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*
Section of Retroviral Immunology, Center for Biologics and Evaluation Research, Food and Drug Administration;
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This work examines whether genomic DNA released by injured cells contributes to APC maturation. Although DNA was historically believed to be immunologically inert, it is now appreciated that DNA can be recognized by the immune system. For example, unmethylated "CpG motifs" expressed at high frequency in bacterial DNA activate lymphocytes, NK cells, and macrophages to proliferate and secrete Ig and/or cytokines (14, 15), whereas dsDNA triggers stromal cells to up-regulate surface expression of MHC molecules (16). DNA is normally sequestered in the nucleus, but can be released into the systemic circulation when cells undergo necrosis/apoptosis (10, 11, 12). Exposure to DNA has been implicated in the development of autoimmune disease, as in DNase-I-deficient mice (17). This set of findings led us to hypothesize that double-stranded genomic DNA released by injured host cells may act as a "danger signal," promoting APC maturation or other forms of immune stimulation (13, 18).
Results indicate that introducing dsDNA into the cytoplasm of macrophages and bone marrow-derived dendritic cells (BMDC)4 induces them to mature phenotypically and functionally. These APCs support the development of primary immune responses against coadministered Ag in vivo. These findings are consistent with the hypothesis that genomic DNA may promote host survival by improving immune recognition of pathogens at sites of tissue damage/infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c and C57BL/6 mice (6- to 10-wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME). All studies were approved by the Center for Biologics Evaluation and Research Animal Care and Use Committee. BMDC were obtained by collecting bone marrow and depleting T, B, and NK cells by MACS (Miltenyi Biotec, Indianapolis, IN) using mAbs against Thy1.2, B220, and DX-5 conjugated to magnet microbeads as previously described (19, 20). The remaining cells were incubated for 5 days in complete medium (DMEM supplemented with 5% FBS, 2 mM L-glutamine, 100 nM nonessential amino acids, 1 mM sodium pyruvate, and 20 mM HEPES) supplemented with 10 ng/ml recombinant mouse GM-CSF (BD PharMingen, San Diego, CA). At day 5, cells were treated with 3 mM EDTA for 5 min and harvested. More than 96% of the cells isolated by this technique were immature dendritic cells (CD11c+, I-Addim/high, CD40low, and CD80low), whereas the others were macrophages (CD11b+/CD11c-).
OVA323–339-specific T cells were generated from BALB/c DO11.10 TCR transgenic mice (National Institute of Allergy and Infectious Diseases-Taconic, Bethesda, MD) as previously described (21). Briefly, CD4+ T cells were enriched by negative selection using magnetic beads (R&D Systems, Minneapolis, MN). Cells were maintained in vitro by repeated stimulation with irradiated BALB/c spleen cells pulsed with OVA323–339 peptide (ISQAVHAAHAEINEAGR) in medium supplemented with 10 ng/ml IL-12, 10 µg/ml anti-IL-4, and 10 U/ml rIL-2 (all obtained from R&D Systems). Viable cells were isolated from resting cultures by density gradient centrifugation over Lympholyte M (Cedarlane Laboratories, Bethesda, MD) immediately before assay. The RAW 264.7 (H-2d) mouse macrophage cell line was purchased from American Type Culture Collection (Manassas, VA) and maintained in complete DMEM supplemented with 10% FBS.
Preparation of cell lysates
Primary fibroblasts were obtained from newborn BALB/c mice as previously described (10). These cells were passaged several times and used in log growth phase. Necrosis was induced by aspirating washed cells through a 30-gauge needle or by suspending cells in distilled water for 10 min at 4°C. Lysis was confirmed by microscopic examination. Some lysates were treated with 400 µg/ml proteinase K (Sigma, St. Louis, MO) for 18 h and/or 100 U/ml DNase-I (Boehringer Mannheim, Indianapolis, IN) for 1 h. The enzymes were inactivated by incubating samples at 65°C for 15 min. BMDC and fibroblasts were mixed at a 1:1 ratio.
