1Unité Mixte de Recherche 7001 Centre National de la Recherche Scientifique/Ecole Nationale de Chimie de Paris/Aventis PharmaGencell Société Anonyme, 94403 Vitry-sur-Seine Cedex, and 2Unité de Pharmacologie CellulaireInstitut Pasteur, 75015 Paris, France
Submitted 25 February 2003 ; accepted in final form 9 September 2003
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ABSTRACT |
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murine interleukin-10; gene transfer; gene therapy; electroporation; lipopolysaccharide; lung inflammation
In earlier studies, we showed that the intra-airway administration of LPS to mice induces inflammation with neutrophil recruitment to lungs and to the bronchoalveolar lavage fluid (BALF), the generation of TNF- in the BALF, and bronchoconstriction (BC), which starts 7090 min after the delivery of LPS and peaks around 3 h, followed by BHR in response to aerosolized methacholine (23, 24). By contrast, the systemic administration of LPS induces neutrophil margination to the endothelium in the absence of migration to the parenchyma and, a fortiori, to the BALF. Even though no BC is noted, BHR is present (23). Pharmacological responses also distinguish the effects of intranasal from those of systemic LPS, since the glucocorticosteroid dexamethasone inhibits BHR induced by the intravenous administration of LPS only at or above the relatively high dose of 5 mg/kg, whereas the production of TNF-
is suppressed at doses as low as 0.625 mg/kg (24). By contrast, when LPS was administered into the airways, dexamethasone only marginally affected BHR, whatever the doses used. This shows that some effects of LPS are corticoresistant, a property reminiscent of their ineffectiveness in suppressing septic shock, and particularly one of its major consequences, acute respiratory distress syndrome (20).
IL-10 is an anti-inflammatory cytokine (28) that, once injected intravenously to mice, reduces grain dust-induced airway inflammation and BHR (32). It suppresses allergen-induced lung inflammation, i.e., eosinophil recruitment and T helper 2 cytokine production (38). The IL-10 cytokine is produced during experimental sepsis and might thus show protective anti-inflammatory effects by downregulating the production of proinflammatory cytokines (35). For those reasons, we studied the interference of exogenous murine IL-10 (mIL-10) and of endogenously produced mIL-10 with LPS-induced inflammation. We have previously shown that electrotransfer allows for an intense secretion of transgenic proteins into the murine circulation (4, 21, 30) and that the electrotransfer of muscle tissue with a plasmid coding for mIL-10 results in the presence of free mIL-10 in the circulation for more than a week (7), i.e., it is much more prolonged than after the intravenous injection of recombinant mIL-10 [rmIL-10; half-life (t1/2) <1 h] (18). We presently demonstrate that mIL-10 can reduce LPS-induced airways and lung inflammation, but only when it is present in the airways.
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MATERIALS AND METHODS |
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LPS (Escherichia coli 055:B5) was from Difco Laboratories (Detroit, MI). Hexadecyltrimethylammonium bromide (HTAB), EDTA, O-dianisidine dihydrochloride, and hydrogen peroxide (H2O2) were from Sigma Chemical (St. Louis, MO). Hanks' balanced salt solution (HBSS) was from GIBCO Life Technology (Paisley, UK). Methacholine (acetyl--methylcholine chloride) was from Aldrich-Chemie (Steinheim, Germany). Murine recombinant TNF-
was from Biosource International (Camarillo, CA), and rmIL-10 was from Immugenex (Los Angeles, CA).
Plasmids and DNA Preparation
The pCOR mIL-10 plasmid (pXL 3458) was constructed as previously described (36). It contains the cytomegalovirus (CMV) promoter (nucleotide -522/+72) inserted upstream of the coding sequence of the mIL-10. The simian virus 40 late poly(A)+ signal was placed downstream of the mIL-10 cDNA. The plasmid pXL 3296 (empty pCOR) is the similar construct devoid of coding sequence. Plasmids were prepared using standard procedures (3, 33).
General Experimental Procedure
Mice were electrotransferred (see Muscle Transfection by Electrotransfer of mIL-10-Encoding Plasmid) with the plasmid pCMV mIL-10 or with the empty plasmid and/or treated with rmIL-10. Treated and control mice were then exposed to LPS (aerosol or intravenously or intraperitoneally). Last, bronchial hyperactivity was evaluated from BC induced by methacholine.
