Eur. J. Immunol. 1999.

29: 1021–1031 Mucosal superantigen exposure model for non-allergic asthma 1021

Airway exposure to bacterial superantigen (SEB)

induces lymphocyte-dependent airway
inflammation associated with increased airway
responsiveness – a model for non-allergic asthma
Udo Herz1, René Rückert1, Kathrin Wollenhaupt2, Thomas Tschernig2, Ulrich
Neuhaus-Steinmetz1, Reinhard Pabst2 and Harald Renz1
1
Department of Laboratory Medicine and Pathobiochemistry, Charité, Campus Virchow-Clinic,
Berlin, Germany
2
Department of Anatomy, Medical School of Hannover, Hannover, Germany

Although immunological consequences of systemic superantigen administration have been

extensively studied, the effects of local mucosal exposure to superantigens are not well
defined. The purpose of this study was to delineate the type of immune response triggered
by superantigen exposure to the airway mucosa in mice. In dose-response experiments we
determined a low dose of staphylococcal enterotoxin B (SEB) that triggered an inflammatory
response characterized by mucosal and airway recruitment of lymphocytes, eosinophils and
neutrophils together with elevated levels of IL-4, but not IFN- + , in bronchoalveolar lavage
(BAL) fluids. TCR V g analysis revealed that superantigen-responsive and -non-responsive
T cells were equally recruited into the airways. SEB markedly enhanced the frequency of
TNF- § -positive BAL macrophages as well as the amount of TNF- § in BAL fluids. These
responses were associated with the development of increased airway responsiveness (AR)
in SEB-treated mice. This effect occurred in an antibody-independent fashion. Furthermore,
this type of response was observed in IgE-high responder BALB/c as well as in IgE-low/
intermediate responder C57BL/6 mice. The development of increased AR was CD4+ T cell
dependent as shown by transfer experiments into BALB/c nu/nu mice. These results sug-
gest that the local immune response following mucosal superantigen administration triggers
a unique inflammatory response in the airways resembling many features of “intrinsic
asthma”. Received 19/6/98
Revised 27/11/98
Accepted 27/11/98
Key words: Bronchial asthma / Superantigen / Airway inflammation / Intrinsic asthma

1 Introduction patients BA is caused by environmental allergens. This

Bronchial asthma (BA) is a chronic inflammatory disease
with airway obstruction due to mucosal edema, mucus
secretion and airway smooth muscle constriction. Char-
acteristic of BA is the development of increased airway
responsiveness (AR), which is measured as an increased
airway smooth muscle contractility following provocation
with specific and unspecific stimuli. In a large group of
[I 18442]
Abbreviations: AR: Airway responsiveness BAL: Bron-
choalveolar lavage DL: Detection limit EFS: Electrical
field stimulation MNC: Mononuclear cells SAg: Superan-
tigen SEB: Staphylococcal enterotoxin B TMS: Tracheal
smooth muscle segment
type of BA is defined by a unique sort of airway inflam-
mation with influx of IL-4- and IL-5-producing T cells and
eosinophils [1, 2]. To further analyze the immunopatho-
genic basis of the disease and to devise novel treatment
strategies, animal models have been established that
demonstrated that this type of BA is dependent on (1)
IgE and B cells [3, 4], (2) CD4+ T cells [5], (3) IL-4 and IL-5
[6–9], (4) eosinophils and (5) that it can be transferred
into naive mice using defined V g -expressing T cell sub-
sets [10]. On the other hand, inflammation and increased
AR were abrogated by IFN- + , soluble IL-4R or allergen-
specific immunotherapy, and CD8+ T cells [11–14].
In addition to allergic BA, the existence of an IgE/
allergen-independent type of BA is also well known.
Non-allergic BA is triggered by viral and bacterial infec-

© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999 0014-2980/99/0303-1021$17.50 + .50/0

