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,
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Division of Basic Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206;
Department of Dermatology, University of Colorado Health Sciences Center, Denver, CO 80262;
Department of Comparative Medicine, University of Washington, Seattle, WA 98195;
Megabios Corporation, Burlingame, CA 94010;
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Department of Pharmacology, University of Wisconsin, Madison, WI 53706; and
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Department of Immunology and the Cancer Center, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. The antitumor activity induced by
CLDC injection was dependent on both NK cells and IFN-. Thus, DNA
complexed to cationic liposomes becomes highly immunostimulatory
and capable of inducing strong antitumor activity when
administered systemically. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in lung tissues (8, 9). Observations made during systemic gene delivery studies using CLDC prompted us to more closely examine the immunologic effects of systemically administered CLDC. We observed that i.v. injection of CLDC, even using noncoding plasmid DNA vectors, consistently induced strong antitumor effects in mice with experimental lung tumor metastases. We also observed adverse effects in CLDC-injected mice that resembled those induced by immune activation (depression, piloerection, dehydration) in the absence of any remarkable pulmonary pathology. Thus, i.v. injection of CLDC appeared to induce systemic effects suggestive of strong, nonspecific immune stimulation.
Therefore, we investigated immunologic responses to systemic CLDC
administration, in particular immune activation, cytokine release, and
antitumor activity. We report here that CLDC injected i.v. triggered
release of high levels of IL-12 and IFN-, as well as accumulation
and activation of NK cells in lung and spleen tissues. Thus, CLDC
administered i.v. appear to be extremely potent inducers of innate
immune responses. Immune activation by CLDC is also responsible for
inhibiting the growth of established tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice used in these studies were either 8- to 12-wk-old female
C57BL/6 mice, 8- to 12-wk-old female BALB/c mice, or 8- to 12-wk-old
ICR mice purchased from either The Jackson Laboratory (Bar Harbor, ME)
or Harlan Sprague-Dawley (Indianapolis, IN). IFN- gene-disrupted
mice, bred on a C57BL/6 background, were obtained from The Jackson
Laboratory. Mice with a disrupted and nonfunctional recombinase
activating gene type 2 (RAG-2 KO) were a kind gift from Dr. Andre
Augustin (National Jewish). Protocols for these experiments were
approved by the Institutional Animal Care and Use Committee at the
National Jewish Medical and Research Center.
Reagents
FBS, LPS, and poly(I:C) were purchased from Sigma (St. Louis, MO). Cell culture medium (modified Eagle’s medium) was prepared by Life Technologies (Gaithersburg, MD).
DNA preparation
The eukaryotic expression vector PCR3.1 (Invitrogen, San Diego, CA) without a gene insert was used as a source of plasmid DNA. The plasmid was purified from Escherichia coli, as described previously, using modified alkaline lysis and polyethylene glycol precipitation (2). The endotoxin content of the plasmid DNA used in these experiments was between .04 and .25 EU per µg DNA. DNA for injection was resuspended in distilled water before use. In some experiments, plasmid DNA was further purified by column chromatography to assure LPS-free conditions. In other experiments, plasmid DNA was methylated in vitro with Sss I methylase (New England Biolabs, Boston, MA) according to the manufacturer’s instructions. Mock-methylated plasmid was prepared by incubation of the plasmid in the absence of the Sss I enzyme, but in the presence of the recommended enzyme reaction solution. Plasmid DNA for methylation experiments was then subsequently purified by column chromatography (Qiagen, Chatsworth, CA) before complexation with liposomes.
