T cell activation in a murine model of asthma

STEPHEN J. KRINZMAN, GEORGE T. DE SANCTIS,

MANUELA CERNADAS, LESTER KOBZIK, JAMES A. LISTMAN,
DAVID C. CHRISTIANI, DAVID L. PERKINS, AND PATRICIA W. FINN
Department of Medicine, Pulmonary Divisions, Massachusetts General Hospital,
Brigham and Womens Hospital and Beth Israel Hospital; Nephrology Division,
Brigham and Womens Hospital; Pathology Department, Brigham and Womens Hospital,
Harvard University School of Medicine, Boston, Massachusetts 02112

Krinzman, Stephen J., George T. De Sanctis, Manuela Although allergic asthma is clearly the result of an
Cernadas, Lester Kobzik, James A. Listman, David C. immunological process, prior work has shown that
Christiani, David L. Perkins, and Patricia W. Finn. T much of the inflammatory response is localized to the
cell activation in a murine model of asthma. Am. J. Physiol. airways and regional lymph nodes (6). Several investi-
271 (Lung Cell. MoZ. PhysioZ. 15): L476-L483, 1996.-To gators have developed murine models of asthma, which

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determine the mechanisms by which inhaled antigens pro- provide the opportunity to study the inflammatory
duce pulmonary inflammation and bronchial hyperreactivity,
responses in an animal whose immune system has been
we have developed a murine model of asthma. BALB/c mice
well characterized. Inbred strains of mice have been
are sensitized and challenged with ovalbumin (OVA). Com-
pared with mice treated with phosphate-buffered saline (PBS), sensitized and challenged with a variety of antigens,
OVA-treated mice developed increased lung resistance, de- including ovalbumin (OVA; l&25), picryl chloride (ll),
creased dynamic compliance, and greater methacholine reac- and sheep erythrocytes (16).
tivity. Bronchoalveolar lavage fluid revealed significant in- These models have facilitated a more detailed analy-
creases in the proportion of neutrophils and eosinophils. sis of the role of lymphocytes in allergic airway reactiv-
Tissue sections of OVA-treated mice demonstrated goblet cell ity. Kennedy and colleagues (17) demonstrated in-
metaplasia and focal perivascular and peribronchial infil- creased numbers of CD4+ and CD8+ lymphocytes in
trates composed of lymphocytes, neutrophils, and eosino- bronchoalveolar lavage (BAL) fluid after administra-
phils. Analysis of thoracic lymphocytes via flow cytometry tion of OVA aerosols, including an increase in memory
revealed an expansion of both CD4+ and B cell populations, cell phenotypes. Other investigations have provided
with increased expression of interleukin-2 receptor on CD4+ evidence that this recruitment and activation of lympho-
T cells, indicated increased activation. There was also in- cytes are mediated in part by IL-4 (2) and IL-6 (8) but
creased expression of CD44 on CD4+ and CD8+ lymphocytes, are inhibited by IL-10 (29). Depletion of CD4+ lympho-
suggesting an expansion of the local memory cell population.
cytes has been shown to prevent antigen-induced air-
These findings support the hypothesis that activation of T
lymphocytes mediates allergic pulmonary inflammation and way reactivity and recruitment of lymphocytes and
bronchial reactivity in asthma. eosinophils (12). These models are limited, however, by
their inability to correlate immunological findings with
airway reactivity; asthma; inbred mice; lymphocytes; metha- airway hyperreactivity (2,17,29), pulmonary inflamma-
choline tion (24, 25) allergic immune responses (8), or detailed
analyses of lymphocyte phenotypes (12).
We have developed a detailed and comprehensive
murine model of allergic asthma, including in vivo
ONE OF THE MOST IMPORTANT advances in the study of
airways physiology, analysis of BAL fluid, characteris-
asthma has been the growing recognition that the
tic pulmonary inflammation, and a detailed immunologi-
disease is characterized by both acute and chronic
cal analysis of pulmonary and thoracic lymphocytes.
pulmonary inflammation. The role of mast cells and
Using this model, we have determined the pattern of
eosinophils in the acute or early phase of an asthma lymphocyte activation that follows aerosolized antigen
exacerbation is well established (27). More recently, challenge.
there has been a growing appreciation of the central
role of T lymphocytes in orchestrating the chronic MATERIALS AND METHODS
inflammatory phase of asthma. Increased numbers of AnimaZs. Male BALB/c mice were obtained from Jackson
activated T cells are found in the bronchial mucosa of Laboratories (Bar Harbor, ME) and were 4-5 wk old at entry
asthmatic subjects (1), and the degree of activation of T into the protocol. Mice were allowed free access to water and
cells in peripheral blood correlates with the severity of commercial pelleted mouse feed.
asthmatic symptoms (6). In patients with allergic Experimental design. Four groups were studied (Fig. 1).
asthma, antigen challenge produces a selective recruit- The OVA group was sensitized and challenged with OVA,
ment of T helper (CD4+, Th) cells into the airways (13). whereas the phosphate-buffered saline (PBS) group was
sensitized and aerosol challenged with PBS. A third group
The majority of T helper lymphocytes from these pa-
(unsensitized/OVA) received OVA aerosols without prior sen-
tients secrete interleukin-4 (IL-4) and interleukin-5 sitization. Finally, a fourth group (untreated) was neither
(IL-5), a characteristic pattern of cytokine production sensitized nor challenged with either reagent.
known as the Th2 phenotype. IL-4 stimulates immuno- Sensitization. On day 0, each mouse in the OVA treatment
globulin E production by B cells (5), and IL-5 recruits group was immunized via intraperitoneal injection with 10 ug
and activates eosinophils (23). chicken OVA(Grade III, Sigma Chemical, St. Louis, MO), and