DNA preparation
Genomic DNA was purified from BALB/c liver using the Wizard Genomic DNA purification kit as recommended by the manufacturer (Promega, Madison, WI) and then repurified by repeated phenol-chloroform extraction. Synthetic polynucleotides (PN) were purchased from Amersham Pharmacia (Piscataway, NJ). Double-stranded Escherichia coli DNA was obtained from Sigma (St. Louis, MO). ssDNA was prepared by boiling dsDNA for 5 min followed by immediate cooling on ice. All DNA was repurified by treatment with Triton X-114 to remove endotoxin as previously described (22). Less than 0.01 U/µg endotoxin or protein was present in any of the DNA samples based on a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).
dsDNA fragments (75, 150, and 300 bp) were obtained by PCR amplifying the pBR322 plasmid (Boehringer Mannheim) using the following primers: 75 bp; sense 5'-ACTCGCTGCGCTCGGTCG-3', antisense 5'-CTGTGGATAACCGTATTA-3', 150 bp; sense-same as 75 bp, antisense ACGCGGCCTTTTTACGGT-3', 300 bp; sense CGGATACCTGTCCGCCTT-3', antisense ACTGTCCTTCTAGTGTAG-3'. The DNA fragments were isolated using Sephaglass (Amersham Pharmacia) after electrophoresis in 2% GTG agarose gel (Bioproducts, Rockland, ME).
In some experiments, genomic DNA was methylated using SssI CpG methylase (New England Biolabs, Beverly, MA) in NEB buffer supplemented with 160 µM S-adenosylmethionine. Methylation of genomic DNA was confirmed by the loss of HpaII cleavage susceptibility (data not shown).
Transfection
DNA (1 µg) was mixed with 3 µl of Fugene 6 transfection reagent (nonliposomal; Boehringer Mannheim) in 100 µl of OptiMEM (Life Technologies, Gaithersburg, MD) for 15 min at room temperature. This solution was resuspended in 1 ml of complete DMEM containing 106 cells. The viability of cells was not affected by this treatment.
Flow cytometric analysis of cell surface molecule expression
Cells were washed with PBS, fixed with 4% paraformaldehyde for
5 min at 37°C, and stained with FITC or PE-labeled Abs for 30 min at
room temperature (23, 24). Stained cells
(106) were washed, resuspended in PBS/0.1% BSA
plus azide, and analyzed by FACSort (BD Biosciences, San Jose, CA). Abs
specific for the following molecules were obtained from BD PharMingen:
I-Ad,
I-Ad/I-Ed, CD40, CD54
(ICAM-1), TCR-
, IL-2, IFN-
, control rat IgG2a for
I-Ad/Ed, CD40, and hamster
IgG for CD54.
In vitro analysis of APC function
BMDC were incubated with 50 µg/ml DNA plus 2 ng/ml
OVA323–339 peptide for 24 h. The cells were
then collected, washed, and mixed with an equal number of D11.10 T
cells (specific for OVA323–339 peptide presented
in the context of MHC class II). After 5 h of stimulation at
37°C in the presence of 2 µM monensin (Sigma), activated T cells
were identified by surface staining for TCR- coupled with
intracytoplasmic staining for IL-2 and IFN- (24).
Cytokine-specific ELISA
Immulon 2 plates (96-well) were coated with anti-IL-6 or anti-IL-12 (BD PharMingen) in PBS (pH 7.2) for 4 h as described (15). After the plates were blocked and washed, supernatants from DNA-stimulated RAW 264.7 cells were added for 2 h at room temperature. The plates were then washed and treated with biotinylated anti-cytokine Ab followed by phosphatase-streptavidin (BD PharMingen), as described (15). The concentration of cytokine was determined by comparison to purified control cytokine included in the same experiment.
Immunization and Ag-specific Ab responses
C57BL/6 mice (10/group) were immunized i.p. with 2 µg of soluble OVA (Sigma). In some cases, 100 µg of free DNA was included in the injection mixture. Two weeks after immunization, serum was collected from these mice and stored at -20°C (25). Anti-OVA Abs were detected by Ag-specific ELISA, as described (25, 26).
Ag-specific CTL assays
Spleen cells were collected from C57BL/6 mice 3 wk after immunization as described above. Spleen cells (5 x 106) were cultured in 2 ml of complete medium with 5 µg/ml SIINFEKL peptide as described (23). Recombinant human IL-2 (10 U/ml; R&D Systems) was added after 2 days of culture, and cells were harvested on day 5. EL4 thymoma (H-2b) and P815 mastocytoma (H-2d) target cells were cultured overnight with 51Cr (DuPont-NEN, Boston, MA) ± 1 µM SIINFEKL peptide. Target cells (5 x 103) were cultured with increasing numbers of effectors. After 6 h, released 51Cr was measured by gamma counting (Wallac, Gaithersburg, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dying cells leak factors capable of activating the immune system
of the host (13). To identify the elements responsible for
this activity, fibroblasts were mechanically or osmotically lysed and
then added to BMDC cultures. These cellular lysates stimulated BMDC to
mature, as manifest by a significant increase in the number of
CD11c+ cells expressing the CD40 maturation
marker (p < 0.02, Fig. 1
A). Treating cell lysates
with both proteinase K and DNase-I abrogated this effect, suggesting
that both self-proteins and DNA contributed to DC maturation
(p < 0.01, Fig. 1B). Unfortunately,
nuclear proteins present in these lysates protected genomic DNA from
digestion, complicating efforts to clarify the independent contribution
of DNA to this process.
