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Vol. 282, Issue 2, 995-1004, 1997
Department of Pharmacology and Toxicology (T.E.L., J.S.F.), Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia and Pathology and Physiology Research Branch (L.L.M., D.G.F., 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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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We characterized the localization and prevalence of dendritic cells (DC) in guinea pig airways before and after s.c. sensitization and aerosol challenge with ovalbumin (OVA). DC, eosinophils, macrophages, T cells and B cells in lung and trachea were identified and quantified in frozen sections using monoclonal antibodies and computer-assisted image analysis. Airway reactivity of conscious animals to inhaled methacholine was examined. In unsensitized animals, DC were localized primarily within the lamina propria of the trachea and bronchi, in the submucosa of the trachea and in the adventitia of the bronchi. In contrast to reported studies on rats, few DC were noted in the epithelium. After OVA challenge, sensitized animals demonstrated an early obstructive response and a late-phase response that was well developed by 18 hr. Challenge with OVA increased DC prevalence in the lamina propria and submucosa of the trachea and in the lamina propria and adventitia of the bronchi. There was widespread eosinophilia throughout the airways, but no changes in B cells or T cells were evident. Macrophages were increased in the epithelium of both OVA-treated and saline-treated animals. At 18 hr after challenge, sensitized guinea pigs but not saline-treated controls were hyperreactive to inhaled methacholine. Except for macrophages, none of these effects were observed after saline treatment. Our findings indicate that inflammation in the airways of OVA-sensitized guinea pigs involves infiltration of DC, which is seen at the time animals are hyperreactive to inhaled methacholine.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Asthma
is a chronic lung disease characterized by reversible airway
obstruction and nonspecific airway hyperreactivity (Sears, 1993
).
Pathophysiologic features include desquamation of the respiratory epithelium, goblet cell metaplasia, increase in mucous gland number, hypertrophy and hyperplasia of airway smooth muscle, inflammation of
the airways and airway hyperreactivity (Corrigan and Kay, 1991). In
asthmatic patients, two phases of response to antigen challenge can be
observed (O'Byrne et al., 1987; Busse and Sedgwick, 1992). The immediate or early-phase response is characterized by
bronchoconstriction, which can be inhibited through the administration
of 
2-adrenoceptor bronchodilators. The second, or
late-phase response, occurs 4 to 8 hr after antigen exposure. This
response is less affected by bronchodilators (Busse and Sedgwick, 1992)
and is associated with inflammation in the airways and airway
hyperresponsiveness.
Pulmonary eosinophilia is prominent in asthmatic airways and is
considered important in the pathogenic changes associated with the
disease. Development of bronchial hyperreactivity (Pretolani et
al., 1994) and damage to the respiratory epithelium (Frigas et al., 1991) have been correlated with the release of major
basic protein and other granule constituents from activated
eosinophils. Recruitment of eosinophils into airways during the
late-phase inflammatory response to antigen involves the sensitization
of T lymphocytes to the antigen with subsequent release of cytokines (Kay et al., 1991), particularly interleukin-3,
interleukin-5 and granulocyte-monocyte colony-stimulating factor.
Inflammation of the airways in response to antigen requires the
presentation of the antigen to T lymphocytes and their subsequent activation. Accessory cells present within the lung that are capable of
antigen presentation to T cells include alveolar macrophages (Holt and
Batty, 1980), B lymphocytes (Kammer and Unanue, 1980) and DC (Steinman
and Nussenzweig, 1980). DC are found in various lymphoid and
nonlymphoid tissues (Schuler and Steinman, 1985). They are
characterized by their long, slender processes and high MHC Class II
expression (Steinman, 1991). DC are present in the airways of numerous
species, including rat, guinea pig and human (Holt and Schon-Hegrad,
1987; Lapa e Silva et al., 1993, Nicod et al.,
1987). In vivo studies in the rat airways have revealed a
network of MHC Class II-positive DC in the epithelium (Holt et
al., 1990), predominantly within the upper airways. The prevalence of DC has been shown to increase by 50% after exposure to wood shavings derived from Pinus radiata (Schon-Hegrad et
al., 1991). The location of DC near the air-tissue interface in
the lung suggests a role for the cells as a first line of defense
against pathogenic and nonpathogenic antigens present within inspired
air.
