Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors

Song Li1, Su-Ping Wu1, Mark Whitmore1, Eric J. Loeffert2, Lin Wang1, Simon C. Watkins2, Bruce R. Pitt1, and Leaf Huang1

1 Department of Pharmacology and 2 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cationic lipid-mediated intravenous gene delivery shows promise in treating pulmonary diseases including lung tumor metastases, pulmonary hypertension, and acute respiratory distress syndrome. Nevertheless, clinical applications of cationic lipidic vectors via intravenous administration are limited by their transient gene expression. In addition, repeated dosing is not effective at frequent intervals. In an effort to elucidate the mechanism of gene inactivation, we report in this study that cationic lipid-protamine-DNA (LPD) complexes, but not each component alone, can induce a high level of cytokine production, including interferon-gamma and tumor necrosis factor-alpha . Furthermore, we demonstrate that LPD administration triggers apoptosis in the lung, a phenomenon that may be mediated in part by the two cytokines. Treatment of mice with antibodies against the two cytokines prolongs the duration of gene expression and also improves lung transfection on a second administration of LPD. Although the mechanism underlying LPD-induced cytokine production is unclear, methylation of the DNA significantly decreased the level of both interferon-gamma and tumor necrosis factor-alpha , suggesting that unmethylated CpG sequences in plasmid DNA play an important role. These data suggest that decreasing the CpG-mediated immune response while not affecting gene expression may be a useful therapeutic strategy to improve cationic lipid-mediated intravenous gene delivery to the lung.

cationic lipids; inflammation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RECENTLY, several groups (2, 9-11, 14, 15, 18, 19, 27, 29, 30, 36) have reported systemic gene expression by intravenous administration of cationic lipidic vectors. Several parameters have been identified as important for achieving a high level of gene expression. These include structures of cationic and helper lipids and the charge ratio between the cationic lipid and DNA (2, 9-11, 14, 15, 18, 19, 27, 29, 30, 36). The use of a cationic polymer such as protamine sulfate can also improve intravenous lipofection (14, 15). Although all organs can be transfected, the lung is the organ with the highest level of gene expression after intravenous administration. The cells that are transfected are primarily lung endothelial cells, although some monocytes are also transfected (9, 14, 19). This unique pattern of intravenous lipofection suggests the possibility of using cationic lipidic vectors to deliver a gene to treat pulmonary diseases. Indeed, intravenous delivery of a retinoblastoma (RB) tumor suppressor gene by lipid-protamine-DNA (LPD) resulted in a significant decrease in the number of pulmonary metastases in RB(+/-) mice (20). Delivery of a prostaglandin G/H synthase gene or a neutrophil inhibitory factor gene to pulmonary endothelial cells by cationic liposomes can also protect rabbit lungs from endotoxin injury (3, 35).

Despite this promise, the clinical applications of cationic lipidic vectors via intravenous administration are hampered by their toxicity, including the lethal effects in experimental animals (11). In addition, repeated injections are ineffective at frequent intervals. Typically, 7-14 days are required for recovery before a second injection can be effective (11, 27). Neither the toxicity nor the refractoriness to repeated injections is due to the cationic lipid itself because intravenous injection of cationic liposomes was not associated with lung injury and preinjection of cationic liposomes did not affect transfection of lungs by subsequent administration of cationic liposome-DNA complexes (11, 27). The toxicity is also not related to expressed gene products because injection of degraded DNA fragments formulated in lipidic vectors is as toxic as liposome-intact DNA complexes (11, 27). Clearly, toxicity is largely induced by cationic lipid-DNA complexes, yet the mechanism by which cationic lipid-DNA complexes generate toxicity remains unknown.

In this study, we demonstrate that cationic lipid-DNA complexes, but not each component alone, can induce the production of large quantities of proinflammatory cytokines that play an important role in complex-induced toxicity. This cytokine production is primarily triggered by unmethylated CpG sequences in the plasmid DNA. Thus decreasing the CpG-mediated immune response while not affecting gene expression may represent an important approach to improve cationic lipid-mediated intravenous gene delivery.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. 1,3-Dioleoyl-3-trimethylammonium propane (DOTAP) was purchased from Avanti Lipids (Alabaster, AL). Cholesterol was obtained from Sigma (St. Louis, MO). Protamine sulfate was from Eli Lilly (Indianapolis, IN). Luciferase assay kit was obtained from Promega (Madison, WI). Indocarbocyanine (Cy3)-dCTP and Cy5-dCTP were from Amersham (Arlington Heights, IL). All other chemicals were of reagent grade.

