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

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).

The empirical support for this KER is moderate. There is both temporal and dose-response evidence to suggest that an increased amount of pro-inflammatory immune cells potentiates ACM damage. However, few studies assessing these KEs include multiple concentrations and timepoints, and as such, these KEs are typically reported as occurring together (i.e. damage is detected along with an increase in cell abundance) (Umbright et al., 2017; Zeidler-Erdley et al., 2011; Additional references available in Table 1).

Dose-Response Evidence:

There are some studies that provide dose-response evidence of this KER. For example, in vitro and in vivo studies testing stressors at different doses/concentrations have demonstrated a dose-response relationship; at the higher dose/concentration of the stressor, the recruitment of pro-inflammatory cells increased leading to loss of ACM integrity.

Microvascular endothelial and the human lung adenocarcinoma cell lines in co-culture were exposed to 6-600 µg/ml silica nanoparticles (NPs). After 4 h of exposure and 20 h recovery, Soluble intercellular adhesion molecule-1 (sICAM-1), IL-6, and IL-8 increased in a concentration-dependent manner. These cytokines increased the recruitment and regulation of neutrophils. The study suggests that there is a crosstalk of both cell types in co-culture that leads to basolateral cytokine secretion. Moreover, transepithelial/transendothelial electrical resistance decreased in a concentration-dependent manner after 4 h exposure, and changes in the assembly of cell-cell junctions were observed (Kasper J et al. 2011).

Polyhexamethylene guanidine phosphate is used for the prevention of microorganism growth in humidifiers. To evaluate the inflammation response after the exposure to this chemical, three human lung cells (Calu-3, differentiated THP-1, and HMC-1 cells) were culture and exposed at the air-liquid interface at 2.2, 4.4, 8.8, and 17.6 mg/ml for 1, 6 and 24 h. An increase in chemoattractant cytokine IL-8 release was observed in a concentration- and time-dependent manner. This response preceded the ACM integrity loss, which occurred at 24 h at the highest concentration. These changes were followed by ROS generation, an increase in the levels of Matrix metalloproteinase (MMP)-2, Metalloproteinase inhibitor 1 (TIMP-1), MMP-9, TIMP-2 mRNA expression, and the release of TNF-α, IL-6 and Transforming growth factor beta 1 (TGF-β1). Moreover, inflammatory cell infiltration, fibrosis, and the release of cytokines was observed in lung sections in Sprague-Dawley rats following a 3 week exposure (Kim et al., 2016).

Arras et al. (2001) studied the effect of IL-9 on the development of lung fibrosis after crystalline silica particle (DQ12) exposure. Transgenic Tg5 mice expressing high levels of IL-9 and wild type (WT) FVB mice were exposed to DQ12 particles by intratracheal instillation at 1 or 5 mg, and mice were sacrificed 2, 4, and 6 months after instillation. Recruitment of pro-inflammatory cells and an increase in the level of Lactate dehydrogenase (LDH) and proteins were observed in bronchoalveolar lavage fluid (BALF) after 2 and 4 months at the highest exposure (5 mg). Hydroxyproline content in the lung increased over time with the highest levels seen 4 months after instillation. Moreover, IL-4 increased in a dose-dependent manner after 2 months of exposure. In contrast, Interferon gamma (IFN-γ) decreased after 2 months and 4 months after the highest exposure dose. In Tg5 mice, fibrosis was less severe than in WT mice. Moreover, intraperitoneal injection of IL-9 in C57BL/6 reduced the amplitude of silica-induced lung fibrosis associated with a recruitment of B lymphocytes in the lung parenchyma.

Morimoto et al. (2015) studied the inflammatory response of cerium oxide NPs in the acute and chronic phases. Male F344 rats were exposed once to 0.2 mg or 1 mg of NPs. Total cell counts in BALF increased at 1 mg after one week, and decreased after 1 and 3 months. Only neutrophils increased at the lowest dose at 3 days and 1 week after exposure and dropped after 1, 3, and 6 months. LDH activity in BALF increased after 3 days of exposure and decreased over time (1 week to 3 months). In the chronic phase, the total cell counts increased 3-days post-exposure, with no effects after 1 and 3 months. LDH increased after 3 days of administration and decreased in a time-dependent manner. The effects were more severe at the highest dose.

Temporal Evidence:

There is strong evidence of a temporal relationship between the two KES.  In vitro and in vivo studies have demonstrated that the recruitment of pro-inflammatory cells increased prior to loss of ACM integrity.

Mice exposed to aerosolized multi-walled carbon nanotube (MWCNT) at 10 mg/m³ (5 h per day for 2, 4, 8 or 12 days) developed pulmonary inflammation 1-day post-exposure. There was an increase in the recruitment of polymorphonuclear cells, neutrophil chemoattractant, albumin concentration, and LDH activity in the whole lung fluid in a time-dependent manner (Porter et al., 2013).