Pulmonary DC have been studied extensively in other species, such as
the rat (Holt et al., 1990; McWilliam et al.,
1994; Nelson et al., 1994). However, there is a paucity of
information about the localization and role of DC in guinea pig
airways, despite the widespread use of the guinea pig for studies on
mechanisms of inflammation and airway hyperreactivity in asthma. We
hypothesized that a DC response in the airways would accompany
inflammation and airway hyperreactivity. Therefore, in the present
study, we characterized and quantified the distribution of DC in guinea pig trachea and bronchi and examined the airways for changes in DC
location and number in guinea pigs that were sensitized and challenged
with OVA. Alterations in DC were examined in relation to changes in
other inflammatory cells and to changes in basal pulmonary function and
reactivity to inhaled MCh aerosol in conscious animals.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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OVA sensitization and challenge.
The method for sensitizing
and challenging guinea pigs with OVA in this study was chosen after
systematically comparing and evaluating four different protocols
(Warner et al., 1995). Dunkin Hartley guinea pigs (Harlan;
Indianapolis, IN) (300-350 g) were sensitized with OVA (Sigma Chemical
Co.; St. Louis, MO) on day 0 and day 14 by s.c. injection, in the
nuchal region, of 10 µg OVA and 1 mg Al(OH)3 dispersed in
0.5 ml of 0.9% NaCl. On day 21, animals were challenged with aerosols
of OVA in saline until desired pulmonary obstructive endpoints were
reached (see below). Animals were placed either in a whole-body
plethysmograph or in a two-chamber plethysmograph (see below). Control
animals received saline aerosol on day 21. Another group of animals,
the naive animals, were not exposed to any aerosol.
Unrestrained whole-body plethysmography.
Two-chamber
plethysmography is not suitable for long-term pulmonary function
measurements, because the animal must be restrained, and food and water
are not available. Therefore, to evaluate obstructive responses over 24 hr, bias-flow-ventilated whole-body plethysmography (Chand et
al., 1992, 1993) was utilized on conscious, unrestrained guinea
pigs to examine the time course of pulmonary function changes after
inhalation challenge with inhaled saline or OVA aerosols. Whole-body
plethysmographs (Buxco Electronics Inc., Troy, NY; XA software)
provided flow-derived parameters from which enhanced pause (Penh), an
indirect parameter of pulmonary obstruction, was calculated by the
software according to the relation
![Penh<IT>=</IT>[(Te)<IT>/</IT>(RT)<IT>−1</IT>][(PEF)<IT>/</IT>PIF)]](/content/vol282/issue2/fulltext/995/img001.gif)
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Two-chamber whole-body plethysmography.
In order to examine
further the obstructive responses of conscious animals in response to
inhaled OVA aerosol or to develop dose-response relationships for
obstructive responses to inhaled MCh aerosol, we measured SRaw changes
using a two-chamber whole-body plethysmograph (Thorne and Karol, 1988)
connected to a noninvasive airway mechanics analyzer (Model LS-20;
Buxco). This method is utilized for short time periods with little
stress on the animal. All animals had been acclimated to the apparatus
before the experiment. SRaw data were logged at 6-sec intervals; the
mean of 10 consecutive interval averages was calculated as the
measurement at each time-point. Calculation of SRaw was based on the
previous mathematical description by Pennock et al. (1979).
SRaw provides an indirect index of obstruction in the lower
airways (Finney and Forsberg, 1994).
Procedures and endpoints during OVA aerosol challenge. When animals were placed in the two-chamber plethysmograph for challenge, the OVA aerosol was delivered until a sign of obstruction or altered breathing was observed. The signs occurred at different times in different animals, and they consisted of cough, increases in breathing frequency, increases in SRaw and/or changes in the breath waveform displayed on the computer screen. When one of these events occurred, the aerosol was stopped and the head chamber was flushed with air. Saline-exposed control animals were administered saline aerosol for 3 min.
When the whole-body plethysmography was employed to examine the time course of obstructive responses to OVA, sensitized animals were challenged with nebulized 0.1% OVA solution, generated by a nebulizer (Ultra Neb 99) and driven to the chamber, until the breath waveform was altered on the computer screen or for 4 min, whichever occurred first. The chamber was flushed with air for 1 min. If the animal did not respond to the challenge with 0.1% OVA, the procedure was repeated after 5 min with nebulized 0.5% OVA solution. By this concentration, all animals except one responded. (We observed that precipitous responses and high mortality occurred if exposures were begun with 0.5% or 1% OVA solutions.) After 30 min to 1 hr, food was added to the chamber, having been withheld earlier; water was always available. Data were recorded for 23 to 24 hr. Saline-exposed controls were exposed to nebulized saline aerosol for 3 min, followed by flushing of the chamber with air for 1 min.In vivo reactivity to inhaled MCh.
