Ceruloplasmin potentiates nitric oxide synthase activity and cytokine secretion in activated microglia

BACKGROUND: Ceruloplasmin is a ferroxidase expressed in the central nervous system both as soluble form in the cerebrospinal fluid (CSF) and as membrane-bound GPI-anchored isoform on astrocytes, where it plays a role in iron homeostasis and antioxidant defense. It has been proposed that ceruloplasmin is also able to activate microglial cells with ensuing nitric oxide (NO) production, thereby contributing to neuroinflammatory conditions. In light of the possible role of ceruloplasmin in neurodegenerative diseases, we were prompted to investigate how this protein could contribute to microglial activation in either its native form, as well as in its oxidized form, recently found generated in the CSF of patients with Parkinson’s and Alzheimer’s diseases. METHODS: Primary rat microglial-enriched cultures were treated with either ceruloplasmin or oxidized-ceruloplasmin, alone or in combination with lipopolysaccharide (LPS). Production of NO and expression of inducible nitric oxide synthase (iNOS) were evaluated by Griess assay and Western blot analysis, respectively. The productions of the pro-inflammatory cytokine IL-6 and the chemokine MIP-1α were assessed by quantitative RT-PCR and ELISA. RESULTS: Regardless of its oxidative status, ceruloplasmin by itself was not able to activate primary rat microglia. However, ceruloplasmin reinforced the LPS-induced microglial activation, promoting an increase of NO production, as well as the induction of IL-6 and MIP-1α. Interestingly, the ceruloplasmin-mediated effects were observed in the absence of an additional induction of iNOS expression. The evaluation of iNOS activity in primary glial cultures and in vitro suggested that the increased NO production induced by the combined LPS and ceruloplasmin treatment is mediated by a potentiation of the enzymatic activity. CONCLUSIONS: Ceruloplasmin potentiates iNOS activity in microglial cells activated by a pro-inflammatory stimulus, without affecting iNOS expression levels. This action might be mediated by the activation of a yet unknown Cp receptor that triggers intracellular signaling that cross-talks with the response elicited by LPS or other pro-inflammatory stimuli. Therefore, ceruloplasmin might contribute to pathological conditions in the central nervous system by exacerbating neuroinflammation.