L476 1040-0605/96 $5.00 Copyright o 1996 the American Physiological Society

 T CELL ACTIVATION IN A MURINE MODEL OF ASTHMA L477

Day 0 7 14 15 16 17 18 19 20 21 centrifuged at 2,000 rpm for 5 min, the supernatant was

discarded, and cell pellets were resuspended in Hanks bal-
t, t t/ anced salt solution (JRH Biosciences, Lenexa, KS). Slides
Inject IP Aerosols 18 min per day & were prepared by spinning samples at 800 rpm for 10 min
[Sensitization] [Challenge] (Cytospin 2, Shandon, Pittsburgh, PA). BAL specimens were
Fig. 1. Experimental design. For sensitization, each injection con- fixed (Leukostat Fixative Solution, Fisher Diagnostics) and
tains 10 ug ovalbumin (OVA) and 1 mg Al(OH)s in 0.2 ml phosphate- stained with methylene blue and eosin-Y (leukostat solutions
buffered saline (PBS) (OVA group), or Al(OH)s in PBS alone (PBS I and II, Fisher Diagnostics). BAL differentials were deter-
group). For challenge, OVA group received 6% OVA in 0.5 X PBS, and mined by counting cells with a hemacytometer, based on two
PBS group received 0.5 X PBS. IP, intraperitoneal; M, measurements counts of 100 cells each for each sample. The investigator
performed. counting the cells was blinded to the treatment groups.
Lymphocyte isoZation and quantification. Lymph nodes
1 mg Al(OH)s (alum; J. T. Baker Chemical, Phillipsburg, NJ) were obtained by dissection from the paratracheal, perihilar,
in 0.2 ml of PBS (Sigma), followed by a boosting injection on and peribronchial regions and placed in chilled RPM1 1640
day 7 with the identical reagents. PBS control mice were medium (Bio-Whittaker, Walkersville, MD). Single-cell sus-
immunized and boosted with 1 mg alum in 0.2 ml of PBS. pensions were created by gently pressing the lymph nodes
through a steel mesh (stainless steel screen 60 mesh) with a

