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*
Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; and
Pulmonary Center, Boston University School of Medicine, Boston, MA 02118
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specified pathogen-free male BALB/c mice (6–8 wk) were obtained from the breeding colony of the National Institute for Public Health and the Environment, Bilthoven, The Netherlands. The mice were housed in Macrolon cages and provided with food and water ad libitum. Active sensitization was performed by seven i.p. injections of 10 µg of OVA (grade II) in 0.5 ml of pyrogen-free saline on alternate days (one injection per day). This sensitization procedure induces high titers of total IgE Abs in serum of which 80% was OVA-specific IgE (14). Four weeks after the last injection, mice were exposed either to eight OVA (2 mg/ml) or eight saline aerosols, on consecutive days (one challenge per day). The aerosol was generated with an ultrasonic nebulizer (Medix 8001; particle size, 3–5 µm) connected to a Plexiglas exposure chamber (5 liters). Animals were exposed for 5 min in maximal groups of six. All experimental procedures were approved by the Dutch Committee of Animal Experiments.
Treatment with Abs to IL-16
To verify the effectiveness of neutralizing anti-human IL-16
Abs for mouse IL-16, human lymphocytes were incubated with supernatant
of Con A-stimulated (3 µg/ml, 37°C, 24 h) mouse splenocytes in
the presence of various anti-human IL-16 Abs or control Abs. In a
migration assay (described below), it appeared that the migratory
response of lymphocytes was significantly inhibited by all
anti-IL-16 Abs tested (Table I
),
indicating that anti-human IL-16 Abs are capable of neutralizing
mouse IL-16.
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Cell preparation and migration assay
Human lymphocytes were isolated from heparinized venous blood samples of healthy normal volunteers by density centrifugation on Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ). The resulting cell layer containing PBMC was recovered and washed three times in Medium 199 supplemented with 25 mM HEPES buffer, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were incubated on a nylon wool column at 37°C and 5% CO2 for 45 min. The cell population eluted from the column contained >97% T lymphocytes as determined by fluorescent staining with anti-CD3 mAb (Becton Dickinson, Mountain View, CA). Migration of lymphocytes was assessed using a modified Boyden chemotaxis chamber as described by Cruikshank et al. (5). The lymphocytes, 1 x 107 cells in 50 µl of Medium 199 enriched with 0.4% BSA, were loaded into the upper well of the chamber and 30 µl of cell-free BALF derived from OVA or saline-challenged mice were placed in the lower well. For blocking experiments, rabbit anti-human IL-16 polyclonal Ab was added to the lower well as well. In previous experiments, it was shown that 5 µg/ml anti-IL-16 Ab neutralizes 0.1 nM recombinant human IL-16 protein. The upper and lower well were separated by a nitrocellulose filter with a pore size of 8 µm. The chamber was incubated for 3 h, and afterward the filter was fixed and stained with hematoxylin. Migration was quantified by counting the number of cells that migrated beyond a depth of 50 µm utilizing an Optomax automated image analyzer (Burlington, MA). All migration data are expressed as the number of cells per high power field (hpf). All samples were performed in triplicate. On average 14 to16 cells/hpf were counted under control conditions.
Immunohistochemistry
Nonsensitized mice and OVA-sensitized animals challenged with OVA or saline were anesthetized with a mixture of Ketalar (35 mg/ml), xylazine (0.6%/ml), and atropine (0.1 mg/ml), of which 0.2 ml was injected i.m. Thereafter, abdomen and chest were opened, and the abdominal aorta was incised. The vascular bed of the lungs was perfused with PBS (37°C), through injection via the right heart ventricle. The lungs were removed and filled intratracheally with 1 to 2 ml of fixation solution (0.8% formalin, 4% acetic acid). Subsequently, the trachea was tied off with a ligature, and the lungs were immersed in the fixative for at least 24 h. Then the tissues were dehydrated and embedded in Paraplast (Monoject, Kildare, Ireland). Transverse sections of 5 µm were prepared, subsequently deparaffinized, and hydrated by submerging in xylenes followed by reagent grade alcohol. The sections were rinsed for 5 min and incubated with 0.3% H2O2 for 30 min to quench endogenous peroxidase activity. After a washing in PBS for 5 min, the sections were incubated with goat serum (1:100 diluted in PBS) for 20 min, and afterward the excess of serum was blotted from the sections. Then the sections were incubated with mouse anti-human IL-16 mAb (clone 14.1, IgG2a, 20 µg/ml) or an isotype control Ab for 30 min. The sections were washed in PBS for 5 min, and a goat anti-mouse Ig secondary Ab (1:100 diluted in PBS) was applied for 30 min. After a washing, sections were incubated with Vectastain ABC reagent (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) for 30 min. The sections were washed again and stained with peroxidase substrate solution until the desired intensity was reached. After a rinsing in running water, the sections were counterstained with hematoxylin. The used reagents and protocol were derived from the commercially available Vectastain Elite ABC kit. At least five mice per group were examined.
