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024 MCP-1 DRIVES CEREBRAL RECRUITMENT OF ACTIVATED MONOCYTES IN CHOLESTATIC MICE C D'Mello, M Ajuebor, T Le, P Kubes, M Swain METHODS: For these studies, we used the well characterized model of cholestasis due to bile duct resection (BDR). Experiments were performed in BDR mice and Sham resected controls on day 10 post-surgery (when BDR mice are overtly cholestatic). Cerebral mononuclear cells were isolated using Percoll gradients and characterized by flow cytometry.
Recently, we observed increased cerebral recruitment of monocytes in cholestatic mice. Immune cells are known to enter the brain in diseases like HIV and MS and cause behavioral alterations, suggesting similar effects may occur in cholestasis. Therefore, we performed a series of experiments to determine which mediator(s) drive the cerebral recruitment of monocytes in cholestatic mice.
RESULTS: We measured whole brain levels of common monocyte chemoattractants (eg, RANTES, MCP-1, MIP-1alpha) by ELISA and found Monocyte Chemoattractant Protein (MCP)-1 levels, a potent chemoattractant for monocytes, to be 2-fold higher in BDR (10.6 pg/mg ± 2.0) vs. Sham mice (5.2 pg/mg ± 0.70)(p=0.01). In order to respond to MCP-1, cells must express CCR2, the cognate receptor for MCP-1. Examination of CCR2 expression revealed that a larger proportion of peripheral blood monocytes and monocytes infiltrating the brain in cholestatic mice (#CCR2 positive cells Sham: 2049±218 vs. BDR: 5278±944, p=0.01) were expressing CCR2. Interestingly, there was a 3-fold reduction in the number of microglia (resident brain immune cells) expressing CCR2 in BDR mice (Sham: 49009±13284 vs. BDR: 16196±3278 cells/brain, p<0.05), likely mediated by elevated cerebral MCP-1 levels seen in these mice. Therefore, to further investigate the role of MCP-1/CCR2 in recruiting monocytes into the brain in cholestasis, studies were repeated using MCP-1 KO and CCR2 KO mice. There was a significant reduction in cerebral monocyte recruitment in these mice to levels similar in sham mice (MCP-1KO BDR: 3050±415, WT BDR: 5807±812 cells/brain, p=0.02; CCR2KO BDR: 2322±360, WT BDR: 4934±581 cells/brain, p=0.009). Furthermore, to identify the cellular sources of MCP-1 in brains of BDR mice, MCP-1 production was examined in infiltrating monocytes and microglia and was found to be significantly increased in both of these cell types.
CONCLUSIONS: These findings suggest that MCP-1, via its actions upon CCR2, plays a key role in recruiting monocytes into the brain in cholestasis. Recently, we also observed that brain infiltrating monocytes, as well as resident microglia, produce TNFalpha. TNFalpha is known to cause changes in neurotransmission and could potentially contribute to the behavioral alterations seen in cholestasis.