1022 U. Herz et al. Eur. J. Immunol. 1999. 29: 1021–1031

tions and other causes [15–17]. The pathogenesis of this
type of asthma is not nearly as extensively studied as the
allergic type of BA. Recent studies of bronchoalveolar
lavage (BAL) material and mucosal biopsiy samples indi-
cate some similarities between allergic and non-allergic
BA including the role of T cells and eosinophils,
enhanced production of IL-4 and IL-5 [18]. In addition,
there are also unique changes in non-allergic BA includ-
ing a large proportion of CD8+ T cells, marked macro-
phage activation with TNF- § and GM-CSF production
[19, 20]. Although animal models are extensively used to
study the IgE/allergen-dependent phenotype, models
resembling the non-allergic type of BA are rare [21].
thelial or mucosal SAg exposure [22, 23]. In contrast,
numerous studies have analyzed the events after sys-
temic SAg administration that are defined by T cell acti-
vation followed by peripheral T cell anergy/depletion
[24–27]. In this study we demonstrate that repetitive
local administration of SEB into the nose triggers a
unique type of airway inflammation with development of
increased AR resembling many features of non-allergic/
non-IgE-mediated bronchial asthma.
2 Results

Since it is well known that allergic individuals are chroni-
cally colonized on skin and nose with Staphylococcus
aureus, many of those secreting bacterial superantigens
(SAg), we developed a mouse model of antibody-
independent mediated BA by intranasal administration of
the prototypic SAg staphylococcal enterotoxin B (SEB).
Except for some recently published data, there is only
limited information available on the effects of local epi-
2.1 Intranasal SEB application induces airway
inflammation
In dose-response experiments the lowest SEB concen-
tration that induced an inflammatory response in lung
and airways was determined. Groups of C57BL/6 mice
received concentrations of SEB ranging from 0.05 ng to
500 ng/application; control animals were treated with

Figure 1. Influx of immune cells into the airway lumen triggered by SEB in a dose-dependent manner. Absolute numbers of leu-
kocytes in BAL fluid/mouse were determined in groups of mice that received SEB solution intranasally, ranging from 50 pg/appli-
cation up to 500 ng/application. BAL was performed 24 h after the last application. Presented are means ± SD cells/BAL of five
animals per study group. (A) Lymphocytes; (B) eosinophils; (C) neutrophils; (D) macrophages. Statistical significance (p p 0.05)
is indicated by (*).
Eur. J. Immunol. 1999. 29: 1021–1031 Mucosal superantigen exposure model for non-allergic asthma 1023

Table 1. Leukocyte infiltration and cytokine production after intranasal SEB application in C57BL/6 and BALB/c micea)

C57BL/6 BALB/c

PBS SEB PBS SEB

cells / BAL Lymphocytes 2.0 ± 1.3 49 ± 8* 0.5 ± 0.2 23 ± 10*

[× 103 ]b)
Eosinophils 0.5 ± 0.3 27 ± 9* 0.4 ± 0.1 84 ± 23*
Neutrophils 2.2 ± 1.6 52 ± 16* 12 ± 8 59 ± 43*
BAL cytokines IL-4 143 ± 15 204 ± 20* 80 ± 48 388 ± 94**
[pg/ml]c)
IFN- + 208 ± 58 183 ± 27 204 ± 20 223 ± 50

a) C57BL/6 and BALB/c mice were treated intranasally with 50 ng SEB/application on days 1, 3 and 6. Controls received PBS
only. Analysis was performed on day 7.
b) Absolute numbers of leukocytes in BAL fluid/mouse were determined according to standard morphology.
c) Cytokine production in BAL fluids was measured by ELISA as described in Sect. 4.7. Presented are mean ± SD for four to six
mice per study group. Statistical analysis: comparison between PBS and SEB group; * p p 0.01.

