The Role of Nuclear Macromolecules in Innate Immunity David S. Pisetsky1 1 Durham VA Medical Center and Duke University Medical Center, Durham, North Carolina Nuclear macromolecules, in addition to their intracellular role in THE IMMUNE PROPERTIES OF DNA IN SLE regulating cell function, can translocate into the extracellular space where they can activate innate immunity. This translocation can The recognition of DNA’s immune properties dates back half occur in various settings and reflects the dynamic nature of nuclear a century to its discovery as a target antigen in systemic lupus structure. Of nuclear molecules, DNA and the DNA-binding protein, erythematosus (SLE). This prototypic autoimmune disease is HMGB1, display distinct patterns of immune activity. For DNA, im- characterized by the production of antinuclear antibodies, with mune activity depends on sequence, base methylation, and context. antibodies to DNA (anti-DNA) serving as markers of diagnostic While bacterial DNA is an immune activator, mammalian DNA is and prognostic signiﬁcance. As shown in studies on patients with either inert or inhibitory when free. In contrast, mammalian DNA SLE as well as murine models, these antibodies can also mediate in the form of immune complexes can trigger immune cell activa- tissue injury, with DNA–anti-DNA immune complexes impor- tion. As shown in in vivo and in vitro studies, DNA can exit cells tant inducers of glomerulonephritis (1–3). during apoptotic as well as necrotic cell death in a process that As an antigen in SLE, DNA most likely functions in the form may depend on the presence of macrophages. Like DNA, HMGB1 of the nucleosome, a highly organized structure comprised of can exit cells and acquire immune properties. For HMGB1, the trans- DNA wrapped around a histone core. Consistent with the role location occurs in macrophages that have been stimulated by Toll- of nucleosomes as the driving antigen for autoantibody induc- like receptor (TLR) ligands as well as cytokines; HMGB1 release can tion, sera from patients with lupus contain antibodies to nucleo- also occur with apoptotic as well as necrotic death. While HMGB1 somes, the DNA and histone components, and other structures alone can display cytokine activity, it may also activate cells in con- comprised of DNA and histones. In this conceptualization, DNA junction with other immune stimulators such as TLR ligands. For behaves as an epitope or surface of a larger structure that impacts both DNA and HMGB1, the immune properties may therefore re- on the immune system; the use of the term DNA does not flect the array of other endogenous as well as exogenous molecules imply that this molecule is free in solution and devoid of other present. nucleosomal components (1, 3). Because of the importance of DNA as antigen in SLE, efforts Keywords: innate immunity; DNA; HMGB1; apoptosis; necrosis to replicate this disease in induced animal models focused pri- The nucleus is the central organelle in the cell because it is the marily on immunization with DNA. With a few possible excep- location for replication, transcription, and the regulation of gene tions, these experiments failed, with DNA, even if attached to expression. While often viewed as uniform in composition, the a carrier and presented in adjuvant, unable to induce an apprecia- nucleus is a dynamic structure with a regional anatomy that can ble autoantibody response. In its immunologic inertness, DNA appeared fundamentally different from proteins and carbohy- vary during the cell cycle. Furthermore, the nucleus can serve drates, the other major classes of macromolecule antigens (3). as a repository for molecules that transit into and out of the The failure of DNA to induce a speciﬁc autoimmune response cytoplasm and even exit the cell entirely. Remarkably, once in contrasts sharply with the situation of other autoimmune dis- the extracellular space, some of these nuclear molecules can eases, such as rheumatoid arthritis or multiple sclerosis, where serve as immune mediators and trigger the innate immune sys- animal disease models can be created in animals by immuniza- tem in settings of injury and death. As such, the translocation tion with self antigens such as collagen or myelin basic protein of nuclear macromolecules from the inside to the outside of the (4). Together, these considerations suggested that SLE reﬂects cell is a central event in innate immunity. a fundamental disturbance in the immune system that allows a Among endogenous molecules stimulating innate immunity, response to an essentially immunologic blank molecule. The two nuclear molecules have attracted the most attention. Thus, alternative explanation for the anti-DNA responses in SLE is both DNA and the DNA-binding protein, HMGB1, can exit cells the existence of a more immunogenic form of DNA that could during death processes and, in the extracellular space, stimulate induce