of October 27, 2015.
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RGS16 Attenuates Pulmonary Th2/Th17
and Kirk M. Druey
Margaret M. Mentink-Kane, Zhihui Xie, Thomas A. Wynn
Sucharita P. Shankar, Mark S. Wilson, Jeffrey A. DiVietro,
2012; 188:6347-6356; Prepublished online 16
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The Journal of Immunology
RGS16 Attenuates Pulmonary Th2/Th17 Inflammatory
Sucharita P. Shankar,* Mark S. Wilson,†Jeffrey A. DiVietro,* Margaret M. Mentink-Kane,†
Zhihui Xie,* Thomas A. Wynn,†and Kirk M. Druey*
The regulatorsofGproteinsignaling(RGS)proteinsuperfamily negativelycontrolsGprotein-coupledreceptorsignaltransduction
pathways. RGS16 is enriched in activated/effector T lymphocytes. In this paper, we show that RGS16 constrains pulmonary in-
flammation by regulating chemokine-induced T cell trafficking in response to challenge with Schistosoma mansoni. Naive Rgs162/2
mice were “primed” for inflammation by accumulation of CCR10+T cells in the lung. Upon pathogen exposure, these mice
developed more robust granulomatous lung fibrosis than wild-type counterparts. Distinct Th2 or putative Th17 subsets expressing
CCR4 or CCR10 accumulated more rapidly in Rgs162/2lungs following challenge and produced proinflammatory cytokines
IL-13 and IL-17B. CCR4+Rgs162/2Th2 cells migrated excessively to CCL17 and localized aberrantly in challenged lungs.
T lymphocytes were partially excluded from lung granulomas in Rgs162/2mice, instead forming peribronchial/perivascular
aggregates. Thus, RGS16-mediated confinement of T cells to Schistosome granulomas mitigates widespread cytokine-mediated
pulmonary inflammation.The Journal of Immunology, 2012, 188: 6347–6356.
chemokine receptors, and gene-targeting studies have implicated
specific chemokines and receptors in leukocyte activation, dif-
ferentiation, and lymphoid organ development (1). Schistosomi-
asis, induced by infection with the helminth Schistosoma mansoni
(among other species), represents a global disease burden because
of resultant hepatic fibrosis and portal hypertension (2). S. man-
soni cercariae breach host skin and develop into adult male–fe-
male worm pairs that generate hundreds of eggs per day (3, 4).
Eggs then enter circulation or embolize in host tissues such as
liver, intestine, and lung, where a granulomatous reaction and fi-
brosis develop in an effort to sequester the foreign eggs Ags (3).
In published mouse models of schistosomiasis, the pulmonary
granulomatous response is initiated by CD4+Th2 cells and their
secreted cytokines, particularly IL-13 (5). Although chemokine
factors mediating Th2 recruitment to lungs acutely challenged
with S. mansoni eggs have been suggested (e.g., CCL17/CCL22
and CCR4/8), signaling pathways involved in pulmonary inflam-
mation have not been fully defined (6).
hemokines dictate coordinated movement of leukocytes
through lymphoid organsand sites ofinflammation. Naive
and activated leukocyte populations express unique
Chemokine receptors are G protein-coupled receptors (GPCRs)
linked to Gai and possibly Gaq to induce chemotaxis (7). The
primary signal transducer of GPCRs, the heterotrimeric G protein
complex of a, b, and g subunits, induces pathway activation
through GDP–GTP exchange on Ga and stimulation of numerous
effectors including kinases, phospholipases, and ion channels (8,
9). The intrinsic GTPase activity of the a subunit, which promotes
Ga reassociation with bg to form an inactive heterotrimer, ter-
minates ligand-induced signaling. The regulators of G protein
signaling (RGS) superfamily, which has .30 members in mam-
malian cells, negatively regulates G protein activity (10). All RGS
proteins contain a characteristic 120-aa “RGS box,” which facil-
itates binding to Ga subunits and GTPase-accelerating protein
(GAP) activity (11). RGS GAP activity hastens GPCR pathway
inactivation by catalyzing the GTPase reaction. Although molec-
ular determinants of RGS activity have been elucidated over the
past decade, most physiological functions of RGS proteins in
mammals remain unknown. Gai inactivation by pertussis toxin
disrupts physiological hematopoietic cell trafficking including thy-
mic emigration, transendothelial leukocyte migration into lymph
nodes (LNs), and Ag-induced recruitment of cells to inflamed tissue
(7). Because RGS proteins are physiologically relevant inhibitors of
Gai, they are poised to regulate chemokine-mediated responses
in vivo (7).
RGS16 was initially discovered as an IL-2–dependent activa-
tion gene in human T lymphocytes (12). RGS16 may control
Th2 lymphocyte migration in vivo because it is upregulated in
activated human Th1 and Th2 cells relative to naive T cells, and
RGS16 overexpression inhibits Th lymphocyte chemotaxis in vitro
(13). To explore intracellular regulation of chemokine pathways
in pulmonary inflammation, we generated Rgs162/2mice and
studied their response to sensitization with S. mansoni egg Ag
followed by an i.v. bolus of live S. mansoni eggs (14). These
studies revealed that RGS16 inhibits Th2 chemotaxis to che-
mokines including CCL17, which constrains T cell localization
to Schistosome egg granulomas, thereby reducing the tissue-
damaging effects of Th2-induced pulmonary inflammation by
confining cytokines to specific regions(s) of the lung.
