title: CCL2 upregulation triggers hypoxic preconditioning-induced protection from stroke.
abstract: BACKGROUND
  A brief exposure to systemic hypoxia (i.e., hypoxic preconditioning; HPC) prior to transient middle cerebral artery occlusion (tMCAo) reduces infarct volume, blood-brain barrier disruption, and leukocyte migration. CCL2 (MCP-1), typically regarded as a leukocyte-derived pro-inflammatory chemokine, can also be directly upregulated by hypoxia-induced transcription. We hypothesized that such a hypoxia-induced upregulation of CCL2 is required for HPC-induced ischemic tolerance.
  
  
  METHODS
  Adult male SW/ND4, CCL2-null, and wild-type mice were used in these studies. Cortical CCL2/CCR2 message, protein, and cell-type specific immunoreactivity were determined following HPC (4 h, 8% O2) or room air control (21% O2) from 6 h through 2 weeks following HPC. Circulating leukocyte subsets were determined by multi-parameter flow cytometry in naïve mice and 12 h after HPC. CCL2-null and wild-type mice were exposed to HPC 2 days prior to tMCAo, with immunoneutralization of CCL2 during HPC achieved by a monoclonal CCL2 antibody.
  
  
  RESULTS
  Cortical CCL2 mRNA and protein expression peaked at 12 h after HPC (both p < 0.01), predominantly in cortical neurons, and returned to baseline by 2 days. A delayed cerebral endothelial CCL2 message expression (p < 0.05) occurred 2 days after HPC. The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2-null mice. HPC did decrease the number of circulating CCR2+ monocytes (p < 0.0001) in a CCL2-dependent manner, but immunohistochemical analyses at this 12 h timepoint indicated that this leukocyte subpopulation did not move into the CNS. While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2-null mice subjected to tMCAo were not protected by HPC. Moreover, administration of a CCL2 immunoneutralizing antibody prior to HPC completely blocked (p < 0.0001 vs. HPC-treated mice) the development of ischemic tolerance.
  
  
  CONCLUSIONS
  The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.
authors.affiliation: Department of Neurological Surgery, Washington University School of Medicine, 660 S, Euclid Ave,, Box 8057, St, Louis, MO 63110, USA.
authors.forename: Ann M
authors.initials: AM
authors.lastname: Stowe
authors.forename: Bradley K
authors.initials: BK
authors.lastname: Wacker
authors.forename: Petra D
authors.initials: PD
authors.lastname: Cravens
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Perfater
authors.forename: Min K
authors.initials: MK
authors.lastname: Li
authors.forename: Ruilong
authors.initials: R
authors.lastname: Hu
authors.forename: Angela B
authors.initials: AB
authors.lastname: Freie
authors.forename: Olaf
authors.initials: O
authors.lastname: Stüve
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Gidday
mesh_terms: D000818:Animals; D020520:Brain Infarction / Q000209:etiology* / Q000517:prevention & control*; D002490:Central Nervous System / Q000378:metabolism / Q000473:pathology; D018932:Chemokine CCL2 / Q000172:deficiency / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000627:therapeutic use; D020244:Infarction, Middle Cerebral Artery / Q000150:complications* / Q000473:pathology; D019194:Ischemic Preconditioning / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009000:Monocytes / Q000502:physiology; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D054390:Receptors, CCR2 / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation / Q000502:physiology*
chemical_list: C491768:Ccl2 protein, mouse; C515335:Ccr2 protein, mouse; D018932:Chemokine CCL2; D005904:Glial Fibrillary Acidic Protein; D007074:Immunoglobulin G; C098483:NADH dehydrogenase subunit 4; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D012333:RNA, Messenger; D054390:Receptors, CCR2; D049452:Green Fluorescent Proteins; D009245:NADH Dehydrogenase; D010751:Phosphopyruvate Hydratase