BiP induces IL-4-dependent regulatory cells

Background

Previously we have described the stress protein BiP as a putative autoantigen in RA [1]. The administration of BiP intravenously (i.v.) prior to induction of collagen-induced arthritis (CIA) resulted in almost complete amelioration of disease [1]. Our studies now indicate that BiP has several immunomodulatory actions including the skewing of T-cell differentiation towards Th2 [2].

Aim

This study focused on the therapy of CIA and investigated subcutaneous administration of BiP in comparison with intravenous administration and whether adoptive transfer of BiP-treated cells could successfully inhibit the onset of disease. Finally, IL-4 knockout mice were used to determine the importance of the T cell in the therapeutic role of BiP

Methods

CIA was induced in DBA-1 mice by injection of bovine collagen type II (CII) in complete Freund's adjuvant followed by a booster injection in incomplete Freund's adjuvant at day 21. At the first appearance of swollen joints, the mice were injected subcutaneously (s.c.) with BiP, a control protein, BSA or vehicle control (PBS). Disease progression was followed by measurement of swollen joints. At termination, splenocytes and draining lymph node cells were removed and T-cell cytokine secretion was assessed. Mixed spleens and lymph nodes from groups of DBA-1 mice that had been immunized s.c. with BiP or BSA (200 μg), or i.v. with BiP or BSA (10 μg), were collected 12 days after immunization. Cell cultures (2.5 × 10⁶ cells/ml) were set up with 20 μg of the respective protein (BiP or BSA) for 5 days. Cells were then washed and injected intraperitoneally into DBA/1 mice (20 × 10⁶ cells/mouse) that had received the first CII immunization 24 days previously. DR1^+/+ IL-4 knockout mice, (n = 20) developed by backcrossing C57Bl/6 IL4^-/- mice to DR1^+/+ mice, were immunized with CII, and on day 24 after immunization were given 10 μg BiP i.v. Wild-type HLA-DR1^+/+ transgenic mice (n = 20/group) were administered 10 μg BiP or PBS i.v.

Results

Administration i.v. or s.c. of BiP significantly reduced (P < 0.05) the incidence and severity of CIA when given at the onset of disease. A lower incidence of arthritis was also recorded from groups of mice treated with BiP s.c. and i.v. (37.5% and 64%, respectively) as compared with 100% recorded from the control group by day 70. T cells removed from mice that had been treated with BiP via both routes were shown to secrete IL-4 in response to in vitro BiP stimulation. In response to CII, results indicated upregulation of IL-5 and IL-10 production from BiP-treated groups compared with the arthritic control group. In the adoptive transfer studies the mice receiving subcutaneous BiP-primed cells had a significant suppression of arthritis by day 48, and by day 66 in those receiving intravenous BiP-primed cells, compared with mice that had received BSA-primed T cells (P < 0.05). When the IL-4^-/- mice were scored for disease severity the IL-4^-/- mice treated with BiP were no different from wild-type mice treated with PBS whereas wild-type mice treated with BiP had a significant suppression of arthritis (P ≤ 0.05, Students test).

Conclusion

These findings suggest that treatment of CIA with BiP is mediated at least in part by induction of IL-4-dependent regulatory T cells.