Antigen-Induced Airway Hyperresponsiveness, Pulmonary Eosinophilia,
and Chemokine Expression in B Cell–Deficient Mice
James A. MacLean, Alain Sauty, Andrew D. Luster, Jeffrey M. Drazen, and George T. De Sanctis
Clinical Immunology and Allergy Unit and Infectious Disease Unit, Department of Medicine, Massachusetts General
Hospital; Pulmonary and Critical Care Divisions, Department of Medicine, Brigham and Women’s Hospital; Partners
Asthma Center; and Harvard Medical School, Boston, Massachusetts
Murine models of allergen-induced pulmonary inflammation share many features with human asthma, including the development of antigen-induced pulmonary eosinophilia, airway hyperresponsiveness, antigen-specific cellular and antibody responses, the elaboration of Th2 cytokines (interleukin [IL]-4 and IL-5),
and the expression of chemokines with activity for eosinophils. We examined the role of B cells and antigen-specific antibody responses in such a model by studying the histopathologic and physiologic responses of B cell–deficient mice compared with wild-type controls, following systemic immunization and
airway challenge with ovalbumin (OVA). Both OVA-challenged wild-type and B cell–deficient mice developed (1) airway hyperresponsiveness, (2) pulmonary inflammation with activated T cells and eosinophils, (3) IL-4 and IL-5 secretion into the airway lumen, and (4) increased expression of the eosinophil active chemokines eotaxin and monocyte chemotactic protein-3. There were no significant differences in
either the pathologic or physiologic responses in the B cell–deficient mice compared with wild-type mice.
These data indicate that B cells and antigen-specific antibodies are not required for the development of airway hyperresponsiveness, eosinophilic pulmonary inflammation, and chemokine expression in sensitized
mice following aerosol challenge with antigen. MacLean, J. A., A. Sauty, A. D. Luster, J. M. Drazen,
and G. T. De Sanctis. 1999. Antigen-induced airway hyperresponsiveness, pulmonary eosinophilia,
and chemokine expression in B cell–deficient mice. Am. J. Respir. Cell Mol. Biol. 20:379–387.
In murine models of allergen-induced pulmonary inflammation, CD41 T lymphocytes and the Th2 cytokines interleukin (IL)-4 and IL-5 are important regulators of
eosinophilic pulmonary inflammation and airway hyperresponsiveness (1–8). The role of antigen-specific antibodies
in these models is less clearly defined. Either immunoglobulin (Ig)E or IgG1 antigen-specific antibodies appear to be
sufficient to induce pulmonary inflammation and airway
hyperresponsiveness in immunized mice following antigen
challenge (9, 10). However, whether the presence of antigenspecific antibodies is required to obtain all aspects of this
asthma-like phenotype remains to be fully established. B
cell–deficient mice developed eosinophil-rich inflammation
(Received in original form December 22, 1997 and in revised form May 27,
1998)
Address correspondence to: James A. MacLean, M.D., Bulfinch 422, Massachusetts General Hospital, 50 Blossom St., Boston, MA 02114. E-mail:
[email protected]
Abbreviations: bronchoalveolar lavage, BAL; BAL fluid, BALF; effective
dose required to increase RL to 200% of control values, ED200RL; enzymelinked immunosorbent assay, ELISA; fetal calf serum, FCS; immunoglobulin, Ig; interleukin, IL; monocyte chemotactic protein-3, MCP-3; ovalbumin, OVA; phosphate-buffered saline, PBS; pulmonary resistance, RL.
Am. J. Respir. Cell Mol. Biol. Vol. 20, pp. 379–387, 1999
Internet address: www.atsjournals.org
in the lung following systemic immunization and airway challenge with antigen (11, 12). However, B cell–deficient mice
sensitized via the airways did not develop airway hyperresponsiveness, despite the accumulation of eosinophils and
Th2 cytokines in the bronchial tissue (12). Reconstitution
of these B cell–deficient mice with antigen-specific IgE resulted in the development of airway hyperresponsiveness,
suggesting an important role for antigen-specific IgE in
this process. In our own studies, we have found that in the
absence of T cells (eliminated by anti-CD3 monoclonal antibody), the presence of antigen-specific IgE and IgG antibodies was not sufficient to induce pulmonary eosinophilia
and the expression of the eosinophil-specific chemokine
eotaxin in sensitized mice following antigen challenge (7).