*Molecular Signal Transduction Section, Laboratory of Allergic Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
MD 20892; and
National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD 20892
†Immunopathogenesis Section, Laboratory of Parasitic Diseases,
Received for publication December 23, 2011. Accepted for publication April 16,
This work was supported by the Intramural Research Program of the National Insti-
tute of Allergy and Infectious Diseases, National Institutes of Health (Grant
AI000746; to K.M.D.).
Address correspondence and reprint requests to Dr. Kirk M. Druey, Laboratory of
Allergic Diseases, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, 10 Center Drive, Room 11N242, Bethesda, MD 20892. E-mail
The online version of this article contains supplemental material.
Abbreviations used in this article: CT, cycle threshold; GAP, GTPase-accelerating
protein; GPCR, G protein-coupled receptor; LN, lymph node; MLN, mediastinal
lymph node; qPCR, quantitative PCR; RGS, regulator of G protein signaling; SEA,
Schistosome egg Ag; Socs2, suppressor of cytokine signaling 2; WT, wild-type.
by guest on October 27, 2015
Materials and Methods
Generation of Rgs162/2mice
C57BL/6 wild-type (WT) mice were purchased from The Jackson Labo-
ratory. Rgs162/2mice were generated directly onto a C57BL/6 back-
ground as outlined in Fig. 1. All mice were housed in pathogen-free
conditions and research performed in accordance with protocols (LAD3e
and LPD16e) approved by the National Institutes of Health Institutional
Animal Care and Use Committee. Male or female mice between 6 and 12
wk of age were used for all experiments.
S. mansoni lung challenge model
Mice were sensitized i.p. with 5000 S. mansoni eggs that were derived
previously from sterile, LPS-free BALB/c mice harboring a liver infection
(3). Fourteen days later, mice were challenged i.v. with 5000 live eggs of
a Puerto Rican strain of S. mansoni (NMRI) as described elsewhere (3).
The challenged mice were euthanized at 4 or 7 d post i.v. injection by CO2
inhalation. Lungs, spleens, and mediastinal LNs (MLNs) were harvested
from both groups of challenged mice for analysis.
Histopathology and immunohistochemistry
Organs were fixed in 10% neutral-buffered formalin (EMD Chemicals) and
embedded in paraffin. Tissue sections (5 mm) were evaluated for granuloma
volume and diameter by H&E staining, and lung fibrosis was scored, based
on Picrosirius red stain for collagen using published scoring methods (15).
Giemsa stains were used to quantify eosinophil numbers. The individual
who scored all histological features was blinded to the experimental
design. Lungs harvested at 7 d post-i.v. challenge were stained for the
following markers: anti-CD3 (1:100) (DakoCytomation) or anti-CCR10
(1:500) (Abcam). A polyconal anti-RGS16 Ab (Dru-4) was generated by
immunizing rabbits with an N-terminal RGS16 peptide (MCRTLATFPNTC-
amide) and collection of serum. The Ab was affinity purified using a pep-
tide-conjugated resin (Spring Valley Laboratories). Slides were deparaffi-
nized and washed twice in distilled water for 5 min at room temperature.
After pretreatment for Ag retrieval (20–40 min, 75–90˚C), the slides were
washed again, and endogenous peroxidase activity was inhibited with
H2O2for 10 min at room temperature. Nonspecific binding was blocked
using 5% BSA (Sigma-Aldrich) in PBS for 20 min at room temperature.
Sections were incubated with primary Ab or matched isotype controls
(1:500) overnight at room temperature, washed, and incubated with the
biotinylated secondary Ab (1:500, 30 min at room temperature) and finally
incubated with streptavidin-HRP (1:400, 30 min at room temperature)
(Alpha Diagnostics). The signal was detected with 3,39-diaminobenzidine
(1–20 min at room temperature). Sections were counterstained in Carazzi
hematoxylin (Histoserv) for 2 min at room temperature, followed by de-
hydration in graded alcohols (75, 95, and 100%) for 1 min each at room
temperature and xylene (Sigma-Aldrich). Images were obtained with a
Leica DMI 4000B microscope and quantified using Image Pro Plus soft-
ware (Media Cybernetics).
Real-time PCR and gene expression arrays
Total RNA was isolated using the RNeasy mini kit (Qiagen), followed by
DNase I treatment (Invitrogen). cDNA was synthesized from 0.25 to 1 mg
RNA using the Superscript III First-Strand Synthesis kit (Invitrogen). Real-
time RT-quantitative PCR (qPCR) was performed using the TaqMan
strategy (Applied Biosystems). No reverse transcriptase controls were in-
cluded to verify DNAse digestion. Gene expression (Rgs16 [NM_011267.3],
suppressor of cytokine signaling 2 [Socs2] [NM_007706.3], Ccr10
[NM_007721.4], Ccl28 [NM_020279.3], Il13 [NM_016971.2], Il17b
[NM_019508.1], or the internal reference control b-actin [NM_007393.3])
was measured by multiplex PCR using probes labeled with FAM. The
simultaneous measurement of target gene-FAM and b-actin-FAM allowed
for normalization of the amount of cDNA added per sample. Duplicate
PCRs were performed using the TaqMan Universal PCR Master Mix and
the ABI 7500 Standard or Step One Plus sequence detection systems
(Applied Biosystems) according to the following thermal cycle routine:
95˚C for 10 min, followed by 40 cycles of 95˚C for 1 min and 60˚C for 20
min. A comparative cycle threshold (CT) method was used to determine
gene expression relative to the no-sample control (calibrator). Steady-state
mRNA levels were expressed as an n-fold difference relative to the cali-
brator. For each sample, the RGS16 CTvalue was normalized with the
formula: DCT= CT RGS162 CT b-ACTIN. To determine relative expression
levels, the following formula was used: DDCT= DCT sample2 DCT calibrator.