Plasmids. Plasmids pCMVL and pEGFP-N1, which contain, respectively, the cDNA of firefly luciferase and enhanced green fluorescence protein (GFP) driven by a human cytomegalovirus (CMV) immediate-early promoter, were amplified in the DH5alpha strain of Escherichia coli, isolated by alkaline lysis, and then purified by cesium chloride centrifugation (23). The purified DNA was further removed from endotoxin by passing it through a QIAGEN-tip 500 column (QIAGEN, Valencia, CA). The endotoxin level in all plasmids used in this study was <0.01 endotoxin unit/µg DNA.

Fluorescence labeling of plasmid DNA. Labeling of pCMVL with Cy3-dCTP or Cy5-dCTP was performed with the nick translation system (Promega). The labeled DNA was extracted with phenol-chloroform, precipitated with ethanol, and further purified by being passed through a Bio-Gel P-30 spin column (Bio-Rad Laboratories, Hercules, CA).

Plasmid methylation. pCMVL was methylated with Sss I CpG methylase (New England Biolabs, Beverly, CA) with 2 U enzyme/µg DNA for 2 h and incubated for an additional 2 h after the further addition of S-adenosylmethionine substrate with the use of buffer conditions suggested by the manufacturer. DNA was extracted with phenol-chloroform, precipitated with ethanol, and further purified by being passed through a Bio-Gel P-30 spin column (Bio-Rad Laboratories). The efficiency of methylation was confirmed to be >95% by enzymatic digestion.

Preparation of liposomes. Liposomes containing DOTAP in a 1:1 molar ratio with cholesterol were prepared as follows. The lipid mixture in chloroform was dried as a thin layer in a 100-ml round-bottomed flask that was further vacuum desiccated for 2 h. The lipid film was hydrated in 5% dextrose in water to give a final concentration of 10 mg DOTAP/ml. Preparation of small unilamellar vesicles by extrusion was performed as follows. The lipid solution was briefly sonicated, followed by incubation at 50°C for 10 min and then sequentially extruded through polycarbonate membranes with pore sizes of 1.0, 0.6, and 0.1 µm.

Preparation of LPD. For preparation of LPD, diluted DNA was added to the mixture of DOTAP-cholesterol liposomes and protamine at a final ratio of 0.6 µg of protamine to 12 nmol DOTAP to 1 µg of DNA (15). The mixture was incubated at room temperature for 10 min before use.

Simultaneous detection of Cy3-DNA and GFP in lungs after a single injection of LPD. Female CD-1 mice, 4-6 wk of age, were purchased from Charles River Laboratories (Wilmington, MA) and housed in accordance with institutional guidelines. The mice were injected intravenously with LPD containing 25 µg each of Cy3-labeled pCMVL DNA and pEGFP-N1 DNA. At different intervals after injection, mice were killed, and the lungs were perfused intravascularly with 2% paraformaldehyde and inflated with this solution to near total lung capacity. After the lungs were rinsed with cold PBS, the lungs were quickly frozen in optimum cutting temperature embedding medium with dry ice. Ten-micrometer lung cryosections were then prepared. To quantify the number of cells binding Cy3-tagged DNA or expressing the enhanced GFP encoded by pEGFP-N1 DNA, it was necessary to develop quantitative algorithms that identify DNA binding or expression at the single-cell level in sectioned material. Cells were defined by the presence of nuclei that were counterstained with the DNA-specific dye 4',6-diamidino-2-phenylindole. Nuclear profiles were collected with fluorescence microscopy (Olympus Provis) and imaged with an integrating Sony 3 chip color charge-coupled device camera, a Coreco frame grabber board (Coreco), and Optimas software (Optimas). The final images were collected with a ×40 Planapo objective, numerical aperture 1.0, with an image resolution of 700 × 600 pixels. The image was converted to gray scale and passed through a 3 × 3 Gaussian filter to remove noise, and nuclear profiles were extracted by selection of an appropriate threshold. The subsequent image was then converted to a binary image. Because EGFP expression is located in the cytoplasm and DNA binding and passaging are predominantly seen at the cell surface and in the cytoplasm, it was necessary to transform the nuclear image such that the EGFP and DNA association with each cell could be measured. We have found that a dilation of individual nuclear profiles such that these compartments are included was the most direct and simple method to do this. Thus Cy3 and EGFP images were collected as described above, and rendered to binary images. Subsequently, each binary image was compared with the nuclear images with an AND comparison, which will extract pixels that are only positive for the nuclear dye and either Cy3 or EGFP. For each specimen, ten randomly collected fields in the distal lung were collected.

