Modulation of allergic inflammation in mice deficient in
TNF receptors
Daniel G.
Rudmann1,2,
Mark W.
Moore3,
Jeffery S.
Tepper1,
Melinda C.
Aldrich1,
Juergen W.
Pfeiffer1,
Harm
Hogenesch2, and
Daniel B.
Tumas1
1 Departments of Pathology and Immunology, Genentech,
Incorporated, South San Francisco 94080; 3 Department of
Molecular Biology, Deltagen, Incorporated, San Carlos, California
94070; and 2 Department of Pathobiology, Purdue University,
West Lafayette, Indiana 47907
 |
ABSTRACT |
Tumor necrosis factor-
(TNF) is implicated as an
important proinflammatory cytokine in asthma. We evaluated mice
deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(
/
) mice] in a
murine model of allergic inflammation and found that TNFR(
/
) mice
had comparable or accentuated responses compared with wild-type
[TNFR(+/+)] mice. The responses were consistent among
multiple end points. Airway responsiveness after methacholine challenge
and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil
numbers in TNFR(
/
) mice were equivalent or greater than those
observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung
interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion
scores were comparable or greater in TNFR(
/
) mice compared with
those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with
anti-murine TNF antibody had BAL fluid leukocyte numbers and lung
lesion scores comparable to control antibody-treated mice. These
results suggest that, by itself, TNF does not have a critical
proinflammatory role in the development of allergic inflammation in
this mouse model and that the production of other cytokines associated
with allergic disease may compensate for the loss of TNF bioactivity in
the TNFR(
/
) mouse.
knockout mice; asthma; animal models; T cell subset 2 cytokines
 |
INTRODUCTION |
ASTHMA IS A
MULTIFACTORIAL DISEASE that has had escalating morbidity and
mortality in the last 20 years (45, 52). The mRNA and/or
protein levels of several cytokines, including tumor necrosis
factor-
(TNF), are increased in the sputum or serum of asthmatic
patients, but the exact roles of these cytokines in disease
pathogenesis are not well defined (4-6, 11, 29, 34, 39,
53). TNF is generally considered a potent proinflammatory cytokine, and in the lung, TNF is synthesized and stored primarily in
mast cells and alveolar macrophages (4, 17, 39).
TNF may have multiple proinflammatory roles in the pathogenesis of asthma, including upregulation of endothelial cell integrin receptors; activation of mast cells, macrophages, and eosinophils; promotion of
epithelial cytotoxicity; and aggravation of airway hyperreactivity (15, 27, 32, 37, 38, 47, 48).
Acute blockade of TNF bioactivity in sensitized animals can attenuate
pulmonary eosinophilic inflammation and/or airway hyperresponsiveness after a single aerosol challenge with antigen (33, 42, 51, 54). These studies have not examined the role of TNF after
multiple aerosol challenges nor have they examined the response in
sensitized mice after chronic TNF blockade [i.e., antibody-treated
mice or TNF receptor (TNFR) knockout mice]. We were interested in
studying the role of TNF in a model of pulmonary eosinophilic
inflammation that was CD4+ T cell dependent (14,
16). We chose a model that used multiple aerosol challenges to
avoid evaluating only the acute effects of altering TNF bioactivity. We
used a CD4+ cell-dependent model because CD4+
cells are thought to be important in orchestrating the chronic inflammatory response in asthma, and recently, TNF has been
demonstrated to downregulate CD4+ T cell function (2,
7, 8, 49). We modified TNF bioactivity both by using
mice deficient in TNFR1 and TNFR2 [TNFR(
/
)] and by treating
wild-type [TNFR(+/+)] mice with anti-TNF neutralizing antibody. Our
results demonstrate that persistent loss of TNF receptor signaling
results in comparable or accentuated allergic responses and that
blockade of TNF bioactivity does not abrogate allergic lung
inflammation in this mouse model.
 |
METHODS |
Gene-deleted mice.
TNFR(
/
) mice were generated by backcrossing TNFR1(
/
) and
TNFR2(
/
) mice, the phenotypes of which have been described previously (12, 13, 28, 31, 35, 36, 41, 43). The genotype
was confirmed in vitro with TNFR1- and TNFR2-specific DNA probes and
oligonucleotide primers in Southern blot and PCR experiments
(12). In vivo, TNFR(
/
) mice had no clinical signs associated with systemic administration of recombinant murine TNF
(Genentech, South San Francisco, CA) given at a 10-fold 100% lethal
dose (Rudmann, unpublished data). All TNFR(
/
) mice were on a
C57BL/6 (Charles River, Gilroy, CA) and 129/Sv (Taconic, Boston, MA)
background, and TNFR(+/+) mice were derived from the littermates of
TNFR(
/
) mice. In select experiments, responses to ovalbumin (Ova)
were also studied in the background strains C57BL/6 and 129/Sv for the
TNFR(+/+) and TNFR(
/
) mice. All mice were housed in microisolator
caging under laminar flow conditions and were 6-10 wk old at the
start of the experiments. Sentinel mice were housed in the same room
and serologically evaluated on a regular basis for murine viral and
bacterial agents. All experiments were approved by Genentech's Animal
Protocol Review Committee.
Murine Ova model of allergic inflammation.
TNFR(
/
), TNFR(+/+), and C57BL/6 and/or 129/Sv mice were ear tagged
and injected intraperitoneally on day 1 with Dulbecco's PBS
(nonsensitized control groups; HyClone, Logan, UT) or 10 µg of grade
V Ova (Sigma, St. Louis, MO) and 1 mg of aluminum hydroxide (Alum,
Intergen, Purchase, NY) in 0.1 ml of Dulbecco's PBS (sensitized groups). In a 3-day Ova challenge model, mice received an additional Ova-Alum immunization or PBS injection (nonsensitized groups) on
day 8 followed by an aerosol challenge with sterile Ova (10 mg/ml) in PBS with 0.01% Tween 20 (Sigma). After 1 wk, mice were aerosol challenged for 3 consecutive days and necropsied ~18 or 36 h after the last aerosol challenge. In a 7-day model, mice were
immunized only on day 1, were Ova aerosol challenged for 7 consecutive days beginning on day 15, and were necropsied
~18 h after the last aerosol challenge.