OVA-specific IgE ELISA
Using an ELISA as described by De Bie et al. (16), the presence
of OVA-specific IgE was measured in serum samples derived from OVA or
saline-challenged mice at 24 h after the last challenge. In short,
96-well microtiter plates were coated with recombinant human
Fc
R1-IgG fusion protein (2 µg/ml, 4°C, 24 h). After a
washing, plates were blocked with ELISA buffer, which contained 2 mM
EDTA, 137 mM NaCl, 50 mM Tris, 0.5% BSA, and 0.05% Tween-20 (pH 7.2)
(room temperature, 1 h). After removal of the ELISA buffer, plates
were incubated with serum samples and duplicate dilution series of an
OVA-specific IgE reference standard (room temperature, 2 h). The
standard was obtained by i.p. immunization of mice with OVA according
to previously published methods (18) and arbitrarily assigned a value
of 10,000 U/ml OVA-specific IgE. After washing, OVA (10 µg/ml) was
added to each well and the mixture was incubated at room temperature
for 1 h. This step was followed by washing and incubation with
horseradish peroxidase-conjugated goat anti-OVA mAb (room
temperature, 1 h). Then, o-phenylenediamine (10 mM) was
added, and after 15 min at room temperature the reaction was stopped by
addition of 4 M H2SO4. The optical density was
read at 492 nm using a Titertek Multiskan (Flow Laboratories, Irvine,
U.K.). Serum samples were compared with the OVA-specific IgE reference
standard, and values were expressed in units per milliliter.
FcR1-IgG, OVA-specific IgE reference standard, and horseradish
peroxidase-conjugated goat anti-OVA mAb were generously provided by
Dr. P. M. Jardieu (Genentech Inc., South San Francisco,
CA).
Airway responsiveness in vivo
Airway responsiveness to methacholine was measured in vivo
24 h after the last aerosol exposure using the air-overflow
pressure method as previously described (19). With this method,
bronchial resistance to inflation is measured. Mice were anesthetized
by i.p. injection of urethan (2 g/kg) and placed on a heated blanket
(30°C). Then the trachea was cannulated, and a small polyethylene
catheter was placed in the jugular vein for i.v. administrations.
Spontaneous breathing was suppressed by i.v. injection of tubocurarine
chloride (3.3 mg/kg). When it stopped, the tracheal cannula was
attached to a respiration pump (C. F. Palmer, London, U.K.). The
inflation volume of the pump was 0.8 ml of which the mouse inhales
0.1 ml per breath with a rate of 190 breaths per min. The
ventilation circuit contained a pressure transducer (MPB-6207, Depex,
Bilthoven, The Netherlands). Any increase in airway tone causes a
reduction of the amount of air floating into the lungs, and
subsequently the remainder overflows, resulting in an increase in
air-overflow pressure. Pressure signals were recorded breath by breath
on a Graphtex thermal arraycorder (Ankersmit, Breda, The Netherlands).
At intervals of at least 4 min and after the response had returned to
baseline level, doubling doses of methacholine ranging from 40 to 1280
µg/kg were administered. Concentrations of methacholine were prepared
in saline and kept on ice for the duration of the experiment. At the
end of the dose-response curve, the maximal response was determined by
clamping the tracheal cannula. The increase in air-overflow pressure
was measured at its peak and expressed as percentage increase of the
maximal response. At least six mice were evaluated per group.