responses under conditions in which experimental DNA– responses via a variety of receptors to signal danger. While both protein complexes fail. DNA and HMGB1 reside primarily in the nucleus and, indeed, may interact, they differ in intranuclear and intracellular mobility THE INDUCTION OF IMMUNE RESPONSES TO DNA as well as physical state during death processes. This review will therefore consider the roles of DNA and HMGB1 in innate The mystery of DNA’s immune activity in SLE is now clearing immunity and propose an integrated picture for their movement because of studies in two seemingly disparate areas that have and function during death processes. converged to provide a picture of the triggering of innate immu- nity by nuclear molecules. The ﬁrst area of this research concerns the immune properties of bacterial DNA or, as it is often called, CpG DNA. As shown in studies conducted over the last 20 years, bacterial DNA, unlike mammalian DNA, is immunologi- (Received in original form January 23, 2007; accepted in final form March 1, 2007 ) cally active and can induce cytokine production and B cell mito- Supported by VA Medical Research Service and the Lupus Research Institute. genesis. As demonstrated in elegant molecular studies, these Correspondence and requests for reprints should be addressed to David S. Pisetsky, responses result from stimulation of the TLR9 receptor which, M.D., Ph.D., Durham VA Medical Center, Box 151G, 508 Fulton St., Durham, in contrast to some other Toll-like receptors (TLRs), resides on NC 27705. E-mail: firstname.lastname@example.org the inside of cells as opposed to the membrane (5, 6). Proc Am Thorac Soc Vol 4. pp 258–262, 2007 DOI: 10.1513/pats.200701-027AW The induction of immune responses by bacterial DNA reﬂects Internet address: www.atsjournals.org structural microheterogeneity and its content of sequence motifs Pisetsky: Nuclear Molecules in Immunity 259 that center on unmethylated CpG dinucleotides. These se- regard, immune complexes with ribonucleoproteins can also quences occur much more commonly in bacterial than mamma- stimulate IFN- , perhaps related to their content of RNA mole- lian DNA because of differences in the patterns of base methyla- cules which can trigger TLR3 or TLR7 (14). tion as well as a phenomenon known as CpG suppression. The activity of DNA in immune complexes contrasts with Because of the differential display of the CpG motifs, bacterial the activity of free mammalian DNA which is either inert or DNA, like LPS, can act as a pathogen-associated molecular inhibitory. As shown in vitro studies, free mammalian DNA can pattern (PAMP). Importantly, in the studies on the structure– inhibit the activity of bacterial DNA, raising the possibility that function relationships of immune stimulation by DNA, intact high concentrations of extracellular DNA can attenuate stimula- mammalian DNA has been consistently inactive in in vitro or tion by bacterial DNA and possibly down-regulate innate immu- in vivo systems. These observations point to the exquisite speci- nity. This inhibitory activity can be termed “safety” in contrast ﬁcity for base recognition in the triggering of innate immunity to “danger” elicited by TLR ligands (17). Thus, as these experi- via TLR9. ments indicate, the formation of immune complexes can radically The demonstration of the intrinsic immune activity of bacte- transform the activity of mammalian DNA to allow stimulation, rial DNA is notable since it joins DNA with proteins and carbo- rather than inhibition, of innate immunity. hydrates in the family of immune active macromolecules. Studies Since even high concentrations of mammalian DNA are inac- on the antibody response to mammalian and bacterial DNA tive, it appears unlikely that the role of the complex is solely to in humans conﬁrmed and extended this role. As this serologic deliver DNA to TLR9. Even if mammalian DNA contains some analysis showed, the sera of normal human subjects (NHS) have CpG motifs, the presence of nonstimulatory or inhibitory motifs signiﬁcant levels of antibody directed to bacterial DNA. These appears predominant. An interaction of active motifs within antibodies bind with high avidity and speciﬁcity to some, but not mammalian DNA may nevertheless cause activation of TLR9, all, bacterial DNA antigens. While the antibodies in NHS differ although the triggering of other internal receptors appears more in isotype from those in SLE, their presence nevertheless sug- plausible. The operation of such receptors can be inferred from gests that, during the ordinary encounter with bacteria, bacterial the activity of mammalian DNA in complexes with transfection DNA triggers responses and can drive responses to sequential reagents known as cytofectins (18). These reagents promote DNA epitopes (7–9). DNA internalization and, while the mode of action of cytofectins Together, these observations raise the possibility that bacte- and