For each 20-min aerosol challenge, a PARI IS-2 nebulizer (PARI,
Richmond, VA) was used at 22 psi. Mice were challenged individually with Ova in a Plexiglas pie chamber (Braintree Scientific, Braintree, MA) that housed 12 mice simultaneously. Under these conditions, the
mass mean aerodynamic diameter of the fully saturated particle was 2.27 µm, with a span of 2.04 µm as measured by a Malvern Mastersizer (Malvern, Malvern, UK). The estimated lung dose of Ova per mouse at
each daily exposure was 10.4 µg.
The Ova used for aerosolization contained 1 ng endotoxin/ml Ova as
determined by the Limulus amebocyte lysate assay (Associates of Cape Cod, Boston, MA). This endotoxin concentration is at least 105 times lower than the concentration necessary to produce
lung inflammation as evaluated by bronchoalveolar lavage (BAL) and histology (Rudmann, unpublished data).
Anti-murine TNF-treated mice.
Neutralizing anti-murine TNF monoclonal antibody (MAb) was used
to abrogate TNF bioactivity in groups of nonsensitized control or
Ova-sensitized TNFR(+/+) mice. For these experiments, 100 µg of an
anti-TNF MAb (46) or 100 µg of control antibody (hamster IgG, Cappel, Durham, NC) were administered to TNFR(+/+) mice by intraperitoneal injection every other day starting 2 wk before sensitization and continuing until the end of the study. Control groups
of nonsensitized TNFR(+/+) mice received anti-TNF MAb or control
antibody. The dose of anti-murine TNF MAb given has been demonstrated
(46) to completely neutralize TNF bioactivity and has a
half-life of ~7 days in mice. The anti-TNF MAb has been administered
to mice for up to 3 mo with no evidence of a neutralizing antibody
response (46).
Airway reactivity measurements.
Breathing mechanics during methacholine (MCh) provocation were
evaluated 18-24 h after the third or seventh Ova aerosol
challenge. Initial experiments were performed with the 7-day challenge
protocol. Briefly, these mice were anesthetized, tracheal and
esophageal cannulas were placed, and the spontaneously breathing mice
were challenged with inhaled MCh. This method was determined to be unacceptable because the sensitized TNFR(
/
) mice were severely impaired (showing dyspnea, mucus accumulation, and gasping) at the time
of pulmonary function testing. Other than this observation (see
RESULTS), the data from this experiment are not included.
Mice receiving 3 days of Ova challenge were anesthetized (25 mg/kg of
pentobarbital sodium and 1.8 g/kg of urethane), the jugular vein was
catheterized (PE-10 connected to PE-50 tubing), and a cannula (1.7 cm
of PE-160 tubing connected to a 19-gauge tubing adapter) was inserted
into the trachea. The resistance of the tracheal cannula was ~0.92
cmH2O · ml
1 · s. Mice were
placed on a heated cradle and ventilated with a four-port manifold. Two
of the three ports were used for the inspiratory and expiratory lines
of the solenoid-actuated constant-flow ventilator. A third port was
connected to a differential pressure transducer for measurement of
airway opening pressure. The fourth port was connected to the tracheal
cannula. The mice were ventilated with 100% O2 at a
frequency of 170 breaths/min and a tidal volume of 9 ml/g. The cradle
was enclosed in a Plexiglas volume-displacement plethysmograph
(Century, Philadelphia, PA) for measurement of thoracic flow with a
differential pressure transducer (SenSym, Milpitas, CA). The two
pressure transducers (airway opening and thoracic flow) were linear in
frequency and phase over the normal tidal breathing range. Breathing
mechanics (respiratory system resistance and dynamic compliance) were
calculated from the digitally converted pressure and flow signals with
the use of the covariance method (44). Before baseline
measurements, mice were paralyzed with pancuronium (0.2 mg/kg), given a
volume history of 22.5 ml/g, and allowed to stabilize for 3 min.
Immediately after baseline measurements were obtained, a
computer-controlled syringe pump (pump 22, Harvard Apparatus, Boston,
MA) delivered 35 µg/kg of intravenous MCh (stock concentration 100 µg/ml) over 10 s. Five additional MCh doses were automatically
delivered every 5 min by doubling the flow rate until a final dose of
1,120 µg/kg was achieved. All pulmonary function parameters were
continuously monitored with a computerized (Buxco Electronics, Sharon,
CT) data acquisition program and were reported at 15-s intervals. An
integrated measurement of the MCh response at each dose was obtained by
averaging the 15-s logged data over the 5-min period. Log-linear
dose-response curves were constructed for each animal, and the
provocative concentration (µg MCh/kg body wt) that causes a 20%
increase over baseline resistance (PC120) was determined by
linear interpolation. The data are reported as means ± SE.
BAL and cytology.
Approximately 18 h after the last Ova aerosol challenge, mice were
anesthetized with halothane and euthanized by exsanguination. The
diaphragm was incised, and the trachea was exposed and cut just below
the larynx. A polyurethane flexible tube (0.040-cm outer
diameter; Braintree Scientific, Braintree, MA) ~4 cm in length and
attached to a blunt 23-gauge needle (Sherwood Medical, St. Louis, MO)
was placed 6 mm into the trachea, and the lung was lavaged three times
with the same bolus of sterile saline. The amount of saline used for
BAL was 30 µl/g mouse wt. This bolus filled the lung to ~60% of
total lung capacity. BAL fluid return averaged 80% and did not vary by
treatment group.
