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Center for Allergy, Asthma, and Immunology, Creighton University School of Medicine, Omaha, NE 68178
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rather unambiguous suppression of preexisting IgE responses in OVA-sensitized mice, as alluded to above, has been reported (7). However, previously published work by others and us (14) have suggested that mycobacterial Ags may not require down-regulation of IgE production to achieve suppression of airway eosinophilia and airway hyperresponsiveness (AHR) (5). These observations have called into question (in the mouse model) the absolute necessity of IgE suppression to attenuate the underlying pathology in asthma, that of airway narrowing. Mycobacterium vaccae, a nonpathogenic mycobacterial species, was specifically included in these experiments to test, within the same animals, its effects upon IgE and pulmonary function.
In this study, we investigated the ability of mycobacteria to suppress preexisting AHR and airway resistance as measured by enhanced pause (Penh) associated with the early allergic response (EAR) and late allergic response (LAR). We hypothesized that treatment with mycobacterial Ags in Ag-presensitized and -challenged mice would reverse and suppress existing allergic airway inflammation and associated clinical correlates of established asthma in mice, excluding factors associated with the EAR.
We observed, for the first time, that previously existing AHR and airway resistance during the LAR in sensitized mice can be reversed with BCG treatment and, to a lesser extent, with M. vaccae treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four- to 5-wk-old female BALB/c mice were obtained from Harlan Laboratories (Indianapolis, IN) and housed according to the National Institutes of Health guidelines. The research protocol of this study was approved by the Animal Research Committee of Creighton University (Omaha, NE). Mice were housed in separate cages according to treatment. Food and water were provided ad libitum.
Mycobacteria preparation
Lyophilized BCG (Tice; Organon, West Orange, NJ) and M. vaccae (no. 29678; American Type Culture Collection, Manassas, VA) were cultured in Lowenstein-Jensen medium (REMEL, Lenexa, KS) at the Creighton University Pathology Lab. Three to 4 wk after the cultures were begun, the vials were centrifuged and resuspended in PBS. Logarithmic dilutions of the cells into sterile vials were made for both bacteria. Agar plates (7-H10; REMEL) corresponding to logarithmic dilutions were incubated for an additional 2 wk. The stock vials were frozen at -80°C. CFUs on the culture plates were counted and an estimation of the number of viable organisms in the stock vials was then made. Aliquots of 1 x 105 organisms (50 µl) in medium were made and refrozen at -80°C until needed.
Sensitization
Mice were sensitized on days 0 and 14 with an i.p. injection of
20 µg grade V chicken egg OVA (Sigma-Aldrich, St. Louis, MO) and 2 mg
alum (Imject Alum; Pierce, Rockford, IL) suspended in PBS to a total
volume of 100 µl. This was followed by a daily administration of
nebulized 1% OVA for 20 min from day 28 through day 30. Nonsensitized
control animals received only the PBS (Fig. 1
).
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Single-chamber whole-body plethysmographs (Buxco Electronics, Troy, NY), without the use of anesthesia or restraint, were used to measure pulmonary functions. This method has been demonstrated to accurately reflect airway resistance (14, 15), expressed as the Penh units (16).
Initial allergen challenge
All mice were placed in individual plethysmograph chambers on day 32 and baseline Penh readings were taken. Subsequently, mice were challenged to 5% aerosolized OVA for 20 min. This was followed by recording of pulmonary functions during the EAR (0–30 min) and the LAR (1–7 h). The results of Ag challenges are expressed as the area under the curve (AUC), which takes into account the complete breadth of either the EAR or LAR. An individual mouse’s baseline Penh value served as the reference to which subsequent increases in Penh were compared.
Initial methacholine challenge
Mice were challenged on day 33, 24 h post-OVA challenge, with increasing doses of aerosolized methacholine, and pulmonary functions were recorded using the Buxco whole-body plethysmograph system. An aerosol challenge at each dose was administered via an Ultra Neb-90 (DeVilbiss, Sommerset, PA) with the highest setting for exactly 1 min. A 1-min wash-out period followed. Immediately thereafter, data were recorded for 5 min and a mean of this time period, in terms of Penh, was made. After the recording period, the Penh values for each mouse were allowed to return to baseline before the next higher dose of methacholine was administered.