PBS alone. Total and differential cell counts were
assessed in BAL fluids from individual mice. The num-
bers of total cells/BAL recovered from mice treated with
at least 50 ng SEB/application were significantly higher
(280 496 ± 47 839 total cells/BAL, p p 0.005) than from
control animals (130 200 ± 14 797 total cells/BAL). Differ-
entiation of cell subpopulations revealed that the major-
ity of the cells in BAL fluids consisted of macrophages
but there were no significant differences between ani-
mals treated with PBS and SEB for this cell type. In con-
trast, the influx of lymphocytes, neutrophils and eosino-
phils differed in a dose-dependent fashion. Whereas
lymphocytes leveled off at 50 ng SEB, neutrophils
started to rise at 50 ng SEB and continued to increase
with higher doses. Eosinophils peaked at 50 ng of SEB
(Fig. 1). Similar results were obtained in BALB/c and
C57BL/6 animals (Table 1). Histological examination of
lung tissue after intranasal application of 50 ng SEB/
application showed a mild to moderate perivascular leu-
kocyte infiltration, whereas after application of 500 ng
SEB/application a marked inflammatory immune
response was detected (Fig. 2).
2.2 Intranasal SEB application triggers IL-4 and
TNF- > production
Based on these dose-response experiments, a concen-
tration of 50 ng SEB/application was selected for further
experiments, since it induced high numbers of eosino-
phils and was the lowest dose for induction of airway
inflammation. Increased influx of lymphocytes, eosino-
phils and neutrophils into the airway lumen was associ-
ated with a relative increase in IL-4 and TNF- § produc-
tion in BAL fluids, whereas IFN- + production remained
unchanged in SEB-treated mice (Table 1, Fig. 3).
2.3 TNF- > is produced by alveolar macrophages
One possible source for TNF- § are alveolar macro-
phages. Twenty-four hours after the last intranasal PBS
or SEB application, BAL cells were immunolabeled with
anti-TNF- § . The frequency of TNF- § -positive macro-
phages in SEB-treated animals was twice that of mice
that received PBS intranasally (Fig. 3A). In addition, TNF-
§ production in BAL fluids from mice that received SEB
intranasally increased about threefold (Fig. 3B). No TNF-
§ -positive lymphocytes or eosinophils were detected in
PBS- or SEB-treated mice (Fig. 4).
2.4 Assessment of the V I repertoire of T cells in
BAL fluids
To determine whether the increase in lymphocytes was
due to the activation of SEB-responsive T cells, the V g
repertoire of the T cells infiltrating the airway lumen was
analyzed. Although SEB exposure triggered T cell influx
into the airways, SEB-responsive (V g 8.1 and V g 8.2) and
SEB-non-responsive (V g 6) T cells were distributed as in
peripheral blood, indicating nonselective T cell accumu-
lation following SEB application (Fig. 5).
1024 U. Herz et al. Eur. J. Immunol. 1999. 29: 1021–1031

Figure 2. Influx of immune cells into the lung triggered by SEB in a dose-dependent manner. Histology of the lung from mice that
received SEB solution intranasally, ranging from 0.05 ng/application up to 500 ng/application. BAL was performed 24 h after the
last application. Representative examples are shown from one out of three experiments. (A) Control; (B) PBS nasally; (C) 0.05 ng/
application; (D) 0.5 ng/application; (E) 5 ng/application; (F) 50 ng/application; (G) 500 ng/application. Magnification 20×.
Eur. J. Immunol. 1999. 29: 1021–1031 Mucosal superantigen exposure model for non-allergic asthma 1025

2.5 Intranasal SEB application triggers increased

AR

To determine whether this inflammatory immune

response in the lung was associated with the develop-
ment of increased AR, the response of tracheal smooth
muscle segments (TMS) to electrical field stimulation
(EFS) was measured. SEB-treated BALB/c and C57BL/6
mice developed increased AR as indicated by a
decrease in ES50 values (see Sect. 4.11) compared to
mice that received PBS alone (Fig. 6).

Figure 3. Evaluation of TNF- § -producing cells and TNF- §

production in BAL fluids from SEB-treated mice. C57BL/6
mice received PBS or SEB (50 ? l/application) via the anterior
nose as described in Sect. 4.2. (A) Cells were incubated with
an antibody directed against TNF- § and immunolabeled
using the APAAP technique as described in Sect. 4.8.
Shown are mean percentage ± SD of immunolabeled mac-
rophages in PBS (open bar)- and SEB (black bar)-treated
mice. (B) TNF- § production was assessed in cell-free super-
natants by ELISA technique. Shown are mean percentage ±
SD in PBS (open bar)- and SEB (black bar)-treated mice.
2.6 Airway inflammation and increased AR
evolve independently of anti-SEB antibodies
To test whether SEB induced a specific antibody
response, anti-SEB antibody titers were measured in
serum samples from PBS- and SEB-exposed mice. After
a short-term exposure to SEB via the anterior nose, no
anti-SEB antibodies were detectable in both PBS- and
SEB-exposed mice (below detection limit, X DL). To fur-
ther explore whether SEB can induce mast cell degranu-
lation, PBS- or SEB-exposed mice were skin tested with
various concentrations of SEB to determine the capacity
of SEB to trigger immediate-type hypersensitivity
responses. As shown in Table 2, none of the PBS- or
SEB-exposed animals developed positive immediate-

