The Journal of Immunology
IL-17A and TNF-a Exert Synergistic Effects on Expression of
CXCL5 by Alveolar Type II Cells In Vivo and In Vitro
Yuhong Liu,*,1Junjie Mei,*,1Linda Gonzales,*,1Guang Yang,* Ning Dai,*
Ping Wang,* Peggy Zhang,* Michael Favara,* Kenneth C. Malcolm,†Susan Guttentag,*,‡
and G. Scott Worthen*,‡
CXCL5, a member of the CXC family of chemokines, contributes to neutrophil recruitment during lung inflammation, but its
regulation is poorly understood. Because the T cell-derived cytokine IL-17A enhances host defense by triggering production of
chemokines, particularly in combination with TNF-a, we hypothesized that IL-17A would enhance TNF-a–induced expression
of CXCL5. Intratracheal coadministration of IL-17A and TNF-a in mice induced production of CXCL1, CXCL2, and CXCL5,
which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-a and IL17Awere
greatly attenuated in Cxcl52/2mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild-
type mice. Bone marrow chimeras produced using Cxcl52/2donors and recipients demonstrated that lung-resident cells were the
source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A
enhanced TNF-a–induced CXCL5 transcription and stabilized TNF-a–induced CXCL5 transcripts. Whereas expression of
CXCL5 required activation of NF-kB, IL-17A did not increase TNF-a–induced NF-kB activation. Apical costimulation of IL-
17A and TNF-a provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimu-
lation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized
fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial
injury.The Journal of Immunology, 2011, 186: 3197–3205.
pattern recognition receptors. This recognition triggers the acti-
vation of transcription factors, release of early response cytokines
(particularly TNF-a) (1–3) and chemokines, recruitment of neu-
trophils, and consequent clearance of microorganisms (4–6). Al-
though alveolar macrophages (AMs) have been accepted as an
important component of innate immunity in the lung, alveolar type
II (ATII) cells have recently been demonstrated to be an active
participant in the innate immune response. ATII cells participate
in lung host defense not only by secreting surfactant lipids (7) and
surfactant-associated proteins (8) to block and opsonize bacteria
and virus, but also by releasing chemokines to facilitate movement
of immune cells (9, 10). Despite these insights, the role of specific
ung host defense requires both innate and adaptive im-
mune responses acting in a highly coordinated manner.
Innate responses are initiated by microbial recognition via
signals in induction of ATII cell responses, and which cytokines
and chemokines are released, remains unclear.
In addition to innate responses, adaptive immune responses,
T and B lymphocytes, are important in host defense (11, 12).
Recent evidence suggests that a specific subset of CD4+T cells
secreting IL-17 plays a nonredundant role in directing innate
responses (12). IL-17A is the prototype member of IL-17 family,
which is associated with increased neutrophil influx through in-
duction of proinflammatory cytokines and chemokines during
bacterial infection (13–16). Which lung cell responds to IL-17A,
however, and the signals released that promote neutrophil accu-
mulation is not known. The best-known candidates, however, are
glutamic acid-leucine-arginine (ELR)+CXC chemokines.
CXCL5 (known as LPS-induced CXC chemokine [LIX] in mice
and epithelial cell-derived neutrophil-activating peptide-78 [ENA-
78] in human) is a unique member of ELR+CXC family of
chemokines, which are responsible for mediating neutrophil re-
cruitment into tissues during inflammation and infection (17).
ENA-78 has been found to be involved in a variety of human
inflammatory diseases (18–27), such as chronic obstructive pul-
monary disease, exacerbation of asthma and chronic obstructive
pulmonary disease, acute coronary syndromes, diabetes mellitus,
and inflammatory bowel diseases. Murine Cxcl5 was first cloned
as a LPS response gene that could be attenuated by glucocorti-
coids (28). In comparison with other CXC chemokines, specifi-
cally CXCL1 (also referred to as keratinocyte chemoattractant
[KC]) and CXCL2 (also referred to as MIP-2), CXCL5 was shown
to have distinct induction kinetics, tissue distribution, and sensi-
tivity to glucocorticoids in an acute endotoxemia model (29). We
have previously shown (10) that CXCL5 was expressed by ATII
cells through immunohistochemical analysis and primary rodent
ATII cell culture, in response to LPS stimulation, whereas KC and
*Division of Neonatology, Children’s Hospital of Philadelphia, Philadelphia, PA
Denver, CO 80206; and‡University of Pennsylvania, Philadelphia, PA 19014
†Department of Medicine, National Jewish Medical and Research Center,
1Y.L., J.M., and L.G. contributed equally to this work.
Received for publication June 29, 2010. Accepted for publication December 23,
This work was supported by National Institutes of Health Grant HL068876.
Address correspondence and reprint requests to Dr. G. Scott Worthen, Division of
Neonatology, Abramson Research Center, Room #414C, Children’s Hospital of Phil-
adelphia, Philadelphia, PA19014. E-mail address: firstname.lastname@example.org
Abbreviations used in this article: AM, alveolar macrophage; ATII, alveolar type II
cell; BALF, bronchoalveolar lavage fluid; BM, bone marrow; CHX, cycloheximide;
DCI, dexamethasone plus 8-Br-cAMP plus isobutylmethylxanthine; ELR, glutamic
acid-leucine-arginine; ENA-78, epithelial cell-derived neutrophil-activating peptide-
78; i.t., intratracheal; KC, keratinocyte chemoattractant; LIX, LPS-induced CXC
chemokine; qPCR, quantitative PCR; rm, recombinant mouse; WT, wild-type.
