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Vol. 284, Issue 1, 222-227, 1998
Department of Pharmacology and Toxicology (T.E.L., J.S.F.), Robert C. Byrd Health Sciences Center, West Virginia University, and Pathology and Physiology Research Branch (L.L.M., J.S.F.), Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia
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Abstract |
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Abstract
Introduction
Methods
Results
Discussion
References
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In this study, we examined the effects of fluticasone propionate (FP) and pentamidine isethionate (PI) on antigen-induced lung inflammation and airway hyperreactivity in guinea pigs. Male guinea pigs were sensitized on days 0 and 14 with 10 µg of ovalbumin (OVA) plus 1 mg of Al(OH)3. On day 21, animals were challenged with a 2% OVA aerosol inhalation until they developed pulmonary obstruction. Animals were treated with aerosol inhalation of FP (2 ml of 0.5 mg/ml, five consecutive doses at 12-hr intervals with the last dose given 6 hr before OVA challenge) or PI (30 mg/ml for 30 min 1 hr before OVA challenge), and control animals received no drug before OVA challenge. Airway reactivity to methacholine (MCh) was assessed before sensitization and 18 hr after OVA challenge. At 18 hr after challenge, histological sections of trachea and lung were examined for eosinophil, dendritic cell (DC) and macrophage cell densities in the airways. In control animals, OVA evoked airway hyperreactivity to MCh in conjunction with pulmonary eosinophilia and increases in DC prevalence in the trachea and bronchi. Treatment with FP or PI abolished the OVA-induced hyperresponsiveness and significantly reduced the OVA-induced increases in eosinophils and DCs in the airways. FP and PI had no effect on saline-treated animals. Our study indicates that both inhaled FP and inhaled PI reduce antigen-induced airway hyperreactivity and pulmonary inflammation in guinea pigs. The results also suggest that the DC is a target of the anti-inflammatory effects of these drugs in the airways.
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Introduction |
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Abstract
Introduction
Methods
Results
Discussion
References
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Asthma
is a common disease that is estimated to affect as many as 12 million
persons in the United States alone (Weiss et al., 1992
). It
is characterized by episodes of reversible airway obstruction,
increased mucus production and infiltration of eosinophils and
mononuclear cells into the airways (Corrigan and Kay, 1991; Sears,
1993). One particular form of the disease, allergen-induced asthma, is
linked to airway hyperresponsiveness (Busse and Sedgwick, 1992).
Patients with allergen-induced asthma exhibit an immediate or
early-phase response that is characterized by the abrupt onset of
bronchoconstriction. A second obstructive response, the late-phase response, occurs 8 to 24 hr after allergen exposure in conjunction with
inflammation in the airways and airway hyperresponsiveness.
This late-phase allergic response is associated with the influx of
eosinophils into the airways (Busse and Sedgwick, 1992). The release of
several toxic proteins, leukotrienes and free radicals from activated
eosinophils are capable of producing significant lung tissue injury
(Frigas et al., 1980, 1991). It has been shown that the
severity of asthma can be correlated to the degree of inflammation in
the airways, in which eosinophils are the predominant inflammatory cell
type (Frew and Kay, 1990). Inflammation in response to allergen
exposure is dependent on the presentation of the allergen for T
lymphocyte activation (Hamacher and Schaberg, 1994). In the lung, there
are three types of accessory cells capable of antigen presentation:
alveolar macrophages (Holt and Batty, 1980; Pretolani et
al., 1988), B lymphocytes (Kammer and Unanue, 1980) and DCs
(Steinman and Nussenzweig, 1980; Sunshine et al., 1980).
DCs are hemapoietic cells derived from the same lineage as epidermal
Langerhans cells (Schuler and Steinman, 1985; Tew et al.,
1982). They are characterized by their long, slender processes (Steinman and Nussenzweig, 1980) and high MHC class II expression (Steinman, 1991). The interaction of the MHC class II molecule with the
T cell receptor, as well as the B7/CTLA-4 costimulatory pathway, is
highly important in the initiation and maintenance of the
allergen-induced inflammatory cascade (Reiser and Stadecker, 1996).