Figure 4. Immunocytological staining of TNF- § -producing cells in BAL fluids from SEB-treated mice. Immunocytological stain-
ing for TNF- § on a cytospin from the BAL of the SEB (a, b; bar j 50 ? m) and the PBS group (c, d). Positive cells are stained red,
indicated by arrows, in the bright-field illumination (a, c). The same area is shown in phase-contrast microscopy (b, d).
1026 U. Herz et al.
Figure 5. Assessment of T cell subpopulations in BAL fluid.
C57BL/6 mice received PBS or SEB (50 ? l/application) via
the anterior nose on days 1, 3 and 6. On day 7 BAL fluid was
recovered. Cells were immunolabeled with antibodies
directed against CD3, TCR V g 8 and TCR V g 6 and analyzed
by FCM.
type skin test responses to SEB. These results indicate
that airway inflammation and increased AR occurred in
the absence of anti-SEB antibodies in this model.
2.7 SAg-induced increased AR is CD4+ T cell
dependent
To analyze the relationship between increased AR and
T cell recruitment in this system, T cell-deficient BALB/c
nu/nu mice were treated intranasally with PBS or SEB
and AR was measured by in vitro electrical field stimula-
tion (Fig. 7A) and, in addition, by in vivo body plethys-
mography (Fig. 7B). No significant difference (p G 0.05)
between PBS-treated BALB/c nu/nu (3.5 ± 0.2 Hz) and
SEB-treated BALB/c nu/nu mice (3.2 ± 0.5 Hz) was
Eur. J. Immunol. 1999. 29: 1021–1031
Figure 6. Assessment of AR in SEB-treated mice. C57BL/6
or BALB/c mice received PBS or SEB (50 ? l/application) via
the anterior nose on days 1, 3 and 6. AR was analyzed 1 day
later by EFS of tracheal smooth muscle preparations. Indi-
cated are mean ± SD of electrical frequencies that resulted
in 50 % of maximum smooth muscle contractility.
observed (Fig. 7A). When BALB/c nu/nu mice were
reconstituted with CD4-positive T cells derived from
naive wild-type (wt) BALB/c mice, these reconstituted
BALB/c nu/nu mice developed increased AR to the same
degree as BALB/c wt mice after intranasal SEB applica-
tion (Fig. 7A, B). In contrast, transfer of CD4+ T cells
Table 2. Immediate cutaneous hypersensitivity (ICHS) reac-
tions in SEB-treated C57BL/6 micea)
C57BL/6 mice treated intranasally
with:
ICHS following i.d. PBS SEB
injection of SEB
PBS 0/8 0/8
Compound 48/80 8/8 8/8
0.5 ? g/ml SEB 0/8 0/8
5 ? g/ml SEB 0/8 0/8
50 ? g/ml SEB 0/8 0/8
a) C57BL/6 mice received PBS or SEB intranasally as
described in Sect. 4.2. Intracutaneous skin testing was
performed with compound 48/80 as a positive control for
mast cell degranulation, PBS as a negative control and
SEB as described in Sect. 4.3. All solutions were injected
i.d. in a volume of 50 ? l. After 15 min vascular leakage
was assessed on the backside of the skin in a blinded
fashion. Indicated are numbers of mice that developed
wheals n 3 mm.
Eur. J. Immunol. 1999. 29: 1021–1031 Mucosal superantigen exposure model for non-allergic asthma
Figure 7. Assessment of AR in BALB/c nu/nu and reconsti-
tuted BALB/c nu/nu mice after SEB treatment. BALB/c nu/
nu or BALB/c nu/nu mice reconstituted with CD4-positive
T cells received PBS or SEB (50 ? l/application) via the ante-
rior nose on days 1, 3 and 6. AR was analyzed 1 day later by
(A) EFS of tracheal smooth muscle preparations and (B)
body plethysmography. Indicated are mean ± SD of electri-
cal frequencies that resulted in 50 % of maximal smooth
muscle contractility (A) and concentration of methacholine
that caused a 50 % reduction in expiratory airflow (MCh50 )
(B). Statistical significance (p p 0.01) is indicated by (**).
followed by nasal PBS application had no affect on AR.
These results indicate that the capacity to develop
increased AR following SEB application is fully restored
in BALB/c nu/nu mice by the transfer of CD4-positive
T cells.
1027
3 Discussion
In this study we have analyzed the immunological conse-
quences of mucosal SAg exposure. Intranasally applied
SEB induces a pattern of airway inflammation character-
ized by increased numbers of lymphocytes and eosino-
phils at the cellular level and by elevated local production
of the Th2 cytokine IL-4 together with marked up-