The effect of
OVA sensitization and challenge on reactivity to MCh was assessed
before the animals were sensitized and again 18 hr after challenge with
OVA. That is, each animal served as its own control for the effect of
OVA or saline treatment; this made possible a within-animal paired
experimental design. (The 18-hr time point was established from the
time courses of changes in Penh after OVA challenge over a 24-hr period
(see fig. 1), which showed that the
animals were experiencing the late-phase response at this time.) Each
guinea pig was placed in a two-chamber plethysmograph and, after a
10-min acclimation period, was exposed to aerosolized saline and,
subsequently, to increasing concentrations of aerosolized MCh (Sigma
Chemical Co., St. Louis, MO; 0.1-80 mg/ml) 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 exposure to saline and the
individual MCh aerosols. MCh aerosols were administered in increasing
concentrations until SRaw was raised to approximately 3 times the
baseline reading, which was taken as that which occurred after exposure
to saline aerosol.
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Lung removal and fixation.
Microscopic examination of the
airways and lungs was done entirely with animals that had been
challenged in the two-chamber plethysmograph. Eighteen hours after
challenge, guinea pigs were anesthetized i.p. with sodium pentobarbital
(The Butler Co., Columbus, OH; 100 mg/kg) and exsanguinated. For
histochemical and immunohistochemical studies, a 3-cm length of trachea
was dissected out. The lungs were inflated with Histocon (Polysciences
Inc., 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 by
liquid nitrogen. Sections of the trachea were prepared by cutting
tangentially through the tracheal wall parallel to the longitudinal
axis; lung lobes were cut such that the largest airways were included
in the sections. Tissue sections 6 µm in thickness (Hacker-Bright
Micro Cryostat 2122, Hacker Instruments Inc., Fairfield, NJ) were cut
at 
20°C, collected on Vectabond-coated slides (Vector Laboratories
Inc., Burlingame, CA) and air-dried.
Detection of dendritic cells, B cells, T cells and macrophages in
histological sections.
Because there are no available antibodies
specific for guinea pig DC, the cells were characterized according to
the following criteria: 1) positive staining with the anti-guinea pig
monoclonal antibody Cl.13.1 (Harlan Bioproducts for Science,
Indianapolis, IN), a marker for MHC Class II; 2) negative staining with
MR-1 (Kraal et al., 1988) (Harlan), a marker for phagocytic
tissue macrophages; 3) dendritic morphology. These criteria were
assessed routinely by preparing consecutive sections of trachea and
lung from the same animal and comparing the localization of Cl.13.1 in
relation to MR-1-positive cells.
; Antoniou et al., 1986).
Monoclonal antibodies were diluted in PBS, pH 7.55. After fixing in
cold acetone for 10 min, consecutive sections of the lung and trachea
were incubated for 30 min with 200 µl of monoclonal antibody
solution. The slides were rinsed in PBS and postfixed in a 1:1
acetone/formalin mixture, pH 6.6, for 1 min. Sections were then placed
in Tris-buffered saline, pH 7.6. The primary antibodies were localized
by utilization of the APAAP staining technique (DAKO Kit K670, Dako
Corp., Carpinteria, CA) (Cordell et al., 1984). Positive
cells were visualized by incubation with Fast Red TR substrate for 20 min, which produced a red reaction product. Sections were
counterstained with Gill's III hematoxylin (Polysciences Inc.) and
mounted with Crystal Mount (Biomeda Corp, Foster City, CA).
Negative controls were processed routinely, and they consisted of
sections that were incubated without primary antibody. Spleen sections
were processed routinely with antibody as positive controls for cell
selectivity because of the natural localization of cells expressing the
cell surface markers of interest within the spleen. It was observed
that T cells were localized adjacent to central arteries in the white
pulp. B cells were also localized in the white pulp, immediately
outside the T lymphocyte-enriched region. Together, the B lymphocytes
and the T lymphocytes formed what is commonly known as the
periarteriolar lymphatic sheath. Macrophages and DC were localized
primarily within the red pulp and in the marginal zone between the red
and white pulp.
