ORIGINAL ARTICLE EXPERIMENTAL ALLERGY AND IMMUNOLOGY

Neutrophils from allergic asthmatic patients produce and

release metalloproteinase-9 upon direct exposure to
allergens

n1, C. Chamorro1, R. Aroca1, E. Go
I. Ventura1,*, A. Vega1,*, P. Chaco
mez1, V. Bellido1, Y. Puente1,
M. Blanca & J. Monteseir
ın
2 1,3

1
Servicio Regional de Inmunolog
ıa y Alergia, Hospital Universitario Virgen Macarena, Sevilla; 2Research Laboratory, Carlos Haya Hospital-
Fundacion IMABIS, M
alaga; 3Departamento de Medicina, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain

n P, Chamorro C, Aroca R, Go
To cite this article: Ventura I, Vega A, Chaco
mez E, Bellido V, Puente Y, Monteseir
ın J. Neutrophils from allergic asthmatic
patients produce and release metalloproteinase-9 upon direct exposure to allergens. Allergy 2014; 69: 898–905.

Keywords Abstract
allergen; asthma; IgE; metalloproteinase-9;
Background: Asthma is characterized by airway inflammation and remodelling in
neutrophils.
which matrix metalloproteinases (MMPs) play an important role. MMP-9 is the
Correspondence major MMP found in the bronchoalveolar lavage fluids and bronchial biopsies

n 27, 3° Izda,
Dr. J. Monteseir
ın, c/Asuncio from patients with allergic asthma after allergen challenge, where it correlates
41011 Sevilla, Spain. with the count of neutrophils and macrophages. However, the cellular sources of
Tel./Fax: +34-954-282-075 MMP-9 in this inflammatory condition have not yet been clearly identified. This
E-mail: fmonteseirin@us.es work was undertaken to analyse whether neutrophils may be a source of MMP-9
in the allergic asthma condition upon allergen challenge.
*These authors contributed equally to this Methods: Neutrophils from allergic asthmatic patients were in vitro stimulated,
work.
and the levels of MMP-9 release were measured in the cell culture supernatants
using enzyme-linked immunosorbent assay (ELISA) and zymography.
Accepted for publication 19 March 2014
Results: We show that MMP-9 is released by neutrophils, but not by eosinophils
from allergic asthmatic patients in response to allergens to which the patients
DOI:10.1111/all.12414
were sensitized. Neutrophils also released MMP-9 in response to anti-IgE Abs,
Edited by: Marek Sanak and agonist Abs against FceRI, FceRII/CD23 and galectin-3. Inhibitors of tran-
scription and translation, actinomycin D and cycloheximide, partially cancelled
this process, suggesting that MMP-9 is also de novo synthesized in response to
stimuli. We also show evidence that the MAPKs, p38 and extracellular signal-reg-
ulated kinase, as well as the transcription factor NF-jB, are involved, as specific
chemical inhibitors of these cell-signalling pathways abolished the anti-IgE/aller-
gen-dependent MMP-9 release.
Conclusions: These data demonstrate that the exposure of neutrophils to allergens
leads to generation of MMP-9, which may then lead to remodelling in asthma.

Metalloproteinase-9 (MMP-9, gelatinase B) is an important
member of the MMP family of endopeptidases responsible
for the remodelling of extracellular matrix (ECM) compo-
nents, involved in tumour invasion, metastasis and tissue
remodelling, a characteristic feature of asthma (1). MMP-9
activity is under strict control at various levels: transcription,
activation of the pro-enzyme and regulation by specific tissue
inhibitors of MMPs (TIMPs) (1). Of the metalloproteinases,
MMP-9 is of particular relevance to asthma. Compared with
normal subjects, increased concentrations of MMP-9 have
been detected in the bronchoalveolar lavage fluid (BALF) (2,
3), sputum (4) and serum (5) of asthmatic subjects. Increased
MMP-9 immunoreactivity (6) has been observed in bronchial
biopsies of asthmatics, and enhanced enzymatic activity has
been reported in BALF after allergen (Ag) challenge (7),
which highly correlates with the count of neutrophils. This
enzyme is expressed and secreted by endothelial cells, mono-
cytes/macrophages, mast cells, eosinophils and neutrophils
(7–10).
There are three defined types of IgE receptors, all previ-
ously described in neutrophils (FceRI, FceRII/CD23 and
galectin-3) (11). We have previously shown that neutrophils
isolated from allergic patients produce a functional response
to those Ags that produce clinical symptoms (11). There is

