Interleukin-17 as a Recruitment and Survival Factor for
Airway Macrophages in Allergic Airway Inflammation
Svetlana Sergejeva, Stefan Ivanov, Jan Lo ¨tvall, and Anders Linde ´n
The Lung Pharmacology Group, Department of Respiratory Medicine and Allergology, Institute of Internal Medicine, Sahlgrenska Academy
at Go ¨teborg University, Gothenburg, Sweden; and The Unit for Lung Investigations, National Institute for Health Development,
(IL)-17, stimulates certain effector functions of human macrophages.
We evaluated whether IL-17 mediates allergen-induced accumula-
to the control of macrophage recruitment and survival. BALB/c
mice were sensitized and challenged with ovalbumin. Three hours
before challenge an anti-mouse IL-17 mAb (a-IL-17) was adminis-
tered. Sampling was conducted 24 h after the allergen challenge.
In vitro chemotaxis assay for blood monocytes and culture of airway
macrophages, immunocytochemistry for Fas-antigen, and matrix
metalloproteinase-9 (MMP-9) were used to determine the effect
of IL-17 on the recruitment, survival, and activity of airway macro-
phages. A-IL-17 reduced the number of airway neutrophils and
macrophages after allergen challenge. In vitro, recombinant IL-17
induced migration of blood monocytes and prolonged survival of
airway macrophages. A-IL-17 also increased the expression of Fas-
antigen in airway macrophages in vivo. Finally, the expression of
MMP-9 by airway neutrophils and macrophages in vivo was down-
regulated by a-IL-17. This study indicates that endogenous IL-17
mediates the accumulation of macrophages during allergen-
induced airway inflammation. IL-17 exerts its effects by acting di-
rectly on airway macrophages by promoting their recruitment and
activity of macrophages and neutrophils in allergen-induced airway
Keywords: mice; asthma; neutrophil; matrix metalloproteinase-9; apo-
increase in the number of various inflammatory cells including
T-lymphocytes, eosinophils, neutrophils, and macrophages (1, 2).
In allergic asthma there is also activation of T-lymphocytes,
cells that via release of proinflammatory cytokines mediate the
mobilization of other inflammatory cells in the airway (1, 3). It
is also known that persistent airway inflammation, such as in
asthma, leads to injury and abnormal repair of airway tissue (4).
Macrophages are predominant cells in bronchoalveolar space
in individuals with and without asthma (5, 6). Moreover, the
in asthma (7). This accumulation of macrophages in asthma may
tion of macrophage chemotactic factors such as monocyte che-
motactic protein (MCP)-1, macrophage inflammatory protein
(Received in original form July 5, 2004 and in final form May 17, 2005)
Correspondence and requests for reprints should be addressed to Svetlana
Sergejeva, M.D., Ph.D., The Lung Pharmacology Group, Department of Respira-
tory Medicine and Allergology, Institute of Internal Medicine, Sahlgrenska Academy
at Go ¨teborg University, Gothenburg, Sweden. Current address: Puuvilla 19A-5,
10314 Tallinn, Estonia. E-mail: Svetlana.Sergejeva@lungall.gu.se
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2004-0213OC on May 18, 2005
Internet address: www.atsjournals.org
Vol 33. pp 248–253, 2005
(MIP)-1?, and granulocyte/macrophage colony-stimulating fac-
tor (GM-CSF) is increased in asthmatic airway (8, 9). However,
it remains to be determined whether these cytokines are partici-
pating in allergen-induced enhanced accumulation of the macro-
phages in asthmatic airway. Furthermore, there is now evidence
of enhanced survival of airway macrophages in the asthmatic
airway (10), and this may be regulated by survival factors such
as interleukin (IL)-1?, tumor necrosis factor (TNF)-?, and
GM-CSF (11). Yet another possibility is that the enhanced sur-
vival of airway macrophages in asthma relates to the degree of
stimulation of the Fas receptor (12), which engagement by its
ligand leads to cell death through apoptosis (13).
