Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M2 muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.
- airway responsiveness
- neural control of airways
- M2 muscarinic autoreceptor
inflammatory processes within the airways of humans may alter airway function, leading not only to obstruction of the airways but also to increases in the level of airway responsiveness. This concept has evolved in part because of clinical studies (5, 12,23, 24, 47) demonstrating that the number of eosinophils in bronchoalveolar lavage fluid or endobronchial biopsies correlate with the level of airway responsiveness. Although these associations do not prove a cause-and-effect relationship, studies in models of airway dysfunction have more clearly shown the potential of inflammatory cells to alter airway function. For example, depletion of polymorphonuclear leukocytes (neutrophils and eosinophils) with nitrogen mustard in an allergen-driven model in rabbits prevented late asthmatic responses and increased airway responsiveness after allergen challenge (30). More importantly, infusion of highly purified polymorphonuclear leukocytes into nitrogen mustard-treated allergic rabbits shortly before allergen exposure restored late responses and led to marked increases in airway responsiveness to histamine (30). In a murine model, treatment of mice with anti-interleukin-5 during the sensitization period abolished the infiltration of eosinophils into the lung tissue normally seen in this model and prevented the development of airway hyperresponsiveness (18).
The mechanisms via which inflammatory cells alter airway function are likely multiple. These include release of inflammatory mediators and various proteases as well as generation of reactive oxygen species. Of these potential mechanisms, the latter has attracted the least attention in terms of scientific studies. Yet work in both models and humans suggests that this may be an important avenue of investigation. In this respect, work involving a canine model of allergen-induced lung dysfunction (44) as well as observations involving allergen challenge in allergic humans (38) suggests that superoxide is one of the most important reactive oxygen species generated at the site of challenge. Superoxide dismutase (SOD) is part of an antioxidant defense system that should prevent or limit injury from oxygen radicals. Yet patients with asthma have reduced levels of SOD in lung cells compared with those in normal subjects (42). Within the respiratory epithelium, cytosolic copper/zinc-containing SOD (Cu/Zn SOD) is the antioxidant that is decreased (9).
Transgenic (TG) mice with expression of elevated levels of Cu/Zn SOD in the lungs have been used to study the importance of this antioxidant system in the face of hyperoxia. On exposure to > 99% oxygen at 630 Torr, White et al. (48) reported that Cu/Zn SOD TG mice had increased survival, decreased edema and hyaline membrane formation, and reduced inflammatory cells within their lungs. This strain of mice has not been used to assess the importance of Cu/Zn SOD in terms of airway function. In this study, we addressed the hypothesis that TG mice that overexpress Cu/Zn SOD are more resistant to allergen-induced changes in neural control of airways than wild-type mice.
MATERIALS AND METHODS
TG mice harboring the human Cu/Zn SOD gene were originally produced by microinjection of fertilized eggs with a linear 14.5-kb fragment of human genomic DNA containing the entire Cu/Zn SOD gene including its regulatory sequences (1, 10). TgHS-51 progeny as used in these initial studies contained five copies of the human Cu/Zn SOD gene in their genomes, resulting in a 2.5-fold increase in Cu/Zn SOD activity measured in the lungs (48). The mice were derived from outbred matings between CBYB/6 [(BALB/c × C57BL/6J)F1] and B6D/2 [(C57BL/6J × DBA)F1] mice. These mice from the Weizmann Institute of Science (Rehovot, Israel) were used to found the colony in Colorado. This colony has now been used in previously published studies from this institution (48, 49).
TG mice with stable expression of elevated levels of human Cu/Zn SOD were bred in a virus-free animal facility at National Jewish Medical and Research Center (Denver, CO). TgHS-51 homozygous, heterozygous, and wild-type mice were identified by SOD activity gel electrophoresis (1, 4). TG heterozygous mice were bred with known male heterozygous and female wild-type parents. Each mouse used in these experiments (heterozygotes with the transgene as well as wild-type mice without the transgene) had their status verified by SOD activity gel electrophoresis. Both male and female mice were used in this study.
