Cellular responses induced by multi-walled carbon nanotubes: in vitro study on the medicinal leech macrophages

The intrinsic characteristics of engineered nanoparticles, such as multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology, however their environmental dispersion could be potentially hazardous to animal and human health. Since the production and use of MWCNTs is steadily increasing, intentional or unintentional environmental discharges may occur. For this reason, the development of new methods and the identification of reliable models to completely understand MWCNTs effects are critical. Here we propose a freshwater invertebrate, the medicinal leech, as alternative model to assess the effects of MWCNTs on immune system. Our previous studies have already demonstrated that in vivo MWCNT treatment induces the activation of leech’s macrophages. In this study, we focus on the direct effects of MWCNTs on these cells. We used the consolidated experimental approach, based on the injection in the leech body wall of the biomatrice Matrigel (MG) added with the cytokine Allograft inflammatory factor-1 (HmAIF-1), to specifically chemoattract macrophages. Cells extracted from MG were cultured and characterized with the specific markers CD45 and CD68, confirming their belonging to the monocyte-macrophage lineage. Primary macrophage cultures were then subjected to an in vitro treatment with MWCNTs at different concentrations (2.5, 5, 10, 25, 50 and 100 μg/ml). Our results indicate that leech macrophages, once in close contact with MWCNTs, actively produce amyloid material to encapsulate the foreign bodies. We also demonstrated that MWCNTs in vitro treatment cause the decrease of cell proliferation rate and the increase of the apoptotic rate. Furthermore, since oxidative stress is linked with inflammation and amyloid production, reactive oxygen species has been evaluated, confirming that their production rate increases after MWCNT treatment. Our combined experimental approaches, not only attest the ability of MWCNTs in inducing a potent inflammatory response, but also confirm the medicinal leech as a good alternative model that can be improved and successfully used to study the possible harmful effects of any nanomaterial. Moreover, since autophagic cell death pathway activation is emerging as a possible consequence of MWCNT treatment, in the future we will attempt to clarify this aspect in order to completely understand MWCNT-induces toxicity.