898 Allergy 69 (2014) 898–905 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Ventura et al. Allergen-induced metalloproteinase-9 production by neutrophils

increasing evidence of the participation of neutrophils in aller- Table 2 Results of the skin prick test and serum-specific IgE of
gic processes in general and in asthma in particular (11–13). the studied asthmatic patients
As it is unknown whether neutrophils can act as source of
Skin test/serum-specific IgE
MMP-9 in allergic asthma upon direct Ag challenge and
therefore to be responsible at least in part of the pathophysi- Patient No D1 G3 T9 W6
ology of the disease, in this study, we have investigated the
expression and release of MMP-9 in response to Ag chal- 1 +
2 +
lenge, and the cell-signalling pathways involved in this pro-
3 +
cess as an attempt to identify molecular targets to modulate
4 +
this important inflammatory mediator.
5 +
6 +
Materials and methods 7 +
8 +
For details and associated references, please refer to the Data 9 +
S1 section. 10 +
11 +
Materials 12 +
13 +
The allergens (Ags) were commercially available Ag extracts 14 +
from Bial-Ar
ıstegui (Bilbao, Spain). All culture reagents 15 +
(including Ags) used in this work had endotoxin levels of 16 +
≤ 0.01 ng/ml, as verified by the Coatest Limulus lysate assay 17 +
(Chromogenix, M€ olndal, Sweden). 18 +
19 +
20 +
Patients and controls 21 +
Two groups were examined and compared: adult atopic 22 +
23 +
patients with intermittent bronchial asthma (n = 25) (Tables 1
24 +
and 2) and healthy nonatopic volunteer controls (n = 10). The
25 +
Hospital Universitario Virgen Macarena ethics committee
approved the study, and each donor gave informed consent.

Cell isolation and culture

Highly purified human peripheral blood neutrophils were iso-
lated and further purified, using a magnetic cell-sorting system
(MACS). The purity of neutrophils was on average >99%.
Highly purified human eosinophils were isolated using the
Eosinophil Isolation Kit (Miltenyi Biotec S.L.). Cells were cul-
tured in RPMI 1640 under standard cell culture conditions.
Analysis of released MMP-9
MMP-9 was determined in the culture supernatant using an
ELISA kit from Calbiochem (Darmstadt, Germany), follow-
ing manufacturer’s instructions.
Zymography
The gelatinase activity of the supernatants (1 9 106 cells/ml)
was analysed by zymography using 5% SDS-PAGE gels con-
taining 1 mg/ml of gelatin.
Nucleic acid extraction and real-time PCR
Highly purified neutrophils were processed for gene expres-
sion by RT-qPCR. The expression levels of MMP-9 were
quantified by RT-qPCR using the ABI 7500 Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA) and
reported to those of b-actin housekeeping gene.

Table 1 Demographic characteristics of the study groups Dissociation of neutrophil-bound Igs

Healthy subjects Asthmatic patients Ig molecules were dissociated from the cell surface of neu-
Parameter (n = 10) (n = 25) trophils using a solution of 0.13 M NaCl, 0.005 M KCl and
0.01 M lactic acid, which was adjusted to pH 3.9.
Age* 37.2  12.4 40.6  14.1
Age (max/min) 64/20 65/19
Gender (male/female) 4/6 13/12 Statistical analysis
Specific IgE* (KU/L) 0 110  22
FEV1 (% predicted)* 100.4  2.9 96.6  4.1 Data are expressed as means  SEM. A one-way ANOVA was
used to make comparisons between groups. A level of
*Mean  SD. P < 0.05 was considered significant.

