Anti-IL-6-receptor antibody promotes repair of spinal cord injury by inducing microglia-dominant inflammation

Abstract

We previously reported the beneficial effect of administering an anti-mouse IL-6 receptor antibody (MR16-1) immediately after spinal cord injury (SCI). The purpose of our present study was to clarify the mechanism underlying how MR16-1 improves motor function after SCI. Quantitative analyses of inflammatory cells using flow cytometry, and immunohistochemistry with bone marrow-chimeric mice generated by transplanting genetically marked purified hematopoietic stem cells, revealed that MR16-1 dramatically switched the central player in the post-traumatic inflammation, from hematogenous macrophages to resident microglia. This change was accompanied by alterations in the expression of relevant cytokines within the injured spinal cord; the expression of recruiting chemokines including CCL2, CCL5, and CXCL10 was decreased, while that of Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF), a known mitogen for microglia, was increased. We also showed that the resident microglia expressed higher levels of phagocytic markers than the hematogenous macrophages. Consistent with these findings, we observed significantly decreased tissue damage and reduced levels of myelin debris and Nogo-A, the axonal growth inhibitor, by MR16-1 treatment. Moreover, we observed increased axonal regeneration and/or sprouting in the MR16-1-treated mice. Our findings indicate that the functional improvement elicited by MR16-1 involves microglial functions, and provide new insights into the role of IL-6 signaling in the pathology of SCI.

Introduction

In the pathology of spinal cord injury (SCI), the primary mechanical injury is followed by post-traumatic inflammation, in which inflammatory cells such as neutrophils, hematogenous macrophages (blood-borne macrophages), and resident microglia accumulate at the lesion site. These inflammatory cells release reactive oxygen, nitrogen radicals, and proteases, which exacerbate tissue damage (Hausmann, 2003). Because the inflammation is regulated by pro-inflammatory cytokines, such as TNFα, IL-1β, and IL-6, these cytokines have been targets for potential pharmaceutical interventions for SCI (Nesic et al., 2001, Okada et al., 2004, Sharma et al., 2003, Tuna et al., 2001). Among these cytokines, IL-6 is known to promote the activation and infiltration of macrophages/microglia (Hurst et al., 2001, Van Wagoner and Benveniste, 1999), which are the major inflammatory cells in the injured spinal cord, and the overexpression of IL-6 extends the inflammation to worsen the tissue injury (Klusman and Schwab, 1997, Lacroix et al., 2002). Hypothesizing that a blockade of IL-6 signaling might reduce the extension of injury by post-SCI inflammation, we previously administered the anti-mouse IL6-receptor antibody (MR16-1) after SCI and demonstrated reduced inflammation, decreased astrogliosis, and enhanced tissue sparing, leading to improved functional recovery (Okada et al., 2004). As a humanized antibody for the human IL-6 receptor (MRA; tocilizumab) is already in clinical use for the treatment of Castleman's disease and rheumatoid arthritis (Choy et al., 2002, Nishimoto et al., 2000, Sato et al., 1993), this drug might represent a new option for the treatment of SCI.

However, recent studies using gene-knockout animals revealed that the continuous inhibition of IL-6 signaling is detrimental to functional recovery, by inhibiting axonal regeneration or causing failed gliosis, implying that IL-6 may also have a beneficial function in spinal cord repair (Cafferty et al., 2004, Okada et al., 2006). Furthermore, numerous studies suggest that inflammation is beneficial or even essential for spinal cord repair, because it clears tissue debris and involves the secretion of various neurotrophic factors (Donnelly and Popovich, 2008, Hashimoto et al., 2005, McTigue et al., 2000). This discrepancy prompted us to investigate how the administration of an anti-IL-6 receptor antibody immediately after SCI promotes the repair process.

One of the important determinants of the extent of secondary damage by inflammation is the nature of the recruited inflammatory cells. For example, the transplantation of macrophages that have been co-incubated with peripheral nerves or skin improves spinal cord repair (Bomstein et al., 2003, Schwartz et al., 1999). On the other hand, zymosan-activated macrophages have neurotoxic properties, although they can also have proregenerative effects (Gensel et al., 2009, Popovich et al., 2002, Steinmetz et al., 2005). Previous reports have shown that these differences in the characteristics of inflammatory cells depend not only on their state of activation, but also on their origin. A subpopulation of hematogenous macrophages is more cytotoxic than microglia, and their excessive infiltration into a lesion is detrimental to spinal cord repair (Gris et al., 2004, Popovich et al., 1999). Since IL-6 is known to promote macrophage infiltration after central nervous system (CNS) trauma (Klusman and Schwab, 1997, Lacroix et al., 2002), here we focused on the effect of the temporary inhibition of IL-6 signaling by MR16-1 on macrophages and microglia after SCI. The administration of MR16-1 reduced the infiltration of macrophages into the injured spinal cord, but increased the number of microglia residing there, thus switching the major inflammatory cell type at the lesion from hematogenous macrophages to resident microglia. A comparison of the expression of phagocytic markers by hematogenous macrophages and microglia revealed that the microglia had greater phagocytic ability against myelin debris after SCI. Consequently, this switch in major inflammatory cell type resulted in improved tissue sparing and debris clearance, which promoted neural repair after SCI.