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Antigen challenge. All nebulizer solutions were dissolved in
sterile, pH balanced (7.35-7.45) solutions consisting of PBS rubber plunger. Cell suspensions were centrifuged at 1,800
diluted 1:l with distilled water (0.5X PBS). Mice underwent rpm for 5 min at 4C the supernatant was discarded, and the
aerosolized antigen challenge with 6% OVA, dissolved in PBS cells were resuspended in PBS with 3% bovine calf serum
(pH 7.35-7.45), for 18 min per day for 7 consecutive days. (BCS; Irvine Scientific, Santa Ana, CA). Lymphocytes were
This series of aerosols began 14-17 days after the initial counted with a hemacytometer.
immunization. Mice were pla&d in a plastic chamber (23 X MonocZonaZ antibodies. Fluorescein isothiocyanate (FITC)-
23 X 11 cm), and the OVA solution was aerosolized via an conjugated anti-CD4 (L3T4), phycoerethrin (PE)-conjugated
ultrasonic nebulizer (model 5000, DeVilbiss, Somerset, PA) anti-B220 (CD45R), and biotinylated anti-IL-2 receptor (anti-
attached directly to the chamber. Continuous air flow (< 1.0 CD25; o-chain) monoclonal antibodies (MAb) were obtained
l/min) was supplied to the nebulizer to drive the aerosol into commercially (Pharmingen, San Diego, CA). Biotinylated
the chamber, and small ventilation holes were created in the antibodies were incubated in a second step (see Flow cytome-
opposite side of the chamber. Control mice (PBS) were try) with Streptavidin-Red 613 (R613; GIBCO, Grand Island,
challenged with 0.5~ PBS alone. In separate experiments, NY). The MAb specific for CD8 was purified from 53.6
one group of mice (unsensitized, OVA challenged) were chal- hybridoma supernatant and conjugated to Cy5 (Biological
lenged with OVA aerosols for 7 days without prior OVA Detection Systems).
sensitization. FZow cytometry. Four-color flow cytometry was performed
Determination of lung resistance and airway reactivity. on thoracic lymphocytes after isolation (see Eksue collection).
Mice were studied 1 day after the final aerosol. Each mouse Cells were washed twice in PBS with 3% BCS (wash buffer).
was anesthetized with an injection of pentobarbital sodium After counting, samples were pooled as needed, to obtain 5 X
(70-80 mg/kg ip; Anthony Products, Arcadia, CA). After lo5 cells per well times the number of wells in each experi-
acceptable anesthesia was achieved, the metal portion of a ment. Each cell sample was suspended in 50-ul aliquots of
19-gauge tubing adapter was inserted into the trachea and wash buffer and incubated for 30 min at 4C with saturating
secured in place with sutures. An internal jugular vein was concentrations of fluorochrome-labeled MAb. Cell samples
cannulated with a saline-filled Silastic catheter (0.06 cm OD, were washed twice with 200 ul wash buffer and resuspended
6-8 cm in length, co.005 ml vol) attached to a O.l-ml in 50 ul wash buffer. Saturating concentrations of streptava-
Hamilton microsyringe (Hamilton, Reno, NV) and used to din R613 were added to those suspensions with biotinylated
administer acetyl-P-methylcholine chloride [methacholine antibodies, and samples were incubated for an additional 30
(MCh), Sigma]. Pulmonary resistance (RL) and dynamic min. Samples were then washed twice more with wash buffer,
compliance were determined as previously described (7). fixed by resuspension in 0.5% paraformaldehyde, and stored
Briefly, dose-response curves to MCh were obtained by admin- in the dark at 4C. Samples were analyzed on a Coulter Epics
istering sequentially increasing doses of MCh (33-3,300 Elite fluorescence-activated cell sorter (FACS), using 488
ug/kg) in a 20- to 35-ul volume. The peak response to each (FITC, PE, and R613) and 633 (Cy5) excitation wavelengths.
dose was obtained within the first minute after MCh injec- Lymphocytes were gated according to size in a forward and
tion. Enough time was allowed to elapse between MCh doses side scatter plot. Fluorescence was detected at 525 (FITC),
so that predose measurements of RL returned to within 10% 590 (PE), 613 (R613), and 670 (Cy5) nm. Listmode data were
of the value obtained before the preceding dose of MCh. Each analyzed with Coulter Elite Software. When possible, popula-
animals dose-response curve was subjected to regression tions were counted based on discrete histogram populations.
analysis and then log-transformed to calculate the dose In cases where discrete populations were not evident [interleu-
required for a twofold increase in RL (log ECzoO RL). kin-2 receptor (IL-2-R) and CD44], high and low popula-
Tissue collection. Mice were removed from the plethysmo- tions were identified based on both negative controls and
graphic chamber, a cervical dislocation was performed, and single positive controls for the other fluorochromes in the
blood was obtained via cardiac puncture. Lungs were re- sample, such that not >2% of either fell into the bright
moved from the thoracic cavity and immersed in K2 fixative region of the antibody/fluorochrome in question. Positive
for 24 h, transferred into sodium cacodylate (pH 7.3), stained controls for each fluorochrome were also used to correct for
with hematoxylin and eosin, and examined by light micros- differences in fluorochrome brightness between analysis days.
copy. The remaining mice in each group underwent BAL and StatisticaL anaZyses. All data are reported as means t SE.
lymph node dissection immediately after completion of air- For measurements of pulmonary physiology, data were ana-
way measurements. BAL fluid was obtained by instilling and lyzed using the JMP 3.0 statistical package (SAS Institute,
withdrawing lavage solution (1.0 ml PBS with 0.6 M EDTA) Cary, NC). Parametric data was analyzed by the Tukey-
three times via the tracheal cannula. BAL samples were Kramer test, and nonparametric data was analyzed by the
L478 T CELL ACTIVATION IN A MURINE MODEL OF ASTHMA

WilcoxonKruskal-Wallace rank-sum test. All other measure-

ments were analyzed, using the Sigma Stat statistical pack-
age (Jandel Scientific, Corte Madera, CA), with paired t-tests
used for parametric data and the Mann-Whitney rank-sum
test used for nonparametric data. A value of P < 0.05 was
taken as the threshold for significance.

