Citrullinated isomer of myelin basic protein can induce inflammatory responses in astrocytes

Abstract

Purpose

During the course of demyelinating inflammatory diseases, myelin-derived proteins, including myelin basic protein(MBP), are secreted into extracellular space. MBP shows extensive post-translational modifications, including deimination/citrullination. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than unmodified MBP. This study investigated the effect of the deiminated/citrullinated isomer of MBP(C8) and the unmodified isomer of MBP(C1) on cultured primary astrocytes.

Methods

MBP charge isomers were isolated/purified from bovine brain. Primary astrocyte cultures were prepared from the 2-day-old Wistar rats. For evaluation of glutamate release/uptake a Fluorimetric glutamate assay was used. Expression of peroxisome proliferator-activated receptor-gamma(PPAR-γ), excitatory amino acid transporter 2(EAAT2), the inhibitor of the nuclear factor kappa-B(ikB) and high mobility group-B1(HMGB1) protein were assayed by Western blot analysis. IL-17A expression was determined in cell medium by ELISA.

Results

We found that MBP(C8) and MBP(C1) acted differently on the uptake/release of glutamate in astrocytes: C1 increased glutamate uptake and did not change its release, whereas C8 decreased glutamate release but did not change its uptake. Both isomers increased the expression of PPAR-γ and EAAT2 to the same degree. Western blots of cell lysates revealed decreased expression of ikB and increased expression of HMGB1 proteins after treatment of astrocytes by C8. Moreover, C8-treated cells released more nitric oxide and proinflammatory IL-17A than C1-treated cells.

Conclusions

These data suggest that the most immunogenic deiminated isomer C8, in parallel to the decreases in glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B(NF-kB).

Summary statement

The most modified-citrullinated myelin basic protein charge isomer decreases glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B in astrocytes.