The results of methacholine challenges were transformed into the Penh index, where increasing Penh units for a given mouse are expressed in terms of the fold increase from the baseline Penh unit. Data from the methacholine challenges were compared in two different ways. First, the PC200, the dose of methacholine at which a 200% increase in Penh units was observed, was calculated for each animal and compared as a measure of airway hypersensitivity. Second, the maximum Penh index for each animal was also compared as a measure of airway hyperreactivity.
Randomization and treatment
Following methacholine challenge, nonsensitized mice were
equally and randomly divided into two groups (intranasal (i.n.) or i.p.
treatment with vehicle) and sensitized mice were equally and randomly
divided into six groups (i.n. or i.p. treatment with either vehicle,
BCG or M. vaccae) and treated with the appropriate
mycobacterial Ag (Fig. 1
). Mice in i.n. experimental groups were
anesthetized with ketamine and xylazine (20:1) followed by immunization
with 50 µl of 1 x 105 CFUs of the
appropriate organism. Mice in i.p. experimental groups were injected
with 50 µl of 1 x 105 CFUs of the
appropriate organism. Nonsensitized and sensitized control mice were
treated only with the vehicle (PBS) via the appropriate route.
Final Ag and methacholine challenges
On day 40, Ag challenge was conducted exactly as previously described. A final methacholine challenge (as described) was administered on day 41.
BAL collection
Immediately following the final methacholine challenge the mice were euthanized with a lethal dose of pentobarbital. Tracheas were cannulated and lungs were washed with 1 ml PBS. Cytospin slides were prepared from each sample following lavage cell counting using a Coulter counter (Beckman Coulter, Fullerton, CA). Slides were stained with DiffQuik (Baxter Healthcare, McGaw Park, IL) for analysis of differential cell populations using standard morphological criteria.
Serum Ig analysis
Blood collected after sacrifice on day 41 was immediately centrifuged and serum was collected and stored at -70°C for later analysis. ELISA for both total and Ag-specific IgE was conducted as previously described (17) and according to the manufacturer’s recommendations using rat anti-mouse IgE (BD PharMingen, San Diego, CA), standard IgE (BD PharMingen), and rat anti-mouse IgE-HRP (Southern Biotechnology Associates, Birmingham, AL) for the total IgE assay, with the substitution of biotinylated OVA (Immunoprobe biotinylation kit; Sigma-Aldrich), followed by addition of streptavidin-HRP (BD PharMingen), for the Ag-specific assay. Both cytokine and Ig assays were developed with 3,3',5,5'-tetramethylbenzidine substrate and read at 450 nm using a Bio-Rad microplate reader and software (Bio-Rad, Hercules, CA). Sensitivity for total IgE was 1 ng/ml. Ag-specific IgE results are expressed in units of absorbance (OD).
Cytokine analysis
Cytokines were measured in the supernatants of bronchoalveolar
lavage (BAL) fluid and/or serum. Ab pairs and protein standards for
IL-4, IL-5, and IFN-
(BD PharMingen), as well as IL-3 and
TGF-
1 (R&D Systems, Minneapolis, MN) were used
according to the manufacturer’s recommendations. Sensitivities for the
assays were 12, 8, 9, 8, and 5 pg/ml, respectively.
Histology
Whole lungs were removed, set in tissue freezing medium
(Triangle Biomedical Sciences, Durham, NC), and frozen immediately in
liquid nitrogen. Sections of 8-µm thickness were prepared and stained
with H&E. Slides were analyzed under low power (x10) for a
determination of total peribronchial inflammation. Semiquantitative
analysis was achieved by assigning a value of 0 for no inflammation, 1
for mild inflammation, 2 for moderate inflammation, and 3 for severe
inflammation (see Fig. 2). Under higher
power magnification (x40), eosinophilic infiltration was determined by
counting the number of eosinophils within the inflamed peribronchial
region and expressing this as a percentage.