Cationic lipids and preparation of DNA-lipid complexes
Cationic lipids were prepared as multilamellar vesicles for in vivo use as described previously (10). Briefly, DOTAP (1, 2 dioleoyl-3-trimethylammonium-propoane; Avanti Polar Lipids, Alabaster, AL) and cholesterol (Sigma) were mixed in a 1:1 molar ratio, dried down in round-bottom tubes, then rehydrated in 5% dextrose solution by heating at 50°C for 6 h, as described previously (10). All experiments were done with DOTAP-cholesterol liposomes, unless otherwise noted. For some experiments, DOTMA ((N-[1-(2, 3-dioleyloxy)propyl]-N,N,N-triethylammonium) (Syntex, Palo Alto, CA) was substituted for DOTAP, and DOPE (diolyl phosphatidylethanolamine; Avanti Polar Lipids) was substituted for cholesterol. For in vivo injection, CLDC were prepared immediately before injection by gently mixing cationic lipids with plasmid DNA at a ratio of 32 nmol total lipid to 1.0 µg DNA, to a final concentration of 100 µg DNA per ml in a sterile solution of 5% dextrose in water.
Injection of CLDC
Lipid-DNA complexes (100 µg/ml DNA in 5% dextrose in water) were injected i.v. via the lateral tail vein. For tumor studies, injections were repeated once 7 days after the first injection.
Flow cytometric analysis of cellular activation
Early cellular activation was assessed by flow cytometric
measurement of CD69 expression on T cells, B cells, monocytes, and NK
cells. Single cell suspensions were prepared from spleens of mice
by the NH4Cl lysis procedure. Lung
mononuclear cells were prepared from lung tissues by collagenase
digestion. Briefly, lung tissues were minced, then digested in 1.0
mg/ml Type 1A collagenase in complete medium, along with 10 µg/ml
DNase and 100 µg/ml soybean trypsin inhibitor, for 1 h at 37°C
with occasional shaking. The lung tissues were then triturated and
mononuclear cells isolated by Ficoll gradient centrifugation. For each
experiment, spleen and lung cells were prepared from three to four
animals per treatment group. Cells were analyzed using a Becton
Dickinson (Mountain View, CA) FACScalibur flow cytometer, with analysis
gates set by first gating on unstained spleen lymphocytes. Between
10,000 and 30,000 gated events were analyzed for each cell type. For
analysis of cell activation, three-color flow cytometric analysis was
done, using anti-CD69 PE (PharMingen, San Diego, CA) to quantitate
the number of CD69-positive cells. T cells were labeled with an
anti-
TCR Ab (biotin H57.597; PharMingen) plus Abs to
either CD4 (FITC RM4–5; PharMingen) or CD8 (FITC 53–6.7;
PharMingen); B cells were dual-labeled with anti-B220 (biotin
RA3-6B2; PharMingen) and either anti-IAb
(FITC 3F12.35; provided by Dr. John Freed, National Jewish) or
anti-IAd (FITC 14.4.4); NK cells were
dual-labeled using anti-NK cell Abs (either anti-NK 1.1 (biotin
PK136; PharMingen) or DX5-biotin (PharMingen)) and anti-CD3 (FITC
2C11); macrophages were evaluated using anti-CR3 (biotin Mac-1;
PharMingen) and FITC anti-IAb or
anti-IAd. The mean peak channel intensity and
percentage of CD69-positive cells was determined for each cell type,
and the mean percentage (±SD) of CD69+ cells was
plotted.
Cytotoxicity assay
A standard 4-h 51Cr-release assay was used to quantitate cytotoxic activity present in freshly isolated lung and spleen mononuclear cells, using YAC-1 cells as targets. Briefly, effector cells from lung or spleen were added in decreasing concentrations to duplicate wells of a Linbro plate, to which was then added 5 x 103 target cells that had been previously labeled for 1 h with 51Cr. The plates were incubated at 37°C for 4 h, then supernatants from each well were harvested and the amount of radioactive 51Cr present was determined by automated gamma counter. The percentage specific lysis was calculated as: [(observed 51Cr release) - (spontaneous 51Cr release)]/[(maximum 51Cr release) - (spontaneous release)] x 100.