To evaluate further the role of antigen-specific antibody
responses in the development of pulmonary eosinophilia,
airway hyperresponsiveness, and chemokine expression in
response to antigen, we evaluated B cell–deficient mice following systemic immunization and repeated airway challenge with ovalbumin (OVA). Our findings demonstrate
that airway hyperresponsiveness, pulmonary inflammation
with activated T cells and eosinophils, and the expression
of eosinophil-active chemokines can develop in the absence
of B cells and antigen-specific antibody responses, to a degree that is comparable to that in wild-type mice.
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Materials and Methods
Mice
C57BL/6 wild-type mice ( 1/1) and C57BL/6-Igh-6tm1Cgn
B cell–deficient mice (2/2) (13) were obtained from The
Jackson Laboratory (Bar Harbor, ME). The B cell–deficient mice had been backcrossed onto the C57BL/6 strain
for eight generations. The mice were studied between 7 and
10 wk of age. The mice were housed in a pathogen-free animal facility and were given food and water ad libitum.
Immunization and Challenge Protocol
Mice were immunized with 10 mg of OVA (Sigma Chemical Co., St. Louis, MO) and 1 mg aluminum hydroxide intraperitoneally (i.p.) on Days 0, 7, and 14. Mice underwent
aerosol challenge with either OVA (5% in phosphatebuffered saline [PBS]) or PBS for 20 min/d for 5 d, 7 d after the final immunization. Aerosol challenge was performed
by placing mice in a Plexiglas box (dimensions: 22 3 23 3
14 cm) and aerosolizing OVA using a DeVilbiss nebulizer
(Somerset, PA) driven by compressed air. Mice were studied between 18 and 24 h after the last aerosol challenge.
Pulmonary Resistance and Airway Responsiveness
Airway responsiveness was measured as previously described (14, 15). In brief, dose-response curves to methacholine were obtained 18 to 24 h after the last aerosol challenge in anesthetized, ventilated mice. Methacholine was
administered intravenously via the internal jugular vein,
sequentially in increasing doses (33 to 1,000 mg/kg). From
the relationship between the dose administered and pulmonary resistance (RL), without controlling for lung volume, the effective dose of methacholine that resulted in a
doubling of RL was determined by log-linear interpolation.
This dose is referred to as the effective dose required to
increase RL to 200% of control values (ED200RL) and was
used as a measure of airway responsiveness. This index
was log-transformed to reflect the log-linear relationship
between the rise in resistance and the methacholine dose.
Bronchoalveolar Lavage
Bronchoalveolar lavage (BAL) was performed after the
physiologic measurements, as previously described (7). In
brief the lungs were lavaged with eight 0.5-ml aliquots of
PBS containing 0.6 mM ethylenediamenetetraacetic acid
(EDTA). Recovered live cells (trypan blue exclusion)
were enumerated in a hemocytometer. Cell differential
counts were determined by enumerating macrophages, neutrophils, eosinophils, and lymphocytes on Wright-stained
(LEUKOSTAT; Fisher Scientific, Pittsburgh, PA) cytocentrifuge preparations of cells recovered by BAL.
Antibodies
Rat antimouse monoclonal antibodies used for tissue staining included: culture supernatants for anti-CD3 (clone
YCD3), anti-CD4 (clone GK1.5), and anti-CD8 (clone
53-6.7) raised in our laboratory. Commercial antibodies
included anti-CD19, anti-B220, anti-CD11b (Mac-1), and
anti-Ly-6G (Gr-1) (all from PharMingen, San Diego, CA).
Commercial conjugated antibodies used for flow cytometric analysis included anti-CD3-FITC (PharMingen), anti-
CD4-PE (Becton Dickinson, San Jose, CA), anti-CD8Red 613 (GIBCO BRL, Grand Island, NY), and antiCD25 (PharMingen).