tion may be important for determining the severity of airway
inflammation. First, airway macrophages constitute a potentially
powerful source of pro- and anti-inflammatory cytokines as well
with asthma (14–17). Second, airway macrophages are involved
in the removal of cells undergoing apoptosis. Importantly, re-
moval of apoptotic cells occurs before cell lysis (18), and so
release of intracellular contents in the surrounding tissue is
avoided (19, 20). Thus, any endogenous factor that controls
the accumulation of airway macrophages bears the potential to
determine the severity of airway inflammation such as in allergic
The proinflammatory cytokine IL-17 has previously been for-
warded as a link between activated T-lymphocytes and the re-
inflammation (21). Recently it has been shown that the concen-
tration of IL-17 is increased in bronchoalveolar lavage fluid
(BALF), sputum, and blood from patients with asthma (22, 23).
In a mouse model of allergic airway inflammation, systemic
blockade of IL-17 inhibits the allergen-induced accumulation of
neutrophils in the airway (24). In IL-17 knockout mice, the
allergen-induced airway hyperreactivity to methacholine is sig-
nificantly reduced (25). In line with this, increased immunoreac-
tivity for IL-17 in the airway submucosa is associated with im-
paired lung function in patients with asthma (23).
Interestingly, there is now evidence that recombinant IL-17
protein stimulates the release of cytokines such as TNF-?, IL-1?,
and IL-6 in human blood–derived macrophages in vitro (26). In
enzyme matrix metalloproteinase-9 (MMP-9) in the same type
macrophages (27, 28). However, it is not known whether IL-17 is
involved in control of allergen-induced accumulation of macro-
phages and their activity in allergic airway inflammation. In the
current study, we therefore evaluated whether IL-17 mediates
in mice and, if so, whether such an effect relates to the control
of macrophage recruitment and survival.
MATERIALS AND METHODS
This study was approved by the Ethical Committee for Animal Studies
Sergejeva, Ivanov, Lo ¨tvall, et al.: IL-17 Mediates Accumulation of Airway Macrophages249
from B&K Universal AB (Sollentuna, Sweden). The mice were 5–6 wk
old and were maintained under conventional animal housing conditions
and provided with food and water ad libitum.
Allergen Sensitization, Challenge, and IL-17 Blockade
BALB/c mice were sensitized by intraperitoneal injections of 0.5 ml
aluminum-precipitated antigen containing 8 ?g of ovalbumin (OVA;
Sigma Aldrich Sweden AB, Tyreso ¨, Sweden) bound to 4 mg of aluminum
hydroxide (Sigma) in phosphate-buffered saline (PBS) twice, 5 d apart.
tized using isoflurane (Schering-Plough, Welwyn Garden City, UK), and
challenged intranasally with 5 ?g of OVA dissolved in 25 ?l of PBS.
Three hours before OVA challenge, 200 ?g of either an anti-mouse
IL-17 mAb (a-IL-17, clone 50104.11; R&D Systems, Abingdon, UK) or
its isotype control antibody rat IgG2a(clone R35–95; BD Biosciences
Europe, Erembodegem, Belgium) were administrated intravenously
into the lateral tail vein. The total volume of injected solution was
matched between active treatment and control group, being 200 ?l.
Collection and Processing of Cell Samples
were anesthetized with a mixture of xylazin (130 mg/kg, Rompun; Bayer,
Leverkusen, Germany) and ketamine (670 mg/kg, Ketalar; Pfizer AB,
Ta ¨by, Sweden). First, blood was obtained by puncture of the heart
right ventricle. Second, bronchoalveolar lavage (BAL) was performed
through the tracheal cannula by instillation of 0.25 and 0.20 ml of PBS.
Finally, bone marrow (BM) was harvested by excising one femur, which
was cut at the epiphyses and flushed with 2 ml of PBS.
Blood. A mixture was made of 200 ?l of blood with 800 ?l of 2 mM
ethylenediaminetetraacetic acid (Sigma) in PBS. Red blood cells were
solution for 15 min at 4?C. The white blood cells were resuspended in
PBS containing 0.03% bovine serum albumin (BSA; Sigma).
BALF and BM. BALF and BM samples were centrifuged at 300 ? g
for 10 min at 4?C and cell pellet was resuspended in 0.03% BSA in
PBS. The cell-free BAL supernatant was collected for cytokine mea-
surements using commercial ELISA kits (R&D Systems).