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Data were analyzed using GraphPad PRISM statistical analysis and graphing software (GraphPad, San Diego, CA). For pulmonary function assays, the analysis of variance using all treatment groups was used to determine differences between both the experimental or control groups and the prerandomized sensitized animals (on day 40/41 vs day 32/33), as well as between nonsensitized and sensitized control groups (on day 40/41). All other assays were also compared using analysis of variance. Values given are means ± SEM from at least six animals in each group unless otherwise noted. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One week following mycobacterial Ag inoculation to presensitized
mice, the animals were again challenged with aerosolized 5% OVA and
monitored for changes in airway resistance as described. There was a
significant increase in the EAR and LAR in OVA-sensitized and
-challenged group (Figs. 3 and 4). None of the treatment groups
exhibited a significant change in the EAR (Fig. 4A). Only
those animals that received i.n. BCG showed a significantly suppressed
response in the LAR (Fig. 4B). Statistical comparison
between the nonsensitized and sensitized control mice for the airway
resistance during the EAR and LAR on both days 32 and 40 showed
significant differences in the EAR (p < 0.001)
as well as the LAR (p < 0.001), while no
significant changes after the 8 days were seen within each control
group.
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There was a significant increase in both airway hypersensitivity
and airway hyperreactivity in OVA-sensitized and -challenged mice (Fig. 5). The PC200
values to methacholine were used as a measure of airway
hypersensitivity in this experiment, as mice typically reach this point
early in the dose response curve. Highly significant suppression was
observed for each experimental group (Fig. 5B). As a measure
of airway hyperreactivity, the maximum Penh index
was used, which reflected the highest degree of airway resistance as
measured by this assay. Only the animals treated i.n. with BCG showed
significant reduction in maximum Penh index (Fig. 5C). Differences between nonsensitized and sensitized
control mice were significant for both parameters on either of day 32
and day 40 (p < 0.01), while no significant
changes were observed after 8 days within each control group.
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Lavage fluid was collected following methacholine challenge on day
41. Eosinophils were significantly reduced only in the i.n.
experimental groups, to statistically the same degree in each (Table I). Lymphocytes, the only other group of
white cells to be attenuated in the experiment, were reduced only in
the M. vaccae-treated animals, regardless of Ag delivery
route. Finally, while both i.n. treatment groups yielded apparent
reductions in total BAL leukocytes, only the M.
vaccae-treated animals showed a significant reduction.
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While very significant increases were seen in sensitized control
animals compared with nonsensitized animals, no reductions compared
with sensitized animals were seen with any experimental group for
either total IgE or OVA-specific IgE (Table II).
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levels
Supernatants collected from BAL fluid were analyzed for the
presence of several cytokines. IL-5 levels were significantly reduced
in all treatment groups, except for i.n. M. vaccae (Table III). Interestingly, IFN- was only
detected in the BAL fluid from mice treated with i.n. BCG. IL-4 levels,
while tending toward lesser amounts in all treatment groups, were not
found to be significantly decreased after mycobacterial Ag exposure.
TGF- was observed at insignificantly increased levels in almost all
experimental groups compared with sensitized controls. IL-3 levels
could not be detected in our BAL assays. There was no significant
difference in serum IFN- levels when compared between the treatment
and sensitized control groups. Assays for serum IL-3, IL-4, and IL-5
were, unfortunately, not sufficiently sensitive.
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Lung sections were inspected for both an overall semiquantitative
score of inflammation as well as a measure of the degree of
eosinophilic infiltration. Postsensitization treatment with
mycobacterial Ags did not have any effect on the degree of
peribronchial inflammation (Figs. 1 and 6A). However, the relative
number of eosinophils infiltrating the peribronchial region was
significantly reduced only with BCG treatment of either route, while
apparent, yet insignificant, reductions were also seen with M.
