Published ahead of print on July 19, 2007, doi:10.1165/rcmb.2007-0067OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0067OC Divergent Effects of Biolistic Gene Transfer in a Mouse Model of Allergic Airway Inflammation1 Department of Dermatology, Clinical Research Unit Allergology; 2 I. Medical Clinic, Asthma Core Facility SFB 548; and 3 III. Medical Clinic, University of Mainz, Mainz, Germany Correspondence and requests for reprints should be addressed to Stephan Sudowe, Ph.D., Clinical Research Unit Allergology, Department of Dermatology, Johannes Gutenberg-University Mainz, Obere Zahlbacher Str. 63, D-55131 Mainz, Germany. E-mail: sudowe{at}mail.uni-mainz.de
Particle-mediated epidermal delivery (PMED) of allergen genes efficiently prevents systemic sensitization and suppresses specific immunoglobulin E synthesis. We investigated in a mouse model of allergic airway disease the effect of PMED on the elicitation of local inflammatory reactions in the lung. BALB/c mice were biolistically transfected with plasmids encoding β-galactosidase (βGal) as model allergen under control of the DC-targeting fascin promoter and the ubiquitously active cytomegalovirus promoter, respectively. Mice were challenged intranasally with βGal-protein with or without intermediate sensitization with βGal adsorbed to aluminiumhydroxide. Subsequently, local cytokine production and recruitment of IFN-  –producing CD8+ effector T cells into the airways were determined, and inflammatory parameters such as cellular infiltration in the bronchoalveolar lavage (BAL) and airway hyperresponsiveness (AHR) were measured. PMED of βGal-encoding plasmids before sensitization significantly reduced frequencies of eosinophils in the BAL and shifted the local T helper (Th) cell response from a distinct Th2 response toward a Th1-biased response. However, AHR triggered by allergen challenge via the airways was not alleviated in vaccinated mice. Most important, we show that PMED using βGal-encoding DNA without subsequent sensitization recruited Tc1 cells into the lung and caused a Th1-prone local immune response after subsequent intranasal provocation, accompanied by neutrophilic infiltration into the airways and elicitation of AHR. We conclude that robust Th1/Tc1 immune responses, although highly effective in the counter-regulation of local Th2-mediated pathology, might as well trigger local inflammatory reactions in the lung and provoke the induction of AHR in the mouse model of allergic airway disease.
Key Words: bronchial asthma • immunologic model • DNA vaccines • gene gun technique • Th1/Tc1 cells
Allergen gene transfer (i.e., transfection of somatic cells with allergen-encoding DNA vectors) has been shown in animal models to be effective in the suppression of specific immunoglobulin (Ig)E production (1–9), a crucial event in the pathogenesis of allergic diseases (10). Needle injection via the intramuscular (2, 3, 5) or the intradermal (1, 4, 6) route is the mode of DNA application that was almost exclusively used for successful intervention in these experimental studies. Recently we demonstrated in a mouse model of type I allergy for the first time that an alternative method of DNA vaccination—namely, particle-mediated epidermal delivery (PMED) of allergen-encoding plasmids using the gene gun device—prevented allergic sensitization and interfered with the establishment of systemic T helper cell (Th)2 responses (8, 9). Moreover, PMED transiently protected presensitized mice against the boost in IgE production caused by subsequent challenge with allergen (9). The suppressive effect of gene gun–mediated DNA vaccination was exerted irrespective of the use of plasmid vectors encoding allergen genes under control of the promoter of the murine fascin gene (pFascin) or the ubiquitously active cytomegalovirus (CMV) promoter (pCMV). We have previously shown that biolistic transfection with pFascin resulted in gene expression preferentially targeted to DC (11) and elicited a strong systemic Th1 response, whereas particle bombardment with pCMV initiated a mixed Th1/Th2 response (12). In addition, biolistic transfection with pFascin as well as pCMV generated large numbers of antigen-specific IFN- –producing CD8+ T cells, displaying a typical Tc1 cell phenotype, in the spleen (11, 12). In contrast to substantial knowledge of the effects of vaccination with allergen-encoding plasmid DNA on the systemic allergen-specific response, the impact of DNA vaccination on the elicitation of local allergen-specific inflammation, as being manifest in allergic asthma, has rarely been investigated. Because asthmatic symptoms such as infiltration of eosinophils into the bronchial tissue and the airway lumen as well as the elicitation of airway hyperreactivity (AHR) are orchestrated by allergen-specific Th2 cells in the lung (13), down-regulation of Th2 responses appears to be a logical rationale also for asthma immunotherapy. Therefore, we investigated the effect of PMED in a mouse model of allergic airway inflammation. Given the differences in systemic immune responses elicited by biolistic transfection with pCMV and pFascin, respectively, we compared the efficiency of PMED using the two vectors to inhibit the recruitment of allergen-specific Th2 cells into the lung and to alleviate bronchial inflammation after allergen challenge. We show that administration of pFascin and pCMV, respectively, led to considerably reduced local Th2 cytokine production, which correlated with substantially decreased frequencies of eosinophils in the bronchoalveolar lavage (BAL). In contrast, PMED without subsequent sensitization induced potent pulmonary Th1/Tc1 responses as well as neutrophilic infiltration into the lung and AHR after provocation with allergen.