Histology and Immunohistochemistry
Lungs were harvested after BAL and either fixed in 10%
formalin and embedded in paraffin blocks for regular histology, or embedded in cryopreservative and frozen for
immunohistochemistry. Paraffin-embedded lung sections
were stained with hematoxylin and eosin (H&E) using standard protocols. Frozen tissue sections were air-dried and
fixed in acetone. The sections were blocked with normal
serum from the species in which the biotinylated secondary antibody was raised (i.e., rabbit serum). Endogenous
biotin was blocked using an Avidin-Biotin Blocking Kit
(Vector Labs, Burlingame, CA). The sections were incubated with the primary monoclonal antibodies for 45 min,
followed by washing in PBS. The sections were then incubated with the biotinylated secondary antibody for 45 min,
followed by washing in PBS. The slides were developed using a standard avidin–biotin enzyme complex method that
uses alkaline phosphatase in the enzyme complex (Vectastain ABC-AP Kit; Vector), followed by the alkaline phosphatase substrate that produces a red reaction product.
Cytokine Assays
Quantification of IL-4 and IL-5 in BAL fluid (BALF) was
performed by capture enzyme-linked immunosorbent assay (capture-ELISA) kits according to the protocols provided by the manufacturers (IL-4/PharMingen; IL-5/Endogen, Cambridge, MA). The limit of detection of the IL-4
assay was 10 pg/ml, and for the IL-5 assay 5 pg/ml.
RNA Extraction and Northern Blotting
Lungs excised for RNA extraction were perfused with
sterile saline via the pulmonary circulation to remove
blood, and then frozen on dry ice and stored at 2708C
before processing. Total cellular RNA was isolated from
the lungs by homogenizing the tissue with a polytron in the
presence of 4 M guanidine hydrochloride and pelleting the
RNA through a 5.7 M CsCl2 cushion (16). Total RNA, 15
mg, was fractionated on a 1.2% agarose gel containing
0.2 M formaldehyde, transferred to GeneScreen (DuPont,
Wilmington, DE), and hybridized with 32P-[dCTP] Klenow-labeled random primed mouse eotaxin, mouse monocyte chemotactic protein-3 (MCP-3), or mouse ribosomal
protein (rpL32) complementary DNA (cDNA) probes.
The ribosomal probe is a 279-base pair fragment encoding
the ribosomal protein rpL32 and was used to control for
RNA loading (17). Hybridization was performed in 50%
formamide, 10% dextran sulfate, 53 saline sodium citrate
(SSC), 13 Denhardt’s solution (0.0002% [wt/vol] polyvinylpyrrolidone, 0.0002% [wt/vol] bovine serum albumin,
0.0002% [wt/vol] Ficoll 400), 100 mg/ml of heat-denatured
herring sperm DNA solution, 1% (wt/vol) sodium dodecyl
sulfate (SDS), and 20 mM Tris (pH 7.5) at for 42 8C for
18 h. Blots were washed with 23 SSC/0.1% SDS at 428C
for 40 min and then in 0.23 SSC/0.1% SDS at 558C for 40
min. RNA was quantified using a PhosphorImager (BioRad, Hercules, CA) and normalized to the signal for total
RNA loaded (i.e., rpL32).
MacLean, Sauty, Luster, et al.: Antigen-Induced Pulmonary Inflammation in B Cell–Deficient Mice
381
Flow Cytometry
Flow cytometry was performed as previously described
(18). In brief, cell suspensions were resuspended in staining buffer (PBS with 10% mouse serum) and stained with
the conjugated antibodies at 48C for 30 min. After washing
with PBS, the cells were fixed with 1% paraformaldehyde.
Single and three-color flow cytometry was performed on a
FACScan cytofluorimeter (Becton Dickinson) and analysis
was performed using Lysys software (Hewlett-Packard,
Palo Alto, CA).
OVA-Specific IgG and IgE Levels and Total Serum IgE
OVA-specific IgG levels were measured as previously described (7). In brief, ELISA plates were coated with OVA
and blocked with PBS–10% fetal calf serum (FCS) (Hazelton Biologicals, Inc., Lenexa, KS). Serum samples were
added to the wells after serial dilution in PBS–10% FCS
and were allowed to incubate for 1 h at room temperature.
The plates were washed five times with PBS–Tween before the addition of the secondary antibody, a goat antimouse IgG alkaline phosphatase (Vector), for 45 min at
room temperature. The plates were washed 5 times with
PBS–Tween and 10 times with distilled water before development with phosphatase substrate (Kirkegaard &
Perry Laboratories, Inc., Gaithersburg, MD). The reaction
was allowed to proceed for 20 min and was stopped by the
addition of EDTA. The plates were read in an ELISA
plate reader at an optical density (OD) of 405 nm.
