Journal of Neuroimmunology 159 (2005) 215–224 www.elsevier.com/locate/jneuroim Evidence for systemic immune system alterations in sporadic amyotrophic lateral sclerosis (sALS) Rongzhen Zhanga, Ron Gascona, Robert G. Millerb, Deborah F. Gelinasb, Jason Massb, Ken Hadlockc, Xia Jinc, Jeremy Reisa, Amy Narvaeza, Michael S. McGratha,* aUniversity of California, San Francisco, San Francisco General Hospital, 995 Potrero Avenue, Building 80, Ward 84, Box 0874, San Francisco, CA 94110, USA bCalifornia Pacific Medical Center, San Francisco, CA 94115, USA cPathologica LLC, Burlingame, CA 94010, USA Received 12 February 2004; received in revised form 7 September 2004; accepted 12 October 2004 Abstract Sporadic amyotrophic lateral sclerosis (sALS) is a progressive neuroinflammatory disease of spinal cord motor neurons of unclear etiology. Blood from 38 patients with sALS, 28 aged-match controls, and 25 Alzheimer’s disease (AD) patients were evaluated and activated monocyte/macrophages were observed in all patients with sALS and AD; the degree of activation was directly related to the rate of sALS disease progression. Other parameters of T-cell activation and immune globulin levels showed similar disease associated changes. These data are consistent with a disease model previously suggested for AD, wherein systemic immunologic activation plays an active role in sALS. D 2004 Elsevier B.V. All rights reserved. Keywords: Amyotrophic lateral sclerosis (ALS); Alzheimer’s disease (AD); Motor neuron disease; Immune activation; Monocyte/macrophage 1. Introduction 1995; Fray et al., 1998), and autoimmunity (Appel et al., 1993). In addition, several recent studies have suggested Amyotrophic lateral sclerosis (ALS) is a devastating that the immune system may be actively involved in the neurological disease characterized by gradual degeneration disease process of ALS, with observations of activated of spinal cord motor neuron cells leading to progressive microglia, IgG deposits, and dysregulation of cytokine weakness, paralysis of muscle and death. Almost 90% of expression in the spinal cord of ALS patients (Troost et ALS patients are characterized as having sporadic ALS al., 1989; Engelhardt and Appel, 1990; Schiffer et al., 1996; (sALS) with 10% having a familial form, a subset of whom Hayashi et al., 2001). However, despite intensive inves- have point mutations in their superoxide dismutase (SOD) tigation ALS has no known cause or effective therapy. gene. The pathogenesis of ALS is incompletely understood, Inflammatory mechanisms and immune reactivity have although different hypotheses have been suggested, includ- been hypothesized to play a role in the pathogeneses of ing mitochondria dysfunction (Richter et al., 1988; Wong et central nervous system (CNS) disease (Akiyama et al., al., 1995; Curti et al., 1996), mutation in the superoxide 2000; Minagar et al., 2002; Bar-Or et al., 2003; Filion et al., dismutase gene (Deng et al., 1993; Durham et al., 1997), 2003, Hirsch et al., 2003; Floris et al., 2004; Ringheim and defects in neuronal glutamate transports (Rothstein et al., Conant, 2004), including ALS (Alexianu et al., 2001; McGeer and McGeer, 2002; Henkel et al., 2004; Simpson et al., 2004). Furthermore, various studies have suggested * Corresponding author. Tel.: +1 415 476 4082x415; fax: +1 415 476 that monocyte/macrophage activation may play a significant 6017. role in the pathogenesis of many neurological diseases, such E-mail address: [email protected] (M.S. McGrath). as Alzheimer’s disease (AD) (Smits et al., 2000; Fiala et al., 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.10.009 216 R. Zhang et al. / Journal of Neuroimmunology 159 (2005) 215–224 2002), multiple sclerosis (MS) (Minagar et al., 2002; Filion criteria (Brooks, 1994) at the Forbes Norris MDA/ALS et al., 2003), Parkinson’s disease (McGeer et al., 1988), and Research Center (San Francisco, CA, USA) had blood HIV-associated dementia (HAD) (Pulliam et al., 1997; drawn in accordance with the CPMC (California Pacific Diesing et al., 2002). Indeed, the best pathological correlate Medical Center) and UCSF committees on human research for HIV-associated neurologic disorders, especially HAD, is guidelines, coordinated by the UCSF AIDS and Cancer the number of activated mononuclear phagocytes present in Specimen Resource (ACSR) program. The Revised ALS the brain (perivascular and parenchymal blood-derived Functional Rating Scale (ALSFRS-R), scored 0–48, was macrophages and microglia), and not the absolute level of used to evaluate overall patient functional status (Cedar- HIV in the brain (Glass et al., 1995; Adamson et al., 1999). baum et al., 1999). All scores were updated within a month Similar findings have been reported for simian