This Key Event Relationship is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

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 pulmonary resident cell membrane components leading to pulmonary fibrosis adjacent Moderate Moderate Sabina Halappanavar (send email) Under development: Not open for comment. Do not cite WPHA/WNT Endorsed

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 alveolar capillary membrane (ACM) results in rapid resolution of the tissue injury and restoration of tissue integrity and function. The irreversible loss of ACM 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 alveolar epithelial cells (AEC1s) and endothelial cells, the collapse of alveolar structures and fusion of basement membranes, and persistent proliferation of type II alveolar epithelial cells (AEC2s) on a damaged extracellular matrix, mark this phase (Barosova et al., 2020; Blum et al., 2014; Inoue et al., 2009; Janga et al., 2018; Marcus et al., 1997; Nemmar et al., 2016; Strieter and Mehrad, 2009). The lung tissues from patients diagnosed with idiopathic pulmonary fibrosis (IPF) 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, reactive oxygen species (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. Tumor necrosis factor alpha [TNF-α], Interleukin [IL]-1, IL-6), activation of transcription factors (e.g. Nuclear factor kappa B [NF-κB], Activator protein [AP-1]), and other cell signalling pathways including Mitogen-activated protein kinases (MAPKs) and Extracellular signal-regulated kinases [ERKs] (Fubini and Hubbard 2003; Hubbard et al., 2001; Hubbard et al., 2002). 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 (Cassel et al.,2008; Kawasaki et al., 2015; Shi et al.,1998). 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 antioxidant treatment attenuates the bleomycin-induced oxidative burden and subsequent pulmonary fibrosis (Punithavathi, et al., 2000; Serrano-Mollar et al., 2003; Wang et al., 2002).

Mice deficient in NLR family pyrin domain containing 3 (Nalp3) showed reduced inflammation, lower cytokine production and dampened fibrotic response following exposure to asbestos or silica (Dostert et al., 2008). Single-walled carbon nanotube 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 Nuclear factor erythroid 2-related factor 2 (Nrf2) (Cho et al., 2004). Multi-walled carbon nanotube (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 that is associated with mounting antioxidant 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 NPs 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

C-X-C motif chemokine ligand 1/keratinocyte-derived chemokine (CXCL1/KC)

1- and 3-days post-exposure

Neutrophil content

1 and 3 days post-exposure

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, Huaux F, Vink A, Delos M, Coutelier JP, Many MC, Barbarin V, Renauld JC, Lison D. Interleukin-9 reduces lung fibrosis and type 2 immune polarization induced by silica particles in a murine model. Am J Respir Cell Mol Biol. 2001 Apr;24(4):368-75. doi: 10.1165/ajrcmb.24.4.4249.

  2. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. 

  3. Bhalla DK, Hirata F, Rishi AK, Gairola CG. Cigarette smoke, inflammation, and lung injury: a mechanistic perspective. J Toxicol Environ Health B Crit Rev. 2009 Jan;12(1):45-64. doi: 10.1080/10937400802545094. 

  4. Blum JL, Rosenblum LK, Grunig G, Beasley MB, Xiong JQ, Zelikoff JT. Short-term inhalation of cadmium oxide nanoparticles alters pulmonary dynamics associated with lung injury, inflammation, and repair in a mouse model. Inhal Toxicol. 2014 Jan;26(1):48-58. doi: 10.3109/08958378.2013.851746. 

  5. Cassel SL, Eisenbarth SC, Iyer SS, Sadler JJ, Colegio OR, Tephly LA, Carter AB, Rothman PB, Flavell RA, Sutterwala FS. The Nalp3 inflammasome is essential for the development of silicosis. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9035-40. doi: 10.1073/pnas.0803933105.

  6. Castranova V. Signaling pathways controlling the production of inflammatory mediators in response to crystalline silica exposure: role of reactive oxygen/nitrogen species. Free Radic Biol Med. 2004 Oct 1;37(7):916-25. doi: 10.1016/j.freeradbiomed.2004.05.032.

  7. Cho HY, Reddy SP, Yamamoto M, Kleeberger SR. The transcription factor NRF2 protects against pulmonary fibrosis. FASEB J. 2004 Aug;18(11):1258-60. doi: 10.1096/fj.03-1127fje.

  8. Chou CC, Hsiao HY, Hong QS, Chen CH, Peng YW, Chen HW, Yang PC. Single-walled carbon nanotubes can induce pulmonary injury in mouse model. Nano Lett. 2008 Feb;8(2):437-45. doi: 10.1021/nl0723634. 

  9. Dong J, Ma Q. Suppression of basal and carbon nanotube-induced oxidative stress, inflammation and fibrosis in mouse lungs by Nrf2. Nanotoxicology. 2016 Aug;10(6):699-709. doi: 10.3109/17435390.2015.

