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Editor,—Heat shock proteins (HSPs) are highly conserved immunogenic intracellular molecules, and are induced by heat, inflammatory mediators, and physiological stress.1 The presence of antibodies to HSPs or heat shock cognate protein (HSC) has been reported in several autoimmune diseases.1 However, despite the prevalence of these antibodies in these autoimmune diseases, their significance is not fully understood.
In this report, we investigated antibodies to HSC71 specific antibody levels in sera from patients with Vogt–Koyanagi–Harada disease (VKH), a systemic disorder that affects various organs that contain melanocytes and is believed to be an autoimmune disease.2
Serum samples were obtained from eight patients with VKH, and from nine unaffected volunteers. All samples were obtained from VKH patients during the acute phase of the illness with severe uveitis and were taken before steroid administration. Mean ages were 43 and 40 for VKH and controls, respectively. Informed consent was obtained from all of the patients and volunteers. All sera were stored at −70°C until use.
Cloning and expression of the recombinant HSC71 (rHSC71) were performed as previously described.3 Anti-HSC71 antibody levels of sera were assayed by enzyme linked immunosorbent assay. In brief, flat bottomed microtitre plates were coated with 10 μg/ml rHSC71 in phosphate buffered saline (PBS). After incubation overnight, non-specific binding sites were blocked with PBS containing 0.05% Tween 20, 1 mM EDTA, 0.25% BSA, and 0.05% NaN3. Wells were incubated with serum diluted 1:200 for 2 hours at room temperature. The plates were then washed and incubated with alkaline phosphatase conjugated anti-human polyvalent immunoglobulins (Sigma BioSciences, St Louis, MO, USA) diluted 1:40 000 for 2 hours at room temperature. For the colour reaction, the washed wells were incubated with p-nitrophenyl phosphate substrate solution and analysed by measuring the optical density (OD) at 405 nm. Results were analysed using the two tailed Student'st test. A p value of 0.05 or less was considered significant. Levels of anti-HSC71 antibody were significantly raised in patients with VKH (p<0.00005) compared with healthy volunteers (Fig 1).
Molecular analysis of specificity of anti-HSC71 antibody of VKH patient serum was done by western blot analysis as described previously.3 The antiserum from VKH patients reacted strongly with rHSC71, while the sera from a toxoplasmic retinochoroiditis patient and a healthy volunteer did not (Fig 2).
In postnatal ocular maturation, the level of HSC70 mRNA expression was still high in the retina, which suggests some important role of HSC70 protein in structural or functional maintenance of this tissue.4 HSP70 proteins bound to the immunodominant epitope of interphotoreceptor retinoid binding protein indicates that they may play a part in the processing and presentation of antigens by antigen presenting cell.5
There are homologies between HSPs and unrelated self proteins as well as antibody cross reactivities.1 It has been proposed that HSP reactive T cells or antibodies serve as amplifiers of ongoing autoimmune destruction. Stressed cells at the sites of inflammation produce elevated HSP levels and thus become targets of HSP reactive T cells. Currently, it appears more likely that self HSP reactive T cells are physiological constituents of the immune system, and that their activities are regulated by numerous factors including cross reactive peptides which are constantly taken up by food and occasionally introduced through infection. Increased HSC71 antibody levels in patients with VKH indicate that HSC71 may play an important role in pathogenesis of VKH. Alternatively, as anti-HSC71 antibodies may merely be an epiphenomenon reflecting tissue damage, it would be necessary to know serum antibody levels in disease controls—that is, in patients who have similar inflammation which is not of an autoimmune nature (infective retinitis/choroiditis). According to our data of serum from a patient with toxoplasmic retinochoroiditis, this possibility was shown to be unlikely although a definitive conclusion will be reached by analysing a larger number of cases and various types of similar inflammatory diseases. Further investigation is needed to elucidate how this autoimmune response is related to the pathogenesis and pathophysiology of VKH.
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