OVA-specific IgE levels were measured by captureELISA as previously described (7). ELISA microtiter
plates were coated with a purified antimouse IgE monoclonal antibody (Pharmingen) at a concentration of 2 mg/
ml and blocked with PBS–10% FCS. Serum samples were
diluted in PBS–10% FCS and incubated in the wells for
2 h. After washing with PBS–Tween, biotinylated OVA
(10 mg/ml) was added to the wells and incubated for 1 h.
The plates were washed with PBS–Tween, followed by the
addition of avidin alkaline phosphatase (Sigma) for 1 h.
The plates were washed with PBS–Tween and distilled water before the addition of the phosphatase substrate. The
plates were allowed to develop for 30 min. The plates were
read in an ELISA plate reader at an OD of 405 nm.
Total serum IgE was measured by capture-ELISA in a
manner similar to the detection of OVA-specific IgE. A
biotinylated rat antimouse IgE (Pharmingen) was used to
detect captured IgE in place of the biotinylated OVA.
Statistical Analysis
Student’s t test (unpaired, two-tailed) was used to calculate significance levels between treatment groups.
Results
Airway Responsiveness in OVA-Immunized/OVAChallenged (OVA/OVA) Wild-Type and B Cell–
Deficient Mice Compared with OVA-Immunized/
PBS-Challenged (OVA/PBS) Control
OVA/OVA wild-type mice had significantly increased
methacholine responsiveness (as reflected by a lower log
ED200RL) compared with OVA/PBS wild-type control mice
(P , 0.01) (Figure 1). Similarly, OVA/OVA B cell–deficient
Figure 1. Airway responsiveness in OVA/PBS and OVA/OVA
wild-type (1/1) and B cell–deficient (2/2) mice. OVA/OVA
wild-type (1/1) and B cell–deficient (2/2) mice were significantly more responsive to methacholine challenge than were
OVA/PBS-treated mice (P , 0.01). There was no significant difference in responsiveness between the OVA/OVA (1/1) and
(2/2) treated mice. Data are presented as means 6 SEM of the
ED200RL; minimum of six mice per group.
mice developed significantly enhanced methacholine responsiveness compared with OVA/PBS B cell–deficient
mice (Figure 1). There was no significant difference in airway responsiveness in OVA/OVA wild-type mice compared
with OVA/OVA B cell–deficient mice. These findings indicate that airway hyperresponsiveness develops in B cell–
deficient mice and that the increased responsiveness is not
significantly different from that of similarly treated wildtype animals.
Inflammatory Cell Recruitment and Th2 Cytokine
Levels in the BAL of OVA/OVA Wild-Type and
B Cell–Deficient Mice
The recovery of inflammatory cells from the airway lumen
(BAL) and the harvesting of lung tissue were performed
immediately following the physiologic assessments. There
was no significant difference in BAL cell recoveries (total
or specific cell types) between OVA/OVA wild-type and
B cell–deficient mice, indicating that B cell–deficient mice
can recruit inflammatory cells into the airway lumen as effectively as wild-type control mice can following antigen
challenge. Recovery of cells from BAL of OVA/PBS wildtype and B cell–deficient mice revealed a predominance of
alveolar macrophages in both groups (Table 1). OVA/
OVA wild-type mice showed a significant increase in total
cells, eosinophils, and lymphocytes compared with OVA/
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TABLE 1
BALF cell differential counts and cytokine levels
Differential Counts (% total cells)
Mouse
1/1
2/2
1/1
2/2
Exposure
Cell Count
(3 10 6 cells)
Macrophages
Neutrophils
Lymphocytes
Eosinophils
OVA/PBS
OVA/PBS
OVA/OVA
OVA/OVA
P value*
P value†
P value‡
0.46 6 0.02
0.53 6 0.05
2.19 6 0.41
2.63 6 0.28
, 0.01
, 0.01
NS
99.75 6 0.025
81.67 6 5.93
13.40 6 2.54
23.57 6 6.71
, 0.01
, 0.01
NS
0.25 6 0.25
8.00 6 3.79
6.00 6 1.26
4.86 6 0.55
, 0.01
5 0.23
NS
0.00
5.67 6 1.33
7.40 6 0.87
10.29 6 1.29
, 0.01
, 0.05
NS
0.00
4.67 6 1.45
73.20 6 3.2
61.43 6 7.0
, 0.01
, 0.01
NS
IL-5
(pg/ml)
IL-4
(pg/ml)
, 5 pg/ml
, 5 pg/ml
43.43 6 10.53
70.77 6 25.31
, 10 pg/ml
, 10 pg/ml
35.23 6 5.80
45.27 6 7.13
NS
NS
Values are means 6 SEM. NS 5 not significantly different.