Gene expression in the lung after repeated injections. pCMVL was used as a reporter gene in this experiment. Groups of five mice received intravenous administration of LPD at a dose of 50 µg DNA/mouse. At different times after the first injection, mice were given a second injection of LPD. Twenty-four hours later, mice were bled from the retroorbital sinuses under anesthesia and killed by cervical dislocation. Lungs were collected and washed twice with cold saline. Lungs were homogenized with lysis buffer (0.05% Triton X-100, 2 mM EDTA, and 0.1 M Tris, pH 7.8) with a tissue tearor (BioSpec Products, Bartlesville, OK). After two cycles of freezing and thawing, the homogenates were centrifuged at 14,000 g for 10 min at 4°C, and 20 µl of the supernatant were analyzed for luciferase activity with an automated LB 953 luminometer equipped with an automated injector (Berthod, Bad Wildbad, Germany). Gene expression is expressed as relative light units per milligram of protein.

Biodistribution of LPD in the lung after repeated injections. Groups of three mice received intravenous injection of LPD containing Cy5-labeled DNA at a dose of 25 µg/mouse. At indicated times after the first injection, mice were given an injection of LPD containing Cy3-labeled DNA at the same dose. Thirty minutes later, mice were killed. Lungs were fixed by cardiac perfusion with 2% paraformaldehyde in PBS, dissected out, inflated to normal size through the trachea with the same fixative and held in this condition, immersed in fixative for 1 more h, and then washed in 0.1 M PBS. Subsequently, the lung was cut into thick (5-mm) slices and cryoprotected by overnight immersion in 10% sucrose in PBS. Samples were then shock frozen in liquid nitrogen-cooled isopentane and stored at -80°C. Detection of Cy3- and Cy5-labeled DNA was performed as described in Simultaneous detection of Cy3-DNA and GFP protein in lungs after a single injection of LPD.

Immunoassays. At different times after the injection of free liposomes, free DNA, free protamine, or LPD, mice were bled from the retroorbital sinuses under anesthesia. The blood was allowed to stay at 4°C for 4 h and then centrifuged at 14,000 g for 10 min at 4°C. Serum was collected and kept at -80°C until used. Cytokine levels in mouse serum were determined with specific immunoassay kits for mouse interferon (IFN)-gamma and mouse tumor necrosis factor (TNF)-alpha (Genzyme, Cambridge, MA). Samples were analyzed in triplicate on a plate reader, and cytokine levels were calculated by linear regression analysis on the basis of values obtained from a standard curve. To test the effect of DNA methylation on cytokine production, a group of five mice was given 50 µg of methylated pCMVL formulated in LPD. Collection of serum samples and immunoassays were performed as described above. The result was compared with that with unmethylated DNA.

Effect of antibody treatment on intravenous lipofection. To test whether treatment of mice with antibodies against cytokines can prolong the duration of gene expression, a group of four mice received a daily intravenous administration of a mixture of hamster anti-IFN-gamma and anti-TNF-alpha antibodies (Genzyme), each at a dose of 100 µg/mouse for 3 days, starting the day before the administration of LPD. Control mice were treated with normal hamster IgG. At 2 and 3 days after injection of LPD (25 µg DNA/mouse), mice were killed and lungs were collected. Gene expression was assayed as described in Gene expression in the lung after repeated injections. To test the effect of antibody treatment on repeated injections, mice were treated with anti-cytokine antibodies as described above. Four days after the first injection of LPD (50 µg DNA/mouse), mice were given another injection of LPD at the same dose. Gene expression was assayed 24 h later as described in Gene expression in the lung after repeated injections.