All procedures employed in this study were approved by the Animal Care and Use Committee of the National Jewish Medical and Research Center and conformed to National Institutes of Health guidelines.
SOD activity gel electrophoresis.
Tissue homogenates (∼10–20 μg of tissue) or red blood cell hemolysates (1:20 vol/vol in 50 mM potassium phosphate, pH 7.8; 20 μl) were loaded onto a 10% acrylamide gel and electrophoresed for ∼3 h at 150 V. Staining for SOD enzymatic activity was done as described elsewhere (4) except that the gels were soaked in potassium phosphate buffer containing nitro blue tetrazolium, riboflavin, and tetramethylethylenediamine for 20 min. Distinct bands representing mouse SOD, mouse-human SOD heterodimer, human SOD, and hemoglobin were identified on these gels as previously described (49).
Passive immunization and airway challenge.
Mice were passively immunized via intravenous administration of murine monoclonal anti-ovalbumin (OVA) IgG1 (clone OVA-14; lot 014H4836; Sigma, St. Louis, MO). The concentration of antibody was 8.1 mg/ml before dilution in normal saline. Each mouse received a total of 2 μg of anti-OVA IgG1 in a total volume of 200 μl by tail vein injection as previously described (32). Groups of mice received an injection of the antibody on 2 consecutive days. Control mice received the same volume of saline given in an identical fashion. Subsequently, the mice were exposed to either PBS or OVA by nebulization for 20 min on 2 consecutive days starting 3 days after the last intravenous injection. The equipment employed for aerosol delivery and the characteristics of the aerosol generated for the challenges are outlined in Active immunization. Mice were killed by cervical dislocation 2 days after the last airway challenge.
Mice were immunized by ultrasonic nebulization of 1% OVA (chicken OVA, grade V; Sigma) diluted in sterile PBS as previously reported (27, 37). The osmolarity of the OVA solution and PBS was 285 mosM. For this process, up to eight mice were placed in a clear plastic box (22 × 23 × 14 cm) with a removable top. The OVA solution was aerosolized into one end of the box with an ultrasonic nebulizer (Aerosonic model 5000 D, DeVilbiss, Somerset, PA) and a continuous pressure of 5 psi. At the other end of the chamber were two small airholes (0.5 cm in diameter) to ensure a continuous cross-current flow of air. As defined by laser nephelometry (6), >90% of the particles were in the 1- to 5-μm range.
Immunization was carried out with a daily 20-min exposure to OVA over a 10-day period. Previous work (37) has shown this to be the optimal immunization schedule for the selective production of allergen-specific IgE. Age-matched nonimmune (control) mice were exposed to PBS alone with the same protocol. Animals were killed 2 days after the last exposure to PBS or OVA.
Assessment of airway smooth muscle responsiveness.
Airway smooth muscle responsiveness was assessed in vitro in two ways: through electrical field stimulation (EFS) and by exposure to increasing concentrations of methacholine (MCh) or acetylcholine (ACh). Tracheal smooth muscle (TSM) segments ∼0.5 cm in length were obtained and cleaned of loose connective tissue. The segments were placed in 3-ml polypropylene organ baths and supported longitudinally by stainless steel wire triangular supports. The lower support was attached to a stainless steel hook at the base of the organ bath, and the upper support was attached via a gold chain to a Grass FT.03 isometric force transducer (Grass Instruments, Quincy, MA) mounted on a rack-and-pinion clamp so that the resting length of the TSM segment could be adjusted. The tissues were bathed in a Krebs-Henseleit solution composed of (in mM) 118 NaCl, 25 NaHCO3, 2.8 CaCl2 · 2H2O, 1.17 MgSO4, 4.7 KCl, 1.2 KH2PO4, and 2 g/l of dextrose. The baths were aerated with a 95% O2-5% CO2 gas mixture, and a pH of 7.43 ± 0.03 was established for the duration of each experiment. The temperature of the baths was maintained at 37°C. Each TSM segment was equilibrated in the bath for 90 min at a tension of 1.5 g. During this time, the tissue was challenged three times at ∼15-min intervals with a final concentration of 120 mM KCl in the bath. Tissues that did not respond to this stimulus with a contractile response were excluded from further study. Responding tissues were rinsed with fresh buffer and allowed to relax to their initial tension after reaching maximal contraction. Recordings of resting tensions as well as all TSM contractile responses were made on a SensorMedics Dynagraph recorder equipped with type 9853A couplers, 461D preamplifiers, and 412 amplifiers (SensorMedics, Anaheim, CA).
