A review of pulmonary neutrophilia and insights into the key role of neutrophils in particle-induced pathogenesis in the lung from animal studies of lunar dusts and other poorly soluble dust particles

Abstract

The mechanisms of particle-induced pathogenesis in the lung remain poorly understood. Neutrophilic inflammation and oxidative stress in the lung are hallmarks of toxicity. Some investigators have postulated that oxidative stress from particle surface reactive oxygen species (psROS) on the dust produces the toxicopathology in the lungs of dust-exposed animals. This postulate was tested concurrently with the studies to elucidate the toxicity of lunar dust (LD), which is believed to contain psROS due to high-speed micrometeoroid bombardment that fractured and pulverized lunar surface regolith. Results from studies of rats intratracheally instilled (ITI) with three LDs (prepared from an Apollo-14 lunar regolith), which differed 14-fold in levels of psROS, and two toxicity reference dusts (TiO₂ and quartz) indicated that psROS had no significant contribution to the dusts' toxicity in the lung. Reported here are results of further investigations by the LD toxicity study team on the toxicological role of oxidants in alveolar neutrophils that were harvested from rats in the 5-dust ITI study and from rats that were exposed to airborne LD for 4 weeks. The oxidants per neutrophils and all neutrophils increased with dose, exposure time and dust's cytotoxicity. The results suggest that alveolar neutrophils play a critical role in particle-induced injury and toxicity in the lung of dust-exposed animals. Based on these results, we propose an adverse outcome pathway (AOP) for particle-associated lung disease that centers on the crucial role of alveolar neutrophil-derived oxidant species. A critical review of the toxicology literature on particle exposure and lung disease further supports a neutrophil-centric mechanism in the pathogenesis of lung disease and may explain previously reported animal species differences in responses to poorly soluble particles. Key findings from the toxicology literature indicate that (1) after exposures to the same dust at the same amount, rats have more alveolar neutrophils than hamsters; hamsters clear more particles from their lungs, consequently contributing to fewer neutrophils and less severe lung lesions; (2) rats exposed to nano-sized TiO₂ have more neutrophils and more severe lesions in their lungs than rats exposed to the same mass-concentration of micron-sized TiO₂; nano-sized dust has a greater number of particles and a larger total particle-cell contact surface area than the same mass of micron-sized dust, which triggers more alveolar epithelial cells (AECs) to synthesize and release more cytokines that recruit a greater number of neutrophils leading to more severe lesions. Thus, we postulate that, during chronic dust exposure, particle-inflicted AECs persistently release cytokines, which recruit neutrophils and activate them to produce oxidants resulting in a prolonged continuous source of endogenous oxidative stress that leads to lung toxicity. This neutrophil-driven lung pathogenesis explains why dust exposure induces more severe lesions in rats than hamsters; why, on a mass-dose basis, nano-sized dusts are more toxic than the micron-sized dusts; why lung lesions progress with time; and why dose-response curves of particle toxicity exhibit a hockey stick like shape with a threshold. The neutrophil centric AOP for particle-induced lung disease has implications for risk assessment of human exposures to dust particles and environmental particulate matter.

Keywords: Lunar dust; PM; PSP; ROS; diesel particles; inhalation toxicity; intratracheal instillation; lung pathogenesis; mechanism of toxicity; moon dust; neutrophilia; neutrophils; particulate matter.

Figure 1.

Hydroxyl free radicals •OH in lunar dusts and reference dusts. Specific chemical reactivity was assessed by measuring the amount of •OH/10 mg of dust generated in the presence of terephthalate. The content of •OH in the dust was estimated by the amount of terephthalate consumed. Data are the mean ± SD of •OH production (average from three runs) in pmol/10 mg test dust. Normalization: LD-ug, 100%; LD-jm, 193%; LD-jm, 1367%; TiO₂ 121%; and quartz 76%. Graph plotted from data from Lam et al. (2022).

Figure 2.

(A) Mutagenic effect of alveolar neutrophils or macrophages from quartz-instilled rats on epithelial cells in culture. (B) Relationship between the hprt mutation frequency in alveolar epithelium cells and severity of particle elicited inflammation (% neutrophils) in lungs of rats instilled with the test dusts at 10 mg/kg or 100 mg/kg. Standard deviations are not plotted; refer to original data for accuracy. Data courtesy of Driscoll, Deyo, et al. (1997).

Figure 3.