BAL fluid samples were centrifuged at 1,000 g at 4°C for
10 min. The supernatants were decanted and immediately frozen at
80°C. The cell pellets were resuspended in 250 µl of PBS with 2%
BSA (Sigma), then enumerated with an automated counter (Baker Instruments, Allentown, PA) and recorded as total number of BAL fluid
cells per milliliter. The cell suspension was adjusted to 200 cells/µl; 100 µl were centrifuged onto coated Superfrost Plus microscope slides (Baxter Diagnostics, Deerfield, IL) at 800 g for 10 min (Cytospin, Shandon, Pittsburgh, PA). Slides
were air-dried, fixed for 1 min in 100% methanol, and stained with
Diff-Quik (Baxter Health Care, Miami, FL). At least 200 cells/slide
were evaluated to obtain a differential leukocyte count.
Histopathology.
To evaluate and compare the severity and character of pathological
changes in lungs between experimental groups, the left lungs of mice
were fixed in 10% neutral buffered Formalin and embedded in paraffin,
and 3-µm sections were stained with hematoxylin and eosin or periodic
acid-Schiff (PAS). Lung sections were taken along the primary bronchus.
The entire section of lung was evaluated blindly by a board-certified
veterinary pathologist and scored based on the severity of the
inflammation around the airways and blood vessels and in the alveoli
and on the extent of airway epithelial cell and mucus-secreting cell
hyperplasia/hypertrophy with a scale from 0 (no inflammation or airway
changes) to 5 (severe inflammation and airway changes). For evaluation
of mucus-secreting cell hyperplasia/hypertrophy, the number and size of
mucus-secreting cells were qualitatively evaluated in sections of lung
stained with PAS.
Cytokine and IgE ELISA.
To quantitate and compare cytokine production in lung tissue in mice
from different experimental groups, supernatants from lung homogenates
were prepared as previously described (33). Briefly, the
right lung was placed in 1 ml of sterile chilled PBS with 0.05% Triton
X-100 (Sigma) and homogenized for 3-5 s. Lung homogenates were
centrifuged at 10,000 g at 4°C for 5 min. Supernatants
were decanted, filtered through a 1.2-µm syringe filter (Whatman,
Clifton, NJ), and immediately frozen at
80°C for later use in
cytokine quantitation.
Interleukin (IL)-2, IL-4, IL-5, and interferon (IFN)-
were
quantitated in lung homogenate supernatants and lymphocyte culture supernatants with a sandwich ELISA procedure (1, 21).
Briefly, 0.5 µg/ml (for IL-2, PharMingen, San Diego, CA) or 1.0 µg/ml (for IL-4 and IL-5, PharMingen; for IFN-
, Genentech
hybridoma 22.1) of anti-murine cytokine MAbs were used to coat
high-binding 96-well ELISA plates (Nalge Nunc, Rochester, NY). Plates
were coated at 4°C for 12-24 h and blocked for 30-60 min
with ELISA buffer (PBS, 0.5% BSA, 2 mM EDTA, and 0.05% Tween 20).
Samples were added undiluted or diluted 1:2 to 1:16 to the coated
plates in duplicate. The recombinant standard was serially diluted 1:2
in duplicate starting at a concentration of 1,000 pg/ml. All incubation
steps were done at room temperature on an orbital shaker. Plates with
sample and standard were incubated for 2-4 h. Next, 1 µg/ml of
biotinylated anti-murine IL-2, IL-4, or IL-5 (PharMingen) or rabbit
anti-murine IFN-
(lot 18183-73, Genentech) MAbs were added, and the
plates were incubated for 1-1.5 h. The plates were washed and
incubated for 0.5-1 h with 1:3,000 to 1:6,000 dilutions of
horseradish peroxidase (HRP)-streptavidin (Zymed, San Francisco, CA)
for the IL-2, -4, and -5 assays or HRP-donkey anti-rabbit Ig (Amersham,
Arlington Heights, IL) for the IFN-
assay. HRP development was done
for 15 min in the dark with o-phenylenediamine
dihydrochloride substrate solution (5 mg of
o-phenylenediamine, 12.5 ml of PBS, and 5 µl of 30%
H2O2). Plates were read at 490 and 405 nm on a
spectrophotometer. The adjusted optical density (OD) was
OD490
(OD405 + ODblank). The absolute concentration of the samples was calculated with the
standard curve and adjusted OD.
To quantitate lung TNF, the L-M cell bioassay was used with the
following modifications (30). Recombinant murine TNF
(Genentech) was used as a standard at a starting concentration of 4 ng/ml, in duplicate, on plated L-M cells. The limit of detection in
this assay was 31.25 pg TNF/ml. Included on each plate was a positive control, recombinant murine TNF-
.
The procedures for the total IgG, IgE, and Ova-specific IgE sandwich
ELISAs were comparable to those for the cytokine ELISAs except that BAL
fluid or serum either undiluted or diluted 1:2 to 1:20 (for BAL fluid)
or 1:25 to 1:200 (for serum) in ELISA buffer was used as the sample.
For total IgE and total IgG, the capture antibodies were rabbit
anti-mouse IgE (Genentech) and goat anti-mouse IgG (Cappel, Durham,
NC), respectively (2 µg/ml in PBS), and plates were coated for
24-48 h at 4°C. The standards were murine IgE
chain
(PharMingen), or IgG (Cappel), which were diluted serially 1:2 starting
with a 100 ng/ml concentration. The detection antibodies, biotinylated
FcER1-IgG (Genentech) for IgE and goat anti-mouse IgG-HRP (Boehringer
Mannheim, Indianapolis, IN) for IgG, were used at a dilution of 1:2,000
for 1-1.5 h. HRP-streptavadin and enzyme development steps were
identical to those used for the cytokine assays.