Several techniques used in other species to optimize DC detection were
found in preliminary experiments to be ineffective in guinea pig
airways. For example, cold ethanol fixation before freezing, suggested
by Schon-Hegrad et al. (1991) to provide superior antigen
preservation, provided poor structural preservation and thus was not
used. Additionally, the immunoperoxidase technique used by others (Holt
et al., 1988; Maarten et al., 1994) for
visualization of inflammatory cells could not be utilized in the guinea
pig airway tissues because of the presence of large amounts of
endogenous peroxidase activity associated with eosinophils in guinea
pigs.
Detection of eosinophils in histological sections.
Cyanide-resistant eosinophil peroxidase activity was employed to stain
sections for eosinophils (Yam et al., 1971). Frozen sections
were incubated in 0.1 M Tris · HCl buffer, pH 7.2, containing 0.5 mg/ml diaminobenzidine (DAB), 0.015% H2O2
and 0.1 M KCN for 5 min at room temperature and counterstained with
Harris hematoxylin (Polysciences Inc.).
Quantitative (immuno)histochemistry. No epithelial shedding or obvious damage was observed in paraffin sections of lung and trachea from OVA-treated and control animals at 18 hr. Computer-assisted image analysis (Optimas Corp., Edmonds, WA) was employed either to count individual cells (DC, macrophages, T cells and B cells) or to quantify the cellular response by area of staining (eosinophils; see below). After rigorous preliminary experiments indicated that it was justified to do so, several compartments in the trachea and lung were designated as regions of interest for localizing and quantitating inflammatory cells, according to the criteria described earlier. Slides were coded and read in a "blind" fashion. For cell counting, regions of the airway wall were chosen at random in the sections. With the assistance of a cursor, a field of interest was outlined, the cells that yielded a red stain for the cell marker of interest within the field were marked and the cells in a given field were automatically counted via the image analysis program. This yielded in each region of interest the number of cells per square millimeter. At least three replicate measurements were made for each region of interest from each slide.
OVA treatment brought about such a robust influx of eosinophils into the tracheal epithelium that the boundaries of cells could not be discriminated. Being unable to count individual cells, we quantitated the eosinophil response with computer-assisted image analysis by setting color parameters and thresholds for the brown DAB product indicative of eosinophils and calculating the percentage of the area of epithelium that stained positively for DAB. The region of interest bracketed the entire epithelial cell layer from apical to basolateral surfaces. This was an advantageous method for quantifying the eosinophil response, because the prevalence of the cells was thus normalized for the entirety of the section through the epithelium for any section, irrespective of any variation in the angle of cutting to prepare the tangential section. The percentage of the epithelial region of interest, defined with a cursor on the computer screen, that stained positively for DAB is referred to as the "eosinophil influx index." The eosinophil response in bronchial epithelium was likewise robust; in this case, eosinophil influx response was not enumerated either by counting of individual cells or by calculation of DAB-positive area, because there was extensive folding of the bronchial epithelium. For quantification of DC localized in the trachea, positive cells were localized and enumerated in 1) the epithelium, between the lumen and the basement membrane, 2) the lamina propria, between the basement membrane and the smooth muscle, and 3) the submucosa, between the smooth muscle and the cartilage. Positive DC in the bronchi (Bai et al., 1994) were localized and enumerated in 1) the lamina
propria, between the basement membrane and the smooth muscle, and 2)
the adventitia, outside the smooth muscle. Counting of DC in the
bronchial epithelium was prevented by extensive folding of the
epithelial layer, which obscured the individual cells; in addition, the
DC were not stained intensely enough to allow calculation of DC area,
the approach that had been used successfully with eosinophils. Because
the APAAP staining techniques did not lead to clearly defined cell
borders, quantitation of DC cell number was determined by counting the
nuclei that were contained within cells that were APAAP-positive. The
area of the analyzed regions and the number of positive cells in those
areas were determined by computer-assisted image analysis. The results
were expressed as number of positive cells per square millimeter of
region area.
The remaining inflammatory cells were easily visualized with APAAP
staining. They were outlined within random fields of interest in the
airways and counted.