MIP-2 are mainly expressed by alveolar macrophages. We further
generated Cxcl5-deficient mice and demonstrated the nonre-
dundant but opposing roles of CXCL5 in mediating neutrophil
influx to the lung in a localized LPS inhalation model and a severe
Escherichia coli pneumonia model (30). Upon LPS inhalation,
CXCL5 expression in bronchoalveolar lavage fluid (BALF) was
much more persistent than KC and MIP-2, which contributed to its
dominant role in mediating neutrophil influx to the lung. Fur-
thermore, in a model of severe E. coli pneumonia, CXCL5 release
induced high levels of KC and MIP-2 in both BALF and plasma
at least partially through inhibiting chemokine scavenging and
thereby modifying the inflammatory response. Although our group
has described CXCL5 as a product of ATII cells in mice and rats,
and as a critical player in lung inflammation and pulmonary host
defense, whether human ATII cells respond similarly is unknown.
Furthermore, the stimuli that induce CXCL5 in the ATII cell are
poorly characterized. Because previous reports have indicated that
IL-17A in combination with TNF-a increased synergistically the
expression of chemokines (16, 17), we hypothesized that IL-17A
combined with TNF-a would exert synergistic effects on CXCL5
expression during lung inflammation. We further tested whether
such synergistic effect would be manifested in primary human
ATII cells, well known to be critical for development as well as
In this study, we first showed the expression of IL-17A, TNF-a,
and CXCL5 in severely inflamed mouse lungs. A synergistic ef-
fect on neutrophil influx was induced by intratracheal (i.t.) co-
administration of IL-17A and TNF-a, and this was associated with
increased expression of CXCL1 and CXCL5. In contrast to
CXCL1, CXCL5 played a dominant role in neutrophil influx
stimulated with IL-17A and TNF-a coinstillation at a later phase
of influx. In differentiated primary human fetal ATII cells, we
demonstrated, for the first time to our knowledge, expression of
CXCL5 by human ATII cells and confirmed the synergistic effect
of IL-17A and TNF-a on induction of CXCL5. The mechanism of
this effect was due to both transcription and mRNA stability and
required NF-kB activation and new protein synthesis. We also
showed, to our knowledge for the first time, a unique polarization
both of stimulation and release of CXCL5 in ATII cells, which
may have an important role in acute lung injury during lung in-
flammation and infection.
Materials and Methods
E. coli strain (ATCC 25922) was purchased from American Type Culture
Collection. Mouse rIL-17A (421-ML/CF), TNF-a (410-MT/CF), human
rIL-17A (317-IL), TNF-a (210-TA), mouse CXCL5/LIX (DY443), and
human CXCL5/ENA-78 ELISA kit (DY254) were obtained from R&D
Systems. IkBa phosphorylation inhibitor BAY-11-7082 was obtained from
Calbiochem. Stock solutions of the inhibitors were prepared in DMSO.
The actinomycin D (A9415) was from Sigma-Aldrich.
Generation of Cxcl52/2mice has been described (30). Cxcl52/2mice
(backcrossed with C57BL/6J mice for six generations) and their control
C57BL/6 mice were kept in specific pathogen-free conditions in the animal
facility of the Children’s Hospital of Philadelphia. All experimental pro-
cedures were in compliance with guidelines approved by the Institutional
Animal Care and Use Committee of the Children’s Hospital of Phila-
delphia. Eight- to 12-wk-old and sex- and age-matched mice were used for
E. coli pneumonia mouse model
The procedures for the E. coli pneumonia (ATCC 25922; American Type
Culture Collection) mouse model have been previously described (31).
Briefly, the mice were anesthetized with ketamine (100 mg/kg) and
xylazene (10 mg/kg), and a midventral incision was performed. After
isolation of muscles, the trachea was exposed, and each mouse was in-
oculated with 106or 107CFU E. coli in 50 ml in 0.9% saline. The mice
were humanely sacrificed at 8 and 24 h postinfection, and BALF was col-
Intratracheal administration of recombinant mouse IL-17A
and/or recombinant mouse TNF-a
C57BL/6 mice were divided into four groups as follows: 1) PBS alone; 2)
recombinant mouse (rm)IL-17A (625 ng/mouse) alone; 3) rmTNF-a (500
ng/mouse) alone; and 4) rmIL-17A plus rmTNF-a. All treatments were
administered in a volume of 50 ml. Mice were euthanized at 4, 8, and 24 h.
Cxcl52/2mice were compared with C57BL/6 mice receiving rmIL-17A
plus rmTNF-a at 8 and 24 h.
Generation of chimeric mice by bone marrow reconstitution
The 6–8-wk-old recipient mice were lethally irradiated in two doses of 800
and 400 rad with an interval of 3 h. Bone marrow (BM) from donor mice
was harvested from both the tibiae and femora. After lysis of red cells, ∼5
million cells were i.v. injected into recipient mice after lethal irradiation.