Apoptosis assay. Groups of three mice were given intravenous administration of free liposomes, free protamine, free DNA, or LPD. Control mice received only 5% dextrose. Twelve hours after the injection, mice were killed. Lungs were collected and cyrosections were prepared as described in Simultaneous detection of Cy3-DNA and GFP in lungs after a single injection of LPD. Detection of apoptotic cells in each tissue section with ApopTag peroxidase kit (Oncor, Gaithersburg, MD) was performed according to a protocol described by the manufacturer.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transient gene expression is not due to rapid elimination of plasmid DNA from transfected cells. As shown in Fig. 1A, gene expression can be detected as early as 2 h after the injection of LPD. Gene expression peaked at 6 h and gradually declined thereafter. The fluorescence intensity in transfected cells is much weaker at 24 h compared with the fluorescence intensity at 6 h. This pattern of gene expression is similar to that when a luciferase plasmid was used as previously reported (14). To determine whether transient gene expression is due to rapid elimination of plasmid DNA from transfected cells, we simultaneously quantified DNA-Cy3- and GFP-positive cells at different times after the injection of LPD. As shown in Fig. 1B, the number of GFP-positive cells decreased rapidly, whereas the percentage of DNA-Cy3-positive cells remained at a high level over 24 h, suggesting that inactivation of gene expression is unlikely due to rapid elimination of plasmid DNA from transfected cells. We are aware of the possibility that the number of DNA-Cy3-positive cells might be overestimated due to the fact that Cy3 can still be detected even after DNA is degraded and/or inactivated inside cells. Indeed, this is a difficult limitation in the current technology in detecting intact DNA inside living cells.



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Fig. 1.   Simultaneous detection of indocarbocyanine (Cy3)-DNA and green fluorescence protein (GFP) in lungs after a single injection of lipid-protamine-DNA (LPD). Mice were injected intravenously with LPD containing 25 µg each of Cy3-labeled pCMVL DNA and pEGFP-N1 DNA. At different times after injection, mice were killed, lungs were collected, and cryosections were prepared. Simultaneous detection of Cy3-DNA and GFP in lungs was performed as described in MATERIALS AND METHODS. A: representative lung sections showing Cy3- and enhanced GFP (EGFP)-positive cells at 2 (a and d), 6 (b and e), and 24 h (c and f) after administration of LPD. B: summary of number of Cy3- and EGFP-positive cells in lung at different times after administration of LPD (n = 10 sections).

Repeated injections are not effective at frequent intervals. To test whether long-term gene expression can be achieved by repeated injections of LPD, mice were given a second injection at different times after the first administration of LPD, and gene expression was assayed 24 h later. The result is shown in Fig. 2. The second injection is not effective until 2 wk after the first injection. The length of refractory period is closely related to the dose of DNA. Decreasing the dose of DNA from 50 to 25 µg/mouse shortened the duration of unresponsiveness from 10-14 days to ~1 wk (data not shown).


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Fig. 2.   Gene expression in lung after repeated injections of LPD. Groups of 5 mice received intravenous injection of pCMVL-containing LPD at dose of 50 µg/mouse. At indicated times after 1st injection, mice were given a 2nd injection of LPD at same dose. Twenty-four hours later, mice were killed and lungs were collected. Lungs were homogenized in lysis buffer. Samples were centrifuged at 14,000 g for 10 min, and supernatant was assayed for luciferase activity and protein concentration; n = 5 animals. RLU, relative light units.

