Cell Stress and Chaperones (2012) 17:281–292 DOI 10.1007/s12192-011-0311-5 PERSPECTIVE AND REFLECTION ARTICLE A case of mistaken identity: HSPs are no DAMPs but DAMPERs Willem van Eden & Rachel Spiering & Femke Broere & Ruurd van der Zee Received: 9 November 2011 /Accepted: 15 November 2011 /Published online: 3 December 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Until recently, the immune system was seen What are DAMPs? solely as a defense system with its primary task being the elimination of unwanted microbial invaders. Currently, In the 1990s, Polly Matzinger introduced the concept of however, the functional significance of the immune system immunological “danger” as a further refinement of has obtained a much wider perspective, to include among Burnet’s original clonal selection theory which is the others the maintenance and restoration of homeostasis basis of self–nonself discrimination by the immune following tissue damage. In this latter aspect, there is a system (Fuchs and Matzinger 1996). In her “danger” growing interest in the identification of molecules involved, concept of immune function, she proposed immune such as the so-called danger or damage-associated molec- responses result from detecting and addressing danger ular patterns (DAMPs), also called alarmins. Since heat and not just nonself. In this context, the molecules shock proteins are archetypical molecules produced under associated with distress and damage (including self stressful conditions, such as tissue damage or inflammation, molecules) are regarded as the prime stimuli for immuno- they are frequently mentioned as prime examples of logical reactivity. DAMPs (Bianchi, J Leukoc Biol 81:1–5, 2007; Kono and Self-danger signals were postulated to originate from, for Rock, Nat Rev Immunol 8:279–289, 2008; Martin-Murphy instance, necrotic cells that succumb to infection or et al., Toxicol Lett 192:387–394, 2010). See for instance ischemia. Subsequently, several molecules were ascribed also a recent review (Chen and Nunez, Science 298:1395– “danger”-associated molecule (DAMPs) functions, such as 1401, 2010). Contrary to this description, we recently high-mobility group box-1 (HMGB1) protein, the most presented some of the arguments against a role of heat abundant member of the HMG family of DNA-binding shock protein as DAMPs (Broere et al., Nat Rev Immunol proteins. Other candidates include cytosolic calcium- 11:565-c1, 2011). With this perspective and reflection binding proteins of the S-100 family, nucleosomes, uric article, we hope to elaborate on this debate and provide acid, and antibacterial peptides (Harris and Raucci 2006). It additional thoughts to further ignite this discussion on this was logical and attractive to incorporate into this concept critical and evolving issue. families of heat shock proteins (HSPs) as they are upregulated during stressful cellular events such as in tissue Keywords DAMP. HSP. Treg . Inflammation . HSP70 . necrosis. HSPs have since often been categorized as Danger . Arthritis . Peptides . Autoimmunity prototypic “danger” associated molecular patterns or DAMPs. As a brief aside, the terminology in this field : : : W. van Eden (*) R. Spiering F. Broere R. van der Zee exemplifies the continuing maturation of the danger Department of Infectious Diseases and Immunology, concept since its original inception. The term DAMPs is Faculty of Veterinary Medicine, Utrecht University, given in analogy with the earlier coined PAMPs (pathogen- Yalelaan 1, 3584 CL, Utrecht, Netherlands associated molecular patterns), which were proposed to be e-mail: [email protected] structures recognized by so-called pattern recognition recep- 282 W. van Eden et al. tors. At this moment, however, DAMPs is more known as with gram-negative bacteria led to the production of high “damage”-associated molecular patterns. The latter was levels of antibodies specific for this common antigen proposed for the situation in clinical organ transplantation, (Thole et al. 1988). Since the mycobacterial HSP60 where the transplant does not reflect a danger but rather a molecule was cloned (Thole et al. 1985), Kaufmann and benefit to the recipient. The upregulated HSPs resulting from others have performed immunization studies with myco- reperfusion injury were therefore viewed as a typical DAMP bacteria and with recombinant mycobacterial HSP60 in the sense of “damage” and not danger-associated molecular demonstrating the resulting frequency of HSP60-specific pattern (Land 2005). T cells can be very high. After immunization with In the more narrow definition, DAMPs are intracellular Mycobacterium tuberculosis, approximately one out of five nonmicrobial molecules normally hidden from recognition of the M. tuberculosis-reactive