title: Resolution of central nervous system astrocytic and endothelial sources of CCL2 gene expression during evolving neuroinflammation.
abstract: BACKGROUND
  The chemokine CCL2 is a critical mediator of neuroinflammation in diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 drives mononuclear cell infiltration into the central nervous system (CNS), alters expression and distribution of microvascular endothelial tight junction proteins, and disrupts the blood-brain and blood-spinal cord barriers. Immunohistochemistry has consistently revealed astrocytes to be a source of this chemokine during neuroinflammation, while providing less uniform evidence that CNS endothelial cells may also express CCL2. Moreover, the relative contributions of these cell types to the CNS pool of CCL2 during MS/EAE are unclear and the aim of this study was to investigate this further.
  
  
  METHODS
  CCL2 gene expression was determined by qRT-PCR in different populations of CNS cells at different times following EAE induced by immunization with MOG35-55 peptide and adjuvants, or after injection with adjuvants alone. CNS cells types were isolated by two different protocols: bulk isolation to yield crude microvascular and parenchymal fractions (containing astrocytes, other glia, and neurons), or laser capture microdissection (LCM) to acquire more precisely microvascular endothelial cells, astrocytes or other parenchymal cells.
  
  
  RESULTS
  Both CNS microvessel and parenchymal populations prepared by crude bulk isolation showed up-regulation of CCL2 mRNA following MOG immunization or injection of adjuvants alone. More exact dissection by LCM revealed microvascular endothelial cells and astrocytes to be the specific sources of CCL2 gene induction following MOG immunization, while only astrocytes showed elevated CCL2 mRNA in response to just adjuvants. Astrocytes displayed the greatest degree of stimulation of CCL2 gene expression following EAE induction.
  
  
  CONCLUSIONS
  High-precision LCM affirmed both microvascular endothelial cells and astrocytes as the major CNS sources of CCL2 gene expression during EAE. Given the high accessibility of the CNS microvascular endothelium, endothelial-derived CCL2 could prove a viable target for therapeutic intervention in neuroinflammatory disease.
authors.forename: Bandana
authors.initials: B
authors.lastname: Shrestha
authors.forename: Shujun
authors.initials: S
authors.lastname: Ge
authors.affiliation: Blood-brain Barrier Laboratory, Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA. pachter@nso1.uchc.edu.
authors.forename: Joel S
authors.initials: JS
authors.lastname: Pachter