*P value 1/1 OVA/OVA versus 1/1 OVA/PBS.
†
P value 2/2 OVA/OVA versus 2/2 OVA/PBS.
‡
P value 1/1 OVA/OVA versus 2/2 OVA/OVA.
PBS mice. OVA/OVA B cell–deficient mice also demonstrated a significant increase in total cells, eosinophils, and
lymphocytes recovered by BAL. Flow cytometric analysis
of BAL cells demonstrated an increase of CD41 T cells
bearing the activation marker CD25 a (IL-2 receptor a
chain) in both the OVA/OVA B cell–deficient mice and
wild-type mice compared with the corresponding OVA/
PBS mice (data not shown). IL-4 and IL-5 levels were
measured in the first milliliter of recovered BAL fluid
from OVA/OVA wild-type and B cell–deficient mice, and
similar levels were found in both groups (Table 1). Levels
were below the limit of detection in OVA/PBS-treated
mice (Table 1).
Interestingly, we noted that OVA/PBS B cell–deficient
mice had an increase in the percentage of lymphocytes and
granulocytes in the BAL compared with similarly treated
wild-type mice. The total cell counts in the OVA/PBStreated mice, however, were not different between wildtype and B cell–deficient mice. Importantly, histologic
analysis of the lungs of OVA/PBS B cell–deficient mice
did not show any evidence of pneumonia or pneumonitis
(not shown).
Lung Histology from OVA/OVA Wild-Type
and B Cell–Deficient Mice
Histopathologic examination of lung tissue from OVA/
OVA wild-type mice revealed a pleomorphic peribronchial and perivascular infiltrate consisting of eosinophils,
lymphocytes, macrophages, and neutrophils (Figure 2A)
that was not seen in OVA/PBS mice (not shown). A similar histopathologic picture was seen in OVA/OVA B cell–
deficient mice (Figure 2B). Immunohistochemical staining
of lung tissue demonstrated increased numbers of CD31
cells in the lungs of OVA/OVA wild-type (Figure 2C) and
B cell–deficient (Figure 2D) mice. CD4 1 and CD81 T
lymphocytes, granulocytes (Gr-11), and monocyte/macrophages (Mac11) were also increased in both OVA/
OVA wild-type and B cell–deficient mice (not shown).
The only differential staining noted was in the number of
B cells in the lungs of OVA/OVA wild-type mice (Figure
2E) that, as expected, were not identified in OVA/OVA B
cell–deficient mice (Figure 2F).
Chemokine Messenger RNA (mRNA) Expression in
OVA/OVA Wild-Type and B Cell–Deficient Mice
Compared with OVA/PBS-Treated Control Mice
The expression of eotaxin and MCP-3 mRNA was assessed by Northern analysis of RNA recovered from
whole-lung extracts, as previously described (7, 19). Eotaxin and MCP-3 mRNA expression was induced in OVA/
OVA wild-type and B cell–deficient mice compared with
OVA/PBS-treated control animals (Figure 3A). Quantification of the mRNA signal was achieved using a PhosphorImager (Figure 3B). There was a significant increase
in eotaxin and MCP-3 mRNA expression in OVA/OVA
wild-type and B cell–deficient mice compared with the respective OVA/PBS-treated mice. There was no significant
difference in the expression of either eotaxin or MCP-3
between OVA/OVA wild-type and B cell–deficient mice.
Total IgE, OVA-Specific IgE, and OVA-Specific IgG in
OVA/OVA Wild-Type and B Cell–Deficient Mice
Immunization with OVA was associated with a significant
increase in total IgE, OVA-specific IgE, and OVA-specific IgG in wild-type mice (Figure 4). As expected, B cell–
deficient mice had undetectable levels of total IgE and did
not develop measurable leveIs of OVA-specific antibodies
of either the IgE or IgG class (Figure 4).