Bronchoalveolar lavage
Three and 24 hours after the last aerosol, mice were anesthetized by i.p. injection of 0.25 ml of sodium pentobarbitone (60 mg/ml). The abdomen and chest were opened, and the abdominal aorta was incised. Below the larynx, a small incision was made, and a flexible polyethylene cannula (PE 50, Intramedic, Clay Adams, NJ) was inserted into the trachea and fixed with a ligature. Subsequently, the mice were lavaged five times with 1-ml aliquots of pyrogen-free saline warmed to 37°C. The first milliliter of saline was supplemented with aprotinine (2 µg/ml) and was centrifuged after withdrawal (400 x g, 4°C, 5 min). The supernatant was immediately frozen at -70°C. Cells derived from this first milliliter of BALF were pooled with the other BAL cells collected. Then all cells were washed with cold PBS (400 x g, 4°C, 5 min), and the pellet was resuspended in 200 µl of cold PBS. Total numbers of BAL cells were counted in a Bürker-Türk chamber. For differential BAL cell counts, cytospin preparations were made and stained with Diff-Quick (Merz and Dade AG, Düdingen, Switzerland). After coding, all cytospin preparations were evaluated by one observer using oil immersion microscopy. Cells were identified and differentiated into mononuclear cells, neutrophils, and eosinophils by standard morphology. Per cytospin preparation, at least 400 cells were counted and the absolute number of each cell type was calculated. To evaluate differences between OVA and saline-challenged groups and to evaluate the effects of treatment with Abs to IL-16, total BAL cell numbers and the numbers of the various BAL cell types were tested with an analysis of variance. For cell types with a very low number in control animals (i.e., eosinophils or neutrophils) a Poisson distribution was assumed.
Eosinophil counts in tracheal tissue
Twenty-four hours after the last aerosol exposure, mice were anesthetized by i.p. injection of 0.25 ml of sodium pentobarbitone (60 mg/ml). The abdomen and chest were opened, and the abdominal aorta was incised. The vasculature of the lungs was perfused with a mixture of paraformaldehyde (2%) and glutaraldehyde (2.5%) in cacodylate buffer (0.1 M, pH 7.4) injected via the right heart ventricle. The trachea was isolated and immersed in the same paraformaldehyde-glutaraldehyde mixture as used for perfusion. After 2 wk, the trachea was cut into pieces and embedded in Paraplast. Before embedding in Paraplast, the tissues were thoroughly rinsed in tap water and routinely processed. Transverse sections of 5 µm were prepared and stained according to Luna’s method for eosinophil staining (20). Using light microscopy, the sections were examined for the number of eosinophils. Per mouse two to three tracheal sections were evaluated, and a mean number per section was calculated. Differences in eosinophil numbers were tested with a nonparametric Mann-Whitney U test. Four to six mice were examined per group.
Chemicals
OVA, aprotinine, ExtrAvidin peroxidase, 2,2'-azinobis(3-ethylbenzthiazoline 6-sulfuric acid) and o-phenylenediamine were purchased from Sigma Chemical Company (St. Louis, MO), urethan and methacholine from Janssen Chimica (Beerse, Belgium), Ketalar from Aesculaap (Boxtel, The Netherlands), xylazine from Bayer (Leverkusen, Germany), atropine from Brocacef (Utrecht, The Netherlands), tubocurarine chloride from Nogepha (Alkmaar, The Netherlands), sodium pentobarbitone (Nembutal) from Abbott Laboratories (North Chicago, IL), and Medium 199 from M.A. Bioproducts (Wakersville, MD).
Data analysis
Unless stated otherwise, data are expressed as mean ± SEM and evaluated using an analysis of variance followed by post hoc comparison between groups. A difference was considered to be significant when p < 0.05. Statistical analyses were conducted using SYSTAT, version 5.03 (SYSTAT Inc., Evanston, IL) or GLIM, version 4.0 (NaG Inc., Oxford, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When BALF samples were tested in a migration assay, it was
observed that BALF derived from OVA-sensitized and OVA-challenged mice
induced a significantly enhanced migration of lymphocytes when compared
with the migratory response induced by BALF from OVA-sensitized and
saline-challenged mice (Fig. 1). This was
observed both with BALF collected at 3 and 24 h after the last
aerosol exposure. BALF derived from saline-challenged mice inhibited
migration in comparison with the migratory response observed in the
presence of medium, which was 15.3 cells per high power field. When
BALF samples of OVA-challenged mice were incubated with rabbit
anti-human IL-16 polyclonal Ab, a significant inhibition of
migration was observed (Fig. 2),
demonstrating the presence of IL-16 in these samples. Inhibition of
migration was less at 24 h than at 3 h. This observation
indicates that the IL-16-induced lymphocyte motility is transiently
expressed at early time points. Incubation of BALF samples of
saline-challenged mice with Abs to IL-16 had no effect on the migratory
response. Neither did Abs to IL-16 affect migration in response to
medium alone (data not shown).