EFS was delivered by a Grass S44 stimulator connected to a stimulus isolation unit (Grass SIU5). The stimulus was applied transmurally across the tissues by means of parallel platinum electrodes (each 0.3 cm2). The optimal resting length for each tissue was established by assessing its maximal contractile response to the following EFS: 4 V, 1-ms pulse duration, and 20-Hz stimulus frequency. Studies in mice showed no change in optimal resting length in the immune state whether produced by repeated airway exposure to the allergen or produced by parenteral administration of allergen-specific antibody (data not shown). After optimal lengths were established, noncumulative stimulus-response curves were generated by varying the stimulus frequencies from 0.5 to 30 Hz. Each EFS was maintained until a peak contractile response was obtained. An ∼2-min recovery time elapsed between each successive stimulation. Studies within this laboratory have found that in murine airways, 30 Hz is the frequency at which the contractile responses were always maximal and tissue damage due to EFS minimal. At higher current densities or voltages, the reproducibility of contractile responses diminished.
To evaluate postsynaptic cholinergic sensitivity of tissues, cumulative dose-response curves to the cholinergic agonist MCh (10−8to 10−4 M) were obtained separately for each TSM segment. After a plateau response with each administered dose was obtained, the next addition of MCh was added to the bath. Each dose was in half-log increments.
To evaluate for possible differences in acetylcholinesterase (AChE) activity that may account for increases in responsiveness, cumulative dose-response curves to ACh (10−8 to 10−4 M in half-log increments) were obtained separately for several TSM segments. ACh is more sensitive to AChE activity than MCh, so a similar response to ACh in immune and nonimmune tissues would suggest that AChE activity was unchanged as a result of the allergen exposure and immune state.
At the end of each experiment, the TSM segments were blotted on a gauze pad and weighed. All tensions are expressed as either grams of isometric tension per gram of TSM weight (g/g) or a percentage of the maximal contractile response (Tmax) to EFS or MCh. The frequency that caused 50% of the maximal contractile response (ES50) was calculated from linear plots of the contractile response versus the frequency of EFS. Likewise, the molar concentration of MCh that caused 50% of the maximal contractile response (EC50,MCh) was calculated from MCh concentration-response curves. All tissues were assessed in terms of their in vitro responsiveness by one investigator (J. Loader) who was blinded to the immunologic and treatment status of each airway.
ACh release in the tissue bath.
Tissues were prepared and equilibrated as in Assessment of airway smooth muscle responsiveness. A priming stimulus of 10 Hz was then delivered to the tissue before the addition of a new buffer containing 3 × 10−5 M physostigmine and 5 × 10−6 M choline in Krebs-Henseleit solution that was filtered through a 0.22-μm Millipore filter. The tissue was incubated in 0.5 ml of the buffer for 30 min, after which the buffer was removed for baseline (unstimulated) ACh measurement. Next, 0.5 ml of the buffer was added to the tissue bath, and the tissue was subjected to EFS at 10 Hz for 15 min. All samples taken from the bath were filtered through a 0.22-μm Millipore filter into a 1.5-ml microcentrifuge container for storage at a temperature of −70°C before ACh release was measured.
Measurement of ACh.
