Gene expression profiling in peripheral blood mononuclear cells from patients with sporadic amyotrophic lateral sclerosis (sALS)
Introduction
Amyotrophic lateral sclerosis (ALS) is a debilitating neurological disorder in which neurodegeneration occurs in concert with an ongoing inflammatory process (McGeer and McGeer, 2002). ALS manifests as muscle weakness and paralysis with death ensuing for most patients within 5 years of symptom onset. Almost 90% of ALS patients are characterized as having sporadic ALS (sALS) with 10% having a familial form, a subset of whom have point mutations in their superoxide dismutase (SOD) gene. Evidence accumulating over the past decade has indicated that inflammation and immune activation may be commonly involved in the pathogenesis of ALS (Alexianu et al., 2001, Graves et al., 2004, Henkel et al., 2004, McGeer and McGeer, 2002, Simpson et al., 2004, Zhang et al., 2005). Neuropathologically, ALS is characterized by motor neuron degeneration with ubiquinated inclusions in the spinal cord and motor cortex. The neuropathologic lesions are surrounded by activated microglia in both the familial and sporadic forms of ALS disease (Kawamata et al., 1992, McGeer and McGeer, 2002), which may play an active role in ALS disease progression (Beers et al., 2006, Boillee et al., 2006).
Along with activated resident microglia, large numbers of infiltrating macrophages are present in sALS spinal cord lesions. Abnormal secretion of macrophage activation products and proinflammatory cytokines, such as monocyte chemoattractant protein-1 (MCP-1) (Baron et al., 2005, Henkel et al., 2004, Simpson et al., 2004, Wilms et al., 2003, Zhang et al., 2006), and interleukin-6 (IL-6) (Ono et al., 2001, Sekizawa et al., 1998) have been reported in cerebral spinal fluid (CSF) and sera in patients with ALS. Enhanced levels of tumor necrosis factor-α (TNF-α) have also been seen in the blood of ALS patients (Babu et al., 2008, Cereda et al., 2008, Poloni et al., 2000). Patients with sALS exhibit elevated levels of CD16+ monocytes in peripheral blood (Zhang et al., 2005). These CD16+ ALS associated monocytes are characterized by high expression of monocyte activation marker HLA-DR and low expression of the MCP-1 receptor, CCR2 (Zhang et al., 2006). In addition, our more recent study found significantly increased levels of plasma endotoxin/lipopolysaccharide (LPS), a systemic macrophage activator, in sALS patients, and that increased LPS levels correlated with degree of abnormally activated monocyte/macrophages in the peripheral blood. Finally, chronic stimulation of innate immunity with LPS has been shown to accelerate the disease course in SOD1 transgenic mice (Nguyen et al., 2004).
Endotoxin/lipopolysaccharide is a potent inflammatory stimulus and immunostimulatory product (Takeda et al., 2003) and induces its effects through stimulation of CD14-bearing inflammatory cells (Flo et al., 2000, Tobias et al., 1999). LPS associated toxicity is mediated through systemic monocyte/macrophage and endothelial cell activation with release of inflammatory cytokines such as TNF-α and IL-6 (Beutler et al., 1985, Danner et al., 1991, Okusawa et al., 1988, Tracey et al., 1986). The effects of LPS are mediated primarily through toll-like receptor 4 (TLR4). LPS/TLR4 signal transduction can be separated into MyD88-dependent and MyD88-independent pathways; the MyD88-dependent pathway was shown to be responsible for proinflammatory cytokine expression, while MyD88-independent pathway mediates the induction of type I interferons and interferon-inducible genes (Lu et al., 2008, Takeda and Akira, 2007, Tanimura et al., 2008).
Thus, sALS is characterized by evidence of systemic monocyte activation, in addition to local activation within disease affected spinal cords. In an effort to better characterize the systemic immune activation present in ALS patients we undertook a microarray-based gene expression analysis of peripheral blood mononuclear cells from patients with sALS. After short-term culture, peripheral blood mononuclear cells from sALS patients upregulated a different set of genes relative to cells from equivalently-aged healthy individuals, encoding type I interferons, interferon-inducible genes, and genes associated with the secreted activation proteins. Quantitative RT-PCR (qRT-PCR) analysis of gene transcription found that signals obtained from patients positively correlated with degree of abnormally activated monocyte/macrophages and plasma levels of LPS in sALS. This study shows for the first time a linkage between systemically activated monocyte/macrophages in sALS patients and the LPS/TLR4 signaling pathways.
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Subjects
Patients with sALS, diagnosed using the El Escorial criteria (Brooks et al., 2000), provided informed consent in accordance with guidelines established by the California Pacific Medical Center and University of California San Francisco (UCSF) committees on human research, coordinated by the UCSF AIDS and Cancer Specimen Resource (ACSR) program. The Revised ALS Functional Rating Scale (ALSFRS-R), scored 0–48, was used to evaluate overall patient functional status (Cedarbaum et al., 1999). All
Characteristics of study populations
Patients with sALS and healthy controls were recruited for this study over a period of 21 months. Basic demographic and clinical information about the patients and healthy control samples is provided in Table 1. Twenty sALS patients were examined in the microarray analysis of gene expression and the PBMCs of an additional 42 sALS patients were analyzed in subsequent experiments. The average time since the participating individuals were diagnosed with probable or definite ALS according to the El
Discussion
Sporadic ALS is characterized by large numbers of activated microglia/macrophages in the regions of the spinal cord undergoing neurodegeneration (Graves et al., 2004, Henkel et al., 2004). Recent studies in ALS disease models have implicated important roles for activated microglia/macrophages in ALS disease progression in vivo (Beers et al., 2006, Boillee et al., 2006). sALS is also characterized by elevated levels of abnormally activated monocyte/macrophages in peripheral blood (Zhang et al.,
Acknowledgements
This work was supported in part by National Institutes of Health's (NIH) grant number U01-CA 66529 (MSM), National Cancer Institute's West Coast AIDS and Cancer Specimen Resource (ACSR) Consortium, University of California, San Francisco (UCSF). We are grateful to the patients with ALS who contributed samples for this study. The authors also wish to acknowledge WJ Rutter for his support and insights.
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