title: Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice.
abstract: BACKGROUND
  Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice.
  
  
  METHODS
  MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-γ and tumor necrosis factor-α) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-γ and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia.
  
  
  RESULTS
  LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-γ-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior.
  
  
  CONCLUSIONS
  The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.
authors.affiliation: Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui, Matsuoka-Shimoaizuki 23, Eiheiji, Fukui 910-1193, Japan.
authors.forename: Alexander Rodriguez
authors.initials: AR
authors.lastname: Guerrero
authors.forename: Kenzo
authors.initials: K
authors.lastname: Uchida
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nakajima
authors.forename: Shuji
authors.initials: S
authors.lastname: Watanabe
authors.forename: Masaya
authors.initials: M
authors.lastname: Nakamura
authors.forename: William Eb
authors.initials: WE
authors.lastname: Johnson
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Baba
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology / Q000627:therapeutic use; D015703:Antigens, CD / Q000378:metabolism; D004195:Disease Models, Animal; D019922:GAP-43 Protein / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D007211:Indoles; D007371:Interferon-gamma / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D016900:Neurofilament Proteins / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D019947:Receptors, Interleukin-6 / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013119:Spinal Cord Injuries / Q000188:drug therapy / Q000473:pathology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D019922:GAP-43 Protein; D007211:Indoles; D015850:Interleukin-6; D016900:Neurofilament Proteins; D019947:Receptors, Interleukin-6; D014409:Tumor Necrosis Factor-alpha; C051774:neurofilament protein H; C018588:Luxol Fast Blue MBS; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; D019204:GTP-Binding Proteins; C472530:Mpa2 protein, mouse