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
regulating cell function, can translocate into the extracellular space
where they can activate innate immunity. This translocation can
occur in various settings and reflects the dynamic nature of nuclear
structure. Of nuclear molecules, DNA and the DNA-binding protein,
HMGB1, display distinct patterns of immune activity. For DNA, im-
mune activity depends on sequence, base methylation, and context.
While bacterial DNA is an immune activator, mammalian DNA is
either inert or inhibitory when free. In contrast, mammalian DNA
in the form of immune complexes can trigger immune cell activa-
tion. As shown in in vivo and in vitro studies, DNA can exit cells
during apoptotic as well as necrotic cell death in a process that
may depend on the presence of macrophages. Like DNA, HMGB1
can exit cells and acquire immune properties. For HMGB1, the trans-
location occurs in macrophages that have been stimulated by Toll-
like receptor (TLR) ligands as well as cytokines; HMGB1 release can
also occur with apoptotic as well as necrotic death. While HMGB1
alone can display cytokine activity, it may also activate cells in con-
junction with other immune stimulators such as TLR ligands. For
both DNA and HMGB1, the immune properties may therefore re-
flect the array of other endogenous as well as exogenous molecules
present.
Keywords: innate immunity; DNA; HMGB1; apoptosis; necrosis
The nucleus is the central organelle in the cell because it is the
location for replication, transcription, and the regulation of gene
expression. While often viewed as uniform in composition, the
nucleus is a dynamic structure with a regional anatomy that can
vary during the cell cycle. Furthermore, the nucleus can serve
as a repository for molecules that transit into and out of the
cytoplasm and even exit the cell entirely. Remarkably, once in
the extracellular space, some of these nuclear molecules can
serve as immune mediators and trigger the innate immune sys-
tem in settings of injury and death. As such, the translocation
of nuclear macromolecules from the inside to the outside of the
cell is a central event in innate immunity.
Among endogenous molecules stimulating innate immunity,
two nuclear molecules have attracted the most attention. Thus,
both DNA and the DNA-binding protein, HMGB1, can exit cells
during death processes and, in the extracellular space, stimulate
responses via a variety of receptors to signal danger. While both
DNA and HMGB1 reside primarily in the nucleus and, indeed,
may interact, they differ in intranuclear and intracellular mobility
as well as physical state during death processes. This review will
therefore consider the roles of DNA and HMGB1 in innate
immunity and propose an integrated picture for their movement
and function during death processes.
(Received in original form January 23, 2007; accepted in final form March 1, 2007 )
Supported by VA Medical Research Service and the Lupus Research Institute.
Correspondence and requests for reprints should be addressed to David S. Pisetsky,
M.D., Ph.D., Durham VA Medical Center, Box 151G, 508 Fulton St., Durham,
NC 27705. E-mail: dpiset@acpub.duke.edu
Proc Am Thorac Soc Vol 4. pp 258–262, 2007
DOI: 10.1513/pats.200701-027AW
Internet address: www.atsjournals.org
THE IMMUNE PROPERTIES OF DNA IN SLE
The recognition of DNA’s immune properties dates back half
a century to its discovery as a target antigen in systemic lupus
erythematosus (SLE). This prototypic autoimmune disease is
characterized by the production of antinuclear antibodies, with
antibodies to DNA (anti-DNA) serving as markers of diagnostic
and prognostic significance. As shown in studies on patients with
SLE as well as murine models, these antibodies can also mediate
tissue injury, with DNA–anti-DNA immune complexes impor-
tant inducers of glomerulonephritis (1–3).
As an antigen in SLE, DNA most likely functions in the form
of the nucleosome, a highly organized structure comprised of
DNA wrapped around a histone core. Consistent with the role
of nucleosomes as the driving antigen for autoantibody induc-
tion, sera from patients with lupus contain antibodies to nucleo-
somes, the DNA and histone components, and other structures
comprised of DNA and histones. In this conceptualization, DNA
behaves as an epitope or surface of a larger structure that impacts
on the immune system; the use of the term DNA does not
imply that this molecule is free in solution and devoid of other
nucleosomal components (1, 3).