The BM reconstitution was performed in four groups of mice: 1) BM from
wild-type (WT) mice into WT mice; 2) BM from WT mice into Cxcl52/2
mice; 3) BM from Cxcl52/2mice into WT mice; and 4) BM from Cxcl52/2
mice into Cxcl52/2mice. Eight weeks after irradiation, mice were used for
106E. coli i.t. inoculation. Analysis of blood genotypes after reconstitution
indicated no admixture of recipient cells (data not shown).
Analysis of leukocytes in BALF
At specified times after treatment, the mice were anesthetized and then
of 3.2 ml containing 100 mM diethylenetriamine pentaacetic acid. Leuko-
by cytospin. Slides were fixed and stained with Diff-quik staining kit
(Sigma-Aldrich), and the neutrophil counts were calculated by multiplying
neutrophil percentage by total number of leukocytes in the lavage.
Human fetal lung tissue (16–20 wk gestation) was obtained from Advanced
Bioscience Resources (Alameda, CA) under a protocol approved by
Children’s Hospital of Philadelphia. Explants of the fetal lung were cul-
tured 4 d in dexamethasone (10 nM) plus 8-Br-cAMP (0.1 mM) plus
isobutylmethylxanthine (0.1 mM) (DCI) at 37˚C in 5% CO2incubator to
induce a type II cell phenotype as described (32). A highly enriched
population of type II cells was isolated as described (33) and cultured in
DCI to maintain the type II phenotype.
Nuclear run-on assay
For the measurement of gene transcription, nuclear run-on assay was
performed following the protocol of Hildebrandt and colleagues (34).
Briefly, nuclei were isolated from human fetal ATII cells treated with or
without TNF-a (10ng/ml) in the presence of IL-17 (25 ng/ml) for 24 h.
Transcripts that were initiated in the cells were allowed to continue in the
presence of NTPs for 15 min at 22˚C. RNA was extracted and reverse-
transcribed using the Superscript First-Strand System (Invitrogen)
according to the manufacturer’s instructions. Real-time quantitative PCR
(qPCR) was performed using TaqMan Univeral PCR Master Mix kit
(Applied Biosystems) with the TaqMan Gene Expression Assays (Applied
ENA-78 mRNA stability
Human alveolar type II cells were treated with TNF-a (10 ng/ml) in the
absence or presence of IL-17A (10 ng/ml) for 10 h. Cells were then washed
and incubated with actinomycin D (5 mg/ml) to inhibit further transcrip-
tion. Total RNA was extracted at 0, 4, 8, and 12 h, and CXCL5/ENA-78
mRNAwas quantified by qPCR. The percentages of the remaining mRNA
at different time points as compared with 0 h were calculated.
Luciferase reporter assay
Human ATII cells were seeded onto 96-well plates at a cell density of 5 3
104cells/well in Waymouth’s medium (11220; Life Technologies) with
DCI and incubated at 37˚C in 5% CO2incubator. On day 4, the cells were
infected with adenovirus vectors that had NF-kB–driven firefly luciferase
element (3 3 105PFU/well) and pRL-SV40-driven renilla luciferase el-
ement (3 3 105PFU/well) (Vector Biolaboratories, Philadelphia, PA).
3198SYNERGISTIC EFFECTS OF IL-17A AND TNF-a ON CXCL5 EXPRESSION
Twenty-four hours postinfection, the cells were then treated with IL-17A,
TNF-a, or both for 24 h in fresh medium. The cells were assayed for firefly
and renilla luciferase activities using Dual-Luciferase Reporter Assay
System (Promega). Luciferase activity was measured by the IVIS Imaging
System (Series 100; Caliper Life Sciences). Data was normalized as in-
Human ATII cell monolayers on transwell filters
Human ATII cells were cultured on transwell filters (collagen-coated
transwell, 12-mm diameter, 0.4 mm pore size; Corning). The human
ATII cells were seeded at 2.5 3 105cells/cm2in a volume of 500 ml/filter.
In the lower compartment, 1.5 ml medium were added, thereby leveling
the height of the liquid levels to prevent hydrostatic pressure. Trans-
epithelial resistance was measured was measured at .300 V/cm2prior to
stimulation of the cells.
Multiplex 42-bead array assay
The 96-well filter plate was prewetted with 200 ml assay buffer per well,
then the buffer was removed using a vacuum manifold. Each standard or
control or sample (25 ml) was added into the appropriate wells. Assay
buffer was used for background. A total of 25 ml assay buffer was added to
the sample wells, and the same amount of cell culture medium was added
to the rest of the wells. The premixed beads (25 ml) were loaded into each
well and incubated overnight at 4˚C with shaking. The next day, the beads
were washed two times by vacuum, and detection Abs were added. After
incubation for 1 h at room temperature, 25 ml streptavidin-PE was pipetted
into each well. The beads were washed twice and then resuspended in 150
ml sheath fluid. The filter plate was run on Luminex 200.
Data are presented as means 6 SEM. We used one-way ANOVA or two-
way ANOVA to compare datasets.