immune complexes may differ, the potency of DNA in rial DNA can initiate or sustain anti-DNA production in SLE cytofectin complexes provides further evidence that the context because of its intrinsic immunologic activity as well as display of DNA determines its activity on innate immunity (18–20). of the double-stranded B DNA antigen conformation. According The immunostimulatory activity of DNA-containing immune to this model, the lesion in SLE would reﬂect antigen recognition, complexes has attracted great interest as a mechanism for pro- not responsiveness, with abnormalities in the immune repertoire moting nonspeciﬁc immune activation in the pathogenesis of in SLE providing an array of B cell precursors that can be SLE. While such complexes can sustain or intensify immune triggered by bacterial DNA antigen to produce autoantibodies activation, they do not explain the initial generation of the anti- (10). This possibility is supported by studies showing that bacte- DNA antibody which is critical to the activation by DNA. Fur- rial DNA can induce an autoantibody response by immunization thermore, the necessity for complex formation suggests that for of pre-autoimmune NZB/NZW mice under conditions in which endogenous DNA to activate innate immunity, it must exist in mammalian DNA is inactive (11). While supporting the ability association with another moiety or structure to allow access to of bacterial DNA to stimulate responses, these studies attest to internal DNA receptors. These considerations (Table 1) thus the paucity of immunologic activity in mammalian DNA. focus attention on the mechanisms by which DNA is released from cells and the molecules to which it is attached during this THE ACTIVITY OF IMMUNE COMPLEXES process. The second line of research on the immune activity DNA devel- oped originally in efforts to characterize a factor in the sera of RELEASE OF DNA FROM DEAD AND DYING CELLS patients with lupus that could stimulate the in vitro production As shown using a variety of assays, DNA appears at high levels of interferon- (IFN- ). As shown in seminal experiments, this in the blood in many conditions including SLE, pulmonary in- factor is an immune complex comprised of DNA and anti-DNA. farction, malignancy, and trauma among others. These condi- While either component alone is devoid of activity, the complex tions are all associated with cell activation or cell death, although can potently stimulate IFN- production by plasmacytoid den- death has been considered the most likely source of blood DNA dritic cells. Furthermore, this stimulatory activity can be mim- (21, 22). As now conceptualized, cell death can be divided into icked by mixing antibody preparations with supernatants of apo- mechanistically distinct processes, with apoptosis and necrosis ptotic cells. Studies on the mechanisms of B cell activation in a representing the prototype pathways. Apoptosis, or programmed rheumatoid factor transgenic system in the mouse also showed cell death, is a highly regulated process characterized by cellular that immune complexes with DNA have activity not present collapse and cleavage of nuclear molecules. In contrast, necrosis with DNA alone (12–14). Subsequent studies on the stimulation by plasmacytoid DCs by DNA immune complexes established a role for both Fc recep- tors and TLR9 receptor; non–TLR receptor mechanisms may TABLE 1. ROLE OF DNA IN INNATE IMMUNITY also operate, however. Since TLR9 resides on the inside of the References cells in an endosomal compartment, immune complexes may allow DNA internalization, where interaction with TLR9 may Stimulation of B cells, macrophages, and dendritic cells 3, 5 occur. The interaction with the Fc receptor could lead to addi- Vary with sequence, base methylation, and backbone structure 3, 5 tional signaling or provide an alternative mechanism for DNA Vary with context and mode of cell entry 18 Stimulation of TLR and non-TLR receptors 19, 20 entry into the cell (15, 16). While the identity of the DNA in Induction of specific autoantibodies in normal humans 3, 7–10 immune complexes from sera has not been characterized exten- sively, endogenous human DNA is the likely source. In this Definition of abbreviations: TLR Toll-like receptor. 