Muscle Transfection by Electrotransfer of mIL-10-Encoding Plasmid
Six- to eight-week-old female C57Bl/6 mice (IFFA Credo, L'Isle d'Arbesle, France) were anesthetized using ketamine and xylazine (113.75 mg/kg and 3.5 mg/kg, respectively), and both legs were shaved. DNA (0.4 or 40 µg in 30 µl of 0.9% NaCl) was injected in the tibial cranial muscle, longitudinally, by the means of a Hamilton syringe. About 20 s after DNA injection, an electric field was applied, as previously described (26, 27). Briefly, the transcutaneous electric pulses (8 square-wave electric pulses of 200 V/cm, 20 ms each, at 2 Hz) were applied through two stainless steel plate electrodes placed 3.74.2 mm apart at each side of the leg. Electrical contact with the shaved leg skin was ensured by means of conductive gel. Electric pulses were generated by an ECM 830 BTX electropulsator (Genetronics, San Diego, CA). A minimum of four mice was included in each experimental group.
Animal experiments followed the recommendations for animal experimentation of the National Institutes of Health and Aventis Local Ethic Committee on Animal Care and Experimentation.
Administration of rmIL-10
rmIL-10 was administered intranasally (1 µg/50 µl) or intraperitoneally (13 µg/50 µl).
LPS Challenge
In the first series of experiments, LPS was administered intranasally (10 µg/50 µl). In other experiments, 6- to 8-wk-old male or female C57Bl/6 mice (IFFA Credo or Centre d'Elevage R. Janvier, Le Genest Saint-Isle, France) were placed in a glass container that allowed aerosolization of LPS (0.3 or 3 mg/ml in 0.9% NaCl solution, saline) or saline (10 ml, 10 min) using an Aldrich apparatus. In the last series of experiments, mice were injected intraperitoneally or intravenously with either LPS (330 µg/kg body wt) or an equivalent volume of saline, the solvent for LPS.
Determination of BC and BHR
Unrestrained conscious mice, preexposed to LPS (see LPS Challenge), were placed in a whole body plethysmographic chamber (Buxco Electronics) to analyze the respiratory waveforms. After 4 min of stabilization, mice received methacholine by aerosolization for 20 s (100 mM in the aerosolator's reservoir). The resulting BC was expressed as Penh = [Te (expiratory time)/40% of Tr (relaxation time) - 1] x Pef (peak expiratory flow)/Pif (peak inspiratory flow) x 0.67 according to the manufacturer's instructions. Results were expressed as Penh, corresponding to differences between the basal and maximal values.
Analysis of Inflammation
Cells in BALF. To collect BALF, animals were anesthetized with 50 mg/kg ip of urethane, the tracheae were cannulated, and the lungs were washed five times with 0.5 ml of saline to provide 2.2 ml of BALF. Aliquots were used to count the total cell numbers using a Coulter Counter (model ZBI; Coulter Electronics, Hialeah, FL). Differential cell numbers were assessed by morphological analysis of cells after cytocentrifugation of BALF aliquots and May-Grünwald-Giemsa coloration.
Measurement of TNF-. BALF supernatants were collected on ice, and TNF-
levels were determined within 23 h. Blood was also collected, serum was prepared by centrifugation, and TNF-
in the serum was measured within 23 h by an enzyme immunometric assay. This method relies on the reaction of thiol groups of monoclonal antibody (MAb) Fab' fragments with maleimido groups previously introduced into acetylcholinesterase (AchE) as previously described (12). Anti-murine TNF-
MAb MP6-XT22 and MP6-XT3 were purified from ascetic fluids (cloned hybridomas kindly provided by Dr. P. Minoprio, Institut Pasteur, Paris, France) using the affinity chromatography method on a protein G column (HiTrap affinity columns; Pharmacia Biotechnology, Uppsala, Sweden) after precipitation by ammonium sulfate. Characteristics of these rat anti-murine TNF-
MAb were described in detail elsewhere (1).
