Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophagelocalized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1 (pro-IL-1)-degrading caspase-1 to lead to IL-1 release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.

Metal ions released from implantable biomaterials have been associated with adverse biological reactions that can limit implant longevity. Previous studies have shown that, in macrophages, Co2+, Cr3+, and Ni2+ can activate the NLR family pyrin domain-containing protein 3 (NLPR3) inflammasome, which is responsible for interleukin(IL)-1β production through caspase-1. Furthermore, these ions are known to induce oxidative stress, and inflammasome priming is known to involve nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. However, the mechanisms of inflammasome activation by metal ions remain largely unknown. The objectives of this thesis were to determine if, in macrophages: 1. IL-1β release induced by metal ions is caspase-1-dependent; 2. caspase-1 activation and IL-1β release induced by metal ions are oxidative stress-dependent; and 3. IL-1β release induced by metal ions is NF-κB signaling pathway-dependent. Lipopolysaccharide (LPS)-primed murine bone-marrow-derived macrophages were exposed to Co2+, Cr3+, or Ni2+, with or without an inhibitor of caspase-1, oxidative stress, or NF-κB. Culture supernatants were analyzed for active caspase-1 (immunoblotting) and/or IL-1β (ELISA). Overall, results showed that while both Cr3+ and Ni2+ may be inducing inflammasome activation, Cr3+ is likely a more potent activator, acting through oxidative stress and the NF-κB signaling pathway. Further elucidation of the activation mechanisms may facilitate the development of therapeutic approaches to modulate the inflammatory response to metal ions, and thereby increase implant longevity.