Expression of IL-17A, TNF-a, and CXCL5 in mouse models of
To investigate the expression patterns of IL-17A, TNF-a, and
CXCL5 in pulmonary inflammation, we challenged C57BL/6
mice with 107CFU E. coli via i.t. inoculation. Mice exposed to
E. coli challenge demonstrated increased IL-17A protein in BALF
8 h postinoculation that was sustained to 24 h (Fig. 1A). TNF-a
protein level was markedly increased 8 h after E. coli inoculation
and was decreased to 24 h (Fig. 1B). CXCL5, though increased at
8 h, continued to increase through 24 h postinoculation (Fig. 1C).
Thus, expression of CXCL5 is associated with protein production
of IL-17A and TNF-a in BALF after E. coli challenge.
Exogenous administration of IL-17A and TNF-a into the lung
exerts synergistic effect on the expressions of ELR+CXC
chemokines and neutrophil influx
To further explore the role of IL-17A and TNF-a in pulmonary
inflammatory responses, we administered IL-17A and TNF-a,
alone or in combination, via i.t. instillation to C57BL/6 mice. Lung
lavage was performed at 4, 8, and 24 h. IL-17A and TNF-a acted
synergistically to promote neutrophil influx to the lung at 8 and
24 h (Fig. 2F). IL-17A and TNF-a together had a synergistic effect
on CXCL5 expression at later time points (Fig. 2A). By comparison,
the synergistic effect of IL-17A and TNF-a to increase CXCL1
(Fig. 2B) and CXCL2 (Fig. 2C) was evident only at early time
points. The combination of two cytokines also synergistically
increased G-CSF expression (Fig. 2D) that was detectable at all
CXCL5 in mouse lung in response to E. coli i.t. chal-
lenge. C57BL/6 mice were subjected to 107CFU E.
coli via i.t. inoculation; BALF was collected at differ-
ent time points. The protein levels of IL-17A (A), TNF-
a (B), and CXCL5 (C) in the BALF were measured by
ELISA. Values are mean 6 SEM (n = 3–6). *p , 0.05
versus saline control group, **p , 0.01, ***p , 0.001.
Expression of IL-17A, TNF-a, and
neutrophil influx. C57BL/6 mice were i.t. instilled with PBS, IL-17A (625 ng/50 ml), TNF-a (500 ng/50 ml), or IL-17A + TNF-a ([625+500] ng/50 ml).
Whole lung lavage was performed at 4, 8, and 24 h. The protein levels of CXCL5 (A), CXCL1 (B), CXCL2 (C), and G-CSF (D) in the BALF were measured
by ELISA. The number of WBCs (E) and neutrophils (F) per milliliter of BALF were determined by light microscopy. Values are mean 6 SEM (n = 3–4).
*p , 0.05 versus PBS, **p , 0.01, ***p , 0.001.
Exogenous administration of IL-17A and TNF-a into the lung exerts synergistic effect on expression of ELR+CXC chemokines and
The Journal of Immunology3199
time points. None of the CXC chemokines, CXCL5, CXCL1, or
CXCL2, were expressed in response to IL-17A alone, whereas the
expression of CXCL5 and CXCL1 was increased by TNF-a
stimulation alone at 24 and 4 h, respectively (Fig. 2A, 2B).
CXCL5 plays a dominant role in neutrophil influx after i.t.
administration of IL-17A and TNF-a by 24 h after exposure
Because significant enhancement of neutrophil recruitment (Fig.
2F) upon coadministration of IL-17A and TNF-a was associated
with upregulated CXCL5 expression (Fig. 2A) in the mouse lung at
8 and 24 h, we hypothesized that CXCL5 in the lung may play
a dominant role in neutrophil influx at later stages. To test this
hypothesis, we coadministered IL-17A and TNF-a into Cxcl52/2
and C57BL/6 WT mice. Although CXCL1 expression (Fig. 3B)
was higher in the Cxcl52/2mice than that of the WT mice after 8 h
of coadministration of IL-17A and TNF-a, there was no signifi-
cant difference in neutrophil influx (Fig. 3E) between the two
groups. In contrast, in the Cxcl52/2mice receiving IL-17A and
TNF-a for 24 h, the total WBCs and neutrophils in the BALF
were significantly reduced as compared with the WT mice (Fig.
3D, 3E). Despite the absence of CXCL5 in the targeted mice (Fig.
3A), CXCL1 (Fig. 3B) and CXCL2 (Fig. 3C) in the BALF were not
different from WT animals receiving IL-17A and TNF-a for 24 h.
The response to deletion of CXCL5 in the setting of similar
amounts of CXCL1 and CXCL2 at this time point indicates that
CXCL5 plays a dominant role in neutropil influx to the lung in
response to i.t. coadministration of IL-17A and TNF-a at this later
Pulmonary-resident cells are the primary source of CXCL5
upon E. coli challenge
To determine which cells were responsible for CXCL5 production,
we generated BM-reconstituted chimeric mice using WT and
Cxcl52/2mice as both donors and recipients. After lethal irradi-
ation of both WT (denoted as W in the second letter) and Cxcl52/2
recipients (denoted as K in the second letter), these mice were
reconstituted with BM of healthy WT (denoted as W in the first
letter) or Cxcl52/2donors (denoted as K in the first letter). After
challenge of these mice with i.t instillation of 106CFU E. coli,
only WT recipient mice showed detectable CXCL5 in BALF, in-
dicating that pulmonary-resident cells, not recruited hematopoietic
cells or alveolar macrophages, express CXCL5 in the lung during
lung inflammation (Fig. 4). Together with our previous findings
that rat ATII cells expressed CXCL5 in vitro and in vivo upon LPS
stimulation (10), these data suggest that ATII cells may be a major
source of CXCL5 in the alveolar space during lung inflammation.