RESULTS
Airway physiology and bronchial reactivity. To deter-
mine the effects of OVA sensitization and aerosol
challenge, in vivo measurements of pulmonary physiol-
ogy were performed on living, anesthetized mice. Air-
Methacholine Dose @g/kg)
way resistance was measured via plethysmography
(Fig. 2). OVA-sensitized and challenged mice had a Fig. 3. Reactivity to intravenous methacholine. Peak resistances
significantly higher baseline airway resistance than produced by administering increasing intravenous methacholine
doses to mice sensitized and challenged with OVA (n = 9), PBS (n =
PBS controls (1.78 t 0.18~s. 1.58 ? 0.13 cn~H~O~rnl-~s-~,

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lo), untreated mice (n = 5), and OVA aerosol-challenged mice that
respectively. P < 0.05). had not previously been OVA sensitized [unsensitized OVA (U-OVA),
After measurement of baseline airway resistance, n = 61 are expressed as %each animals baseline resistance before
mice were challenged with increasing doses of intrave- each injection. NSal, normal saline. *P < 0.05 for OVA vs. all other
groups; TP < 0.05 for OVAvs. U-OVA.
nous MCh (Fig. 3). Before each injection, baseline
resistance was measured, and the subsequent response
to MCh was measured and expressed as a percent of the
prior baseline. The MCh responses of the two groups treated mice had significantly lower threshold doses
above were measured, as well as those of mice chal- than controls (1.92 t 0.09 vs. 2.63 t 0.12, OVAvs. PBS;
lenged with OVA without prior sensitization, and age- P < 0.001).
matched BALB/c mice that were not sensitized or PathoZogy. To correlate in vivo pulmonary physiology
challenged. At 100, 330, and 1,000 ug/kg, OVA-treated with pathology, lung tissue specimens were obtained
mice had significantly greater responses than PBS- immediately after measurement of airway resistance
treated mice. There were no significant differences on mice from the PBS, OVA, and unsensitized OVA-
between untreated mice, PBS-treated mice, and unsen- challenged groups (Fig. 4). The lungs from the PBS
sitized OVA-challenged mice at any MCh dose. group and the unsensitized OVA-challenged group had
OVA-treated mice had a parallel fall in dynamic no abnormal features, except for mild leukosequestra-
compliance, with significant decreases compared with tion in one PBS specimen. Lungs from the OVA-treated
saline controls at MCh doses of 330 pg/kg (12 5 4 vs. mice demonstrated moderate focal perivascular and
41 t 4%, OVAvs. PBS; P < O.Ol), 1,000 ug/kg (11 t 3 vs. peribronchiolar infiltrates composed of lymphocytes,
37 2 3%, OVAvs. PBS; P < 0.05). Expressed as the log neutrophils, and a lesser number of eosinophils, as well
of the MCh dose required to produce a twofold increase as bronchiolar goblet cell metaplasia.
in resistance (EC&, OV&treated mice had signifi- BAL. BAL fluid was examined to provide a quantita-
cantly lower threshold doses (ECzoo = 1.75 t 0.06 vs. tive assessment of intrapulmonary leukocytes (Fig. 5).
2.18 2 0.19, OVA vs. PBS, respectively; P < 0.001). OVA-sensitized and -challenged mice had a marked
Similarly, when expressed as the log of the dose re- increase in the proportion of inflammatory cells in BAL,
quired to cause a 35% fall in compliance (EC&, OVA- with significant increases in granulocytes (0.4 t 0.4 vs.
29.2 t 10.4%, PBS vs. OVA; P < 0.05) and eosinophils
(0 k 0 vs. 2.33 -+ 0 .75% PBS vs. OVA; P < O.OS>,and a
2.2,
trend toward an increase in the proportion of lympho-
v cytes (6 6 + 2 1 vs. 11.3 t 2.2%, PBS vs. OVA; P = 0.16).
OVA mice-also had a corresponding decrease in the
rn proportion of alveolar macrophages (92.8 t 2.1 vs.
b 2.0-
rI 58.4 t 12.0%, PBS vs. OVA; P < 0.05).
-
E A Lymphocyte subsets and activation. To directly assess
vv
6 thoracic lymphocytes, mice from the OVA-treated and
I 1.8- v the PBS-treated groups were dissected, and lymph
E nodes were harvested from the paratracheal, hilar,
0 .:
t v peribronchial, and mediastinal regions. OVA-treated
d 1.6- mice had a significantly higher number of thoracic
:A* lymphocytes obtainable by dissection than did PBS
v
: controls (3.09 + - 0 .75 vs 1.46 t 0.22 x 106, OVA vs.
1A 1 I I
PBS). Analysis via flow cytometry revealed that this
PBS OVA increase was due to expansions of both B cell and CD4+
populations; however, due to variance in cell numbers
Fig. 2. Pulmonary resistance (RL) after sensitization and challenge
with OVA (n = 9) or PBS (n = 10). RL was measured via plethysmog- between animals, these differences were not statisti-
raphy on living, anesthetized, mechanically ventilated mice, before cally significant (in B cells, 304 t 69 vs. 829 t 343, PBS
methacholine challenges. P < 0.05 for comparison of groups. vs. OVA, P = 0.07; in CD4 cells, 777 t 139 vs. 1,042 t
 Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on November 15, 2017
Fig. 4. Lung histology. Lungs were inflated
and fixed with K2 fixative overnight. Sec-
tions were embedded in paraffin, cut onto
slides, stained with hematoxylin and eosin,
and examined by light microscopy (x200). r4:
representative lung biopsy from PBS-sensi-
tized and challenged mouse. B: section from
an OVA mouse, showing peribronchial infil-
trate with neutrophils, lymphocytes, and
eosinophils. C: section from an OVA-sensi-
tized and -challenged mouse, showing goblet
cell metaplasia.
L480 T CELL
80
AM Poly Lymph
Fig. 5. Proportion of leukocytes in bronchoalveolar