NK cell depletion
Mice were depleted of NK cells in vivo by a single i.p. injection of 50 µl rabbit anti-asialoGM1 antiserum (Wako BioProducts, Richmond, VA). Control animals were injected with 50 µl nonimmune rabbit serum. Treatment with the asialo GM1 Ab eliminated detectable NK cells in spleen and lung (by flow cytometry) and also eliminated cytotoxic activity in spleen cells.
Cytokine assays
Cytokine release was measured in spleen cell supernatants after
either in vivo or in vitro stimulation, or in serum after in vivo
injection of CLDC. For assay of cytokine release after in vivo
stimulation, spleen or lung mononuclear cells were prepared from mice 6
or 24 h postinjection, then cultured at a concentration of 5
x 106 cells/ml for an additional 18 h
before supernatants were harvested. For in vitro stimulation of
cytokine release, spleen cells were incubated in vitro with DNA, lipid,
or DNA plus lipid at a final DNA concentration of 1.0 µg/ml for
18 h, at which time the supernatants were harvested for cytokine
assays. Serum was obtained by tail vein bleed at various time points
postinjection. IFN- concentrations inserum or tissue culture
supernatants were assayed using a sandwich ELISA that consisted of two
mAbs (XMG1.1 and biotinylated R45G). IL-10 and IL-4 concentrations in
supernatants were quantitated using specific ELISA kits (PharMingen).
TNF-, total IL-12, and IL-6 concentrations were quantitated using
ELISA kits obtained from Genzyme (Boston, MA) according to the
manufacturer’s instructions.
Tumor lines
B16 (clone F10) cells were obtained from Dr. Isiah Fidler (M D Anderson, Houston, TX); MCA-205 cells were provided by Dr Jack Routes (National Jewish); CT-26 cells were provided by Dr. Nicholas Restifo (National Cancer Institute, Bethesda, MD). All cell lines were maintained at 37°C in MEM supplemented with essential and nonessential amino acids, penicillin and glutamine, and 5% FBS, and were treated periodically with ciprofloxacin (10 µg/ml) to maintain mycoplasma-free conditions.
Tumor challenge and quantitation of lung tumor burden
To establish experimental pulmonary metastases, mice (four per treatment group) were injected once via the lateral tail vein with 2.5 x 105 tumor cells. Treatment with CLDC was initiated 3 days after tumor injection, and was repeated once on day 10 after tumor injection. Control mice were injected with 5% dextrose in water. Mice were sacrificed on days 17–20 after tumor injection, and the number of tumor nodules per lung was determined by manual counting, as described previously (11).
Statistical analysis
Statistical analyses were done using SAS Institute (Cary, NC) software. Lung tumor counts were evaluated for statistically significant differences between treatment groups. For experiments involving two treatment groups, Student’s t test was used for analysis, whereas the Tukey-Kramer test was used to compare differences between multiple treatment groups in a given experiment. Kaplan-Meier survival and Log-Rank analysis was used to compare survival times. Significance was determined for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of CLDC injection on CD69 expression by various
immunologically relevant cell types was evaluated in C57BL/6 mice (Fig. 1
). Mice were injected i.v. with 100 µl
of CLDC solution (which delivered 10 µg total plasmid DNA), or with
5% dextrose in water. Spleen cells were harvested 24 h
postinjection, and five different cell populations
(CD4+/TCR+,
CD8+/TCR+,
NK1.1+/CD3-,
B220+/IAb high, and
Mac-1+/IAb low) were immunostained and evaluated
by three-color flow cytometry for CD69 expression. The mean peak
channel and percentage of CD69+ cells (±SD) was
determined for each cell population. Flow cytometric analysis
demonstrated strong up-regulation of CD69 expression on
CD8+ and CD4+ T cells, NK
cells, B220+ B cells, and
Mac-1+ cells (macrophages) following CLDC
injection (Fig. 1a). The mean percentage of
CD69+ cells was significantly up-regulated
(p < 0.001, compared with sham-treated mice)
24 h after CLDC injection in all five different cell populations
evaluated, compared with control animals. Similar results were obtained
in repeated experiments with C57BL/6 mice, as well as with other
strains of mice, including BALB/c, 129, and ICR (data not shown). The
up-regulation of CD69 expression was maximal by 6 h postinjection
and declined nearly to baseline levels by 3 days postinjection. Thus,
systemic administration of CLDC induced rapid and marked activation of
multiple different immune effector cells.