Because of the importance of DNA as antigen in SLE, efforts
to replicate this disease in induced animal models focused pri-
marily on immunization with DNA. With a few possible excep-
tions, these experiments failed, with DNA, even if attached to
a carrier and presented in adjuvant, unable to induce an apprecia-
ble autoantibody response. In its immunologic inertness, DNA
appeared fundamentally different from proteins and carbohy-
drates, the other major classes of macromolecule antigens (3).
The failure of DNA to induce a specific autoimmune response
contrasts sharply with the situation of other autoimmune dis-
eases, such as rheumatoid arthritis or multiple sclerosis, where
animal disease models can be created in animals by immuniza-
tion with self antigens such as collagen or myelin basic protein
(4). Together, these considerations suggested that SLE reflects
a fundamental disturbance in the immune system that allows a
response to an essentially immunologic blank molecule. The
alternative explanation for the anti-DNA responses in SLE is
the existence of a more immunogenic form of DNA that could
induce responses under conditions in which experimental DNA–
protein complexes fail.
THE INDUCTION OF IMMUNE RESPONSES TO DNA
The mystery of DNA’s immune activity in SLE is now clearing
because of studies in two seemingly disparate areas that have
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-
cally active and can induce cytokine production and B cell mito-
genesis. As demonstrated in elegant molecular studies, these
responses result from stimulation of the TLR9 receptor which,
in contrast to some other Toll-like receptors (TLRs), resides on
the inside of cells as opposed to the membrane (5, 6).
The induction of immune responses by bacterial DNA reflects
structural microheterogeneity and its content of sequence motifs
Pisetsky: Nuclear Molecules in Immunity
that center on unmethylated CpG dinucleotides. These se-
quences occur much more commonly in bacterial than mamma-
lian DNA because of differences in the patterns of base methyla-
tion as well as a phenomenon known as CpG suppression.
Because of the differential display of the CpG motifs, bacterial
DNA, like LPS, can act as a pathogen-associated molecular
pattern (PAMP). Importantly, in the studies on the structure–
function relationships of immune stimulation by DNA, intact
mammalian DNA has been consistently inactive in in vitro or
in vivo systems. These observations point to the exquisite speci-
ficity for base recognition in the triggering of innate immunity
via TLR9.
The demonstration of the intrinsic immune activity of bacte-
rial DNA is notable since it joins DNA with proteins and carbo-
hydrates in the family of immune active macromolecules. Studies
on the antibody response to mammalian and bacterial DNA
in humans confirmed and extended this role. As this serologic
analysis showed, the sera of normal human subjects (NHS) have
significant levels of antibody directed to bacterial DNA. These
antibodies bind with high avidity and specificity to some, but not
all, bacterial DNA antigens. While the antibodies in NHS differ
in isotype from those in SLE, their presence nevertheless sug-
gests that, during the ordinary encounter with bacteria, bacterial
DNA triggers responses and can drive responses to sequential
DNA epitopes (7–9).
Together, these observations raise the possibility that bacte-
rial DNA can initiate or sustain anti-DNA production in SLE
because of its intrinsic immunologic activity as well as display
of the double-stranded B DNA antigen conformation. According
to this model, the lesion in SLE would reflect antigen recognition,
not responsiveness, with abnormalities in the immune repertoire
in SLE providing an array of B cell precursors that can be
triggered by bacterial DNA antigen to produce autoantibodies
(10). This possibility is supported by studies showing that bacte-
rial DNA can induce an autoantibody response by immunization
of pre-autoimmune NZB/NZW mice under conditions in which
mammalian DNA is inactive (11). While supporting the ability
of bacterial DNA to stimulate responses, these studies attest to
the paucity of immunologic activity in mammalian DNA.