IL-17A and TNF-a act synergistically to induce
CXCL5/ENA-78 expression in differentiated ATII cells derived
from human fetal lung
To determinewhether human ATII cells express CXCL5 in response
to TNF-a and IL-17A, epithelial cells isolated from second tri-
mester (16–20 wk gestation) human fetal lung and differentiated
in vitro to an ATII phenotype (33) were exposed to IL-17 and/or
TNF-a for 24 h. Consistent with our in vivo data, TNF-a alone
increased CXCL5/ENA-78 mRNA (Fig. 5A) and protein (Fig. 5B)
to a greater extent than did IL-17A alone in human ATII cells, but
in both cases were dose dependent. The combination of IL-17A
IL-17A and TNF-a ([625+500] ng/50 ml) into the tracheas of both WT and Cxcl52/2mice, whole lung lavage was performed at 8 and 24 h. The protein
levels of CXCL5 (A), CXCL1 (B), CXCL2 (C), and G-CSF in the BALF of WT and Cxcl52/2mice were measured by ELISA. The number of WBCs (D)
and neutrophils (E) per ml of BALF were determined by light microscopy. Values are mean 6 SEM (n = 3–8). ***p , 0.001. N.D., not detected.
CXCL5 plays a dominant role in mediating neutrophil influx to the lung induced by coinstillation of IL-17A + TNF-a. After coinstillation of
E. coli challenge. The BM chimeric mice were i.t. challenged with 106
CFU E. coli. Eight hours after challenge, the BALF was collected and
measured for CXCL5 level by ELISA. KK, BM from Cxcl52/2donor mice
into Cxcl52/2recipient mice; KW, BM from Cxcl52/2donor mice into
WT recipient mice. Values are mean 6 SEM (n = 3–4). N.D, not detected;
WK, BM from WT donor mice into Cxcl5–/–recipient mice; WW, BM
from WT donor mice into WT recipient mice.
Lung-resident cells are the primary source of CXCL5 upon
3200SYNERGISTIC EFFECTS OF IL-17A AND TNF-a ON CXCL5 EXPRESSION
and TNF-a induced a synergistic response on CXCL5/ENA-78
mRNA (Fig. 5A) and immunoreactive protein expression (Fig. 5B).
We then examined the time course of CXCL5/ENA-78 mRNA
and proteinexpressioninresponse toIL-17A andTNF-a compared
with TNF-a alone. TNF-a alone induced peak expression (still far
below that of TNF-a plus IL-17A) of CXCL5/ENA-78 mRNA by
8–10 h, which then declined toward baseline by 24 h. The addi-
tion of IL-17A to TNF-a greatly enhanced CXCL5/ENA-78
mRNA expression, which peaked by 10 h and remained elevated
through 24 h (Fig. 5C). By contrast, CXCL5/ENA-78 protein was
not detected in the media in either case until 8 h and then in-
creased exponentially between 8 and 24 h, especially in response
to the combination of IL-17A and TNF-a (Fig. 5D).
IL-17A enhances TNF-a–induced CXCL5/ENA-78 gene
expression through both transcriptional and
Gene upregulation is commonly due to the induction of gene
transcription, stabilization of mRNA, or the presence of both
mechanisms. To investigate the mechanisms of the marked up-
regulation ofCXCL5/ENA-78expression upon IL-17A and TNF-a
costimulation, we performed nuclear run-on assay in human ATII
cells treated with IL-17A (25 ng/ml) and/or TNF-a (10 ng/ml) for
24 h to examine the transcription rate. Intact nuclei were isolated,
and the RNA transcripts that had been initiated in vivo were
allowed to complete in vitro. The relative levels of CXCL5/ENA-
78 transcription were normalized to that of GAPDH. As shown in
Fig. 6A, IL-17A or TNF-a treatment increased novel CXCL5/
ENA-78 mRNA expression by ∼3-fold or 13-fold, respectively.
A synergistic increase in CXCL5/ENA-78 transcription of ∼50
fold was observed in the presence of IL-17A plus TNF-a.
The modulation of mRNA stability has emerged as an important
means of regulation for a number of proinflammatory genes (35).
To understand whether this mechanism contributes to the syner-
gistic effect of IL-17A and TNF-a on CXCL5/ENA-78 message,
we tested stability of CXCL5/ENA-78 mRNA in human ATII cells
treated with TNF-a (10 ng/ml) in the presence or absence of IL-
17A (10 ng/ml) for 10 h. After incubation with actinomycin D,
Human ATII cells were stimulated with IL-17A (10, 50 ng/ml) or TNF-a (2, 10 ng/ml) alone or in combination for 24 h. Total RNA was isolated, and
CXCL5/ENA-78 mRNA level determined by qPCR and normalized to 18S rRNA (representative result from separate experiments with three donors). B,
CXCL5/ENA-78 protein level in cell medium was measured by ELISA. Time course of CXCL5/ENA-78 mRNA (C) and protein (D) expression induced by
TNF-a and its augmentation by IL-17A. ATII cells were treated with TNF-a (10 ng/ml) or TNF-a (10 ng/ml) + IL-17A (10 ng/ml). Cell media and cell
contents were collected at different time points. Values are mean 6 SEM. Data represent results from three independent donors. *p , 0.01 TNF-a + IL-
17A–treated cells versus TNF-a–treated cells, ***p , 0.001.