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). At 24 h, the number of
eosinophils was 3.5 times higher then at 3 h. When the absolute
numbers of eosinophils in BALF of OVA-challenged mice were compared
with the percentage of lymphocyte migration induced in vitro by BALF
samples derived from the same mice, no significant correlation was
observed (r = 0.529, p =
0.178).
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IL-16 immunoreactivity was identified in airway sections of
OVA-sensitized and OVA-challenged mice (Fig. 3A). In these mice,
IL-16 was localized predominantly in the epithelium (Fig. 3A), but also IL-16-positive cells were detected in
the cellular infiltrates surrounding bronchi and blood vessels (Fig. 3C). In sections of OVA-sensitized and
saline-challenged mice but not in sections of nonsensitized mice, some
IL-16 immunoreactivity was found in epithelial cells, suggesting
that the sensitization procedure already induced the expression of
IL-16 (Fig. 3, E and F, respectively). No
staining could be detected in sections of OVA-sensitized and
OVA-challenged mice incubated with control Ab (Fig. 3, B and
D).
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BALF derived from OVA-challenged mice treated with control Ab
induced an increased migratory response compared with the response
induced by BALF from control Ab-treated saline-challenged mice
(p < 0.001, Fig. 4). After in vivo treatment with Abs to
IL-16, this enhanced migratory response toward BALF of OVA-challenged
mice was significantly (p < 0.01, Fig. 4),
although not completely, inhibited. When Abs to IL-16 were added in
vitro, no additional blocking of migration was observed, indicating
that IL-16 generated in vivo was completely inhibited (data not shown).
Migration induced by BALF from saline-challenged mice was not affected
by in vivo treatment with Abs to IL-16.
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In control Ab-treated mice, repeated inhalation of OVA induced a
marked up-regulation of the amount of OVA-specific IgE in serum
compared with their saline-challenged controls
(p < 0.001, Fig. 5). Interestingly, when mice were treated
with Abs to IL-16 and challenged with OVA this up-regulation was
significantly decreased (55%, p < 0.01). OVA-specific
IgE levels measured in the anti-IL-16-treated OVA-challenged mice
were still increased compared with anti-IL-16-treated
saline-challenged mice; however, this did not reach the level of
significance (p = 0.06). No effect of treatment
with Abs to IL-16 on the amount of OVA-specific IgE detected in
saline-challenged mice was observed.
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The responsiveness to methacholine was significantly increased in
control Ab-treated OVA-challenged mice compared with control Ab-treated
saline-challenged mice (Fig. 6A). The maximal
potentiation in responsiveness observed in OVA-challenged animals was
44% at a dose of 320 µg/kg methacholine. After treatment with Abs to
IL-16, the responsiveness to methacholine measured in OVA-challenged
mice was not significantly different from the responsiveness measured
in saline-challenged mice, indicating that the development of
airway hyper-responsiveness in the OVA-challenged group was
prevented (Fig. 6B). When anti-IL-16-treated
OVA-challenged mice were compared with control Ab-treated
OVA-challenged mice the responsiveness in anti-IL-16-treated mice
was significantly decreased at doses of 320 and 640 µg/kg
methacholine. Methacholine dose-response curves measured in the
saline-challenged groups were not significantly different among the
different treatment groups. Irrespective of the treatment, no changes
in ED50 were observed and there was no difference in basal
bronchial resistance to inflation between the various groups (data not
shown).