Relative expression is presented as 22DDCT. Multiplex qPCR was per-
formed using a mouse chemokines and receptors qPCR array (SA Bio-
sciences), according to the manufacturer’s instructions.
Single-cell suspensions were prepared from MLNs harvested 7 d following
i.v. challenge; 0.5 3 106cells/well were plated in triplicate wells of 96-
well round polystyrene plates (Corning) in RPMI 1640 medium plus 10%
FBS (Invitrogen). Cells were left untreated or stimulated with Schistosome
egg Ag (SEA) from S. mansoni in PBS (10 mg/ml final concentration) for
3 d at 37˚C. Cell supernatants were collected, and cytokine levels were
measured with a fluorescence-based Bio-Plex Pro mouse cytokine Th1/Th2
assay (Bio-Rad), according to the manufacturer’s protocols. Cells were
fixed by the addition of PBS containing 2% BSA and 4% paraformalde-
hyde (Electron Microscopy Sciences). ELISAs were performed to quantify
secretion of mouse cytokines IL-4, IL-13, IFN-g, IL-17A, or IL-17B (R&D
Systems) in accordance with the manufacturer’s protocols.
Flow cytometry and intracellular staining
Organs were harvested 4 or 7 d postchallenge and converted to single-cell
suspensions followed by fixation in PBS containing 2% BSA and 4%
paraformaldehyde. Surface markers were analyzed with the following Abs:
CD3 (17A2), B220 (RA3-6B2), CD11c (N418) (eBioscience), CD4 (RM4-
5), CD8 (53-6.7), CCR3 (83103), CXCR4 (2B11) CXCR3 (CXCR3-173),
(248918) (R&D Systems), and cytokines were detected with anti–IL-5
(TRFK5) (BD Biosciences), anti–IL-13 (ebio13A), anti–IFN-g (XMG1.2)
(eBioscience), or anti–IL-17B (R&D Systems). For intracellular cytokine
staining, cells were activated with PMA (50 ng/ml) and ionomycin
(0.5 mM) (Sigma-Aldrich) for 6 h at 37˚C in the presence of brefeldin A
(BD Biosciences). Cells were permeabilized with PBS/0.1% saponin
(Sigma-Aldrich) and blocked with 5% PBS/milk (Santa Cruz Biotech-
nology) (15 min, 4˚C) and Fc block (BD Biosciences). Fixed per-
meabilized cells were stained in the dark with fluorescently conjugated
Abs (1 h, 4˚C) and washed twice with FACS buffer. Samples were ana-
lyzed by flow cytometry using an LSR Fortessa (BD Biosciences), and data
were analyzed using FlowJo (Tree Star).
In vitro Th1, Th2, or Th17 culture
Single-cell suspensions were generated from peripheral LNs from naive
mice. Naive T cells (CD4+CD62L+) were isolated using the naive T cell
isolation kit II (Miltenyi Biotec). A total of 3 3 105cells/well were plated
in 6-well plates precoated with anti-CD3 (1 mg/ml) and anti-CD28 (3 mg/
ml) containing RPMI 1640 medium plus 10% FBS, recombinant mouse
IL-4 (10 ng/ml) (PeproTech), anti-mouse IFN-g (10 mg/ml) (BD Bio-
sciences), and 50 mM 2-ME for 3 d at 37˚C. For differentiation into a Th1
phenotype, naive T cells were cultured in media containing IL-2 (10 ng/
ml), IL-12 (10 ng/ml) (R&D Systems), and anti–IL-4 (10 mg/ml) (eBio-
science), referred to as the Th1 mixture. For differentiation into a Th17
phenotype, naive T cells were cultured in media containing IL-23 (5 ng/
ml), IL-21 (100 ng/ml), IL-6 (10 ng/ml), human TGF-b (5 ng/ml), anti–IL-
4 (10 mg/ml), anti–IFN-g (10 mg/ml) (R&D Systems), and anti–IL-12
(10 mg/ml) (BD Biosciences), referred to as the Th17 mixture. The dif-
ferentiated activated Th1 or Th2 cells were then expanded in medium
containing recombinant mouse IL-2 (10 ng/ml) and IL-7 (5 ng/ml)
(PeproTech) or in IL-2 alone for Th17 cells.
Generation and purification of TAT fusion proteins
The coding region of human RGS16 was amplified by PCR using
pcDNA3.1-RGS16 as a template and subcloned in-frame into the plasmid
vector pTat-H6HA-GFP, which results in an N-terminally tagged GFP-
RGS16 (16). Recombinant TAT fusion proteins were expressed in Escher-
ichia coli and purified by nickel-affinity chromatography as described pre-
viously (17). TAT proteins (TAT-GFP-RGS16 or TAT-GFP control) were
added directly to T cell cultures for 2 h prior to stimulus addition.
Differentiated Th2 cells (0.5 3 106cells/well) were plated in upper wells
of 5-mm-pore polycarbonate membrane Transwell migration chambers
containing RPMI 1640 medium plus 0.5% BSA (Corning). The bottom
wells contained chemokines (CXCL12, CCL21, CCL17, CXCL9, or
CCL20) (R&D Systems). Control wells, in which upper and lower chambers
had equivalent chemokine concentrations, were used to determine chemo-
kinesis. Cells migrated to the lower chamber were counted after 3 h by flow
cytometry using a FACSCalibur (BD Biosciences).
CFSE assay for analysis of cell proliferation
Cells were labeled using the CellTrace CFSE cell proliferation kit (Invi-
trogen) plated in 96-well plates left untreated or precoated with anti-CD3/
6348RGS16 INHIBITS LUNG INFLAMMATION
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anti-CD28. Cells were harvested 4 d later, and CSFE dilution profiles were
evaluated by flow cytometry.
