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* Hart and Louise Lyon Laboratory, Division of Clinical Immunology and Allergy, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024-1680
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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levels were significantly
inhibited. These results suggest that ETS can induce allergic
sensitization to a normally harmless Ag, and they may explain why
secondhand smoke is a major risk factor for the development of allergy
in children. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we investigated whether ETS can induce sensitization to OVA (i.e., induce a primary response). In most murine models, repeated aerosolized exposure to protein Ag normally induces tolerance. Although some studies have shown that certain exposure regimes can sensitize mice, in the absence of an adjuvant, to aerosolized OVA (normally an innocuous Ag), no eosinophilia or cytokine changes are observed in these animals. (12) Additionally, increases in Ab responses only occur in IgE high-responder strains of mice (e.g. BALB/c). Sensitization of IgE low-responder strains of mice (e.g. C57BL/6), without prior i.p. immunization, normally requires modulation of the immune system such as overexpression of GM-CSF (13).
We and others (4, 14) have previously shown the potential of particulate pollutants to alter immune function. Thus, the model airborne pollutant, diesel exhaust particles, can induce allergic sensitization in murine models (15, 16, 17, 18) and in the human upper airways. (19) In this study we show that ETS can induce sensitization to OVA in both high and low IgE-responder strains of mice via the airway in a manner highly relevant to normal exposures. We demonstrate that not only did ETS induce Ag-specific IgE and IgG1 responses, but also it could promote cytokine changes and airway eosinophilia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were housed in the UCLA vivarium under specific pathogen-free conditions. The study was approved by the Chancellor’s Animal Research Committee in adherence to guidelines set forth by the National Institutes of Health. Mice were 6–8 wk of age at the onset of each experiment. Additionally, study animals were fed an egg-free chow to preclude them from any environmental exposure to OVA.
Ag and ETS exposure
OVA (Grade V; Sigma-Aldrich, St. Louis, MO) exposure was used as the Ag in this study. Exposure was to a nebulized 1% (w/v) solution of OVA dissolved in PBS for 20 min following a 15-min chamber equilibration. Nebulization was achieved by the Schuco 2000 (Allied Health Care Products, St. Louis, MO) with a flow rate of 6 L/min at the nebulizer cup yielding particle sizes within 0.5–4.0 µm. A control group was exposed to saline alone with no OVA in an identical fashion.
ETS exposure was achieved from the side-stream smoke from 1R4F cigarettes (University of Kentucky Tobacco and Health Research Institute, Lexington, KY). ETS is composed primarily (95%) of side-stream smoke (emitted from the burning zone) and also (4%) smolder stream smoke (emitted from the puffing zone) (20). The reference-filtered cigarette is known to contain 9.2 mg of tar and 0.8 mg of nicotine. (21) It was stored in a sealed plastic bag at 4°C to hold proper moisture levels in the cigarettes. Fifteen minutes before use, they were immediately brought to room temperature and used in an RM G1 Borgwaldt smoking machine (Hamburg, Germany) set at one inhalation/55 s (where an inhalation/exhaust cycle is of a 5-s duration) to conform to Federal Trade Commission guidelines for generation of side-stream smoke. No mainstream smoke entered the system. ETS was mixed with saline administered as before through a nebulizer delivering an airflow of 6 L/min. This acted as a pressurizing agent carrying ETS into and out of the exposure chamber, and it diluted fatally anoxic concentrations of CO. Mice were challenged with the smoke from 5 cigarettes administered over the duration of 1 h.
Both ETS and OVA exposures were performed in a sealed system plexiglas exposure chamber (Ejay International, Glendora, CA) with an internal exposure area of 18 x 9 x 9 in. Chambers were cleaned between exposures to each agent to ensure no accidental contamination.
Exposure protocol
Groups of six BALB/c or C57BL/6 mice were used. Mice received exposures as detailed above to either: saline, 1% OVA for 20 min, 1 h ETS, or 1 h ETS followed by 1% OVA for 20 min. This procedure was repeated for 10 consecutive days. It is important to note that mice receiving either ETS alone or ETS plus OVA were placed together in the same chamber and were challenged simultaneously to ensure identical ETS exposure to both groups. Similarly, the second and fourth groups received simultaneous OVA exposure.