Treatment of mice with LPD does not affect the uptake of LPD by lungs after the second injection. To test whether the refractoriness to repeated injections was due to the inability of lungs to take up LPD on a second injection, we prepared two different LPDs containing Cy5- or Cy3-labeled DNA and injected both LPDs into the same mice at different intervals. The distribution of Cy5- and Cy3-labeled DNA in lungs is shown in Fig. 3. When the two LPDs were injected at short intervals, such as 0 or 30 min, Cy5- and Cy3-labeled DNAs were intensively colocalized in lungs of treated mice (Fig. 3, A and B). With the prolongation in the interval between the two injections, Cy3-labeled DNA became dominant in lungs of treated animals. Yet colocalization of the two plasmids was still observed even when the interval between two injections was 16 h (Fig. 3C). The above results suggest that treatment of mice with LPD does not affect the uptake of LPD by lungs after a second injection.


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Fig. 3.   Biodistribution of DNA in lung after repeated injections of LPD. Groups of 3 mice received intravenous injection of LPD containing Cy5-labeled DNA at dose of 25 µg/mouse. A: 0 min. B: 30 min. C: 16 h. D: 24 h. At indicated times after 1st injection, mice were given intravenous injection of LPD containing Cy3-labeled DNA at same dose. Thirty minutes later, mice were killed, lungs were collected, and lung cryosections were prepared. Detection of Cy3- and Cy5-labeled DNAs in lung sections was performed as described in MATERIALS AND METHODS.

Intravenous administration of LPD induces a high level of cytokine production. Figure 4A shows the level of IFN-gamma in mouse serum 5 h after the injection of free liposomes, free DNA, free protamine, protamine-DNA complexes, or LPD. Administration of LPD resulted in a high level of IFN-gamma production, whereas free liposomes, free DNA, free protamine, or protamine-DNA complexes had only a minimal effect in inducing the production of IFN-gamma . The LPD-induced IFN-gamma was detected as early as 30 min after injection, peaked at 5 h, and declined thereafter (Fig. 4B). IFN-gamma was hardly detectable in mouse serum 48 h after the administration of LPD (Fig. 4B). LPD also triggered a high level of TNF-alpha production (Fig. 5). Again, TNF-alpha is mainly induced by LPD complexes because each component alone was not effective in inducing TNF-alpha production. The kinetics of cytokine expression after intravenous administration of LPD is similar to that when CpG-containing phosphorothioate oligonucleotide was intraperitoneally injected into mice (34). Cationic lipid-DNA complexes without protamine are as active as LPD in inducing the production of IFN-gamma and TNF-alpha (data not shown).


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Fig. 4.   Interferon (IFN)-gamma levels in serum after intravenous injection of LPD. A: groups of 3 mice received intravenous injection of free liposomes, free protamine, free DNA, protamine-DNA complexes, or LPD. Control mice received only 5% dextrose. Six hours after injection, blood was collected from injected mice. B: in separate experiment, groups of 3 mice received intravenous injection of LPD at a dose of 50 µg DNA/mouse. At indicated times after injection, blood was collected from injected mice. IFN-gamma levels in serum were determined with immunoassay kit specific for mouse IFN-gamma (n = 3 animals).



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Fig. 5.   Tumor necrosis factor (TNF)-alpha levels in serum after intravenous injection of LPD. A: groups of 3 mice received intravenous injection of free liposomes, free protamine, free DNA, protamine-DNA complexes, or LPD. Control mice received only 5% dextrose. Two hours after injection, blood was collected from injected mice. B: in a separate experiment, groups of 3 mice received intravenous injection of LPD at dose of 50 µg DNA/mouse. At indicated times after injection, blood was collected from injected mice. TNF-alpha levels in serum were determined with an immunoassay kit specific for mouse TNF-alpha (n = 3 animals).

Intravenous administration of LPD induces apoptosis in lungs. Previous studies (8, 17) have shown that cytokines, especially TNF-alpha , can cause apoptosis of vascular endothelial cells including lung endothelial cells. To determine whether LPD-induced cytokines have a similar effect on lung endothelial cells, we collected lungs at different times after intravenous administration of LPD, and the presence of apoptotic cells was examined by indirect immunohistochemistry. The results are shown in Fig. 6. About 10% of the cells in lungs were identified to be apoptotic 12 h after intravenous injection of LPD. In contrast, apoptotic cells were barely detectable in lungs of mice treated with free liposomes, free DNA, or free protamine. This is in agreement with the fact that LPD, but not each component alone, induces a high level of cytokine production. DOTAP-cholesterol liposome-DNA complexes without protamine are as active as LPD in inducing apoptosis in the lung (data not shown).