Data sets were compared by Student t test for two groups or one-way
ANOVA for multiple groups using Graph Pad Prism software. Differ-
ences with p # 0.05 were considered significant.
Generation of Rgs162/2mice
Rgs16 was targeted by flanking exons 2–4 with loxP sites (Fig. 1A).
We generated knockouts by crossing mice with floxed Rgs16 allele
with Rosa-Cre mice, which have the gene encoding Cre recombi-
nase inserted into the ubiquitous Rosa26 locus. Correct targeting of
the Rgs16 allele was confirmed by Southern blot analysis (Fig. 1B).
Homozygous mice were viable and fertile and exhibited no gross
phenotypic abnormalities. Architecture of the spleens, lungs, and
peripheral LNs in naive Rgs162/2mice was comparable to WT
mice by light microscopy (Fig. 1C). Percentages of CD4+and CD8+
T cells, B cells, and dendritic cells in the spleen, lungs, and LNs
were similar in WT and Rgs162/2mice (Table I).
RGS16 inhibits Th1 and Th2 chemotaxis
To investigate function(s) of RGS16 in T cell-dependent inflam-
mation, we examined its expression pattern in mouse effector
T cells and their chemotactic responses. Compared with naive
(CD4+CD62L+) T cells isolated from peripheral LNs, activated
T cells were polarized to a Th2 phenotype by IL-4, and anti–IFN-
g Ab expressed 5-fold more Rgs16 mRNA, whereas those polar-
ized to Th1 phenotype by a Th1 mixture or Th17 phenotype by
a Th17 mixture expressed 2-fold more Rgs16 mRNA (Fig. 2A).
We measured chemotaxis of naive CD4 T cells to gradients of
CCL21 and CXCL12 because these chemokines have an impor-
tant function in homeostatic lymphocyte trafficking in lymphoid
organs (18). Consistent with relatively low Rgs16 expression in
naive CD4 T cells, CXCL12 and CCL21 induced equivalent
chemotaxis of naive splenocytes or naive splenic CD4 T cells from
unchallenged WT and Rgs162/2mice (Fig. 2B). Th2 cells up-
regulate several “inflammatory” chemokine receptors not typically
expressed by naive cells including CCR4 and CCR8, which me-
diate trafficking to inflamed tissues containing CCL17 (19). Ac-
cordingly, in vitro-differentiated Th2 cells from mice lacking
RGS16 migrated much more toward CCL17 gradients than WT
counterparts (Fig. 2C). At the CCL17 concentration inducing a
peak response (50 nM), nearly double the number of RGS16-
deficent Th2 cells than WT cells migrated to the lower chamber
(∼80 versus 40%).
In contrast to Th2 cells, Th1 and Th17 cells express a distinct set
of chemokine receptors including CXCR3 and CCR5 (Th1) or
Rgs162/2mice. (A) A targeting vector was generated
by flanking a floxed-Neorcassette (PGKNeo) by two
homologous arms of the endogenous Rgs16 gene.
Shown are restriction maps and exons (solid vertical
bars) of the endogenous and targeted Rgs16 loci
(blue bars). (B) WTand floxed alleles were identified
by Southern blotting of BglII-digested genomic DNA
with a 59 probe (red), yielding 10.6- or 5.5-kb frag-
ments, respectively (middle). Mice homozygous for
the floxed allele were crossed with Rosa-Cre strain,
resulting in deletion of PGKNeo and exons 2–4,
which was identified as an 8.3-kb fragment.
(C) Architecture of spleens, lungs, and peripheral LNs
in naive WT or Rgs162/2mice was evaluated by
H&E staining. Original magnification 35.
Generation and characterization of
Table I.Cellular composition of spleens, LNs, or lungs of naive WT or Rgs162/2mice
Tissue MiceCD3+(%) CD3+CD4+(%) CD3+CD8+(%)B220+(%)CD11c+(%)
WT41.32 6 15.42
48.69 6 5.47
60.27 6 6.32
63.23 6 4.27
18.14 6 3.81
23.79 6 10.17
59.13 6 5.6
58.39 6 1.55
55.52 6 4.84
54.06 6 1.4
24.12 6 10.99
21.06 6 18.24
5.06 6 6.33
6.31 6 6.56
0.105 6 0.06
49.04 6 5.51
47.8 6 6.59
30.53 6 4.54
32.08 6 4.62
6.99 6 2.10
7.29 6 5.85
8.24 6 8.17
4.90 6 4.05
6.54 6 2.18
6.92 6 3.99
B lymphocytes, T lymphocytes, and dendritic cell percentages in lymphoid organs and lungs were determined by means of flow cytometry using the
indicated markers. Results are mean 6 SEM from six mice of each genotype.
N.D., Not detectable.
The Journal of Immunology6349
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CCR6 (Th17) (20–22). Similar to the behavior of RGS16-deficient
Th2 cells, Th1 cells differentiated from Rgs162/2mice migrated
significantly more toward a gradient of CXCL9 (CXCR3 ligand)
than did WT counterparts (Fig. 2C). For unclear reasons, we did
not observe significant chemotaxis of murine Th17 cells toward
CCL20 gradients in Transwell assays, independent of genotype
(Supplemental Fig. 1). WT and Rgs162/2T cells migrated to
a similar extent in the presence of equivalent chemokine con-
centrations in the upper and lower chambers, indicating that the
loss of RGS16 affected chemotaxis rather than chemokinesis (Fig.
