Background

The role of B-cells in the pathogenesis of myocarditis is not yet well understood. Their role is mainly seen in their ability to co-stimulate T-cells and to produce high-affinity pathogenic auto-antibodies (Ab). B-cells produce cytokines in high amounts and possess the ability to actively interfere with the immune system. The success of B-cell-depleting therapies in rheumatic diseases proves their pathogenic role. Our work with human B-cells, suggested that interaction with T-cells is essential for their regulatory effect. No single marker or phenotype exists for regulatory B-cells. This suggests that they belong to different subsets which are generated during immune response and induce immune tolerance via different mechanisms. The aim of this study was to investigate the role of B-cells during the development of EAM.

Methods

Spleen cells from A/J mice with or without EAM were cocultivated, stimulated with TnI and the T-cells, B-cells or a combination were transferred to Irradiated animals (Scheme1). Furthermore, 5-6 weeks old wild type (muMT^+/+) and B-cell deficient (muMT^-/-) A/J mice were treated with CD20Ab in order to deplete B-cells. For controls untreated mice and IgG Ab treated mice were used. Mice were immunized three times every week with TnI peptide (TnI) or control to induce EAM. Histopathology was used to determine the degree of inflammation via haematoxylin eosin (HE) staining. To investigate cardiac damage, hsTnT-level as well as Auto-antibody titer against TnI peptide was measured in serum.

Scheme1 Experimental setup of cell transfer.

Results

Combination of stimulated T- and B-cells from animals with EAM showed increased inflammation as well as antibody formation against TnI peptide (Ø=24560±38888, p=0.0002) in B-cell deficient mice in contrast to T- as well as B-cells which alone did not cause inflammation (Fig.1A/B). Depletion of B-cells lead to lower hsTnT levels, muMT^-/- mice treated with TnI (Ø=3.838±5.111) vs. muMT^+/+ mice (Ø=356.7±406.6, p=0.0282, Fig.1A). The inflammation was higher in muMT^+/+ (Ø=33.58±21.93) than muMT^-/- animals (Ø=6.250±6.944, p<0.0001, Fig.1B). Mice without B-cell depletion (muMT^+/+, IgG Ctrl.) showed higher myocardial inflammation (Ø=26.50±25.96, Fig.2B). Ab formation against TnI was also increased in these animals (Fig.2D).

Fig.1: Results of A/J mice after cell transfer from donor mice with EAM. A) Histopathology of hearts via HE staining B) Ab titer against TnI

Fig.2 Results of muMT^-/-/^+/+ mice immunized with TnI or Ctrl. as well as untreated or treated with CD20Ab, IgG Ctrl. A) hsTnT level in serum B-C) Histopathology of hearts via HE staining D) Ab titer against TnI

Conclusion

Transfer of cells of mice with EAM can cause inflammation in B-cell deficient animals. The combination of T- and B-cells showed the greatest effect, whereas T-cells also led to inflammation and B-cells did not. Genetic depletion of B-cells leads to decreased hsTnT level, as well as less infiltration of immune cells into the myocardium after TnI immunization and lower auto-Ab formation against TnI. This indicates a proinflammatory effect of B-cells during progression of EAM. Further studies should elucidate possible therapeutic approaches of these results.