Immunohistochemical analysis of guinea pig bronchi at 2 and 24 hr after
intraperitoneal OVA challenge of subcutaneous OVA-sensitized animals
revealed no increases in eosinophils, T lymphocytes or DC at 2 hr.
However, at 24 hr, significant influxes of eosinophils and MHC class
II-positive cells were seen, whereas the number of T lymphocytes
remained unchanged (Lapa e Silva et al., 1994). These data
suggest a possible role for DC in the maintenance of airway
inflammation after antigen presentation. In a recent study by
Keane-Myers et al. (1997), it was demonstrated that
administration of CTLA-4-Ig in a murine model of asthma resulted in the
abolition of airway hyperresponsiveness and pulmonary eosinophilia
produced by allergen challenge, suggesting that the interaction between DC and T lymphocytes is critical for the development of airway inflammation.
Mast cells have been suggested to play a significant role in
allergen-induced eosinophil recruitment (Kung et al., 1995)
independent of the involvement of antigen presenting cells. However,
Hom and Estridge (1994) have shown in mast cell-deficient mice a
significant eosinophil migration into the airways in response to
allergen challenge. In fact, products of activated mast cells in
culture (de Pater-Huijsen et al., 1997) produced
proliferation of CD8+ T cell clones, suggesting that mast
cells may decrease Th2-type responses seen in asthma through increases
in a CD8+-predominant Th1-type response. Thus, although
many different cell types take part in allergen-induced inflammation,
the activation of T cells by antigen presenting cells, such as DCs, are
integral to the initiation and maintenance of an inflammatory response.
The purpose of this investigation was to assess the effects of two anti-inflammatory compounds, FP and PI, on airway inflammation and airway hyperreactivity in the late-phase response of guinea pigs that were sensitized and challenged via inhalation. Specifically, we wanted to characterize the relationship among pulmonary DC prevalence, airway inflammation and hyperreactivity and the ability of the drugs to inhibit the development of these responses. The hypothesis of this study was that DCs play a central role in stimulating antigen-induced lung inflammation and airway hyperreactivity in asthma. The significance of such a role is that DCs could represent new cellular targets for pharmacological manipulation to reduce the obstructive responses that accompany the onset and exacerbation of asthma.
FP and PI were chosen for study because they exert anti-inflammatory
effects by two different mechanisms. By far, the most effective
treatment for patients with asthma is the administration of
glucocorticoids (Cronstein and Weissmann, 1995). Administration of one
glucocorticoid, budesonide, twice a day for 3 months has been shown to
significantly reduce base-line airway obstruction, airway
hyperresponsiveness to histamine, and the total number of T lymphocytes
and RFD1+ macrophages and HLA-DR expression in bronchial
biopsies (Burke et al., 1992). Glucocorticoids bind to
cytosolic glucocorticoid receptors, and the glucocorticoid-receptor
complexes translocate to the nucleus. The steroid/receptor complexes
bind to the glucocorticoid-responsiveness elements of genes to either
increase or decrease transcription (Barnes, 1995) involved in the
regulation of gene expression for inflammatory cytokines (Ray and
Prefontaine, 1994). FP is currently used in the United States as a
nasal spray for the treatment of allergic rhinitis. In clinical trials,
FP has been shown to have greater anti-inflammatory potency in
asthmatics than other inhaled steroids, such as beclomethasone and
budesonide (Holliday et al., 1994).
Although currently not indicated for the management of asthma, PI is a
nonsteroidal aromatic diamidine that is used for the treatment of
Pneumocystis carinii pneumonia in patients with AIDS (Wispelwey and Pearson, 1991). Its mechanism of action is not certain.
In vitro, PI has been shown to reduce tumor necrosis factor-
(Corsini et al., 1992) and interleukin-1
(Rosenthal et al., 1991) release from alveolar macrophages
and inhibit B lymphocyte responses (Ferrante and Secker, 1985).