A modification of a previously utilized system (7,26) employing high-performance liquid chromatography (HPLC) with biosensor detection was used to quantitate ACh release into the tissue baths [Bioanalytical Systems (BAS) Peroxidase Electrode Kit Instruction Manual, West Lafayette, IN]. HPLC columns were obtained from BAS and arranged proximal to distal as follows: a lipophilic column to remove physostigmine, a choline oxidase with catalase column to remove choline, an ACh analytic column to separate residual choline from ACh, and finally the ACh- immobilized enzyme reactor with AChE-choline oxidase, which produces 2 molecules hydrogen peroxide/molecule ACh. The mobile phase consisted of a 10 mM sodium phosphate buffer (pH 8.0 ± 0.05) with 5 ml/l of Kathon CG reagent (1% vol/vol; BAS) added as a bacteriostatic agent. Liquid chromatography-grade deionized water that had been passed through a 0.2-μm filter was employed (Milli-Q system, Millipore, Bedford, MA). A sample volume of 100 μl (Hamilton syringe, Hamilton, Reno, NV) was injected into a Waters (Milford, MA) injector. The mobile phase was delivered by a Waters solvent delivery system at a flow rate of 1.0 ml/min at room temperature. Detection of hydrogen peroxide was accomplished with a Waters electrochemical detection unit with a glassy carbon electrode coated with a redox polymer film containing horseradish peroxidase (BAS) versus a Ag-AgCl reference electrode. Results were recorded on an Omni Scribe recorder (Houston Instruments, Austin, TX) at a chart speed of 0.25 cm/min. The amount of ACh within a sample was determined by comparing the peak heights of the samples with the peak heights of the standard curves, with the latter generated by injecting ACh standards (1–10 pmol) before and after each experimental run. Peak heights (vertical distance from the peak maximum to the baseline) rather than areas were used in the assessments as recommended by the manufacturer of the ACh assay kit (BAS). When baseline drift was present, this was corrected for by extrapolating the baseline between the front and the end of the peak tracing. For the standard curves, Krebs-Henseleit solution was the diluent for ACh.
Assessment of M2 muscarinic autoreceptor function.
To assess the function of M2 muscarinic autoreceptors on the airway smooth muscle of mice, tracheal segments were obtained from both nonimmune and immune wild-type and TG mice for assessment of ACh release within the tissue baths by the methods outlined in ACh release in the tissue bath. However, for these studies, an antagonist (gallamine, 30 μM) of the M2 muscarinic receptor was present within the bath at the time of EFS. The concentration of gallamine was based on amounts used by Yang and Biggs (50) to study M2 receptor function in isolated innervated tracheae from guinea pigs and previous work with murine tissues in our laboratory (26). All tissues for this part of the study were subjected to EFS at 10 Hz for 15 min.
Analysis of eosinophil influx into the lungs.
Eosinophils within the lungs after repeated exposure to either PBS (control) or OVA were assessed in both wild-type and TG mice. Cells containing eosinophilic major basic protein (MBP) were identified by immunohistochemical staining as previously described with rabbit anti-mouse MBP (18). After perfusion via the right ventricle, the lungs were inflated through the trachea (2 ml) and fixed in 10% Formalin. Blocks of lung tissue were cut around the main bronchi and embedded in paraffin. Tissue sections 5 μm thick were affixed to microscope slides, deparaffinized, and incubated in normal rabbit serum for 2 h at 37°C. The slides were subsequently stained with either rabbit anti-mouse MBP or normal rabbit serum and incubated overnight at 4°C. After a wash and incubation in 1% chromotrope 2R (Harlesco, Gibbstown, NJ) for 30 min, the slides were placed in fluorescein-labeled goat anti-rabbit IgG for 30 min at 37°C. The slides were examined with a Zeiss microscope equipped with a fluorescein filter system.
