Abstract
BackgroundThe chemokine (C-C motif) ligand 2 (CCL2) is a monocyte chemoattractant protein that mediates macrophage recruitment and migration during peripheral and central nervous system (CNS) inflammation.MethodsTo determine the impact of CCL2 in inflammation in vivo and to elucidate the CCL2-induced polarization of activated brain microglia, we delivered CCL2 into the brains of wild-type mice via recombinant adeno-associated virus serotype 9 (rAAV-9) driven by the chicken β-actin promoter. We measured microglial activation using histological and chemical measurement and recruitment of monocytes using histology and flow cytometry.ResultsThe overexpression of CCL2 in the CNS induced significant activation of brain resident microglia. CD45 and major histocompatibility complex class II immunoreactivity significantly increased at the sites of CCL2 administration. Histological characterization of the microglial phenotype revealed the elevation of “classically activated” microglial markers, such as calgranulin B and IL-1β, as well as markers associated with “alternative activation” of microglia, including YM1 and arginase 1. The protein expression profile in the hippocampus demonstrated markedly increased levels of IL-6, GM-CSF and eotaxin (CCL-11) in response to CCL2, but no changes in the levels of other cytokines, including TNF-α and IFN-γ. Moreover, real-time PCR analysis confirmed increases in mRNA levels of gene transcripts associated with neuroinflammation following CCL2 overexpression. Finally, we investigated the chemotactic properties of CCL2 in vivo by performing adoptive transfer of bone marrow–derived cells (BMDCs) isolated from donor mice that ubiquitously expressed green fluorescent protein. Flow cytometry and histological analyses indicated that BMDCs extravasated into brain parenchyma and colabeled with microglial markers.ConclusionTaken together, our results suggest that CCL2 strongly activates resident microglia in the brain. Both pro- and anti-inflammatory activation of microglia were prominent, with no bias toward the M1 or M2 phenotype in the activated cells. As expected, CCL2 overexpression actively recruited circulating monocytes into the CNS. Thus, CCL2 expression in mouse brain induces microglial activation and represents an efficient method for recruitment of peripheral macrophages.
Highlights
The chemokine (C-C motif) ligand 2 (CCL2) is a monocyte chemoattractant protein that mediates macrophage recruitment and migration during peripheral and central nervous system (CNS) inflammation
Chemokine ligand 2 (CCL2) expression was exacerbated in rAAV9-CCL2 injected mice To assess the role of CCL2 in activation of resident brain glial cells and infiltration of peripheral macrophages, we constructed a recombinant adeno-associated virus serotype 9 (rAAV-9) pTR2-CCL2 vector (Figure 1A)
Basal levels of CCL2 in rAAV9-green fluorescent protein (GFP)-injected animals were in the low range (0.13 ng/ml); as expected, CCL2 expression was significantly higher in the hippocampi of rAAV9CCL2-injected animals (406 ng/ml)
Summary
The chemokine (C-C motif) ligand 2 (CCL2) is a monocyte chemoattractant protein that mediates macrophage recruitment and migration during peripheral and central nervous system (CNS) inflammation. Transgenic mice that overexpress CCL2 in the central nervous system (CNS) exhibit a robust accumulation of macrophages and activation of microglia in the brain [8,9], whereas mice deficient in the CCL2 gene show reduced leukocyte infiltration after autoimmune encephalomyelitis, traumatic brain injury, spinal cord injury and HIV-associated dementia [10,11,12] In neurodegenerative diseases such as AD, recent evidence derived from genetically modified animal models suggests that infiltrating macrophages contribute to reduced amyloid pathology [13,14,15,16,17,18]. CCL2 levels are reportedly increased in the brain, serum and cerebrospinal fluid of AD patients [20,21,22], further supporting a central role for this chemokine in neuroinflammation
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