fluid of OVA-treated (n = 6) and PBS-treated
alveolar macrophages; Poly, polymorphonuclear
lymphocytes; Eos, eosinophils. '%P < 0.05.
252, PBS vs. OVA, P = 0.33). Examination of the
relative proportion of each lymphocyte subset (Fig. 6)
revealed a significant increase in the proportion of B
cells and a significant decrease in the proportion of
CD4+ cells. There was no significant difference in the
proportion of CD8+ cells, or in the CD4KD8 ratio (2.04 t
0.14 vs. 1.80 t 0.12, PBS vs. OVA).
To further characterize the functional phenotype of
these cells, activation of lymphocyte subsets was exam-
ined using MAb for IL-2-R, an acute activation marker,
and CD44, a chronic activation marker (Fig. 7). OVA-
treated mice had significant increases in expression of
IL-2-R on CD4+ T cells (17 t 2.3 vs. 11 t 0.6%, P <
0.01) and significantly increased expression of CD44 on
CD4+ and CD8+ T cells (CD4+, 13 t 1.1 vs. 9 ? 0.8%;
CD8+, 11 t 1.3 vs. 7 ? 0.5%; P < 0.05 for both
comparisons).
DISCUSSION
The role of lymphocytes in allergic asthma remains
controversial. We believe that murine models can pro-
vide important insights into the pathogenesis of aller-
gic asthma, because the disease is mediated by immuno-
logical phenomena, and mice provide an immune system
that has been very well studied and correlated with
human findings. Our model has the additional advan-
tage of analyzing the immune response to aerosolized
antigen challenge in the context of characteristic in
vivo physiological and pathological changes. All murine
models of asthma differ from the human disease in that
allergic airway inflammation and hyperreactivity only
occur after experimental sensitization to antigen and
%CD4 %CD8
Fig. 6. Percent of thoracic lymphocyte subsets after sensitization
challenge with OVA (n = 6) or PBS (n = 11). Samples
lymphocytes were incubated with monoclonal antibody/fluorochrome
conjugates for CD4, CD8, and B220 (a B cell marker)
flow cytometry. +'P< 0.05.
ACTIVATION IN A MURINE MODEL OF ASTHMA
q PBS not because of normal environmental exposure or inher-
n OVA ent immunological dysregulation.
OVA-treated mice demonstrated a mean 13% in-
crease in the in vivo airway resistance (Fig. 2). In-
creased airway resistance is one of the hallmarks of
acute asthma and to our knowledge has only been
demonstrated in one prior murine model (ll), which
used nasal challenge with picryl chloride to stimulate a
Eos delayed-type hypersensitivity reaction.
lavage (BAL) Increased responsiveness to intravenous MCh is
(n = 5) mice. AM, considered a defining feature of most forms of asthma
lymphocytes; Lymph,
(14) and was clearly demonstrated in OVA-treated mice
(Fig. 3). BALBI c mice that are not antigen challenged
do not demonstrate unusual airway reactivity com-
pared with other strains of mice (19). In OVA-treated
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mice, examination of the dose-response curve to intrave-
nous MCh reveals a very high peak response after 330
ug/kg, followed by a decrease in responsiveness to
higher doses. This corresponds to the observation that
individual mice that achieve X2- to X-fold increase in
resistance usually become less reactive to all subse-
quent MCh doses, regardless of which dose causes this
high response. PBS-treated mice do not demonstrate
this phenomenon, because they did not achieve these
high levels of airway resistance. We confirm that all
mice are alive throughout the airway measurements, so
the mechanism of this eventual decreasedreactivity is not
due to decreased viability One potential explanation why
this pattern of decreased reactivity has not been observed
in prior animal and human studies of airways reactivity is
that the prior studies have not consistently achieved
reponses as high as we report in this study.
The determinants of airway resistance and dynamic
compliance are complex and multifactorial (9, 28). A
greater increase in resistance after a given dose of MCh
may indeed reflect more intense contraction of airway
smooth muscle, due to mediators such as histamine and
leukotrienes or due to damage to the bronchial epithe-
lium, removing a barrier between the smooth muscle
and the bronchoconstrictor. Airways responsiveness
may also be due to mechanical and geometric changes
in the lung. Because resistance in an airway is inversely
proportionate to the fourth power of the radius of the
airway, edema or inflammation that decreasethe radius of
the lumen can cause a similar degree of smooth muscle
contraction to cause a markedly greater increase in resis-
tance (15). Asthmatics may have compromised airway
lumens due to acute factors, including peribronchial
q IPBS inflammation and endobronchial mucus, and chronic
n OVA factors such as smooth muscle hyperplasia (15).
In our model, any of these factors may have contrib-
uted to the development of airway hyperresponsiveness
in OVA-treated mice, but pulmonary edema from the
fluid content of the aerosol or endobronchial accumula-
tion of the protein could cause similar results. To
determine the specific contributions of antigen-induced
% B220
inflammation, fluid volume and protein content, three
and additional experimental groups were studied (Fig. 3).
of thoracic
One group was not treated with either OVA or PBS, and
and analyzed by a second group (PBS) received sham immunization and
nebulization with PBS to control for fluid effects. A
 T CELL ACTIVATION IN A MURINE MODEL OF ASTHMA L481