Discussion
In B cell–deficient mice, systemic immunization with antigen followed by repeated aerosol challenge resulted in the
development of (1) airway hyperresponsiveness to methacholine, (2) pulmonary inflammation with activated T cells
and eosinophils, (3) Th2 cytokine secretion in the lung,
and (4) the induction of chemokines (eotaxin and MCP-3)
with activity for eosinophils. The development of these parameters was not significantly different from similarly
treated wild-type mice. These findings suggest that in the
absence of B cells and antigen-specific antibody responses,
the pathologic and physiologic changes that characterize
this murine model can develop.
Pulmonary inflammation with eosinophilia in systemically immunized and airway challenged B cell–deficient
mice has been previously reported; however, airway physi-
MacLean, Sauty, Luster, et al.: Antigen-Induced Pulmonary Inflammation in B Cell–Deficient Mice
383
Figure 2. Lung histopathology and immunohistochemistry from OVA/OVA wild-type (1/1) and B cell–deficient (2/2) mice. H&E
stain of formalin-fixed lung tissue from OVA/OVA wild-type (1/1) (A) and B cell–deficient (2/2) (B) mice showed a peribronchial
and perivascular accumulation of inflammatory cells including eosinophils. Immunohistochemical staining revealed increased numbers
of CD31 cells (red staining, arrows) in both OVA/OVA wild-type (C) and B cell–deficient (D) mice that were not seen in OVA/PBS
control animals (not shown). B cells (B2201) were identified in the lungs of OVA/OVA wild-type mice (E) (red staining, arrows) but
not in B cell–deficient mice (F). Sections shown are from a representative experiment. Magnification: A–D, 3320; E and F, 3200.
ology was not examined by these investigators (11). In a
separate study, Hamelmann and colleagues examined pathologic and physiologic lung responses in B cell–deficient mice
following repeated airway challenge (12). In this study,
mice developed eosinophilic pulmonary inflammation, antigen-induced T cell-proliferation, and increased production of Th2 cytokines (IL-4 and IL-5) from bronchial
lymph nodes stimulated with antigen in vitro. Interestingly, airway hyperresponsiveness was not seen in sensi-
tized B cell–deficient mice, but could be elicited following
the passive transfer of antigen-specific IgE into immunized
mice. These findings suggest that T cell responses to antigen were present in the B cell–deficient mice but that airway hyperresponsiveness could develop only when antigen-specific IgE was present.
In the current experiments, mice were immunized systemically (i.p.) and challenged by aerosol. This method of
sensitization and challenge may account for our ability to
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Figure 3. Chemokine mRNA expression in OVA/PBS- and OVA/OVA-treated wild-type (1/1) and B cell–deficient (2/2) mice. RNA
extracted from whole lungs 24 h after the last aerosol challenge was analyzed by Northern blotting. (A) The blot was sequentially hybridized with eotaxin, MCP-3 32P-labeled cDNA probes, and a probe for total RNA rpL32 (L32). (B) The RNA signal was quantified
using a PhosphorImager and normalized to the rpL32 signal. Constitutive expression of eotaxin and MCP-3 mRNA was seen in OVA/
PBS (1/1) and (2/2) mice. There was a significant increase in the expression of eotaxin in both the ( 1/1) and (2/2) OVA/OVA
mice (*P , 0.005 and †P , 0.03, respectively). Similarly, there was a significant increase in the expression of MCP-3 in both the (1/1)
and (2/2) OVA/OVA mice (both *P , 0.03). There was no significant difference in the expression of eotaxin or MCP-3 between the
OVA/OVA wild-type and B cell–deficient mice. Each lane represents a different mouse. Data represent mean eotaxin or MCP-3 counts
adjusted for RNA load 6 SEM (OVA/PBS, three mice/group; OVA/OVA, minimum of five mice/group).
