1 Divisions of Pediatric Pulmonary Medicine and Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206; and 2 Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
acetylcholine; airway responsiveness; neural control of airways; M2 muscarinic autoreceptor
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. 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.
Active immunization. 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 (10ACh 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 × 105 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.
Statistical analysis. 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 ES50 was 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).
|
|
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 104 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 · g1 · 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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M2 receptor 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- 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.
![]() |
ACKNOWLEDGEMENTS |
---|
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.
![]() |
FOOTNOTES |
---|
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.
Received 22 October 1999; accepted in final form 17 March 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Avraham, KB,
Schickler M,
Sapoznikov D,
Yarom R,
and
Gruner Y.
Down's syndrome: abnormal neuromuscular junction in tongue of transgenic mice with elevated levels of human Cu/Zn-superoxide dismutase.
Cell
54:
823-829,
1988[ISI][Medline].
2.
Ayala, LE,
and
Ahmed T.
Is there loss of a protective muscarinic receptor mechanism in asthma?
Chest
96:
1285-1291,
1989[Abstract].
3.
Barnes, PJ.
Muscarinic receptor subtypes: implications for lung disease.
Thorax
44:
161-167,
1989[ISI][Medline].
4.
Beauchamp, C,
and
Fridovich I.
Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.
Anal Biochem
44:
276-287,
1971[ISI][Medline].
5.
Bentley, AM,
Menz G,
Storz C,
Robinson DS,
Bradley B,
Jeffery PK,
Durham SR,
and
Kay AB.
Identification of T lymphocytes, macrophages, and activated eosinophils in the bronchial mucosa in intrinsic asthma. Relationship to symptoms and bronchial responsiveness.
Am Rev Respir Dis
146:
500-506,
1992[ISI][Medline].
6.
Clay, MM,
Pavia D,
Newman SP,
and
Clarke SW.
Factors influencing the size distribution of aerosols from jet nebulization.
Thorax
38:
755-759,
1983[Abstract].
7.
Colasurdo, GN,
Loader JE,
Graves JP,
and
Larsen GL.
Modulation of acetylcholine release in rabbit airways in vitro.
Am J Physiol Lung Cell Mol Physiol
268:
L432-L437,
1995
8.
Costello, RW,
Schofield BH,
Kephart GM,
Gleich GJ,
Jacoby DB,
and
Fryer AD.
Localization of eosinophils to airway nerves and effect of neuronal M2 muscarinic receptor function.
Am J Physiol Lung Cell Mol Physiol
273:
L93-L103,
1997
9.
De Raeve, HR,
Thunnissen FBJM,
Kaneko FT,
Guo FH,
Lewis M,
Kavuru MS,
Secic M,
Thomassen MJ,
and
Erzurum SC.
Decreased Cu,Zn-SOD activity in asthmatic airway epithelium: correction by inhaled corticosteroid in vivo.
Am J Physiol Lung Cell Mol Physiol
272:
L148-L154,
1997
10.
Epstein, CJ,
Avraham KB,
Lovett M,
Smith S,
Elroy-Stein O,
Rotman G,
Bry C,
and
Groner Y.
Transgenic mice with increased Cu/Zn-superoxide dismutase activity: animal model of dosage effects in Down syndrome.
Proc Natl Acad Sci USA
84:
8044-8048,
1987[Abstract].
11.
Evans, CM,
Fryer AD,
Jacoby DB,
Gleich GJ,
and
Costello RW.
Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.
J Clin Invest
100:
2254-2262,
1997
12.
Ferguson, AC,
Whitelaw M,
and
Brown H.
Correlation of bronchial eosinophil and mast cell activation with bronchial hyperresponsiveness in children with asthma.
J Allergy Clin Immunol
90:
609-613,
1992[ISI][Medline].
13.
Fryer, AD,
El-Fakahany EE,
and
Jacoby DB.
Parainfluenza virus type 1 reduces the affinity of agonists for muscarinic receptors in guinea-pig lung and heart.
Eur J Pharmacol
181:
51-58,
1990[ISI][Medline].
14.
Fryer, AD,
and
Jacoby DB.
Parainfluenza virus infection damages inhibitory M2 muscarinic receptors on pulmonary parasympathetic nerves in the guinea-pig.
Br J Pharmacol
102:
267-271,
1991[Abstract].
15.
Fryer, AD,
and
Maclagan J.
Muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig.
Br J Pharmacol
83:
973-978,
1984[Abstract].
16.
Gahtan, E,
Auerbach JM,
Groner Y,
and
Segal M.
Reversible impairment of long-term potentiation in transgenic Cu/Zn-SOD mice.
