Mice lacking the VIP gene show airway hyperresponsiveness
and airway inflammation, partially reversible by VIP
Anthony M. Szema1, 4, Sayyed A. Hamidi1, 4, Sergey Lyubsky2, 4, Kathleen G.
Dickman1,4, Suni Mathew1,4, Tarek Abdel-Razek1,4, John J. Chen3, James A.
Waschek5, and Sami I. Said1,4
1Departments of Medicine, 2Pathology, and 3Preventive Medicine, State University of
New York at Stony Brook, NY, USA.
4Northport VA Medical Center, Northport, NY, USA.
5Department of Psychiatry, University of California at Los Angeles, LA, California,
Address correspondence to Dr. Said at: Pulmonary and Critical Care Medicine, SUNY
Health Sciences Center, Stony Brook, NY 11794-8172
Tel #: 631-444-1754; Fax #: 631-444-7502
MCP5: Macrophage/Monocyte Chemotactic Protein-5
MMP: Matrix Metalloprotease
TARC: Thymus Activation Regulated Chemokine
Page 1 of 32
Articles in PresS. Am J Physiol Lung Cell Mol Physiol (June 16, 2006). doi:10.1152/ajplung.00499.2005
Copyright © 2006 by the American Physiological Society.
The mechanisms leading to asthma, and those guarding against it, are yet to be fully
defined. The neuropeptide VIP is a co-transmitter, together with nitric oxide (NO), of
airway relaxation, and a modulator of immune and inflammatory responses. NO-storing
molecules in the lung were recently shown to modulate airway reactivity, and were
proposed to have a protective role against the disease. We report here that mice with
targeted deletion of the VIP gene spontaneously exhibit airway hyperresponsiveness to
the cholinergic agonist methacholine, as well as peribronchiolar and perivascular cellular
infiltrates and increased levels of inflammatory cytokines in broncho-alveolar lavage
(BAL) fluid. Immunologic sensitization and challenge with ovalbumin generally
enhanced the airway hyperresponsiveness and airway inflammation in all mice.
Intraperitoneal administration of VIP over a 2-week period in KO mice virtually
eliminated the airway hyperresponsiveness, and reduced the airway inflammation in
previously sensitized and challenged mice. The findings suggest that: a) VIP may be an
important component of endogenous anti-asthma mechanisms; b) deficiency of the VIP
gene may predispose to asthma pathogenesis; and c) treatment with VIP or a suitable
agonist may offer potentially effective replacement therapy for this disease.
Page 2 of 32
The pathogenesis of bronchial asthma remains incompletely understood. Atopy, cellular
and humoral mediators of inflammation, and abnormal neurogenic influences are
recognized factors (3). One postulated mechanism is an imbalance between pro-asthma,
e.g., histamine, leukotrienes (11), and potential anti-asthma mediators (1). Among the
latter compounds is VIP, a neuropeptide with potent bronchodilator, immunomodulator,
and anti-inflammatory properties (8, 31, 39). Together with NO and carbon monoxide,
VIP is a co-transmitter of the dominant neurogenic relaxant system of airway smooth
muscle (31, 33), the natural defense mechanism against airway constriction. VIP also
suppresses airway smooth muscle proliferation (25), an important component of airway
remodeling in chronic asthma, and thus has biological properties that are capable of
counteracting all major features of the asthmatic response.
Identification of endogenous anti-asthma defenses should provide useful insights into the
pathogenesis of the disease and its management. Depletion of S-nitrosothiols, key NO-
carrying molecules in the lung, was recently found to correlate with increased airway
constriction in mice, leading to the conclusion that these compounds form a natural
defense against asthma (29). We hypothesized that VIP may provide another such
defense mechanism, and reasoned that, if this hypothesis is correct, then absence of VIP
from the airways should be associated with features commonly identified with the human
disease -- namely, bronchial hyperresponsiveness and airway inflammation. We report
that mice with targeted deletion of the VIP gene do exhibit such hallmarks of asthma. To
Page 3 of 32
confirm the interpretation that the absence of the VIP gene was responsible for the
asthma-like phenotype, we administered VIP for a 2-week period, following which the
airway hyperresponsiveness was nearly abolished, and the airway inflammation was
attenuated. These observations support the conclusions that: a) VIP serves an important
physiological role as a modulator of airway constriction and other asthmatic responses, b)
its deficiency may be a causative factor in the pathogenesis of the disease, and c) agonists
of the peptide or its receptors may represent a new, targeted therapeutic approach.
