Increased, recruitment of inflammatory cells

Pro-inflammatory cells originate in bone marrow and are recruited to the site of infection or injury via circulation following specific pro-inflammatory mediator (cytokine and chemokine) signalling. Pro-inflammatory cells are recruited to lungs to clear the invading pathogen or the toxic substance. Monocytes (dendritic cells, macrophages, and neutrophils) are subsets of circulating white blood cells that are involved in the immune responses to pathogen or toxicant stimuli. They are derived from the bone marrow. They can differentiate into different macrophage types and dendritic cells. They can be categorised based on their size, the type of cell surface receptors and their ability to differentiate following external or internal stimulus such as increased expression of cytokines. Monocytes participate in tissue healing, clearance of toxic substance or pathogens, and in the initiation of adaptive immunity. Recruited monocytes can also influence pathogenesis (Ingersoll et al., 2011). Sensing or recognition of pathogens and harmful substances results in the recruitment of monocytes to lungs (Shi and Pamer, 2011). Activated immune cells secrete a variety of pro-inflammatory mediators, the purpose of which is to propagate the immune signalling and response, which when not controlled, leads to chronic inflammation, cell death and tissue injury. Thus, Event 1496 and Event 1497 act in a positive feedback loop mechanism and propagate the proinflammatory environment.

Literature evidence for its perturbation:

Macrophages accumulate in bronchoalveolar fluid (BALF) post-exposure to bleomycin (Phan et al., 1980; Smith et al., 1995). NM-induced inflammation is predominantly neutrophilic (Shvedova et al., 2005; Rahman L et al., 2016; Rahman et al., 2017; Poulsen et al., 2015). An increased number of neutrophils (Reynolds et al., 1977) is observed in the BALF of patients with idiopathic pulmonary fibrosis. Eosinophils are a type of white blood cells and a type of granulocytes (contain granules and enzymes) that are recruited following exposure to allergens, during allergic reactions such as asthma or during fibrosis (Reynolds et al., 1977). MWCNTs induce increased eosinophil count in lungs (Købler C et al., 2015). MWCNTs act as allergens and induce lung infiltration of eosinophils and cause airway hypersensitivity (Beamer et al., 2013).

It is important to note that the stressor-induced Event 1495, Event 1496, and Event 1497 are part of the functional changes that we collectively consider as inflammation, and together, they mark the initiation of acute inflammatory phase. Event 1495 and Event 1496 occur at the cellular level. Event 1497 occurs at the tissue level.

In vivo, recruitment of pro-inflammatory cells is measured using BALF cellularity assay. The fluid lining the lung epithelium is lavaged (BALF) and its composition is assessed as marker of lung immune response to the toxic substances or pathogens. BALF is assessed quantitatively for types of infiltrating cells, levels and types of cytokines and chemokines. Thus, BALF assessment can aid in developing dose-response of a substance, to rank a substances’ potency and to set up no effect level of exposure for the regulatory decision making. For NMs, in vivo BALF assessment is recommended as a mandatory test (discussed in ENV/JM/MONO(2012)40 and also in OECD inhalation TG for NMs). Temporal changes in the BALF composition can be prognostic of initiation and progression of lung immune disease (Cho et al., 2010).

In vitro, it is difficult to assess the recruitment of pro-inflammatory cells. Thus, a suit of pro-inflammatory mediators specific to cell types are assessed using the same techniques mentioned above (qRT-PCR, ELISA, immunohistochemistry) in cell culture models, as indicative of recruitment of cells into the lungs. Alternatively, the use of precision cut lung slices can allow for limited assessment of recruitment of tissue resident inflammatory cells, based on the repertoire of cells remaining in the specific slice following harvesting. This method was used to show that there is a histological increase in inflammatory foci following treatment with bleomycin and MWCNTs (Rahman et al., 2020). Finally, more complicated microfluidic lung-on-a-chip devices can be used to assess the migration of select immune cells and fibroblasts toward a simulated epithelium following treatment with a pro-fibrotic compound (He et al., 2017). However, this method is limited to two cell types, and it lacks the reservoirs of immune cells present in the body in vivo.