260 PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 4 2007 is a random process, provoked by physical or chemical trauma immune system, the distinction between apoptosis and necrosis that culminates in cell lysis (23). According to current paradigms, may not be as stark as sometimes portrayed. Thus, in the intact necrosis is pro-inﬂammatory while apoptosis is anti-inﬂamma- animal or in mixed cell cultures, both apoptotic and necrotic tory, perhaps related to release or spillage of internal molecules. cells can release DNA that is similar in properties as measured To investigate nuclear molecule dynamics during death, our by size distribution. These considerations do not imply that the laboratory has used in vitro and in vivo system to assess the extracellular DNA from apoptotic and necrotic cells are equiva- amount and properties of extracellular DNA released from dying lent immunologically, since the immune activity of DNA de- cells. In addition, we have investigated the inﬂuence of macro- pends on context and the presence of other molecules that may phages on the release of DNA, since macrophages can scavenge affect its intracellular trafﬁcking. It is important therefore to dead cells by phagocytosis and therefore potentially modify the consider other molecules that may be released in death settings. amount of DNA arising from such cells. In these experiments, we have measured DNA by both an ELISA for nucleosomes as THE IMMUNE ACTIVITY OF HMGB1 well as a direct chemical measurement using the dye PicoGreen. This dye binds speciﬁcally to double-stranded DNA and allows HMGB1 is a nonhistone nuclear protein that has also been sensitive detection by a ﬂuorometric assay. implicated in the stimulation of innate immunity during death Results of these studies have provided a novel perspective processes. Structurally, HMGB1 is 214 amino acids long and can on DNA release during cell death as well as highlighted differ- be divided into an A box, B box, and a C-terminal tail domain, ences in the in vitro and in vivo settings. Thus, during in vitro with the A and B boxes responsible for DNA interaction. This culture, Jurkat leukemia T cells made apoptotic by chemical protein can bind DNA in a non–sequence-speciﬁc manner to agents release DNA in a time-dependent process. This extracel- bend DNA, although it binds preferentially to distorted DNA lular DNA shows laddering by gel electrophoresis, indicating structures such as strand junctions. In addition to binding DNA, cleavage into nucleosomal fragments. In contrast to cells under- HMGB1 can interact with nuclear proteins. While the precise going apoptosis, cells made necrotic by treatment with heat or function of HMGB1 is not known, this protein can regulate ethanol fail to release DNA even after prolonged culture and transcription, perhaps by altering the architecture of DNA to despite changes in cellular permeability, as demonstrated by promote interactions with other factors. Depending on cell type, propidium iodide staining (24). HMGB1 can also appear in the cytoplasm (29, 30). As our experiments showed, in the in vitro setting, macro- Like DNA, HMGB1 can leave the cell where, in the extracel- phages can display important but divergent effects on DNA lular space, it can serve as an alarmin and promote immune release. With apoptotic cells, the presence of macrophages (ei- activation. An alarmin is an intracellular molecule that, when ther the RAW264.7 cell line or murine bone marrow–derived released from cells during death processes, can stimulate innate macrophages) can reduce the amount of DNA released, whereas immune processes (31). In another terminology, an alarmin is macrophages can increase the amount of DNA released by ne- a DAMP or damage (or death)-associated molecular pattern. crotic cells. For necrotic cells co-cultured with macrophages, the In the case of HMGB1, the release processes was ﬁrst detected DNA present in the medium showed laddering suggestive of in the setting of macrophage activation rather than death. In nuclease digestion. While indicating the importance of macro- these experiments, supernatants of cultures stimulated with LPS phages in mediating DNA release, these ﬁndings also showed were characterized to identify other molecules that could medi- that the size of the DNA in the extracellular space is not itself ate septic shock and therefore represent novel targets of therapy. a measure of whether death occurs by apoptosis or necrosis (25). As these experiments showed, HMGB1 appears in high concen- In vivo systems can also help dissect the DNA release process. trations in the medium of macrophages activated by LPS as well Thus, in normal mice, the intraperitoneal administration of apo- as pro-inﬂammatory mediators such as TNF- , IFN- , and nitric ptotic or necrotic Jurkat cells leads to a prompt rise in the amount oxide. Furthermore, HMGB1 levels are high in the serum of of DNA in the blood that returns to baseline after 24 h. With patients with shock and antibodies to HMGB1 can block shock both types of dying cells, DNA shows laddering with a similar in mice (32). A series of insightful experiments established a size distribution by gel electrophoresis. This result is similar to broad range of immunostimulatory activities of HMGB1, consis- that obtained in vitro using mixed cultures of Jurkat cells and tent with its role as a late mediator of LPS and its function as macrophages. Other studies with this model showed that the a cytokine. This stimulation appears dependent on the RAGE extent of DNA release from transferred dead cells can be