Analysis of results. The results are presented as mean ± S.E.M. Differences in inflammatory cell prevalence among OVA-treated, saline-treated and nontreated animals were determined by the Mann-Whitney ranked-sum test for nonparametric data. Changes in eosinophil prevalence were evaluated for significance by one-way ANOVA with post-hoc comparisons by the Student-Newman-Keuls test. Treatment effects on basal SRaw were analyzed using one-way ANOVA for repeated measures. For in vivo studies on reactivity to MCh, SRaw values for each aerosolized MCh concentration were averaged over 1 min, and the average value was plotted vs. the MCh concentration. The MCh concentration that doubled SRaw (the PC200 concentration) was determined by linear interpolation. Changes in reactivity to MCh were analyzed by a paired t test. n is the number of separate experiments. P < .05 was considered significant.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Time course of pulmonary response to inhaled OVA; whole-body plethysmography. Monitoring Penh over a 23- to 24-hr period in 14 animals revealed two patterns of response to OVA challenge. An initial, rapid increase in Penh lasting approximately 1 to 2 hr occurred in every animal. In 7 of the 14 animals, a late-phase response also was observed, whereas in the remaining animals, the late-phase response did not develop. Figure 1 shows a typical experiment from one of the animals in which both phases appeared. As shown in the figure, the late response typically was less severe, the obstruction ensued at approximately 8 to 12 hr, it was well developed by 18 hr and it lasted until the end of the recording period. No responses were elicited by saline aerosol in nonsensitized animals. On the basis of these results, all subsequent experiments utilized the 18-hr post-exposure period for examination of histological and functional changes.
Effect of OVA challenge on SRaw; double-chamber
plethysmography.
Before challenge with saline, saline-treated
animals had a prechallenge basal SRaw of 1.8 ± 0.2 cm
H2O · s. Before OVA challenge, the SRaw values of
OVA-sensitized animals were not different from these values.
Aerosolized saline had no effect on basal SRaw immediately after or 18 hr after challenge (data not shown; n = 6), this
parameter increased significantly immediately after OVA challenge (fig. 2) and returned to prechallenge levels by
18 hr.
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Effect of OVA challenge on reactivity to inhaled MCh.
Airway
reactivity to MCh was measured in saline-treated and OVA-treated
animals; representative concentration-response curves are shown in
figure 3. Three general observations were
made during the development of these curves. 1) The first effective
dose of a series of MCh concentrations often evoked a very large SRaw response. 2) The curves were very "steep." Administering more than
two or three effective doses led to asphyxia and death, even though the
concentration increment between doses was small and the maximal
response was not achieved. Because EC50 values could not,
therefore, be calculated, linear interpolation was used to calculate
the MCh concentration that produced a 2-fold increase in SRaw above
basal level, which was designated [MCh] PC200. 3) There
was appreciable variability in the control level of reactivity to MCh
between animals, even from a given shipment. However, reactivity in
each animal was observed to be reproducible during the treatment period, so each animal could serve as its own control.
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Dendritic cells.
Cells that stained positively for MHC Class
II antibody, stained negatively for tissue macrophage antibody and had
a dendritiform appearance were scored as DC. In naive, untreated
animals, the cell bodies of these cells were seen primarily within the
lamina propria of trachea and bronchi, as well as in the submucosa of the trachea and in the adventitia of the bronchi, particularly clustering around blood vessels (fig. 5).
Comparatively few DC were seen in the guinea pig epithelium, which in
rats appears to be their primary location (Holt et al.,
1990). In the trachea, the order of abundance normalized to area was
lamina propria = submucosa > epithelium. Endothelial cells
of blood vessels and Type I and Type II alveolar cells expressed weak
MHC Class II positivity.
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Macrophages, T cells, B cells.
Cells staining positively for
the tissue macrophage antibody were localized primarily in the
epithelium near the basement membrane (fig. 5E). Tissue macrophage
density was found to increase in the tracheal epithelium after OVA
treatment. However, saline challenge also increased macrophage cell
number in the tracheal epithelium (fig.
8). T helper cells were found in all
regions of interest in the trachea and bronchus. However, OVA treatment had no effect on T cell density in any region of the trachea or bronchus (fig. 9). No B cells were
detected in tracheal or bronchial sections from naive animals. A few
cells were noted in the saline-treated and OVA-treated animals, but no
differences in density were noted (data not shown).
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Eosinophils.
OVA challenge resulted in a significant increase
in the degree of eosinophilia within the tracheal epithelium (fig.