The results of the in vitro analyses of airway responsiveness and ACh release are reported as means ± SE. Student's unpaired two-tailed t-test was used to determine the level of difference between two groups (33). An analysis of variance with the Tukey-Kramer honestly significant difference multiple comparison procedure was employed to determine the level of difference between more than two groups (19). A P value of <0.05 was considered significant.
In vitro airway responsiveness to EFS.
The responses of TSM from wild-type and TG mice that were either nonsensitized or passively sensitized to OVA are shown in Fig.1. With the parameters used to stimulate the tissues, the responses were both neurally and cholinergically mediated in that either tetrodotoxin (an inhibitor of neural transmission) or atropine (a muscarinic receptor antagonist) abolished EFS-induced contractions in all groups of mice (data not shown). In wild-type (n = 11) and TG (n = 14) mice that were not sensitized but were challenged with PBS or OVA, neither the ES50 values nor the maximum tensions generated in response to EFS (data below) were significantly different between these two groups. When passively sensitized wild-type mice (n= 13) were exposed to OVA, a significant decrease in ES50was seen. The value of this parameter was 4.2 ± 0.3 Hz in the nonsensitized group after aerosol exposure and 2.3 ± 0.3 Hz when sensitized wild-type mice were exposed to OVA (P = 0.0001). In addition, the percent maximal response was significantly greater at 0.5, 1, 3, 5, 7, and 20 Hz in the sensitized versus the nonsensitized wild-type mice after aerosol exposure (P< 0.05 at all frequencies; Fig. 1). Conversely, TSM segments from TG mice (n = 7) were protected from changes in ES50 (3.9 ± 0.3 and 4.0 ± 0.4 Hz for nonsensitized and sensitized TG mice, respectively). There were also no significant differences in the percent maximal responses at any frequency of stimulation in the TG groups. For both wild-type and TG groups, the Tmax values were not significantly altered by passive sensitization and OVA challenge. These values were 105 ± 17 and 110 ± 14 g/g in nonsensitized and sensitized wild-type mice, respectively (not significant). For nonimmune and immune TG mice, the values were 117 ± 15 and 88 ± 18 g/g, respectively (not significant).
Wild-type and TG mice were also studied in terms of the consequences of active sensitization via repeated aerosol exposure to OVA. The same pattern was evident in terms of contractile responses to EFS (Table1). Thus wild-type mice demonstrated a significant decrease in ES50 (P = 0.016), whereas the TG strain did not. Tmax values did not differ among any of the groups.
In vitro airway responsiveness to MCh.
The postsynaptic cholinergic sensitivity of TSM from wild-type and TG mice was measured under control and experimental conditions involving both passive and active sensitization. This was accomplished by assessing the in vitro responses of TSM to increasing concentrations of MCh. The responses are expressed in terms of both Tmax and EC50,MCh. There were no significant differences in these in vitro responses to this cholinergic agonist. Responses of the tissues to MCh in the studies employing passive sensitization are shown in Fig.2, and EC50,MCh values in the studies employing active sensitization are shown in Table 1.
In vitro airway responsiveness to ACh.
The postsynaptic cholinergic sensitivity of TSM from wild-type and TG mice was also measured under control and experimental conditions involving both passive (Fig. 3) and active (Table 1) sensitization. Because ACh is more subject to degradation by AChE than MCh, these studies were conducted to determine whether a decrease in AChE activity might explain the differences in ES50 seen in the wild-type mice when either actively or passively sensitized and challenged with OVA. These in vitro characteristics are expressed in terms of the molar concentration of ACh causing 50% of the contractile response with 10−4 M ACh (EC50,ACh). This method of assessing ACh dose-response curves has been employed by others (45) because the contractile response does not reach a plateau, even at 10−3 M ACh. There were no significant differences in the EC50,ACh values in any of the control and experimental groups. The response to ACh in studies involving passive sensitization is shown in Fig. 3, whereas Table 1 summarizes the results when active sensitization was employed. The absolute contractile responses in terms of tension generated were not significantly different between these groups of animals at any concentration of ACh (data not shown).