PBS OVA

Fig. 7. A: representative flow cytometry histograms

e w demonstrating
receptor (IL-2-R)
upregulated expression
on CD4+ cells in OVA-treated
of interleukin-2
mice.
CD4+

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Only cells that fall within the viable region on forward
and side scatter analysis, and that also express CD4, are
included in these histograms. Ordinate, fluorescence of
Cy5conjugated anti-CD4; abscissa, fluorescence of red
20- * q PBS 613-conjugated IL-2-R. Cells that fall within the box in
n OVA
each histogram
ers. B: expression
are positive
of activation
for both cell surface mark-
and memory cell surface
16.- markers on thoracic CD4+, CD8+, and B (B220+) lympho-
* cytes after sensitization and challenge with OVA (n = 6)
or PBS (n = 11). *P < 0.05.
*
12.- -5-

8--

4--

0, t
CD4+ CD4+ CD8+ B220+
I I I
IL2-R+ CD44+

third group received the OVA aerosols without prior response during the aerosol challenge or to nonspecific
immunization or boosting, to control for nonimmunologi- effects of the fluid or protein load.
cal effects of the protein, including mechanical effects. These patterns of parenchymal inflammation were
There were no significant differences between these accurately reflected in the BAL fluid, where there were
three control groups, indicating the MCh hyperrespon- increased proportions of neutrophils, eosinophils, and a
siveness in our model was not due to nonspecific fluid or trend toward increased lymphocytes. This increase in
protein effects. OVA-challenged mice that had not been inflammatory cells has also been observed in other
previously immunized did not develop increased respon- murine models (2, 17, 29) and in human asthmatics
siveness to MCh, providing strong evidence that air- after aerosolized antigen challenge (21). Data from
ways reactivity in our model is due to a secondary both human asthmatics and animal models reveal that
immunological response to antigen. the cellular response to antigen challenge is dependent
The pattern of lung pathology demonstrated in our in large part on the interval after exposure to the
model reflects many of the features of human asthma. antigen. In both human asthmatics (20-22) and mu-
Goblet cell metaplasia is a prominent feature of asthma rine models (291, several investigators have found that
as well as other chronic inflammatory diseases of the neutrophils are markedly increased after a single anti-
lung (10). Lung biopsies and autopsy studies of asthmat- gen challenge and often outnumber lymphocytes and
ics frequently find dense peribronchial infiltration with eosinophils after 24-48 h, whereas lymphocytes and
lymphocytes, macrophages, eosinophils, and in some eosinophils dominate after 96 h. Our model utilizes
cases neutrophils (10). The benign appearance of the BAL 24 h after the final antigen challenge and repro-
lungs of unsensitized OVA-challenged mice further duces the neutrophilic predominance seen in many
reinforces the conclusion that both airway reactivity human asthmatics at this time point.
and pulmonary inflammation are due to a secondary To determine the effects of inhaled antigen on local
immunological response to OVA and not to a primary pulmonary lymphocyte activation and proliferation,
L482 T CELL ACTIVATION IN A MURINE MODEL OF ASTHMA