Immunometric assays were performed in 96-well microtiter plates (MaxiSorp; Nunc, Roskilde, Denmark) coated with 10 µg/ml of the rat anti-murine TNF- MAb MP6-XT3, as described previously (31). The one-step procedure used for immunometric assays involved the simultaneous addition of 100 µl of TNF-
standards (15.61,000 pg/ml) or samples and 100 µl of the second rat anti-murine TNF-
MAb MP6-XT22-AchE conjugated at a concentration of 10 U/ml of Ellman's medium. After being incubated for 18 h at 4°C, the plates were extensively washed, and solid phase-bound AchE activity was determined colorimetrically by adding 200 µl of Ellman's medium. Absorbance was read at 405 nm with an automatic microplate reader (Dynatech MR 5000; Dynatech Laboratories, Saint-Cloud, France). The lower limit of detection of this assay was
15 pg of TNF-
/ml sample.
Determination of lung myeloperoxidase activity. After bronchoalveolar lavage, lung vessels were flushed to discard circulating blood. Thus, the left atrium was open, and 10 ml of saline were gently perfused into the right ventricle. The lungs were then removed from the thorax, blotted with gauze to remove blood, and frozen at -20°C until assay. Lung tissue myeloperoxidase (MPO) activity was determined following a previously described method (15) with minor modifications. Collected lungs were homogenized for 30 s (Potter-Elvehjem glass homogenizer; Thomas, Philadelphia, PA) at 4°C in 1 ml of PBS. The corresponding extracts were centrifuged (10,000 g, 10 min, 4°C), and supernatants containing hemoglobin were discarded. The pellets were resuspended in 1 ml of PBS supplemented with HTAB (0.5%) and EDTA (5 mM) and homogenized again. After centrifugation, 50 µl of supernatants were placed in a test tube with 200 µl PBS-HTAB-EDTA, 2 ml HBSS, 100 µl O-dianisidine dihydrochloride (1.25 mg/ml), and 100 µl H2O2 (0.05% = 0.4 mM). After 30 min of incubation while shaking at 37°C, the reaction was stopped by the addition of 100 µl of NaN3 (1%). The MPO activity was determined as change in absorbance at 460 nm.
Measurement of mIL-10 Concentrations
Peripheral blood samples were collected from retroorbital sinus or from intracardiac puncture at different time points. Concentrations of circulating mIL-10 protein were measured using a commercial ELISA kit specific for mIL-10 (Biosource International) according to the manufacturer's instructions.
Reactivity of Lung Tissue to Inflammatory Stimulation
Mice were treated with pCMV mIL-10 or with empty plasmid electrotransfer. Two days later, they were instilled intranasally with 1 or 3 µg of rmIL-10 protein. One hour later, lungs were washed with PBS and removed as described in Determination of Lung Myeloperoxidase Activity. Parenchyma was cut in 18 regular fragments that were distributed in a 24-well plate. Fragments were cultured in AIM V medium (Life Technologies, Grand Island, NY) containing L-glutamine (1%, Life Biotechnologies) and gentamicin (10 µg/ml, Sigma). In some cases, rmIL-10 was added to the medium (100 ng/ml final concentration). One hour later, lung fragments were challenged with LPS (1 µg/ml final concentration). After 5 h at 37°C under a humidity-saturated atmosphere containing 5% CO2, supernatants were collected for TNF- measurement.
Statistical Analysis
Variance analysis on log values of the measured parameters and a protected least significance test of Fisher for comparison between treatments have been used. Results have been presented with linear scale for clarity.
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RESULTS |
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As shown in Fig. 1, lung resistance after the intranasal instillation of LPS (10 µg/mouse, 50 µl) started to increase by 6070 min, peaked by 2 h, and persisted for 3 or more hours. When rmIL-10 was instilled with LPS, lung resistance peaked at lower values (P < 0.05) and returned to basal levels by 160 min. Aerosolized methacholine (100 mM, 20 s) induced a submaximal BC in naive mice, whereas in those exposed to LPS alone, the responses were enhanced (BHR, data not shown). BHR was next evaluated 24 h after LPS challenge. In this group of animals, Penh was calculated for 3 min, 3 h after LPS to confirm that intranasal rmIL-10 protects mice from BC (P < 0.001, Fig. 2A). BHR, in response to methacholine, was suppressed in animals that received LPS with rmIL-10 24 h earlier (P < 0.0001 at the time of peak value, Fig. 2B). The intranasal administration of LPS induced the recruitment of neutrophils to the BALF and their sequestration in the lungs, as shown by the increased MPO titers (Fig. 3A). This recruitment was decreased when rmIL-10 was associated with LPS (P < 0.05). Finally, the TNF- content in BALF increased when mice were exposed to LPS alone and remained at background levels if rmIL-10 was administered with LPS (P < 0.0001, Fig. 3B). Twenty-four hours later, MPO showed values similar to those observed 3 h after LPS challenge (Fig. 4) and was also only partially reduced in mice exposed to intranasal rmIL-10 with LPS.