MATERIALS AND METHODS
Animals. VIP KO mice, backcrossed to C57BL/6, were prepared as described (5). We
bred the mice locally and genotyped them to confirm the absence of the VIP gene (5). We
mated homozygous KO males with homozygous KO females or, if necessary, with
heterozygous KO females. For genotyping, we extracted DNA from 1 cm long tail snips
using a DNA isolation kit (Qiagen, Inc., Valencia, CA). DNA (100 ng) was subjected to
PCR using primers to detect both VIP and the neomycin cassette. Control, wild-type
(WT) C57BL/6 mice were from Taconic Labs. (Germantown, NY).
Airway Responsiveness. To assess bronchial reactivity to a standard airway constrictor,
12 male VIP KO and 9 male control mice were anesthetized with pentobarbital,
tracheostomized and mechanically ventilated at a constant tidal volume. After baseline
values were established, we delivered methacholine as an aerosol at 1, 10, 102& 500
mg/ml, using an Aeroneb Nebulizer System for mice (Buxco; Troy, NY), placed in-line
Page 4 of 32
with the tracheostomy tube. We recorded airway pressure continuously, and evaluated the
degree of bronchoconstriction by increases in peak airway pressure which, at constant
breath volumes, reflected increases in pulmonary resistance. Airway responsiveness was
again examined in 4 KO and 5 WT mice after sensitization and challenge with
ovalbumin, as described below.
A) Histologic evidence. To test for the presence of airway inflammation, a corollary of
airway hyperresponsiveness in asthma (42), we subjected the lungs from 5 male VIP KO
and 6 male WT mice to histologic examination by a pathologist who was blinded to the
identities of the samples. All abnormalities were graded 0, 1, 2, 3 or 4, based on the
intensity and extent of peribronchiolar and perivascular cellular infiltration.
B) Lung Cytokines. As added evidence of possible airway inflammation, we performed
bronchoalveolar lavage (BAL) in 5 knockout and 5 WT mice. The lungs of each mouse
were lavaged 3 times with 1 ml phosphate-buffered saline (PBS), including an EDTA-
free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The BAL fluid was
centrifuged at 400 g for 5 min at 21oC, and the supernatant was analyzed by a
quantitative ELISA assay (Pierce Biotechnology, Woburn, MA) for selected
inflammatory cytokines, chemokines, and a matrix metalloprotease (MMP): IL-2, IL-5,
IL-6, IL-10, IL-13, Thymus Activation Regulated Chemokine (TARC), osteopontin
(OPN), interferon γ, and MMP2.
Page 5 of 32
C) Cell Counts. We measured differential cell counts in BAL fluid in cytospin
preparations, and by flow cytometry; the latter also permitted analysis of intracellular
cytokines (37). After harvesting cells from BAL fluid, they were resuspended in PBS
with azide to 1 x 106per ml after counting in a Coulter 21 Particle Counter (Hialeah, FL).
The cells were then permeabilized with BD Cytofix/cytoperm and labeled with various
monoclonal antibodies according to the manufacturer’ s recommendations (BD
Biosciences, San Diego, CA). Following labeling, the cells were washed and resuspended
in 500 ml of 1% formalin in BSA. Cells were refrigerated until acquisition and analysis
with a BD FACSCaliber (Becton-Dickinson, Mountain View, CA). For analysis, the cells
were gated on individual fluorescence positivity for CD11c (dendritic cells and
macrophages), CCR3 (eosinophils, mast cells, and Th2 cells) and then examined for
reactivity to anti IL-5 and anti IL-6 antibodies. Neutrophils were gated by CD11c low
intensity and CCR3 negativity, and confirmed by light scatter backgating characteristics.
CCR3 and CD11c negative lymphocytes were gated and confirmed by light scatter
backgating. Reactivities to anti-IL-5 and IL-6 were then examined. The production of
intracellular cytokines was expressed in “ geometric mean fluorescent channels” .
Immunologic Sensitization & Challenge
To compare the immunologic airway responses in KO mice with those in control WT
mice, we sensitized 4 KO and 5 WT mice with ovalbumin (20 µg, Grade VI, Sigma),
emulsified in 2.25 mg Alum (Pierce), and injected intraperitoneally, on days 0 and 14.
On days 21 and 28, we challenged the mice with an aerosol of 100 mg ovalbumin in 10
Page 6 of 32
ml PBS, delivered via a DeVilbiss ultrasonic nebulizer over 20 min. On day 29, the mice
were tested for airway reactivity, euthanized with pentobarbital, and their lungs removed
for histologic examination.