thoracic lymphocytes were obtained by dissection. There 5. Chretien, I., J. Pene, F. Briere, R. D. W. Malefijt, F. Rousset,
and J. V. DeVries. Regulation of human IgE synthesis. I.
was a significant increase in the total number of
Human IgE synthesis in vitro is determined by the reciprocal
lymphocytes in OVA-treated mice, and analysis by flow antagonistic effects of interleukin 4 and interferon. Eur. J.
cytometry revealed that this regional lymphocyte prolif- Immunol. 20: 243-251,199O.
eration was due to increases in both the number of B 6. Corrigan, C. J., and A. B. Kay. CD4 T lymphocyte activation in
cells and CD4+ T cells (Fig. 6). Through analysis of cell acute severe asthma.Am. Rev. Respir. Dis 141: 970-977,1992.
surface markers, we further characterized the pattern 7. De Sanctis, G. T., M. Merchant, D. R. Beier, R. D. Dredge,
of lymphocyte activation (Fig. 7). Expression of IL-Z-R J. K. Grobholz, T. R. Martin, E. S. Lander, and J. M. Drazen.
Quantitative locus analysis of airway hyperresponsiveness in A/J
was increased on CD4 lymphocytes, indicating in-
and C57BL/6J mice. Nature Genet. 11: 150-154,1995.
creased activation. In human asthmatics, IL-Z-R expres- 8. DiCosmo, B. F., G. P. Geba, D. Picarelli, J. A. Elias, J. A.
sion on CD4+ lymphocytes is elevated in both periph- Rankin, B. R. Stripp, J. A. Whitsett, and R. A. Flavell.
eral blood (6) and BAL fluid (26), and it has been shown Airway epithelial cell expression of interleukin-6 in transgenic
to correlate with several indexes of disease severity (6). mice. J. Clin. Invest. 94: 2028-2035, 1994.
There was also increased expression of CD44 on both 9. Drazen, J. M. Physiological basis and interpretation of indices
of pulmonary mechanics. Environ. HeaZth. Perspect. 56: 3-9,
CD4+ and CD8+ cells. Although the function of CD44 on
1984.

Downloaded from http://ajplung.physiology.org/ by 10.220.33.3 on November 15, 2017

T cells has not been well defined, there is evidence to 10. Dunnill, M. S. The pathology of asthma with special reference to
suggest that it mediates lymphocyte migration into an changes in the bronchial mucosa. J. CZin. Pathol. 13: 27-33,
area of inflammation (4) and may serve as a marker for 1960.
memory cell populations (3). A recent model of OVA 11. Garssen, J., F. P. Nijkamp, H. H. Van Der Vliet, and H. V.
challenged mice noted increased expression of CD44 on Loveren. T-cell-mediated induction of airway hyperreactivity in
mice.Am. Rev. Respir. Dis. 144: 931-938,1991.
CD4+ T cells in BAL fluid (17). These findings suggest
12. Gavett, S. H., X. Chen, F. Finkelman, and M. Wills-Karp.
that the recruitment of T lymphocytes into the lung Depletion of murine CD4 T lymphocytes prevents antigen-
that was observed after antigen challenge may be induced airway hyperreactivity and pulmonary eosinophilia.
mediated by augmented expression of CD44. Am. J. Respir. CeZZ MOL. BioZ. 10: 587-593, 1994.
The data presented confirm that aerosolized antigen 13. Gerblich, A. A., H. Salik, and M. R. Schuyler. Dynamic T-cell
challenge and the resulting airways hyperreactivity changes in peripheral blood and bronchoalveolar lavage. Am.
Rev. Respir. Dis. 143: 533-537,199l.
are associated with expansion of the intrathoracic 14. Hargreave, F. E., G. Ryan, N. C. Thomson, P. M. OByrne, K.
lymphocyte population and activation of B cells as well Latimer, E. F. Juniper, and J. Dolovich. Bronchial responsive-
as CD4+ and CD8+ T cells. Furthermore, the increased ness of histamine or methacholine in asthma: measurement and
baseline lung resistance is consistent with baseline clinical significance. J. AlZergy Clin. Immunol. 68: 347-355,
airway narrowing, indicating a degree of obstruction 1981.
even in the absence of MCh challenge. These character- 15. James, A. L., P. D. Pare, and J. C. Hogg. The mechanics of
airway narrowing in asthma. Am. Rev. Respir. Dis. 139: 242-246,
istics indicate parallels between human asthma and 1989.
our murine model which may reflect the contributions 16. Kaltreider, H. B., J. L. Curtis, and S. M. Arraj. The mecha-
of both T and B lymphocytes to the pathogenesis of this nism of appearance of specific antibody-forming cells in lungs of
disorder. inbred mice after immunization with sheep erythrocytes intratra-
cheally.Am. Rev. Respir. Dis. 135: 877-892, 1978.
We thank Drs. Maria Lara-Marquez, Tom Martin, and Jeffrey 17. Kennedy, J. D., C. A. Hatfield, S. F. Fidler, G. E. Winter-
Drazen for helpful discussions. rowd, J. V. Haas, J. E. Chin, and I. M. Richards. Phenotypic
This work was supported in part by grants from National Insti- characterization of T lymphocytes emigrating into lung tissue
tutes of Health Grants HL-36110 (to G. T. De Sanctis), ES-ROl- and the airway lumen after antigen inhalation in sensitized
06568 and ES-00002 (to P. W. Finn), and AI-31527 (to D. L. Perkins). mice.Am. J. Respir. CeZZ. MOL. BioZ. 12: 613-623, 1995.
G. T. De Sanctis is the recipient of the Medical Research Council of 18. Kung, T. T., D. Stelts, J.A. Zurcher, H. Jones, S. P. Umland,
Canada Award. W. Kreuter, R. W. Egan, and R. W. Chapman. Mast cells
Address for reprint requests: P. Finn, Respiratory Div., Brigham & modulate allergic pulmonary eosinophilia in mice. Am. J. Respir.
Womens Hospital, 75 Francis St., Boston, MA 02115. CeZZ. MOL. BioZ. 12: 404-409, 1995.
Received 1 September 1995; accepted in final form 27 March 1996. 19. Levitt, R. C., and W. Mitzner. Autosomal recessive inheritance
of airway hyperreactivity to 5-hydroxytryptamine. J. AppZ.
Physiol. 67: 1125-1132, 1989.
REFERENCES
20. Liu, M. C., W. C. Hubard, D. Proud, B.A. Stealey, S. J. Galli,
Azzawi, M., B. Bradley, P. K. Jeffery, A. J. Frew, B. Assoufi, A. Kagey-Sobotka, E. R. Bleeker, and L. M. Lichtenstein.
J. V. Collins, S. Durham, and A. B. Kay. Identification of Immediate and late inflammatory response to ragweed antigen
activated T lymphocytes and eosinophils in bronchial biopsies in challenge of the peripheral airways in allergic asthmatics. Am.
stable atopic asthma. Am. Rev. Respir. Dis. 142: 1407-1413, Rev. Respir. Dis. 144: 51-58,199l.
1990. 21. Metzger, W. J., H. B. Richerson, K. Worden, M. Monick, and
Brusselle, G. G., J. C. Kips, J. H. Tavernier, J. G. Van Der G. W. Hunninghake. Bronchoalveolar lavage of allergic asth-
Heyden, C. A. Cuvelier, R. A. Pauwels, and H. Bluethmann.
matic patients following allergen bronchoprovocation. Chest 89:
Attenuation of allergic airway inflammation in IL-4 deficient
477-483,1986.
mice. CZin. Exp. Allergy 24: 73-80, 1994.
22. Metzger, W. J., D. Zavala, H. B. Richerson, P. Moseley, P.
Butterfield, K., C. G. Fathman, and R. C. Budd. A subset of
memory CD4+ helper T lymphocytes identified by expression of Iwamota, M. Monick, K. Sjoerdsma, and G. W. Hunning-
pgp-1. J. Exp. Med. 169: 1461-1466, 1989. hake. Local allergen challenge and bronchoalveolar lavage of
Camp, R. L.,A. Scheynius, C. Johanssin, and E. Pure. CD44 allergic asthmatic lungs. Am. Rev. Respir. Dis. 135: 433-440,
is necessary for optimal contact allergic responses but is not 1987.
required for normal leukocyte extravasation. J. Exp. Med. 178: 23. Nakajima, H., I. Iwamoto, S. Tomoe, R. Matsumura, H.
497-507,1993. Tomioka, K. Takatsu, and S. Yoshida. CD4+ T lymphocytes
 T CELL ACTIVATION IN A MURINE MODEL OF ASTHMA L483