vaccae treatment (Figs. 1 and 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intranasal BCG treatment (one dose, 1 x 105
CFUs) was the most effective combination in reversing Ag-induced asthma
in this study. It was effective in reducing Penh,
which correlates well with airway resistance and intrapleural pressure
(16) during the LAR (Fig. 4B), both airway
hypersensitivity and hyperreactivity to methacholine (Fig. 5), BAL
eosinophilia (Table I), BAL IL-5 (Table III), and peribronchial
eosinophilia (Fig. 6). Other combinations, while variably effective in
terms of cellularity and cytokines, did not differ among each other in
terms of their effects on pulmonary functions. While these data cannot
discern the exact mechanism, the observations of highly significant
reductions in peribronchial eosinophilic infiltration and BAL fluid
IL-5 concentrations in samples from animals treated i.n. with BCG do
implicate the role of the eosinophil. Indeed, many reports have also
positively correlated eosinophils and/or IL-5 with AHR
(18, 19, 20). However, there have been negative reports as
well, suggesting that eosinophils are neither required nor sufficient
to induce AHR (21). In other reports, treatment with mAb
to IL-5 had no effect on the reversal of established AHR in mice
(22) and humans (23) despite complete
suppression of eosinophil accumulation of airway tissue. Further
studies, therefore, are clearly warranted to understand the role of
eosinophils in AHR.
None of the treatments abrogated IgE (total or OVA-specific), IL-4, or OVA-induced airway resistance associated with the EAR. These findings are consistent with our previous observations that mycobacterial pretreatment also failed to prevent this set of parameters (14).
In addition to us, others have demonstrated a suppressive effect on various parameters of allergic inflammation in mouse models after mycobacteria pretreatment (4, 5, 8, 9). However, except for one report from the work of Wang and Rook (7), there is no information as to the potential for these Ags in attenuating a preexisting allergic state. This is not an insignificant detail, as envisioning future immunotherapies for allergic asthma must certainly take into account presensitized individuals in addition to aiming to prevent sensitization altogether.
In light of the potential clinical application of the literature in this area, the stark contrast of our findings on the effect of mycobacterial Ags on serum IgE to those of Wang and Rook (7) becomes all the more important. Similarities between the study designs include very relevant factors of gender and genetic strain, while the differences may account for the discrepancies. Wang and Rook (7) found total IgE suppression (with neither OVA-specific IgE nor IgG1 being suppressed) with 107–109 CFUs of M. vaccae. The differences in our results from those of Wang and Rook (7) could be due to the differences in the type and amount of M. vaccae used. For example, they used attenuated M. vaccae and 100–10,000 times more Ag than our protocol. Erb et al. (5), using BCG treatment at the outset of OVA sensitization, found no change in either IgE or IgG1 with doses on the order of 105 CFUs, while other allergic parameters were nonetheless suppressed. We did not include such a high range of concentrations, as this lower dose has consistently suppressed the central parameters of AHR, airway resistance during the LAR, and eosinophilia (14, 24).
One of the hypotheses we have proposed as a result of our research is that mycobacterial Ags suppress asthma-like parameters independent of IgE. This is consistent with the reports of other investigators who have shown that IgE is not required for the development of eosinophilic airway inflammation and AHR in mice (21, 25, 26, 27). This could also be true in humans as observed by Haselden et al. (28). These investigators demonstrated that an intradermal injection of a linear peptide sequence within an allergen at a high dose can directly initiate a MHC-restricted, T cell-dependent late asthmatic reaction, without the requirement for an early IgE mast cell-dependent response in sensitized asthmatic subjects (28). These data support our model of airway inflammation and AHR. Furthermore, should this prove true in most of the asthmatic subjects, the entire breadth of the type 2 response, such as IL-4 and IgE, may not necessarily demand suppression, easing concerns of the notion of a pendulum swinging too far to the type 1 T cell response side (and unintended effects that may accompany this) and enabling novel approaches toward conventional immunotherapy. Indeed, the current therapy of choice for moderate to severe asthmatics is inhaled glucocorticoids, which themselves do little to suppress IgE levels (29, 30, 31).
Attempts to reverse or suppress a preexisting type 2 cytokine-weighted
state raises questions of the stability and plasticity of Th cytokine
secretion profiles. Ohta et al. (32) demonstrated a
delayed-type hypersensitivity response in BALB/c mice adoptively
transferred with Th1 cells primed in vitro, a response that lasted
several months. Infants, too, are believed to possess a Th2-weighted Th
repertoire (33), and recent evidence supports the
hypothesis that in utero allergic sensitization occurs
(34). Marchant et al. (35) demonstrated a
lasting proliferative response and IFN- release in response to
purified protein derivative challenge at 1 year of age in
infants immunized with BCG at birth. Interestingly, a significant
suppression of IL-4 was not concurrently observed (35).