Rats were intratracheally instilled with lunar dusts (LD), TiO₂, and quartz (SiO₂) at 0 (control), 1, 2.5, or 7.5 mg/rat. The lungs were lavaged 1 or 4 weeks after the dust instillation. Numbers of alveolar macrophages (A) and neutrophils (B) were determined. ROS of total BAL cells was assessed by chemiluminescence (C). X-axis labels: jet-milled LD: LD-jm (green); unground LD: LD-ug (blue); ball-milled LD: LD-bm (red); bars of lighter color-shades represent 1 week; bars of darker color-shades represent 4 weeks. Each bar is the mean ± SD from six rats.

Figure 4.

Relative ROS levels in BAL cells from rats instilled with lunar dusts (LD), TiO₂, and quartz (SiO₂) at 0 (control), 1, 2.5, or 7.5 mg/rat. The lungs were lavaged 1 or 4 weeks after the dust instillation. ROS in BAL cells were assessed by a chemiluminescence assay. X-axis labels: jet-milled LD: LD-jm (green): unground LD: LD-ug (blue); ball-milled LD: LD-bm (red); bars of lighter color-shades represent 1-week groups; bars of darker color-shades represent 4-week groups. Each bar is the mean ± SD from six rats.

Figure 5.

(A) A replica of Figure 3(C) (levels of ROS in all BAL cells) plotted here to ease comparison. (B) ROS levels in all BAL neutrophils (Figure 4(B)) were multiplied by 2 and plotted here (Figure 5(B)) to compare with the ROS levels of all BAL cells (Figure 5(A)). Cells were lavaged from rats 1 or 4 weeks after they instilled with lunar dusts (LD), TiO₂, and quartz (SiO₂) at 0 (control), 1, 2.5, or 7.5 mg/rat. Note, we initially assumed that the level of oxidants generated by a neutrophil = the level of oxidants generated by a BAL cell; however, data from Driscoll, Deyo, et al. (1997) showed that the oxidative capacity of a neutrophil is twofold of that of a BAL cell from rats exposed to quartz. X-axis labels: jet-milled LD: LD-jm (green): unground LD: LD-ug (blue); ball-milled LD: LD-bm (red); bars of lighter color-shades represent 1-week groups; bars of darker color-shades represent 4-week groups.

Figure 6.

Rats were exposed to lunar dust by inhalation at 0, 2.1, 6.8, 20.8, or 60.6 mg/m³ for 4 weeks and the right lungs were lavaged thereafter at 1 d and 1, 4, and 13 weeks. BAL cells were harvested; macrophages and neutrophils were counted and plotted in (A) and (B), respectively. ROS generated by all BAL cells were assessed by chemiluminescence. Each bar is the mean ± SD from five rats.

Figure 7.

Rats were exposed to lunar dust by inhalation at 0, 2.1, 6.8, 20.8, or 60.6 mg/m³ for 4 weeks. The right lungs were lavaged thereafter at 1 d and 1, 4, and 13 weeks. (A) The relative amount of ROS from all BAL cells or neutrophils (estimated by a chemiluminescence assay) and (B) the relative amount of ROS detected in all neutrophils from each of the groups of animals. Each bar is the mean ± SD from five rats.

Figure 8.

(A) Relative amount of ROS in all BAL cells of rats one day or 1, or 13 weeks after they were exposed to airborne lunar dust (a replica of Figure 6(C) plotted here to ease comparison. (B) ROS levels in all BAL neutrophils were multiplied by 2 and plotted here to compare with the ROS levels of all BAL cells (Figure 8(A)). Note, we initially assumed that the level of oxidants generated by a neutrophil = the level of oxidants generated by a BC; however, data from Driscoll, Deyo, et al. (1997) showed that the oxidative capacity of a neutrophil is twofold of that of a BAL cell from rats exposed to quartz.

Figure 9.

Rats were exposed to lunar dust by inhalation at 0, 2.1, 6.8, 20.8, or 60.6 mg/m³ for 4 weeks and the right lungs were lavaged thereafter at 1 d and 1, 4, and 13 weeks. BALFs were assessed for cytokines. (A) IL-1β levels. (B) TNF-α levels. Each data point contains the mean ± SD from five rats.

Figure 10.

Rats were exposed to lunar dust by inhalation at 0, 2.1, 6.8, 20.8, or 60.6 mg/m³ for 4 weeks and the right lungs were lavaged thereafter at 1 d and 1, 4, and 13 weeks. Post-lavage lung tissues were assessed for CXCL2 gene expression (A). Relative levels of ROS from all BALF neutrophils were estimated by a chemiluminescence assay (B). Each point represents the average data from five rats.