IL-17A and TNF-a act synergistically to induce CXCL5/ENA-78 expression in differentiated ATII cells derived from human fetal lung. A,
ATII cells. A, Nuclei were isolated from human fetal ATII cells treated with or without TNF-a (10 ng/ml) in the presence of IL-17A (25 ng/ml) for 24 h.
Transcripts that were initiated in the cells were allowed to continue in vitro. The relative expression levels of novel CXCL5/ENA-78 mRNAwas determined
by qPCR and normalized to GAPDH. *p , 0.05 versus control, **p , 0.01. B, Human ATII cells were treated with TNF-a (10 ng/ml) or TNF-a + IL-17A
(10 ng/ml) for 10 h. Cells were then incubated with actinomycin D to inhibit further transcription. CXCL5/ENA-78 mRNA expression over the next 12 h
was quantified by qPCR and normalized to 18S rRNA. Values are mean 6 SEM. Data in A and B represent results for three independent donors.
IL-17A and TNF-a synergistically enhance CXCL5/ENA-78 mRNA transcription rate and stabilize CXCL5/ENA-78 mRNA in human fetal
The Journal of Immunology3201
RNA was extracted at various time points and CXCL5/ENA-78
measured using qPCR. In cells treated with TNF-a alone, CXCL5
t1/2was determined to be 3.8 h, whereas in cells treated with both
IL-17A and TNF-a, t1/2was much longer (Fig. 6B). Therefore,
these data demonstrated that IL-17A enhances TNF-a–induced
CXCL5/ENA-78 expression by effects on both increased tran-
scription rates and prolonged stability of mRNA.
Inhibition of de novo protein synthesis abolishes the synergistic
effect of IL-17A and TNF-a on CXCL5/ENA-78 expression
To determine whether synthesis of an intermediate mediator (such
as a transcription factor or RNA-binding protein) might be in-
volved in expression of CXCL5, we pretreated human ATII cells
with 5 mg/ml cycloheximide (CHX), a protein synthesis inhibitor
for 1 h prior to the stimulation with IL-17A and/or TNF-a for
10 h. CXCL5/ENA-78 mRNA levels were determined by qPCR.
As shown in Fig. 7, the synergistic effect of IL-17A and TNF-a
on CXCL5/ENA-78 expression was abolished by treatment with
CHX (Fig. 7, black bars), even though CHX by itself increased
CXCL5/ENA-78 in control. Overall, these results indicate the
synergistic effect of combined IL-17A and TNF-a on CXCL5/
ENA-78 expression depends on new protein synthesis.
TNF-a, but not IL-17A, activates NF-kB signaling on
CXCL5/ENA-78 expression in human ATII cells
NF-kB is a critical mediator of the cellular response to a variety of
extracellular stimuli such as stress, cytokines, and bacteria. Al-
though TNF-a is well known to activate NF-kB activity, and the
Cxcl5 promoter contains several NF-kB binding sites, the role of
IL-17A in inducing NF-kB is controversial (35–37). To determine
the relative effects of TNF-a and IL-17A on NF-kB activation, we
transduced primary human ATII cells with recombinant adenoviral
vectors containing an NF-kB–driven firefly luciferase reporter and
a pRL-SV40–driven renilla luciferase reporter (the latter repre-
senting a control for transduction efficiency). As seen in Fig. 8A,
TNF-a activated NF-kB activity, whereas IL-17 did not. The
combination of TNF-a and IL-17 did not induce synergistic ac-
tivation of NF-kB, suggesting the enhancement afforded by IL-
17 treatment was not through the NF-kB pathway. To further veri-
fy whether NF-kB signaling was involved in TNF-a–induced
CXCL5/ENA-78 expression, we pretreated human ATII cells with
BAY-11-7082, an inhibitor of IkBa phosphorylation, for 30 min
and then stimulated them with IL-17A and/or TNF-a for 24 h. As
seen in Fig. 8B, CXCL5/ENA-78 expression was greatly attenu-
ated upon NF-kB inhibition in either stimulation with TNF-a
alone or TNF-a plus IL-17A. These data demonstrated that TNF-
a, but not IL-17A, activated NF-kB signaling, which was neces-
sary, but not sufficient, for IL-17A enhancement of CXCL5/ENA-
78 expression by human ATII cells.
Human ATII cells secrete CXCL5/ENA-78 in a polarized and
The alveolar lining layer has developed a highly organized cellular
polarity: surfactant components are exclusively secreted to the
apical sides; and fibrinogen is mainly secreted basolaterally (38).
To understand whether CXCL5/ENA-78 was secreted in a polar-
ized fashion in human ATII cells, a monolayer of cells were plated
on transwell filters and stimulated with either IL-17A or TNF-a
alone, the combination of both from either the upper or the lower
compartments, or both compartments of the transwell (Fig. 9A).