regulation of TNF- § production by macrophages. This
inflammatory response is associated with increased AR
and occurs in BALB/c as well as in C57BL/6 mice. There-
fore, airway mucosa exposure to SEB stimulates a type
of immune response that mirrors many events observed
in non-allergic, “intrinsic” bronchial asthma.
SAg stimulate immune cells by binding to the MHC class
II molecule and the TCR. Conventional processing and
presentation by APC is not required to activate these
cells, since SAg can bind directly to the TCR-V g element
outside the conventional binding groove, and, similarly,
to the MHC class II g chain [28]. Therefore, one of the
major cell types in the airways that first encounters SAg
following local administration are MHC class II-
expressing macrophages and dendritic cells. These cells
can then be triggered to release potent pro-inflammatory
mediators including TNF- § and IL-1 with pluripotent
inflammatory effects. Indeed, following intranasal appli-
cation of SEB, alveolar macrophages were identified as a
major cell subpopulation producing large amounts of
TNF- § . TNF- § has been described as an important effec-
tor molecule in the pathogenesis of allergic respiratory
reactions, since it can promote mediator and cytokine
release, expression of adhesion molecules, and lympho-
cyte and eosinophil recruitment. In addition, TNF- §
induces increased AR in rats following aerosolization of
recombinant human TNF- § [29]. It is important to note
that BALB/c nu/nu mice exposed to SEB did not develop
increased AR, despite the presence of normal macro-
phages and dendritic cells. The major difference
between BALB/c nu/nu and wt animals is the absence of
functional, mature T cells in the nude strain. The absence
of increased AR in SEB-exposed nude mice clearly indi-
cates T cell dependency of the development of
increased AR. In addition, it is likely that TNF- § contrib-
utes to the development of increased AR in an indirect
fashion, for example through the recruitment of inflam-
matory cells in the airway mucosa and lung. Indeed, anti-
TNF- § markedly reduces both neutrophil and eosinophil
accumulation in the lung of allergen-sensitized and chal-
lenged animals [30, 31].
A parallel mode of action for SEB could be through the
direct activation of T cells. Numerous studies have ana-
lyzed the effects of SAg on T cell activation [24–27]. Pro-
posed mechanisms for SAg activation are (1) direct acti-
1028 U. Herz et al.
vation via the binding to the V g chain of the TCR, (2)
bridging between MHC class II molecules on one T cell
and the TCR on another cell, (3) bridging between MHC
class II on APC and the TCR. Either way, T cell activation
would be the result of such an interaction. SAg actions
on T cells are dose and time dependent. i.p. injection of
100 ? g SEB into mice resulted in depletion of the V g 8-
positive T cells [24]. At lower concentrations SEB
induced T cell anergy. In our experimental setup we
chose another route of administration (local versus sys-
temic) and we used much lower concentrations as com-
pared to others [26, 27]. Dose-response experiments
defined a concentration of 50 ng/application as the low-
est dose causing mucosal infiltration and airway influx of
eosinophils, lymphocytes, and to a lesser extent neutro-
phils.
Following systemic SAg administration, a biphasic T cell
response has been well established [25, 27, 32–34].
Dependent on the dose of SAg, the initial events are
characterized by activation and expansion of the V g -
restricted SAg response T cell pool. This effect is fol-
lowed by clonal deletion of the T cells. This type of T cell
response differs from the one observed after mucosal
SAg exposure in our model. The T cell response was
polyclonal and not restricted in terms of V g usage. SAg
responsive as well as non-responsive T cells were simul-
taneously recruited into the airways. This type of
response makes it unlikely that T cell recruitment
resulted from direct interaction with the SAg. It is more
likely that T cell recruitment and activation resulted from