Allergy 69 (2014) 898–905 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 899
Allergen-induced metalloproteinase-9 production by neutrophils
Results
MMP-9 is released in response to allergens by human
neutrophils from allergic asthmatic patients
The effect of specific Ags to which the patients were sensitized
and anti-IgE Abs upon MMP-9 release into the culture super-
natant was examined by ELISA and zymography, to deter-
mine the form of MMP-9 present in the supernatant. As
shown in Fig. 1A, the specific Ags to which the patients were
sensitized and anti-IgE Abs stimulated MMP-9 release by
neutrophils as confirmed by ELISA and zymography. fMLP
was used as positive control (14), and a nonspecific goat IgG
Ab was used as negative control of the goat anti-IgE Ab. As
shown in Fig. 1B, while neutrophils released MMP-9 in
response to Ags to which the patients were sensitized, eosin-
ophils from the same patients did not respond to those Ags.
However, they responded to the combination of TNF-a plus
granulocyte–macrophage colony-stimulating factor (GM-
CSF) as previously described (8). As shown in Fig. 1B, a
prominent band of approximately 92 kDa which correspond
to the proMMP-9 form of MMP-9, and a faint band of
approximately 130 kDa which correspond to proMMP-9
A C
B D
Figure 1 MMP-9 is released in response to allergens by neutroph-
ils from allergic asthmatic patients. (A) Neutrophils from allergic
patients (n = 8) were left unstimulated (us) or incubated with fMLP
(100 nM), an allergen (Ag) to which the patient was sensitized
(20 lg/ml), anti-IgE (a-IgE) (20 lg/ml) or IgG (20 lg/ml) for 24 h.
MMP-9 release was measured in the culture supernatant by ELISA,
and a representative zymography is shown. (B) Neutrophils or eo-
sinophils isolated from the same allergic patients (n = 5) were left
unstimulated (us) or incubated, where indicated, with fMLP
(100 nM), an allergen (Ag) to which the patient was sensitized
900 Allergy 69 (2014) 898–905 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Ventura et al.
bound to a neutrophil gelatinase-associated lipocalin (15)
were detected in the culture supernatant. Purified MMP-9
was used as positive control. As shown in Fig. 1C and 1D,
neutrophils released MMP-9 in a time- and dose-dependent
manner after exposure to the same Ags that induce clinical
allergic symptoms in the patients, and anti-IgE Abs. MMP-9
release was detectable from 1 h and continued to increase up
to 24 h. MMP-9 release was detected at an Ag/anti-IgE Ab
dose of 1 lg/ml, reaching the maximum at a dose of 20 lg/
ml. No effect was observed at any dose of goat IgG. Fig. 2A
shows that neutrophils from allergic asthmatic patients cul-
tured with an Ag to which the patients were sensitized (e.g.
neutrophils from a T9 allergic patient cultured with T9 protein
extract) released a significantly higher MMP-9 amount than
neutrophils from healthy donors or from asthmatic patients
cultured with an Ag to which the patients were not sensitized
(P < 0.001). No statistical differences were observed between
unstimulated cells, cells from healthy donors and cells from
allergic subjects cultured in the presence of Ags to which the
patients were not sensitized (Fig. 2A). Nevertheless, MMP-9
release was clearly detected when neutrophils from donors
from all groups were treated with fMLP (Fig. 2A).
(20 lg/ml), anti-IgE (a-IgE) (20 lg/ml) or GM-CSF (G) (10 ng/ml)
plus TNF-a (T) (10 ng/ml) for 24 h. MMP-9 release was measured
in the culture supernatant by ELISA, and a representative zymogra-
phy is shown. MMP-9 purified from human neutrophils is shown
as a control (*). Neutrophils from allergic patients (n = 10) were
incubated with 20 lg/ml of an Ag to which the patient was sensi-
tized, a-IgE or IgG, where indicated, for the indicated times (C) or