Figure 11.

Molecular and genomic mechanism of pathogenesis. Dust lands on the alveolar epithelial cells and triggers the cells to up-regulate the proinflammatory genes to produce chiefly CXCL2 chemokine (cytokine-induced neutrophil chemoattractant 2 (CINC-2)) and TNFα, IL-1β, IL6 cytokines. CINC chemokine recruits neutrophils while TNFα and IL-1β activate neutrophils to produce oxidants, leading to alveolar neutrophilia. Neutrophilic oxidants damage the lung and DNA. Persistent neutrophilia in chronically and heavily dust exposed rodents (particular the rats) could result in lung lesions including fibrosis, hyperplasia, metaplasia, and even cancer. OS: oxidative stress. Total amount of neutrophilic oxidants: $\sum [\mathrm{O}\mathrm{x}{]}_{\mathrm{N}\mathrm{u}}=[\mathrm{O}\mathrm{x}{]}_{\mathrm{N}\mathrm{u}}\times \eta$.

Figure 12.

Rats were exposed to 15.3 mg/m³ quartz for up to 116 days. (A) number of neutrophils (value × million/rat) and NOx (reactive nitrogen species) levels (treated minus control) in BALF and lung fibrosis increased with duration of dust exposure. Data courtesy of Castranova et al. (2002). (B) BAL neutrophils (values in million) and fibrosis were assessed in a subset of the animals exposed for 20, 40, or 60 days, 36 days thereafter. Data courtesy of Porter et al. (2004).

Figure 13.

Lung tumor incidence in rats that were chronically exposed to poorly soluble dusts. Doses were estimated based on surface particle area (m²) of the dust that deposited in the lung of an exposed rat. Data are courtesy of Dr. Driscoll. Refer to original data (Driscoll 1996) for accuracy.

Figure 14.

(A) TiO₂ burden in the lung of rats and hamsters that were exposed to pigment TiO₂ for 13 weeks. (B) Percentage of neutrophils in the BAL cells the lung of TiO₂-exposed rats and hamsters. Each point is the average data from five animals. Black arrows indicate the Y-axis with the values for the curve. Data (no standard deviation) presented here are courtesy of Dr. Warheit and were replotted from data presented in Bermudez et al. (2002).

Figure 15.

(A) Comparison of toxicity biomarkers (neutrophil counts and LDH) and (B) oxidant production (superoxide anion, nitric oxide anion, and malonaldehyde) in BAL cells collected from rats and hamsters seven days after they were instilled with saline, or quartz (at 0.2, 2.0, or 20 mg). N = 4–6 animals. Data courtesy of Dr. Driscoll (Carter and Driscoll 2001).

Figure 16.

Diagram illustrating a proposed mechanism of particle-induced pathogenesis and carcinogenesis in the lungs that is mediated by epithelial cells and neutrophils. Sequence of events leading to dust-induced pathogenesis: (1) particles enter the alveoli and deposit on alveolar epithelial cells (AECs, light blue), causing some irritation or injury. (2) The dust-inflicted AECs turn on gene processes that produce elevated levels of cytokines, which recruit blood leukocytes (chiefly monocytes (brown) and neutrophils (orangep) to the airspaces. Some of the cellular mediators are also released by resident alveolar macrophages (AM or Mu, green). (3) The incoming leukocytes, especially neutrophils, are activated by cytokines to increase their oxidant levels; monocytes become activated macrophages (AM, green ) and neutrophils become activated neutrophils (redp). (4) Neutrophils are short-lived; apoptotic neutrophils and dust particles are phagocytized by AMs. (5) In animals exposed heavily to dust, AMs are overloaded with particles. Some of the dust-laden and dead AMs cannot leave the lung; they aggregate to produce granulomas. (6) The unphagocytized apoptotic neutrophils undergo necrosis (marked with x) and eventually release their oxidants and harmful enzymes onto the alveolar epithelium. (7) Some oxidants are destroyed by antioxidants and protective enzymes. (8) Oxidants and proteases released from neutrophils damage AECs. (9) If particles are below a threshold level, they can be removed by AMs and the inflammatory/damage cycle ends leading to repair. Above a threshold dust burden, persistent and chronic neutrophilic inflammation causes lung lesions to progress with time. (10) Fibroblasts proliferate on the wall of damaged epithelial areas, resulting in fibrosis. (11) Persistent and chronic oxidative damage to AECs can cause proliferation of type II AECs; some of these cells may eventually undergo hyperplasia, metaplasia, and, in the worst case, carcinogenesis.