Ultrafine Particle-Mediated Activation of Macrophages: Intracellular Calcium Signaling and Oxidative Stress

Abstract

Various intracellular pathways are known to activate gene expression of proinflammatory mediators such as cytokines; one such pathway involves calcium. Recently we presented data that show that ultrafine (uf) carbon black (CB)(14 nm diameter), but not fine CB (260 nm diameter) is able to induce a 2.6-fold increase in the calcium response to stimulation by thapsigargin in a human macrophage cell line (Mono Mac 6). The present study aimed to investigate whether other uf particles could invoke similar alterations in calcium influx in both macrophage cell lines and primary macrophages. Treatment of MM6 cells with uf latex beads (64 nm diameter) for 1500 s induced a 2.3-fold (p < .01) increase in the response to thapsigargin, whereas fine latex (202 nm diameter) did not have any significant effect. Similarly, in primary rat bronchoalveolar lavage (BAL) cells (>80% macrophages), ufCB (33 μg/ml, 1500 s) induced a 2.6-fold (p < .001) increase in the response to thapsigargin, whereas fine CB had no significant effect. The effects of ufCB on the enhanced response to thapsigargin in the MM6 cells were significantly attenuated by the antioxidants mannitol (p < .05) and nacystelin, indicating that the effect of ultrafine particles on calcium influx was in part mediated by reactive oxygen species. Support for a role for reactive oxygen species was obtained in MM6 cells using the dye dichlorofluorescin diacetate. Ultrafine latex induced a significant increase in fluorescence of 133.0 ± 6.5 fluorescence units (p < .001), whereas fine latex did not have any significant effect. In conclusion, effects on calcium fluxes induced by thapsigargin were seen with two very different ultrafine particles — ultrafine latex beads and ultrafine CB—and were seen in both the human MM6 cell line and rat BAL cells. Finally, the induction of an oxidative stress by the ultrafine particles was supported by the ability of ultrafine latex beads to induce ROS production. In addition, ultrafine carbon black was found to induce enhanced calcium influx, partly through oxidative stress.