Eur J Neurosci
10:
538-544,
1998[ISI][Medline].
17.
Gambone, LM,
Elbon CL,
and
Fryer AD.
Ozone-induced loss of neuronal M2 muscarinic receptor function is prevented by cyclophosphamide.
J Appl Physiol
77:
1492-1499,
1994
18.
Hamelmann, E,
Oshiba A,
Loader J,
Larsen GL,
Gleich G,
Lee J,
and
Gelfand EW.
Antiinterleukin-5 antibody prevents airway hyperresponsiveness in a murine model of airway sensitization.
Am J Respir Crit Care Med
155:
819-825,
1997[Abstract].
19.
Hayter, AJ.
A proof of the conjecture that the Tukey-Kramer multiple comparisons procedure is conservative.
Ann Stat
12:
61-75,
1984[ISI].
20.
Jacoby, DB,
Gleich GJ,
and
Fryer AD.
Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.
J Clin Invest
91:
1314-1318,
1993[ISI][Medline].
21.
Jacoby, DB,
Xiao H-Q,
Lee NH,
Chan-Li Y,
and
Fryer AD.
Virus- and interferon-induced loss of inhibitory M2 muscarinic receptor function and gene expression in cultured airway parasympathetic neurons.
J Clin Invest
102:
242-248,
1998
22.
Kanazawa, H,
Kurihara N,
Hirata K,
and
Takeda T.
The role of free radicals in airway obstruction in asthmatic patients.
Chest
100:
1319-1322,
1991[Abstract].
23.
Kirby, JG,
Hargreave FE,
Gleich GJ,
and
O'Byrne PM.
Bronchoalveolar cell profiles of asthmatic and nonasthmatic subjects.
Am Rev Respir Dis
136:
379-383,
1987[ISI][Medline].
24.
Laitinen, LA,
Laitinen A,
Heino M,
and
Haahtela T.
Eosinophilic airway inflammation during exacerbation of asthma and its treatment with inhaled corticosteroid.
Am Rev Respir Dis
143:
423-427,
1991[ISI][Medline].
25.
Lansing, MW,
Ahmed A,
Cortes A,
Sielczak MW,
Wanner A,
and
Abraham AM.
Oxygen radicals contribute to antigen-induced airway hyperresponsiveness in conscious sheep.
Am Rev Respir Dis
147:
321-326,
1993[ISI][Medline].
26.
Larsen, GL,
Fame TM,
Renz H,
Loader JE,
Graves J,
Hill M,
and
Gelfand EW.
Increased acetylcholine release in tracheas from allergen-exposed IgE-immune mice.
Am J Physiol Lung Cell Mol Physiol
266:
L263-L270,
1994
27.
Larsen, GL,
Renz H,
Loader JE,
Bradley KL,
and
Gelfand EW.
Airway response to electrical field stimulation in sensitized inbred mice. Passive transfer of increased responsiveness with peribronchial lymph nodes.
J Clin Invest
89:
747-752,
1992[ISI][Medline].
28.
Meltzer, S,
Goldberg B,
Lad P,
and
Easton J.
Superoxide generation and its modulation by adenosine in the neutrophils of subjects with asthma.
J Allergy Clin Immunol
83:
960-966,
1989[ISI][Medline].
29.
Minette, PA,
Lammers JW,
Dixon CM,
McCusker MT,
and
Barnes PJ.
A muscarinic agonist inhibits reflex bronchoconstriction in normal but not asthmatic subjects.
J Appl Physiol
67:
2461-2465,
1989
30.
Murphy, KR,
Wilson MC,
Irvin CG,
Glezen LS,
Marsh WR,
Haslett C,
Henson PM,
and
Larsen GL.
The requirement for polymorphonuclear leukocytes in the late asthmatic response and heightened airways reactivity in an animal model.
Am Rev Respir Dis
134:
62-68,
1986[ISI][Medline].
31.
Okayama, M,
Shen T,
Midorikawa J,
Lin J-T,
Inoue H,
Takishima T,
and
Shirato K.
A muscarinic agonist inhibits reflex bronchoconstriction in normal but not in asthmatic subjects.
J Appl Physiol
67:
2461-2465,
1989
32.
Oshiba, A,
Hamelmann E,
Takeda K,
Bradley KL,
Loader JE,
Larsen GL,
and
Gelfand EW.
Passive transfer of immediate hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin (Ig) E and IgG1.
J Clin Invest
97:
1398-1408,
1996
33.
Ott, L.
An Introduction to Statistical Methods and Data Analysis. North Scituate, MA: Duxbury, 1977, p. 408-457.
34.
Oury, TD,
Day BJ,
and
Crapo JD.