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Bacterial DNA has been shown previously to have immune stimulatory
properties, including stimulation of IL-12 and IFN- release and
activation of NK cells and B cells (12, 13, 14, 15). Therefore,
experiments were done to determine the relative contributions of
plasmid DNA and cationic liposomes to the systemic immune activation
induced by CLDC in vivo. C57BL/6 mice (three per group) were injected
i.v. with either 10 µg DNA, 320 nmol of DOTAP-cholesterol liposomes,
or with CLDC comprised of 10 µg DA plus 320 nmol liposomes.
Twenty-four hours postinjection, CD69 up-regulation by
NK1.1+ spleen cells was measured flow
cytometrically (Fig. 1
b). Intravenous injection of CLDC
induced marked up-regulation of CD69 expression on NK cells, whereas
CD69 expression was unchanged after i.v. injection of DNA alone or
lipid alone. Similar results were obtained for T cells, B cells, and
macrophages (data not shown). These data, plus cytokine data (see Fig. 3, below), indicated clearly that the complex of DNA and cationic
liposomes was much more immunostimulatory than either DNA or liposome
alone.
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The dose-responsiveness of immune activation (CD69 up-regulation)
by CLDC was determined in C57BL/6 mice. Mice (three per group) were
injected with 100 µl of a CLDC solution containing 100, 10, or 1.0
µg/ml plasmid DNA (Fig. 1c). Twenty-four hours later, the
percentage of CD69+/NK1.1+
spleen cells was determined for each group of treated mice and compared
with control mice. A dose-dependent increase in CD69 up-regulation was
observed. Intravenous administration of as little as 100 ng DNA (in the
form of CLDC) was sufficient to induce a significant increase
(p < 0.05) in CD69 expression by NK cells.
Intravenous injection of LPS and poly(I:C) also induced strong CD69
up-regulation on multiple cell types (data not shown). Injection of
CLDC formulated using any of three different cationic lipid
formulations (DOTAP-cholesterol, DOTAP-DOPE, or DOTMA-cholesterol, see
Materials and Methods) all stimulated CD69 up-regulation to
an equivalent degree (data not shown). Thus, the immune-activating
properties of CLDC were not dependent on use of a specific
liposome.
Intravenous injection of CLDC induces release of IFN- and IL-12
The effect of CLDC injection on cytokine release was assessed by
culturing spleen or lung cells obtained from in vivo-treated mice
24 h postinjection (Fig. 2). Mice were
injected with either plasmid DNA only (10 µg), liposomes only, or
CLDC. Single cell suspensions of spleen and lung mononuclear cells were
prepared (see Materials and Methods) and then cultured in
vitro for an additional 18 h. Release of cytokines into the
supernatants was quantitated by specific cytokine ELISA.
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a) and IFN- (Fig. 2b), whereas
there was minimal cytokine release from spleens of control mice or mice
injected with equivalent amounts of DNA only or cationic liposomes
only. Similar results were observed with cultured lung mononuclear
cells (data not shown). In vitro addition of CLDC (1.0 µg DNA per ml
medium) to naive spleen cells in vitro also induced release of IL-12
and IFN- (see Fig. 5, and data not shown). Injection of CLDC also
induced release of high levels of IFN- into the serum, with the peak
of release occurring 8 h postinjection and then declining
thereafter (Fig. 2c). Spleens from CLDC-injected mice
released high levels of IL-6, but equivalent levels of IL-2, TNF-,
IL-4, or IL-10, compared with control mice (data not shown). Thus, i.v.
injection of CLDC elicited strong systemic release of Th1 cytokines in
naive mice. The complex of DNA plus a cationic lipid was a much
stronger stimulus for cytokine release than DNA or lipid alone.
