Therapeutic effects of systemic administration of chaperone αB-crystallin associated with binding proinflammatory plasma proteins

Abstract

The therapeutic benefit of the small heat shock protein αB-crystallin (HspB5) in animal models of multiple sclerosis and ischemia is proposed to arise from its increased capacity to bind proinflammatory proteins at the elevated temperatures within inflammatory foci. By mass spectral analysis, a common set of ∼70 ligands was precipitated by HspB5 from plasma from patients with multiple sclerosis, rheumatoid arthritis, and amyloidosis and mice with experimental allergic encephalomyelitis. These proteins were distinguished from other precipitated molecules because they were enriched in the precipitate as compared with their plasma concentrations, and they exhibited temperature-dependent binding. More than half of these ligands were acute phase proteins or members of the complement or coagulation cascades. Consistent with this proposal, plasma levels of HspB5 were increased in patients with multiple sclerosis as compared with normal individuals. The combination of the thermal sensitivity of the HspB5 combined with the high local concentration of these ligands at the site of inflammation is proposed to explain the paradox of how a protein believed to exhibit nonspecific binding can bind with some relative apparent selectivity to proinflammatory proteins and thereby modulate inflammation.

FIGURE 1.

Exploration of therapeutic effects of HspB5. A and B, clinical scores of mice with active EAE treated with daily intraperitoneal injection of 50 μg of HspB5 for 10 days beginning at peak symptoms (A) and Th17-EAE induced via adoptive transfer of Th17 polarized T cells (B). The bar represents the duration of the treatment. Values in the graph represent mean ± S.E. #, p < 0.05 by Mann-Whitney U test. CD4+ T cells stimulated with and without anti-CD3/CD28 (C) or MOG_35–55 peptide (0, 5, 10, and 20 μg/ml; D) were treated with varying amounts of HspB5 (0–40 μg/ml). E, fluorescence profiles of the expression of activation markers CD80, CD83, and CD86 on the surface of A20 B cells after stimulation with LPS at 0.5 μg/ml alone (black line) or incubated with HspB5 at 50 μg/ml (shaded region) for 24 h. The gray line represents unstimulated A20 cells. F, the concentration of HspB5 in the plasma of MS patients (n = 9) and mice with active EAE (n = 13) and Th17 EAE (n = 4) is elevated as compared with healthy controls (Normal, n = 5) and wild type mice (Control, n = 5), respectively. Values in graph represent mean ± S.E. *, p < 0.03, **, p < 0.004, and ***, p < 0.016 by Mann-Whitney U test.

FIGURE 2.

Comparison of relative concentrations of proteins precipitated by either HspB5 or TCA from plasma from two multiple sclerosis patients and effect of incubation temperature on binding. A and C, the relative concentrations of the proteins precipitated by either HspB5 or TCA are compared with those whose relative concentration was enhanced (black bars) in the HspB5 precipitate as distinguished from those that were not (gray histograms). B and D, incubation with HspB5 at increasing temperatures results in the greater amount of some, but not all, proteins in the precipitate. The proteins that did not exhibit temperature dependence (*) correspond to highly expressed plasma proteins, such as albumin, serotransferrin, fibrinogen, and the immunoglobulins. Error bars correspond to S.D. between triplicate measurements of two different plasma samples (six separate measurements). Insulin-like grow fact bind prot, insulin-like growth factor-binding protein.

FIGURE 3.

Precipitation with HspB5 reduces plasma concentration of several acute phase proteins in MS and RA plasma. The amount precipitated with HspB5 from plasma from MS (A–H) and RA patients (I–P) at each temperature and the amount remaining in the supernatant are shown for C-reactive protein (A, B, I, and J), serum amyloid A (C, D, K, and L), serum amyloid P (E, F, M, and N), and apolipoprotein E (G, H, O, and P). In some cases, the protein was not identified in the mass spectral analysis of the HspB5 precipitate and was labeled B.D. for ”below detection.“ conc., concentration.

FIGURE 4.

Precipitation with HspB5 reduces plasma concentration of several acute phase proteins in plasma of mice with EAE. The relative increase in the amount of C-reactive protein (A), serum amyloid A (C), serum amyloid P (E), apolipoprotein E (G), and complement factor H (I) as a function of incubation temperature with HspB5 is compared with depletion of each of the proteins in the resultant sample of plasma (B, D, F, H, and J). conc., concentration.

FIGURE 5.

HspB5 administration reduces plasma concentration of IL-6. A, mice challenged with 50, 100, and 200 μg of LPS with co-administration of 200 μg of HspB5 at 0 and 3 h reduced the plasma levels of IL-6 as compared with mice without treatment after 6 h (n = 3–5 mice per group). B, mice challenged with 100 μg of LPS were treated with 0 (n = 20), 50 (n = 8), 100 (n = 15), 200 (n = 17), and 300 (n = 11) μg of HspB5 at 0 and 3 h and increasingly reduced the plasma concentration of IL-6 measured at 6 h. Results from 2–3 separate experiments were combined. Values in the graph represent mean ± S.E. *, p < 0.028, **, p < 0.0483, ***, p < 0.0001, significantly different from 0 μg of HspB5 by Student's t test.