10;
11B). Eosinophils also were observed in
the adventitia between smooth muscle fascicles (data not shown). In the
bronchial epithelium, eosinophilia was observed after OVA challenge
(fig. 11), but the obvious influx was not amenable to quantitation (see
"Materials and Methods").
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In recent years, the significant role of the DC as an
antigen-presenting cell (Levin et al., 1993) and its
potential role in asthma have become increasingly appreciated.
Therefore, our study utilized quantitative immunohistochemical analysis
of trachea and bronchi to gain insight into the DC response in the
context of other inflammatory cell changes after OVA treatment of
guinea pigs. We examined these changes in animals that had been
determined at 18 hr after OVA challenge to be hyperreactive to inhaled
MCh. At this time, a late-phase obstructive response was also observed in many of the animals. That it did not occur in all of the animals is
similar to a pattern of human response to occupational sensitizers (Perrin et al., 1991).
There have been numerous studies of the localization and function of DC
in the lungs of the rat, but this cell has received little attention in
guinea pigs. We observed many differences between rat and guinea pig
airways with respect to DC. For example, in the rat, pulmonary DC are
localized primarily within the tracheal epithelium at a density of
~600/mm2 (Schon-Hegrad et al., 1991; Holt
et al., 1994). The large number of DC present in the
extensive network in rat airway epithelium is readily visualized in
tangential sections (Holt et al., 1990). In contrast, in the
guinea pig trachea, DC were localized largely in the lamina propria
(203/mm2). In tangential sections of the guinea pig
trachea, very few MHC Class II-positive cells were detected within the
epithelium. These differences are not due to methodological or
technical differences, however, because we have used the same
procedures on tracheas from the Brown Norway rat and confirmed the
localization of numerous DC in the epithelium (unpublished
observations). Therefore, the paucity of DC in guinea pig epithelium
reflects a species difference. Our findings are in agreement with the
previous qualitative study of DC in guinea pigs (Lapa e Silva et
al., 1993). The distribution and density of DC in guinea pigs are
very similar to those obtained from human bronchial biopsy material
(Maarten et al., 1994). In human airways, the density of MHC
Class II-positive cells in the epithelium (245/mm2) was
considerably less than that in rat, and the dense network of DC seen in
the rat (Schon-Hegrad et al., 1991) was not observed in the
human airways. The greatest density of DC in the human biopsy material
(320/mm2) was found in the subepithelial tissue (Maarten
et al., 1994), which is similar to what was observed in the
lamina propria and submucosa of trachea in the present study. Overall,
the density of DC was lower in guinea pig airways than in rat and human
airways. It should be noted that the studies on airways involved human biopsy material from smokers (Maarten et al., 1994); the
effect that smoking could have had on DC density in these patients is not known.
The observation that the cell bodies of pulmonary DC in guinea pigs and humans reside predominantly in the subepithelial regions, whereas dendritic extensions are not observed in the epithelium, is of interest in the context of these cells playing an important role as a first line of defense against incoming antigens. The presence of dendritic extensions throughout epithelium in rats implies that antigen uptake and processing must occur very close to the air interface. Perhaps this different spatial relationship connotes different mechanisms and sites of antigen delivery and processing in human and guinea pig airways, with antigen having to cross the epithelium to be taken up and processed by DC. Alternatively, the dendritic extensions are present in the epithelium but are not visualized by the APAAP procedure in frozen sections. The advent of more suitable antibodies for guinea pig DC may increase resolution of dendritic processes and assist in clarifying these alternatives.
Although OVA treatment results in an increase in DC density, our
experiments do not enable us to distinguish whether the number of DC in
the airways was increased or whether MHC Class II expression on
resident DC had increased. Although these alternatives have never been
directly assessed, studies on airway epithelial DC after irradiation in
rats (Holt et al., 1994) have shown that resident DC have a
turnover rate of approximately 3 days and that there is a renewal from
incoming DC precursors derived from the bone marrow. The migration of
DC into the airways could thus increase their numbers. A study of DC
recruitment after bacterial exposure (McWilliam et al.,
1994) showed that an increase in the number of MHC Class II-positive
cells with rounded morphology was seen 2 hr after exposure. Between 8 and 24 hr after exposure, the morphology of the cells changed to a more
dendritic nature without any further increase in number. This suggests
that immature DC are recruited into the airways and undergo maturation
after reaching the pulmonary tissue. At 18 hr after OVA treatment, we
observed a clustering of DC around pulmonary blood vessels, which could
suggest migration of DC into the airways. It is therefore likely that
the increase in MHC Class II-positive cells in the airways of the
guinea pig reflects, at least in part, homing of DC to the airways.