stimulation of airway-resident APC that secreted media-
tors able to up-regulate endothelial adhesion molecules
necessary for T cell recruitment.
These responses occurred in the absence of anti-SEB
antibodies. In humans several entities of BA have been
described. In addition to the allergen-, IgE- and mast
cell-dependent allergic type of asthma, asthma can also
be triggered in an allergen/IgE-independent fashion.
Most prominent triggers are viral infections, cold air,
physical exercise and others. In any case the clinical
phenotype of asthma is accompanied by airway inflam-
mation and development of increased AR. Recent stud-
ies have compared the nature of airway inflammation in
allergic and non-allergic asthmatic patients [35–37]. Sev-
eral important similarities have been identified between
these two types of asthma. The inflammatory compo-
nents are dominated by T cell and eosinophil infiltration
of airway mucosa and influx into the airway lumen. The
phenotype of effector T cells is predominantly of the Th2
type with IL-4 and IL-5 production. Although most of the
T cells express CD4 on the cell surface, in non-allergic
asthma a considerable proportion of T cells is CD8 posi-
tive. A striking difference between allergic and non-
Eur. J. Immunol. 1999. 29: 1021–1031
allergic asthma is the activation of bronchoalveolar mac-
rophages, which has been shown at different cellular lev-
els [19, 20]. Comparison of the immunological character-
istics of non-allergic BA with our model reveals several
important similarities, including predominant Th2 cell
activation and recruitment with local eosinophilia, and
most importantly marked macrophage activation with
TNF- § release.
In conclusion, the results presented illustrate that local
application of SAg to the airways favors development of
a unique inflammatory immune response. This response
is characterized by influx of T cells and eosinophils,
together with increased levels of IL-4, resembling a typi-
cal Th2 immune response. In addition, marked macro-
phage activation with TNF- § production was detected.
SEB induced increased AR in a T cell-dependent fash-
ion. Development of this immunological and respiratory
phenotype occurred in the absence of anti-SEB anti-
bodies. Therefore, local mucosal SEB administration
may serve as a model for non-allergic “instrinsic” asth-
matic responses.
4 Materials and methods
4.1 Animals
C57BL/6, BALB/c and BALB/c nu/nu mice (all obtained from
Bomholtgard, Ry, Denmark) aged between 6–8 weeks were
maintained under pathogen-free conditions.
4.2 Intranasal SEB administration
Mice were slightly anesthetized with Ketanest and Xylazin
(Sigma, Deisenhofen, Germany) and 50 ? l of an SEB/PBS
solution (0.05 to 500 ng SEB/application) (Serva, Heidel-
berg, Germany) was instilled into the anterior nose on days
1, 3 and 6. On day 7 mice were killed. Controls received PBS
alone.
4.3 Assessment of immediate cutaneous
hypersensitivity reactions
Intracutaneous skin testing was performed using a modifi-
cation of a previously described technique [38]. Before skin
testing 100 ? l 0.5 % (w/v) Evans blue (Sigma) solution was
injected i.v. The following skin test solutions were injected
i.d.: PBS as a negative control; compound 48/80 (5 ? g/ml)
(Sigma) as a positive control, SEB at different concentrations
(0.5, 5. 50 ? g/ml) (Serva). After 15 min, development of blue
flare reactions was assessed on the inverted skin by an inde-
pendent investigator and scored as positive if the reaction
was G 0.3 cm.
Eur. J. Immunol. 1999. 29: 1021–1031 Mucosal superantigen exposure model for non-allergic asthma
4.4 Determination of anti-SEB antibody titers
Anti-SEB antibody titers were measured by ELISA. Briefly,
microtiter plates were coated with serum (diluted 1:10 in
0.1 M carbonate buffer, pH 8.2, overnight at 4 °C), followed
by incubation with 10 ng/ml SEB (Serva) for 2 h at 37 °C.
After washing the plates, 10 ? g/ml anti-SEB horseradish
peroxidase conjugate (Serva) was added. The reaction was
developed with tetramethylbenzidine (TMB) and stopped
with 2 N sulfuric acid. Plates were measured at 450/490 nm.