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(data not shown) or IL-12 (Fig. 2d). Nonetheless,
these doses of LPS and poly(I:C) did induce in vivo immune activation,
as revealed by pronounced CD69 up-regulation (data not shown).
Thus, systemically injected CLDC were more potent activators of innate
immunity on a per weight basis than LPS or poly(I:C) and induced high
circulating levels of IFN-. The immunogenicity of CLDC is partially dependent on the immunogenicity of bacterial DNA
Bacterial DNA is immunogenic in mammals. The immunogenicity of
bacterial DNA stems in part from the high content of unmethylated CpG
motifs in bacterial DNA, compared with eukaryotic DNA (12, 13, 15). Therefore, experiments were done to determine whether the
marked immunogenicity of CLDC was mediated in part by increasing the
immunogenicity of the plasmid DNA component of the complex. CLDC
were prepared using either unmodified plasmid DNA or plasmid DNA that
had been methylated in vitro (Fig. 3).
Controls included plasmid DNA incubated with the appropriate buffers
but without the methylase enzyme. Naive spleen cells were incubated for
18 h with modified or unmodified CLDC, and the induction of IL-12
release was quantitated.
Compared with CLDC prepared with unmodified plasmid DNA, CLDC prepared with methylated DNA elicited only 25% as much IL-12 release from spleen cells. The level of IL-12 release triggered by methylated CLDC was reduced to the levels induced by incubation with control DNA alone. Lipid alone did not induce IL-12 release (data not shown). These results suggest that formation of the CLDC greatly amplifies the inherent immunogenicity of the plasmid DNA, possibly by increasing DNA entry into cells and their nuclei. However, we have also observed immune activation by CLDC formulated with eukaryotic DNA (S.W. Dow, unpublished observations), and a recent publication suggests that any double-stranded DNA sequence is capable of inducing immune activation (16). Thus, the immune-stimulatory properties of CLDC may involve more than just immune stimulation by bacterial DNA.
Intravenous injection of CLDC controls the growth of established metastatic lung tumors
Others have reported that systemic injection of CLDC
containing noncoding (empty vector) plasmid DNA can induce antitumor
activity (17). Therefore, we investigated in greater
detail the mechanism(s) by which systemic injection of CLDC might
induce antitumor activity. Three different tumor lines (fibrosarcoma
(MCA-205), melanoma (B16.F10, and colon carcinoma (CT26)) were used to
assess the effect of CLDC treatment on a variety of established
pulmonary metastatic tumors (Fig. 4,
a–d). Mice (four per treatment group) were each injected
i.v. with 2.5 x 105 tumor cells. C57BL/6
mice were injected with MCA-205 cells or B16.F10 cells, and BALB/c mice
were injected with CT26 cells. Mice were treated by i.v. injection of
CLDC solution beginning 3 days after tumor injection. The CLDC
injection was repeated once 7 days later, and the mice were sacrificed
7–10 days after the second CLDC injection (day 17–20 posttumor
injection). The lung tumor burden was quantitated by counting the
number of lung tumor nodules, as described previously
(11). In all three tumor models, injection of CLDC induced
highly significant reductions (p < 0.0001) in
the lung tumor burden, compared with control animals.
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d).
Mice (eight per treatment group) were treated three times with i.v.
injection of CLDC on days 3, 10, and 17 posttumor injection, and then
treatment was discontinued. Control mice were sham-treated with
diluent. The survival time in CLDC-treated animals (mean = 35
days) was significantly increased (p = .0001)
compared with control animals (mean survival = 15 days). Thus,
injection of CLDC induced a sustained antitumor effect in animals with
established lung tumor metastases.