  10. Dostert C, Pétrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science. 2008 May 2;320(5876):674-7. doi: 10.1126/science.1156995. 

  11. Fubini B, Hubbard A. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic Biol Med. 2003 Jun 15;34(12):1507-16. doi: 10.1016/s0891-5849(03)00149-7. 

  12. Gautam N, Hedqvist P, Lindbom L. Kinetics of leukocyte-induced changes in endothelial barrier function. Br J Pharmacol. 1998 Nov;125(5):1109-14. doi: 10.1038/sj.bjp.0702186. 

  13. Grande NR, Peao MN, de Sa CM, Aguas AP. Lung Fibrosis Induced by Bleomycin: Structural Changes and Overview of Recent Advances. Scanning Microsc. 1998 12(3), 487–494.

  14. Hubbard AK, Timblin CR, Rincon M, Mossman BT. Use of transgenic luciferase reporter mice to determine activation of transcription factors and gene expression by fibrogenic particles. Chest. 2001 Jul;120(1 Suppl):24S-25S. doi: 10.1378/chest.120.1_suppl.s24. 

  15. Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001.

  16. Inoue H, Shimada A, Kaewamatawong T, Naota M, Morita T, Ohta Y, Inoue K, Takano H. Ultrastructural changes of the air-blood barrier in mice after intratracheal instillation of lipopolysaccharide and ultrafine carbon black particles. Exp Toxicol Pathol. 2009 Jan;61(1):51-8. doi: 10.1016/j.etp.2007.10.001.

  17. Janga H, Cassidy L, Wang F, Spengler D, Oestern-Fitschen S, Krause MF, Seekamp A, Tholey A, Fuchs S. Site-specific and endothelial-mediated dysfunction of the alveolar-capillary barrier in response to lipopolysaccharides. J Cell Mol Med. 2018 Feb;22(2):982-998. doi: 10.1111/jcmm.13421.

  18. Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC. Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures. Part Fibre Toxicol. 2011 Jan 27;8(1):6. doi: 10.1186/1743-8977-8-6. 

  19. Kawasaki H. A mechanistic review of silica-induced inhalation toxicity. Inhal Toxicol. 2015;27(8):363-77. doi: 10.3109/08958378.2015.1066905.

  20. Kim HR, Lee K, Park CW, Song JA, Shin DY, Park YJ, Chung KH. Polyhexamethylene guanidine phosphate aerosol particles induce pulmonary inflammatory and fibrotic responses. Arch Toxicol. 2016 Mar;90(3):617-32. doi: 10.1007/s00204-015-1486-9. 

  21. Kim SN, Lee J, Yang HS, Cho JW, Kwon S, Kim YB, Her JD, Cho KH, Song CW, Lee K. Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice. Toxicol Res. 2010 Sep;26(3):217-22. doi: 10.5487/TR.2010.26.3.217.

  22. Ma B, Whiteford JR, Nourshargh S, Woodfin A. Underlying chronic inflammation alters the profile and mechanisms of acute neutrophil recruitment. J Pathol. 2016 Nov;240(3):291-303. doi: 10.1002/path.4776.

  23. MacNee W. Oxidative stress and lung inflammation in airways disease. Eur J Pharmacol. 2001 Oct 19;429(1-3):195-207. doi: 10.1016/s0014-2999(01)01320-6. 

  24. Marcus BC, Hynes KL, Gewertz BL. Loss of endothelial barrier function requires neutrophil adhesion. Surgery. 1997 Aug;122(2):420-6; discussion 426-7. doi: 10.1016/s0039-6060(97)90035-0.

  25. Mo Y, Jiang M, Zhang Y, Wan R, Li J, Zhong CJ, Li H, Tang S, Zhang Q. Comparative mouse lung injury by nickel nanoparticles with differential surface modification. J Nanobiotechnology. 2019 Jan 7;17(1):2. doi: 10.1186/s12951-018-0436-0. 

  26. Morimoto Y, Izumi H, Yoshiura Y, Tomonaga T, Oyabu T, Myojo T, Kawai K, Yatera K, Shimada M, Kubo M, Yamamoto K, Kitajima S, Kuroda E, Kawaguchi K, Sasaki T. Pulmonary toxicity of well-dispersed cerium oxide nanoparticles following intratracheal instillation and inhalation. J Nanopart Res. 2015;17(11):442. doi: 10.1007/s11051-015-3249-1.

  27. Nathan C. Points of control in inflammation. Nature. 2002 Dec 19-26;420(6917):846-52. doi: 10.1038/nature01320. 

  28. Nemmar A, Al-Salam S, Yuvaraju P, Beegam S, Yasin J, Ali BH. Chronic Exposure to Water-Pipe Smoke Induces Alveolar Enlargement, DNA Damage and Impairment of Lung Function. Cell Physiol Biochem. 2016;38(3):982-92. doi: 10.1159/000443050. 

