Autoantibodies directed against the protease inhibitor calpastatin in psoriasis

Abstract

Psoriasis is believed to be a T cell-mediated autoimmune disease, but also exhibits autoantibody production. Calpastatin is an endogenous inhibitor of calpain, a ubiquitous protease that regulates inflammatory processes. Anti-calpastatin autoantibody was first identified as an autoantibody specific to rheumatoid arthritis, but has been also detected in other autoimmune diseases. In this study, we examined the presence and levels of anti-calpastatin antibody in 77 psoriasis patients by enzyme-linked immunosorbent assay. Compared with normal controls, psoriasis patients exhibited significantly elevated IgG anti-calpastatin antibody levels that were similar to those found in rheumatoid arthritis patients. Remarkably, IgG anti-calpastatin autoantibody in sera from psoriasis patients inhibited calpastatin activity. Calpain II expression was up-regulated in psoriasis skin lesions compared with normal skin while calpastatin expression was normal. The results of this study reveal the presence of anti-calpastatin autoantibody in psoriasis.

Fig. 1

Anti-calpastatin antibody levels in patients with psoriasis. IgG or IgM anti-calpastatin antibody levels were determined by ELISA using human recombinant calpastatin domain I in patients with psoriasis vulgaris patients, those with psoriatic arthritis, those with generalized pustular psoriasis (GPP), those with RA and normal controls (CTL). The short bar indicates the mean value in each group. A broken line indicates the cut-off value (mean ± 2 s.d. of the control samples). Values in parenthesis represent the dilutions of pooled sera giving half-maximal OD values in ELISA, which were determined by linear regression analysis to generate arbitrary units per millilitre that could be directly compared between each group of patients and normal controls.

Fig. 2

Inhibition of calpastatin activity by IgG anti-calpastatin antibody from patients with psoriasis. IgG was purified from serum samples of psoriasis patients positive for IgG anti-calpastatin antibody by ELISA [anti-calpastatin antibody (+)], those negative for IgG anti-calpastatin antibody [anti-calpastatin antibody (–)], RA patients with IgG anti-calpastatin antibody and normal control (CTL). Purified IgG or control blocking monoclonal antibody to calpastatin was incubated with calpastatin and the calpastatin activity was assessed by the inhibition of calpain activity that can proteolyse its substrate, casein. Inhibition of calpastatin activity by isolated IgG is shown compared with that by control blocking anti-calpastatin monoclonal antibody that was defined as 100%. Each histogram shows the mean (± s.d.) results obtained for five patients in each group.

Fig. 3

Expression of calpain II and calpastatin mRNA in skin lesions of psoriasis. Relative mRNA amount of calpain II and calpastatin was assessed by real-time PCR using the ΔΔCt method in lesion skin of psoriasis and normal skin (CTL). Total RNA was isolated from frozen skin tissues, reverse transcribed into cDNA, and then amplified using primers specific for calpain II and calpastatin. One of the control samples was chosen as a calibrator sample. Each histogram shows the mean (± s.d.) results obtained for five patients in each group.

Fig. 4

Immunohistochemical expression of calpain II and calpastatin in skin lesions of psoriasis. Calpain II (a) and calpastatin (b) expression in normal skin and lesion skin of psoriasis was evaluated by immunohistochemistry using anti-calpain II and anti-calpastatin antibody. Sections were counterstained with methyl green. Magnification ×200.