In vivo, PI inhibits contact hypersensitivity through a
decrease in MHC class II-positive Langerhans cells (Blaylock et
al., 1991) and blocks the pathophysiological effects of
endotoxemia through inhibition of tumor necrosis factor- and
interleukin-6 release (Rosenthal et al., 1992). These
studies suggest that PI possesses an anti-inflammatory action through the inhibition of cytokine release. Such effects, were they to occur in
the lungs, could reduce the late-phase response.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
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OVA sensitization and challenge protocols. Male Dunkin-Hartley guinea pigs (Harlan, Indianapolis, IN) (300-350 g) were sensitized and challenged with OVA (Sigma Chemical, St. Louis, MO) as follows: animals were sensitized by subcutaneous injection of 0.5 ml of 0.9% NaCl containing 10 µg of OVA dispersed in 1 mg of Al(OH)3 in the nuchal region on days 0 and 14. On day 21, the animals were challenged with OVA aerosol (2% dissolved in saline) until an obstructive airway response appeared. The onset of obstruction was indicated on the basis of increases in BF and SRaw (see below). Control animals received saline aerosol for 3 min on day 21.
Aerosols were generated with an Ultra Neb 99 Devilbiss nebulizer (Devilbiss, Somerset, PA). The details of the method for aerosol generation and regulation of the aerosols have been described previously (Lawrence et al., 1997).
Pulmonary function measurements. Basal SRaw and BF were measured in conscious guinea pigs 1 min before OVA challenge, 1 min after challenge and 18 hr after challenge using a two-chamber whole-body plethysmograph (Buxco Electronics, Troy, NY) connected to a noninvasive airway mechanics analyzer (model LS-20; Buxco). SRaw and BF data were logged at 6-sec intervals. The mean of 10 consecutive interval averages was calculated as the measurement at each time point. BF was calculated as the number of breaths taken in 1 min (BPM), and SRaw was calculated as airway resistance multiplied by the thoracic gas volume (cm H2O · sec).
Lung removal and fixation. At 18 hr after OVA challenge, guinea pigs were anesthetized intraperitoneally with sodium pentobarbital (The Butler Co., Columbus, OH; 50 mg/kg) and exsanguinated by severing the abdominal aorta. A 3-cm length of trachea was marked, and an incision was made in the lower trachea through which a 1-cm piece of polyethylene tubing placed on a 15-gauge needle was inserted and tied. The trachea was then dissected out and stretched to 3 cm. The lungs were inflated with Histocon (Polysciences, Warrington, PA) at a pressure of 30 cm H2O and removed en bloc. The trachea and right diaphragmatic and left cardiac lobes of the lung were snap-frozen in isopentane cooled with liquid nitrogen. The tracheal sections were cut tangentially parallel to the longitudinal axis; lung lobes were cut so the largest airways were included in the sections. Tissue sections, 6 µm thick (Hacker-Bright Micro Cryostat 2122; Hacker Instruments, Fairfield, NJ), were collected on Vectabond-coated slides (Vector Laboratories, Burlingame, CA) and air-dried.
Immunohistochemistry: tissue macrophages and DCs.
Tissue
macrophage prevalence and distribution were identified after incubation
of sections with the anti-guinea pig monoclonal antibody MR-1 (Harlan
Bioproducts for Science, Indianapolis, IN), a marker for phagocytic
tissue macrophages (Kraal et al., 1988).
), as
described previously (Lawrence et al., 1997).
Eosinophil detection.
A histochemical method for
cyanide-resistant eosinophil peroxidase activity was used to stain for
eosinophils present within the respiratory tissue (Yam et
al., 1971; Zucker-Franklin and Grusky, 1976).
In vivo reactivity to MCh. Changes in reactivity to MCh were assessed by measuring SRaw in conscious animals before sensitization and 18 hr after OVA challenge. Each guinea pig was placed in the plethysmograph box for a 10-min acclimation period. An aerosol of saline followed by increasing concentrations of MCh (Sigma) from 0.1 to 80 mg/ml in increments of 2-fold were given for 3 min via a side port in the head chamber of the plethysmograph. SRaw readings were taken at 6-sec intervals for 1 min after each exposure. Increasing MCh concentrations were given until SRaw was approximately three times the base-line reading measured after delivery of saline aerosol to the animal.
Administration of drugs.
