Myelin basic protein charge isomers change macrophage polarization

Abstract

Purpose: During a neuronal injury, a variety of immune cells infiltrate into the local microenvironment at the demyelination site. After the destruction of the intact myelin sheath, its major constituent myelin basic protein (MBP) dissociates from the plasma membrane and acts as a free ligand on the infiltrated immune cells. MBP exhibits charge microheterogeneity as a result of post-translational modifications, but the effect of various isomers of MBP on the activity of macrophages is not known.

Materials and methods: MBP was isolated and purified from bovine brain white matter. RAW 264.7 macrophages were cultured in DMEM supplemented with heat-inactivated fetal bovine serum. For evaluation of macrophage polarization following treatment of RAW 264.7 cells with MBP charge isomers, inducible nitric oxide synthase (iNOS) expression (M1 phenotype marker) and arginase-1 expression (M2 phenotype marker) were determined in cell lysates by ELISA. To assess Rac activity, G-LISA Rac Activation Assay system was used. The expression of receptor for advanced glycation end-products (RAGE) and high mobility group box 1 (HMGB1) protein were assayed by Western blot analysis.

Results: Our results have shown that minimally modified C1 component of MBP increases the expression of arginase-1 in cells, decreases the expression of iNOS, does not change the secretion of HMGB1 protein, but significantly elevates surface expression of RAGE, and in parallel, increases the activity of small GTPase Rac. On the other hand, highly modified deiminated isomer C8-MBP increases the secretion of HMGB1 protein but does not change the expression of arginase-1 or the content of RAGE.

Conclusion: These data indicate that deiminated C8 isomer of MBP tends to polarize RAW macrophages into M1 phenotypes, whereas C1 enhances the activity of M2 phenotype markers.

Keywords: Arginase-1; HMGB1; RAGE; iNOS.

Figure 1

Chromatography of MBP acid-soluble material on the CM-52 cellulose cation-exchange column and PAGE results. Abbreviations: CM-52, carboxymethyl cellulose-52; MBP, myelin basic protein.

Figure 2

Changing arginase-1 and iNOS expressions by action of C8 and C1 isomers in RAW 264.7 macrophages. Notes: (A) Arginase-1 expression: RAW 264.7 cells (5–10⁵ cells per well) were treated for 24 hours with MBP C8 and C1 isomers, followed by the determination of arginase-1 expression as described in the “Methods” section. (B) iNOS expression: RAW 264.7 cells (5–10⁵ cells per well) were treated for 24 hours with MBP C8 and C1 isomers, followed by the determination of iNOS expression as described in the “Methods” section. Data represented are mean ± SEM of results from four separate experiments performed in duplicate. ^*P<0.05 vs corresponding control cells. Abbreviations: Contr, control; iNOS, inducible nitric oxide synthase; MBP, myelin basic protein; SEM, standard error of mean.

Figure 3

Change of arginase-1 expression by action of C8 and C1 isomers in IL-4/IL-10 and LPS/IFN-γ stimulated macrophages. Notes: RAW 264.7 cells (5–10⁵ cells per well) were treated for 24 hours with IFN-γ (20 ng/ml) and LPS (100 ng/ml) to induce the M1 phenotype, and with IL-4 (20 ng/ml) and IL-10 (10 ng/ml) to induce the M2 phenotype in the presence of MBP C8 and C1 isomers followed by the determination of arginase-1 expression as described in the “Methods” section. Data represented are mean ± SEM of results from four separate experiments performed in duplicate. ^*P<0.05 vs naive cells. Abbreviations: IFN-γ, interferon-gamma; LPS, lipopolysaccharide; MBP, myelin basic protein; SEM, standard error of mean.

Figure 4

Chang of iNOS expression by action of C8 and C1 isomers in IL-4/IL-10 and LPS/IFN-γ stimulated macrophages. Notes: RAW 264.7 cells (5–10⁵ cells per well) were treated for 24 hours with IFN-γ (20 ng/ml) and LPS (100 ng/ml) to induce the M1 phenotype, and with IL-4 (20 ng/ ml) and IL-10 (10 ng/ml) to induce the M2 phenotype in the presence of MBP C8 and C1 isomers followed by the determination of iNOS expression as described in the “Methods” section. Data represented are mean ± SEM of results from four separate experiments performed in duplicate. ^*P<0.05 vs naive cells. Abbreviations: IFN-γ, interferon-gamma; iNOS, inducible nitric oxide synthase; lPs, lipopolysaccharide; MBP, myelin basic protein; SEM, standard error of mean.

Figure 5

The effects of C8 and C1 isomers on HMGB1 and Rage expressions in control RAW 264.7 macrophages. Notes: (A) The expression of HMGB1: RAW 264.7 macrophages were incubated with MBP C1 and C8 isomers (0.5 mM) for 24 hours followed by the determination of HMGB1 expression by Western blot analysis, as described in the “Methods” section. β-actin was also determined by Western blotting to confirm equal loading of the fractions. Data shown are representative of three independent experiments. (B) Quantification of HMGB1 blots are shown, ^*P<0.05 vs corresponding control cells. (C) The expression of Rage: RAW 264.7 macrophages were incubated with MBP C1 and C8 isomers (0.5 µM) for 24 hours followed by the determination of Rage expression by Western blot analysis, as described in the “Methods” section. β-actin was also determined by Western blotting to confirm equal loading of the fractions. Data shown are representative of three independent experiments. (D) Quantification of Rage blots are shown, ^*P<0.05 vs corresponding control cells. Abbreviations: Contr, control; HMGB1, high mobility group box 1; MBP, myelin basic protein; Rage, receptor for advanced glycation end-products.

Figure 6

The effects of C8 and C1 isomers on the Rac-activation signal in control RAW 264.7 macrophages. Notes: RAW 264.7 macrophages were incubated with MBP C1 and C8 isomers (0.5 µM) for 24 hours followed by the determination of Rac activity as described in the “Methods” section. Data represented are mean ± SEM of results from four separate experiments performed in duplicate.^*P<0.05 vs corresponding control cells. Abbreviations: Contr, control; MBP, myelin basic protein; SEM, standard error of mean.