1. Mechanisms involved in cytokine responses induced by carbon black nanoparticles in epithelial lung cells Annike Irene Totlandsdal Department of Air Pollution and Noise, Norwegian Institute of Public Health

2. Outline Introduction - Nanoparticles (carbon black) - Particle-induced inflammation in the lung - cellular release of cytokines - activation of intracellular signaling pathways Results - Carbon black-induced responses of primary lung cells Concluding remarks

3. Nanoparticles - intentionally produced particles with at least one dimension below 100 nm Nanoparticles Kilometer (1000 m) Mountains Millimeter (0.001 m) Ant Micrometer (0.000001 m) Bacteria Nanometer (0.000000001 m) Proteins, DNA Meter (1 m) Child

4. Printex 90: - primary particle size ≈ 14 nm Photo: E. Namork,FHI Carbon black nanoparticles Applications: strengthen and color rubber, color inks and leather, insulate electrical equipment

5. Carbon black nanoparticles Exposure: - primarily occupational (carbon black manufacturing industry) - primarily via inhalation Inhaled nanoparticles are likely to deposit in the upper as well as lower parts of our respiratory system

6. Fate of inhaled particles Type II cell Type I cell ALVEOLAR SPACE alveolar macro- phage CAPILLARY Inhaled nanoparticles may enter the capillaries and gain access to extrapulmonary organs

7. Particle-induced inflammation Type II cell Type I cell ALVEOLAR SPACE alveolar macro- phage CAPILLARY ROS Cellular release of inflammatory mediators Dilation & increased permeability of capillary Recruitment and infiltration of immune cells Production of reactive oxygen species (ROS) Four key events

8. Aims of this study 2. Investigate the role of intracellular signaling pathways in the particle-induced cytokine responses - focus on mitogen-activated protein kinases (MAPKs) - focus on the nuclear transcription factor-kappa B (NF-κB) 1. Investigate cytokine responses of alveolar epithelial cells to carbon black nanoparticles - focus on the cytokines IL-1 and IL-6

9. Particle-induced release of cytokines IκBIκB NF-κB P Binding to cytokine promotor regions Cytokine mRNA Cell membrane Cytoplasm Nucleus MKKK MKK MAPKs NF-κB IκBIκB NIK IKK Activation of transcription factors/ post-transcriptional regulation CytokineCytokine release

10. Alveolar epithelial type II cells IL-6 IL-1α IL-1β Methods - stimulation of cells carbon black nanoparticles induce a concentration-dependent release of IL-6, IL-1α and IL-1β

11. Results – cytokine release Is the particle-induced release of IL-6 regulated by IL-1 cytokines? control cellsexposed cells (100 ug/ml) carbon black nanoparticles induce a time-dependent increase in the release of IL-6, IL-1α and IL-1β Particle-induced release of IL-1α, IL-1β and IL-6 in time

12. Treatment with IL-1ra attenuates the IL-6 responses of lung cells to the carbon black nanoparticles Results – role of IL-1 in release of IL-6 Responses of untreated cells Responses of treated cells Experiments with cells treated with IL-1 receptor antagonist (IL-1ra)

13. Particles IL-6 IL-1 IL-1ra Summary of findings_ part 1/5 What is the role of MAPKs in this response?

14. carbon black nanoparticles induce a concentration-dependent activation of all three investigated MAPKs Results – role of MAPKs C Particle-induced phosphorylation (activation) of MAPKs How does this activation influence the release of cytokines?

15. Treatment with MAPK inhibitors attenuates the carbon black-induced release of IL-1 and IL-6 Results – role of MAPKs Experiments with cells treated with MAPK inhibitors Responses of untreated cells (ERK) (p38) (JNK)

16. Particles IL-6 IL-1 IL-1ra Are the MAPKs also involved in the IL-1-induced release of IL-6? MAPKs Inhibitors Summary of findings_ part 2/5

17. Results – role of IL-1 Treatment with IL-1ra seems to attenuate the particle-induced activation of MAPKs Effects of treatment with the IL-1 receptor antagonist (IL-1ra) on particle-induced phosphorylation (activation) of MAPKs

18. Particles IL-6 IL-1 IL-1ra Are the MAPKs also involved in the IL-1-induced release of IL-6? MAPKs Inhibitors MAPKs The MAPKs are probably also involved in the IL-1-induced release of IL-6 What about NF-κB? Summary of findings_ part 3/5

19. Carbon black nanoparticles seem to induce a concentration-dependent activation of NF-κB Results – role of NFκB Particle-induced degradation of IkBα (activation of NF-κB) How does this influence the release of cytokines?

20. Treatment with PDTC attenuates the carbon black-induced release of IL-6, but not IL-1 Results – role of NFκB Experiments with cells treated with PDTC (inhibitor of NFκB) Responses of untreated cells

21. Particles IL-6 IL-1 IL-1ra MAPKs Inhibitors MAPKs The particles seem to activate NF-κB, but NF-κB is probably only involved in the release of IL-6 What about NF-κB? MAPKs (NFκB) Is NF-κB involved in the IL-1-induced release of IL-6? Summary of findings_ part 4/5

22. Results – role of IL-1 Treatment with IL-1ra seems to attenuate the particle-induced degradation of IκBα Effects of treatment with the IL-1 receptor antagonist (IL-1ra) on particle-induced particle-induced degradation of IκBα

23. Particles IL-6 IL-1 IL-1ra MAPKs Inhibitors MAPKs NFκB is probably involved in the IL-1- induced release of IL-6 Is NF-κB involved in the IL-1-induced release of IL-6? MAPKs (NFκB) MAPKs/ NFκB MAPKs Summary of findings_final

24. The alveolar epithelial cells respond to carbon black nanoparticles by releasing mediators (cytokines). The present findings provide more insight into the largely unknown mechanisms involved in the release of cytokines from lung cells in response to nanosized particles. The particle-induced release of IL-6 seems to strongly depend on IL-1 and involves activation of MAPKs and NFkB. Concluding remarks

25. Funding: The Research Council of Norway (Project: Stress responses induced by particulate matter in the lung and heart: significance of ultrafine particles and metals) Co-workers: Marit Låg Magne Refsnes Tonje Skuland Hans-Jørgen Dahlman Edel Lilleås Per Schwarze Thanks to…