Mice and Sensitization Protocol Female BALB/c mice were bred and maintained in the Central Animal Facilities of the University of Mainz under specific pathogen–free conditions on a standard diet. The "Principles of Laboratory Animal Care" (NIH publication no. 86–23, revised 1985) were followed. The experiments were approved by the Ethics Commission according to the German Law on the Protection of Animals. The basic immunization protocol for the induction of AHR and airway inflammation was the following: 6- to 8-week-old mice were sensitized on Days 0 and 10 by intraperitoneal injection of 1 µg recombinant β-galactosidase (βGal; Sigma-Aldrich, Deisenhofen, Germany), dissolved in 0.1 ml PBS, and adsorbed to an equal volume of Imject alum (Pierce, Rockford, IL) as adjuvant. On Days 17, 18, and 19, βGal-protein (1 mg/ml in PBS) was applied intranasally in a volume of 0.05 ml to mice that were anesthetized by intraperitoneal injection of 0.2 ml Avertine (1 g/ml of tribromoethylalcohol in tertiary amylalcohol, diluted 1:40 in PBS). Control mice received PBS alone intranasally. Subsequently sera were recovered, airway reactivity was determined, and mice were killed to prepare lungs and bronchial lymph nodes (LN) and to perform BAL.
Plasmid Vectors and DNA Vaccination Genetic immunization was performed by biolistic transfection of plasmid DNA (4 µg) using the helium-driven Helios gene gun system (Bio-Rad, Munich, Germany) as described (8). In the airway inflammation model mice were vaccinated before sensitization with βGal/alum by three consecutive administrations of plasmid DNA (Days –21, –14, –7). Likewise, to assess whether gene gun–mediated DNA immunization primed for airway inflammatory reactions, mice were treated by three weekly administrations of plasmid DNA and challenged 1 week later by intranasal application of βGal-protein as described.
Determination of βGal-Specific Serum Ab Titers
Assessment of Airway Function Alternatively, airway responsiveness was assessed by forced oscillation method (15). Anesthetized (pentobarbital sodium, 70–90 mg/kg intraperitoneally), tracheostomized (18G cannula) mice were placed on a computer-controlled piston ventilator (flexiVent; SCIREQ Inc., Montreal, PQ, Canada) and were ventilated at a tidal volume of 0.2 ml with a positive end-expiratory pressure of 5 cm H2O. The computer-controlled piston ventilator was programmed to deliver a 2-second pseudorandom perturbation that consisted of waveforms of mutually prime frequencies. Multiple linear regression was used to fit impedance spectra derived from measured pressure and volume in each individual mouse to the constant phase model of the lung and airway resistance was determined (R). Mice were then challenged with increasing concentrations of aerosolized methacholine as indicated. After each dose, the time course of response was followed by applying a 2-second perturbation as described above every 10 seconds for a total of 3 minutes. The peak response for airway resistance was determined and the relative change from baseline measured at the beginning of the protocol was calculated. To determine AHR, provocative concentration value was calculated for every individual mouse by intrapolation of the dose–response curve as the methacholine dose causing 100% increase above baseline in Penh for noninvasive measurements (PC100 Penh) or lung resistance for invasive measurements (PC100 RL).
