Elsevier

Molecular Immunology

Volume 48, Issues 1–3, November–December 2010, Pages 219-230
Molecular Immunology

CNS-specific expression of C3a and C5a exacerbate demyelination severity in the cuprizone model

https://doi.org/10.1016/j.molimm.2010.08.007Get rights and content

Abstract

Demyelination in the central nervous system (CNS) is known to involve several immune effector mechanisms, including complement proteins. Local production of complement by glial cells in the brain can be both harmful and protective. To investigate the roles of C3a and C5a in demyelination and remyelination pathology we utilized the cuprizone model. Transgenic mice expressing C3a or C5a under the control of the glial fibrillary acidic protein (GFAP) promoter had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to WT mice. C3a and C5a transgenic mice had increased cellularity in the corpus callosum due to increase activation and/or migration of microglia. Oligodendrocytes migrated to the corpus callosum in higher numbers during early remyelination events in C3a and C5a transgenic mice, thus enabling these mice to remyelinate as effectively as WT mice by the end of the 10 week study. To determine the effects of C3a and/or C5a on individual glial subsets, we created murine recombinant C3a and C5a proteins. When microglia and mixed glial cultures were stimulated with C3a and/or C5a, we observed an increase in the production of proinflammatory cytokines and chemokines. In contrast, astrocytes had decreased cytokine and chemokine production in the presence of C3a and/or C5a. We also found that the MAPK pathway proteins JNK and ERK1/2 were activated in glia upon stimulation with C3a and C5a. Overall, our findings show that although C3a and C5a production in the brain play a negative role during demyelination, these proteins may aid in remyelination.

Introduction

Activation of any of the complement pathways results in the downstream production of two small protein cleavage products, C3a and C5a, collectively known as anaphylatoxins. C3a and C5a are known to induce proinflammatory functions such as chemotaxis, upregulation of cytokine and chemokine production, and enhanced phagocytosis (Haas and van Strijp, 2007). C3a and C5a mediate their effects by binding to their receptors, C3aR and C5aR (CD88), which are expressed on a wide variety of cell types, including glial cells of the CNS. Anaphylatoxins also bind C5a receptor-like 2 (C5L2), whose functions have not been clearly defined, but may act as a decoy or scavenger receptor (Klos et al., 2009). Previous studies have shown that astrocytes, microglia, neurons and oligodendrocytes express anaphylatoxin receptors (Davoust et al., 1999, Gasque et al., 1997, Gavrilyuk et al., 2005, Nataf et al., 2001). C3a and C5a signaling in the CNS can induce a wide range of functions depending upon the cell type and local environmental stimuli. The overall effect of C3a and/or C5a signaling on glial cell subsets and during CNS disease pathology can be complex, because these proteins have proven to be both protective and harmful. For instance, C5a has been shown to protect neurons against glutamate-mediated apoptosis both in vitro and in vivo (Mukherjee et al., 2008, Osaka et al., 1999b). Additionally, C3a has been implicated in neural stem cell regeneration and migration (Rahpeymai et al., 2006, Shinjyo et al., 2009). In contrast, C3a and C5a have been shown to have exacerbating effects on CNS disease severity in mouse models of Alzheimer's (Fonseca et al., 2009), amyotrophic lateral sclerosis (ALS) (Woodruff et al., 2008) and lupus (Jacob et al., 2010a, Jacob et al., 2010b). Several studies have also suggested that complement may play a role in the neurodegenerative disease, multiple sclerosis (MS) (Ingram et al., 2009).

MS is a chronic, inflammatory disease that targets the myelin sheath surrounding nerves, affecting nerve conduction (Trapp and Nave, 2008). There have been several studies showing that complement proteins, including C3a, contribute to the pathogenesis of demyelination. Glial cells are able to produce complement proteins, so it is possible for localized activation of complement pathways to occur in the CNS (Sospedra and Martin, 2005). C3aR and C5aR are expressed in areas of inflammation in the CNS of MS patients (Gasque et al., 1997, Gasque et al., 1998, Muller-Ladner et al., 1996). In EAE, a mouse model of multiple sclerosis, C3a and C3aR have been shown to play a role in exacerbating disease severity (Boos et al., 2004). Interestingly, it has been shown that C5a and C5aR do not play a role in EAE pathology (Reiman et al., 2005, Reiman et al., 2002). Results from our lab have determined that complement plays a role in the murine cuprizone-mediated demyelination and remyelination model. When a soluble CNS-specific form of the rodent complement regulatory protein, Crry (sCrry) was expressed, mice were protected from demyelination during cuprizone treatment, implicating a role for C3 and downstream complement proteins in this model (Briggs et al., 2007). In our current studies, we utilized the murine cuprizone model to assess the role of local C3a and C5a production in the brain during demyelination and remyelination.