The total cell numbers in blood, BALF, and BM were determined
using standard hematologic procedures. Cytospins of blood, BALF, and
BM samples were prepared and stained according to the May-Gru ¨nwald-
Giemsa method for differential cell counting, performed by counting
400 cells, using a conventional light microscope (Zeiss Axioplan 2;
Carl Zeiss, Jena, Germany). The cells were identified using standard
All immunocytochemistry (ICC) procedures were performed at room
temperature unless otherwise stated. Cells were determined by count-
ing 400 cells using a light microscope (magnification ?1,000, Zeiss
Axioplan 2; Carl Zeiss).
ICC for Fas extracellular domain. Cytospin preparations were fixed
with 2% formaldehyde during 30 min, followed by incubation in pre-
heated basic antigen retrieval reagent (R&D Systems) at 94?C for 4 min.
Unspecific binding was blocked using 10% donkey serum (Jackson
ImmunoResearch Laboratories, West Grove, PA), and endogenous
biotin was blocked with Biotin Blocking System (DAKO Corporation,
Glostrup, Denmark). Slides were incubated with a purified anti-mouse
Fas/TNFRSF6 mAb (R&D Systems) over night at 4?C. As sec-
ondary antibody a biotinylated F(ab?)2-fragment donkey anti-goat IgG
(Jackson ImmunoResearch Laboratories) was used, followed by alka-
line phosphatase–conjugated streptavidin (DAKO). Bound antibodies
were visualized with Vector Red alkaline substrate kit (Vector Labora-
tories, Burlingame, CA). Mayer’s Hematoxylin (Sigma) was used for
ICC for MMP-9. Cytospin preparations were fixed with 2% formal-
dehyde during 30 min. Unspecific binding and endogenous biotin were
blocked as described above. Slides were incubated with a purified anti-
mouse MMP-9 mAb (R&D Systems) during 1 h, followed by the same
secondary antibody and detection system as described previously.
In Vitro Experiments
For in vitro experiments, mice were sensitized and challenged with
OVA or PBS as described above. BAL was performed by instillation
of 1 ml of PBS four times. Blood was obtained by puncture of the heart
right ventricle. For each single experiment, BALF or blood from 15–20
mice was pooled to obtain a sufficient cell number. Cell enrichment was
(Miltenyi Biotec, Bergisch Gladbach, Germany). BALF macrophages
or blood monocytes were enriched using a biotinylated Griffonia
simplicifolia lectin 1 antibody (Vector Laboratories) (29). Results from
in vitro experiments are shown as the average from three separate and
Chemotaxis assay. Blood monocytes were brought to the final con-
centration of 0.5 ? 106/ml in RPMI medium containing 1% BSA. The
chemotaxis assay was performed in a 48-well microchemotaxis chamber
(Neuroprobe, Cabin John, MD) as previously described (30). Briefly,
the solutions and equipment were brought to 37?C before onset of the
experiment. The bottom wells of the chamber (at least triplicate for
each condition in each experiment) were filled with 28 ?l of fluid
containing either the 1% BSA/RPMI (negative control), the 10 ng/ml
or recombinant MCP-1 (positive control; R&D Systems), or 10 ng/ml
of recombinant IL-17 (R&D Systems). A polycarbonate filter with pore
size of 8 ?m (Nucleopore, Pleasanton, CA) was placed over the bottom
wells. The silicon gasket and upper piece of the chamber were applied,
and 50 ?l of monocyte suspension was pipetted into upper wells. The
chamber was incubated in humidified air with 5% CO2at 37?C for
1 h, then disassembled, and the filter was removed. The filter was then
fixed in methanol, stained according to the May-Gru ¨nwald-Giemsa
method, and mounted on a glass slide. In each well, monocytes that com-
(magnification ?1,000; Zeiss Axioplan 2, Carl Zeiss). The chemotactic
response was expressed as a migration index. For each chemotactic
stimulus (recombinant MCP-1 or IL-17 protein), a migration index was
calculated by dividing the number of migrated cells in response to the
cytokine by the number of cells that migrated randomly, that is, in
response to 1%BSA/RPMI alone. Thus, a reference index exceeding
1 indicates chemotaxis.