For the Ova-specific IgE (Ova IgE) and IgG (Ova IgG) ELISAs, the
capture reagent was grade V Ova (Sigma) coated at a concentration of 2 µg/ml in PBS on 96-well plates for 12-24 h. In experiments that
used a reference standard, the standard was pooled Ova-immunized mouse
serum (Genentech). Standard and samples were incubated for 2 h in
duplicate. For the Ova IgE ELISA, the samples were diluted 1:10 to
1:640. For the Ova IgG ELISA, the samples were diluted 1:50. The
detection antibody was HRP-conjugated goat anti-Ova (Genentech lot
16542-51) and was used at 1:4,000. Enzyme development and OD reading
steps for both total IgE and Ova-specific IgE and IgG were identical to
those used in the cytokine ELISA protocols. Ova-specific IgE and IgG
concentrations were reported in OD (Ova IgG) or in units per milliliter
(Ova IgE). When reported in units per milliliter, the OD at the highest
concentration of standard, a 1:10 dilution of pooled serum from
Ova-immunized mice, was considered 100 U/ml.
Flow cytometric analysis.
To obtain lung cells for fluorescence-activated cell sorter (FACS)
analysis of lymphocytes, lungs from selected mice were digested by a
previously described method (25) with modifications. While
under anesthesia, mice were exsanguinated from the abdominal aorta. One
or both lungs were removed, cut into small pieces, and digested at
37°C for 45 min in 4 mg of collagenase D, 8 U of dispase, and 1 mg of
DNase (Boehringer Mannheim) dissolved in 10 ml of RPMI 1640 medium with
0.05 M HEPES and 10% controlled process serum replacement-2 (Sigma).
After incubation, lungs were aspirated 20 times through a 30-ml syringe
with an 18-gauge needle. The lung digest was filtered through nylon and
spun at 1,500 g at 4°C for 5 min. Erythrocytes were
eliminated with lysis buffer (0.14 M NH4Cl and 0.017 M
Tris, pH 7.2), the lung cell pellet was washed and centrifuged twice,
and total lung cells were enumerated on a hematocytometer, excluding
those cells that stained with 0.1% trypan blue. Lung cells were stored
on ice and used for immunostaining and FACS analysis.
To evaluate the T and B cell populations by FACS in spleen, lung, and
tracheobronchial lymph nodes, single-cell suspensions were made as
described previously (29).
Cell (1 × 106) suspensions from lung digests of
sensitized and control TNFR(+/+) and TNFR(
/
) mice and regional
lymph nodes and spleen from sensitized TNFR(+/+) and TNFR(
/
) mice
were stained with anti-murine CD4 FITC (Calbiochem, La Jolla,
CA), CD8 phycoerytherin (Calbiochem), B220 phycoerytherin (Boehringer
Mannheim), or the following isotype controls: rat IgG2a FITC, rat IgG
FITC, rat IgG2a phycoerytherin, and hamster IgG FITC. Cells were
incubated with antibody (10 µg) for 30 min, washed, fixed in
paraformaldehyde, and analyzed on a flow cytometer (FACScan) with
Cellquest software (Becton Dickinson, San Jose, CA).
Statistical methods.
With UNISTAT for Excel software (version 4.5, UNISTAT, London, UK), BAL
fluid, flow cytometric, and cytokine ELISA data were compared with a
one-way ANOVA and, if significant (P < 0.05), were
followed with a protected least significant differences test for
between-group comparisons. Pulmonary function measurements from all
groups were compared with a one-way ANOVA, and, if significant (P < 0.05), were followed with an uncorrected
t-test. Histological lesion scores were compared with
Kruskal-Wallis one-way ANOVA and, if significant, were followed with a
multiple comparison test (multiple comparisons with t
distribution) for between-group comparisons.
 |
RESULTS |
Ova-sensitized TNFR(
/
) mice did not have reduced
immune or inflammatory responses to aerosol Ova challenge.
Asthma is associated with an increased number of leukocytes, especially
eosinophils and lymphocytes, in the BAL fluid of patients (3,
9). We quantitated and characterized BAL fluid leukocytes in
mice 18 h after 3 or 7 consecutive days of Ova aerosol exposure to
determine whether BAL fluid leukocyte numbers would be attenuated in
TNFR(
/
) mice. In previous experiments with this model, it was found
that the BAL fluid leukocyte response in sensitized mice was
maximal 18-24 h after the last aerosol exposure (Tepper, unpublished data). After three aerosol exposures with
Ova, sensitized TNFR(
/
) and TNFR(+/+) mice had pulmonary airway
inflammation as reflected by marked increases in BAL fluid leukocytes
compared with those in control nonsensitized mice (Fig.
1). Eosinophils were the predominant cell
population in the BAL fluid; however, lymphocytes were also
significantly increased in sensitized mice (Fig. 1). Surprisingly, loss
of TNFR function did not significantly affect the BAL fluid leukocyte
numbers. Total numbers of BAL fluid total leukocytes, eosinophils, and
lymphocytes were, instead, slightly but not significantly higher in
TNFR(
/
) compared with TNFR(+/+) mice (Fig. 1).

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Fig. 1.
Cell counts in bronchoalveolar lavage fluid (BALF)
measured 18 h after 3 days of ovalbumin (Ova) aerosol challenge.
TNF, tumor necrosis factor- ; TNFR, TNF receptor; TNFR(+/+)S and
TNFR( / )S, sensitized wild-type and TNFR-deficient mice,
respectively; TNFR(+/+)NS and TNFR( / )NS, nonsensitized wild-type
and TNFR-deficient mice, respectively. Values are means ± SE.