The antibody titers of the samples were related to the blank.
A blank value of X 0.05 was arbitrarily set as DL.
4.5 Histological evaluation of lung tissue
For analysis of lung inflammation, lungs were fixed with 4 %
formaldehyde (w/v) via the trachea. The lung was removed
and stored in 4 % formaldehyde. Paraffin-embedded sec-
tions (3 ? m) were stained with hematoxylin and eosin.
4.6 BAL and BAL cell differentiation
BAL and BAL cell differentiation were performed as previ-
ously described [38]. In each study group a similar volume of
BAL fluid could be recovered (1.4 ± 0.2 ml). Fluids were cen-
trifuged and cell-free supernatants were frozen at −20 °C
until measurement of cytokine content.
4.7 Determination of cytokines in BAL fluid
IL-4, IFN- + and TNF- § were measured by ELISA as previ-
ously described [38]. Sensitivities were 10 pg/ml for IL-4,
100 pg/ml for IFN- + and 100 pg/ml for TNF- § .
4.8 Assessment of TNF- > -positive cells
BAL were performed as described above, cytospins were
performed on glass slides (600 rpm, 10 min) and fixed in
acetone (10 min, −20 °C). The alkaline phosphatase-anti-
alkaline phosphatase (APAAP) technique was used to detect
intracellular TNF- § . Sections were incubated with antibodies
directed against TNF- § (Pharmingen, Hamburg, Germany)
for 30 min at room temperature. After washing with 0.05 %
Tween-20 diluted in Tris-buffered saline, incubation with a
bridging antibody (rabbit anti-rat, Dako, Hamburg, Germany)
for 30 min and then the APAAP-complex (30 min) was
applied. To increase the staining intensity, the incubation
with the bridging antibody and the addition of the APAAP-
complex were repeated once. Fast red (Sigma, Munich, Ger-
many) served as substrate for alkaline phosphatase. All sec-
tions were counterstained with hemalum and mounted in
glycerol (Dako).
1029
4.9 Flow cytometric analysis of cell surface marker
expression
The distribution of T cell subpopulations in BAL fluids was
analyzed by flow cytometry. The following FITC-labeled
mAb were used: rat anti mouse -CD3, -V g 8.1/8.2, -V g 6
(Pharmingen).
4.10 Lymphocyte transfer into BALB/c nu/nu mice
Splenic mononuclear cells (MNC) from naive BALB/c were
prepared by density centrifugation. CD4-positive cells were
purified by removing CD11- and CD19-positive cells by
magnetic-activated cell sorter (MACS) technique. Remain-
ing cells contained n 92 % CD4 positive cells as determined
by FCM analysis. Cells were washed with PBS and adjusted
to 4 × 107 cells/ml. BALB/c nu/nu were reconstituted by i.v.
injection of 1 × 107 cells. SEB was applied intranasally on
days 1, 3 and 6 after transfer. On day 7 mice were analyzed.
4.11 Assessment of AR by EFS
Airway smooth muscle responsiveness was assessed by
EFS as previously described [38]. The frequency that caused
50 % of the maximum contraction was calculated from loga-
rithmic plots of the contractile response versus the fre-
quency of EFS, and was expressed as ES50.
4.12 Assessment of AR by body plethysmography
AR was assessed by head-out body plethysmography as
described [39]. Briefly, four mice were placed in four body
plethysmographs attached to a exposure chamber (Crown
Glass, Somerville, NJ). Airflow was measured with a PTM
378/1.2 pneumotachograph (Hugo Sachs Electronics,
March-Hugstetten, Germany) and a 8-T2 differential pres-
sure transducer (Gaeltec, Dunvegan, GB). Airflow in
response to various concentrations of methacholine (10, 15,
20, 25, 30, 40 mg/ml, 1 min) delivered by at jet nebulizer
(Pari-Boy®; Pari-Werke, Starnberg, Germany) was mea-
sured. The concentration of methacholine that caused a
50 % reduction in expiratory airflow (MCh50 ) was deter-
mined.
4.13 Statistical analysis
Results are presented as mean values ± SD, unless stated
otherwise. Mann-Whitney U test was used to determine the
level of difference between animal groups.
1030 U. Herz et al.
Acknowledgments: We thank Christine Seib, Katja Preiss
and Margarita Strozynski for excellent technical assistance.
This work was supported by the Deutsche Forschungsge-
meinschaft.
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