Collection of BAL and Characterization of Cells
Preparation of Cells and Cell Culture For determination of local cytokine production, LN cells (5 x 106/well) were cultured on 24-well tissue culture plates (Corning Costar, Bodenheim, Germany) in a volume of 1 ml culture medium with or without recombinant βGal (25 µg/ml) as described (12). Culture supernatants were collected after 72 hours and frozen at –20°C until thawed for determination of cytokines.
ELISPOT Assay for Enumeration of IFN-
Measurement of Cytokines in BALF and Cell Culture Supernatant
Statistical Analysis of Data
Intranasal Challenge of Sensitized Mice with βGal Induces Bronchial Infiltration of Eosinophils To define the optimal antigen dose for bronchial provocation using β-galactosidase (βGal) as model allergen, BALB/c mice were sensitized by intraperitoneal injection of βGal adsorbed to alum (βGal/alum), which gave rise to a distinctive systemic Th2 response (7). Subsequently, βGal was applied in various doses to mice on three consecutive days via the intranasal route. Because allergen-induced migration of eosinophils into the lung tissue and especially into the bronchial lumen represents an important hallmark for allergic respiratory inflammation, the number and the frequency of eosinophils in the BAL was determined by differential cell counts. The BAL of control mice that received PBS intranasally contained almost no eosinophils (Figure 1). Increasing doses of βGal induced strong eosinophilic infiltration into the airways both in absolute numbers and in relative proportions. Since the total number of BAL cells recovered from individual mice within an experimental group varied to some extent, determination of the frequency of eosinophils was more reliable. Intranasal provocation of naïve mice, even with high doses of βGal, did not induce migration of eosinophils into the lung (data not shown). The frequency of neutrophils in the BAL of mice challenged with βGal was low and not significantly different from that of PBS-treated control mice (Figure 1). On the basis of these data, an antigen dose of 50 µg βGal was administered intranasally in subsequent experiments.
PMED Inhibits Pulmonary Th2 Responses and Reduces Infiltration of Eosinophils, but Does Not Attenuate AHR To evaluate the impact of PMED in the murine model of allergic airway inflammation, plasmid vectors expressing βGal under the control of the fascin promoter (pFascin-βGal) and the CMV promoter (pCMV-βGal), respectively, were administered three times in weekly intervals via the gene gun before sensitization with βGal/alum. Control mice were vaccinated with a plasmid encoding enhanced green fluorescent protein (pCMV-EGFP) to test for antigen specificity. After the last intranasal challenge of mice with allergen, sera were recovered, and titers of βGal-specific Ig isotypes were determined. Mice solely sensitized with βGal/alum revealed only low IgG titers, being predominantly of IgG1 isotype (Figure 2). Preceding gene gun–mediated vaccination with βGal-encoding plasmid DNA, but not with control vector pCMV-EGFP, led to overall stimulation of Ab formation, probably reflecting the activation of memory B cells originating from the primary response after biolistic transfection, because the isotype pattern is concordant with the one initially induced by DNA vaccination (12): IgG2a titers were higher in mice vaccinated with pFascin-βGal, whereas biolistic transfection with pCMV-βGal provoked stronger IgG1 secretion following protein challenge. Likewise, βGal-specific IgE titers in this short-term setting showed a tendency to be higher in DNA vaccinated animals than in control mice (Figure 2). This is in line with previous data obtained by us in a mouse model of type I allergy, demonstrating enhanced levels of IgE and IgG1 shortly after DNA vaccination and sensitization with βGal/alum, but considerably reduced levels at later time points (8, 9). However, the stimulating effect on IgE production was far less pronounced than the strong increase in IgG formation (Figure 2).