FP (a gift from Glaxo,
Hertfordshire, UK) was prepared as a fine suspension in 0.1% ethanol.
FP was given as aerosol (0.5 mg/ml) for 10 min five times at 12-hr
intervals. The last FP dose was given 6 hr before OVA challenge. This
dosing regimen was based on a study on the effects of aerosolized
budesonide on inflammation and airway hyperreactivity in trimellitic
anhydride-sensitized guinea pigs (Hayes et al., 1993).
) and preliminary studies performed in our
laboratory (data not shown).
After drug administration, groups of animals were assessed for in
vivo changes in airway reactivity to MCh or for inflammatory cells
18 hr after OVA challenge, as described previously.
Treatment effects on SRaw: Quantitation and statistics. All data are expressed as arithmetic mean ± S.E.M. A value of P < .05 was considered significant. The effects of treatment on SRaw were analyzed using one-way analysis of variance with repeated measures.
To characterize reactivity to inhaled MCh, SRaw values logged for each MCh concentration were averaged and plotted vs. the MCh concentration. The MCh concentration that doubled SRaw ([MCh] PC200) above the basal level was determined by linear interpolation from concentration-response curves. Changes in reactivity to MCh were analyzed with a paired t test.Immunohistochemistry and enzyme histochemistry: Quantitation and
statistics.
Slides were coded and read in a blinded fashion.
Positive cells in the trachea were enumerated in several regions: the
epithelium between the lumen and basement membrane, lamina propria
between the basement membrane and smooth muscle and the submucosa
between the smooth muscle and cartilage. Positive cells in the bronchi were enumerated in the lamina propria, between the basement membrane and the smooth muscle, and in the adventitia, outside the smooth muscle
(Bai and Prasad, 1994). Cells present in the epithelium of the bronchi
could not be quantified due to the extensive folding of the epithelial
layer. The number of inflammatory cells in the areas of interest was
determined as described previously (Lawrence et al., 1997)
using the Optimas Image Analysis System (Optimas Corp., Edmonds, WA).
Results are expressed as the number of positive cells/mm2
compartment area (mean ± S.E.M.).
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Results |
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Abstract
Introduction
Methods
Results
Discussion
References
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Effects of FP on obstruction, inflammation and airway
hyperreactivity in response to OVA challenge.
Treatment with FP
before OVA challenge did not inhibit the increase in SRaw that is
observed immediately after OVA challenge (Lawrence et al.,
1997).
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Effects of PI on obstruction, inflammation and airway
hyperreactivity in response to OVA challenge.
The early elevation
in SRaw after OVA challenge was not affected by PI treatment (Lawrence
et al., 1997). The administration of PI aerosol before OVA
challenge produced anti-inflammatory effects similar to those seen
after FP administration. OVA-induced eosinophil influx was reduced
significantly by 70% in the tracheal epithelium after PI
administration to a level that remained significantly greater than the
value observed in the saline-treated animals (fig.
5); that is, PI did not completely
inhibit the OVA-induced eosinophil response.
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Inhaled glucocorticoids are effective in preventing the
inflammation observed in asthmatics (Schleimer, 1993). Although these compounds have been shown to act on many of the cell types involved in
antigen-induced lung inflammation (Schleimer, 1990), their effect on DC
in the airways has not been investigated in the guinea pig. FP given by
inhalation before OVA challenge significantly reduced the elevation in
the prevalence of DC that would ordinarily occur in all tracheal and
bronchial compartments. In conjunction with the inhibited DC response
was a decrease in eosinophil influx into the tracheal epithelium. Last,
the development of airway hyperreactivity to MCh was prevented.
Previous studies of glucocorticoid effects on allergen-induced
responses in the guinea pig have focused primarily on alterations of
inflammatory cell influx into the airways with little attention being
paid to concomitant changes in pulmonary function and airway responsiveness. Oral administration of methylprednisolone at 2 and 24 hr before OVA challenge significantly reduced pulmonary eosinophilia
(Chand et al., 1990). Likewise, the administration of
dexamethasone (Tarayre et al., 1991) and betamethasone
(Gulbenkian et al., 1990) to OVA-treated guinea pigs was
shown to decrease eosinophil influx into the airways. The
administration of dexamethasone before OVA challenge decreased both
eosinophil and CD4+ T lymphocyte infiltration of the
bronchial wall (Lapa e Silva et al., 1995).