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). This genomic DNA lost
its ability to induce APC maturation when reduced to single-stranded
form by boiling. In contrast, APC maturation was induced by
CpG-containing bacterial DNA in both single- and double-stranded form
(Fig. 2). To determine whether cell surface binding was required for DC
maturation, Fugene 6 was used to transfect dsDNA directly into the
cytoplasm of target cells. Dose-response experiments showed that
100-fold less DNA was required to optimally up-regulate CD40 expression
following transfection than after adding free DNA, suggesting that
intracytoplasmic dsDNA was responsible for the observed maturation of
APC (Fig. 2 and data not shown).
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Consistent with results involving BMDC, murine dsDNA (but not
ssDNA) stimulated RAW 264.7 murine macrophages to significantly
up-regulate expression of CD40 (Fig. 3).
Genomic dsDNA also increased APC expression of MHC class II (I-A/Ed)
and CD54, with the maximal effect being observed at 24 h and
persisting through 96 h (Fig. 3 and data not shown).
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A). Longer strands of dsDNA
more efficiently induced APC maturation. Only background levels of MHC
class II expression were observed when RAW 264.7 cells were transfected
with dsDNA 
75 bp in length. By comparison, class II expression
increased significantly when cells were transfected with dsDNA 150 bp
long (p < 0.05, Fig. 4B).
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Bacterial DNA contains immunostimulatory CpG motifs that activate
B cells to secrete Ig, NK cells to produce IFN-, and
macrophages/monocytes to up-regulate expression of MHC and
costimulatory molecules (Table I and Ref.
27). Methylation of the CpG motifs in single-stranded
bacterial DNA abrogates this immune stimulation (Table I and data not
shown).
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). Similar APC activation was observed
when freshly isolated spleen cells were used in these experiments (data
not shown). However, unlike bacterial DNA, treating murine DNA with CpG
methylase did not reduce its effect on APC maturation (Table I). An
additional difference between CpG DNA and double-stranded murine DNA
was the inability of the latter to induce cytokine production. These
findings suggest that 1) unmethylated CpG motifs are not responsible
for the immune activation triggered by genomic dsDNA and 2) the nature
of the immune response elicited by genomic DNA differs from that
induced by CpG motifs.
To confirm these conclusions, PN of known sequence were synthesized
and tested. Mock transfected cells, and cells transfected with
single-stranded PN (ssPN) (poly(dG) + poly(dC); poly(dA) + poly(dT))
did not induce RAW 264.7 cells to up-regulate expression of MHC class
II, CD40, or CD54 (Fig. 5). However, when
the same ssPN were mixed together to form double-stranded structures,
stimulation was observed. Of note, dsPN lacking CpG motifs
(poly(dA)-poly(dT); poly(dI)-poly(dC); poly(dA-dT)-poly(dA-dT))
activated APC to the same extent as dsPN that were high in C/G content
(poly(dG)-poly(dC); poly(dG-dC)-poly(dG-dC)) (Fig. 5 and data not
shown). These findings confirm that the activity of dsDNA was not CpG
dependent. They also provide evidence that the activity of dsDNA was
not due to endotoxin contamination, because the same PN that were
inactive in single-stranded form induced APC maturation when mixed to
form dsPN.
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The effect of dsDNA on the ability of APC to present Ag was
investigated. BMDC were cultured in vitro with
OVA323–339 (an immunogenic peptide of OVA)
and/or 50 µg/ml dsDNA. The cells were harvested 24 h later and
mixed with DO11.10 T cells (these cells are specific for
OVA323–339 presented in the context of the
I-Ad MHC class II molecule). BMDC pulsed with
OVA323–339 effectively presented Ag to DO11.10
cells (21, 29), resulting in a 2- to 3-fold increase in
the number of T cells secreting IFN- and IL-2 (Fig. 6). When dsDNA was combined with
OVA323–339, a further 3-fold increase in T cell
activation was observed (Fig. 6). This improved ability to present
exogenous Ag indicates that the APCs induced to mature by dsDNA
were functionally active. This effect was Ag-specific and
MHC-restricted; no increase in T cell activation was induced by BMDC
treated with ssDNA, pulsed with irrelevant Ag, or by cells that present
OVA323–339 in the context of MHC class I (Fig. 6
and data not shown).