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No eosinophils were observed in BALF of both control Ab and
anti-IL-16-treated mice that were exposed to saline aerosols. In
contrast, after repeated OVA inhalations in control Ab-treated mice, a
significant number of eosinophils was present
(p < 0.05, Fig. 7A). Treatment with Abs
to IL-16 did not affect the number of eosinophils present in the BALF
after repeated OVA exposure (Fig. 7A). In addition,
no effects of treatment with Abs to IL-16 were observed on the total
amount of BAL cells, the number of mononuclear cells, or the number of
neutrophils (Table III). Eosinophils were
also counted in tracheal tissue. After exposure to repeated OVA
aerosols, the number of eosinophils observed in the trachea was
significantly increased in control Ab-treated mice compared with their
saline-challenged controls (p < 0.05, Fig. 7B). In OVA-challenged animals treated with Abs to
IL-16, the eosinophil number was also increased in comparison with
their saline-challenged controls, indicating that treatment with Abs to
IL-16 had no effect on the number of eosinophils in the trachea.
Compared with OVA-challenged animals treated with control
Ab, the number of eosinophils in the
anti-IL-16-treated OVA-challenged group was higher; however, this
was not significant.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALF derived from OVA-sensitized and challenged mice at 3 and 24 h after the last OVA inhalation stimulated human lymphocyte migration in vitro. This migratory response was inhibited by Abs to IL-16, clearly demonstrating the presence of IL-16 in BALF of the OVA-challenged animals. From our experiments, it appeared that the amount of IL-16 present in BALF was higher at 3 h than at 24 h, suggesting that IL-16 is transiently expressed at early time points. BALF obtained from OVA-sensitized and saline-challenged mice inhibited lymphocyte migration in comparison with migration induced by medium, indicating that BALF contains factor(s) that are inhibitory for migration. BALF samples applied in our experiments were not placed over an IL-16 affinity column and therefore may contain many different factors that can affect overall migration. As a result of this, the observed increase in migration in the OVA group could be caused by production of chemoattractant factors or loss of inhibitory factors. This is why neutralization studies are performed to determine the presence of a specific chemoattractant, IL-16. Our data are very similar to those described for humans (12). BALF obtained from allergen-challenged atopic asthmatics, but not from saline-challenged asthmatics, stimulates migration of human lymphocytes in vitro, and a significant part of this chemoattractant activity is attributable to IL-16 (12). In humans, IL-16 is detected early after antigen challenge, i.e., at 6 h (12), which is comparable to our observations in mice. In addition, in humans it has also been shown that BALF obtained after saline challenge (in both normal subjects and asthmatics) contains factors that inhibit migration of human lymphocytes (12).
Immunohistochemistry data demonstrate that IL-16 immunoreactivity was induced in the airway tissue through OVA sensitization and markedly increased after repeated OVA inhalations. Because the Ab applied in these studies detects both IL-16 and the IL-16 precursor protein, we hypothesize that OVA sensitization induces expression of IL-16 precursor protein in airway epithelial cells, which is cleaved and released into BALF after repeated exposure to OVA. Because we also found IL-16-positive cells within cellular infiltrates after repeated OVA exposure, it seems likely that these cells contribute to the release of IL-16. Our data are comparable with those found in humans. Laberge et al. (10) have shown that airway epithelial cells of asthmatics contain both IL-16 mRNA and protein. Interestingly, in that study, a significant association was demonstrated between epithelial IL-16 immunoreactivity and airway responsiveness to methacholine (10).
IgE cross-linking of human and murine mast cells induces the release of histamine and serotonin, mediators that stimulate IL-16 production by CD8+ T lymphocytes (7, 8). Although not rigorously proved, other in vitro data suggest that epithelial cells also release IL-16 after stimulation with histamine (11). Previously, we have observed mast cell degranulation within 30 min after OVA challenge in sensitized mice (14). It may therefore be hypothesized that in our mouse model mast cell-derived mediators induce the secretion of IL-16 by CD8+ T lymphocytes and/or epithelial cells after repeated OVA inhalations in sensitized mice.
In this study, sensitized mice were treated with Abs to IL-16 during
the challenge period. The migratory response induced by BALF from these
mice was clearly but not completely inhibited. Addition of Abs to IL-16
to the samples in vitro did not further inhibit the migratory activity.
Therefore, it can be concluded that all IL-16 present in the BALF was
blocked by in vivo treatment with Abs to IL-16. Furthermore, these
results indicate that, besides IL-16, BALF obtained after repeated OVA
inhalation also contains other factors that induce migration of
lymphocytes. A possible candidate may be macrophage-inflammatory
protein-1
(MIP1), which is a chemoattractant for lymphocytes (21)
and has been detected in lung tissue in a mouse model of airway
inflammation (22). In humans, it has been shown that the remaining
migration activity after addition of Abs to IL-16 is attributable to
MIP-1 and not to RANTES (12).
