Preconditioning occurs when an organism, tissue, or cell is exposed to a stressful, but non-damaging, stimulus that initiates genomic reprogramming for protection from subsequent injury [1–3]. One well-established model to induce "ischemic tolerance" in the central nervous system (CNS) utilizes a brief exposure to systemic hypoxia as the preconditioning stimulus (hypoxic preconditioning; HPC) to promote neurovascular protection in stroke [4–7]. Mechanistically, hypoxia activates survival-promoting signaling pathways responsible for altering gene expression via the upregulation of hypoxia-inducible factor-1 (HIF-1) . HIF-1 modifies the expression of effector pathways that ultimately come to define the ischemia-tolerant phenotype, in part through the specific upregulation of HIF-1 in cortical neurons . Following ischemia, HIF-1 mediated mechanisms contribute to cortical repair via the homing of progenitor cells to the site of injury , the upregulation of pro-angiogenic molecules [10, 11], and the upregulation of erythropoietin [10, 12].
CCL2, or monocyte chemoattractant protein (MCP)-1, is one of only two chemokines under the direct transcriptional control of HIF-1α regulation . CCL2 is predominantly produced by astrocytes and resident microglia, and is traditionally known for its role in recruiting neutrophils and macrophages , as well as circulating neuroblasts , to sites of cortical injury under multiple pathological states. CCL2 is a full competitive agonist to its receptor, CCR2 , a Gαi-coupled receptor that modulates its signaling based on binding to individual CC-motif chemokines . CCR2 is found on virtually all CNS cell types, including neurons, glial, endothelial, and immune cells [13–16], and is the only known receptor for CCL2 - although CCR2 also binds the chemokines CCL7, CCL8, CCL13, and CCL16 . Several studies suggest a detrimental role for CCL2 in the progression of stroke injury, as both CCL2-/-  and CCR2-/-  mice exhibit reduced infarct volumes compared to wild-type controls.
Given its well-documented pro-inflammatory roles, CCL2 seems an unlikely candidate for inducing neuroprotection. However, since it is well established that harmful stimuli at higher doses can - at lower doses - serve as preconditioning stimuli, a role for chemokines in general, and CCL2 in particular, in the induction of ischemic tolerance is not necessarily unexpected. Indeed, several traditionally pro-inflammatory stimuli, including lipopolysaccharide (LPS) [18, 19], tumor necrosis factor-α (TNF-α) , and even brief ischemia , upregulate signaling pathways that induce stroke tolerance. Evidence for the contribution of CCL2 to upstream cellular signaling during injury and repair shows that CCL2-CCR2 signaling upregulates transcription factors, including MCP-1-induced protein (MCPIP)  and Ets-1 , in monocytes and endothelial cells to initiate angiogenesis, a process that is critical to stroke recovery . In addition, overexpression of CCL2 in cardiac myocytes protects during myocardial ischemia by activation of SAPK/JNK1/2 pathway , although, by implication, the activity of other signal transduction pathways downstream of CCR2 receptor activation (e.g., MAPK, ERK, and phospholipase C) may also participate in this epigenetic response [15, 23, 24].
Because of the direct upregulation of CCL2 by hypoxia and these signaling intermediary roles, we investigated whether CCL2 participates as a mediator of HPC-induced tolerance to stroke. We found that a single exposure to systemic hypoxia (our HPC stimulus) rapidly upregulates CCL2 mRNA and protein early in cortical neurons, with a delayed upregulation of CCL2 message in cortical microvessels. In the periphery, HPC reduced circulating granulocyte, T lymphocyte, and monocyte populations, while increasing B lymphocytes, in a CCL2-independent manner. However, CCL2 regulated the transmigration of CCR2+ monocytes out of the peripheral blood in response to HPC. Moreover, in mice that lack bioavailable CCL2, either through genetic knockout or immunoneutralization, ischemic tolerance to HPC was not achieved, providing causal evidence for CCL2, likely produced by both neurons, cerebral endothelial cells, and circulating leukocytes, as a proximal signaling factor in HPC-induced gene induction pathways. While the fundamental mechanisms of CNS preconditioning have been under investigation for a couple of decades, this is the first evidence of chemokine signaling being critical to the induction of ischemic tolerance.