In vitro measurements of ACh release from TSM.
Neurally mediated release of ACh from airways of wild-type and TG mice was measured with HPLC. ACh release expressed as picomoles per gram of tissue per minute of stimulation for wild-type and TG mice is shown in Fig. 4. ACh release from the normal and TG nonsensitized control mice did not differ (92 ± 8 and 95 ± 13 pmol · g−1 · min−1, respectively). The TSM from sensitized wild-type mice (n = 7) released significantly greater amounts of ACh after OVA challenge (195 ± 19 pmol · g−1 · min−1), whereas TSM segments from TG mice (n = 7) were resistant to OVA-induced changes in ACh release (101 ± 10 pmol · g−1 · min−1).
M2 muscarinic autoreceptor function.
The function of M2 muscarinic autoreceptors on TSM from both nonsensitized and passively sensitized and challenged wild-type and TG mice was assessed with the use of gallamine, which has antagonist activity for this subtype of muscarinic receptor. As shown in Fig. 5, nonsensitized wild-type and TG mice both had significant increases in ACh release in the presence of gallamine (P < 0.05), demonstrating that the muscarinic autoreceptor is functional in both strains of mice. In sensitized wild-type mice challenged with OVA, ACh release was elevated compared with the wild-type nonsensitized group and was not modulated by gallamine. Conversely, sensitized TG mice still demonstrated greater release of ACh in the presence of gallamine (P < 0.05), implying that M2 receptor function remained intact after OVA challenge.
Analysis of eosinophil influx into the lungs.
Eosinophil influx into the lungs was assessed by immunohistochemical staining for eosinophil MBP with rabbit anti-mouse MBP as previously described (18). Studies were done in wild-type and TG mice repeatedly exposed to aerosolized PBS (control) as well as in these same two murine strains repeatedly exposed to OVA. Two separate areas of the lungs from each of two animals in each group were examined. This method of assessing eosinophils failed to show differences between TG and wild-type mice exposed to PBS. When these strains were sensitized by repeated exposure to OVA, both strains demonstrated increased immunofluorescence compared with the PBS-treated control mice (Fig.6). However, there were no obvious differences between the two strains in terms of the magnitude of eosinophil influx into the lungs.
Oxygen radicals produced by infiltrating inflammatory cells, including neutrophils and eosinophils (41), have the potential to injure resident cells within tissues and organs. The experiments performed as part of this study were designed because studies in both animal models (25, 44) and humans (38, 40) suggested that reactive oxygen species may be important in allergen-induced injury to airways. The normal mechanism of defense against superoxide, dismutation of superoxide by SOD, was the focus of this work because superoxide is a reactive oxygen species that has been associated with asthma in humans (22, 28, 38). For example, Kanazawa et al. (22) demonstrated that superoxide radical production by peripheral blood neutrophils was enhanced in asthmatic patients compared with that in healthy control subjects, whereas Sanders et al. (38) demonstrated spontaneous production of superoxide at sites of allergen challenge within the lungs of allergic subjects. In addition, patients with asthma have reduced levels of SOD in lung cells compared with those in normal subjects (42). Within the respiratory epithelium, Cu/Zn SOD, but not Mn SOD, is the antioxidant that is decreased (9). Because extracellular SOD has been localized in connective tissue surrounding smooth muscle in airways (34), it also may play an important role in regulating airway tone. Therefore, investigation of this antioxidant in asthmatic patients and animal models could also be important.