Treatment with Abs to IL-16 decreased up-regulation of OVA-specific IgE
during repeated OVA inhalation in sensitized mice. The production of
IgE is dependent on CD4+ T lymphocytes (23), and since
IL-16 binds to the CD4 molecule (5) it is possible that it may act as a
costimulus. In this way it may be involved in the process of IgE
up-regulation. Alternatively, IL-16-induced production of IFN-
may
up-regulate OVA-specific IgE in previously sensitized mice. IL-16 has
been found to induce the release of IFN- after incubation with T
lymphocytes (W. W. Cruikshank, unpublished observation), and we
have previously demonstrated that memory IgE responses can be
up-regulated by IFN- (22). More experiments are needed, however, to
elucidate the precise mechanism by which IL-16 stimulates up-regulation
of IgE.
Interestingly, treatment of sensitized and OVA-challenged mice with Abs
to IL-16 during the challenge period markedly inhibited the development
of airway hyper-responsiveness. This observation demonstrates the
importance of IL-16 in the induction of airway hyper-responsiveness. We
can only speculate how IL-16 induces airway hyper-responsiveness in our
murine model of allergic asthma. In the present study, treatment with
Abs to IL-16 did not inhibit the process of eosinophil infiltration. In
addition, there was no correlation between the amount of IL-16 in BALF
and the number of eosinophils in BAL. These observations in itself do
not rule out the involvement of eosinophils in the development of
airway hyper-responsiveness because Abs to IL-16 could have acted at
the level of eosinophil activation. However, since it has been
described that IL-16 has no effect on eosinophil activation or priming
(6), this explanation seems unlikely. An alternative explanation for
the importance of IL-16 in the induction of airway hyper-responsiveness
may be that IL-16 acts via other cells. A likely candidate is the
CD4+ T lymphocyte, since depletion of these T cells
inhibits the development of airway hyper-responsiveness in a mouse
model of Ag-induced airway hyper-responsiveness (25). IL-16 activates T
lymphocytes and induces expression of MHC class II molecules and the
IL-2 receptor on human T lymphocytes (5). In addition, as mentioned
above, incubation of T lymphocytes with IL-16 induces the release of
IFN-. We have recently demonstrated that Abs to IFN- could
inhibit airway hyper-responsiveness without any effect on the presence
of eosinophils in BAL (19). It is tempting to speculate that Ag
inhalation in sensitized animals causes mast cell degranulation,
leading to the early release of IL-16 by CD8+ T lymphocytes
and/or epithelial cells, which subsequently induces IFN- production
by lymphocytes. IFN- in its turn may be involved in the development
of airway hyper-responsiveness without affecting eosinophil
infiltration.
Although IL-16 has been described as a potent human eosinophil chemoattractant in vitro (6), treatment with Abs to IL-16 during the challenge period had no effect on the number of eosinophils appearing in BALF or airway tissue in our mouse model. These results indicate that IL-16 plays no crucial role in this process. It is known, however, that there is a great redundancy in cytokines capable of evoking eosinophil infiltration (26). Furthermore, it has been described that chemotactic responses induced by IL-16 are proportional to the amount of cell surface-expressed CD4 (5), and thus an alternative explanation for our findings may be that mouse eosinophils may not express high numbers of CD4 on their surface. We were not able to detect CD4 molecules on mouse eosinophils as determined by flow cytometry (data not shown). On the basis of our results, we conclude that IL-16 may not be a potent chemoattractant factor for mouse eosinophils in vivo.
In summary, IL-16 immunoreactivity is present in the airways after sensitization. After repeated OVA inhalation, IL-16 immunoreactivity is markedly increased and IL-16 is detectable in BALF. Treatment in vivo with Abs to IL-16 suppresses the up-regulation of OVA-specific IgE during the OVA challenge period. Furthermore, Abs to IL-16 markedly inhibit the development of airway hyper-responsiveness without an effect on the infiltration of eosinophils. Altogether, these data suggest a prominent role for IL-16 in this mouse model of allergic asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. E. M. Hessel, Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands. E-mail address:
3 Abbreviations used in this paper: BALF, bronchoalveolar lavage fluid; BAL, bronchoalveolar lavage; hpf, high power field; MIP1, macrophage-inflammatory protein-1.
