Serrano-Mollar, A., Closa, D., Prats, N., Blesa, S., Martinez-Losa, M., Cortijo, J., Estrela, J., Morcillo, E. and Bulbena, O. (2003). In vivoantioxidant treatment protects against bleomycin-induced lung damage in rats. British Journal of Pharmacology, 138(6), pp.1037-1048.
  42. Shi, X., Castranova, V., Halliwell, B. and Vallyathan, V. (1998). Reactive oxygen species and silicainduced carcinogenesis. Journal of Toxicology and Environmental Health, Part B, 1(3), pp.181-197.
  43. Shinozaki S et al. Pulmonary hemodynamics and lung function during chronic paraquat poisoning in sheep. Am Rev Respir Dis, 1992, 146:775-780.
  44. Soehnlein, O., Steffens, S., Hidalgo, A. and Weber, C. (2017). Neutrophils as protagonists and targets in chronic inflammation. Nature Reviews Immunology, 17(4), pp.248-261.
  45. Strieter, R. and Mehrad, B. (2009). New Mechanisms of Pulmonary Fibrosis. Chest, 136(5), pp.1364-1370.
  46. Umbright, C., Sellamuthu, R., Roberts, J. R., Young, S. H., Richardson, D., Schwegler-Berry, D., McKinney, W., Chen, B., Gu, J. K., Kashon, M., & Joseph, P. (2017). Pulmonary toxicity and global gene expression changes in response to sub-chronic inhalation exposure to crystalline silica in rats. Journal of toxicology and environmental health. Part A, 80(23-24), 1349–1368.
  47. Wan et al. Cobalt nanoparticles induce lung injury, DNA damage and mutations in mice. Particle and Fibre Toxicology, 2017, 14:38.
  48. Ward P. A. (2003). Acute lung injury: how the lung inflammatory response works. The European respiratory journal. Supplement, 44, 22s–23s.
  49. Wang, H., Yamaya, M., Okinaga, S., Jia, Y., Kamanaka, M., Takahashi, H., Guo, L., Ohrui, T. and Sasaki, H. (2002). Bilirubin Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats. American Journal of Respiratory and Critical Care Medicine, 165(3), pp.406-411.
  50. Zeidler-Erdely, P. C., Battelli, L. A., Stone, S., Chen, B. T., Frazer, D. G., Young, S. H., Erdely, A., Kashon, M. L., Andrews, R., & Antonini, J. M. (2011). Short-term inhalation of stainless steel welding fume causes sustained lung toxicity but no tumorigenesis in lung tumor susceptible A/J mice. Inhalation toxicology, 23(2), 112–120.
  51. Zemans, R. L., Colgan, S. P., & Downey, G. P. (2009). Transepithelial migration of neutrophils: mechanisms and implications for acute lung injury. American journal of respiratory cell and molecular biology, 40(5), 519–535.