The availability of genetically engineered mice that overexpress Cu/Zn SOD (1, 10) provides a means to study the functional importance of this system within various organs of the body. These mice were originally engineered to address issues relevant to the pathogenesis of Down syndrome in that the gene that encodes Cu/Zn SOD is found on chromosome 21. Thus this strain has been used to address potential deleterious effects of a 1.5-fold increase in the expression of this gene in a mammalian system. Studies have found that these TG mice may suffer from chronic oxidative stress manifesting as enhanced apoptosis of thymocytes and bone marrow cells (35), impaired muscle function (36), and cognitive deficits (16). Yet these mice have also been employed to gauge the potential beneficial effects of this antioxidant on acute forms of lung injury. For example, White et al. (48) exposed TG and wild-type mice to high concentrations of ambient oxygen and found that expression of elevated levels of Cu/Zn SOD decreased pulmonary oxygen toxicity as assessed by both histological damage and mortality. This present study represents the first use of these TG mice to address the possible importance of superoxide and SOD on maintaining or altering airway function.
Based on the previous data demonstrating increased reactive oxygen species in asthmatic patients, the main focus of the present study was to address the hypothesis that lung Cu/Zn SOD may make airways resistant to allergen-induced changes in airway function. Two previously characterized approaches to induction of altered cholinergic responsiveness were used. Wild-type mice as well as TG mice that express elevated levels of human Cu/Zn SOD were either passively sensitized to OVA by intravenous injection of homocytotropic antibody or actively sensitized by repeated (and exclusive) airway exposure to OVA. In both systems, wild-type mice had increases in cholinergic reactivity as shown by a significant decrease in ES50. In contrast, sensitized TG mice were resistant to changes in airway function in both approaches, with ES50 values not significantly altered by airway exposure to the allergen.
The EFS-induced increase in the contractile (cholinergic) response in wild-type mice may be due to one or more mechanisms that can be localized to the myoneural junction or to pre- and/or postjunctional sites. For example, an increase in the airway smooth muscle response to ACh released by neural stimulation may take place because of postjunctional alterations in muscarinic receptors (increased number, affinity for ACh). However, the observation that the in vitro responses to MCh and ACh were not significantly different between tissues from sensitized and control mice (Figs. 2 and 3, Table 1) makes this an unlikely explanation for the demonstrated differences. Junctionally, a decrease in AChE could also lead to an increase in the effect of ACh released from neural tissue. Again, the observation that the in vitro responses with both methods of sensitization were not associated with significant changes in the responses of TSM to ACh (Fig. 3, Table 1) makes this unlikely. Prejunctionally, an increase in the neural release of ACh from parasympathetic nerve endings could lead to enhanced contraction. Stimulation of TSM with EFS to elicit ACh release showed that passively sensitized wild-type mice had increased ACh release after OVA exposure. Larsen et al. (26) found a similar pattern in past work with actively sensitized BALB/c mice. Conversely, TG mice were protected against this change in neurotransmitter release. Thus these observations suggest that the site of injury from oxygen radicals within an allergen-driven system is prejunctional. Furthermore, the results suggest that an increased antioxidant defense may provide resistance to these adverse consequences.
Within airways, release of endogenous ACh is under the local control of muscarinic autoreceptors (M2) on postganglionic parasympathetic nerves (3, 15). Release of ACh normally leads to stimulation of the receptor, with subsequent downregulation of release of this neurotransmitter. A dysfunction of M2 autoreceptors with loss of this inhibitory control has been described in models as a consequence of both infectious and noninfectious insults to the airway (discussed below). In this study, we further localized the prejunctional injury to the M2receptor by showing that sensitized wild-type mice had increases in ACh release, whereas mice that overexpress Cu/Zn SOD were resistant to this change. The M2 muscarinic autoreceptor was functional in the TG mice as shown by the ability to upregulate ACh release with an antagonist of the receptor (Fig. 5). This function was preserved despite allergen sensitization and challenge.
