Glia Maturation Factor and Mast Cell-Dependent Expression of Inflammatory Mediators and Proteinase Activated Receptor-2 in Neuroinflammation

Abstract

Parkinson's disease (PD) is characterized by the presence of inflammation-mediated dopaminergic neurodegeneration in the substantia nigra. Inflammatory mediators from activated microglia, astrocytes, neurons, T-cells and mast cells mediate neuroinflammation and neurodegeneration. Administration of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induces PD like motor deficits in rodents. 1-methyl-4-phenylpyridinium (MPP+), a toxic metabolite of MPTP activates glial cells, neurons and mast cells to release neuroinflammatory mediators. Glia maturation factor (GMF), mast cells and proteinase activated receptor-2 (PAR-2) are implicated in neuroinflammation. Alpha-synuclein which induces neurodegeneration increases PAR-2 expression in the brain. However, the exact mechanisms are not yet understood. In this study, we quantified inflammatory mediators in the brains of MPTP-administered wild type (Wt), GMF-knockout (GMF-KO), and mast cell knockout (MC-KO) mice. Additionally, we analyzed the effect of MPP+, GMF, and mast cell proteases on PAR-2 expression in astrocytes and neurons in vitro. Results show that the levels of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and the chemokine (C-C motif) ligand 2 (CCL2) were lesser in the brains of GMF-KO mice and MC-KO mice when compared to Wt mice brain after MPTP administration. Incubation of astrocytes and neurons with MPP+, GMF, and mouse mast cell protease-6 (MMCP-6) and MMCP-7 increased the expression of PAR-2. Our studies show that the absence of mast cells and GMF reduce the expression of neuroinflammatory mediators in the brain. We conclude that GMF along with mast cell interactions with glial cells and neurons during neuroinflammation can be explored as a new therapeutic target for PD and other neuroinflammatory disorders.

Keywords: Astrocytes; PAR-2; Parkinson’s disease; cytokines; glia maturation factor; mast cells; mouse mast cell proteases.

Fig. 1.

Mast cell deficiency decreases the levels of cytokines TNF-α and IL-1β in the brain lysates of MC-KO mice after MPTP administration. TNF-α and IL-1β were measured by ELISA in the brain lysates of MPTP administered Wt. mice, GMF-KO mice, MC-KO mice and untreated control mice. MPTP-administration significantly increased TNF-α level in the Wt. mice as compared to the control mice (A, n=4–5/group). However, there is no significant increase of TNF-α in both GMF-KO mice and MC-KO mice as compared with control mice. In these groups of mice, TNF-α levels remain significantly reduced (p<0.05) as compared to Wt. mice treated with MPTP (A). Further, MPTP administration significantly increased (p<0.05) IL-1β level in Wt. mice as compared to untreated control mice (B, n=3–4/group). Both GMF-KO mice and MC-KO mice show significantly reduced (p<0.05) IL-1β levels as compared to the Wt. mice after MPTP administration. *,# p<0.05, *Vs Wt. Control, #Vs Wt. + MPTP, ANOVA and Tukey-Kramer post hoc –––––test.

Fig. 2.

Mast cell deficiency decreases chemokine CCL2 level in the brain lysates of MC-KO mice after MPTP administration. CCL2 level was measured by ELISA in the brain lysates of MPTP administered Wt. mice, GMF-KO mice, MC-KO mice and untreated control mice (n=3/group). Our results show that CCL2 level increased in Wt. mice brain administered with MPTP as compared with control mice. Both GMF-KO mice and MC-KO mice show reduced levels CCL2 as compared with Wt. mice with MPTP administration. *p<0.05, Vs. Wt. Control, ANOVA and Tukey-Kramer post hoc test.

Fig. 3.

Mast cell deficiency decreases serum levels of TNF-α and IL-1β in MC-KO mice. We measured serum levels of TNF-α and IL-1β in MPTP administered Wt. mice, GMF-KO mice, MC-KO mice and untreated control mice (n=3–4/group). Wt. mice administered with MPTP show significantly increased (p<0.05) serum levels of TNF-α (A) and IL-1β (B) as compared with serum levels of untreated control mice. Both GMF-KO mice and MC-KO mice show significantly decreased (p<0.05) serum TNF-α level as compared with the serum level in MPTP-administered Wt. mice (A). However, serum IL-1β has been significantly reduced (p<0.05) only in MC-KO mice as compared with Wt. mice administered with MPTP (B). *,# p<0.05, *Vs Wt. Control, #Vs Wt. + MPTP, ANOVA and Tukey-Kramer post hoc test.

Fig. 4.

MPP+ and GMF upregulate the expression of PAR-2 in mouse primary astrocytes as determined by double immunofluorescence staining. Astrocytes were incubated with MPP+ (10 μM) or GMF (100 ng/ml) for 48 hrs and the expression of PAR-2 and GFAP were analyzed by immunofluorescence staining (n=3). The cells were incubated with monoclonal antibody for PAR-2 and polyclonal primary antibodies for GFAP followed by incubation with Alexa Flour 488 goat anti-rabbit IgG and Alexa Flour 568 goat anti-mouse IgG secondary antibodies. Then the cover glass with cells were lifted from the wells and mounted on to the microscope glass slides, dried and viewed using a confocal microscope. Representative images show that astrocytes incubated with MPP+ and GMF increased the expression of PAR-2 (red fluorescence) as compared with untreated control cells. Astrocyte marker GFAP is shown by the green fluorescence. Merged images show co-localization of PAR-2 and GFAP in the astrocytes. Photomicrographs original magnification = 630x.

Fig. 5.

Mast cell proteases MMCP-6 and MMCP-7 upregulate the expression of PAR-2 in mouse primary astrocytes as determined by double immunofluorescence staining. Astrocytes were incubated with MMCP-6 and MMCP-7 for 48 hrs and the expression of PAR-2 and GFAP were analyzed by immunofluorescence staining as mentioned previously (n=3). Representative images show that astrocytes incubated with MMCP-6 and MMCP-7 increased the expression of PAR-2 (red fluorescence) as compared with untreated control cells. Astrocyte marker GFAP is shown with green fluorescence. Merged images show co-localization of PAR-2 and GFAP in the astrocytes. Photomicrographs original magnification = 630x.

Fig. 6.

MPP+ and GMF upregulate the expression of PAR-2 in mouse primary neurons as detected by double immunofluorescence. Mouse primary neurons were grown and incubated with MPP+ (10 μM) or GMF (100 ng/ml) for 48 hrs and the expression of PAR-2 and MAP2 were analyzed by double immunofluorescence staining as mentioned previously. Representative images (n=3) show that neurons incubated with MPP+ or GMF for 48 hrs increased the expression of PAR-2 (red fluorescence) as compared with control untreated cells. MAP2 expression is shown by green fluorescence. Merged images show co-localization of MAP2 and PAR-2. Photomicrographs original magnification = 630x.

Fig. 7.

Mast cell proteases MMCP-6 and MMCP-7 upregulate the expression of PAR-2 in mouse primary neurons as detected by double immunofluorescence. Mouse primary neurons were grown and incubated with MMCP-6 (100 ng/ml) or MMCP-7 (100 ng/ml) for 48 hrs and the expression of PAR-2 and MAP2 were analyzed by double immunofluorescence staining as mentioned previously. Representative images (n=3) show that both MMCP-6 and MMCP-7 increased the neuronal expression of PAR-2 (red fluorescence) as compared with untreated control cells. Neuronal marker MAP2 is shown by green fluorescence. Merged images show co-localization of MAP2 and PAR-2. Photomicrographs original magnification = 630x.