The chemokine MCP-1 and the dendritic and myeloid cells it attracts are increased in the mSOD1 mouse model of ALS

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Abstract

We recently demonstrated increased dendritic cells (potent antigen-presenting cells) and MCP-1 (monocyte, T-cell, and dendritic cell attracting chemokine) levels in ALS spinal cord tissue. Additionally, we presented data suggesting that dendritic cells might be contributing to the pathogenesis. To determine whether MCP-1 and dendritic cells are present in the mSOD1 mouse and how early in the disease process they are involved, we examined mSOD1 and control spinal cord tissue at different ages using real-time RT-PCR and immunohistochemistry. Dendritic cells were present and transcripts elevated in mSOD1 spinal cord beginning at 110 days. MCP-1 mRNA and immunoreactivity were upregulated in mSOD1 neuronal and glial cells as early as 15 days, prior to any evidence of microglial activation. CD68+ cells were present at 39 days of age. Although it is not clear if these responses are protective or injurious, the early increased MCP-1 expression and CD68+ cell presence indicate early preexisting injury.

Introduction

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is characterized by the progressive degeneration of upper and lower motoneurons. While the etiology of motoneuron degeneration remains elusive, increasing evidence indicates that immune-inflammatory reactions contribute to the pathogenesis in ALS. Several groups have demonstrated the infiltration of T-cells and macrophages and the presence of IgG and activated microglia in ALS spinal cord tissue (Henkel et al., 2004, Ince et al., 1996, Engelhardt et al., 1993, Kawamata et al., 1992, Engelhardt and Appel, 1990, Troost et al., 1990, Lampson et al., 1990, Troost et al., 1989, McGeer et al., 1988). Additional indications of inflammation include reactive astrocytes (Schiffer et al., 1996, McGeer et al., 1988) and increased IL6 expression (Sekizawa et al., 1998), increased COX-2 (Maihofner et al., 2003, Yasojima et al., 2001, Almer et al., 2001), increased prostaglandin E2 (Almer et al., 2002), increased iNOS (Sasaki et al., 2000), as well as other indications of inflammation (Grewal et al., 1999, Kawamata et al., 1992). Thus, there is compelling evidence for an immune-inflammatory response in the spinal cords of ALS patients.

Recently, we demonstrated the presence of dendritic cells in ALS spinal cord tissue that was not present in control tissue (Henkel et al., 2004). Additionally, we determined that the chemokine that attracts dendritic cells, monocyte chemoattractant protein 1 (MCP-1 or CC-chemokine ligand 2), was upregulated in glial cells in ALS spinal cord tissue and not in control. Importantly, those ALS patients that progressed the most rapidly expressed more dendritic cell transcripts than patients that progressed more slowly. This finding was significant because dendritic cells control both the innate and adaptive immunity and are able to prime naïve T-cells or to eliminate or suppress autoactive T-cells.

A mouse model of familial ALS has been used to investigate ALS pathology. The pathology observed in these mSOD1 transgenic mice resembles that seen in the ALS patients. All of the mice appear normal at birth and begin to show motor dysfunction as they age, although the age of onset depends on the number of copies of the mSOD1 gene, on the specific mutation (human SOD1 G93A, G85R, and G37R mutations, and mouse SOD1 G86R mutation) (Gurney et al., 1994, Bruijn et al., 1997, Wong et al., 1995, Ripps et al., 1995, respectively), and on the genetic background of the mouse. Their symptoms progress from hind limb weakness to muscle atrophy, paralysis, and finally death. Evidence indicating immune-inflammatory activation in the mSOD1 mouse includes activated microglia, infiltrating T-cells, and IgG present in mSOD1 mouse spinal cord tissue (Alexianu et al., 2001, Hall et al., 1998). Additional evidence includes activated astrocytes (Alexianu et al., 2001, Levine et al., 1999, Hall et al., 1998), increased iNOS expression (Sasaki et al., 2001, Almer et al., 1999), increased p38 activation (Tortarolo et al., 2003, Hu et al., 2003), and increased TNFα, IL-1β, TGFβ, Il-6, and MCP-1 expressions (Hensley et al., 2002, Hensley et al., 2003, Nguyen et al., 2001, Elliott, 2001). Additionally, in the mSOD1 mouse, immune-inflammatory responses are present early in disease prior to any evidence of dysfunction (Alexianu et al., 2001, Almer et al., 2001). Thus, there are immune-inflammatory reactions present in both ALS and mSOD1 mouse spinal cord tissue.

To determine if increased MCP-1 and dendritic cells are present in the mSOD1 mouse model of ALS, as they are in ALS tissue, we examined mRNA expression and immunoreactivity of dendritic cell proteins, myeloid proteins, cytokines, and MCP-1. We used real-time RT-PCR and immunohistochemistry to evaluate mSOD1 and control spinal cord tissue at several ages before and during disease. We demonstrate that dendritic cells and transcripts were increased at 110 days and end stage in the mSOD1 mouse, that CD68+ phagocytic cells were present as early as 39 days of age, and that there was increased MCP-1 expression as early as 15 days of age, well before any evidence of generalized microglial activation or disease.

Section snippets

Dendritic cell transcripts increased in mSOD1 spinal cord tissue

To determine if dendritic cell transcripts are increased in mSOD1 mouse spinal cord tissue as they were in ALS tissue (Henkel et al., 2004), we examined a variety of different dendritic markers. Specifically, we examined the mRNA expression of CD11c, the integrin αx-chain and a myeloid dendritic cell marker; CD123, the IL-3 receptor α-chain and a lymphoid dendritic cell marker; and CD86, an activation and costimulatory protein and a mature dendritic cell marker. While some of these transcripts

Discussion

Dendritic cells control both innate and adaptive immunity and are able to either prime naïve T-cells or to eliminate autoactive T-cells, in both the thymus and the periphery (Liu, 2001, Banchereau et al., 2004). We had previously demonstrated the presence of dendritic cells in ALS spinal cord tissue that were not present in control tissue (Henkel et al., 2004), and that those ALS patients that progressed the most rapidly expressed more dendritic cell transcripts than patients that progressed

Mutant SOD1 and control mice

Fifteen-, 40-, 80-, and 110-day-old (±2 days) and end stage mSOD1 mice and 15-, 40-, and 80-day-old mSOD1 and hSOD1 mice were used for analyses. Age-matched littermates, without the mutant or human SOD1 genes, were used as controls. For RNA analyses, the spinal cords were isolated from lethally anesthetized mice (4 mSOD1 mice and 4 control mice at each age group) and snap frozen. For immunohistochemistry, lethally anesthetized mice were perfused with paraformaldehyde, and the spinal cords were

Acknowledgments

This study was supported by the following grants: JSH: the Muscular Dystrophy Association, The Methodist Research Institute; SHA: NIH-NINDS# R01 NS048950-01, the Muscular Dystrophy Association, the Hamill Foundation. We thank Dr. Wei-dong Le and members of his laboratory for allowing us the use of their iCycler iQ Real-time PCR Detection System and for their advice prior to obtaining our iQ5 Multicolor Real-time PCR Detection System.

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