THE ACTIVITY OF IMMUNE COMPLEXES
The second line of research on the immune activity DNA devel-
oped originally in efforts to characterize a factor in the sera of
patients with lupus that could stimulate the in vitro production
of interferon-␣ (IFN-␣). As shown in seminal experiments, this
factor is an immune complex comprised of DNA and anti-DNA.
While either component alone is devoid of activity, the complex
can potently stimulate IFN-␣ production by plasmacytoid den-
dritic cells. Furthermore, this stimulatory activity can be mim-
icked by mixing antibody preparations with supernatants of apo-
ptotic cells. Studies on the mechanisms of B cell activation in a
rheumatoid factor transgenic system in the mouse also showed
that immune complexes with DNA have activity not present
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
also operate, however. Since TLR9 resides on the inside of the
cells in an endosomal compartment, immune complexes may
allow DNA internalization, where interaction with TLR9 may
occur. The interaction with the Fc receptor could lead to addi-
tional signaling or provide an alternative mechanism for DNA
entry into the cell (15, 16). While the identity of the DNA in
immune complexes from sera has not been characterized exten-
sively, endogenous human DNA is the likely source. In this
259
regard, immune complexes with ribonucleoproteins can also
stimulate IFN-␣, perhaps related to their content of RNA mole-
cules which can trigger TLR3 or TLR7 (14).
The activity of DNA in immune complexes contrasts with
the activity of free mammalian DNA which is either inert or
inhibitory. As shown in vitro studies, free mammalian DNA can
inhibit the activity of bacterial DNA, raising the possibility that
high concentrations of extracellular DNA can attenuate stimula-
tion by bacterial DNA and possibly down-regulate innate immu-
nity. This inhibitory activity can be termed “safety” in contrast
to “danger” elicited by TLR ligands (17). Thus, as these experi-
ments indicate, the formation of immune complexes can radically
transform the activity of mammalian DNA to allow stimulation,
rather than inhibition, of innate immunity.
Since even high concentrations of mammalian DNA are inac-
tive, it appears unlikely that the role of the complex is solely to
deliver DNA to TLR9. Even if mammalian DNA contains some
CpG motifs, the presence of nonstimulatory or inhibitory motifs
appears predominant. An interaction of active motifs within
mammalian DNA may nevertheless cause activation of TLR9,
although the triggering of other internal receptors appears more
plausible. The operation of such receptors can be inferred from
the activity of mammalian DNA in complexes with transfection
reagents known as cytofectins (18). These reagents promote
DNA internalization and, while the mode of action of cytofectins
and immune complexes may differ, the potency of DNA in
cytofectin complexes provides further evidence that the context
of DNA determines its activity on innate immunity (18–20).
The immunostimulatory activity of DNA-containing immune
complexes has attracted great interest as a mechanism for pro-
moting nonspecific immune activation in the pathogenesis of
SLE. While such complexes can sustain or intensify immune
activation, they do not explain the initial generation of the anti-
DNA antibody which is critical to the activation by DNA. Fur-
thermore, the necessity for complex formation suggests that for
endogenous DNA to activate innate immunity, it must exist in
association with another moiety or structure to allow access to
internal DNA receptors. These considerations (Table 1) thus
focus attention on the mechanisms by which DNA is released
from cells and the molecules to which it is attached during this
process.