TNFR(+/+)NS and TNFR( / )NS mice (n = 9/group) and
TNFR(+/+)S (n = 5) and TNFR( / )S
(n = 9) mice had significantly increased total
leukocyte (P < 0.03), lymphocyte (P < 0.002), and eosinophil (P < 0.03) counts compared with
control mice. TNFR( / )S mice tended to have more total
leukocytes than TNFR(+/+)S mice (P = 0.12).
|
|
After seven aerosol exposures with Ova, the pulmonary inflammatory
response as measured in the BAL fluid was at least threefold greater in
sensitized TNFR(
/
) and TNFR(+/+) mice compared with that in mice
challenged for 3 days (Fig. 2). Again,
loss of TNFR function did not abrogate the pulmonary inflammatory
response as measured by lavage leukocyte numbers. In contrast, the
numbers of BAL fluid leukocytes, eosinophils, and lymphocytes were more than twofold higher in TNFR(
/
) mice compared with TNFR(+/+) mice
(Fig. 2).

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Fig. 2.
Cell counts in BALF measured 18 h after 7 days of
Ova aerosol challenge. Values are means ± SE. TNFR(+/+)NS and
TNFR( / )NS (n = 6/group), TNFR(+/+)S
(n = 6) and TNFR( / )S (n = 6) mice had significantly increased total leukocyte (P < 0.03) and eosinophil (P < 0.03) counts compared
with control mice. TNFR( / )S mice also had significantly increased
BALF lymphocytes compared with their control counterparts
(P = 0.001). TNFR( / )S mice had significantly
greater numbers of total leukocytes (P = 0.0035),
eosinophils (P = 0.0009), and lymphocytes
(P = 0.0068) than TNFR(+/+)S mice.
|
|
A prominent component of the histological lesions in sensitized mice
that were aerosol challenged with Ova was interstitial (perivascular
and peribronchiolar) leukocyte infiltrates. To determine whether
differences in interstitial leukocyte numbers were present in the lung,
mice were exsanguinated while under anesthesia, and total and
differential leukocyte counts from digested lungs were done on
sensitized mice after three Ova aerosol challenges. The number of total
and individual leukocytes was two- to threefold higher in lungs from
sensitized TNFR(
/
) and TNFR(+/+) mice compared with the respective
nonsensitized control mice (Fig. 3).
However, there were no significant differences in the number of total
lung leukocytes or in individual leukocyte populations between
TNFR(
/
) and TNFR(+/+) mice (Fig. 3).

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Fig. 3.
Cell counts from lung digests measured 18 h after 3 days of Ova aerosol challenge. Values are means ± SE. Compared
with TNFR(+/+)NS and TNFR( / )NS (n = 4/group) mice,
TNFR(+/+)S (n = 4) and TNFR( / )S (n = 4) mice had increased total leukocyte (P = 0.07),
eosinophil (P = 0.09), and neutrophil
(P < 0.03) counts. There were no differences
between interstitial cell counts in TNFR( / )S and TNFR(+/+)S mice.
|
|
Approximately 60% of the interstitial lymphocytes in the lungs of
sensitized mice were B220 positive, and the remaining cells were mostly
T cell receptor-
positive, indicating a B and T cell lineage,
respectively (Table 1). In the
tracheobronchial lymph nodes, T cell receptor-
-positive cells
were the predominant cell population in sensitized mice. IgE- and
IgG-positive B220 cells accounted for ~60% of the pooled lymph node
and lung lymphocytes. The immunophenotype of lung and regional lymph
node lymphocyte subpopulations, as determined by flow cytometry,
did not differ between the TNFR(
/
) and TNFR(+/+) mice (Table 1).
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Table 1.
Immunophenotype of lung and tracheobronchial lymph node mononuclear
cells in Ova-sensitized TNFR(+/+) and TNFR( / )
mice
|
|
The mice used in the above experiments were exsanguinated while under
anesthesia; however, the lungs were not perfused with saline before
digestion. The predominant peripheral blood leukocyte in the mouse is
the lymphocyte, and lymphocytes from the blood remaining in the lung
would contribute to the cell population analyzed with FACS. Other
leukocyte populations in the peripheral blood (i.e., neutrophils) would
also be identified in the interstitial cell differential counts. In
effect, interstitial cell differentials or the FACS interstitial
lymphocyte immunophenotype may have been influenced by the circulating
leukocytes that remained in the lung after exsanguination.
Human asthma is associated with elevated levels of T cell subset
2-type cytokines (2, 4, 5, 11) and IgE (26, 40). Lung concentrations of IL-2, IL-4, and IL-5 in sensitized TNFR(
/
) and TNFR(+/+) mice after 7 Ova aerosol challenges were 2- to 15-fold higher than those in nonsensitized control mice (Table
2). TNFR(
/
) animals did not
have lower concentrations of these lung cytokines. Instead, the
differences for IL-4 and IL-5 between TNFR(
/
) mice and their
control counterparts were approximately twofold greater than the
differences between TNFR(+/+) mice and their controls (Table 2). At the
time point examined (18 h after the final Ova aerosol exposure), lung
homogenate concentrations of TNF and IFN-
were below the level of
detection in sensitized TNFR(
/
) and TNFR(+/+) mice.
There were higher BAL fluid concentrations of IgG and
IgE and higher serum concentrations of IgE and
Ova-specific IgE in sensitized TNFR(
/
) and TNFR(+/+) mice
compared with those in control mice (Table
3). As was the case for the other end
points, sensitized TNFR(
/
) mice did not have an attenuated response
compared with TNFR(+/+) mice. Lavage fluid IgG was 20-fold higher in
sensitized TNFR(+/+) mice and 200-fold higher in sensitized TNFR(
/
)
mice compared with that in their respective nonsensitized controls (Table 3). Lavage fluid IgE was ~30-fold higher in sensitized TNFR(+/+) mice and 300-fold higher in sensitized TNFR(
/
) mice compared with that in their respective nonsensitized controls (Table
3).
