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.

Conclusion: 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.

Figure 1

Histological evaluation and locomotor function after anti-interleukin (IL)-6-receptor (MR16-1) treatment. (A) Compared with the rat IgG control group, axial sections at the epicenter of the injury site obtained at 42 days post-injury and stained with luxol fast blue (LFB) showed a remarkable reduction in the area of demyelination in (B) the MR16-1-treated group. (C) Quantification of LFB-positive spared myelin areas in the ventrolateral funiculus at the lesion site showed a significant difference between the two groups at 42 days, but not at 14 days post-injury. Representative images of injury epicenter mid-sagittal sections at 42 days after spinal cord injury in (D, I) the rat IgG control group and (F, K) MR16-1-treated group: in the high-magnification photomicrographs of the respective boxed area, a greater abundance of neurofilament heavy 200 kDa-positive and growth-associated protein nerve fibers were seen in (G, L) the MR16-1-treated group compared (E, J) with the rat IgG control group. (H, M) Note the significant differences in the GAP-43-positive and NF-H positive areas at 42 days after injury between the two groups. (N) Analysis of the locomotor Basso Mouse Scale (BMS) score after SCI. A significant improvement in hind-limb motor function was seen in the MR16-1-treated group compared with the rat IgG control group from 7 days after injury. Scale bar (A, B) 200 μm, (D, F, I, K) 500 μm, (E, G, J, L) 50 μm. (D-G) GAP-43 conjugated to Alexa fluor 568 (red); (I-J) NF-H conjugated to Alexa fluor 568 (red). Data are expressed as mean ± SD. (C, H, M) n = 3 for each group; (N) n = 5 for each group. (C, H, M) paired t-test; (N) ANOVA test. *P <0.05, **P <0.01

Figure 2

Immunoblot analysis of T helper (Th)1 and Th 2 cytokines after MR16-1 treatment. (A, B) Interferon (IFN)-γ levels were consistently higher in the control groups, and the difference with the MR16-1-treated group was significant up to 7 days post-injury. (C, D) Tumor necrosis factor (TNF)-α levels were enhanced in the control groups, and were significantly different compared with the MR16-1-treated group from 1 to 14 days post-injury. (E, F) Interleukin (IL)-4 levels remained significantly higher in the MR16-1-treated group from 1 to 7 days post-injury compared with the controls. (G, H) IL-13 levels were increased in the MR16-1-treated group, and were significantly different to the control groups between 1 and 4 days post-injury. No difference was found in the cytokine expression between the control groups (saline and Rat IgG). Each graph indicates relative band intensity normalized to that of β-actin. (B, D, F, H) Data are expressed as mean ± SD, n = 3 for each group. *P ≤ 0.05, **P ≤ 0.01 by ANOVA

Figure 3

Distribution of classically activated (M1) and alternatively activated (M2) phenotype macrophages in the injured spinal cord at 3 days after MR16-1 treatment. A common finding in the MR16-1-treated group was (C, D) a focal injury site with centralized presence of CD11b-positive cells, whereas (A, B) in the rat IgG control group, there was a larger injury site with cephalic and distal expansion of CD11b-positive cells. (E, F) Inducible nitric oxide synthase (iNOS)-positive cells were distributed over the same CD11b-positive area in the rat IgG control group, whereas a weaker reaction to iNOS was seen in (G, H) the MR16-1-treated group. (K, L) Arginase 1-positive cells were localized more specifically to the injury site in the MR16-1-treated group, whereas arginase 1-positive cells were rare in (I, J) the rat IgG control group. (A-D) CD11b conjugated to Alexa fluor 568 (red); E-H) iNOS conjugated to Alexa fluor 488 (green);(I-L) arginase 1 conjugated to Alexa fluor 488 (green); (A-L) DAPI used for nuclear counterstaining (blue). Scale bar: (A, C, E, G, I, K) 200 μm,(B, D, F, H, J, L) 500 μm

