A case of mistaken identity: HSPs are no DAMPs but DAMPERs HMGB-1 to promote inflammation and was taken as proof that RAGE binds a pro-inflammatory HMBG-1 (Luan et al. 2010). However, despite the fact that HMBG-1 may be taken as a prototypic DAMP, its role in directly promoting inflammation is also clouded in controversy (Bianchi 2007). In fact, the discussions here are similar to those concerning HSPs as HMBG-1 can also complex with other molecules such as LPS, and these associations are incrim- inated as the possible sources of the pro-inflammatory stimuli. Highly purified recombinant HMGB-1 was demon- strated to have a very weak direct pro-inflammatory activity at best (Rouhiainen et al. 2007). To quote another paper: “Thus, for full activity, HMGB1 may need to form a complex with another component to activate inflammation by enhancing effects stimulated by a PAMP, a DAMP, or a pro- inflammatory cytokine. Importantly, HMGB1 can bind avidly to immuno-stimulatory molecules such as LPS, DNA, or interleukin (IL)-1β and promote their activity in a synergistic fashion” (Pisetsky et al. 2008). As mentioned, no specific or unique receptors are known for HSPs. And the most credited TLR receptors are not strictly pro-inflammatory. Interestingly, TLR2 and TLR4, which are possibly the primary receptors for HSP60 and HSP70, were found not to be essential in the host response to DAMPs derived from necrotic cells (Chen and Nunez 2010). Most relevant in this discussion are the results of experiments in which DCs were cultured in the presence of HSPs. For self-HSP, there is a lack of compelling evidence that HSP can directly activate dendritic cells, which are expected to express the whole panoply of DAMP receptors. When exorbitant concentrations (200–300 μg/ml) of self- HSP70 (purified from mouse liver using standard protocols under sterile conditions and with endotoxin-free reagents) were applied, no stimulatory effects on murine DCs were observed (Wallin et al. 2002). As an aside, the same authors have seen responses to low concentrations (50–100 ng/ml) of the same liver-derived HSP70 contaminated with minute amounts of endotoxin. Given the role of dendritic cells as key initiators of the immune response and their ability to give direction to the ensuing adaptive response, one may wish to see full activation of DCs for a pro-inflammatory adaptive immune response reaction. In even more careful analyses, human HSP70 has shown a lack of direct human DC activation qualities (Bendz et al. 2008). Despite earlier claims (Floto et al. 2006), even mycobacterial HSP70 exhibited an absence of activation (no calcium signaling) of human DCs (Bendz et al. 2008). And possibly more telling, the group of Bonorino has shown that highly purified mycobacterial HSP70 impaired the maturation of murine bone marrow-derived DCs, leading to inhibition of T cell proliferation (Motta et al. 2007). Finally, HSPs seem to exert immune-dampening effects in model systems, as we will discuss in the following section (Table 2). 285 Table 2 Features of HSPs which disqualify them as DAMPs Absence of molecular patterns Main receptors TLR2 and TLR4 are not always pro-inflammatory TLR2 and TLR4 are not needed for signaling by DAMPs from necrotic cells Some HSP receptors are downregulating inflammatory signaling HSP-incubated DCs adopt a tolerogenic phenotype HSP immunizations are anti-inflammatory The immune-“dampening” activity of HSP Initial studies of the immunological effect of HSP were carried out with recombinant mycobacterial HSP60 in the rat model of adjuvant arthritis. HSP60 immunizations were found to inhibit disease development in this T cell-mediated autoimmune disease model (van Eden et al. 1988). This disease ameliorative effect was T cell mediated since transfer of HSP60-specific T cells into naïve recipients also imparted protection against arthritis in the recipients. By analyzing the exact antigen specificity of the protective T cells, it was discovered that disease suppression was mediated by T cells that responded to a particularly conserved HSP60 sequence. This sequence was so con- served that these T cells cross-recognized the homologous sequence present in the mammalian HSP60 molecule. Immunization with a synthetic peptide comprising this protective T cell epitope induced a fully protective T cell response against arthritis (Anderton et al. 1995). Cytokine analyses of the protective T cells demonstrated their capacity to produce suppressive cytokines such as IL-10 (Prakken et al. 2001). Since then, similar protective effects have been shown for microbial HSPs, including members of the HSP70 family, in various experimental models of autoimmunity (summarized in (van Eden et al. 2005). A recent study by Borges et al. further demonstrated the immunosuppressive potential of mycobacterial HSP70 in two allograft survival models, an allogeneic melanoma and a regular allogeneic skin graft. Very interestingly, rejection was inhibited by HSP70 incubation of the melanoma cells or the skin prior to grafting. Concurrent injection of an anti- CD25 antibody removed Treg cells and fully abolished the rejection inhibitory effect (Borges et al. 2010). Altogether, astonishing findings for a molecule derived from a microbe—mycobacterium—which is known to have such strong immune-potentiating effects! In some studies, immunizations with mammalian HSP have also been shown to inhibit disease. This was done in arthritis for HSP60 (Paul et al. 2000) and HSP70 (Chandawarkar et al. 2004; Corrigall et al. 2001; Wendling et al. 2000) and in diabetes with an HSP60-derived peptide (Bockova et al. 1997). In in vitro studies, Gp96 (glucose- regulated protein 94), a member of the HSP90 family of