After 24 h stimulation, the CXCL5/ENA-78 levels in the super-
natants from either the upper or lower chambers were measured by
ELISA. Apical stimulation by TNF-a provoked apical secretion of
ENA-78, whereas basolateral stimulation by TNF-a led to both
apical and basolateral secretion of CXCL5/ENA-78 by human
ATII cells. This polarized response was also synergistically en-
hanced by IL-17A costimulation (Fig. 9B).
Effect of IL-17A and TNF-a on cytokine and chemokine
secretion in human ATII cells
Because coinstillation of IL-17A and TNF-a into murine lungs
synergistically increased not only CXCL5, but also CXCL1, -2
kB signaling on CXCL5/ENA-78 expression in human
ATII cells. A, human ATII cells were transfected with
adenovirus vectors that had NF-kB–driven firefly lu-
ciferase element and pRL-SV40–driven renilla lucif-
erase element. Twenty-four hours postinfection, the
cells were then treated with IL-17A (25 ng/ml), TNF-a
(10 ng/ml), or both for 24 h in fresh medium. The cells
were assayed for firefly and renilla luciferase activities
using Dual-Luciferase Reporter Assay System. Lucif-
erase activities were measured by the IVIS Imaging. B
and C, Human ATII cells were treated with or without
phosphorylation IkBa inhibitor (BAY-11-7082) for 30
min and then stimulated with IL-17A and/or TNF-a for
24 h. The CXCL5/ENA-78 mRNA expression was
measured by PCR (B) and protein levels were mea-
sured by ELISA (C). Values are mean 6 SEM. Data
represent results for three independent donors. ***p ,
0.001 versus BAY-treated group.
TNF-a, but not IL-17A, activates NF-
ergistic effect of IL-17A and TNF-a on CXCL5/ENA-78 mRNA expres-
sion. Human ATII cells were pretreated with CHX (5 mg/ml) for 1 h, and
then they were stimulated with IL-17A (25 ng/ml) and/or TNF-a (10 mg/
ml). Ten hours later, cells were collected, and total RNA was isolated.
qPCR were performed to quantify the relative expression of CXCL5/ENA-
78 mRNA normalized to 18S rRNA. *p , 0.05 versus control, ***p ,
0.001,&NS. Values are mean 6 SEM. Data represent results for three
Inhibition of de novo protein synthesis abolishes the syn-
3202 SYNERGISTIC EFFECTS OF IL-17A AND TNF-a ON CXCL5 EXPRESSION
and G-CSF, we further explored whether IL-17A and TNF-a
exerted a synergistic effect on the secretions of other cytokines
and chemokines in human ATII cells. We used multiplex bead
technology to measure the protein levels of a variety of cytokines
and chemokines in media of human ATII cells that were treated
with IL-17A and/or TNF-a for 24 h. As shown in Fig. 10, IL-17A
alone induced only minimal cytokine and chemokine secretion,
but it synergized with TNF-a in the induction of cytokine (IL-6,
G-CSF, GM-CSF), and chemokine (IL-8, GRO, MCP-1, MIP-1b,
MCP-3) expression (Fig. 10), suggesting that the synergism of
TNF-a with IL-17A may play an important role in orchestrating
pulmonary inflammatory responses through ATII cells.
During lung inflammation and infection, neutrophils generated in
the BM are quickly recruited to inflamed sites to form the first line
of lung host defense. ELR+CXC chemokines play an important
role in directing this event and, at least in part, determine the
nature and severity of the resulting inflammatory response. The
sources of these chemokines are less clear. Although traditionally
the AM has been considered to secrete most of the ELR+CXC
chemokines, we have shown murine AM do not express CXCL5
during lung inflammation (10). In the present work, we suggest
that the ATII cell contributes to the lung inflammatory response
by virtue of its ability to secrete CXCL5 and a variety of other
chemokines in response to TNF-a and IL-17A, a frequently
detected combination of cytokines.
The ATII cell has been described for many years, since the
original work of Mason and Williams (39), as the defender of the
alveolus, based on its ability to secrete surfactants to prevent al-
veolar collapse. More recently, a body of work indicates that ATII
cells participate as well in host defense (7, 8, 40, 41). In this ar-
ticle, we extend the function of ATII cells in inflammation by
focusing on a unique member of the ELR+CXC chemokine
family, CXCL5. This chemokine, as we previously demonstrated,
is mostly produced by AT II cells during lung inflammation (10)
and plays a unique role in chemokine scavenging in blood and
regulating neutrophil influx to the lung (30).