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Fig. 6.   Detection of apoptotic cells in lungs after intravenous injection of LPD. Groups of 3 mice received intravenous administration of free liposomes, free DNA, free protamine, or LPD. Control mice were given 5% dextrose only. Twelve hours after injection, mice were killed and lungs were collected. Cryosections were then prepared, and presence of apoptotic cells was analyzed with an ApopTag peroxidase kit.

Treatment of mice with anti-cytokine antibodies improves intravenous lipofection by LPD. To determine whether gene inactivation after intravenous injection of cationic lipidic vectors is due to the toxic effect of cytokines, mice were given a daily intravenous administration of a mixture of anti-IFN-gamma and anti-TNF-alpha antibodies for 3 days, starting the day before the administration of LPD. Gene expression in the lung on days 2 and 3 was then evaluated and compared with that in mice treated with irrelevant IgG. The result is shown in Fig. 7. The levels of lung transfection in the mice treated with anti-cytokine antibodies were significantly higher than those in mice treated with irrelevant IgG (Fig. 7). Treatment of mice with anti-cytokine antibodies also improves lung transfection on repeated injections. As shown in Fig. 8, a low level of gene expression was found in the lungs of mice that received nonspecific antibody treatment. However, mice that received specific antibody treatment showed an ~20-fold increase in the level of lung transfection. The above results indicate that the two cytokines play an important role in inactivation of gene expression after intravenous injection of LPD.


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Fig. 7.   Effect of anti-cytokine antibody treatment on duration of gene expression after a single injection of LPD. A group of 4 mice received a daily intravenous injection of a mixture of hamster anti-mouse IFN-gamma and anti-mouse TNF-alpha antibodies, each at dose of 100 µg/mouse for 3 days, starting 1 day before 1st injection of LPD (25 µg DNA/mouse). Mice in control group received normal hamster IgG at a dose of 200 µg/mouse. At days 2 (D2) and 3 (D3) after injection of LPD, mice were killed and lungs were collected. Gene expression was assayed as described in Fig. 2. * P < 0.01 vs. group treated with irrelevant IgG.



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Fig. 8.   Effect of anti-cytokine antibody treatment on transfection of lungs after a 2nd injection of LPD. A group of 4 mice received a daily intravenous injection of a mixture of hamster anti-mouse IFN-gamma and anti-mouse TNF-alpha antibodies (Abs), each at a dose of 100 µg/mouse for 3 days, starting 1 day before 1st injection of LPD (50 µg DNA/mouse). Mice in control group received normal hamster IgG at a dose of 200 µg/mouse. Four days after 1st injection of LPD, mice were given another injection of LPD. Gene expression was assayed 24 h later as described in Fig. 2. * P < 0.001 vs. group treated with irrelevant IgG.

Methylation of plasmid DNA reduces the level of cytokine production. Recent studies (24, 33) have shown that the unmethylated CpG sequences in bacterial plasmid DNA are highly potent in inducing cytokine production including IFN-gamma and TNF-alpha . To determine whether LPD-induced cytokine production is also related to the unmethylated CpG sequences in plasmid DNA, pCMVL plasmid was randomly methylated by CpG methylase, and its ability to induce cytokine was then compared with that of unmodified DNA. The result is shown in Fig. 9. Methylation of plasmid DNA significantly decreased the level of both IFN-gamma and TNF-alpha . Sonicated pCMVL formulated in LPD triggered the cytokine production at a level similar to that of intact pCMVL.