with the indicated doses (D) for 24 h, and MMP-9 release was
measured in the culture supernatant by ELISA. The results shown
are the means  SEM. *P < 0.001, vs fMLP, Ag, anti-IgE, G+T.
Ventura et al.
A A
B
C
Figure 2 MMP-9 release in response to allergens is highly specific.
IgE receptor challenge induces MMP-9 release by neutrophils from
allergic patients. (A) Neutrophils obtained from healthy donors (n = 5)
or allergic asthmatic patients (n = 12) were left unstimulated (us),
treated with 20 lg/ml allergen (Ag) to which the allergic patients
were sensitized or not, or fMLP (100 nM) for 24 h. Sensitized:
P < 0.01, vs fMLP and Ag; nonsensitized: P < 0.01, vs fMLP;
healthy: P < 0.01, vs fMLP. (B) Neutrophils from allergic patients
(n = 4) were left unstimulated or treated with fMLP (100 nM), anti-
IgE (a-IgE) (20 lg/ml), an Ag to which the patient was sensitized
(20 lg/ml), anti-FceRI (5 lg/ml), anti-FceRII (5 lg/ml), anti-galectin-3
(5 lg/ml) or mouse IgG (5 lg/ml) for 24 h. *P ≤ 0.01, vs untreated
and IgG; **P < 0.001, vs FceRI, untreated and IgG; †P < 0.01 vs
fMLP, Ag, anti-IgE. (C) Neutrophils from allergic patients (n = 7) were
left untreated or were treated to elute the surface Igs and then chal-
lenged with an Ag to which the neutrophil donor was sensitized
(20 lg/ml), anti-IgE (a-IgE) (20 lg/ml) or fMLP (100 nM) as a positive
control of MMP-9 release, for 24 h. *P ≤ 0.01, vs Ag, a-IgE without
IgE elution. In all cases, MMP-9 release was measured in the culture
supernatant by ELISA, and the results shown are the means  SEM.
MMP-9 is released in response to Abs against FceRI, FceRII/
CD23 and galectin-3 by neutrophils from allergic asthmatic
patients
Experiments were next performed to examine whether the
engagement of IgE receptor/s could mimic the effect
Allergy 69 (2014) 898–905 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Allergen-induced metalloproteinase-9 production by neutrophils
B
Figure 3 A portion of the released MMP-9 is de novo synthesized
in response to allergens. (A) Neutrophils from allergic patients
(n = 8) were pre-incubated for 1 h with cycloheximide (CHD; 5 lg/
ml) or actinomycin D (AcD; 5 lg/ml) prior to the addition of anti-IgE
(a-IgE) (20 lg/ml) or an allergen (Ag) to which the patient was sen-
sitized (20 lg/ml) for 24 h. MMP-9 release was measured in the
culture supernatant by ELISA, and the results shown are the
means  SEM. *P < 0.001, vs CHD and AcD. (B) Neutrophils from
allergic patients (n = 5) were left untreated (—) or incubated with
an Ag to which the patient was sensitized (20 lg/ml) for 24 h.
Expression of MMP-9 mRNA was analysed by real-time PCR analy-
sis. Expression was standardized against b-actin mRNA. Values
shown are mean  SEM. *P < 0.001, vs untreated.
observed over the MMP-9 release in response to Ags. The
effect of agonist Abs against FceRII (9P.25) or galectin-3
(A3A12) promoted the release of MMP-9 into the culture
medium, while anti-FceRI (CRA1) had a lower effect
(Fig. 2B). A nonspecific mouse IgG Ab was used as a nega-
tive control.
We further investigated the participation of IgE in Ag-
induced MMP-9 release. As shown in Fig. 2C, MMP-9
release was not detected when IgE molecules were stripped
from the neutrophil surface prior to Ag/anti-IgE challenge,
but was detected when neutrophils were incubated with
fMLP.
MMP-9 is constitutively expressed but also de novo
synthesized upon allergen challenge by neutrophils from
allergic asthmatic patients
MMP-9 has been described to be constitutively expressed by
neutrophils because it is quickly released (10–20 min) in
response to some inflammatory stimuli such as IL-8, TNF-a,
LPS and granulocyte colony-stimulating factor (G-CSF) (16,
17). However, we could detect the release of MMP-9 only