Thirty days after the initial challenge (20 days after its completion), mice were re-exposed to 1% OVA for 20 min. Bronchoalveolar lavage (BAL) was performed 24 h later by standard methods as previously described. (22)
Collection of BAL and blood
Bleeds were performed from the periorbital sinus of the eye under methoxyflurane anesthesia (Pitman-Moore, Mundelein, IL) before the initial exposure (day 1) and at several times subsequent to that. Sera were stored at -70°C until ready for use. BAL was spun at 300 x g for 20 min, and the supernatant was used for cytokine determination (see below). A total BAL cell count was performed using a hemacytometer. Cells were fixed onto slides following cytocentrifugation, and differential staining was then performed by Wright-Giemsa staining (Dade Behring, Newark, DE). Two hundred cells were counted under a microscope by two different investigators and the absolute numbers of each cell type were calculated.
Cytokine and Ig determination
Cytokine levels were determined in BAL fluid. Specifically,
IL-2, IL-4, IL-5, IFN-, and GM-CSF were detected using commercial
ELISA kits consisting of paired Abs and standards (BD PharMingen, San
Diego, CA) as per the manufacturer’s instructions. Cytokine levels
were measured from standard curves constructed from serial dilutions of
the reference standard provided with each kit. The threshold of
detection for IL-2 was 10 pg/ml, IL-was 4.1 pg/ml, IL-5 was 15 pg/ml,
IFN- was 5 pg/ml, and GM-CSF was 7 pg/ml.
Both total IgE and IgG1 were assayed by sandwich ELISA in the
peripheral blood sera of the mice. Briefly, 96-well ELISA plates
(Corning Glass, Corning, NY) were coated overnight with 5
µg/ml Fc region, isotype-specific Abs (anti-IgG1 and
anti-IgE; BD PharMingen). After washing and blocking with 1%
BSA-PBS, sera samples were placed in wells overnight. The sera was
removed from the plate and washed. Biotinylated, Fc-specific detecting
Abs were added to the corresponding wells in question after being
diluted 1/500 in a PBS-Tween buffer containing 0.1% bovine
globulin and 0.5% BSA plus streptavidin alkaline phosphatase at
1/500. The detection mixture was allowed an incubation of 4
h at room temperature before detection with PNPP (10 mg/ml). The
resultant color was read at 405 nm on a microplate reader (DPC Cirrus,
Randolph, NJ). For a comparison standard, purified Abs (polyclonal IgG1
and IgE; PharMingen) were serially diluted and plated to establish a
reference curve. OD readings were compared with these curves and were
given appropriate values.
A similar method was used to quantify allergen-specific Abs. Plates were coated with the specific allergen used in the study; OVA at 50 µg/ml in PBS. Reference curves for OVA-IgG1 were constructed using an OVA-specific IgG1 mAb (Sigma-Aldrich). The detection limit was 5 µg/ml. Pooled sera from hyperimmunized BALB/c mice was obtained and the amount of OVA-IgE in the pool was given an arbitrary value of 100,000 U/ml. Reference curves for OVA-IgE were then constructed from serial dilutions from this pool, and the concentration of OVA-IgE in the test samples were estimated by comparison to this curve. Similarly, reference curves for OVA-IgG2a were constructed from LPS-immunized mice.
Statistical analysis
The Statview II computer package (Abacus Concepts, Berkeley, CA) for the Macintosh was used for all analysis. Comparisons of Ig levels at different times within a group were calculated using a paired t test. Comparisons of Ig and cytokine levels between groups were analyzed using the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ETS induced sensitization to OVA in our murine model. When the low
(C57BL/6) IgE-responder mice were exposed to aerosolized OVA alone for
10 days, no OVA-specific IgE was observed at any of the time points
studied. In contrast, in the group exposed to ETS plus OVA for 10 days,
Ag-specific IgE was apparent 12 days (day 12) after the initial
exposure (mean = 318 ± 108 U/ml) and persisted until day 25
(Fig. 1
A). Exposure to OVA
alone did result in a significant increase on day 12 in the high
responder BALB/c strain (Fig. 1B) compared with baseline,
but this response was transient and levels were back to baseline by day
25. BALB/c mice exposed to both ETS/OVA made significantly higher
OVA-IgE levels than those receiving OVA alone at day 18, and this
response was still significantly above baseline levels at day 30. ETS
also synergized with OVA to significantly elevate total serum IgE in
the low responder C57BL/6 strain (Fig. 1C). Mice exposed to
ETS or OVA alone did not exhibit any change in IgE levels, nor did the
control group that was exposed to saline. In contrast, total IgE levels
in the ETS/OVA groups were significantly elevated from baseline and
were higher than in OVA-exposed animals at days 12, 18, and 25.