Interstitial macrophages were present within the epithelium, but they
did not show MHC Class II-positivity. This suggests these cells did not
play a role in OVA presentation to T lymphocytes. Alveolar macrophages,
on the other hand, did react with MHC Class II antibodies in our
experiments. It has been previously reported (Gorenberg and Daniele,
1978; Lipscomb et al., 1981) that guinea pig alveolar
macrophages can stimulate T lymphocyte proliferation in
vitro. This interaction is different from that in rat and mouse (Thepen et al., 1989; Holt et al., 1993), in
which alveolar macrophages are poor antigen-presenting cells and can
down-regulate the accessory cell activity of DC. However, the previous
experiments on guinea pigs (Gorenberg and Daniele, 1978; Lipscomb
et al., 1981) were performed with lymph node T lymphocytes.
In a study (Hill and Burrell, 1979) investigating the stimulation of T
lymphocytes isolated from lung tissue by guinea pig alveolar
macrophages, a suppression of antigen-stimulated proliferation of T
lymphocytes was observed. In addition, the previous guinea pig studies
utilized an isolation technique for alveolar macrophages that involves harvesting cells that are adherent to plastic Petri dishes. However, DC
also are loosely adherent to plastic (Nicod et al., 1987;
Wilkes and Weissler, 1994), and some DC may have been present in
Gorenberg and Daniele's (1978) and Lipscomb et al.'s
(1981) alveolar macrophage preparations.
T helper cells present in the airways were not significantly increased
18 hr after OVA challenge. This is in agreement with the findings of
Lapa e Silva et al. (1993), who noted no change in numbers
of CD4+ T lymphocytes 24 hr after antigen challenge. This
result differs from a significant increase in CD3+ T-cells
17 hr after antigen challenge observed in other studies in the guinea
pig (Frew et al., 1990). It can be hypothesized that the
peak T lymphocyte response, i.e., activation and migration to regional lymph nodes (Holt et al., 1991), may have
already occurred by the 18-hr time-point used in our experiments.
We observed a large influx of eosinophils into the epithelium of the
trachea and bronchi 18 hr after OVA challenge that is characteristic of
the late-phase inflammatory response in guinea pigs (Hutson et
al., 1988; Underwood et al., 1992). Thus the elevations in DC number seen in the present study occur at a time of profound epithelial eosinophilia. The guinea pig has been widely used to investigate allergen-provoked changes in pulmonary function and inflammatory cell influx because of the similarities that exist with
the human asthmatic response, the mediators involved and the reactivity
of the airways to bronchoactive agents. The localization of DC in the
guinea pig airways, which agrees with pulmonary DC distribution in
humans, and the increases in DC density within the airways of
OVA-treated animals suggest that the guinea pig may provide much
insight into the role of DC in the development of inflammation in human
airways. Further quantitative studies on the time courses of DC changes
vis à vis changes in other inflammatory cells will
provide more insight into key sequential intercellular interactions
between DC, T cell and B cells, which will help clarify the
relationship between DC and pulmonary dysfunction.
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Acknowledgments |
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We wish to thank Dr. Daniel Lewis for comments on the manuscript.
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Footnotes |
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Accepted for publication April 17, 1997.
Received for publication December 16, 1996.
1 This paper is partially supported by National Institutes of Health grant 5 T32 GM07039.
Send reprint requests to: Jeffrey S. Fedan, NIOSH, 1095 Willowdale Road, Morgantown, WV 26505.
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Abbreviations |
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DC, dendritic cells; OVA, ovalbumin; PBS, phosphate-buffered saline; APAAP, alkaline phosphatase anti-alkaline phosphatase; MCh, methacholine; SRaw, specific airway resistance.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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) and 18 hr after challenge with
saline or OVA (
). Both curves in each panel were obtained from the
same animal, the first before treatment and the second 18 hr after challenge, i.e., each animal served as its own control.
There was no change in MCh reactivity in the saline-treated animal. However, there was a leftward shift of the MCh dose-response curve in
the OVA-treated animal 18 hr after OVA challenge.