Research has shown that crystalline silica induces pulmonary toxicity. Rats exposed to this stressor at 15 mg/m³, 6 h/day, 5 days/week for 3, 6, and 12 weeks, showed an accumulation of macrophages and neutrophils in BALF. Moreover, an increase in BALF LDH activity and albumin content was also observed (Umbright et al., 2017).

Gautam et al. (1998) studied the effect of a chemotactic stimulant, N-formylmethionine-leucyl-phenylalanine (fMLP), on polymorphonuclear cells resting on an endothelial cell monolayer in the upper compartment. Bovine aorta endothelial cells exposed to 10^-7 M fMLP in the upper compartment induced adhesion of polymorphonuclear (PMN) cells followed by a decrease in transendothelial electrical resistance and an increase in protein permeability for 10-50 min in a time-dependent manner. The findings indicate that PMN are activated by fMLP and adhere to the endothelium which induces an increase in cytosolic free Ca²⁺. This lead to a decrease in electrical resistance, leading to a structural rearrangement of endothelial cells which impairing their barrier integrity.

Paraquat is a herbicide that induces pulmonary toxicity. Chronic exposure to Paraquat results in inflammation, damage to AECs, and fibrosis. In sheep exposed to Paraquat at 5 mg/Kg (intramuscular), there was an increase in the number of granulocytes after 1, 2, and 3 weeks in a dose-dependent manner. After 3 weeks of paraquat administration alveolar wall thickening was observed, and the concentration of lung malondialdehyde increased as an indicator of lipid peroxidation. These results indicate that the recruitment of pro-inflammatory cells precedes alveolar damage (Shinozaki et al., 1992).

Exposure to nickel NPs can induce oxidative stress and lung inflammation. In a dose-response study, mice were intratracheally instilled with 0, 10, 20, 50, and 100 µg per mouse of nickel NPs and sacrificed at day 3 post-exposure. There was an increase in the neutrophil count in BALF, LDH activity, and total protein in BALF in a dose-dependent manner; the highest response was observed at 50 µg per mouse (Mo et al., 2019). Mice were intratracheally instilled with 50 µg per mouse of nickel NPs and sacrificed at days 1, 3, 7, 14, 28, and 42 post-instillation.  The recruitment of neutrophils increased at day 1 and 3 post-exposure. The levels of thiobarbituric acid reactive substances (TBARS) and deoxyguanosine (8-OHdG), which are biomarkers for oxidative stress, increased after 3 days of instillation. LDH activity and total protein in BALF increased at day 3 and 7 post-exposure. These responses decreased after 14 days post-exposure. However, at 42-day post-exposure, an increase in the level of hydroxyproline content was observed in lung tissues exposed to NPs (Mo et al., 2019).

Park et al. (2009) used BALF indicators as a tool for evaluating radiation-induced lung damage. Sprague-Dawley rats received 20 Gray (Gy) of radiation to the right lung. At 3, 7, 14, 28, and 56 days after radiation, rats were sacrificed. Total cells in the BALF increased at 14 and 28 days, with the highest levels at day 56. Meanwhile, total protein in BALF increased after 7 days of radiation and peaked at 28 days post-radiation. The levels of TGF-β increase after 56 days of radiation.

Sapoznikov et al. (2019) studied the role of neutrophils in the early disruption of the alveolar-capillary barrier in a ricin-induced acute respiratory distress syndrome mouse model. Female CD-1 mice were administered intranasally with crude ricin (50 ml; 7 µg/Kg diluted in phosphate buffered saline), and after 3, 6, 24, 48, and 72 h animals were sacrificed. The neutrophil count increased at 24, 48, and 72 h post-exposure. ACM integrity loss was evaluated as Evans blue dye extravasation and the protein expression of Vascular endothelial cadherin (VE-cadherin), claudin 18, claudin 5, connexin 43, and occludin. After 6 h post-exposure, alveolar permeability increased in a time-dependent manner. From 3 h post-exposure, the decrease of junction proteins was evident. Animals treated with anti-ricin antibody, anti-Ly6G (neutrophil depletion), and marismat (MMP inhibition) showed less severity in alveolar membrane integrity loss.

Wan et al. (2017) studied the genotoxic effects of cobalt NPs and their capacity for causing oxidative stress and inflammation. Gpt delta transgenic mice were exposed to 50 μg/mouse of cobalt NPs by intratracheal instillation, and animals were sacrificed at days 1, 3, 7, and 28 post-exposure, as well as 4 months post-exposure. The levels of C-X-C motif chemokine ligand (CXCL)1/Keratinocyte chemoattractant (KC) and neutrophils increased at 1, 3, and 7 days post-exposure, but they decreased at day 28. LDH activity and protein content in BALF also increased, but their levels dropped after 28 days post-exposure. Four months after instillation, 8-OHdG levels were measured and were found at high levels. Moreover, histological changes were observed 7 days (infiltration of a large amount of neutrophils and macrophages in the alveolar space and septa, focal alveolar epithelial cell hyperplasia, and thickening of the alveolar wall) and 4 months (interstitial fibrosis, bronchiolization of the alveoli and collagen deposition in the alveolar septa) after the exposure. 

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.

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.