RELEASE OF DNA FROM DEAD AND DYING CELLS
As shown using a variety of assays, DNA appears at high levels
in the blood in many conditions including SLE, pulmonary in-
farction, malignancy, and trauma among others. These condi-
tions are all associated with cell activation or cell death, although
death has been considered the most likely source of blood DNA
(21, 22). As now conceptualized, cell death can be divided into
mechanistically distinct processes, with apoptosis and necrosis
representing the prototype pathways. Apoptosis, or programmed
cell death, is a highly regulated process characterized by cellular
collapse and cleavage of nuclear molecules. In contrast, necrosis
TABLE 1. ROLE OF DNA IN INNATE IMMUNITY
References
Stimulation of B cells, macrophages, and dendritic cells 3, 5
Vary with sequence, base methylation, and backbone structure 3, 5
Vary with context and mode of cell entry 18
Stimulation of TLR and non-TLR receptors 19, 20
Induction of specific autoantibodies in normal humans 3, 7–10
Definition of abbreviations: TLR ⫽ Toll-like receptor.
260
is a random process, provoked by physical or chemical trauma
that culminates in cell lysis (23). According to current paradigms,
necrosis is pro-inflammatory while apoptosis is anti-inflamma-
tory, perhaps related to release or spillage of internal molecules.
To investigate nuclear molecule dynamics during death, our
laboratory has used in vitro and in vivo system to assess the
amount and properties of extracellular DNA released from dying
cells. In addition, we have investigated the influence of macro-
phages on the release of DNA, since macrophages can scavenge
dead cells by phagocytosis and therefore potentially modify the
amount of DNA arising from such cells. In these experiments,
we have measured DNA by both an ELISA for nucleosomes as
well as a direct chemical measurement using the dye PicoGreen.
This dye binds specifically to double-stranded DNA and allows
sensitive detection by a fluorometric assay.
Results of these studies have provided a novel perspective
on DNA release during cell death as well as highlighted differ-
ences in the in vitro and in vivo settings. Thus, during in vitro
culture, Jurkat leukemia T cells made apoptotic by chemical
agents release DNA in a time-dependent process. This extracel-
lular DNA shows laddering by gel electrophoresis, indicating
cleavage into nucleosomal fragments. In contrast to cells under-
going apoptosis, cells made necrotic by treatment with heat or
ethanol fail to release DNA even after prolonged culture and
despite changes in cellular permeability, as demonstrated by
propidium iodide staining (24).
As our experiments showed, in the in vitro setting, macro-
phages can display important but divergent effects on DNA
release. With apoptotic cells, the presence of macrophages (ei-
ther the RAW264.7 cell line or murine bone marrow–derived
macrophages) can reduce the amount of DNA released, whereas
macrophages can increase the amount of DNA released by ne-
crotic cells. For necrotic cells co-cultured with macrophages, the
DNA present in the medium showed laddering suggestive of
nuclease digestion. While indicating the importance of macro-
phages in mediating DNA release, these findings also showed
that the size of the DNA in the extracellular space is not itself
a measure of whether death occurs by apoptosis or necrosis (25).
In vivo systems can also help dissect the DNA release process.
Thus, in normal mice, the intraperitoneal administration of apo-
ptotic or necrotic Jurkat cells leads to a prompt rise in the amount
of DNA in the blood that returns to baseline after 24 h. With
both types of dying cells, DNA shows laddering with a similar
size distribution by gel electrophoresis. This result is similar to
that obtained in vitro using mixed cultures of Jurkat cells and
macrophages. Other studies with this model showed that the
extent of DNA release from transferred dead cells can be modi-
fied by treatment of recipient mice with clodronate (which elimi-
nates macrophages), dexamethasone, or the induction of perito-
neal exudates (26–28).
Together, these studies provide evidence that macromolecule
release from dead cells is a modifiable process that may depend
on the presence of more than one cell population, especially
macrophages (Table 2). Furthermore, they suggest that, to the
extent that extracellular DNA can be a player in the innate
TABLE 2. ORIGIN AND PROPERTIES OF EXTRACELLULAR DNA
References
Increased blood levels in settings of cell death 21
DNA laddering 21, 22
In vitro release by apoptotic cells 24
In vivo release with apoptosis and necrosis 26
Role of macrophages 25, 27
PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 4 2007
immune system, the distinction between apoptosis and necrosis
may not be as stark as sometimes portrayed. Thus, in the intact
animal or in mixed cell cultures, both apoptotic and necrotic
cells can release DNA that is similar in properties as measured
by size distribution. These considerations do not imply that the
extracellular DNA from apoptotic and necrotic cells are equiva-
lent immunologically, since the immune activity of DNA de-
pends on context and the presence of other molecules that may
affect its intracellular trafficking. It is important therefore to
consider other molecules that may be released in death settings.