To analyze the impact of PMED on the recruitment and activation of local Th cell subsets, triggered by application of allergen via the airways, lymphocytes from the draining bronchial LN were restimulated with βGal-protein in vitro and cytokine release was assessed. Provocation of sensitized mice with βGal induced the production of high levels of IL-5 and IL-13, but only small amounts of IFN- , indicating the activation of Th2 cells (Figure 3A). Production of these cytokines was strictly dependent on challenge with βGal, because intranasal treatment with PBS did not result in significant secretion of any of the cytokines. In contrast to preceding vaccination with the control vector pCMV-EGFP, which moderately increased IFN- synthesis by restimulated LN cells, but did not affect IL-5 and IL-13 production as compared with unvaccinated controls, biolistic transfection with pFascin-βGal or pCMV-βGal led to a profound shift in Th cell polarization: synthesis of IL-5 and IL-13 was substantially reduced, whereas IFN- production was considerably enhanced, suggesting the prevalent activation of Th1-biased cells. In accordance, analysis of IL-5 and IFN- content in the BALF revealed the activation of divergent Th cell populations: unvaccinated and pCMV-EGFP treated control mice showed predominant production of IL-5 over IFN-, whereas on the contrary IL-5 levels were strongly decreased and IFN- levels were elevated in animals vaccinated with βGal-encoding plasmids (Figure 3B). In general, this effect was more prominent after vaccination with pFascin-βGal than with pCMV-βGal.
In addition, we measured eosinophilia in the airway lumen of provocated mice as a parameter for allergic inflammation in the lung. The rate of eosinophilic infiltration was very efficiently reduced by biolistic transfection with pFascin-βGal (92.3%) and pCMV-βGal (76.5%), respectively (Figure 4A). In contrast, gene gun–mediated vaccination with pCMV-EGFP did not alter the frequency of eosinophils in the BAL, documenting the antigen specificity of the regulatory mechanism. On the other hand, we observed a significant increase in the occurrence of neutrophils in the BAL of allergen-challenged mice previously vaccinated with βGal-encoding plasmids. Again, as compared with unvaccinated control mice the enhancement was stronger after vaccination with pFascin-βGal (636.8%) than with pCMV-βGal (344.7%). To determine functional consequences of the reduction of airway eosinophilia, we challenged mice with increasing doses of methacholine on the day after the last intranasal application of allergen and measured airway reactivity in conscious, freely moving mice by noninvasive whole-body plethysmography, a method that has documented a tight correlation between AHR and eosinophil number in the BAL (17). Airway responsiveness was expressed as relative change of the enhanced pause (Penh) measured for each concentration of methacholine in comparison to baseline Penh values (no vaccination, PBS-treated 0.652 ± 0.086; no vaccination, βGal-challenged 0.727 ± 0.087; pFascin-βGal, βGal-challenged 0.792 ± 0.143; pCMV-βGal, βGal-challenged 0.732 ± 0.133; pCMV-EGFP, βGal-challenged 0.818 ± 0.159). Whereas respiratory reactivity of PBS-treated mice was only slightly increased after application of methacholine, sensitized mice challenged with βGal exhibited considerably enhanced Penh values after methacholine provocation (P < 0.05) (Figure 4B). Vaccination with EGFP-encoding vectors did not result in a reduction of the magnitude of pulmonary reactivity. In addition, the PC100 Penh values, indicating the provocative concentration of methacholine, which produced a 100% increase in Penh above baseline, were significantly higher in PBS-treated animals (50.3 ± 39.6 mg/ml) than in unvaccinated βGal-challenged mice (18.4 ± 20.1 mg/ml; P < 0.05) or mice vaccinated with the control vector (14.8 ± 8.5 mg/ml; P < 0.01). Altogether, the data denote the establishment of AHR in βGal-challenged animals. Notably, despite impaired eosinophilia, mice vaccinated with βGal-encoding vectors also showed high respiratory sensitivity for methacholine challenge (PC100 Penh: pFascin-βGal 16.4 ± 16.5 mg/ml; pCMV-βGal 12.7 ± 9.0 mg/ml) and thus developed considerable AHR, which was not significantly different from that of control mice exhibiting severe symptoms of allergic inflammation.
