Hsp40 proteins modulate humoral and cellular immune response in rheumatoid arthritis patients

Abstract

Recent research on the heat shock proteins (Hsps) in chronic inflammatory diseases indicates that Hsps may have disease-suppressive activities. Our aim was to characterize immune response directed to bacterial (DnaJ) and human Hsp40s in patients with rheumatoid arthritis (RA). We found elevated levels of anti-DnaJ, anti-Hdj2, and anti-Hdj3 (but not ant-Hdj1) serum antibodies in the RA patients (P < or = 0.001) compared to healthy controls. In peripheral blood mononuclear cells (PBMCs) culture, all tested Hsp40 proteins significantly inhibited the divisions of CD4+ and CD8+ T cells of the RA patients but not those of the controls. Both DnaJ and Hdj2 stimulated secretion of the main anti-inflammatory cytokine IL-10 by PBMCs of the RA patients (P < 0.05), and of IL-6 by PBMCs of the RA (P < 0.001) and control (P < 0.01) groups. DnaJ reduced TNFalpha secretion (P < 0.05) by both groups of PBMCs. Our results show for the first time that the RA patients have an increased humoral response to human Hsp40 proteins Hdj2 and Hdj3. This is also the first description of immunomodulatory effect of human Hsp40s on T cells and cytokine secretion in RA, suggesting that Hsp40s act as natural anti-inflammatory agents in RA.

Fig. 1

Schematic outline of the wild-type structure of E. coli DnaJ protein. Black lines below the E. coli DnaJ schematic protein represent the mutant proteins containing the N- and C-terminal domains of DnaJ and human Hdj2. Deleted amino acids are indicated in parentheses (Krzewski et al. , modified)

Fig. 2

The levels of anti-Hsp40 antibodies (IgG) in sera of rheumatoid arthritis (RA) patients (n = 48) and age-matched healthy controls (n = 50), assayed by ELISA test. The E. coli DnaJ (a) and human Hdj1 (b), Hdj2 (c) and Hdj3 (d) proteins or N-terminal J domains of DnaJ (N-DnaJ) (e) and Hdj2 (N-Hdj2) (f), and the C-terminal domains of DnaJ (C-DnaJ) (g) and Hdj2 (C-Hdj2) (h) were used as antigens. The results are presented as mean values (±SD). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 3

Proliferative responses of CD4 T (b) and CD8 T (c) cells from patients with RA (n = 22) and age-matched healthy controls (n = 15) in the presence of Hsp40 proteins: bacterial DnaJ and human Hdj1, Hdj2 and Hdj3. PBMCs were stained with CFSE, cultured for 72 h in the presence or absence of an antigen, labeled with anti-CD4+ or CD8+ antibodies and assayed by flow cytometry. An example of the flow cytometry result showing T CD4+ cells of a representative RA patient untreated with antigen or treated with DnaJ (a). M1 marks cells which have not divided while M2 marks cells which have divided at least once. A ratio of the M2 cells to the total cells (M1 + M2), expressed in %, was used for further analyses and is shown in the graphs b and c. Graphs present median values of the percentage of dividing cells; the bottom and the top of boxes represent the 75th and 25th percentiles, respectively, and the ends of the vertical lines show min–max. Responses of the antigen-treated T cells are compared to those of the antigen-untreated cells. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 4

Evaluation of IL-6, IL-10, TNFα, IL-2, IL-4, and IFNγ secretion by PBMCs from patients with RA and healthy control. The results are presented as mean values (±SD). The PBMCs’ responses to Hsp40 antigens are compared to those of the untreated cells, unless otherwise indicated. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001