  29. Palecanda A, Kobzik L. Receptors for unopsonized particles: the role of alveolar macrophage scavenger receptors. Curr Mol Med. 2001 Nov;1(5):589-95. doi: 10.2174/1566524013363384.

  30. Park KJ, Oh YT, Kil WJ, Park W, Kang SH, Chun M. Bronchoalveolar lavage findings of radiation induced lung damage in rats. J Radiat Res. 2009 May;50(3):177-82. doi: 10.1269/jrr.08089.

  31. Porter DW, Hubbs AF, Chen BT, McKinney W, Mercer RR, Wolfarth MG, Battelli L, Wu N, Sriram K, Leonard S, Andrew M, Willard P, Tsuruoka S, Endo M, Tsukada T, Munekane F, Frazer DG, Castranova V. Acute pulmonary dose-responses to inhaled multi-walled carbon nanotubes. Nanotoxicology. 2013 Nov;7(7):1179-94. doi: 10.3109/17435390.2012.719649.

  32. Punithavathi D, Venkatesan N, Babu M. Curcumin inhibition of bleomycin-induced pulmonary fibrosis in rats. Br J Pharmacol. 2000 Sep;131(2):169-72. doi: 10.1038/sj.bjp.0703578.

  33. Sapoznikov A, Gal Y, Falach R, Sagi I, Ehrlich S, Lerer E, Makovitzki A, Aloshin A, Kronman C, Sabo T. 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 Jan 1;316(1):L255-L268. doi: 10.1152/ajplung.00300.2018.

  34. Serrano-Mollar A, Closa D, Prats N, Blesa S, Martinez-Losa M, Cortijo J, Estrela JM, Morcillo EJ, Bulbena O. In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats. Br J Pharmacol. 2003 Mar;138(6):1037-48. doi: 10.1038/sj.bjp.0705138.

  35. Shi X, Castranova V, Halliwell B, Vallyathan V. Reactive oxygen species and silica-induced carcinogenesis. J Toxicol Environ Health B Crit Rev. 1998 Jul-Sep;1(3):181-97. doi: 10.1080/10937409809524551.

  36. Shinozaki S, Kobayashi T, Kubo K, Sekiguchi M. Pulmonary hemodynamics and lung function during chronic paraquat poisoning in sheep. Possible role of reactive oxygen species. Am Rev Respir Dis. 1992 Sep;146(3):775-80. doi: 10.1164/ajrccm/146.3.775.

  37. Strieter RM, Mehrad B. New mechanisms of pulmonary fibrosis. Chest. 2009 Nov;136(5):1364-1370. doi: 10.1378/chest.09-0510.

  38. Umbright C, Sellamuthu R, Roberts JR, Young SH, Richardson D, Schwegler-Berry D, McKinney W, Chen B, Gu JK, Kashon M, Joseph P. Pulmonary toxicity and global gene expression changes in response to sub-chronic inhalation exposure to crystalline silica in rats. J Toxicol Environ Health A. 2017;80(23-24):1349-1368. doi: 10.1080/15287394.2017.1384773. 

  39. Wan R, Mo Y, Zhang Z, Jiang M, Tang S, Zhang Q. Cobalt nanoparticles induce lung injury, DNA damage and mutations in mice. Part Fibre Toxicol. 2017 Sep 18;14(1):38. doi: 10.1186/s12989-017-0219-z. 

  40. Wang HD, Yamaya M, Okinaga S, Jia YX, Kamanaka M, Takahashi H, Guo LY, Ohrui T, Sasaki H. Bilirubin ameliorates bleomycin-induced pulmonary fibrosis in rats. Am J Respir Crit Care Med. 2002 Feb 1;165(3):406-11. doi: 10.1164/ajrccm.165.3.2003149. 

  41. Ward PA. Acute lung injury: how the lung inflammatory response works. Eur Respir J Suppl. 2003 Sep;44:22s-23s. doi: 10.1183/09031936.03.00000703a.

  42. Zeidler-Erdely PC, Battelli LA, Stone S, Chen BT, Frazer DG, Young SH, Erdely A, Kashon ML, Andrews R, Antonini JM. Short-term inhalation of stainless steel welding fume causes sustained lung toxicity but no tumorigenesis in lung tumor susceptible A/J mice. Inhal Toxicol. 2011 Feb;23(2):112-20. doi: 10.3109/08958378.2010.548838. 

  43. Zemans RL, Colgan SP, Downey GP. Transepithelial migration of neutrophils: mechanisms and implications for acute lung injury. Am J Respir Cell Mol Biol. 2009 May;40(5):519-35. doi: 10.1165/rcmb.2008-0348TR.