Perivascular and peribronchial inflammation and airway mucus cell
hyperplasia are characteristic findings in biopsies from human asthma
patients (3). We performed blind evaluations on sections
of lung from mice to determine the extent of airway, perivascular, and
interstitial inflammation; airway epithelial hypertrophy; and airway
mucus plugging. A total lesion score (0 = no lesions to 5 = severe changes) was derived from each section. Histological changes in
sensitized TNFR(+/+) and TNFR(
/
) mice included peribronchiolar,
peribronchial, and perivascular lymphocytic and eosinophilic
inflammation, airway mucus cell hypertrophy, and alveolar eosinophils
and histiocytes (Fig. 4). PAS-stained lung sections confirmed that the extent of hypertrophy and hyperplasia of the mucus-secreting cells corresponded to the lesion severity scores
(data not shown). Whereas the histological characteristics of the
lesions were comparable between sensitized TNFR(
/
) and TNFR(+/+)
mice, lesions were equal or more severe in TNFR(
/
) mice than in
TNFR(+/+) mice challenged with Ova for 3 or 7 days (Fig.
5).

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Fig. 4.
Histological lesions in lungs of mice examined 18 h
after 7 days of Ova aerosol challenge. NS control mice had no
histological changes (A). Histological lesions in TNFR(+/+)
(B) and TNFR( / ) (C) mice were characterized
by airway mucus (B, arrow), airway epithelial hypertrophy
(C, arrow), accumulation of peribronchial and perivascular
eosinophils and lymphocytes, and intra-alveolar macrophages and
eosinophils. Original magnification of hematoxylin- and eosin-stained
sections, ×200. Bars, 100 µm.
|
|

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Fig. 5.
Histological lesion scores after 3 or 7 days of Ova
aerosol challenge. Lungs were graded for extent of
perivascular/peribronchiolar inflammation, airway epithelial
hypertrophy, and alveolar leukocytic infiltrates (0, no inflammation or
airway changes; 5, severe inflammation and airway changes). In both the
3- and 7-day experiments, lesion scores were greater in TNFR( / )S
mice than in TNFR(+/+)S mice.
|
|
Ova-sensitized TNFR(
/
) mice did not have reduced
airway hyperreactivity compared with Ova-sensitized
TNFR(+/+) mice.
We next determined whether differences in airway hyperreactivity would
correspond to inflammatory and immune responses in TNFR(
/
) and
TNFR(+/+) mice. In the first experiment, we attempted to measure
baseline breathing mechanics and airway hyperreactivity in
spontaneously breathing anesthetized mice challenged with inhaled MCh
after seven Ova aerosol challenges. This protocol produced severe
allergic inflammation, elevating baseline respiratory system resistance
in sensitized TNFR(+/+) mice (2.58 ± 0.71 cmH2O·ml
1·s;
n = 5) compared with nonsensitized TNFR(+/+) mice
(1.93 ± 0.25 cmH2O · ml
1 · s,
n = 6). Although the baselines were high, only
one mouse in the sensitized TNFR(+/+) group was so severely impaired
that changes in airway reactivity could not be evaluated. In contrast, four of six mice from the sensitized TNFR(
/
) group had dyspnea, mucus accumulation, or gasping that prohibited airway reactivity testing. The two remaining TNFR(
/
) mice were more reactive to inhaled MCh than either the sensitized TNFR(+/+) group or the nonsensitized controls (data not shown).
Because of the difficulties in measuring airway reactivity in
sensitized TNFR(
/
) mice, a similar experiment that used three rather than seven Ova inhalation challenges was performed in an attempt
to reduce the severity of the inflammatory response. To better control
the ventilatory status of the mice, the methods for measuring breathing
mechanics and delivering MCh were changed. Rather than delivering
inhaled MCh to anesthetized spontaneously breathing mice, MCh was
delivered intravenously to paralyzed, ventilated mice.
Less severe lung inflammation as measured by BAL fluid leukocyte
numbers (Figs. 1 and 2) was observed with the three-inhalation challenge protocol. However, like the seven-inhalation challenge protocol, the BAL fluid inflammatory response was greater in sensitized TNFR(
/
) compared with sensitized TNFR(+/+) mice. Baseline
respiratory system resistance was significantly larger in the
sensitized TNFR(
/
) mice (2.08 ± 0.04 cmH2O·ml
1·s)
compared with the sensitized TNFR(+/+) mice (1.92 ± 0.13 cmH2O·ml
1·s;
P = 0.05), whereas both of the sensitized groups had
greater baseline respiratory system resistance than their respective
nonsensitized control counterparts. When challenged with intravenous
MCh, only the sensitized TNFR(
/
) mice were hyperreactive
(P < 0.001) compared with the nonsensitized
TNFR(
/
) group, with no significant difference in PC120
observed between sensitized TNFR(+/+) and nonsensitized TNFR(+/+) mice (Fig. 6).

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Fig. 6.
Airway hyperreactivity in TNFR(+/+)S, TNFR( / )S,
TNFR(+/+)NS, and TNFR( / )NS mice (n = 4/group) after
3 days of Ova aerosol challenge. The provocative concentration that
causes a 20% increase over baseline resistance (PC120) was
significantly decreased in TNFR( / )S mice compared with that in
TNFR(+/+)S mice, *P < 0.001.
|
|
Anti-murine TNF treatment of Ova-sensitized
TNFR(+/+) mice did not reduce immune
or inflammatory responses to aerosol Ova challenge.