2D). Rgs16 expression correlated with chemokine resistance. WT
T cells retained in the upper chamber in the presence of a CCL17
gradient for 3 h (“nonmigratory”) expressed significantly more
Rgs16 than cells migrating to the lower chamber during that time
period (“migratory”) (Fig. 2E). Finally, because WT and Rgs162/2
Th-polarized subsets expressed similar levels of surface che-
mokine receptors including CXCR3 for Th1 cells, CXCR4,
CCR10, or CCR4 for Th2 cells, and CCR6 for Th17 cells, these
outlined in Materials and Methods. Relative Rgs16 expression was measured by real-time PCR (**p = 0.005, *p , 0.01; unpaired t test). (B) Whole
splenocytes or splenic CD4 T cells C57BL/6 and Rgs162/2mice were exposed to chemokines in Transwell plates for 3 h at 37˚C, followed by enumeration
of migrated cells by flow cytometry. (C) Migration of Th1 or Th2 cells toward CXCL9 or CCL17 gradients, respectively, at the indicated concentrations was
measured as in (B) (*p = 0.04, **p , 0.003; unpaired t test). (D) Chemokinesis was measured by incubating cells with equimolar concentrations of CCL17
(50 nM) in the upper and lower chambers of Transwell plates, followed by enumeration of cells by flow cytometry. (E) Rgs16 expression in cells migrated to
the lower chamber of CCL17-containing Transwell plates (“migratory”) was compared with cells retained in the upper chamber (“nonmigratory”) by real-
time PCR (*p = 0.04, paired t test). (F) Th2 cells from Rgs162/2mice were left untreated or preincubated with TAT-GFP or TAT-RGS16 (60–500 nM) for
1 h prior to exposure to CCL17 gradients in Transwell assays (***p , 0.001; one-way ANOVA, TAT-RGS16 compared with untreated or TAT-GFP). All
data are mean 6 SEM of three to four independent experiments using cells from 1 mouse/group in each.
RGS16 inhibits Th2 chemotaxis. (A) Naive T cells were isolated from peripheral LNs and differentiated into Th1, Th2, or Th17 cells as
6350RGS16 INHIBITS LUNG INFLAMMATION
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results indicated that RGS16 inhibits chemotaxis downstream of
receptors (Supplemental Fig. 1). Consistent with this interpreta-
tion, reconstitution of RGS16-deficient Th2 cells with TAT-RGS16
reduced CCL17-evoked chemotaxis compared with untreated cells
or cells incubated with a control TAT protein (GFP) (Fig. 2F).
Collectively, these results suggest that RGS16 directs trafficking of
Th1 or Th2 lymphocytes by curbing their response to chemokine.
Rgs162/2mice have increased granulomatous lung fibrosis
after challenge with S. mansoni
To evaluate Th2 trafficking in vivo in Rgs162/2mice and its
impact on an acute inflammatory response, we sensitized mice
with S. mansoni eggs i.p., followed by i.v. injection of eggs
14 d later (Fig. 3A). Mice were sacrificed 4 and 7 d after the i.v.
inoculation, at which time S. mansoni eggs embolize in the lungs,
resulting in synchronous pulmonary inflammation characterized
by granulomas, infiltration of neutrophils, eosinophils, and Th2
lymphocytes, and collagen deposition/fibrosis (23). Consistent
with a role for RGS16 in the regulation of host responses to
S. mansoni, Rgs16 mRNA expression was increased in the lungs
of sensitized challenged mice 7 d following the i.v. Schistosome
challenge (Fig. 3B). We also detected RGS16 in the lungs of
challenged mice by immunohistochemistry (Fig. 3C). RGS16+
cells localized predominantly in granulomas surrounding lodged
S. mansoni eggs. The lungs of Rgs162/2mice developed signifi-
cantly more inflammation and fibrosis than those of WT mice
(Fig. 3D). Fibrosis scores (Fig. 3E), granuloma volumes (Fig. 3F),
and eosinophil scores (Fig. 3G) were all higher in lungs of knockout
mice compared with WT C57BL/6 mice. These results indicated
that RGS16 constrains acute granulomatous pulmonary fibrosis
induced by S. mansoni infection.
RGS16 deficiency induces anomalous lymphocyte trafficking
To determine which lymphocyte subsets mediated the atypical
fibrotic response of Rgs162/2mice lungs to S. mansoni, we
immunophenotyped pulmonary inflammatory infiltrates by means
of flow cytometry. Although we detected no substantial differ-
ences in the percentages or total numbers of B and T cells in the
lungs of challenged WT and Rgs162/2mice, there was a skewed
Th2 response in knockout mice as evidenced by increased per-
centages of IL-13+T cells in the lungs 4 d following helminth
challenge. The ratio of IL-13+(Th2)/IFN-g+(Th1) frequencies
was significantly increased in Rgs162/2lungs relative to WT (Fig.
4A, 4B). Because Th cells from WTand Rgs162/2mice exhibited
comparable rates of differentiation, proliferation, and survival
in vitro (Supplemental Fig. 2; data not shown), aberrant cell
growth and/or death probably did not account for the differences
in the cellular composition observed.