THE IMMUNE ACTIVITY OF HMGB1
HMGB1 is a nonhistone nuclear protein that has also been
implicated in the stimulation of innate immunity during death
processes. Structurally, HMGB1 is 214 amino acids long and can
be divided into an A box, B box, and a C-terminal tail domain,
with the A and B boxes responsible for DNA interaction. This
protein can bind DNA in a non–sequence-specific manner to
bend DNA, although it binds preferentially to distorted DNA
structures such as strand junctions. In addition to binding DNA,
HMGB1 can interact with nuclear proteins. While the precise
function of HMGB1 is not known, this protein can regulate
transcription, perhaps by altering the architecture of DNA to
promote interactions with other factors. Depending on cell type,
HMGB1 can also appear in the cytoplasm (29, 30).
Like DNA, HMGB1 can leave the cell where, in the extracel-
lular space, it can serve as an alarmin and promote immune
activation. An alarmin is an intracellular molecule that, when
released from cells during death processes, can stimulate innate
immune processes (31). In another terminology, an alarmin is
a DAMP or damage (or death)-associated molecular pattern.
In the case of HMGB1, the release processes was first detected
in the setting of macrophage activation rather than death. In
these experiments, supernatants of cultures stimulated with LPS
were characterized to identify other molecules that could medi-
ate septic shock and therefore represent novel targets of therapy.
As these experiments showed, HMGB1 appears in high concen-
trations in the medium of macrophages activated by LPS as well
as pro-inflammatory mediators such as TNF-␣, IFN-␣, and nitric
oxide. Furthermore, HMGB1 levels are high in the serum of
patients with shock and antibodies to HMGB1 can block shock
in mice (32). A series of insightful experiments established a
broad range of immunostimulatory activities of HMGB1, consis-
tent with its role as a late mediator of LPS and its function as
a cytokine. This stimulation appears dependent on the RAGE
receptor, although activation of TLR2 and TLR4 may also con-
tribute to stimulation (33–37).
As shown subsequently, death processes, in addition to mac-
rophage activation, can lead to HMGB1 release, with this protein
posited as an important mediator of inflammation induced by
necrotic cells. As shown in vitro, cells induced to undergo necro-
sis by freeze-thawing release large concentrations of HMGB1.
This release process differs from that occurring during activation,
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-
plasm into vesicles for secretion (38, 39). In contrast, for HMGB1
release during necrosis, the process appears passive and results
from the diffusion of this protein away from chromatin and out
of the cell. Reflecting its nuclear function, HMGB1 is only weakly
adherent to chromatin, differing markedly from histones in the
strength of its DNA interaction. Thus, unanchored to the nu-
cleus, HMGB1 can leave readily when the permeability barriers
break down during death (38).
Pisetsky: Nuclear Molecules in Immunity
While the release of HMGB1 during necrosis is consistent
with its biophysical properties, its behavior during apoptosis has
been more uncertain. Studies by Scaffidi and coworkers indicated
that apoptotic cells do not release HMGB1, with studies by FLIP
(fluorescence loss of photobleaching) in fact demonstrating that
its nuclear diffusion decreases dramatically during apoptosis,
implying greater adherence to chromatin (40). Since apoptotic
cells are generally considered anti-inflammatory, a retention of
HMGB1 in the nucleus could limit immune activation during
this death process. These results are notable since they suggest
that, while apoptotic cells release DNA, they do not release a
companion DNA-binding protein (i.e., HMGB1).