IL-17 is a proinflammatory cytokine produced predominately by
Th17 cells. Although IL-17 is recognized to participate in host
defense and autoimmunity through the induction of proinflam-
matory gene expression, including CXC chemokines, and subse-
quently leading recruitment of neutrophils (5), the molecular mech-
anisms by which this is achieved remain poorly defined. It has
been reported that IL-17 treatment is a poor stimulus for gene ex-
pression. However, IL-17 can cooperate with other cytokines,
particularly TNF-a, to induce a synergistic response (42). We
developed a new model to test potential interactions between
cytokines. In this model, i.t. instillation of TNF-a produces a mild
inflammatory response, consistent with previous observations in
mice (43). The coadministration of IL-17A, however, which to our
knowledge has not previously been demonstrated, markedly in-
creased neutrophil accumulation. CXCL1 and -2 were upregulated
early, whereas CXCL5 was induced later. Cxcl52/2mice showed
markedly diminished neutrophil influx at later stages, but early
neutrophil accumulation was unaffected. These data indicate
ELR+CXC chemokines, despite their similarities, may be re-
sponsible for different phases of the inflammatory response. Al-
though the model itself is created through exogenous instillation,
it represents conditions that occur in a variety of inflammatory
To study mechanisms of synergism, we used human ATII cells
in vitro as a model. We focused on human CXCL5/ENA-78 as
a bona fide homolog of murine CXCL5/LIX for several reasons.
Although human CXCL5/ENA-78 and human CXCL6/ GCP-2 are
of mouse and human CXCL5 are nearly identical, whereas that of
human CXCL6 is part of a duplicate region containing pseudo-
genes. Furthermore, murine CXCL5/LIX and human CXCL5/
ENA-78 share functional homology as well, as they both are ex-
pressed in platelets (44) and ATII cells (30).
Using human ATII cells, we suggest that the mechanism by
which IL-17A synergizes with TNF-a to induce CXCL5 is appar-
ently complex. IL-17A–induced synergism required new protein
synthesis, suggesting that synthesis of new transcription factor(s)
and regulated fashion. Human ATII cells seeded on transwell filters were
treated with PBS, IL-17A (25 ng/ml), TNF-a (10 ng/ml), or IL-17A+TNF-
a from top or bottom chambers for 24 h. Supernatants were collected for
measurement of CXCL5/ENA-78 protein. Values are mean 6 SEM. Data
represent results for five independent donors.
Human ATII cells secrete CXCL5/ENA-78 in a polarized
or TNF-a (10 ng/ml) for 24 h. Supernatants were collected for Luminex assay. Values are mean 6 SEM. Data represent results for three independent donors.
**p , 0.01, ***p , 0.001.
Effect of IL-17A and TNF-a on cytokine and chemokine secretion in human ATII cells. ATII cells were treated with IL-17A (25 ng/ml) and/
The Journal of Immunology3203
and/or RNA-binding protein(s) was necessary. It was reported
that the binding sites for NF-kB, C/EBP, AP-1, and OCT1 were
overrepresented in IL-17A target genes by a computational anal-
ysis on 18 well-documented IL-17A target promoters; NF-kB was
the major transcription factor on IL-17A–induced CXCL5 in this
study (45). Activation of the transcription factor NF-kB is critical
for the TNF-a–induced inflammatory response (46). In this study,
we found that TNF-a induced NF-kB activity; moreover, NF-kB
signaling appeared necessary for TNF-a–induced CXCL5/ENA-
78 expression in human ATII cells. Hwang et al. (37) demon-
strated that IL-17A induced production of IL-6 and IL-8 in
rheumatoid arthritis synovial fibroblasts via NF-kB–dependent
pathways; IL-17A was the potent stimulus in this study. In con-
trast, in this study, we demonstrated IL-17A was a weak stimulus
for CXCL5/ENA-78 expression in human ATII cells; moreover,
neither direct IL-17A–induced, or IL-17A–enhanced, CXCL5/
ENA-78 gene expression was through an NF-kB signaling path-
way. Furthermore, using an Ad-AP-1–luc vector, we examined
that AP-1 activity was not induced by either treatment (data not
shown). Because C/EBPa, C/EBPb, and C/EBPd are expressed in
ATII cells (47, 48), and because signaling via the IL-17R activates
C/EBPb and C/EBPd (49), future studies will be focused on the
potential role of C/EBP family in CXCL5/ENA-78 gene expres-
sion induced by IL-17A and/or TNF-a.
Both increased transcription and enhanced stability of the
mRNA play a role in the synergistic induction of CXCL5/ENA-78
expression as the 39 untranslted region of CXCL5/ENA-78 has
eleven AUUUAs, which are likely to be involved in regulation of
CXCL5/ENA-78 mRNA decay. Regulation of mRNA decay is
a potential mechanism by which the duration of the inflammatory
response can be limited. However, the ability to prolong the t1/2of
the message is necessary to reach a high level of expression in the
setting of inflammation, and we observed an increase in CXCL5/
ENA-78 mRNA stability by IL-17A plus TNF-a. This finding
further emphasizes the importance of the role of regulated mRNA
stability in the control of proinflammatory gene expression (35).
The lung is uniquely poised in between the external environment
notion that the lung is capable of distinguishing these different
initiating factors and responding to them in distinct fashions is new.
Furthermore, the nature of that response results in expression of
chemokines that may be secreted into different compartments,
where a complexset of binding proteins both shapes and refines the
chemokine milieu. We speculate that control of inflammatory
processes resides, at least in part, by regulation of the direction
(apical versus basal) in which chemokines are secreted. Although
considerable further work is required to address these new insights,
they suggest the potential for much more nuanced intervention in
lung inflammatory processes than heretofore envisioned.
We thank Dr. Eric Rappaport and Dr. Venkat Kolla for important technical
advice. We also thank Dr. Heather Collins for assistance with multiplex ar-
The authors have no financial conflicts of interest.
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