after 1 h and for up to 24 h of cell stimulation with Ag/anti-
IgE, suggesting that MMP-9 is de novo synthesized. We thus
tested the effect of the inhibitor of protein translation, cyclo-
heximide, and the inhibitor of transcription, actinomycin D,
on this process. As shown in Fig. 3A, both compounds par-
tially cancelled the effect of Ags/anti-IgE upon MMP-9
release, suggesting that part of the released MMP-9 is de
novo synthesized. In addition, real-time PCR analysis
revealed that the amount of MMP-9 mRNA was approxi-
mately twofold increased in response to Ag treatment
(Fig. 3B).
901
Allergen-induced metalloproteinase-9 production by neutrophils
Cell-signalling pathways involved in the allergen-dependent
MMP-9 release by human neutrophils
Previous studies have shown that in neutrophils, fMLP and
PMA induce functional responses through the activation of
cell-signalling pathways such as mitogen-activated protein
kinases (MAPKs), p38 and ERK1/2 (18), and Ca2+/calcineu-
rin (18) and that the NF-jB transcription factor is involved
in MMP-9 expression in breast cancer cells (19). Given that
MMP-9 is released in response to fMLP and Ags, we thus
asked whether these two stimuli share some cell-signalling
pathways. To study the implication of these signalling path-
ways on the modulation of MMP-9 release, we tested the
effect of PD098059 on this process, a specific inhibitor of
MEK1/2, the upstream activator of ERK1/2 (20), and the
effect of SB203580, a specific inhibitor of p38 MAPK (21).
Both compounds were found to effectively block Ag/anti-IgE
Ab-promoted induction of MMP-9 release in neutrophils
from allergic patients (Fig. 4A and 4B). In support to these
findings, we have previously found that ERK1/2 and p38
MAPK signalling pathways are activated in response to Ags/
anti-IgE Abs in human neutrophils (22).
NF-jB is anchored to the cytoplasm by a number of inhib-
itory proteins of the I-jB family, which become degraded at
the proteasome upon cell stimulation with concomitant trans-
location of NF-jB to the nucleus (23). We have previously
found that the NF-jB pathway is activated in response to
Ag challenge (22). We therefore tested whether NF-jB is
A B
C D
Figure 4 Cell-signalling pathways involved in the allergen/anti-IgE-
dependent MMP-9 release by neutrophils from allergic patients.
Neutrophils from allergic patients (n = 6) were pre-incubated for
1 h with PD98059 (PD) (A) or SB203580 (SB) (B) at the indicated
doses prior to the addition of anti-IgE (a-IgE) (20 lg/ml) or an aller-
gen (Ag) to which the patient was sensitized (20 lg/ml) for 24 h.
Neutrophils from allergic patients were pre-incubated for 1 h with
902 Allergy 69 (2014) 898–905 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Ventura et al.
involved in the Ag-dependent MMP-9 release using MG-132,
a specific proteasome inhibitor (23), and BAY 11-7082, a spe-
cific inhibitor of IjB-a phosphorylation and degradation
(24). Both compounds strongly attenuated Ag/anti-IgE-
dependent MMP-9 release (Fig. 4C and 4D).
The involvement of the Ca2+-dependent phosphatase calci-
neurin in the activation of neutrophils by fMLP has been
previously documented (18). We thus next analysed the effect
of cyclosporin A (CsA), a specific inhibitor of calcineurin.
CsA did not have any significant effect upon MMP-9 release
induced by Ags/anti-IgE (Fig. 4E). In agreement, IL-8-medi-
ated MMP-9 release by human neutrophils was found to be
independent of Ca2+ (16).
Discussion
In this study, we have shown that in response to Ag chal-
lenge, neutrophils from allergic asthmatic patients produce
and release MMP-9. This is supported by the following evi-
dences: a) in vitro challenge of highly purified neutrophils
from allergic asthmatic patients with Ags induced the release