Administration of VIP
Nine male VIP KO mice, aged 16-20 weeks, received VIP (15 µg in 0.2 ml PBS),
intraperitoneally, every other day, for 2 weeks, for a total of 7 injections, ending the day
before testing for airway reactivity and lung cell infiltration. Another group of 4 male
VIP KO mice of a similar age received 0.2 ml PBS, without VIP, in the same manner and
for the same duration. Our choice of the dosage, duration, frequency, and mode of
administration of VIP was guided by protocols for related studies by other investigators
(7, 22, 34). At the end of this treatment period, we evaluated airway responsiveness to
methacholine in the 9 mice. In addition, lungs from 5 of these mice were examined for
histologic evidence of airway inflammation. Another group of 5 KO mice which were
sensitized and challenged with ovalbumin, were treated with VIP for 2 weeks, as above.
These mice were then examined for airway responses to methacholine and their lungs
were examined for histologic evidence of inflammation.
Immunoreactive VIP Levels in BAL Fluid and Lung Tissue
To examine the degree to which the VIP treatment restored the VIP content in lungs of
VIP KO mice, we measured immunoreactive VIP levels (27) in BAL fluid and lung
tissue of the treated mice 24 hours after completing the VIP treatment. We compared
Page 7 of 32
those levels to corresponding values in WT mice under the same conditions, i.e., with or
without immunologic sensitization and challenge.
All results were expressed as means ± SEM. Cytokine-chemokine data were analyzed by
both parametric (t-test) and non-parametric (Mann-Whitney U test) approaches. Change
in peak airway pressure from baseline was modeled using the repeated-measures analysis
of variance (ANOVA) approach, taking into consideration the dependent measures at
different concentrations of methacholine in the same mouse. The differences in the peak
airway pressure changes among the 3 groups of mice (WT, VIP KO, and VIP KO + VIP)
were further evaluated using post-hoc least significant difference (LSD) tests. For
histologic evidence of lung inflammation, exact tests for contingency tables with ordinal
categories were used to compare scores of WT and VIP KO mice. All statistical analyses
were done using SAS software, with two-tailed P values < 0.05 considered statistically
Page 8 of 32
In unsensitized animals, methacholine elicited a greater bronchoconstrictor response in
the VIP KO mice than in WT mice (Fig. 1). The airway pressure responses were
significantly different among the 3 groups of mice at the 100 and 500 mg/ml dose levels
(P = 0.008 and 0.007, respectively). Using post-hoc LSD tests, the airway pressure in KO
mice was significantly higher than in WT mice at the 100 and 500 mg/ml dose levels (P =
0.003 and 0.001, respectively).
A) Histologic Evidence. Lungs from unsensitized KO mice showed peribronchiolar and
perivascular infiltration with lymphocytes and eosinophils, while lungs from control WT
mice appeared normal (Table 1, Figs. 2a & b). The degree of cellular infiltration varied
among individual KO mice, but at least some infiltration was present in the lungs of 3 out
of 5 mice. More severe inflammation was associated with prominent eosinophilic
infiltration, and occasionally also alveolar edema, but there was no evidence of airway
remodeling. By contrast, none of 5 WT mice showed any evidence of inflammation. The
intensity of inflammatory cell infiltration in lungs from unsensitized, unchallenged VIP
KO mice was significantly higher than for WT mice (P=0.028); but the difference was
insignificant between KO and KO+VIP (P=0.22).
Page 9 of 32
B) Lung Cytokines, Chemokines, and Proteases. Evidence of airway inflammation was
supported by analysis of BAL fluid for 8 inflammatory cytokines and chemokines, and
one matrix metalloprotease: IL-2, IL-5, IL-6, IL-10, IL-13, TARC, OPN, interferon γ,
and MMP2. With the exception of IL-2, mean levels of all cytokines were significantly
higher in 5 KO mice than in 5 control mice (Table 2).
In unsensitized unchallenged mice, mean total cell count in BAL fluid was higher in VIP
KO mice than in WT mice (430,000 ± 250,000 vs. 95,000 ± 12,000, P = 0.019 based on
U-test). The predominant cells in cytospin preparations of BAL fluid from WT mice
were macrophages and lymphocytes; eosinophils were notably present in BAL fluid from
KO mice. Using flow cytometry, neutrophils in BAL fluid from KO mice produced more
IL-5 than corresponding neutrophils from WT mice (22.7 ± 4 vs. 5.3 ± 5, P = 0.067,
approaching significance, based on t-test).