Because TNFR(
/
) mice did not have an attenuated response to Ova, we
examined the effect of administering anti-murine TNF MAb to TNFR(+/+)
mice. We used a well-characterized antibody (TN3) that did not induce a
neutralizing antibody response after long-term administration in the
mouse (46). We administered TN3 in doses that were
comparable to those used in previous studies (8, 46) in
mice with this antibody. We administered this antibody to TNFR(+/+)
mice starting 2 wk before Ova plus Alum sensitization and continued
antibody treatments throughout the Ova aerosol challenges to better
simulate the chronic loss of TNFR signaling in the TNFR(
/
) mice.
Lesion scores were similar between anti-TNF MAb-treated mice and those
treated with the control antibody (Fig.
7). There were no significant differences
between the numbers of BAL fluid leukocytes, eosinophils, lymphocytes,
or macrophages in anti-TNF MAb-treated mice and the control
antibody-treated mice (Fig. 8). There
were also no significant differences in the quantity of serum
Ova-specific IgE (P = 0.433) in anti-TNF MAb-treated
mice and control antibody-treated mice.

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Fig. 7.
Histological lesion scores in NS mice
(a-TNFNS and IgGNS; n = 3/group) and TNFR(+/+)S mice (n = 5-6/group) given
IgG or anti-TNF antibody (a-TNF). Lungs were graded for extent of
perivascular/peribronchiolar inflammation, airway epithelial
hypertrophy, and alveolar leukocytic infiltrates. There were no
differences between the lesion scores in TNFR(+/+)S mice treated with
anti-TNF antibody or IgG.
|
|

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Fig. 8.
Cell counts in BALF in mice treated with anti-TNF
antibody 18 h after 7 days of Ova aerosol challenge. Values are
means ± SE. Control mice, sensitized TNFR(+/+) a-TNF mice
(n = 6) given anti-TNF starting 2 wk before
sensitization (see METHODS) and IgG mice (n = 5) given the control antibody IgG had significantly increased numbers
of total leukocytes, eosinophils, and lymphocytes (P < 0.01) compared with a-TNFNS and IgGNS mice
(n = 3/group). There were no significant differences in
BALF leukocyte numbers between IgG-treated and TNF antibody-treated
mice.
|
|
 |
DISCUSSION |
TNF has been hypothesized to be an important protagonist in the
pathogenesis of asthma (6, 11, 29, 34, 53). For this
reason, we used mice unresponsive to TNF [TNFR(
/
)] or modified TNF bioactivity in mice by chronic treatment with anti-TNF MAb in order
to better understand the role of TNF in allergic inflammation. We
predicted that Ova-sensitized TNFR(
/
) or TNFR(+/+) mice treated with anti-TNF MAb would have reduced immune and inflammatory responses to aerosolized Ova. However, Ova-sensitized and challenged TNFR(
/
) mice had similar or exaggerated airway hyperreactivity and immune and
inflammatory responses to aerosolized Ova compared with responses in
sensitized TNFR(+/+) mice. Ova-sensitized and challenged TNFR(+/+) mice
treated with anti-TNF MAb also had comparable immune and inflammatory
responses to aerosolized Ova compared with those in TNFR(+/+) mice
treated with the control antibody. These data demonstrate that the
elimination of TNF bioactivity alone is not sufficient to abrogate the
murine inflammatory response to aerosolized Ova.
There are several possible explanations for the lack of protection
afforded to the TNFR(
/
) mice and anti-TNF MAb-treated TNFR(+/+)
mice in the Ova model of allergic inflammation. An unlikely explanation
is that TNF may not be involved in the pathogenesis of allergic
inflammation in this model. Although TNF was below detectable levels
18 h after 3 days of aerosol challenge (Table 2), TNF has been
measured at earlier time points after Ova challenge in the BAL fluid
and serum of animals (38, 51, 54). Also, both a TNF
neutralizing MAb and a TNFR1 fusion protein, TNFR1-IgG, have been
demonstrated (42, 54) to attenuate immune responses in a
single-challenge Ova model of allergic inflammation.
What is more likely is that the importance of TNF is different during
specific phases of allergic inflammation. TNF is elevated in
Ova-sensitized mice for only 1 h after Ova aerosol challenge (38). In the experiments in which neutralization of TNF
afforded some benefit to animals sensitized to Ova, the animals were
treated with a neutralizing TNF MAb or TNFR1-IgG before a single
aerosol challenge with Ova (42, 54). These groups did not
examine the benefit or lack of benefit of neutralizing TNF after
additional Ova challenges. Except for the present investigation, only
one study (19) has evaluated the effect of TNF
neutralization after multiple aerosol challenges with Ova, and TNF
neutralization did not have a significant effect on the murine immune
response or airway hyperresponsiveness. Our data from 3-day and 7-day
Ova challenge experiments, combined with those from published
single-day Ova challenge experiments (19, 42, 54), suggest
that neutralizing TNF only transiently abrogates the murine immune
response and airway hyperresponsiveness in models of allergic inflammation.
The immune system has a redundant system of effector molecules. In
allergic inflammation, it is possible that other proinflammatory cytokines are more important than TNF after repeated antigen challenges with Ova. We demonstrated increased levels of IL-2, IL-4, and IL-5 in
the lung homogenates from Ova-sensitized and -challenged TNFR(+/+) and
TNFR(
/
) mice compared with those from control mice (Table 2).
Additionally, we showed that TNFR(
/
) mice had higher lung IL-2,
IL-4, and IL-5 concentrations than their TNFR(+/+) counterparts. Mice deficient in TNFRs may have an
altered immunological feedback mechanism(s), resulting in an
accentuated Th2-type response in Ova-induced allergic inflammation. In
a model of endotoxemia, TNFR(
/
) mice have markedly increased levels
of circulating TNF because of the loss of normal negative feedback from
TNFR signaling (Rudmann, unpublished observations).