Culture of BALf macrophages. BALF macrophages were seeded in
a concentration of 0.625 ? 106/ml in a 96-well plate (BD Biosciences)
from Sigma) at 37?C in an atmosphere containing 5% CO2for 20 h.
The seeded fraction contained 92.7 ? 2.3% macrophages (n ? 3) as
determined using May-Gru ¨nwald-Giemsa staining. For BAL macro-
phages from OVA- and PBS-exposed animals, the following treatment
groups (at least triplicate for each condition in each experiment) were
established: (1) Negative control (i.e., vehicle with no recombinant
mouse IL-17 protein); (2) Recombinant IL-17 protein (R&D Systems)
0.1 ng/ml; and (3) IL-17 protein 1 ng/ml. The trypan blue exclusion
test was performed at baseline before and at the end of the 20-h experi-
ment. The baseline viability of the seeded cells from OVA- and PBS-
exposed animals, respectively, was 76.5 ? 1.9% and 81.3 ? 3.7% (n ?
3). After each experiment, the conditioned medium was collected for
Data are presented as mean with SEM. Statistical analysis was per-
formed using nonparametric ANOVA (Kruskal-Wallis test) to evaluate
variance among all groups. If a significant variance was found, an un-
paired two-group test (Mann-Whitney test) was used to determine
significant differences between individual groups. Spearman Rank cor-
relation test was used for detection of the relationship between two
variables. P ? 0.05 was considered statistically significant.
Pretreatment with a-IL-17 decreased the number of macro-
phages and neutrophils in BALf from sensitized and allergen-
challenged mice (P ? 0.009 and P ? 0.03, respectively; Figure 1).
In contrast, a-IL-17 caused no statistically significant changes
250AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 332005
Figure 1. BALF differential cell counts in ovalbumin-sensitized and chal-
lenged BALB/c mice. Mice received systemic pretreatment with an anti-
mouse IL-17 mAb (a-IL-17, filled columns) or an isotype control antibody
(IgG2a, open columns). Data are shown as mean with SEM from two
independent experiments. *P ? 0.05, n ? 12–13.
in the corresponding number of eosinophils, lymphocytes, or
basophils (Figure 1).
Pretreatment with a-IL-17 increased the expression of Fas
antigen in BALF macrophages (P ? 0.0004; Figure 2), but not
example of the ICC staining for Fas antigen in BALF cytospin
is illustrated in Figure 3.
sion of MMP-9 in BALF macrophages (P ? 0.004; Figure 4)
and in BALF neutrophils (P ? 0.04; Figure 4), but not in BALF
eosinophils (10 ? 7 versus 20 ? 16% for a-IL-17 and isotype
control antibody, respectively, P ? 0.5). A typical example of
the ICC staining for MMP-9 in BALF macrophages and neutro-
phils is illustrated in Figure 5.
The measurements of cytokines in BALF samples did not indi-
cate any significant differences in the concentration of MIP-2
(n ? 7, 27 ? 4 versus 24 ? 2 pg/ml, P ? 0.5), TNF-? (n ? 12–13,
39 ? 4 versus 41 ? 2 pg/ml, P ? 0.6), IL-6 (n ? 5, 269 ? 79
versus 241 ? 57 pg/ml, P ? 0.7), macrophage colony-stimulating
Figure 2. Relative number of Fas-
positive BALF macrophages in
OVA-sensitized and -challenged
BALB/c mice. Mice received sys-
temic pretreatment with a-IL-17
or IgG2a. Data are shown as mean
with SEM. *P ? 0.05, n ? 12–13.
Figure 3. Photograph of ICC staining for Fas antigen in BALF cytospin
(?1,000). Red staining indicates Fas antigen. The number 1 indicates a
Fas?eosinophil, 2 indicates a Fas?macrophage, 3 indicates a Fas?macro-
phage, N indicates cell nucleus, and C indicates cell cytoplasm.
factor (M-CSF) (n ? 5, 55 ? 2 versus 57 ? 2 pg/ml, P ? 0.5),
or MCP-1 (n ? 12–13, 11 ? 3 versus 34 ? 15 pg/ml, P ? 0.2) for
respectively. GM-CSF was not detectable in any of the BALF
samples from either treatment group (n ? 12–13).