modi- receptor, although activation of TLR2 and TLR4 may also con- ﬁed by treatment of recipient mice with clodronate (which elimi- tribute to stimulation (33–37). nates macrophages), dexamethasone, or the induction of perito- As shown subsequently, death processes, in addition to mac- neal exudates (26–28). rophage activation, can lead to HMGB1 release, with this protein Together, these studies provide evidence that macromolecule posited as an important mediator of inﬂammation induced by release from dead cells is a modiﬁable process that may depend necrotic cells. As shown in vitro, cells induced to undergo necro- on the presence of more than one cell population, especially sis by freeze-thawing release large concentrations of HMGB1. macrophages (Table 2). Furthermore, they suggest that, to the This release process differs from that occurring during activation, extent that extracellular DNA can be a player in the innate where acetylation and phosphorylation alter the charge of HMGB1. These modiﬁcations affect the intracellular trafﬁcking of HMGB1, with the modiﬁed molecule transiting to the cyto- TABLE 2. ORIGIN AND PROPERTIES OF EXTRACELLULAR DNA plasm into vesicles for secretion (38, 39). In contrast, for HMGB1 release during necrosis, the process appears passive and results References from the diffusion of this protein away from chromatin and out Increased blood levels in settings of cell death 21 of the cell. Reﬂecting its nuclear function, HMGB1 is only weakly DNA laddering 21, 22 adherent to chromatin, differing markedly from histones in the In vitro release by apoptotic cells 24 strength of its DNA interaction. Thus, unanchored to the nu- In vivo release with apoptosis and necrosis 26 cleus, HMGB1 can leave readily when the permeability barriers Role of macrophages 25, 27 break down during death (38). Pisetsky: Nuclear Molecules in Immunity 261 While the release of HMGB1 during necrosis is consistent HMGB1 can enhance DNA transfection, suggesting its ability with its biophysical properties, its behavior during apoptosis has to shuttle nucleic acid into cells (43). been more uncertain. Studies by Scafﬁdi and coworkers indicated Together, these considerations suggest that immune activity that apoptotic cells do not release HMGB1, with studies by FLIP of nuclear molecules may result from the ensemble of species (ﬂuorescence loss of photobleaching) in fact demonstrating that present, their physical interaction, and their ability to trigger its nuclear diffusion decreases dramatically during apoptosis, more than one receptor type simultaneously. Whether DNA implying greater adherence to chromatin (40). Since apoptotic promotes HMGB1 stimulation or vice versa is a matter of seman- cells are generally considered anti-inﬂammatory, a retention of tics, as both components may have to be present simultaneously HMGB1 in the nucleus could limit immune activation during in a complex to induce cell activation. The rules by which com- this death process. These results are notable since they suggest plexes stimulate immunity are not known, although stimulation that, while apoptotic cells release DNA, they do not release a by organelle fragments or particulates may be more analogous companion DNA-binding protein (i.e., HMGB1). to a cell–cell interaction (where multiple molecular interactions To resolve this seeming contradiction, we reevaluated occur) than the stimulation by a cytokine, a hormone, or other HMGB1 release during death processes, using Jurkat cells as small molecular mediator (e.g., prostanoid), which may act in a models. In these experiments, apoptosis was induced by chemical unitary manner. agents and HMGB1 release measured by Western blotting. Con- While these issues require much further investigation, the focal microscopy with staining with anti-HMGB1 was used to studies on DNA and HMGB1 nevertheless indicate the diversity conﬁrm translocation. Together, these experiments showed of intracellular molecules with immune activity and the impor- clearly that, in apoptosis as well as necrosis, translocation of tance of context in the stimulation of innate immunity by nuclear HMGB1 can occur, with the nucleus of cells showing dramati- macromolecules. Future studies will track the dynamics of nu- cally reduced HMGB1 content in association with nuclear con- clear molecule trafﬁcking during death processes, the role of densation (41). macrophages in determining the extracellular release, and the Since DNA release occurs during apoptosis, HMGB1 release signaling pathways stimulated by the complex mixtures of large is not unexpected and suggests that permeability changes of molecules. Hopefully, this research will elucidate the pathogene- nuclear and cytoplasmic membranes allow the diffusion of a sis of SLE and other autoimmune diseases as well as provide variety of macromolecules into the extracellular space. 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