The relative contributions of DNA and/or liposomes to the antitumor
effects of systemic CLDC injection were determined in mice with
established MCA-205 lung tumor metastases. Treatment with plasmid DNA
alone or liposomes alone did not induce antitumor activity, whereas
injection of an equivalent amount of CLDC induced significant
(p < 0.001) antitumor activity (Fig. 5a). We also assessed whether
the nonspecific immune stimulation induced by injection of LPS or
poly(I:C) could also induce antitumor activity in these experimental
models. In contrast to injection of CLDC, i.v. injection of equivalent
amounts of (10 µg) of either LPS or poly(I:C) did not induce
antitumor activity against established MCA-205 metastases (Fig. 5b). Thus, though LPS, poly(I:C), and CLDC were all capable
of inducing nonspecific immune stimulation when injected systemically,
only CLDC also exerted potent antitumor activity.
Accumulation of NK cells in lungs of CLDC-injected mice
Lung mononuclear cells and spleen cells were collected
from mice 3 days after i.v. injection of CLDC and analyzed by flow
cytometry (Fig. 6). Flow cytometric
analysis revealed a pronounced, 4-fold increase in the percentage of
intrapulmonary NK cells in CLDC-injected mice, compared with
sham-treated control animals (p < 0.0001).
There was also an increase in intrasplenic NK cells in CLDC-injected
mice, though the increase was not as large as in the lungs. Thus, CLDC
administered systemically serve as a stimulus for accumulation of NK
cells in various tissues, particularly the lungs.
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release
The role of NK cells in IFN- release after CLDC injection was
investigated by NK cell depletion experiments. Mice were depleted of NK
cells by treatment with anti-asialo GM1
antiserum. Control mice were treated with nonimmune rabbit serum.
Forty-eight hours after injection of anti-asialo
GM1 antiserum, mice were injected with CLDC.
Twenty-four hours after CLDC injection, spleen and lung mononuclear
cells were harvested, cultured in vitro, and assayed for release of
IFN- (Fig. 7, a and
b). NK cell depletion almost completely eliminated release
of IFN- from both spleen and lung cells, compared with mice treated
with non-immune rabbit serum or untreated control animals. Thus, NK
cells were the major source of IFN- release triggered by CLDC
injection.
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Spleen cells harvested 24 h after i.v. injection of CLDC in
C57BL/6 exhibited high levels of cytotoxic activity, as assessed in a
4-h chromium release assay, using 51Cr-labeled
YAC-1 cells as targets (Fig. 8a). The cytotoxic activity
was not MHC-restricted, as revealed using MHC-mismatched target cells
(data not shown). The cytotoxic activity was markedly reduced in the
spleens of NK cell-depleted mice. Similar results were observed using
lung mononuclear cells as effector cells (data not shown). Furthermore,
cytotoxic activity was not generated by injection of either DNA alone
or lipid alone (data not shown). High levels of cytotoxic activity were
also detected in spleen cells of RAG-2 KO mice injected with CLDC (Fig. 8b). Thus, i.v. administration of CLDC elicited strong
functional activation of NK cells, a response that did not require T or
B cells.
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The role of NK cells in mediating tumor rejection in response to
CLDC injection was investigated by depleting mice of NK cells in vivo.
NK cells were depleted in BALB/c mice (four per group) 1 day after i.v.
injection with CT26 cells. Control mice were treated with nonimmune
rabbit serum or were untreated. On day 3 posttumor injection, the mice
were injected with CLDC. The Ab depletion of NK cells and CLDC
treatment was repeated once 7 days later, and the mice were sacrificed
on day 17 posttumor injection and the lung tumor burden was
quantitated. Depletion of NK cells reduced the antitumor activity
induced by systemic injection of CLDC, such that there was no
significant difference in tumor burden between CLDC-treated and control
mice (Fig. 9a). However, the
tumor burden in mice treated with nonimmune rabbit serum was
significantly reduced by injection of CLDC. Thus, NK cells were a key
mediator of the antitumor activity induced by CLDC injection.