Blood and BM
Pretreatment with a-IL-17 did not lead to any statistically signi-
ficant changes in total or differential cell counts in blood (Figure
6A) or BM (Figure 6B).
Chemotaxis of Blood Monocytes
Recombinant mouse MCP-1 induced migration of blood mono-
cytes from OVA-challenged but not from PBS-exposed animals
the positive control MCP-1, recombinant mouse IL-17 protein
induced substantial migration of blood monocytes harvested from
the OVA-challenged animals (P ? 0.0125; Figure 7).
Culture of BALF Macrophages
Stimulation with recombinant mouse IL-17 protein prolonged
the survival ofBALF macrophages in vitro fromboth OVA- and
Figure 4. Relative number
of MMP-9–positive BALF
and -challenged BALB/c
mice. Mice received sys-
temic pretreatment with
a-IL-17 (filled columns)
or IgG2a(open columns).
Data are shown as mean
with SEM. *P ? 0.05,
n ? 6.
Sergejeva, Ivanov, Lo ¨tvall, et al.: IL-17 Mediates Accumulation of Airway Macrophages251
Figure 5. Photograph of ICC staining for MMP-9 in BALF cytospin
(?1,000). Red staining indicates MMP-9. The number 1 indicates a
significant manner (Rs ? 0.8, P ? 0.0001 and Rs ? 0.6, P ? 0.02,
respectively; Figure 8). The IL-17–induced (1ng/ml) increase in
survival was higher for macrophages from animals challenged
with OVA compared with those from PBS-exposed mice (33.8
versus 20.1%, respectively, P ? 0.0495).
Stimulation of BALF macrophages recovered from OVA-
challenged mice with recombinant IL-17 protein did not mark-
edly change the concentration of GM-CSF, MIP-2, or MCP-1
protein in the conditioned medium (n ? 3–6, data not shown).
Our study shows that intravenous pre-treatment with neutraliz-
ing a-IL-17 decreases the number of BALF macrophages and
neutrophils after allergen challenge in sensitized mice in vivo.
sion on BALF macrophages, but not on other airway cells in vivo.
In addition, recombinant IL-17 protein is chemotactic for blood
monocytes and it prolongs the survival of BALF macrophages
the number of MMP-9–positive macrophages and neutrophils
in BALF in vivo.
Even though it is now well established that the expression of
IL-17 is increased in the airway of patients with asthma (22, 23),
the pathogenetic role of IL-17 in allergic inflammation is poorly
understood. Indeed, there is only one published study on the
effect of systemic blockade of IL-17 during allergic airway in-
flammation in mice (24). The referred study indicated that sys-
temic blockade of IL-17 reduces neutrophil number and in-
creases eosinophil number after allergen exposure in sensitized
airway, and suggested alterations in BM granulocytopoiesis and
chemotaxis of mature granulocytes as plausible mechanisms of
action. In our study, we now confirm a corresponding role of endo-
genous IL-17 in allergen-induced accumulation of neutrophils
in the airway. Moreover, our study expands the role of IL-17
in allergic inflammation by showing that systemic blockade of
IL-17 also attenuates the allergen-induced accumulation of mac-
rophages in sensitized airway. This effect of blocking IL-17 oc-
curs without any substantial changes in the cell counts in blood
or BM in our study. It seems likely that the observed discrepan-
cies in IL-17 blockade-caused alterations in number of airway
macrophages and eosinophils for our study and the study by
Hellings and coworkers are due to differences between the re-
Figure 6. (A) Blood differential cell counts and (B) relative number of
BM eosinophils and neutrophils in OVA-sensitized and -challenged
BALB/c mice. Mice received systemic pre-treatment with a-IL-17 (filled
columns) or IgG2a(open columns). Data are shown as mean with SEM
(n ? 12–13).
tocol for allergen exposure, the dose of neutralizing anti–IL-17
antibody, the route and timing of anti–IL-17 antibody adminis-
tration, and the timing of sample harvest. In agreement with
previous observations on the role of IL-17 in endotoxin-induced
airway inflammation (31), our data imply that endogenous IL-17
primarily exerts an effect on airway neutrophils and macro-
phages via local rather than systemic mechanisms.