Splenic CD4 T cells isolated from challenged Rgs162/2mice
migrated more toward CCL17 gradients than cells from WT mice
(Fig. 4C). Thus, irregular trafficking patterns of fibrosis-inducing
IL-13+Th2 cells might contribute to the enhanced inflammation in
Rgs162/2mice following helminth challenge. Consistent with this
hypothesis, we observed starkly atypical T lymphocyte localiza-
tion patterns in the lungs of RGS16-deficient mice following S.
mansoni inoculation compared with WT. Whereas T cells were
distributed uniformly within fibrotic granulomas surrounding em-
bolized eggs in WT lungs, they were restricted to the periphery of
granulomas in the lungs of Rgs162/2mice (Fig. 5A). In addition,
we detected dense perivascular/peribronchial T cell aggregates
that were largely absent in WT mice (Fig. 5B, 5C). These data
tion in Rgs162/2mice challenged with Schis-
tosoma mansoni. (A) Experiment layout. (B)
Rgs16 expression in naive or S. mansoni-chal-
lenged lungs was measured by real-time PCR
(*p = 0.04, unpaired t test). (C) RGS16 ex-
pression detected by immunohistochemistry
using an RGS16 Ab in the lungs of WT or
knockout mice as indicated. Images are repre-
sentative of three to five mice per group. Orig-
inal magnification 35. (D) Picrosirius red
staining for collagen in the lungs of WT or
Rgs162/2mice 7 d following inoculation with
S. mansoni eggs. Original magnification 35 or
340. (E–G) Fibrosis scores (E), granuloma
volumes (F), and eosinophil scores (G) were
evaluated in WT and Rgs162/2lungs 7 d after
helminth challenge. Data represent eight mice
per group evaluated in two independent ex-
periments (*p , 0.04; unpaired t test).
Enhanced pulmonary inflamma-
The Journal of Immunology6351
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suggest that RGS16 attenuates inflammation in Schistosome egg-
challenged lungs by retaining T lymphocytes within granulomas.
RGS16 regulates lymphocyte trafficking in vivo mediated by
CCR4 and CCR10
Because Th2 cells express CCR4, and RGS16-deficient T cells
migrated excessively toward CCL17, we hypothesized that the
CCL17/22–CCR4 pathway mediated uncharacteristic migration of
a subset of Th2 cells (CCR4+IL-13+) in the challenged lungs of
Rgs162/2mice. Consistent with this hypothesis, we detected
CCR4+IL-13+T cells in the lungs of Rgs162/2much earlier than
in WT mice (4 d after challenge with S. mansoni) (Fig. 6A). To
determine whether other chemokine/receptor pairs contributed to
T lymphocyte mislocalization in the lungs of helminth-challenged
Rgs162/2mice, we analyzed differential gene expression by
qPCR array (full gene list in Supplemental Table I). Notably, we
found selectively increased expression of Il17b and Ccr10 mRNA
in the lungs of Rgs162/2mice compared with WT. CCR10 is
expressed by effector/memory T cells, Langerhans cells, and
plasma cells, among others (24–26), and its ligands CCL27/28 are
produced by epidermal keratinocytes (27) and displayed on the
surface of dermal endothelial cells (28). CCR10 expression on
skin-homing Th2 cells (29) has been implicated in the patho-
genesis of T cell-mediated inflammatory skin diseases including
atopic dermatitis and contact hypersensitivity (26, 27). We
detected CCR10 in the lungs of challenged mice by immunohis-
tochemistry, and its staining pattern mirrored T cell localization
in WT and Rgs162/2mice (predominantly in granulomas or
peribronchial/perivascular areas, respectively) (Fig. 6B). This re-
sult suggests a role for CCR10 in S. mansoni-evoked inflamma-
tion and in the aberrant trafficking of cytokine-producing T cells
we observed in Rgs162/2mice.
Although we found decreased or comparable expression of
Ccl17 and CCR10 ligands Ccl27 and Ccl28 in the lungs of WT
and Rgs162/2mice (in the presence or absence of S. mansoni
challenge) (Fig. 6C, Supplemental Table I; data not shown), the
naive lungs of knockout mice had significantly increased Ccr10
expression (Fig. 6D). We detected CCR10+T lymphocytes in the
naive lungs of Rgs162/2mice but not in WT mice (Fig. 6E),
suggesting that anomalous migration of RGS16-deficient CCR10+
T cells to the lungs accounted for the increased Ccr10 gene ex-
pression. Moreover, lungs from Rgs162/2mice, but not from WT
in Rgs162/2lungs following S. mansoni
challenge. (A and B) Frequencies of IL-
13+or IFN-g+T cells (CD3+) (A) or ratio
IL-13+/IFN-g+T cell percentages (B) in
the lungs 4 d following S. mansoni chal-
lenge were quantified by flow cytometry
(*p = 0.04; unpaired t test). The numbers
in each quadrant represent a percentage
of total T cells. (C) Chemotaxis of
splenic CD4+T cells collected from the
spleens of WT or Rgs162/2mice 4 d
following helminth challenge (mean 6
SEM of four to five mice per group; *p =
0.03; unpaired t test).
Aberrant Th2 migration
Rgs162/2lungs. (A and B) T cell localization in helminth-challenged lungs
evaluated by immunohistochemistry with a CD3 Ab. Images in (A) show
parenchymal T cell accumulation in granulomas, whereas those in (B)
demonstrate perivascular/peribronchial cell aggregates (arrowheads). Origi-
nal magnification 35. Total area containing cellular aggregrates around
airways and vessels was quantified using Image Pro Plus software (*p =
0.04; unpaired t test) as indicated in (C). Images represent eight mice per
group evaluated in two independent experiments.
Anomalous lymphocyte localization in helminth-challenged
6352 RGS16 INHIBITS LUNG INFLAMMATION
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mice, contained cytokine-producing CCR4+or CCR10+T cells
(IL-13 or IL-17B) 4–7 d following S. mansoni challenge (Fig.
6F–H). Collectively, these results indicate that ectopic trafficking
of CCR4+and CCR10+T effector cells to the lungs of Rgs162/2
mice may exacerbate fibrogenesis in response to pulmonary hel-
minth challenge through enhanced cytokine production.
