Mast Cells Release Chemokine CCL2 in Response to Parkinsonian Toxin 1-Methyl-4-Phenyl-Pyridinium (MPP(+))

Abstract

Microglial activation and release of inflammatory cytokines and chemokines are crucial events in neuroinflammation. Microglial cells interact and respond to other inflammatory cells such as T cells and mast cells as well as inflammatory mediators secreted from these cells. Recent studies have shown that neuroinflammation causes and accelerates neurodegenerative disease such as Parkinson's disease (PD) pathogenesis. 1-methyl-4-phenyl-pyridinium ion (MPP(+)), the active metabolite of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine activates glial cells and mediate neurodegeneration through release of inflammatory mediators. We have shown that glia maturation factor (GMF) activates glia and induces neuroinflammation and neurodegeneration and that MPP(+) activates mast cells and release proinflammatory cytokines and chemokines. The chemokine (C-C motif) ligand 2 (CCL2) levels have been shown to be elevated and play a role in PD pathogenesis. In the present study, we analyzed if MPP(+) activates mouse and human mast cells to release chemokine CCL2. Mouse bone marrow-derived mast cells (BMMCs) and human umbilical cord blood-derived cultured mast cells (hCBMCs) were incubated with MPP(+) (10 µM) for 24 h and CCL2 levels were measured in the supernatant media by ELISA. MPP(+)-significantly induced CCL2 release from BMMCs and hCBMCs. Additionally, GMF overexpression in BMMCs obtained from wild-type mice released significantly more CCL2, while BMMCs obtained from GMF-deficient mice showed less CCL2 release. Further, we show that MPP(+)-induced CCL2 release was greater in BMMCs-astrocyte co-culture conditions. Uncoupling protein 4 (UCP4) which is implicated in neurodegenerative diseases including PD was detected in BMMCs by immunocytochemistry. Our results suggest that mast cells may play role in PD pathogenesis.

Keywords: 1-Methyl-4-phenyl-pyridinium; CCL2; Glia maturation factor; Mast cells; Parkinson’s disease.

Fig. 1

Effect of MPP⁺ on CCL2 release from mouse primary mast cells obtained from wild-type mice. BMMCs obtained from wild-type mice were infected with Ad5CMV GMF to overexpress GMF or Ad5CMV cytoLacZ as control. These cells were then incubated with or without MPP⁺ (10 μM) for 24 h at 37°C. CCL2 levels were measured in the supernatant media were by ELISA (n=6). MPP⁺ significantly increased the release of CCL2 from BMMCs. MPP⁺ also increased CCL2 release from LacZ V and GMF-V infected BMMCs when compared with their respective MPP ⁺ untreated conditions. BMMCs released more CCL2 after GMF overexpression with GMF -V when compared to LacZ-V control. *=p<0.05, compared to respective unstimulated control cells, ANOVA and Tukey-Kramer.

Fig. 2

Effect of MPP⁺ on CCL2 release from mouse primary mast cells obtained from GMF-KO mice. We infected primary BMMCs obtained from GMF-KO mice with Ad5CMV GMF to overexpress GMF or Ad5CMV cytolacZ as control. These cells were incubated with or without MPP⁺ (10 μM) for 24 h at 37°C. Then the supernatant media were collected and measured CCL2 release by ELISA (n=4). MPP⁺ significantly increased CCL2 release from GMF-V infected BMMCs when compared with GMF-V control. *=p<0.05, compared to respective unstimulated cells, ANOVA and Tukey-Kramer.

Fig. 3

Effect of MPP⁺ on Mouse primary mast cells and mouse astrocyte co-culture. BMMCs and mouse astrocytes were co-cultured in tissue culture plates to evaluate the effect of MPP⁺ (10 μM) stimulation individually on either BMMCs or astrocytes or in a co-culture system consisting of both BMMCs and astrocytes in the same wells (n=4). Cells were incubated with MPP⁺ for 24 h and CCL2 levels were measured in the supernatant media by ELISA. More CCL2 release was observed from astrocyte and BMMCs co-culture system than released either from astrocytes culture alone or BMMCs culture alone after incubation with MPP⁺. #=p<0.05, compared to astrocytes+MPP⁺ or BMMCs+MPP⁺. *=p<0.05, compared to respective unstimulated control cells, ANOVA and Tukey-Kramer.

Fig. 4

MPP⁺ activates human mast cells and release CCL2. hCBMCs were incubated with MPP⁺ (10 μM) for 24 h and the release of CCL2 was measured in the culture supernatants by ELISA (n=4). MPP⁺ significantly increased (p<0.05) CCL2 release from hCBMCs as compared to the release from control hCBMCs treated only with buffer. *=P<0.05, control Vs MPP⁺, unpaired ‘t’ test.

Fig. 5

UCP4 expression in mouse primary mast cells. Cytospin smears of BMMCs were prepared and immunostained for the expression of UCP4 and UCP2 (n=3). We show the expression of UCP4 (upper panel) by BMMCs in addition to UCP2 (lower panel). Positive immunoreactivity was detected by brown colour. Negative control staining without primary antibodies did not show positive reaction for UCPs. Original magnification 400x.