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In all these experiments, pretreatment of mice by an intraperitoneal injection of rmIL-10 (3 µg the day before LPS challenge) did not improve the effect of rmIL-10 administered intranasally with LPS (data not shown).
In Vitro and In Vivo Administration of rmIL-10 Prevents LPS-Induced Production of TNF- by Lung Fragments: Effect of mIL-10-Encoding Plasmid Electrotransfer
In vitro stimulation of lung fragments with LPS induced TNF- production after 5 h that was inhibited in the presence of rmIL-10 (P < 0.001). In addition, when mice received rmIL-10 (1 µg) intranasally, TNF-
production induced by subsequent in vitro stimulation with LPS was also inhibited (P < 0.001). Nevertheless, in vitro addition of rmIL-10 (100 ng/ml) before in vitro challenge with LPS failed to further reduce the production of TNF-
(Fig. 5). In another experiment (not shown), plasmid electrotransfer was performed. Empty plasmid did not modify the LPS-induced production of TNF-
by lung fragments. This result demonstrated that electrotransfer by itself had no effect on inflammation. mIL-10-encoding plasmid was also electrotransferred. LPS-induced TNF-
secretion by fragments of lungs provided by animals of this group was as low as that of naive mice. Accordingly, when administered into the airways (intranasal administration), rmIL-10 reduced LPS-induced pulmonary inflammation. By contrast, no protective effect was observed after the electrotransfer of mIL-10-encoding plasmid.
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Effects of Transgenic Circulating mIL-10 and Airway rmIL-10 in LPS-Challenged Mice
LPS administration by nebulization. Two days after the electrotransfer of mIL-10-encoding plasmid, elevated titers of circulating mIL-10 were found. However, it failed to inhibit BC and BHR induced by the nebulization of LPS (3 mg/ml or 0.3 mg/ml, 10 ml, 10 min). Similarly, lung MPO and TNF- levels in the BALF were not significantly modified in treated mice challenged with both doses of LPS (Table 1). Paradoxically, only the total number of neutrophils in the BALF was reduced in experiments using the high dose of LPS (P < 0.05). Accordingly, high levels of mIL-10 secreted into the circulation 2 days after the electrotransfer of the mIL-10-encoding plasmid had no protective effect on LPS-induced BC, BHR, and inflammation.
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Because in those experiments LPS was delivered 2 days after electrotransfer, neutrophils and macrophages might not have been exposed to mIL-10 for a sufficient length of time. Accordingly, LPS (3 mg/ml, 10 ml, 10 min) was nebulized 15 days after the electrotransfer of mIL-10-encoding plasmid. Circulating mIL-10 produced by the electrotransferred plasmid protected mice from BHR (P < 0.01) but did not significantly reduce BC or lung MPO, and neutrophil recruitment was not modified (Table 2). TNF- secretion in the BALF was markedly reduced (P < 0.01) only when mice were pretreated with rmIL-10 itself (1 µg intraperitoneally 18 h before LPS and 1 µg intranasally 1 h before LPS). This inhibition probably resulted from the rmIL-10 delivered intranasally, since in those experiments and in others, we observed that systemic rmIL-10 had no suppressive effect. BHR in response to methacholine was also reduced but not significantly.