Cuprizone is a copper chelating drug that when given at low doses in the diet of C57BL/6 mice, leads to apoptosis of oligodendrocytes, the myelin producing cells of the brain (Mason et al., 2000). The death of these cells results in demyelination in predictable, specific axonal tracts of the brain, including the corpus callosum, cerebellum and the superior cerebellar peduncle (Groebe et al., 2009, Matsushima and Morell, 2001). One attractive aspect of the cuprizone model is the relative lack of blood brain barrier breakdown (Bakker and Ludwin, 1987, Kondo et al., 1987), which makes it possible to analyze the local response of glia to C3a or C5a production. The primary effector cells in demyelination during treatment with cuprizone are reactive astrocytes and microglia. Demyelination in this model shares some characteristics with type III and IV lesions described in MS (Lucchinetti et al., 2000), characterized by oligodendrocyte death, microglial infiltration and lack of antibodies present in areas of demyelination. After mice are returned to a normal diet, the affected areas in the brain undergo remyelination, allowing for the investigation of events that occur during remyelination pathogenesis. We utilized transgenic mice expressing C3a or C5a under the control of the CNS-specific GFAP promoter to determine the role of C3a and C5a production on demyelination and remyelination events.

C3a and C5a are able to act as chemotactic factors; therefore, we hypothesized that an increase in the expression of C3a and C5a in transgenic mice being treated with cuprizone would exacerbate demyelination severity by increasing the number of inflammatory cells migrating into the corpus callosum. In contrast to results observed in the EAE model, we found that both C3a and C5a played a role in increased demyelination pathology. Both C3a and C5a transgenic mice had increased microglia in the corpus callosum during demyelination. Interestingly, when cuprizone was removed from the diet, oligodendrocytes returned to the demyelinated corpus callosum in higher numbers in C3a and C5a transgenic mice, thus allowing for complete remyelination of the corpus callosum. When we investigated the effect of C3a and/or C5a on individual subsets of glial cells, we found an increase in proinflammatory cytokine and chemokine production, indicating an ability of C3a and C5a to induce proinflammatory effects in glial cells.

Section snippets

Mice

Previously described C3a-GFAP (Boos et al., 2004) and C5a-GFAP (Reiman et al., 2005) transgenic mice on the C57BL/6 background were provided by S.R. Barnum (University of Alabama). Five-week-old male C57BL/6 mice were purchased from Jackson Laboratories, Bar Harbor, ME.

Induction of demyelination and remyelination

To induce demyelination, 8–12 week old male mice were fed a diet of NIH-31 modified 6% Mouse/Rat Sterilizable Diet-meal form (Harlan Laboratories, Madison, WI), containing 0.2% cuprizone (bis (cyclohexanone) oxaldihydrazone,

Increased levels of C3a and C5a in the brain lead to exacerbated demyelination and delayed remyelination

To determine the role of C3a and C5a in demyelination and remyelination, we used transgenic mice expressing C3a or C5a under the control of the CNS-specific GFAP promoter. Our results show that after 4 weeks on the cuprizone diet, both C3a and C5a transgenic mice had worse demyelination severity compared to C57BL/6 wild type (WT) mice (Fig. 1A and B). Demyelination severity was increased through 7 weeks in both transgenic groups (Fig. 1A). Although these mice showed worse demyelination severity

Discussion

It has been previously established that C3a and C5a receptors are upregulated in patients with MS (Gasque et al., 1998, Muller-Ladner et al., 1996) and complement deposition is present within MS lesions (Ingram et al., 2009, Lucchinetti et al., 2000). These results would suggest that C3a and/or C5a could potentially play a role in demyelination, lesion pathology and subsequent remyelination. In the present study we have shown that the C3a and C5a exacerbate demyelination severity in the murine

Role of the funding source

This work was supported by NIH NINDS grant number 5 R21 NS056364-03 and National Multiple Sclerosis Society grant number RG 3676-A-2 to bkm. The funding sources had no part in the design, interpretation or preparation of these data for publication.

Acknowledgments

The authors would like to thank J. P.-Y. Ting for the BV-2 cells and C.D. Stiles for providing the Olig-2 antibody.

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