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Fig. 9.   Effect of DNA methylation on LPD-induced cytokine production. Methylation of pCMVL by Sss I CpG methylase was performed as described in MATERIALS AND METHODS. A group of 5 mice was given intravenous injection of methylated pCMVL-containing LPD at a dose of 50 µg DNA/mouse. Mice in control group were given LPD containing unmodified pCMVL. Determination of IFN-gamma and TNF-alpha in mouse serum 3 h after injection was performed as described in Figs. 4 and 5.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Despite the potential of intravenous lipofection in treating lung tumor metastases (20) and other pulmonary diseases (3, 35), current vectors need to be improved to resolve two major problems: rapid decrease in gene expression and refractoriness to repeated injections at short intervals. Short duration of gene expression is a general problem for all nonviral vectors; however, gene expression declines much more rapidly in intravenous lipofection than in gene transfer via other routes of administration such as intratissue injection (4, 32). Furthermore, in intravenous lipofection, a duration of ~2 wk is required for lungs to recover before a second injection can be effective, another problem not shared by gene transfer via other routes of administration. These differences suggest a different mechanism of gene inactivation in intravenous lipofection compared with other gene transfer methods.

Biodistribution study (Fig. 3) suggests that unresponsiveness to repeated injections is unlikely due to the inability of lungs to take up lipid-DNA complexes on a second injection. It is also not likely due to lipid-induced damage to lung endothelial cells because injection of free liposomes alone did not prevent lung transfection after a second administration of cationic lipid-DNA complexes. In addition, Li and Huang (14) and others (27) have shown that intravenous injection of LPD, lipoplexes, or free liposomes does not generate noticeable necrosis in the lung. These studies suggest that inactivation of gene expression in lung endothelial cells might be caused by an indirect mechanism rather than by direct toxic effects of cationic lipid-DNA complexes on endothelial cells.

Immune responses have been shown to have an inhibitory effect on in vivo gene transfer. For example, a specific immune response mounted against vectors or gene products can cause elimination of transfected cells or even prevent repeated dosing. Recently, nonspecific immune responses have also been shown to have an inhibitory effect on gene expression (6, 7, 21). In particular, IFN-gamma and TNF-alpha could inhibit gene transcription or decrease the stability of mRNA, resulting in inactivation of gene expression (6, 7, 21). The inhibitory effect is more pronounced when the gene is driven by a viral promoter. Such an inhibitory effect was demonstrated in either an in vitro transfection or an in vivo setting where a viral vector was used. Therefore, it is important to determine whether nonviral vectors can also induce nonspecific immune responses and how these responses affect gene expression.

Results from this study clearly show that cationic lipidic vectors induce nonspecific immune responses: substantial amounts of IFN-gamma and TNF-alpha were induced after intravenous administration of LPD. In separate studies, Scheule et al. (25) and Freimark et al. (5) have shown that intratracheal administration of cationic lipid-DNA complexes also induces cytokine production. Cytokines might cause inactivation of gene expression through two different mechanisms. First, as discussed above, cytokines might directly inhibit transgene expression in lung endothelial cells. This was recently confirmed in an in vitro study where addition of IFN-gamma or TNF-alpha to cultured mouse lung endothelial cells inhibited lipofection even at doses that were not toxic to treated cells (Y. Tan and L. Huang, unpublished data). This is also supported by the fact that neutralization of cytokines by specific antibodies significantly prolonged the duration of gene expression (Fig. 7). Second, many studies (8, 17) have shown that endothelial cells, including lung endothelial cells, are highly sensitive to cytokines, particularly TNF-alpha . Exposure of endothelial cells to TNF-alpha could result in apoptosis in a dose-dependent manner in vitro and in vivo. We also demonstrated in this study that intravenous administration of LPD induces apoptosis of lung endothelial cells. Apoptosis is unlikely due to direct toxic effects from cationic lipids because intravenous administration of cationic liposomes alone did not cause any apoptosis in lungs. This is in agreement with the fact that injection of cationic liposomes alone generated only a low level of cytokine production. All of the results above suggest that cytokines play an important role in inactivation of gene expression in intravenous lipofection. Inactivation of gene expression either caused by direct inhibitory effect of cytokines or subsequent to cell apoptosis could be responsible for both the short duration of gene expression and the unresponsiveness of lungs to repeated injections at short intervals.