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LPS administration by systemic routes. The systemic administration of LPS to mice also induces BHR (24). Accordingly, we studied whether mIL-10 would prevent this effect. Elevated amounts of circulating mIL-10 were generated after the mIL-10 plasmid electrotransfer (Tables 1 and 3). Two days later, LPS was injected intraperitoneally (330 µg/kg, 100 µl). No statistically significant protective effect of electrotransfer was observed against the lung recruitment of granulocytes (MPO) or against BHR. In the presence of mIL-10 generated in the blood by electrotransfer, circulating TNF- titers were markedly decreased (P < 0.05). It is important to note that intraperitoneal LPS led to the endogenous production of mIL-10, since mice electrotransferred with empty plasmid had up to 550 pg/ml of mIL-10 in their blood. As expected (24), no neutrophil recruitment to the BALF was observed when LPS was administered intraperitoneally. The total number of lung cells (essentially macrophages) was not modified by electrotransfer of mIL-10-encoding plasmid (Table 3).
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Mice treated with rmIL-10 intraperitoneally (1 µg 18 h before LPS) and intranasally (1 µg 1 h before LPS) expressed a small reduction of BC and of BHR, associated to a more expressive reduction of circulating TNF- titers (data not shown).
In subsequent experiments, two doses (0.8 and 80 µg/mouse) of mIL-10 plasmid were electrotransferred, LPS having been injected intravenously (Table 4). This experiment confirmed the induction of circulating mIL-10 by LPS. Moreover, mIL-10 titers increased further after the electrotransfer of 80 µg of mIL-10 plasmid/mouse, resulting in a significant decrease in the blood titers of TNF- (P < 0.01). Electrotransfer of 0.8 µg of mIL-10 plasmid did not affect the circulating levels of mIL-10 and failed to reduce the blood titers of TNF-
. Finally, whatever the amount of plasmid used, neither the increased MPO activity nor the BHR was reduced.
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The same experiment was performed using LPS injected intraperitoneally (not shown). In this experiment, we confirmed that the electrotransfer of 0.8 µg of mIL-10 plasmid did not affect the circulating titers of mIL-10 and TNF-. However, BHR was reduced. After the electrotransfer of 80 µg of mIL-10 plasmid, blood mIL-10 titers reached a high value of
6 ng/ml. In this case, BHR was not prevented, and, paradoxically, blood TNF-
was increased twofold compared with untreated mice.
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DISCUSSION |
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As in the case of intranasal instillation of 10 µg of LPS, its aerosolization at 3 mg/ml (10 ml, 10 min) induced BC, BHR, and neutrophil recruitment to the BALF and sequestration to the lungs as well as TNF- production in BALF. Under those conditions as well (see RESULTS), the intranasal administration of rmIL-10 reduced BC and BHR, but not significantly. However, MPO titers were not reduced by this treatment, as they were with respect to the intranasal delivery of LPS.
The results of the experiments in which LPS was administered intranasally or by aerosol are probably not comparable. Indeed, it is likely that the aerosolization of LPS reaches the alveolar compartment more effectively and that, as a consequence, rmIL-10 administered intranasally might be less effi-cient against aerosol than against intranasal LPS. It is important to note that when rmIL-10 was administered intranasally in the first experiment together with LPS, the MPO titers, both after LPS as well as after LPS plus rmIL-10, were higher than after LPS aerosolization. In agreement with both in vivo experiments, TNF- secretion into the culture medium was markedly suppressed when rmIL-10 was administered intranasally 1 h before in vitro stimulation of lung explants with LPS.
We attempted unsuccessfully to enhance the inhibition of the effects of LPS (BC, BHR, TNF-, and MPO titers) by associating systemically delivered and intranasal rmIL-10. However, the additional pretreatment by systemic administration of rmIL-10 did not allow the improvement of inhibitory effects of intranasal rmIL-10 (1 µg). This indicates that circulating rmIL-10 at the dose used has a poor effect. However, the half-life of rmIL-10 is short (<1 h in blood) (18), and, accordingly, we decided to study whether the electrotransfer of a plasmid coding for mIL-10, which induces a prolonged presence of free mIL-10 in the circulation (7), would be more effective. Clearly, the electrotransfer of mIL-10-encoding plasmid also failed to significantly inhibit lung inflammation when performed 2 days before challenge with LPS administered intraperitoneally, intravenously, or by aerosol. In those experiments, only TNF-
production in the blood was inhibited, even though at this time the circulating levels of mIL-10 peaked (7), thus validating the experiment. Accordingly, the TNF-
production by LPS-stimulated lung explant was not reduced. This indicates that circulating mIL-10 was not able to prevent the activation of the involved lung cells or perhaps even failed to reach them.