Cytokine abnormalities in S. mansoni-challenged Rgs162/2
In addition to increased Ccr10 expression, array analysis also
revealed upregulation of cytokines in helminth-challenged Rgs162/2
lungs compared with WT—specifically, Il13 and Il17b. We con-
firmed these results by real-time PCR (Fig. 7A, 7B) and investi-
gated the source(s) of cytokines by restimulating MLN cells from
WTor Rgs162/2mice with SEA. LN supernatants from Rgs162/2
mice had significantly more IL-5, IL-10, and IL-13 than WT
(Fig. 7C). Because effector T cells are generally considered to be
the most common sources of these cytokines, we analyzed
quantities of LN cytokine-producing CD4 T cells in WT and
knockout mice. In contrast to the increased numbers of cytokine+
T cells present in the lungs of S. mansoni-challenged Rgs162/2
mice relative to WT at the earlier time point (4 d postchallenge),
frequencies of IL-13+or IL-17B+T cells and the intensity of IL-13
staining were similar in WT and Rgs162/2MLNs at the 7-d time
point (Fig. 7D, 7E). These results suggested that further charac-
terization of these cell populations is required to determine the
source of increased cytokines in MLN supernatants of Rgs162/2
mice relative to WT following re-exposure to S. mansoni.
We have elucidated a function for a modifier of GPCR signaling,
RGS16, in a Th2-mediated murine pulmonary inflammatory re-
sponse to helminth challenge—specifically, through regulation of
lung T cell trafficking and cytokine production. The loss of
RGS16 in mice triggered an enhanced granulomatous reaction in
the lung following challenge with S. mansoni eggs, resulting in
more fibrosis and eosinophil influx, anomalous localization of
T cells, and increased cytokine production. This study also high-
lights an unanticipated function of the CCR10–CCL27/8 chemo-
kine axis and IL-17B in the pathogenesis of S. mansoni-associated
Several lines of evidence suggest that RGS16 directly controls
differentiated Th/effector T cell migration patterns but does not
regulate trafficking of quiescent, naive T lymphocytes. RGS16
Th1, Th2, and Th17 cells compared with naive CD4 T cells (Fig.
2A) (13). Chemokines involved prominently in the maintenance of
lymphoid compartments through induction of lymphocyte ex-
ulations in Rgs162/2mice following
helminth challenge. (A) CCR4+Th2 cells
(IL-13+) in the lungs of challenged mice
were enumerated by flow cytometry. (B)
CCR10 expression in helminth-challenged
lungs was evaluated by immunohisto-
chemical staining with a CCR10 Ab. (C
and D) Ccl28 (C) or Ccr10 (D) expres-
sion in naive or S. mansoni-challenged
lungs evaluated by real-time PCR (*p =
0.04; unpaired t test). (E–G) CCR10+
T cells in naive lungs (E) or cytokine+
CCR10+(F) or CCR4+(G) T cells in
helminth-challenged lungs were quanti-
fied by means of flow cytometry. (H)
Frequencies of IL-17B+T cells (CD3+)
in lungs 7 d following S. mansoni chal-
lenge were quantified by means of flow
cytometry. Numbers represent percentage
of total T cells. All data were generated
using three naive mice per group, three to
five challenged WT mice, or four to five
challenged Rgs162/2mice. *p , 0.05,
***p , 0.0005.
Lung lymphocyte pop-
The Journal of Immunology6353
by guest on October 27, 2015
travasation through high endothelial venules of the spleen and
LNs (CCL21 and CXCL12) induced comparable chemotaxis of
naive WT and RGS16-deficient T cells. In contrast, Rgs162/2
effector Th2 lymphocytes differentiated in vitro or cells extracted
from S. mansoni-challenged lungs had exaggerated chemotaxis
toward a Th2-associated chemokine (CCL17). The degree of
RGS16 expression correlated inversely with the extent of migra-
tion, and chemotaxis of Rgs162/2T cells was reduced by re-
constitution with RGS16. Spleen and LN lymphocyte populations
and organ architecture in the absence of immune challenge were
unchanged in either RGS16-Tg or Rgs162/2mice (Ref. 13 and
this study), whereas acute inoculation with S. mansoni accentuated
trafficking of differentiated Th2 cells to these sites.
A surprising finding of this study is the anomalous collection of
CCR10+Th2 cells in the lungs of Rgs162/2mice. Although the
CCR10–CCL27 axis has been previously associated with Th2-
mediated inflammation in the skin, its role in pulmonary pathol-
ogy induced by S. mansoni challenge was unknown (20). CCL27
is produced in skin epidermal keratinocytes (27) and presented by
dermal endothelial cells (28). CCL17 also promotes CCL27 in-
duction by keratinocytes in the presence of TNF-a (30). CCL28,
another CCR10 ligand, has been implicated in CCR10-mediated
leukocyte homing to the respiratory tract in a murine asthma
model (31). The presence of similar or reduced amounts of
CCL27/28 ligands in the lungs of Rgs162/2mice compared with
WT suggests that aberrant chemotactic responses of RGS16-
deficient T cells to these ligands underlies their accumulation in
lungs in the absence of immune challenge. However, for unknown
reasons, we did not observe chemotaxis of Th2 cells (WT or
knockout) toward these chemokines in Transwell assays in vitro
despite expression of CCR10.