To resolve this seeming contradiction, we reevaluated
HMGB1 release during death processes, using Jurkat cells as
models. In these experiments, apoptosis was induced by chemical
agents and HMGB1 release measured by Western blotting. Con-
focal microscopy with staining with anti-HMGB1 was used to
confirm translocation. Together, these experiments showed
clearly that, in apoptosis as well as necrosis, translocation of
HMGB1 can occur, with the nucleus of cells showing dramati-
cally reduced HMGB1 content in association with nuclear con-
densation (41).
Since DNA release occurs during apoptosis, HMGB1 release
is not unexpected and suggests that permeability changes of
nuclear and cytoplasmic membranes allow the diffusion of a
variety of macromolecules into the extracellular space. The rea-
sons cells differ in their behavior during apoptosis is unknown,
although this process is not uniform among cells and may be
influenced by both the cell type as well as inducing agent. Fur-
thermore, comparisons between the amount of HMGB1 released
during apoptosis and necrosis can be misleading, since the extent
of macromolecule release during necrosis may also vary de-
pending upon the inducing agent or treatment. Thus, in our
experience, the extent of HMGB1 released from cells undergo-
ing freezing and thawing is much greater than cells that have
been treated with ethanol or heat. The most relevant system for
inducing necrosis is speculative, since necrosis is a random and
unregulated process that may follow a host of damaging agents.
Table 3 summarizes the immune properties of HMGB1.
THE ACTIVITY OF EXTRACELLULAR
NUCLEAR MATERIAL
While original studies suggested that HMGB1 has direct cyto-
kine activity, other studies have shown a more complicated situa-
tion. Thus, studies with purified mammalian HMGB1 show much
lower activity than molecularly cloned material, which may have
contamination by endotoxin or DNA. These considerations sug-
gest that HMGB1 may not be fully active as a cytokine but
rather may act in concert with other endogenous or exogenous
molecules to trigger innate immunity (42). In this conceptualiza-
tion, HMGB1 may tune the system, serving a thermostatic func-
tion to allow other molecules to trigger responses. Furthermore,
HMGB1, like antibody or cytofectin, may serve as a carrier for
DNA to allow triggering of internal DNA receptors. Indeed,
TABLE 3. ROLE OF HMGB1 IN INNATE IMMUNITY
References
Induction of extracellular release by TLR ligands 32
Stimulation of cytokine production 33, 35
Interaction with RAGE 37
Role of acetylation and phosphorylation in translocation 38, 39
Extracellular release with necrosis and apoptosis 40, 41
261
HMGB1 can enhance DNA transfection, suggesting its ability
to shuttle nucleic acid into cells (43).
Together, these considerations suggest that immune activity
of nuclear molecules may result from the ensemble of species
present, their physical interaction, and their ability to trigger
more than one receptor type simultaneously. Whether DNA
promotes HMGB1 stimulation or vice versa is a matter of seman-
tics, as both components may have to be present simultaneously
in a complex to induce cell activation. The rules by which com-
plexes stimulate immunity are not known, although stimulation
by organelle fragments or particulates may be more analogous
to a cell–cell interaction (where multiple molecular interactions
occur) than the stimulation by a cytokine, a hormone, or other
small molecular mediator (e.g., prostanoid), which may act in a
unitary manner.
While these issues require much further investigation, the
studies on DNA and HMGB1 nevertheless indicate the diversity
of intracellular molecules with immune activity and the impor-
tance of context in the stimulation of innate immunity by nuclear
macromolecules. Future studies will track the dynamics of nu-
clear molecule trafficking during death processes, the role of
macrophages in determining the extracellular release, and the
signaling pathways stimulated by the complex mixtures of large
molecules. Hopefully, this research will elucidate the pathogene-
sis of SLE and other autoimmune diseases as well as provide
new approaches to treat the broad range of immune-mediated
diseases.
Conflict of Interest Statement : D.S.P. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript.
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