The data obtained in this study support this. Murphy et al.
(36) present data arguing against the plasticity of Th
populations after long-term (3 wk), polarized stimulation. Such in
vitro experiments almost certainly oversimplify the complex,
heterogeneous stimuli presented to maturing T cells in vivo and must be
very cautiously extrapolated. Nonetheless, the issue is highly relevant
and ongoing studies in our lab aim to address it. Ultimately, the
quality somewhat unique to mycobacterial Ags, to evade complete
eradication by the immune system and thus continue to stimulate local
type 1 cytokine responses, may become realized as a rather useful,
slow-release "capsule" for sustained suppression of some Th2-like
responses.
In the present study, mycobacterial Ags failed to suppress the local
secretion of TGF-1 (Table III). This cytokine
has been implicated in the profibrotic changes occurring in airway
remodeling (37). Investigators have further linked
expression of TGF-1 with eosinophils
(38, 39) and have found significantly increased levels in
BAL fluid after allergen challenge (40) in humans. Our
finding of unaffected TGF-1 concentrations in
murine BAL samples was an unexpected result in light of the suppressed
eosinophil numbers. Although a complete account of the relative role
that airway macrophages may play in TGF-1
levels, and thus remodeling, remains to determined, it is known that
these cells can produce this cytokine (41), and activated
macrophages may have been an important source in our experiment. In any
event, the TGF-1 finding in this study does
encourage a more thorough examination of the effects mycobacteria may
have on airway remodeling.
The blockade of eosinophils through various mechanisms has been
correlated with abrogation of LAR (42) and airway
hyperreactivity (19). There is also considerable evidence
suggesting the eosinophil product major basic protein and its
antagonistic effect on inhibitory M2 receptors
may be at least partly responsible for this acute airway narrowing
(43). Eosinophils, both BAL and peribronchial, were most
significantly inhibited in mice treated with BCG i.n. (Table I and Fig. 5). The most dramatic reduction in both airway hypersensitivity and
hyperreactivity was also observed in this group (Fig. 4). These results
support the hypothesis that eosinophils, and not Ag-specific B cells,
are a more important target of the action of mycobacterial Ags in this
mouse model of asthma.
In summary, we have shown that low, single-dose mycobacterial treatment can suppress the LAR, AHR to methacholine, and BAL IL-5 and eosinophilia in presensitized BALB/c mice without affecting serum IgE levels. While more investigation is needed to define the durability of this effect, these results support the hypothesis that BCG may be an effective immunotherapeutic agent, operating in unique ways to inhibit asthma symptoms.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Devendra K. Agrawal, Center for Allergy, Asthma, and Immunology, Creighton University School of Medicine, CRISS I Room 131, 2500 California Plaza, Omaha, NE 68178. E-mail address: dkagr{at}creighton.edu
3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; EAR, early allergic response; LAR, late allergic response; Penh, enhanced pause; i.n., intranasal(ly); AUC, area under the curve.
Received for publication June 18, 2001. Accepted for publication January 2, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adrenoceptor-mediated responses in human lung mast cells. Br. J. Pharmacol. 121:717.[Medline]
RII/sCD23) in childhood bronchial asthma. Arch. Dis. Child. 75:314. in tissue fibrosis. N. Engl. J. Med. 331:1286. expression in mucosal biopsies in asthma and chronic bronchitis. Am. J. Respir. Crit. Care Med. 156:591.1 mRNA expression and airways fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 17:326.1 in asthma: measurement in bronchoalveolar lavage fluid. Am. J. Respir. Crit. Care Med. 156:642. (TGF-) and fibronectin by alveolar macrophages in airway diseases. Clin. Exp. Immunol. 106:114.[Medline]
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), OVA-sensitized and
challenged (
), i.n. BCG (
), i.p. BCG (
), i.n. M.
vaccae (•), and i.p. M. vaccae (
).