We also found increased Il13 expression in the challenged lungs
of Rgs162/2mice relative to WT, consistent with its central con-
tribution to fibrosis induced by helminth infection (32–33). Al-
though previous work has also defined a role for IL-17A in murine
lung inflammation (34), we observed increased Il17b expression in
the lungs of S. mansoni-challenged Rgs162/2mice relative to WT
but did not detect Il17a. These results suggest an unanticipated
function of IL-17B in helminth immunity. Among other IL-17
family members, neutralization of IL-17B in a collagen-induced
in Rgs162/2mice inoculated with S.
mansoni. (A and B) Expression of Il13
(A) or Il17b (B) was evaluated in naive
or helminth-challenged lungs by real-
time PCR (*p = 0.03, **p = 0.005;
unpaired t test). (C) Cells were har-
vested from lung-draining MLNs 7 d
following inoculation with S. mansoni.
Cells were left untreated or restimu-
lated with SEA for 3 d, followed by
measurement of the indicated cyto-
kine levels in supernatants (*p = 0.005,
***p = 0.0001; unpaired t test). (D)
Frequencies of IL-13+or IFN-g+T
cells (CD3+) in LNs 7 d following S.
mansoni challenge were quantified by
means of flow cytometry Numbers in
each quadrant represent percentage of
total T cells. The graph on the right
shows intensity of IL-13 staining (geo-
metric mean fluorescence intensity,
MFI) in the CD4 T cell population. (E)
Frequencies of IL-17B+T cells (CD3+)
in LNs 4 or 7 d following S. mansoni
challenge were quantified by means of
flow cytometry. All data were generated
using three naive mice per group, three
to five challenged WT mice, or four to
five challenged Rgs162/2mice. For
(C), data are representative of seven to
eight mice per group evaluated in two
6354 RGS16 INHIBITS LUNG INFLAMMATION
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model of murine arthritis suppressed disease progression by re-
ducing cell infiltration and production of proinflammatory cyto-
kine such as IL-1b and TNF-a (35), factors also known to induce
CCL28 in airway epithelial cells (36, 37). Although we detected
IL-17B+T cells in the challenged lungs, it is unclear whether these
cells are conventional Th17 cells. Indeed, in vitro-differentiated
Th17 cells produced IL-17A but not IL-17B (Supplemental Fig.
3). Taken together, these findings suggest the presence of a unique
proinflammatory environment downstream of IL-17B in the lungs
of Rgs162/2mice challenged with S. mansoni. The absence of
RGS16 may promote T cell chemotaxis to CCL27/28 displayed on
the surface of endothelial cells, which could account for the
peribronchial/perivascular accumulation of T cells we observed
in the challenged lungs of Rgs162/2mice.
How RGS16 regulates Ag-induced cytokine production requires
further study. We detected unique populations of IL-13 or IL-17B
cytokine-producing cells expressing CCR10 or CCR4 much earlier
in the lungs of Rgs162/2mice following helminth challenge. Al-
though quantities of cytokine+T cells in the lungs were equiv-
alent in the two strains by day 7, overall lung cytokine expression
was increased in the lungs of knockout mice at this time point
relative to WT. Additional gene array analysis revealed reduced
expression of Socs2 in the lungs of naive Rgs162/2mice com-
pared with WT counterparts but not in RGS16-deficient Th2 cells
differentiated in vitro (Supplemental Fig. 3; data not shown).
These data suggest that lung T cells in Rgs162/2mice are primed
for increased Th2 cytokine production as a result of reduced Socs2
In contrast, although levels of immunoreactive cytokines in
the supernatants of Ag-restimulated MLN cells extracted from
Rgs162/2mice 7 d post-S. mansoni challenge were significantly
higher than those from WT mice, flow cytometric analysis dem-
onstrated equivalent cytokine+T cell frequencies and intensity of
cytokine staining in these same LN T cell populations. Polarized
Th cells from WTand Rgs162/2mice secreted roughly equivalent
cytokine amounts (IFN-g for Th1, IL-4 or IL-13 for Th2, and IL-
17A for Th17) in response to TCR stimulation with anti-CD3
and anti-CD28 in vitro (Supplemental Fig. 3; data not shown).
Increased accumulation of specific populations of cytokine-
producing Th cells in MLNs of Rgs162/2mice compared with
WT (presumably because of altered trafficking patterns) could also
account for the overall increases in secreted cytokines observed.
Thus, on the basis of these data alone, we cannot yet determine
whether Ag-stimulated, RGS16-deficient T lymphocytes generate
more cytokine than WT cells on a per cell basis.
Although our work and that of others have shown that RGS
proteins inhibit chemokine-mediated lymphocyte chemotaxis and
adhesive responses invitro (39, 40), function(s) of RGS proteins in
T cell-mediated immunity have not been explored in detail. Sur-
prisingly, Rgs22/2mice had reduced footpad swelling following
local inoculation with lymphocytic choriomeningitis virus com-
pared with WT, which correlated with impaired proliferation of,
and IL-2 production by, RGS2-deficent T cells. Taken together,
these results and our studies suggest unique, context-dependent
functions of individual RGS proteins in immune cells that may
or may not be predictable, based on their shared biochemical
(GAP) activity. Further exploration of RGS16 in specific T cell
populations and in the setting of immune challenges will be
needed to fully clarify its function(s) in adaptive immunity. Al-
though we present abundant evidence that anomalous Th2 traf-
ficking and increased T cell-derived cytokines contribute to the
enhanced pulmonary inflammation in S. mansoni-challenged,
RGS16-deficient mice relative to WT (Figs. 4–7), non–Th-
dependent factors may also mediate fibrosis in this setting. Ad-
ditional models of inflammation and approaches such as adoptive
T cell transfer and/or generation of bone marrow chimeras will be
needed to determine the relative importance of T cell-intrinsic-
and T cell-independent factors to the immune responses of mice
The authors have no financial conflicts of interest.
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