T cells recognizes HSP60. by the immune system. They are released during necrotic Immunization with recombinant HSP60 in an adjuvant also and not apoptotic cell death, after which the innate immune stimulated an impressively high number of M. tuberculosis- system responds both directly with inflammation and reactive T cells (1 out of 2,000) (Kaufmann et al. 1987). indirectly by recruiting reinforcements from the adaptive Since then, many others have shown the dominant immuno- immune system (Kono and Rock 2008). Kono and Rock genicity of microbial HSPs (Zugel and Kaufmann 1999). have attempted to outline four criteria in terms of biological Given the conserved nature of HSPs, this high level of outcomes that must be fulfilled for a molecule to be immunogenicity is not immediately understood. One would classified as a DAMP (Kono and Rock 2008): predict a low degree of immunogenicity for molecules having extensive sequence similarities with self antigens. A 1. A DAMP should be active as a highly purified healthy immune system is not supposed to respond to self molecule antigens or their closely related homologs. Paradoxically, 2. The biological effect should not be owing to contam- when mapping T cell epitopes for mycobacterial HSP60 in ination with microbial molecules. Caution is particu- Lewis rats, defined epitopes included many conserved larly warranted if the putative DAMP is found to work sequences, and the responding T cells were found to cross- through receptors for PAMPs such as toll-like receptors respond to homologous self-peptide derived from mammalian (TLRs). HSP60 (Anderton et al. 1995). Since then, self-HSP cross- 3. The DAMP should be active at concentrations present reactive T cells have been identified in many models and in pathophysiological situations even in human cord blood (Durai et al. 2004;Pauletal. 4. Selective elimination or inactivation of the DAMP 2000). In children suffering from juvenile idiopathic arthritis, should ideally inhibit the biological activity of dead their strongest T cell responses to self-HSP60 were detected cells in in vitro or in vivo assays. in patients during remitting phases of their disease, sugges- In the case of HSPs, the first two criteria are already tive of an anti-inflammatory mechanism resulting from self- problematic. Given their chaperone nature, HSPs readily HSP-directed T cell reactivity (de Graeff-Meeder et al. 1995; engage other molecular structures, and experience has de Kleer et al. 2003; Prakken et al. 1996). taught us that recombinant HSPs are easily contaminated Furthermore, autoantibodies against HSP have been with microbial products when produced in microbial found to be prevalent in healthy subjects. For HSP60, this expression systems. Criterion 4, by the authors’ own was summarized by Quintana and Cohen (2011). Similar admission, is an ideal effect, and in most cases, this patterns of autoantibodies are observed for self-HSP70, criterion is not fulfilled. To put it bluntly, it seems for example in diabetes and atherosclerosis (Abulafia- questionable whether or not there exists any single DAMP Lapid et al. 2003). Interestingly, antigen microarray which meets all of these criteria. Our brief analysis of the analysis for multiple sclerosis (MS) patients revealed that current literature on DAMPs suggested that convincing HSP70 autoantibodies were a characteristic antibody evidence for single DAMP molecules initiating pro- signature of patients with a remitting and relapsing form inflammatory responses, as proven by the above criteria, of the disease. These antibodies were much less abundant may be lacking so far. in patients with progressive forms of MS, again associat- ing HSP70 immune reactivity with disease remission Microbial HSPs are immunodominant and induce (Quintana et al. 2008). cross-reactivity with self-HSP The immunodominance of HSPs may not depend One of the remarkable characteristics of HSPs is their on interactions with innate receptors immunodominance. HSP60 was already known as the “common antigen” of gram-negative bacteria before the There are no reports of peculiar pro-inflammatory isotype actual recognition of its molecular identity. Immunization (e.g., IgG2a) profiles for HSP-binding antibodies in patients A case of mistaken identity: HSPs are no DAMPs but DAMPERs 283 with autoimmune diseases. And as an aside, direct privileged antigens, so-called homunculus antigens. This polyclonal stimulation of mouse B cells with human would imply that the reactivity to such molecules is a HSP60 induced production of IgG3. These activated B genetically determined inherent quality of the immune cells were also found to stimulate a Th0 profile in T cells as system (Cohen 2007). Thus, there are more ways to explain opposed