A link between the production of reactive oxygen species and dysfunction of M2 receptor function has been suggested in past work. Schultheis et al. (39) reported that ozone-induced airway hyperresponsiveness in guinea pigs was due to loss of neuronal M2 muscarinic receptor function. Additional work from the same laboratory by Gambone et al. (17) suggested that these effects were mediated via inflammatory cells. Studies by Tsukagoshi et al. (46) concluded that superoxide anions released from inflammatory cells in the airways of Brown-Norway rats may be involved in ozone-induced airway hyperresponsiveness. Thus it is possible that production of superoxide was involved in the changes in M2 receptor function described by Schultheis et al. (39) and Gambone et al. (17). The current study more directly links this reactive oxygen species with dysfunction of this muscarinic autoreceptor in an allergen-driven model. This strain of mice has not yet been used to study ozone-induced changes in airway function but might aid in the assessment of the role of superoxide in this airway insult.
Disruption of M2 autoreceptor function may be due to other mechanisms. For example, loss of M2 function in allergen-driven models may be due, at least in part, to MBP acting as an endogenous allosteric antagonist of this receptor (11,20). This mechanism of altering M2 receptor function may not be as pertinent in murine models in that eosinophils that infiltrate the lung appear to retain their granule MBP (43; Gelfand, Larsen, and Joetham, unpublished observations). Other mechanisms may be most pertinent in infectious insults. For example, parainfluenza viral infections are thought to injure this receptor via the actions of viral neuraminidase (13, 14). In addition, parainfluenza as well as interferon-γ can decrease M2 receptor gene expression (21). The work described in this paper adds to the list of mechanisms via which the function of this receptor may be altered. The importance of understanding both the insults and mechanisms through which this occurs is underscored by clinical studies that suggest dysfunction of this receptor occurs in humans with asthma (2, 29,31). Furthermore, eosinophils have been seen in association with airway nerves in patients with fatal asthma (8). Thus this cell has the potential to directly alter receptor function on nerves via release of MBP (and possibly other cationic proteins) and via production of superoxide.
Other important antioxidant systems exist that may also be important in protection against allergen-induced injury. In a sheep model of allergen-induced airway hyperresponsiveness, hydrogen peroxide has been implicated in the process (25). In Ascaris suum-sensitive sheep, treatment with an aerosol of catalase (an enzyme that catalyzes the decomposition of hydrogen peroxide) before and shortly after Ascaris challenge prevented subsequent increases in airway responsiveness to carbachol. This was accomplished without a discernible change in the recruitment of inflammatory cells into the lungs as defined in the bronchoalveolar lavage fluid. It should be noted that a product of the dismutation of superoxide is hydrogen peroxide. Given the observations of Lansing et al. (25) noted above, it is theoretically possible that greater production of this latter reactive oxygen species as well as its breakdown products could have a detrimental effect on airway function. This possibility is further supported by the observations cited above suggesting that these TG mice may suffer from chronic oxidative stress (16, 35, 36). Yet the methods employed to assess airway function in this study do not support this conclusion. It is pertinent to note that when hyperoxia was the stimulus to the lung in a prior study (48), a beneficial and not harmful effect also was observed in this TG strain of mice. Thus the presence of a greater amount of Cu/Zn SOD in the lungs of mice was not detrimental. Rather, increased Cu/Zn SOD was associated with preservation of normal cholinergic mechanisms of airway control.
In summary, we have shown that elevated levels of lung Cu/Zn SOD activity were associated with resistance of airways to allergen-induced changes in neural control. This was seen in both passively and actively sensitized mice. Tracheae from wild-type but not from TG mice developed enhanced cholinergic mechanisms of airway control involving increased release of ACh from neural terminals. This was associated with alterations in M2 receptor function in wild-type mice. Conversely, the TG strain maintained normal function of the receptor. These results demonstrate that Cu/Zn SOD makes neural tissue resistant to oxidant-induced damage in this allergen-driven model.
We acknowledge the contribution of Dr. Karen Avraham in the production of this transgenic strain of mice. Stephanie Park provided valuable assistance in the preparation of this manuscript.
This work was supported in part by National Heart, Lung, and Blood Institute Grant P01-HL-36577.
Address for reprint requests and other correspondence: G. L. Larsen, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206.
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.
- Copyright © 2000 the American Physiological Society