Figure 4

Effects of MR16-1 treatment on macrophage polarization after spinal cord injury (SCI), as delineated by double immunolocalization. (A) At 3 days post-injury, large amounts of inducible nitric oxide synthase (iNOS) colocalized with CD11b (merged)-positive cells were found inthe rat IgG control group), whereas only a few were found in (B) the MR16-1-treated group. (C) The differences in the presence of merged double-immunopositive cells between the two groups were significant from 1 to 7 days after injury. (D) Scarce numbers of arginase 1 colocalized with CD11b-positive (merged) cells were found in the rat IgG control group, in contrast to (E) the MR16-1-treated group; (F) analysis showed predominance of merged double-immunopositive cells in the MR16-1-treated group from 1 to 7 days after injury. (G) At 7 days post-SCI, a larger number of cells immunopositive for CD16/32 and CD11b (merged) was found in the injury epicenter in the rat IgG control group than in (H) the MR16-1-treated group, and (I) these differences were significant from 3 to 14 days after injury. (K) Cells immunopositive for CD206 and CD11b (merged) were prevalent in the MR16-1-treated group, but barely present in (J) the rat IgG control group. (L) The population of CD206/CD11b-immunopositive cells became significantly greater in the MR16-1-treated group compared with the control group from 3 to 14 days after injury. CD11b conjugated to (A, B, D, E, G, H, J, K); Alexa fluor 568 (red) (A, B) iNOS, (D, E) arginase 1, (G, H) CD16/32 and (J, K) CD206 conjugated to Alexa fluor 488 (green). Scale bar = 50 μm. (C, F, I, L) Data are expressed as mean ± SD; n = 3 for each group. *P <0.05, **P <0.01 by paired t-test.

Figure 5

Semi-quantitative flow-cytometry analysis of interferon (IFN)-γ and interleukin (IL)-4 levels in neutrophils, microglia and macrophages after MR16-1 treatment. Representative flow-cytometry plots at 1 day post-injury showed large numbers of neutrophils positive for intracellular IFN-γ in (A) the rat IgG control group with (B) smaller numbers in the MR16-1-treated group. (C, D) The numbers of neutrophils and their levels of IFN-γ, but not IL-4, were significantly lower in the MR16-1-treated group compared with the control group from 1 to 7 days post-injury. (E, F) Representative data at 3 days post-injury showed that the MR16-1-treated group had larger numbers of microglia and greater positivity for IL-4 compared with the rat IgG control group, and this difference was significant from 1 day to 7 days post-injury. (G, H) However, there was no significant difference in IFN-γ expression. (I, J, K) At 3 days post-injury, the total number of macrophages was lower in (I, J) the MR16-1-treated group compared with (K) the control group, and this difference was significant from 1 to 7 days post-injury, with increased IL-4 expression at days 1 and 3, and (L) decreased expression of IFN-γ. (C, D, G, H, K and L) Data are expressed as mean ± SD; n = 5 for each group. (C, G, K) paired t-test; (D, H, L) ANOVA test; *P <0.05, **P <0.01.

Figure 6

Semi-quantitative flow-cytometry analysis of phagocytic and digestive activities of alternatively activated macrophages after MR16-1 treatment. (A, B) Representative flow-cytometry data at 3 days post-injury identified larger numbers of iNOS-positive and CD16/32-positive macrophages in the injured SC of the rat IgG control group, compared with a lower number of such macrophages with an increased arginase 1-positive and CD206-positive sub-population in the MR16-1-treated group. (C, D) The differences in the relative preponderance of iNOS-positive and arginase 1-positive macrophages between the control and treatment group were significant from 1 to 7 days post-injury, whereas (E, F) the differences in CD16/32-positive and CD206-positive macrophages were significant from 3 days post-injury. (G, H) Microglia of both groups showed no major change in Mac-2 and Mac-3 expression; however, the number of Mac-2 and Mac-3-positive cells within the population of arginase 1-positive macrophages was significantly larger in the MR16-1-treated group than in the rat IgG control group. (I) There was no major difference in Mac-2 and Mac-3 expression in microglia, whereas (J) arginase 1-positive macrophages of the MR16-1-treated group showed enhanced expression of both antigens from 3 to 7 days post-injury. (C-F, I, J) Data are expressed as mean ± SD; n = 5 for each group;(C, D, E, F) paired t-test; (I, J) ANOVA. *P <0.05, **P <0.01