Anti-Heat Shock Protein 70 antibody levels are increased in myasthenia gravis and Guillain-Barré syndrome

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Abstract

Myasthenia gravis (MG) is an autoimmune disorder where patients develop autoantibodies towards skeletal muscle proteins (e.g. acetylcholine receptor and muscle specific kinase), causing weakness in striated muscles. Ocular MG (OMG) represents a subtype of (MG) affecting only the periocular muscles. The pathogenesis of this phenotype remains unclear. Heat Shock Protein 70 (Hsp70) plays a role in immune regulation. Antibodies against this protein are associated with several autoimmune diseases, and its biological significance has been shown in vivo. We have therefore examined the concentration of anti-Hsp70 antibodies in sera from 35 OMG patients and 94 patients with generalized MG (GMG) using ELISA assays. The antibody concentrations were compared to those in patients with multiple sclerosis (MS), Guillain-Barré syndrome (GBS) and to healthy controls.

MG patients had significantly higher anti-Hsp70 antibody concentrations than both MS patients and healthy controls. GBS patients had higher antibody levels than all other groups. No difference in antibody levels was found when comparing OMG and GMG. Our results suggest that patients with MG and GBS have a previous or current increased exposure to Hsp70 antigens. The similarity between GMG and OMG strengthens the hypothesis that OMG represents a systemic disease, similar to GMG.

Introduction

Myasthenia gravis (MG) is an autoimmune disorder where patients experience weakness in voluntary muscles, including respiratory function. It is in most cases caused by circulating antibodies targeting the nicotinic acetylcholine receptor (AChR) on skeletal muscle cell membranes, but antibodies towards muscle specific kinase (MuSK) can also have pathogenic significance (Pascale Ter Beek et al., 2009, Vincent and Leite, 2005). A subgroup of MG patients experience only periocular motor symptoms such as diplopia and ptosis, and are hence referred to as ocular MG (OMG). The reasons for the purely periocular involvement are unclear. Approximately 40% of OMG patients are anti-AChR antibody positive in conventional assays, (Luchanok and Kaminski, 2008) but the sensitivity is higher in cell culture based assays. However, this has only been demonstrated for GMG sera, and not in sera from OMG patients (Leite et al., 2008).

Antibodies against Hsp70 have been detected in sera from patients with sensory neuronal hearing loss (Tebo et al., 2006), systemic lupus erythematosus (Tasneem et al., 2001), juvenile idiopathic arthritis (Zlacka et al., 2006) and MG (Munakata et al., 2008). These studies have focused on the presence of antibodies, and have not aimed at providing any etiological evidence. When applying anti-Hsp70 antibodies together with an allergy-inducing agent, mice did not develop the allergic dermatitis, but rather gained systemic antigen specific tolerance which could be adoptively transferred (Yusuf et al., 2009).

The Heat Shock Proteins (Hsp) are divided into subfamilies according to weight (Multhoff, 2007). They play a crucial role in functioning as chaperones to prevent protein misfolding and aggregation (Bukau and Horwich, 1998). While many of the HSPs are expressed at low levels in most eukaryotic cells, they can be induced upon cellular stress such as increased temperature, radiation, exposure to various chemicals, oxidative stress and various physiological and pathological stimuli (Multhoff, 2007, Tsan and Gao, 2004). In addition to being chaperone proteins, the HSPs play a part in antigen presentation and cross-presentation, (Li et al., 2002) and function as cytokines to induce production of pro-inflammatory cytokines and promote dendritic cell maturation (Wang et al., 2002, Asea et al., 2000).

Hsp70 is believed to be involved in the pathogenesis of several autoimmune disorders, including Behcet's disease (Birtas-Atesoglu et al., 2008), Grave's disease (Ratanachaiyavong et al., 1991, Hunt et al., 2001) and multiple sclerosis (MS) (Salvetti et al., 1996). Its ability to augment antigen presentation has been shown in experimentally induced diabetes mellitus (Millar et al., 2003), and increased levels are found locally in experimental autoimmune neuritis, a Guillain-Barré syndrome (GBS) model (Zhang et al., 2009).

Hsp70 therefore plays an important role in antigen presentation and development of tolerance, and antibody-mediated interference of its function can alter the immune response for antigens being presented by antigen presenting cells. In this study we investigated whether patients with generalized and ocular MG had altered anti-Hsp70 levels, as compared to healthy controls and patients with the widespread immune disorders MS and GBS.

Section snippets

Patient and control sera

Sera from 129 patients with MG were provided by one of the authors (GTP, UK), all of them with ocular symptoms as the dominant ones. Patients were referred through an eye casualty department to the neuro-ophtalmology clinic and followed by GTP. MG patients were included using the following inclusion criteria: recent onset (within last 2 years) of fatiguable diplopia, ptosis due to levator dysfunction, or both in the absence of generalized weakness. Clinically supportive criteria were the

Results

The 129 MG patients consisted of 66 males and 63 females. Their mean age at time of inclusion (± SD) was 57.1 years (± 17.7). 94 (73%) patients had generalized MG (GMG), whereas 35 (27%) had purely ocular phenotype (OMG). Patients classified as OMG had neither electrophysiological nor clinical evidence of limb involvement. Of the 94 GMG patients 36 had detectable levels of anti-AChR antibodies, 53 were negative and 5 unknown. The OMG group had 16 anti-AChR positive, 18 negative and 1 unknown. The

Discussion

We have shown that patients with MG and ocular muscle weakness have increased circulating levels of anti-Hsp70 antibodies compared to healthy blood donors and patients with MS. Higher levels of anti-Hsp70 antibodies are likely to reflect increased exposure to Hsp70 antigens, current or previous, in the circulation or bound to cell membranes. Our results therefore indicate increased exposure to Hsp70 in the course of MG development.

OMG affects a few small muscles only, and patients experience

Acknowledgements

This work was supported by “Norwegian Neuromuscular Disorders Foundation” provided by the “Neuromuscular Disorders Association, Norway”.

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