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release in
response to CLDC injection, the role of IFN- in mediating
CLDC-induced tumor rejection was investigated. CLDC-induced antitumor
activity was evaluated in C57BL/6 mice with a targeted disruption of
the IFN- gene (IFN--/-). Wild-type
C57BL/6 mice (IFN-+/+) or
IFN--/- mice with day 3 established MCA-205
tumor cells were treated with either CLDC or were sham-injected
(control). The tumor burden in IFN -/- mice
treated with CLDC was not significantly decreased, compared with
control IFN-/- mice, whereas treatment of
IFN+/+ mice induced a significant reduction in
tumor burden (Fig. 9b). The
IFN--/- mice were therefore impaired in
their ability to respond to CLDC injection and control the growth of
MCA-205 lung tumors. Thus, the antitumor activity induced by
systemically administered CLDC was mediated to a large degree by
IFN- released from activated NK cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). CLDC
were particularly stimulatory for NK cells, inducing functional
activation, IFN- release, and antitumor activity. Bacterial DNA is immunogenic in eukaryotes. Exposure to bacterial DNA triggers innate immune responses and activation of B cell, macrophages, and NK cells (12, 13, 14, 15). B cells and macrophages are activated directly by bacterial DNA, whereas NK cell activation is reported to be mediated indirectly by IL-12 released from DNA-stimulated macrophages (12, 13, 14, 15, 18, 19). The immunogenicity of bacterial DNA is due in part to the increased purine content of bacterial DNA, which is enriched in CpG motifs relative to eukaryotic DNA (12, 13, 14, 15, 20, 21, 22). The hypomethylated state of bacterial DNA is also immunogenic (12, 22). The immunogenicity of bacterial DNA has also been shown to play a role in the effectiveness of DNA vaccines, in part through induction of Th1 cytokines (23).
One surprising finding from our study was the extreme immunogenicity of
lipid-DNA complexes. The cationic liposomes by themselves were without
apparent immune stimulatory activity, as was the plasmid DNA alone at
the low doses employed in these studies (Figs. 1 and 2). However, the
complex of the two was markedly stimulatory. Complexation to liposomes
was reported previously to enhance the immunogenicity of synthetic
oligonucleotides (24). The enhanced immunogenicity of
complexes rela-tive to naked DNA or oligonucleotides is most likely a
result of lipid-facilitated intracellular entry and nuclear
translocation of DNA, inasmuch as immune activation by bacterial DNA
requires DNA entry into the cell nucleus (14, 25). In
vivo, cationic liposomes protect DNA from degradation and prolong
circulation time, which may in turn facilitate binding to monocytes and
macrophages (26).
Previous studies have demonstrated that the formation of complexes with
liposomes serves to increase the immune stimulatory properties of DNA,
both in vitro and in lung tissues in vivo (8, 24). Our
observation that methylation of the plasmid DNA reduced immune
activation by CLDC (Fig. 3) is consistent with these prior
observations. Intravenous injection of high doses (mg) of plasmid DNA
has been reported to induce immune stimulation and IFN- release
(18). However, we observed immune activation at DNA doses
as low as 100 ng when the DNA was complexed to a cationic liposome
(Fig. 1), indicative of the degree to which lipids increased the
immunogenicity of bacterial DNA.
The immunostimulatory properties of CLDC were not due to contaminating
LPS, as evidenced by the failure of DNA alone to induce immune
stimulation, either in vitro or in vivo (Figs. 2 and 5). Injection of
equivalent amounts of purified LPS also failed to elicit the same
pattern of cytokine responses or antitumor responses as CLDC (Figs. 2 and 5). In addition, CLDC formulated with highly purified DNA
(virtually free of detectable LPS) induced immune stimulatory effects
indistinguishable from CLDC prepared with other sources of DNA (data
not shown).