Received for publication October 10, 1996. Accepted for publication November 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP1) in bronchoalveolar lavage fluid of antigen-challenged asthmatics. Am. J. Respir. Cell Mol. Biol. 13:738.[Abstract]
and MIP1ß. Science 260:355. influences eosinophil recruitment in antigen-specific airway inflammation. Eur. J. Immunol. 25:245.[Medline]
in a murine model of allergic asthma. Am. J. Respir. Crit. Care Med. 153:A220.
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K. Reich, A. Heine, S. Hugo, V. Blaschke, P. Middel, A. Kaser, H. Tilg, S. Blaschke, C. Gutgesell, and C. Neumann Engagement of the Fc{epsilon}RI Stimulates the Production of IL-16 in Langerhans Cell-Like Dendritic Cells J. Immunol., December 1, 2001; 167(11): 6321 - 6329. [Abstract] [Full Text] [PDF]
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G. Cheng, T. Ueda, F. Eda, M. Arima, N. Yoshida, and T. Fukuda A549 Cells Can Express Interleukin-16 and Stimulate Eosinophil Chemotaxis Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 212 - 218. [Abstract] [Full Text] [PDF]
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 N. KRUG, W. W. CRUIKSHANK, T. TSCHERNIG, V. J. ERPENBECK, K. BALKE, J. M. HOHLFELD, D. M. CENTER, and H. FABEL Interleukin 16 and T-cell Chemoattractant Activity in Bronchoalveolar Lavage 24 Hours after Allergen Challenge in Asthma Am. J. Respir. Crit. Care Med., July 1, 2000; 162(1): 105 - 111. [Abstract] [Full Text]
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 T. Yoshimoto, C.-R. Wang, T. Yoneto, A. Matsuzawa, W. W. Cruikshank, and H. Nariuchi Role of IL-16 in delayed-type hypersensitivity reaction Blood, May 1, 2000; 95(9): 2869 - 2874. [Abstract] [Full Text] [PDF]
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R. I. Zuberi, J. R. Apgar, S.-S. Chen, and F.-T. Liu Role for IgE in Airway Secretions: IgE Immune Complexes Are More Potent Inducers Than Antigen Alone of Airway Inflammation in a Murine Model J. Immunol., March 1, 2000; 164(5): 2667 - 2673. [Abstract] [Full Text] [PDF]
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 M. Laan, I. Qvarfordt, G. C Riise, B. A Andersson, S. Larsson, and A. Lindén Increased levels of interleukin-16 in the airways of tobacco smokers: relationship with peripheral blood T lymphocytes Thorax, October 1, 1999; 54(10): 911 - 916. [Abstract] [Full Text]
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A. Kaser, S. Dunzendorfer, F. A. Offner, T. Ryan, A. Schwabegger, W. W. Cruikshank, C. J. Wiedermann, and H. Tilg A Role for IL-16 in the Cross-Talk Between Dendritic Cells and T Cells J. Immunol., September 15, 1999; 163(6): 3232 - 3238. [Abstract] [Full Text] [PDF]
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K F Chung and P J Barnes Cytokines in asthma Thorax, September 1, 1999; 54(9): 825 - 857. [Full Text]
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J. Nicoll, W. W. Cruikshank, W. Brazer, Y. Liu, D. M. Center, and H. Kornfeld Identification of Domains in IL-16 Critical for Biological Activity J. Immunol., August 15, 1999; 163(4): 1827 - 1832. [Abstract] [Full Text] [PDF]
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 N. Bannert, A. Avots, M. Baier, E. Serfling, and R. Kurth GA-binding protein factors, in concert with the coactivator CREB binding protein/p300, control the induction of the interleukin 16 promoter in T lymphocytes PNAS, February 16, 1999; 96(4): 1541 - 1546. [Abstract] [Full Text] [PDF]
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) or OVA (
) at
3 or 24 h after the last aerosol exposure. Migration is expressed
as the number of cells per hpf. Samples were tested in triplicate, and
values are expressed as mean ± SEM. *, significantly different
from the response in OVA-sensitized and saline-challenged controls
(p < 0.05).