It is generally regarded that proinflammatory cytokines such as IFN-gamma and TNF-alpha are produced in immune cells including natural killer cells, T cells, and macrophages. Yet the active component that triggers the production of cytokines in intravenous lipofection remains unknown. Li et al. (16) recently showed that cationic lipidic vectors undergo dynamic changes in their biophysical characteristics after exposure to mouse serum. Serum initially causes aggregation of lipidic vectors (16). Prolonged interactions with serum lead to vector disintegration (16). Vector aggregates, due to their relatively large size, can be efficiently taken up by circulating immune cells. Yet the fact that liposomes also form aggregates in serum but are much weaker in inducing cytokine production suggests that aggregates per se are not the major component that is responsible for cytokine production. DNA formulated in lipidic vectors appears to play an important role in inducing cytokine production, although DNA alone is not active.

Recently, it has been shown (1, 12, 24, 26, 28, 33) that nucleic acids including oligodeoxynucleotides and plasmid DNA can serve as a strong immunogen if they are delivered to immune cells in an "intact" form. Further studies showed that the functional motifs in nucleic acids are the unmethylated CpG sequences. Interestingly, CpGs are usually unmethylated in bacterial DNA, whereas in mammalian DNA, ~75% of the CpGs are methylated to 5'-methylcytosine (22). This structural difference between bacterial and mammalian DNAs is a signal for the induction of innate immunity to microbial infections (13, 31). An unmethylated CpG dinucleotide flanked by two 5' purines (optimally a GpA) and two 3' pyrimidines (optimally a TpC or TpT) has strong stimulatory effects on murine and human lymphocytes in vitro and murine lymphocytes in vivo. These stimulatory effects include triggering B-cell proliferation; resistance to apoptosis; release of interleukin (IL)-6 and IL-12; natural killer cell secretion of IFN-gamma and increased lytic activity; and monocyte and/or macrophage secretion of IFN-alpha /beta , IL-6, IL-12, granulocyte-macrophage colony-stimulating factor, chemokines, and TNF-alpha (1, 12, 24, 26, 28, 33). Although these studies suggest that CpG-enriched nucleic acids can be used to purposely stimulate immune response in vaccination, they also point out a potential problem in the use of plasmid DNA in gene therapy. Indeed, the CpG-induced immune response inhibited gene expression in a dose-dependent manner when plasmid DNA was delivered by lipid 67 via intratracheal administration (S. H. Cheng, personal communication). Intratracheal administration of free plasmid DNA also induced the production of proinflammatory cytokines (5, 26), but the immune response was significantly enhanced when lipid-DNA complexes were used (5). To determine whether CpG sequences play an important role in inducing cytokine production in intravenous lipofection, we investigated the effect of methylation on cytokine production. As shown in Fig. 8, methylation of pCMVL resulted in a significant decrease in the level of cytokines. The decrease in cytokine production is not due to the lack of gene expression because intravenous delivery of sonicated plasmid DNA (which is already inactivated) by LPD could give rise to cytokine production at a level similar to that when intact plasmid DNA was used. DNA alone is not active in inducing cytokine production. This might be due to the fact that free DNA is rapidly degraded in blood (16). Random degradation of DNA by nucleases in the blood may result in a decrease in CpG contents. This is in agreement with an in vitro study (12) in which DNase-treated plasmid DNA is much weaker in inducing cytokine production compared with intact plasmid DNA.

In summary, intravenous administration of cationic lipidic vectors is associated, in addition to expression of transgene, with production of cytokines. Cytokines play an important role in inactivation of gene expression. Production of cytokines is mainly due to the unmethylated CpG sequences in plasmid DNA. Thus decreasing the CpG-mediated immune response while not affecting gene expression may represent an important approach to improve cationic lipid-mediated gene transfer.


    ACKNOWLEDGEMENTS

The first two authors contributed equally to this work.


    FOOTNOTES

The work was supported by National Cancer Institute Grants CA-59327, CA-64654, and CA-71731 (to L. Huang); National Heart, Lung, and Blood Institute Grant HL-32154 (to B. Pitt); National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44935; and a contract from Targeted Genetics Corporation (to L. Huang).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Huang, Dept. of Pharmacology, Univ. of Pittsburgh School of Medicine, W1351 Biomedical Science Tower, Pittsburgh, PA 15261 (E-mail: leaf{at}prophet.pharm.pitt.edu).

Received 20 November 1998; accepted in final form 12 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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