Antibodies-Abzymes with Antioxidant Activities in Two Th and 2D2 Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology

Abstract

The exact mechanisms of multiple sclerosis development are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis) in Th and 2D2 mice is associated with the infringement of the differentiation profiles of bone marrow hematopoietic stem cells which are bound with the production of compounds that are harmful for human autoantibodies-abzymes that hydrolyze myelin oligodendrocyte glycoprotein, myelin basic protein, and DNA. It showed that autoimmune patients' antioxidant IgG antibodies oxidise some compounds due to their peroxidase (H₂O₂-dependent) and oxidoreductase (H₂O₂-independent) activities more effectively than those in healthy humans can. It was interesting to identify whether the redox activities of the antibodies change during the development of autoimmune diseases. Here, we analyzed the change in these redox activities of the IgGs from the blood of Th and 2D2 mice, which corresponded to different stages of the EAE development. The peroxidase activity in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in the Th (4-fold) and 2D2 (2-fold) mice IgGs, on average, is higher than the oxidoreductase activity is. The peroxidase activity of the Th (1.9-fold) and 2D2 (3.5-fold) mice IgGs remarkably increased during the 40 days of the spontaneous development of EAE. Forty days after the immunization of the MOG peroxidase activity, the IgGs of the Th and 2D2 mice increased 5.6-6.0 times when they were compared with those that presented no increase (3 months of age). The mice IgGs were oxidized with 3,3'-diaminobenzidine (2.4-4.3-fold) and o-phenylenediamine (139-143-fold) less efficiently than they were with ABTS. However, the temper of the change in the IgG activity in the oxidation of these substrates during the spontaneous and MOG-induced development of EAE was close to that which occurred for ABTS. All of the data show that the IgG peroxidase and oxidoreductase activities of EAE mice can play an important role in their protection from toxic compounds and oxidative stress.

Keywords: EAE model; Th and 2D2 mice; catalytic antibodies; peroxidase and oxidoreductase activities.

Figure 1

SDS-PAGE analysis of 14 µg IgGs electrophoretic homogeneity of four individual Abs (lanes 1–4) and equimolar mixtures of 30 preparations corresponding IgGs from Th (lane m1; Th-IgG_mix) and 2D2 (lane m2; 2D2-IgG_mix) mice using nonreducing 4–15% gradient gel (A). Lane C corresponds to a mixture of proteins with known molecular weights (A). In situ peroxidase activity analysis of m1 and m2 IgG_mix (B). After SDS-PAGE, two longitudinal strips of gel were incubated using special conditions for protein refolding. Then, the peroxidase activity was revealed by incubating gel slices for 10 h in the standard reaction mixture containing DAB and H₂O₂ until a coloured insoluble oxidation product was formed.

Figure 2

Typical examples of the time dependences of different product formations in the reaction of three substrates (ABTS, DAB, and OPD) due to their oxidation by IgG_mix preparations in the presence of H₂O₂. All of the designations are given in Panels (A–D).

Figure 3

Average changes over time in the relative activity in the oxidation of ABTS (A), DAB (B), and OPD (C) before and after Th mice immunization with MOG. Spontaneous development of EAE corresponds to untreated mice. All of the designations are marked in three panels. The mean values of k_cat corresponding to 10 mice of each group are presented.

Figure 4

Average changes over time in the relative activity in the oxidation of ABTS (A), DAB (B), and OPD (C) before and after 2D2 mice immunization with MOG. All of the designations are marked in three panels. The mean values of k_cat corresponding to 10 mice of each group are presented.

Figure 5

Relative values of k_cat characterizing the rate of ABTS oxidation by IgGs from the blood of Th and 2D2 mice in the absence of hydrogen peroxide (oxidoreductase activity) at the zero time of the experiment and 40 days after spontaneous development of EAE and immunization of mice with MOG. To estimate the k_cat values, equimolar mixtures of 10 IgG preparations corresponding to each of the groups were used.