Human, mouse, rat

1. Beamer, C., Girtsman, T., Seaver, B., Finsaas, K., Migliaccio, C., Perry, V., Rottman, J., Smith, D. and Holian, A. (2013). IL-33 mediates multi-walled carbon nanotube (MWCNT)-induced airway hyper-reactivity via the mobilization of innate helper cells in the lung. Nanotoxicology, 7(6), pp.1070-1081

2. Cho, W., Duffin, R., Poland, C., Howie, S., MacNee, W., Bradley, M., Megson, I. and Donaldson, K. (2010). Metal Oxide Nanoparticles Induce Unique Inflammatory Footprints in the Lung: Important Implications for Nanoparticle Testing. Environmental Health Perspectives, 118(12), pp.1699-1706.

3. He, J.; Chen, W.; Deng, S.; Xie, L.Modeling alveolar injury using microfluidic co-cultures for monitoring bleomycin-induced epithelial/fibroblastic cross-talk disorder. RSC Adv. 2017, 7, 42738– 42749.

4. Ingersoll, M., Platt, A., Potteaux, S. and Randolph, G. (2011). Monocyte trafficking in acute and chronic inflammation. Trends in Immunology, 32(10), pp.470-477.

5. Købler, C., Poulsen, S., Saber, A., Jacobsen, N., Wallin, H., Yauk, C., Halappanavar, S., Vogel, U., Qvortrup, K. and Mølhave, K. (2015). Time-Dependent Subcellular Distribution and Effects of Carbon Nanotubes in Lungs of Mice. PLOS ONE, 10(1), p.e0116481.

6. Kolaczkowska, E. and Kubes, P. (2013). Neutrophil recruitment and function in health and inflammation. Nature Reviews Immunology, 13(3), pp.159-175.

7. Kopf, M., Schneider, C. and Nobs, S. (2014). The development and function of lung-resident macrophages and dendritic cells. Nature Immunology, 16(1), pp.36-44.

8. Phan, S., Thrall, R. and Ward, P. (1980). Bleomycin-induced Pulmonary Fibrosis in Rats: Biochemical Demonstration of Increased Rate of Collagen Synthesis1,2. American Review of Respiratory Disease, 121(3), pp.501-506.

9. Poulsen, S., Saber, A., Williams, A., Andersen, O., Købler, C., Atluri, R., Pozzebon, M., Mucelli, S., Simion, M., Rickerby, D., Mortensen, A., Jackson, P., Kyjovska, Z., Mølhave, K., Jacobsen, N., Jensen, K., Yauk, C., Wallin, H., Halappanavar, S. and Vogel, U. (2015). MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicology and Applied Pharmacology, 284(1), pp.16-32.

10. Rahman, L., Williams, A., Gelda, K., Nikota, J., Wu, D., Vogel, U., & Halappanavar, S. (2020). 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small, 16(36), e2000272.

11. Rahman, L., Jacobsen, N., Aziz, S., Wu, D., Williams, A., Yauk, C., White, P., Wallin, H., Vogel, U. and Halappanavar, S. (2017). Multiwalled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 823, pp.28-44.

12. Rahman, L., Wu, D., Johnston, M., Williams, A. and Halappanavar, S. (2016). Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses. Mutagenesis, 32(1), pp.59-76

13. Reynolds, H., Fulmer, J., Kazmierowski, J., Roberts, W., Frank, M. and Crystal, R. (1977). Analysis of cellular and protein content of broncho-alveolar lavage fluid from patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis. Journal of Clinical Investigation, 59(1), pp.165-175.

14. Shi, C., & Pamer, E. G. (2011). Monocyte recruitment during infection and inflammation. Nature reviews. Immunology, 11(11), 762–774.

15. Shvedova, A. A., Kisin, E. R., Mercer, R., Murray, A. R., Johnson, V. J., Potapovich, A. I., Tyurina, Y. Y., Gorelik, O., Arepalli, S., Schwegler-Berry, D., Hubbs, A. F., Antonini, J., Evans, D. E., Ku, B. K., Ramsey, D., Maynard, A., Kagan, V. E., Castranova, V., & Baron, P. (2005). Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. American Journal of Physiology - Lung Cellular and Molecular Physiology, 289(5 33-5), 698–708. 

16. Smith, R. E., Strieter, R. M., Zhang, K., Phan, S. H., Standiford, T. J., Lukas, N. W., & Kunkel, S. L. (1995). A role for C-C chemokines in fibrotic lung disease. Journal of Leukocyte Biology, 57(5), 782–787. https://doi.org/10.1002/JLB.57.5.782