A major finding from this study was that CLDC injected i.v. could
induce high levels of antitumor activity. Treatment of mice with
established pulmonary metastases by CLDC injection induced significant
reductions in lung tumor burden and prolonged survival times (Fig. 4).
The antitumor activity induced by CLDC was unique in that it could not
be reproduced by injection of equivalent low doses of other nonspecific
activators of innate immunity, such as LPS or poly(I:C) (Fig. 5).
NK cells were the major mediators of the antitumor effects induced by
CLDC. For example, NK cells accumulated to high levels in the lungs of
CLDC-injected mice (Fig. 6) became highly cytotoxic (Fig. 8) and
secreted large amounts of IFN- (Fig. 7). Furthermore, depletion of
NK cells almost completely eliminated the antitumor activity of CLDC
injection (Fig. 9). It is likely that NK cell activation occurred in
response to IL-12 released following CLDC injection (Fig. 2). Bacterial
DNA has been shown previously to activate NK cells by triggering
release of IL-12 from macrophages (14, 19). However, we
cannot exclude the possibility that CLDC injection might also induce
production of IL-18, which could in turn trigger release of IFN- and
antitumor activity. It should also be noted that the NK cells that
accumulated in the lungs of CLDC-treated mice (Fig. 6) were classical
NK cells (NK1.1+, CD3-),
as opposed to the recently described natural T
cells (NK1.1+/CD3+)
(27).
IFN- also played a critical role in the antitumor effects of CLDC
injection. Mice with a nonfunctional IFN- gene were significantly
impaired in their ability to control the growth of lung tumors after
treatment with CLDC (Fig. 9). Thus, NK cells activated by CLDC may have
induced inhibition of tumor growth indirectly by releasing IFN-.
Both IFN- and IL-12 exert multiple antitumor activities in vivo,
including inhibition of tumor angio-genesis (28, 29, 30).
Therefore, it is conceivable that CLDC may inhibit tumor growth in part
by stimulating local release of antiangiogenic cytokines within the
tumor vasculature. For example, it was demonstrated recently that CLDC
preferentially bound to the neovascular endothelium of tumors following
i.v. injection (31).
Our findings have important implications for systemic nonviral gene
delivery using CLDC. Widespread immune activation is likely to occur
when CLDC are used to deliver genes systemically to the lungs or other
target organs. However, the immune response to CLDC was dose-dependent
and self-limiting (Figs. 1 and 2), and could therefore be controlled by
dosage adjustments. The immune response elicited by CLDC injection was
not vector-specific, therefore still allowing repeated CLDC
administration. The development of vector-specific immunity is, by
contrast, a major drawback to repeated use of viral vectors in vivo
(6, 7). Continued improvements in plasmid vectors are
likely to improve the duration of gene expression following systemic
gene delivery, thereby reducing the frequency of administration.
Activation of innate immune responses by CLDC may also represent an
unanticipated advantage for immunotherapy of cancer or allergic
diseases. The Th1 cytokines elicited by CLDC may provide a strong
adjuvant effect for DNA vaccines administered using CLDC. For example,
the superiority of CLDC for induction of immune responses to HIV Ags
using DNA vaccines was reported previously (32). The use
of CLDC may prove particularly effective for systemic, therapeutic
vaccination against tumor Ags, given the strong antitumor effects
induced by the plasmid vector alone.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven W. Dow, Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80523. E-mail address:
3 Abbreviation used in this paper: CLDC, cationic lipid-DNA complex.
Received for publication March 17, 1999. Accepted for publication May 18, 1999.
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References |
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. Proc. Natl. Acad. Sci. USA 93:2879. in vivo and increases the toxicity of lipopolysaccharides. J. Immunol. 156:4570.[Abstract]
production is dependent on macrophage secretion of IL-12. Clin. Immunol. Immunopathol. 84:185.[Medline]
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affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity 9:25.[Medline]
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) or CLDC-injected (•) RAG-2 gene
knockout mice, 24 h postinjection.