There was no difference in total IgE levels between BALB/c mice exposed
to either OVA alone or ETS/OVA (Fig. 1D).
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A), and it remained elevated
up to 30 days after initial exposure. OVA alone did induce OVA-IgG1
responses in BALB/c mice, but this response was significantly greater
in the OVA/ETS group at all time points after day 12 (Fig. 2C). In contrast, ETS did not enhance production of a
Th1-driven isotype, so that in both strains of mice, OVA-IgG2a levels
were similar between OVA- and OVA/ETS-challenged animals (Fig. 2, B and D).
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The ability of ETS to induce eosinophilia was investigated in
high- and low-responder mice. Mice were exposed to OVA, ETS, or both
for 10 days as before, and then 20 days later (30 days after initial
exposure), the mice were re-exposed to 1% OVA for 20 min. BAL fluid
obtained 24 h after re-exposure was assessed for airway
eosinophilia (Fig. 3). Confirming
previous studies (12, 23), BALB/c mice exposed to OVA
alone showed no significant changes in the cellular profile. Total
eosinophil, neutrophil, and mononuclear cell counts were similar in
mice groups exposed to saline, OVA, or ETS alone. However, exposure to
the combination of OVA/ETS resulted in significant eosinophilia
following OVA re-exposure (5.98 x 105
cells/ml vs 0 cells/ml saline control group, p <
0.01). This increase in eosinophil cell numbers was accompanied by an
increase in total cell numbers in this OVA/ETS group. Similarly, in the
C57BL/6 strain, only in the BAL from those mice previously exposed to
both OVA/ETS was an accumulation of eosinophils detected. Unlike the
BALB/c strain, this group was also characterized by significant
increases in neutrophil as well as total cell numbers, but this was
less marked than the eosinophil response.
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Airway eosinophilia and allergic Ab responses are normally
associated with a Th2 cytokine milieu (24, 25). We
therefore studied whether ETS exposure could cause a change in the
cytokine profile in BAL obtained from C57BL/6 mice following
re-exposure to OVA (Table I). In
saline-exposed animals, re-exposure to OVA did not result in changes to
any of the cytokines measured (IL-2, IL-4, IL-5, GM-CSF, or IFN-).
Previous exposure to 10-day aerosolized OVA resulted in elevated
IFN- BAL levels upon OVA re-exposure, but no change was seen in
other cytokines. In contrast, increases in IL-5, GM-CSF, and IL-2 were
observed in BAL from mice with prior exposure to OVA/ETS. In addition,
this group also had significantly reduced levels of IFN- as compared
with the OVA alone exposed group. It is noteworthy that in the ETS
alone-exposed animals, compared with the saline control group,
significantly increased levels of GM-CSF and IL-2 could be
detected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of ETS in atopic sensitization is more controversial. Our studies aimed to study the association between tobacco smoke and allergic sensitization by direct experimentation. The production of specific allergic Abs against inhaled protein allergens is the hallmark of the sensitization phase of allergic airway disease (allergic rhinitis and asthma); subsequent seasonal exposure leads to a secondary immune response. Many studies have demonstrated elevated IgE levels in active mild smokers (32, 33, 34, 35). Studies investigating passive smoke exposure have shown differing results. Among others, Wagner et al. (5) have described a significant increase in serum IgE levels in children of smoking parents. Weiss et al. (6) described a 2.2-fold increased risk of being atopic (as defined by the presence of at least one positive skin test) when maternal smoking occurred. Ronchetti et al. (7) reported both increased skin test reactivity, serum IgE, and increased prevalence of eosinophilia in 9-year-old children of smoking parents. In contrast, other studies have failed to find such a link. Ownby and McCullough (36) found no increase in either total or allergen-specific IgE in children aged 1–19 years exposed to parental smoking. Although Osaka et al. (8) reported an increase in mite-specific IgE levels in Japanese children of smoking parents, Ozasa et al. (9) found a negative association between Japanese cedar pollen-specific IgE and passive smoking in the same population.