AIDS related of blood testing. Patients had sALS for 4–93 months with a encephalopathy (SIVE) (Williams et al., 2002). Studies on range of ALSFRS-R scores of 8–43. Demographic infor- blood from patients with HAD (Liu et al., 2000) and mation on sALS patients whose specimens were studied is monkeys with SIVE (Williams et al., 2002) have shown a shown in Table 1, in which 12 patients were using various direct relationship between the presence of activated blood anti-inflammatory medications with standard dose (Cele- macrophages and CNS disease. These activated macro- brex, Vioxx, Naproxyn, Excedrin), and 30 patients were phages are thought to mediate blood–brain barrier (BBB) taking riluzole (50 mg twice daily); 10 patients received breakdown and directly contribute to CNS pathogenesis. both medications. Given evidence of macrophage activation in spinal cords of Two control groups were used in the study. The first patients with ALS disease (Engelhardt and Appel, 1990; control group consisted of 28 age-matched normal blood Appel et al., 1993; Obal et al., 2001; McGeer and McGeer, samples (10 females and 18 males, mean age 56.0F15.1 2002; Wilms et al., 2003; Henkel et al., 2004), a pathogenic years), which were obtained from blood draws at Stanford process similar to these may occur in ALS. University Blood Center and processed in a similar manner In addition to local evidence for tissue associated to the sALS patient blood specimens. Normal control macrophage activation, increasing numbers of studies on samples for IgG and IgM studies consisted of plasma from peripheral immune activation levels in CNS disease patients 80 blood donors and were also obtained from the Stanford such as AD (Shalit et al., 1995; Scali et al., 2002; Casal et University Blood Center. The second control group obtained al., 2003), HIV-associated neurologic disorder (Pulliam et from the same source as sALS, had Alzheimer’s disease al., 1997; Sanchez-Ramon et al., 2003) and MS (Bar-Or et (AD). This group consisted of 25 AD patients (11 females al., 2003; Filion et al., 2003) support the hypothesis of a and 14 males, mean age 77.5F7.9), and was used as peripheral immune reaction which might be correlated with neurological disease controls. the clinical stages of disease. In order to examine whether macrophage activation observed in spinal cords of ALS 2.2. Flow cytometry patients would be reflected in the systemic immune system, the current study was designed: (1) to detect differences, if About 10 ml of peripheral blood was drawn from each any, in the expression of monocyte/macrophage and T-cell patient and normal controls into heparinized tubes and activation markers in peripheral blood mononuclear cells transferred to the laboratory at room temperature for same (PBMC) between sALS patients and controls and (2) to test day immunologic studies. Cellular immunologic activation whether the peripheral immune activation state in sALS was evaluated by quantitating levels of CD38 on T-cell patient blood would correlate with clinical stage of disease. subsets and HLA-DR on CD14 cells. CD16 (Fc gamma III This study shows for the first time that the pattern of receptor) expression on CD14 cells was used as another immunologic activation in blood cells from patients with marker for monocyte differentiation and has been an antigen sALS correlates with ALS disease state and that rate of associated with cytokine expression patterns characteristic disease progression is directly related to the degree of of tissue macrophages (Ziegler-Heitbrock et al., 1993; systemic monocyte/macrophage activation. These data are Frankenberger et al., 1996). The monocyte granularity consistent with a model of sALS that includes systemic associated with its differentiation was measured by CD14- immunologic dysfunction as a cofactor in disease patho- associated bbackgatingQ on side light-scatter characteristics genesis, not dissimilar to pathogenic models for AD, MS (SSC). Whole blood was stained with CD14-fluorescein and HAD as described above. isothiocyanate (FITC), CD16-phycoerythrin (PE) (DAKO, Carpinteria, CA, USA), CD8-FITC, CD38-PE, HLA-DR- PE, and CD4-peridinin chlorophyll protein (PerCP) (Bec- 2. Methods ton-Dickinson, San Jose, CA, USA) for 15 min at room temperature. Negative controls consisted of aliquots stained 2.1. Subjects with isotype IgG-FITC, IgG-PE, and IgG-PerCP; all staining was performed as per manufacturers specifications. Thirty-eight patients diagnosed with sALS (12 females Samples were then lysed with FACS Lysing Solution and 26 males, mean age 59.3F13.4 years) by El Escorial (Becton-Dickinson) for 10 min at room temperature R. Zhang et al. / Journal of Neuroimmunology 159 (2005) 215–224 217 Table 1 Clinical summary of sALS