One of the plausible mechanisms behind a-IL-17–caused re-
duction in the number of airway macrophages after allergen
challenge is the inhibition of cell recruitment into the airway.
To assess this hypothesis, we first evaluated whether recombi-
nant IL-17protein hasan effecton themigration ofblood mono-
cytes harvested from allergen-challenged or vehicle-exposed
mice and subsequently cultured in vitro. Our study shows that
recombinant IL-17 protein directly induces the migration (i.e.,
chemotaxis) of blood monocytes from allergen-challenged mice.
Moreover, the chemotactic effect of IL-17 was of similar magni-
tude as that of positive control—MCP-1. Because, in the case
of neutrophils, the accumulating effect of IL-17 is believed to
be indirect, mediated mainly via secondary mediators (21), we
252AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 332005
Figure 7. Blood monocyte
migration caused by 1-h
stimulation with recom-
binant MCP-1 or IL-17
protein. Blood monocytes
were recovered from PBS-
exposed (open columns)
or OVA-challenged (filled
columns) BALB/c mice.
Data are shown as mean
values from three sepa-
rate experiments, each
licate wells. *P ? 0.05.
also evaluated whether endogenous IL-17 exerts an effect on
the macrophage chemotactic factors after allergen challenge.
However, systemic pretreatment with a-IL-17 did not cause any
substantial changes in the concentration of either MCP-1,
that endogenous IL-17 constitutes a recruitment factor with direct
in allergen-induced airway inflammation.
Another plausible mechanism behind the effect of endoge-
ligand system is recognized as a major extrinsic pathway of
apoptosis (13). Fas antigen belongs to the TNF receptor family,
is expressed in macrophages, and its binding to ligand induces
interest that, in our study, pretreatment with a-IL-17 caused an
increase in the expression of Fas antigen selectively in airway
macrophages in sensitized and allergen-challenged mice. This
via downregulation of programmed cell death mediated by the
Fas–Fas ligand system. In support of these in vivo observations,
in vitro prolonged their survival in concentration-dependent man-
ner. It is noteworthy that macrophages from allergen-challenged
mice were more susceptible to IL-17 than were macrophages
from vehicle-exposed mice. Taken together, these data forward
endogenous IL-17 as a local survival factor for airway macro-
phages during allergic airway inflammation, acting in part through
the inhibition of Fas-mediated cell apoptosis.
MMP-9 is the most abundant MMP present in BALF from
patients with asthma (32). Importantly, the MMP-9 concentra-
tion is increased in subjects with asthma compared with healthy
subjects (32). In our study, we found that pretreatment with
mice. The potential pathogenetic relevance of this finding is
indicated by the fact that in asthma, the immunoreactivity for
MMP-9 in airway tissue is linked to disease severity, including
impaired lung function and increased numbers of tissue neutro-
tissue-degrading collagenolytic and elastolytic activity (34), our
findings prompt for further investigation of the pathogenetic
significance of IL-17 in controlling proteolytic load in the airway
during allergic airway inflammation.
Figure 8. The effect of 20 h stimulation with recombinant IL-17 protein
on the survival of BALF macrophages, recovered from PBS-exposed
(open columns) or OVA-challenged (filled columns) BALB/c mice. Data
are shown as mean values from three separate experiments, each per-
formed in at least triplicate wells.
nized role of endogenous IL-17 in allergic airway inflammation:
IL-17 contributes to the local accumulation of macrophages in
bronchoalveolar space by recruiting macrophage precursor cells
and by increasing the survival of macrophages within the airway.
In addition, IL-17 may indirectly control the local load of poten-
eling in the airway, which is fully compatible with recent data
on IL-17 in the airway of patients with asthma (22, 23).
Conflict of Interest Statement: None of the authors have a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors are grateful to Carina Malmha ¨ll, B.Sc., for techni-
cal assistance during the progress of the study.
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