PMED Primes for Pulmonary Th1/Tc1 Responses and Promotes the Development of AHR We next investigated whether PMED of allergen-encoding DNA per se primes for inflammatory reactions in the lung and subsequently leads to the development of AHR. Therefore mice immunized with βGal-encoding plasmids via the gene gun were challenged intranasally with βGal without intermediate sensitization with βGal/alum. Biolistic transfection with pFascin-βGal led to the recruitment of Th1 cells in the draining LN, secreting high amounts of IFN- but only marginal levels of IL-5 and IL-13 after restimulation in vitro (Figure 5A). Consistently, we solely detected IFN- in the BALF of these animals (Figure 5B). The cytokine profile of draining LN cells from mice immunized with pCMV-βGal was characterized by considerable production of IFN- and moderate secretion of IL-5 and IL-13 (Figure 5A).
Accordingly, we determined substantial levels of IL-5 as well as IFN- in the BALF, suggesting the induction of a local mixed Th1/Th2 response in mice immunized with pCMV-βGal (Figure 5B). In addition, we analyzed the recruitment of CD8+ T cells into the lung and the draining local LN. We have previously shown that PMED of βGal-encoding plasmids triggered the activation of IFN-–secreting CD8+ effector T cells (Tc1 cells) in the spleen (11, 12). Herein we additionally determined ex vivo the frequency of Tc1 cells in the airways using an ELISPOT assay, in which βGal-specific CD8+ T cells were selectively stimulated after incubation of LN and lung cells with the βGal-derived nonamer peptide TPHPARIGL, representing an MHC class I–restricted epitope (7). In contrast to mice injected with βGal/alum, intranasal provocation of biolistically transfected mice with βGal led to considerable recruitment of IFN-–secreting CD8+ effector T cells into the regional draining LN and particularly into the lung (Figure 6).
This recruitment was dependent on local application of allergen because in PBS-instilled gene gun–immunized mice only low numbers of βGal-specific Tc1 cells were detected in the airways. Furthermore, antigen specificity was substantiated by analysis of control mice, which were immunized with the plasmid vector pCMV-EGFP. Although these mice displayed substantial numbers of EGFP-specific IFN-–secreting CD8+ effector T cells in the spleen (data not shown), no migration of Tc1 cells into the regional draining LN or into the lung was detected after intranasal challenge with βGal (Figure 6).
Analysis of the cellular composition of the BAL revealed strong infiltration of neutrophils in the airway lumen after PMED of both pFascin-βGal and pCMV-βGal and subsequent provocation with βGal, whereas the eosinophil number was only slightly augmented in these mice (Figure 7A). On the other hand, animals sensitized with βGal/alum developed strong eosinophilia after provocation. Furthermore, we assessed airway reactivity induced by biolistic transfection and triggered by subsequent allergen provocation. In contrast to mice that were transfected with the EGFP-encoding vector (baseline Penh 0.681 ± 0.067; PC100 Penh 13.4 ± 7.8 mg/ml), mice immunized with pFascin-βGal and pCMV-βGal, respectively, established considerable AHR after methacholine challenge, as was evident from the overall increase in Penh (Figure 7B, upper panel; P < 0.05) (baseline Penh: pFascin-βGal 0.730 ± 0.071; pCMV-βGal 0.724 ± 0.093) as well as from the significantly lower PC100 Penh values (pFascin-βGal 3.6 ± 1.9 mg/ml, P < 0.05; pCMV-βGal 4.8 ± 2.7 mg/ml, P < 0.05). This reaction was not significantly different in its magnitude from AHR induced after sensitization of mice with βGal/alum (baseline Penh: 0.743 ± 0.097; PC100 Penh 3.8 ± 2.0 mg/ml). To rule out a contribution by the nose and the upper respiratory tract to the elicitation of AHR and to ensure that the observed changes in bronchial reactivity were not due to erroneous levels of sensitivity of the experimental methodology, we also used anesthetized, tracheostomized mice to measure airway resistance by forced oscillation method. Consistent with the data obtained by determination of Penh, we verified with this technique that PMED of both pFascin-βGal and pCMV-βGal (baseline airway resistance: pFascin-βGal 0.449 ± 0.084 cm H2O x s/ml; pCMV-βGal 0.479 ± 0.166 cm H2O x s/ml) like sensitization with βGal-protein (baseline airway resistance 0.527 ± 0.113 cm H2O x s/ml) led to