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A, p < 0.01 vs
ssDNA, p < 0.002 vs ssPN). Coadministering OVA plus dsDNA
also led to a significant increase in Ag-specific CTL activity
(p < 0.01 dsDNA vs ssDNA, Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mature APCs play a critical role in the development of Ag-specific immunity (4, 13). Whereas molecules derived from infectious pathogens most commonly stimulate APC maturation (6), recent studies indicate that factors released by dying host cells may also serve this function. Heat shock proteins (HSPs) (including HSP70, 90, and gp96) have been implicated in this immunostimulatory process (30). For example, HSPs released by dying cells up-regulate expression of B7.1 and B7.2 by APC (30). Yet HSPs alone do not account for the range of immunostimulatory activity observed with whole cell lysates, indicating that other factors (alone or in combination with HSP) may contribute to these effects. Shi et al. (31) showed that coadministering OVA with cytoplasmic material from necrotic and/or apoptotic cells enhanced CTL activity. Those studies involved directly conjugating OVA to intracellular proteins, and thus did not examine the role of genomic DNA.
Our data suggest that introducing dsDNA into the cytoplasm of APCs stimulates their maturation. These effects were sequence independent, with dsPN of entirely different sequences showing equal activity. There was no evidence that CpG motifs contributed to this stimulation, because 1) dsPN lacking C or G nucleotides were stimulatory, 2) CpG methylation did not inhibit the activity of dsDNA, and 3) single-stranded genomic DNA was inactive. It is unlikely that bacterial contaminants were responsible for the activity ascribed to dsDNA or dsPN. All DNA preparations were endotoxin-free, and the same PN that were inactive in single-stranded form were stimulatory when combined to form dsDNA.
The mechanism by which dsDNA induces APC maturation is unknown.
Because tissue damage results in the release of dsDNA and other
immunostimulatory factors by dying host cells, we consider it likely
that these agents may synergistically promote a local immune response.
Studies are underway to determine whether Toll-like receptor 9 (the
cell surface receptor responsible for the recognition of CpG DNA) or
DNA-PK (an intracytoplasmic protein linked to CpG-mediated immune
activation) are involved in the signaling cascade activated by dsDNA
(32, 33). We are also investigating whether dsDNA-induced
APC maturation proceeds through intermediates such as NF-
B, Stat-1,
or other cellular factors (16).
Our results support the hypothesis that "factors" released by dying host cells facilitate the induction of immunity against foreign and/or self Ags (13). Several reports indicate that necrotic/apoptotic cellular debris promotes the development of autoimmune disease (17). For example, lupus autoantibodies preferentially react with nuclear Ags complexed to dsDNA (34), and DNase-I-deficient mice commonly develop autoimmune disease (17). These findings underscore the potential role of genomic DNA in the development or persistence of autoimmune disease. Yet, if immune recognition of dsDNA has been conserved, it must provide some benefit to the host. We postulate that dsDNA released following tissue injury/infection promotes APC maturation and thus facilitates the elimination of pathogens at the injury site. This mechanism might also improve host recognition of virus-infected APCs. During viral replication, pro-viral dsDNA commonly accumulates in the cytoplasm of infected cells (35, 36). By improving the presentation of viral Ags, APCs, the immune response elicited by dsDNA would facilitate the elimination of infected APCs and promote host survival.
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Acknowledgments |
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Footnotes |
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2 The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Food and Drug Administration at large.
3 Address correspondence and reprint requests to Dr. Dennis M. Klinman, Building 29A Room 3 D 10, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. E-mail address: Klinman{at}CBER.FDA.GOV
4 Abbreviations used in this paper: BMDC, bone marrow-derived dendritic cells; PN, polynucleotide; HSP, heat shock protein; ssPN, single-stranded PN; dsPN, double-stranded PN.
Received for publication April 4, 2001. Accepted for publication July 6, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 93:2879.
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K. J. Ishii, F. Takeshita, I. Gursel, M. Gursel, J. Conover, A. Nussenzweig, and D. M. Klinman Potential Role of Phosphatidylinositol 3 Kinase, rather than DNA-dependent Protein Kinase, in CpG DNA-induced Immune Activation J. Exp. Med., July 15, 2002; 196(2): 269 - 274. [Abstract] [Full Text] [PDF]
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), OVA plus 100 µg of poly(dT) (
), or
OVA plus poly(dA)-poly(dT) (
). Lysis of H-2-matched OVA
peptide-pulsed EL-4 cells by CTL isolated 3 wk after immunization is
shown. Lysis of EL4 cells without peptide, and of peptide-pulsed H-2
mismatched P815 cells, was uniformly <5\%. |P8, >p
< 0.01; dsPN vs ssPN.