Abstract
BackgroundRecent 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.MethodsMR16-1 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.ResultsLFB-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.ConclusionThe 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.
Highlights
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
Anti-interleukin (IL)6-receptor (MR16-1) treatment increased the area of spared myelin, neurofilament heavy 200 kDa-positive and growth-associated protein-43positive nerve fibers, and locomotor function after spinal cord injury To examine the therapeutic effects of MR16-1 on SC repair, we used Luxol fast blue (LFB) staining to evaluate the sparing of myelin sheaths around axons, and the immunoreactivity of both neurofilament heavy kDa (NF-H)-positive and GAP-43-positive nerve fibers at the lesion epicenter, at 14 and 42 days after SCI
A temporal blockade of IL-6 signaling by MR16-1 antibody could promote the generation of alternatively activated macrophages (M2 phenotype), and modify the inflammatory response after SCI to promote SC regeneration with functional recovery
Summary
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. The pro-inflammatory cytokines are produced by resident microglia, along with infiltrating neutrophils and macrophages, and induce a reactive process of secondary cell death in the tissue surrounding the original site of injury [1,2,3] This secondary damage continues in the days and weeks following SCI, which may lead to increase in cavitation and glial scar formation at the lesion site, exacerbating neurological dysfunction [4,5,6]. Such variation in the effects of macrophages could be the result of the presence of different activation pathways for the locally present macrophages, possibly generating sub-populations of cells with divergent abilities [16,17]
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