Effect of Immunologic Challenge
Airway hyperresponsiveness persisted in VIP KO mice after immunologic sensitization
and challenge. The differences in airway pressure among the 3 groups were significant at
the 10, 100 and 500 mg/ml dose levels (Fig. 3; P =0.029, 0.013, and <0.001). Based on
post-hoc LSD tests, airway pressures were greater in KO than in WT mice at the 10
mg/ml level (P =0.014), the 100 mg/ml level (P=0.007), and the 500 level (P <0.001).
Page 10 of 32
Immunologic sensitization and challenge led to an increase in the degree of lung cellular
infiltration in WT mice (P=0.04). Cellular infiltration was also marginally increased in
VIP KO mice, relative to unchallenged KO mice (P=0.067). Infiltration was marginally
greater in KO compared to WT mice (P=0.056). In some of the KO mice, there were also
areas of focal alveolar edema (Table 3, Fig. 2c).
Judging by cytospin preparations, eosinophils were absent in BAL fluid from
unchallenged WT mice, but were clearly present after challenge (P = 0.004, by Mann-
Whitney test). Eosinophil counts, already present in most unchallenged KO mice, showed
a tendency to increase further following challenge (P =0.057). Analysis by flow
cytometry showed that, in WT mice, mean neutrophil count in BAL fluid increased from
9% ± 2 to 40% ± 11 (P = 0.011), and in VIP KO mice it increased from 7% ± 1 to 32% ±
8 (P = 0.019). Both CCR3- positive cells and neutrophils in BAL fluid from WT mice
produced more IL-5 in sensitized, challenged mice than in unsensitized mice: 22.2 ± 2.0
vs.7.3 ± 7.3 for CCR3-positive cells (P= 0.15, based on U-test) , and 24.6 ± 4.0 vs. 5.3 ±
5.3 for neutrophils (P= 0.035, based on t-test).
CD11c-positive (dendritic) cells in BAL fluid from VIP KO mice produced more IL-5
than dendritic cells in BAL fluid from WT mice:17.3 fluorescent channels ± 4.5 vs. 4.6 ±
1.1, P=0.12 based on t-test, and 0.032 based on U-test. Dendritic cells in BAL fluid from
VIP KO also produced more IL-6 than dendritic cells in BAL fluid from WT mice, but
the difference was not statistically significant (62.0 ± 38.1 vs. 7.8 ± 2.5, P=0.39 based on
t-test, and 0.095 based on U-test).
Page 11 of 32
Administration of VIP to a group of VIP KO mice achieved pulmonary levels of the
peptide that were similar to those in WT mice. Analysis of BAL fluid and lung tissue
from KO mice treated with VIP for 2 weeks showed VIP levels of 20 ± 8.3 pg/ml in BAL
fluid (n=5), and 0.14 ± 0.07 pg/mg protein in lung tissue (n=5) in unsensitized,
unchallenged mice, and 17 ± 12.8 pg/ml (n=5) and 0.14 ± 0.06 pg/mg protein in
sensitized, challenged mice. The corresponding values in WT mice (which did not
receive VIP) were 12.3 ± 8.7 pg/ml (n=6), and 0.04 ± 0.04 pg/mg protein in lung tissue
(n=5). in the absence of sensitization and challenge, and 32.7 ± 10.1 pg/ml (n=5) in BAL
fluid, and 0.12 ± 0.06 pg/mg protein (n=5) in lung tissue, after sensitization and
VIP replacement therapy markedly attenuated airway hyperresponsiveness in KO mice.
In the 9 VIP KO mice treated with VIP, methacholine elicited greatly reduced airway
pressure responses, which closely resembled those of WT mice (Fig. 3). Based on LSD
post-hoc tests, airway pressures in VIP-treated KO mice were significantly lower than in
untreated mice at the 10 (P=0.027), the 100 (P=0.011), and the 500 mg/ml (P< 0.001)
dose levels. Three of the 4 KO mice that received only PBS responded like other
untreated KO mice.
Page 12 of 32
Compared to untreated or buffer-treated KO mice, most lung sections from VIP-treated
KO mice revealed considerably less intense, and more limited, cellular infiltrates (Fig. 4).