significant enhancement in airway resistance after local provocation with βGal (PC100 RL: pFascin-βGal 6.2 ± 3.2 mg/ml, P < 0.05; pCMV-βGal 7.8 ± 9.4 mg/ml, P < 0.05; βGal 5.1 ± 2.9 mg/ml, P < 0.01) as compared with PBS-treated mice (Figure 7B, lower panel; P < 0.05) (baseline airway resistance 0.554 ± 0.113 cm H2O x s/ml; PC100 RL 16.8 ± 10.3 mg/ml). In contrast, mice biolistically transfected with the control vector pCMV-EGFP did not develop increased bronchial reactivity after intranasal challenge with βGal (baseline airway resistance: 0.503 ± 0.116 cm H2O x s/ml; PC100 RL 22.7 ± 23.9 mg/ml), proving the antigen dependency of this phenomenon and excluding the possibility that the procedure of gene gun–mediated immunization favors per se the induction of AHR. The observation that control mice immunized with βGal-encoding plasmids and instilled with PBS did not develop AHR (data not shown) is in line with this argumentation.
In this article we demonstrate in a mouse model of allergic airway disease that PMED of allergen-encoding plasmid DNA is a feasible method to inhibit local allergen-specific Th2 responses via the induction of Th1/Tc1 cells and their recruitment into the lung. Concomitantly Th2-associated inflammatory processes in the lung, in particular eosinophilic infiltration, were considerably reduced by PMED. On the other hand, we point out that the therapeutic approach to initiate strong systemic type 1 immune responses, with the intention to counteract the activation of allergen-specific Th2 cells, might entail detrimental consequences for local inflammatory reactions in the airways. Using gene gun–mediated DNA immunization with βGal-encoding plasmids, we show for the first time in situ that the generation of Th1/Tc1 cells causes accumulation of neutrophils in the airways and triggers AHR after provocation with βGal. We assume that these mechanisms are involved in the maintenance or even in the induction of AHR, which we observed despite considerable reduction of infiltration of eosinophils in βGal/alum-sensitized mice previously vaccinated by PMED. Inhibition of allergic sensitization induced by DNA vaccination with vectors encoding allergens or their antigenic epitopes is often associated with systemic immune deviation from Th2-polarized responses toward nonallergic Th1-orientated responses, irrespective of whether the plasmid DNA was administered by injection via the intradermal (1, 4, 6) or the intramuscular (3, 5, 18) route or by gene gun–mediated transfection (8, 9). The efficiency of DNA vaccination in the suppression of the recruitment of local Th2 cells to the lung and consequently in the attenuation of pulmonary inflammation is less well documented. We herein show in a mouse model of allergic airway disease that the development of local Th2 responses was inhibited by PMED, instead favoring type 1–biased responses in the lung after vaccination with pFascin-βGal and pCMV-βGal, respectively. In addition, Th2-mediated inflammatory infiltration of eosinophils in the airways was considerably reduced by prophylactic DNA vaccination, as has been published before by Spiegelberg and coworkers (19) using intradermal injection as vaccination mode. Recently Darcan and colleagues (20) have shown that allergen-induced pathologic alterations of the lung tissue resembling chronic bronchitis were suppressed by intradermal application of allergen-encoding DNA. Likewise, histologic analysis of random samples of lung sections of biolistically transfected mice that were subsequently sensitized and provocated with βGal revealed a tendency to reduced symptoms of inflammation (eosinophilia, goblet cell hyperplasia, mucus production) as compared with specimens of control mice that were not vaccinated (data not shown). Strikingly, AHR was not ameliorated in mice biolistically vaccinated with βGal-encoding plasmids. These findings contrast with previous data that illustrate the reduction of AHR by intradermal (2) or intramuscular (3) injection of allergen-encoding DNA, implicating that the route of vaccination plays a major role with respect to the inhibitory capacity. The question of whether the induction of type 1 responses for the purpose of controlling Th2 responses is always protective against inflammatory reactions and thus is generally useful remains a matter of debate (21). Passive transfer experiments with antigen-specific Th1 cells in rodents