Extracellular superoxide dismutase in vessels and airways of humans and baboons.
Free Radic Biol Med
20:
957-965,
1996[ISI][Medline].
35.
Peled-Kamar, M,
Lotem J,
Okon E,
Sachs L,
and
Groner Y.
Thymic abnormalities and enhanced apoptosis of thymocytes and bone marrow cells in transgenic mice overexpressing Cu/Zn-superoxide dismutase: implications for Down syndrome.
EMBO J
14:
4985-4993,
1995[Abstract].
36.
Peled-Kamar, M,
Lotem J,
Wirguin I,
Weiner L,
Hermalin A,
and
Groner Y.
Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type Cu/Zn superoxide dismutase.
Proc Natl Acad Sci USA
94:
3883-3887,
1997
37.
Renz, H,
Smith HR,
Henson JE,
Ray BS,
Irvin CG,
and
Gelfand EW.
Aerosolized antigen exposure without adjuvant causes increased IgE production and increased airways responsiveness in the mouse.
J Allergy Clin Immunol
89:
1127-1138,
1992[ISI][Medline].
38.
Sanders, SP,
Zweier JL,
Harrison SJ,
Trush MA,
Rembish SJ,
and
Liu MC.
Spontaneous oxygen radical production at sites of antigen challenge in allergic subjects.
Am J Respir Crit Care Med
151:
1725-1733,
1995[Abstract].
39.
Schultheis, AH,
Bassett DJP,
and
Fryer AD.
Ozone-induced airway hyperresponsiveness and loss of neuronal M2 muscarinic receptor function.
J Appl Physiol
76:
1088-1097,
1994
40.
Sedgwick, JB,
Calhoun WJ,
Vrtis RF,
Bates ME,
McAllister PK,
and
Busse WW.
Comparison of airway and blood eosinophil function after in vivo antigen challenge.
J Immunol
149:
3710-3718,
1992
41.
Sedgwick, JB,
Vrtis RF,
Gourley MF,
and
Busse WW.
Stimulus-dependent differences in superoxide anion generation by normal human eosinophils and neutrophils.
J Allergy Clin Immunol
81:
876-883,
1988[ISI][Medline].
42.
Smith, LJ,
Shamsuddin M,
Sporn PH,
Denenberg M,
and
Anderson J.
Reduced superoxide dismutase in lung cells of patients with asthma.
Free Radic Biol Med
22:
1301-1307,
1997[ISI][Medline].
43.
Stelts, D,
Egan RW,
Falcone A,
Garlisi CG,
Gleich GJ,
Kreutner W,
Kung TT,
Nahrebne DK,
Chapman RW,
and
Minnicozzi M.
Eosinophils retain their granule major basic protein in a murine model of allergic pulmonary inflammation.
Am J Respir Cell Mol Biol
18:
463-470,
1998
44.
Stevens, WHM,
Inman MD,
Wattie J,
and
O'Byrne PM.
Allergen-induced oxygen radical release from bronchoalveolar lavage cells and airway hyperresponsiveness in dogs.
Am J Respir Crit Care Med
151:
1526-1531,
1995[Abstract].
45.
Stuart-Smith, K,
and
Vanhoutte PM.
Airway epithelium modulates the responsiveness of porcine bronchial smooth muscle.
J Appl Physiol
65:
721-727,
1988
46.
Tsukagoshi, H,
Haddad E-B,
Sun J,
Barnes PJ,
and
Chung KF.
Ozone-induced airway hyperresponsiveness: role of superoxide anions, NEP, and BK receptors.
J Appl Physiol
78:
1015-1022,
1995
47.
Walker, C,
Kaegi MK,
Braun P,
and
Blaser K.
Activated T cells and eosinophilia in bronchoalveolar lavages from subjects with asthma correlated with disease severity.
J Allergy Clin Immunol
88:
935-942,
1991[ISI][Medline].
48.
White, CW,
Avraham KB,
Shanley PF,
and
Groner Y.
Transgenic mice with expression of elevated levels of copper-zinc superoxide dismutase in the lungs are resistant to pulmonary oxygen toxicity.
J Clin Invest
87:
2162-2168,
1991[ISI][Medline].
49.
White, CW,
Nguyen DH,
Suzuki K,
Taniguchi N,
Rusakow LS,
Avraham KB,
and
Groner Y.
Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase.
Free Radic Biol Med
15:
629-636,
1993[ISI][Medline].
50.
Yang, Z-J,
and
Biggs DF.
Muscarinic receptors and parasympathetic neurotransmission in guinea-pig trachea.
Eur J Pharmacol
193:
301-308,
1991[ISI][Medline].