The stress protein HSP70 is not only a vitally important chaperone molecule but its involvement has also been described with respect to several immunological phenomena. For example, it has been reported as a carrier of antigens to antigen-presenting cells (Lussow et al. 1991
; Nishikawa et al. 2007
), as pro-inflammatory mediator (Kono and Rock 2008
), or on the contrary, as an anti-inflammatory molecule that inhibits maturation and induces the secretion of IL-10 in DCs (Detanico et al. 2004
; Motta et al. 2007
). However, the idea of HSP70 as a pro-inflammatory mediator has been considered controversial since several groups have shown that bacterial products often present in HSP70 protein preparations were responsible for the detected pro-inflammatory phenotype (Bausinger et al. 2002
; Bendz et al. 2008
; Stocki et al. 2012
). Therefore, for the experiments performed here, HSP70 preparation with less than 2.1 EU LPS/mg was used. In addition, administration of Mt HSP70 has been shown to be protective in several experimental autoimmune disease models (Kingston et al. 1996
; Prakken et al. 2001
; van Eden et al. 2005
; Wieten et al. 2009
Here, we report that Mt- and mouse HSP70-treated BMDC can be functionally tolerogenic and can suppress PGIA. However, mouse HSP70-treated BMDC ameliorated disease to a much larger extent. Differences between mouse and Mt HSP70-treated BMDC are most probably due to differences in protein sequence of the two HSP70 species. Even though both molecules have a sequence conservation of about 50 %, the sequences that interact with the immune system could be very different. As illustrated by two papers of Wang et al., one HSP70 molecule may have stimulatory as well as inhibitory sequences. Stimulatory sequences induced maturation of DCs and the production of pro-inflammatory cytokines. In contrast, inhibitory sequences could inhibit DC maturation and cytokine production after stimulation (Wang et al. 2002
; Wang et al. 2005
). In our experiments, the balance between inhibitory and stimulatory sequences might tilt towards inhibitory sequences more clearly for mouse HSP70 as it does for Mt HSP70.
Since HSP70-treated BMDC had obtained a functionally tolerogenic capacity, we investigated the exact phenotype of Mt- and mouse HSP70-treated BMDC. Although no clear tolerogenic phenotype was induced, mouse HSP70-treated BMDC had obtained a semi-mature phenotype as indicated by the increased expression of CD86 and MHC class II. Furthermore, mouse and Mt HSP70 treatments induced the production of low amounts of IL-6. Both induction of a semi-mature phenotype and production of IL-6 have also been described as tolerogenic DC features (Frick et al. 2010
). Furthermore, levels of IL-12p70 were lower after HSP70 pretreatment in LPS-treated BMDC as compared to BMDC solely treated with LPS. These results indicate an inhibition of LPS-induced maturation and a stable tolerogenic phenotype of the HSP70-treated BMDC. Expression of the earlier-described tolerogenic DC markers IDO, ILT3 and GILZ (Cohen et al. 2006
; Manavalan et al. 2003
; Mellor and Munn 2004
) were not altered after HSP70 treatment. In addition, production of the anti-inflammatory cytokine IL-10 did not reach levels above detection limit after HSP70 treatment. The increased IL-6 production after HSP70 treatment was not caused by contaminants like endotoxins or nucleotides (Bendz et al. 2008
; Goth et al. 2006
; Takeuchi et al. 1999
). BMDC treatment with proteinase K-digested Mt- or mouse HSP70 in order to degrade the protein but not the possible contaminants, showed no increased IL-6 secretion compared to untreated BMDC. Furthermore, endotoxin levels of the Mt- and mouse batches were low and our preparation of Mt HSP70 did not trigger the NF-κB pathway in a NF-κB luciferase reporter construct transfected intestinal crypt epithelial m-ICcl2
cell-line that expresses TLR-2 or TLR-4 (data not shown) (Hornef et al. 2003
). By the group of Bonorino, two papers were published where they showed that Mt HSP70-treated DCs were less mature as shown by CD86 expression, and produced more IL-10 as compared to PBS-treated DCs (Detanico et al. 2004
; Motta et al. 2007
). They suggest that HSP70 binds a yet undefined endocytic receptor and hereby signal via TLR-2. This would eventually result in the production of IL-10 (Borges et al. 2012
). We did not observe the reduced CD86 expression or an induced production of IL-10, although our HSP70 treated BMDC were tolerogenic. However, our DCs were stimulated with a lower HSP70 dose and for a shorter period of time.
As HSP70 treatment induced a semi-mature phenotype in DCs, we investigated possible alterations in the antigen-presenting capacity of HSP70-treated BMDC in vivo. No increase or decrease of pOVA-specific CD4+
T cell proliferation was found. Also, no evidence for an altered expression of the regulatory T cell marker FoxP3 was found in HSP70 BMDC-treated mice when compared with untreated BMDC-treated mice. These results indicated that HSP70-treated BMDC did not induce antigen-specific T cell anergy or the induction of a classic FoxP3+
regulatory T cell phenotype. In contrast, HSP70-treated BMDC induced a small but significant increase of IL-6 secretion and a more pronounced and significant increase of the anti-inflammatory cytokine IL-10 in T cells. As it was earlier demonstrated that administration of Mt HSP70 or a conserved HSP70 sequence was protective in experimental arthritis models in an IL-10-producing T cell-dependent manner (Prakken et al. 2001
; Tanaka et al. 1999
; Wendling et al. 2000
; Wieten et al. 2009
), it is feasible that HSP70-treated BMDC-induced suppression of PGIA is mediated by IL-10-producing antigen-specific Tr1-like cells.
We recently showed that immunization with an Mt HSP70-derived peptide induced HSP70-specific regulatory T cells. These regulatory T cells suppressed PGIA when given prophylactically and decreased disease severity therapeutically in established PGIA (van Herwijnen et al. 2012
). Thus, another possibility for HSP70-treated BMDC to suppress PGIA is via the induction of HSP70-specific regulatory T cells. The BMDC in the PGIA experiments are pulsed with PG; however, HSP70-derived peptides will also be loaded onto MHC class II and possibly presented to HSP70-specific T cells. As suggested by a review of van Eden et al. (van Eden et al. 2005
), these regulatory HSP70-specific T cells can inhibit effector T cells via the secretion of IL-10.
In conclusion, this study shows that Mt- and mouse HSP70-treated BMDC can suppress PGIA; although mouse HSP70-treated BMDC suppressed PGIA to a greater extent. This could be an indication of a more stable tolerogenic phenotype in mouse HSP70-treated BMDC. Amelioration of disease was likely induced by the induction of regulatory T cells: either by IL-10-producing arthritis antigen-specific T cells or possibly via HSP70-specific regulatory T cells.