revealed contradictory results concerning the suppression of local Th2 responses and the initiation of Th1-driven acute lung pathology (22–25). Takaoka and coworkers (25) reported that transferred Th1 cells induced AHR as well as neutrophilic infiltration in the BAL, being dependent on the production of CXC chemokine(s). Cohn and colleagues (26) and Sawicka and coworkers (27) confirmed the recruitment of neutrophils in the lung by activated Th1 cells, but in contrast did not observe the development of AHR. Sugimoto and colleagues (28) demonstrated that antigen-stimulated memory Th1 cells became harmful and were able to induce AHR after administration of IL-18. Recently Cui and coworkers (29) pointed to a pathway whereby Th1 cells mediated AHR independent of neutrophilic infiltration. We show for the first time by in vivo induction of systemic allergen-specific Th1/Tc1-biased responses, instead of by passive transfer of in vitro differentiated TCR-transgenic T cells, that the recruitment of such cells to the lung after allergen challenge is correlated with neutrophilic infiltration and the elicitation of AHR. In the murine model of allergic airway disease PMED of pFascin-βGal and pCMV-βGal, respectively, induced airway neutrophilia 24 hours after allergen provocation of sensitized mice. Although it is known that sensitized mice show a transient rise in neutrophil numbers very early after allergen challenge (2–8 h) (30), this influx is not concordant with the considerable increase we observed in vaccinated mice after 24 hours, since in unvaccinated control mice the frequency of neutrophils at 24 hours was low. We show that in vaccinated mice the local allergen-specific Th2 response was efficiently converted into an allergen-specific type 1 response. Because we noticed no additive or synergistic effects of DNA vaccination and subsequent immunization with βGal-protein on the exacerbation of lung function, it is tempting to speculate that Th2-driven AHR was replaced by Th1/Tc1-driven AHR.
PMED of pFascin-βGal induced highly polarized Th1 cells in the lung, whereas local cytokine production after biolistic transfection with pCMV-βGal indicated the establishment of a mixed Th1/Th2 response. Remarkably, the intensity of neutrophilic infiltration and the strength of AHR after intranasal application of βGal was comparable among the two groups, suggesting that according to our line of argumentation the Th1-biased component of the immune response after vaccination with pCMV-βGal might be strong enough to exert disease promoting activity. Moreover, particle-mediated administration of both vectors induced a strong and robust systemic CD8+ T cell response (9, 12), which has been previously shown to be an outstanding feature of PMED in mice (31) and in humans (32). Therefore, it has to be taken into account that the recruitment of IFN- In summary, we have shown that prophylactic PMED of allergen-encoding DNA before allergenic sensitization suppressed the establishment of a local Th2 response in the lung and consequently efficiently inibited eosinophilic pulmonary inflammation. In contrast, Th1/Tc1 cells and neutrophils were recruited into the lung after allergen challenge of mice subjected to gene gun–mediated DNA vaccination. We conclude that the approach to induce robust allergen-specific type 1 responses to negatively control Th2 responses and to exert inhibitory activity on asthma pathology requires a delicate balance between the conversely polarized T cell subsets, because otherwise the effect of potent local Th1 cell or Tc1 cell recruitment might be detrimental, leading to aggravation of inflammatory reactions. Therefore, it might be necessary to devise other immunomodulatory strategies than the induction of type 1 responses, for example the stimulation of regulatory T cells, which in general suppress the activation of allergen-specific T cells.
The authors thank Evelyn Montermann and Petra Scholtes for excellent technical assistance. This study was done in partial fulfillment of the requirements of the doctoral thesis of E.Z.
This work was supported by funding from the Deutsche Forschungsgemeinschaft, SFB548. Originally Published in Press as DOI: 10.1165/rcmb.2007-0067OC on July 19, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 1, 2007 Accepted in final form July 3, 2007
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Abstract
CLINICAL RELEVANCE