of MMP-9; b) the release of MMP-9 is abrogated by cyclo-
heximide and actinomycin D, and the levels of MMP-9
mRNA are increased in response to Ags, supporting de novo
synthesis of MMP-9; and c) anti-IgE Abs and anti-IgE recep-
tors Abs mimic the effect of Ags upon MMP-9 release, and
Ag/anti-IgE-mediated MMP-9 release is cancelled by strip-
ping IgE molecules from the neutrophil surface, suggesting an
E
MG-132 (MG) (n = 5) (C), BAY 11-7082 (BAY) (n = 7) (D) or cyclo-
sporin A (CsA) (n = 5) (E) at the indicated doses prior to the addi-
tion of anti-IgE (a-IgE) (20 lg/ml) or an allergen (Ag) to which the
patient was sensitized (20 lg/ml) for 24 h. MMP-9 release was
measured in the culture supernatant by ELISA, and the results
shown are the means  SEM. *P < 0.001, vs PD98059 (25 lM),
SB203580 (10 lM), MG132 (5 lM), BAY 11-7082 (10 lM).
Ventura et al.
involvement of IgE/IgE receptors. In this sense, it has been
shown that some Ags such as D1 can alternatively activate
neutrophils through protease-activated receptors (PARs) (25).
However, we discard this possibility because we could not
detect MMP-9 release when culturing with D1 neutrophils
from allergic patients not sensitized to this Ag (data not
shown) or from allergic patients sensitized to this Ag but
stripping IgE from the neutrophil surface prior to stimulation
with D1.
Our results are exclusively due to neutrophils and cannot
be ascribed to a possible eosinophil contamination because,
albeit detected, eosinophils were only 0.001–0.004% of the
final cell population and they did not release MMP-9 in
response to Ags. To the best of our knowledge, none of the
identified cellular sources of MMP-9 were found to produce
and release this important inflammatory mediator upon Ag
challenge. Thus, the present data provide the first evidence
that a human cell produces and releases MMP-9 as a conse-
quence of direct Ag challenge. Interestingly, in line with our
results, a recent report has shown that human basophils
release TNF-a following IgE-dependent activation and that
this cytokine subsequently stimulates MMP-9 release from
monocytes (10). Of the metalloproteinases, MMP-9 is of par-
ticular relevance to asthma. Compared with normal subjects,
increased concentrations of MMP-9 have been detected in
the BALF, sputum and serum of asthmatic subjects, and
increased MMP-9 levels and enhanced enzymatic activity
have been reported after Ag challenge (6, 26, 27). In the con-
text of the allergic asthma, the involvement of neutrophils
remains controversial. Asthma is an inflammatory disease
with a complex immunopathology involving several different
cell types and mediators. Eosinophils have been considered
the most important cells in the pathophysiology of asthma
and other allergic diseases. However, the interest in neu-
trophils as important mediators of the asthmatic airway
inflammation has been renewed because they are the first
cells to enter the airway in response to an allergen challenge
and because the presence of airway eosinophilia does not
fully explain this pathologic process (28). The challenge of
neutrophils with specific Ags to which the patients were sen-
sitized leads to the release of IL-8 (11), a potent neutrophil
chemotactic factor and activator, which also induces the
release of MMP-9 (16). Increased levels of IL-8 have been
noted in noneosinophilic asthma (29). This autocrine effect
of IL-8 on neutrophils may prolong the inflammatory process
started by an acute exacerbation. In this sense, we can
exclude the possibility of paracrine effects of IL-8 upon
MMP-9 release for two reasons: first, Abs against IL-8 did
not affect Ag/anti-IgE-mediated MMP-9 release (data not
shown); and second, CsA which inhibited Ag/anti-IgE-medi-