MacLean, Sauty, Luster, et al.: Antigen-Induced Pulmonary Inflammation in B Cell–Deficient Mice
Figure 4. Total IgE, OVA-specific IgE, and OVA-specific IgG titers from OVA/PBS and OVA/OVA wild-type ( 1/1) and B
cell–deficient (2/2) mice. Serum obtained 24 h after the final
aerosol challenge. Both OVA/PBS- and OVA/OVA-treated
wild-type mice developed increased titers of total serum IgE (A),
OVA-specific IgE (B), and OVA-specific IgG (C) compared with
similarly treated B cell–deficient mice (all *P , 0.01). Data represent means 6 SEM (minimum of four mice/group). OVA-spe-
385
detect significant airway hyperresponsiveness in antigenchallanged B cell–deficient mice in the absence of antigenspecific antibody responses. Takeda and associates have
suggested that the protocol of sensitization and challenge
used in any given murine model may determine the relative importance of antigen-specific antibodies and mast
cells in regulating tissue eosinophilia and airway hyperresponsiveness (20). They showed that in the absence of
mast cells, pulmonary eosinophilia and airway hyperresponsiveness developed after either repeated airway challenge or systemic sensitization followed by airway challenge (20). Similarly, eosinophilic pulmonary inflammation
and airway hyperresponsiveness developed in IgE-deficient mice following repeated airway sensitization (21). In
contrast, however, Kung and coworkers previously reported that mast cells may influence the degree of eosinophil recruitment to the lung following antigen challenge
when the airway challenge was less intensive (22). As such,
mast cells and IgE may play an important role in models in
which sensitization and challenge are relatively attenuated. In contrast, T cells may play a more important role in
models when the sensitization and challenge are repeated
more frequently (23). This hypothesis is intriguing and
may help explain the differences that have been noted between our findings and those of Hamelmann and associates (12). The relative importance of the route of sensitization in the development of airway hyperresponsiveness
still remains to be fully established. Our findings suggest
that the systemic method of sensitization leads to an increase in total IgE and in antigen-specific IgE and IgG antibodies in wild-type mice; however, the presence of these
antibodies does not appear to act in a synergistic fashion
to augment the histopathologic or physiologic changes in
the model when compared with B cell–deficient mice.
Another factor that may have accounted for some of
the differences between our studies in B cell–deficient
mice and those of Hamelmann and associates is the method
used to measure airway hyperresponsiveness. We used an
in vivo method that involves anesthetizing test animals
and measuring airway responses to intravenously administered methacholine, a method we have found to be reproducible in the study of both immunized and naive animals
(15, 24, 25). In contrast, Hamelmann and coworkers used
an in vitro technique that has previously been demonstrated to correlate well with in vivo measurements (26).
Finally, strain differences may have contributed to
some of the differences between our study and that of
Hamelmann and coworkers. They used B cell–deficient
backcrossed to B10.BR mice, whereas we used C57BL/6 B
cell–deficient mice. It has been suggested previously that
airway hyperresponsiveness in C57BL/6 mice may be
more dependent on cells other than mast cells because
these mice are genetically deficient in important mast-cell
mediators (e.g., mast-cell protease 7) (27). To the best of
our current knowledge, genetic differences in airway hyperresponsiveness between B10.BR and C57BL/6 mice
have not been established.
cific IgG was assayed at a dilution of 1:500. Total IgE and OVAspecific IgE were assayed at a dilution of 1:3.
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lnterestingly, we noted that OVA/PBS-treated B cell–
deficient mice had an increased percentage of granulocytes and lymphocytes in the BALF, which was not seen in
similarly treated wild-type mice. The total BALF cell
counts, however, were not significantly different between
these two groups, and we found no evidence of pneumonia
or pneumonitis on gross lung histology in the B cell–deficient OVA/PBS mice. The increased percentage of granulocytes and lymphocytes in the BALF of B cell–deficient
mice following PBS is likely related to the lack of antibodies in these mice. The increased percentage of lymphocytes and granulocytes in the BALF may reflect a compensatory mechanism for the lack of humoral immunity in the
respiratory tract. Importantly, the increased percentage of
lymphocytes and granulocytes in OVA/PBS B cell–deficient mice did not alter the airway responsiveness compared with similarly treated wild-type mice. Furthermore,
it did not interfere with the ability of these mice to respond to an antigenic challenge with the development of
pulmonary inflammation with eosinophils and increased
airway hyperresponsiveness.