The only previous report of the effects of FP on pulmonary DC was
obtained using rats (Nelson et al., 1995). FP pretreatment for 7 days in a rat model of respiratory viral infection decreased DC
number by 50%. This finding, as well as the results of the present
study, reveals that steroids can modify the pulmonary DC response and
supports the idea that DC are an important component in the initiation
of pulmonary inflammation, whether virus or allergen induced.
PI is a compound that is not currently used in the management of asthma
but has been shown in animal models to possess anti-inflammatory properties. The drug inhibits B lymphocyte responses (Ferrante and
Secker, 1985), reduces antigen presentation by Langerhans cells
(Blaylock et al., 1991) and inhibits the production of
numerous cytokines such as tumor necrosis factor-, interleukin-1 and
interleukin-6 (Corsini et al., 1992; Rosenthal et
al., 1991, 1992). The administration of PI reduced OVA
challenge-induced eosinophil influx into the tracheal epithelium,
prevented the development of airway hyperreactivity to MCh and reduced
DC prevalence in the tracheal epithelium and tracheal and bronchial
lamina propria. However, in contrast to FP, PI treatment did not affect
DC number in either the tracheal submucosa or bronchial
adventitia. Why DC prevalence remained unchanged in the deeper
regions of the tracheal and bronchial walls remains to be seen. It
could be related to the limitations on the uptake and availability of
PI into the walls of the airways. Studies in both rats and mice have
shown that aerosolized PI produces high, sustained lung tissue levels
(Debs et al., 1987), but this could reflect absorption
through the walls of smaller airways or alveoli. Therefore, it is
possible that increasing the dose or number of treatments would allow
greater deposition of the drug. On the other hand, the
anti-inflammatory effects of FP and PI may not be the same at any dose
of PI.
In conclusion, we developed an animal model of asthma in which increases in airway DC, airway inflammation and airway hyperreactivity are concurrently observed. The administration of FP, an anti-inflammatory drug, and PI, an antiprotozoal compound with known anti-inflammatory effects, inhibited the OVA-induced increase in DC prevalence in the airways while reducing the levels of other inflammatory cells and preventing the development of airway hyperreactivity. These results suggest a possible correlation between the events leading to the increase in pulmonary DC prevalence and the development of lung inflammation and airway hyperreactivity. Our study suggests that DCs, which are at the top of the inflammation cascade initiated by antigen, may be targets for the effects of currently available and future drugs for the treatment of asthma. Inhibition of DCs may prevent the development of airway hyperresponsiveness.
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Acknowledgments |
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We thank Kathy Fedan and R. Brent Lawrence for comments on the manuscript. (Mention of brand name does not constitute product endorsement.)
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Footnotes |
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Accepted for publication September 26, 1997.
Received for publication May 19, 1997.
1 This work was supported in part by National Institutes of Health Grant 5-T32-GM07039 and Berlex Biosciences.
Send reprint requests to: Dr. Jeffrey S. Fedan, NIOSH, 1095 Willowdale Road, Morgantown, WV 26505. E-mail: jsf2{at}cdc.gov.
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Abbreviations |
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BF, breathing frequency; DAB, diaminobenzidine; DC, dendritic cells; FP, fluticasone propionate; OVA, ovalbumin; PI, pentamidine isethionate; MCh, methacholine; SRaw, specific airway resistance.
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Introduction
Methods
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by alveolar macrophages: Modulation by pentamidine isethionate.
Immunol Lett
34: 303-308[Medline]. 
B and the glucocorticoid receptor.
Proc Natl Acad Sci USA
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) and FP-treated (
) guinea pigs. There was a significant decrease in the
[MCh] PC200 18 hr after challenge in untreated but not
FP-treated animals. There was no significant difference in
PC200 values before OVA challenge. A decrease in
PC200 indicates an increase in airway reactivity.
n = 8 for each treatment group. * P < .05 compared with presensitization values.