ated IL-8 production (22) did not affect Ag/anti-IgE-depen-
dent MMP-9 release.
Another goal of our study was to unveil the potential sig-
nalling pathways triggered by the challenge of neutrophils
from allergic asthmatic patients with Ags. Numerous reports
have indicated a role of MAPK, particularly ERK1/2 and
p38, in the fMLP-dependent MMP-9 release in neutrophils
(18). On the other hand, an association between MAPK
Allergy 69 (2014) 898–905 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Allergen-induced metalloproteinase-9 production by neutrophils
activity and allergic inflammation has been found previously
in the lungs of asthmatic mice (30) or in the bronchoalveo-
lar lavage fluids from asthmatic patients (31). In agreement
with these results, our previous work showed that the expo-
sure of neutrophils from allergic asthmatic patients to spe-
cific Ags promoted p38 and ERK1/2 activation (22).
Moreover, present data show that the p38-specific inhibitor
SB203580 and the MEK inhibitor PD098059, provoked a
strong down-regulation of Ag-induced MMP-9 release by
human neutrophils.
The involvement of the NF-jB transcription factor in the
expression of MMP-9 by breast cancer cells has been docu-
mented (19). Moreover, NF-jB plays a pivotal role in
allergy (32), and an activation of NF-jB has been found
in mononuclear cells of patients with atopic asthma (33). In
agreement with these results, our previous work showed that
the exposure of neutrophils from allergic asthmatic patients
to specific Ags promoted NF-jB activation (22). In this
light, the present work shows evidence of the participation
of NF-jB in Ag-dependent MMP-9 release in human neu-
trophils as both the proteasome inhibitor, MG-132, and a
specific inhibitor of IjB-a phosphorylation and degradation,
BAY 11-7082, abrogate Ag/anti-IgE-induced MMP-9
release.
We also found that calcineurin is not involved in the pro-
cess, which is in agreement with previous data showing a cal-
cineurin-independent MMP-9 release in response to IL-8 by
human neutrophils (16). However, a possible participation of
NFAT in this process cannot be ruled out, as it has been
described a Cot kinase-dependent calcineurin-independent
NFAT activation (34).
In summary, we present evidence of a new mechanism of
MMP-9 production by human neutrophils from allergic asth-
matic patients, elicited by Ags, and provide information on
the intracellular signal transduction pathways involved in this
process. Our data allow us to envision neutrophils as a
source of MMP-9 in Ag-induced asthmatic reactions, and the
extrapolation of these data to known observations in active
asthma strongly supports the hypothesis that neutrophils
may contribute not only to the allergic inflammation, but
possibly to airway remodelling through the generation of
MMP-9 in allergic asthma.
Acknowledgments
This work was supported by grants from the Junta de And-
alucia (Ayudas Grupos de Investigaci
on), Fundaci
on de la
Sociedad Espa~nola de Alergia e Inmunolog
ıa Cl
ınica, Fun-
daci

on Sanitaria Virgen Macarena, FIS-Thematic Networks
and Co-Operative Research Centres RIRAAF (RD07-0064)
and Fundaci
on Alergol, Spain. Javier Monteseirin is under
the Programa de Intensificaci

on de la Actividad Investigador-
a del Sistema Nacional de Salud.
Author contributions
IV, AV and PC performed the research. CC, EG, VB and
YP performed some aspects of the research. MB performed
903
Allergen-induced metalloproteinase-9 production by neutrophils
aspects of the manuscript preparation. AV and JM wrote the
article. JM designed the research and analysed the data.
Conflicts of interest
All the authors listed in this work declare that they have no
conflicts of interest.
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