The accumulating evidence suggests that in murine
models of allergen-induced airway hyperresponsiveness,
the end points of airway hyperresponsiveness and pulmonary eosinophilia can develop by at least two distinct pathways: (1) a mast cell–dependent pathway and (2) a CD41
T cell–dependent pathway (23, 27). The mast cell–dependent pathway is believed to result from the release of inflammatory mediators (e.g., tryptase, leukotrienes) from
mast cells, which act on the airway microenvironment to
increase responsiveness. Mast cell–dependent airway hyperresponsiveness has been demonstrated both in the
presence (12) and absence (28) of a sensitizing antigen or
following the passive transfer of antigen-specific antibodies (9, 10). Other studies, however, clearly demonstrate
that airway hyperresponsiveness can occur independently
of IgE or mast cells (20, 21, 29). In these studies it is presumed that airway hyperresponsiveness results from the
recruitment of eosinophils to the lung by antigen-specific
T cells, with the subsequent release of eosinophil-derived
inflammatory and cytotoxic mediators (e.g., major basic
protein, eosinophil cationic protein, and leukotrienes) in
the bronchial mucosa. A direct role for T lymphocytes in
the development of airway hyperresponsiveness (i.e., independent of eosinophil recruitment) may also be important.
Garssen and colleagues have shown that airway hyperresponsiveness can occur in a model of delayed-type hypersensitivity in the lung (30), and De Sanctis and coworkers
have demonstrated that airway hyperresponsiveness can
be transferred by T cell–selective bone marrow transplantation in naive mice (25). The findings in the current report support the concept that airway hyperresponsiveness
can occur independently of antigen-specific antibody responses and the mast cell–dependent pathway.
The recruitment of cells to sites of inflammation is a
complex process that involves cell adhesion, activation,
chemoattraction, and ultimately transmigration of the endothelial barrier. Chemokines play an important role in
both the activation and chemoattraction phases of cellular
recruitment. Chemokine expression in rodent models of
allergen-induced pulmonary inflammation has been well
described (7, 31–37). In the current experiments, both eotaxin and MCP-3, two chemokines important for eosinophil recruitment in this model (34, 38, 39), were upregulated in both wild-type and B cell–deficient mice after
immunization and challenge with antigen. In previous experiments, we demonstrated that eotaxin expression was
upregulated in a similar fashion following antigen challenge (7). Furthermore, we demonstrated that elimination
of T lymphocytes with anti–T cell antibodies significantly
diminished the expression of eotaxin in challenged mice
without altering the levels of antigen-specific antibody titers. These findings suggest that in the absence of T cells,
antigen-specific antibodies alone are not sufficient to induce the expression of eotaxin. The current findings demonstrate that at least two chemokines with activity for
eosinophils can be upregulated in the absence of antigenspecific antibody responses, and these findings are consistent with our previous results. The importance of this result lies in determining the events that occur following the
introduction of antigen into the airway of a sensitized animal. Humbles and colleagues have examined the kinetics
of eosinophil recruitment and eotaxin expression in the
guinea pig and have suggested that following allergen
challenge, the mast cells may play an important role in the
early events leading to eosinophil recruitment in normal
mice (37). Our findings suggest that chemokine upregulation and eosinophil recruitment can occur independently
of antigen-specific antibodies and that the T cell may play
a central role in the regulation of these molecules. The
current concept that pulmonary eosinophilia and airway
hyperresponsiveness may arise by two separate pathways
(i.e., mast cell and T cell) may also apply for chemokine induction in vivo. Further studies examining the in vivo regulation of chemokine expression in this model are ongoing.
The finding that mice can develop antigen-induced pulmonary inflammation and airway hyperresponsiveness in
the absence of antigen-specific antibodies is of interest.
Whereas T cells are recognized as playing an important
role in regulating asthmatic inflammation in humans, antigen-specific IgE antibodies and mast cells are also recognized as important mediators of allergic respiratory disease. Our study suggests that these are complicated
models and that multiple possible parallels to asthmatic
airway responses exist.
Acknowledgments: The authors thank Zhi Qing Wu and Aiping Jiao for technical assistance. This work was supported by National Institutes of Health Grants
AI01245 (J.A.M.), CA69212 and AI40618 (A.D.L.), and HL 36110 (J.M.D. and
G.T.D.S.); Allen & Hanbury’s Respiratory Diseases Research Award (J.A.M.);
a grant from the Association Suisse Contre la Tuberculosle et les Maladies Respiratoires (A.S.); and a grant from the American Lung Association (G.T.D.S).
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