Cellular infiltration was significantly less severe in KO mice that received VIP treatment
compared to those that did not (P= 0.016), and was no different that in WT mice (P=0.66,
Table 3). Additionally, in contrast to KO mice, neutrophils in BAL fluid from KO mice
treated with VIP did not increase after sensitization and challenge (17% ± 7 vs. 19%, ± 7,
P = 0.85 based on t-test). Thus, the 2-week treatment with VIP almost largely corrected
the two major asthma-like features observed in untreated KO mice.
Our results demonstrate that homozygous VIP KO mice spontaneously express two of the
cardinal features of bronchial asthma: airway hyperresponsiveness and airway
inflammation, manifested by cellular infiltration with lymphocytes and eosinophils. This
infiltration was moderately severe, and was accompanied by an inflammatory cytokine/
chemokine response, consistent with findings in experimental and clinical asthma (2, 9,
23, 41). Lymphocytes, eosinophils, and probably also neutrophils, were present in BAL
fluid, especially after immunologic challenge. The presence of neutrophils in BAL fluid
under these conditions suggests that they may have contributed to the inflammatory
response. A role for neutrophils in the pathogenesis of asthma, particularly severe forms
of the disease, has recently been documented (24a). Airway remodeling, a third feature of
the human disease, especially in its chronic stages (1), was not observed in these mice,
possibly reflecting a relatively short duration of the disease model.
Page 13 of 32
Despite the traditional view that the asthma phenotype is driven by increased T helper 2
(Th2) cytokines, e.g., IL-5 and IL-13, with decreased Th1 cytokines, e.g., IFN γ, studies
of mouse models strongly suggest that simultaneous activation of the Th1 immune
response may promote a more severe airway inflammation (4, 20, 30). Possible
interactions between IL-13 and IFN γ, both of which were elevated in our KO mice, are
of special interest (14): a) Depending on the antigen, IFN γ may actually accentuate the
inflammatory response; and b) in a mouse model of airway inflammation induced by
mixed T cell responses, blockade of IL-13 partially inhibited airway hyperreactivity but
not inflammation (14). The latter observation appears to parallel the greater suppression
of airway hyperresponsiveness than of airway inflammation after treatment of KO mice
with VIP. The phosphodiesterase inhibitor Pentoxifylline, a selective suppressor of Th1
cytokine production, attenuated airway hyperresponsiveness but not airway inflammation
in a mouse model of asthma (13).
The possible role of VIP in asthma has long been under investigation, but a clear answer
has been lacking. Early after its discovery and isolation, VIP was demonstrated to occur
widely in nerve fibers and nerve terminals supplying human and other mammalian
airways (10). Soon afterward, it was shown to be a potent relaxant of airway smooth
muscle (31), and to act as a co-transmitter of neurogenic airway relaxation (33). Later, its
anti-inflammatory and immunomodulator properties were described (7, 15), as well as its
ability to inhibit airway smooth muscle proliferation (25). The picture thus emerged of
VIP as a compound that is potentially capable of counteracting most major components
of the asthmatic phenotype (3, 35). Bolstering this viewpoint, VIP-immunoreactive
Page 14 of 32
nerves were reported absent in the airways of a small group of severely asthmatic patients
(26), raising the possibility that a deficiency of the neuropeptide might even be causally
related to the disease.
Recently, however, a number of immunologic studies have added a different perspective
on what may be a complex relationship between VIP and asthma. Findings typical of
immediate-type hypersensitivity (elevated blood IgE levels and eosinophil counts) were
described in transgenic mice that constitutively and selectively expressed, in CD4T cells,
the normally inducible VPAC2 receptor (38, 40). This is one of 3 receptors common to
VIP and the related pituitary adenylate cyclase - activating peptide (PACAP) that is
normally not constitutively expressed (21). Conversely, VPAC2 - null mice manifested
decreased immediate-type hypersensitivity (17). These and related studies, by themselves,
suggested that VIP plays a significant role in shifting the Th1/Th2 balance in favor of the
Th2 phenotype (8). There is no real conflict between these observations and the present
results. Deletion of the VIP is not equivalent to loss of one of its receptors; the full effects
of VIP are mediated by the combined influence of its 3 receptors, known as VPAC1,
VPAC2 and PAC1 (21).
One of the more remarkable findings in this report is that deletion of the VIP gene did not
merely predispose the mice to asthma-like features; many mice spontaneously exhibited
airway hyperresponsiveness and airway inflammation, even in the absence of
immunologic sensitization and challenge. In this respect, these mice are reminiscent of
those with targeted deletion of the T-bet transcription factor (12). A possible link between
Page 15 of 32