and interleukin-5 mediate antigen-induced eosinophil infiltra- peripheral blood and bronchoalveolar lavage. Am. Rev. Respir.
tion into the mouse trachea. Am. Rev. Respir. Dis. 146: 374-377, Dis. 146: 109-l&1992.
1992. 27. Wardlaw, A. J., S. Dunnette, G. J. Gleich, J. V. Collins, and
24. Renz, H., H. R. Smith, J. E. Henson, B. S. Ray, C. G. Irvin, A. B. Kay. Eosinophils and mast cells in bronchoalveolar lavage
and E. W. Gelfand. Aerosolized antigen exposure without in mild asthma: relationship to bronchial hyperreactivity. Am.
adjuvant causes increased IgE production and increased airway Rev.Respir. Dis. 137:62-69,1989.
responsiveness in the mouse. J. Allergy. CZin. Imunol. 89: 28. Wiggs, B. R., C. Bosken, P. D. Pare, A. James, and J. C.
1127-1138,1992.
Hogg. A model of airway narrowing in asthma and chronic
25. Saloga, J., H. Renz, G. L. Larsen, and E. W. Gelfand.
obstructive pulmonary disease. Am. Rev. Respir. Dis. 145: 1251-
Increased airways responsiveness in mice depends on local
challenge with antigen. Am. J. Respir. Crit. Care. Med. 149: 1258,1992.
65-70,1994. 29. Zauny-Amorim, C., S. Haile, D. Leduc, C. Dumarey, M.
26. Walker, C., E. Bode, L. Boer, T. T. Hansel, K. Blaser, and Huerre, B. B. Vargaftig, and M. Pretolani. Interleukin-10
J. C. Virchow. Allergic and non-allergic asthmatics have dis- inhibits antigen-induced cellular recruitment into the airways of
tinct patterns of T-cell activation and cytokine production in sensitized mice. J. Clin. Invest. 95: 2644-2651, 1995.

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