Numerous studies (2, 4, 5, 11) have suggested that IL-4
and IL-5 are important in the pathogenesis of allergic inflammation. In
a recent study (19) that examined the effects of antibody
neutralization of IFN-
, IL-4, or TNF in Ova-sensitized mice
challenged for 8 consecutive days with aerosolized Ova, only neutralizing IFN-
attenuated airway hyperreactivity and eosinophil influx into the BAL fluid. Taken together, these data suggest that
cytokines other than TNF may have an important role in the allergic
response to repeated antigen challenge. These types of experiments
demonstrate the time-dependent, redundant, and interactive nature of
the cytokine response in allergic disease.
TNF may also have roles other than proinflammatory ones in allergic
inflammation. We demonstrated that Ova-sensitized TNFR(
/
) mice that
were aerosol challenged with Ova had increased BAL fluid eosinophils,
lymphocytes, lung cytokines, IgG, and IgE compared with levels in
TNFR(+/+) mice. Investigations have demonstrated that chronic TNF
administration downregulates murine T cell responses, peritoneal
macrophage Ia expression, and accessory cell function (7, 8, 18,
48, 50). TNF decreases T cell receptor signaling, thereby
downregulating the function of both Th1 and Th2 subtypes of T
lymphocytes (7). Neutralizing TNF by treatment with
anti-TNF MAb reverses this effect on murine T cells and, in fact, can
result in exaggerated T cell function (7, 8). TNF-mediated
downregulation of T cell function may explain why recombinant murine
TNF treatment reduces the severity of inflammation in murine models of
systemic lupus erythematosus, type I diabetes, and delayed-type
hypersensitivity (18, 22-24). T cells are likely important in human asthma (2, 9, 49), and murine Ova
models of allergic inflammation have been shown to be CD4+
T cell dependent (14, 16). It is possible that the
downregulatory effects of TNF on T cells are an important aspect of the
murine response to Ova, especially after repeated Ova aerosol
challenges. Absence of the downregulatory effects of TNF on T cells may
have negated the attenuation afforded by losing the proinflammatory effects of TNF in TNFR(
/
) mice or TNFR(+/+) mice treated with anti-TNF. The elevated inflammatory and immune response in TNFR(
/
) mice in this model may also be a result of the loss of TNF-mediated T
cell regulation.
It has been demonstrated that airway hyperreactivity in mice and the
response of mice in allergy models is influenced by genetic background
(10, 20). We tested whether the TNFR(
/
) (C57BL/6 × 129/Sv) and TNFR(+/+) (C57BL/6 × 129/Sv) background strains 129/Sv and C57BL/6 responded differently in the Ova model of allergic inflammation. We found that when aerosol challenged, the Ova-sensitized 129/Sv mice had an approximately twofold greater number of BAL fluid
leukocytes and elevated histological lesion scores (data not shown)
than identically Ova-sensitized C57BL/6 mice and TNFR(+/+) mice. In
separate experiments, BAL fluid total leukocyte, eosinophil, lymphocyte, and macrophage numbers were ~2.6-, 2.8-, 1.7-, and 1.6-fold greater, respectively, in 129/Sv mice compared with those in
TNFR(+/+) mice. 129/Sv mice also had greater airway hyperreactivity to
MCh challenge than TNFR(+/+) mice (data not shown). Levels of lung
IL-4, lung IL-5, BAL fluid IgE, and serum IgE were not significantly different between the strains. The response
differences in the background strains demonstrated the importance of
using control mice [TNFR(+/+)] derived from
littermates of TNFR(
/
) mice as was done in all of the allergy
experiments described in this report.
To our knowledge, there have been no published reports describing the
immune response to Ova in TNFR(
/
) mice or TNFR(+/+) mice
chronically treated with anti-TNF antibodies. Neutralizing TNF with
MAbs or TNFR1 fusion proteins in mice or other animal species has been
shown to lessen the severity of allergic inflammation (42,
54); however, in those studies, TNF neutralization was done
after Ova sensitization and before a single aerosol challenge with Ova.
We propose that these models selectively evaluated the early effects of
TNF in allergic inflammation. Our data demonstrate that with repeated
antigen challenges, neutralization of TNF does not abrogate the mouse
inflammatory response to Ova. This is the case whether TNF is
chronically neutralized with anti-TNF MAb or whether TNF signaling is
blocked by genetic deletion of TNFR1 and TNFR2. These data may suggest
that persistent blockade of TNF bioactivity may not provide any
clinical benefit in allergic inflammatory diseases such as asthma in
humans. Furthermore, our data demonstrate that chronic loss of TNFR
signaling could result in an exaggerated T cell-dependent murine
response to Ova. In the development of asthma, repeated long-term,
low-dose antigen exposure in humans is likely the rule rather than the
exception. Whether this exposure is sufficient to stimulate chronic TNF
production and whether these low levels of TNF downregulate potential
problematic T cell responses in the pathogenesis of asthma requires
further investigation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Alan Rebar, Greg Stevenson, and Tim Terrell of the
collaborative Purdue University and Genentech Pathology Fellow program
for guidance and support. We also thank Dr. Paula Jardieu for input and
Lorena Cabote, Betty Chan, David Finkle, Gabrielle Hatami, Boyd
Hurtado, Laurie Leong, Robin Taylor, Patty Tobin, and Kim Zioncheck for
expert technical assistance.
 |
FOOTNOTES |
This work was supported by Genentech (D. Rudmann, J. Tepper, D. Tumas,
J. Pfeiffer, and M. Aldrich) and the Purdue University Department of
Veterinary Pathobiology (D. Rudmann and H. Hogenesch).
Address for reprint requests and other correspondence:
D. G. Rudmann, Pharmacia Corp., 7000 Portage Rd., 7270-300-226.1, Kalamazoo, MI 49001-7983 (E-mail:
daniel.g.rudmann{at}am.pnu.com).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 January 2000; accepted in final form 10 June 2000.
 |
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