Others (32) have shown that the intravenous injection of rmIL-10 to mice reduces grain dust-induced airway inflammation and BHR, but the amounts of mIL-10 injected were very high (50 µg/kg, i.e., >300 ng/ml of blood). In that study (32), the authors showed that a lower dose of mIL-10 (10 µg/kg) failed to prevent lung inflammation induced by LPS. Adenoviral vector-mediated IL-10 expression was shown to reduce LPS-induced TNF- mRNA expression in the mouse lung 1 day after transfection (37). In this study, the levels of circulating mIL-10 at this time point were >50 ng/ml, an amount comparable to the unsuccessful injection of 10 µg/kg iv reported by Quinn et al. (32). However, transfection methods allow induction of the presence of mIL-10 in the circulation for longer periods of time. It is possible that, with elevated amounts of circulating mIL-10, the slight amount that would reach the airways becomes sufficient to show effectiveness. In other words, with high doses, the authors possibly observed the effect of airway mIL-10 and not of the circulating mIL-10.
At day 15 after electrotransfer, the levels of circulating mIL-10 were low, and TNF- produced in lung airways after aerosol of LPS was not inhibited. As in earlier experiments, 2 days after electrotransfer, the neutrophil counts in the BALF and MPO were not reduced. However, BHR to methacholine was clearly and significantly reduced. In this case, exposure of inflammatory cells to mIL-10 should have been more effective and/or these exposed cells would have had time to migrate to the airways. This also indicates that TNF-
in the airway, which is not reduced, is not involved in BHR or that target cells are inactivated. More broadly, there is no direct relationship between BHR and lung inflammation. Reciprocally, we have shown that dexamethasone reduces LPS-induced production of TNF-
in the airways, but not BHR (23). Thus TNF-
and related inflammatory cytokines seem not to be implicated in BHR. However, some relationship between inflammatory cells and BHR should exist since depletion of neutrophils suppresses LPS-induced BHR (23). Another cytokine, IL-17, was shown to have a role in LPS-induced airway neutrophilia (10). However, it triggers the second phase of LPS-induced lung neutrophil recruitment, occurring at day 2 after challenge, whenever BHR is detected, as soon as 1 h after LPS challenge. In addition, IL-10 would have no suppressive effect on the production of this cytokine (5).
Difficulties in demonstrating inhibitor effects of circulating mIL-10 may also be linked to its immunostimulatory properties, which have been shown in human subjects (22). However, the dose of IL-10 injected intravenously was very elevated (25 µg/kg).
In conclusion, this study shows that IL-10 suppresses LPS-induced lung inflammation and BHR when delivered into the airway compartment, with the inhibition of BHR being independent from the concomitant reduction of TNF- generation in the airways. Circulating mIL-10 did not prevent activation of lung cells or possibly did not reach them. Even when the blood titers of mIL-10 were elevated 2 days after mIL-10 plasmid electrotransfer, very little mIL-10 was found in the BALF, supporting the concept that the presence of IL-10 in the airway compartment is essential. Consequently, it would be better to stimulate the generation of endogenous anti-inflammatory mediators, such as IL-10, in the airway compartment where inflammation and pathology develop. Thus mIL-10 gene transfer directly to the airway should be the most efficient procedure. This might possibly be achieved by aerosolization of DNA/cationic lipid complexes (8) or by adeno-associated virus (2, 11, 13, 14). However, when LPS challenge into the airway was performed 15 days after mIL-10-encoding plasmid electrotransfer, lung inflammation was also not reduced, but BHR was reduced. After such a delay, does vascular IL-10 have time to reach and modify cells of the lung tissue? Alternatively, the inhibitory effect on BHR might be indirect via the migration to the airway of IL-10-treated blood leukocytes, like monocytes. Further studies of such questions could help to determine efficient treatment of BHR.
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ACKNOWLEDGMENTS |
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Present address of M. F. Bureau: Unité de Pharmacologie Chimique et Génétique, Centre National de la Recherche Scientifique FRE 2463, Faculté de Pharmacie, 4 av de l'Observatoire, 75270 Paris Cedex 06, France.
GRANTS
V. Deleuze was supported by a grant from the Centre National de la Recherche Scientifique and from Gencell S. A.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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