In this study we show that ETS can augment primary sensitization to an innocuous protein. Our studies demonstrate that ETS can initiate de novo responses. Previous reports have studied the ability of ETS to augment pre-established Th2 responses. Seymour et al. (11) sensitized BALB/c mice to OVA and then exposed them to ETS or ambient air. Using this murine model of allergy, this showed that ETS had an adjuvant effect on IgE production and eosinophil numbers in the blood. Moreover, IL-4 and IL-10 was significantly higher in aerosolized allergen-sensitized mice exposed to ETS when compared with those exposed to ambient air. Raised serum IgE levels were found in rats exposed to tobacco smoke twice daily 5 days a week for 8 wk, however, it is unclear whether exposure was limited to only side-stream smoke in these experiments.
Aside from the evidence cited above, the effect of ETS on atopy can be
inferred from experiments using other substances. Tobacco smoke
contains 
6000 known chemical components, as well as nitrogen dioxide
and sulfuric acid (20). Some of these components such as
lead acetate, mercuric chloride, nickel sulfate, and tungsten have been
shown to increase allergenicity in animal models. We have previously
shown that, experimentally, diesel exhaust particles (DEP), a model
environmental pollutant, can induce allergic sensitization in a human
nasal model. DEP shares many characteristics with tobacco smoke
including having a particulate phase and the presence of many similar
polyaromatic hydrocarbons (PAH). Of note are the prototypical PAHs
benzo(a)pyrene (which is particularly high in side-stream smoke) and
phenanthrene, which can also induce IgE and Th2 cytokine responses in
mice and in vitro.
The mechanism by which ETS induce primary sensitization has yet to be
established. One possibility is that ETS may be improving Ag
presentation either by causing structural modifications to the allergen
itself, or by absorption of allergen, thereby causing a more persistent
allergen exposure. Allergen adsorption has been observed in vitro after
mixing of DEP with the allergen Lol p1. (17) However, it
is unlikely that this is the case in these experiments, where ETS and
allergen were administered separately. Of course, it is still possible
that absorption/modification occurs in the airways themselves. It is
more plausible to believe that ETS is altering the environment to one
more conducive to allergic sensitization by increasing induction of
adhesion molecules, cytokine cascades, or proinflammatory cells. PAHs
have been demonstrated to enhance MHC-II gene expression in murine
macrophages and to up-regulate CD80 (B7-1) protein (14).
In addition, they can affect production of inflammatory cytokines such
as GM-CSF, IL-1, and TNF-
(4).
Under normal circumstances, the lungs can be viewed as sites of
immunological homeostasis in which repeated aerosolized exposure to
protein Ag induces a T cell-mediated immunological tolerance (13, 37). In this study, in the low responder C57BL/6 strain IgE and
IgG1, responses were observed following ETS/OVA exposure, but not
following exposure to OVA alone. Similar to previous studies,
aerosolized OVA alone induced OVA-IgE responses in BALB/c mice;
however, this was not accompanied by eosinophilia. In contrast, mice
exposed to both OVA and ETS developed eosinophilia had significantly
less IFN- and had an increase in the Th2 cytokine IL-5. Thus, in our
model we have shown that ETS can disrupt the initial lung homeostatic
mechanism via the airway in a manner highly relevant to normal exposure
to permit allergic sensitization characterized by formation of allergic
Abs, eosinophilia, and a Th2 cytokine response.
In conclusion, this work demonstrating the potential of ETS to interact with allergen and augment allergic sensitization provides experimental evidence to support studies that suggest that maternal smoking as a risk factor in the development of atopy. Because the prevalence of parental smoking in the U.S. is estimated to be from 40 to 60%, (38, 39), these results may have serious public health implications.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Diaz-Sanchez, Division of Clinical Immunology and Allergy, Department of Medicine, 52-175 Center for Health Sciences, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095-1680. E-mail address: ddiazsa{at}mednet.ucla.edu
3 Abbreviations used in this paper: ETS, environmental tobacco smoke; BAL, bronchoalveolar lavage; DEP, diesel exhaust particles; PAH, polyaromatic hydrocarbons.
Received for publication May 21, 2001. Accepted for publication August 14, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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