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α-1 Antitrypsin Promotes Semimature, IL-10
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Tolerogenic Dendritic Cells
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Eyal Ozeri, Mark Mizrahi, Galit Shahaf and Eli C. Lewis
J Immunol published online 25 May 2012
http://www.jimmunol.org/content/early/2012/05/25/jimmun
ol.1101340
http://www.jimmunol.org/content/suppl/2012/05/25/jimmunol.110134
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Supplementary
Material
Published May 25, 2012, doi:10.4049/jimmunol.1101340
The Journal of Immunology
a-1 Antitrypsin Promotes Semimature, IL-10–Producing and
Readily Migrating Tolerogenic Dendritic Cells
Eyal Ozeri, Mark Mizrahi, Galit Shahaf, and Eli C. Lewis
D
endritic cells (DC) undergo transition from immature DC
to mature DC (mDC) under inflammatory conditions.
During this process, surface levels of CD40, CD80,
CD86, and MHC class II (MHCII) increase, and the cells release
inflammatory cytokines (1–4). Inflamed mDC migrate to draining
lymph nodes (DLN) and present Ags to T lymphocytes to facilitate Ag-specific adaptive immune responses, such that would
mediate, for example, acute allograft rejection (reviewed in
Ref. 5).
DC migrate primarily toward CCL19 and CCL21, chemokines
that serve as ligands for inducible CCR7 on the surface of DC (6).
As CCR7 is essential for migration of DC to lymph nodes, it is not
unexpected that CCR7 surface levels increase during inflammatory conditions (7). Indeed, CCR7 overexpression causes immature
DC to migrate to DLN (8), and CCR72/2 mice contain mDC that
fail to migrate and are incapable of promoting adaptive immune
responses (7, 9). Migrating DC, under specific conditions, also
hold the capacity to mediate T regulatory (Treg) cell differentiation
and facilitate Ag-specific immune tolerance. Indeed, CCR72/2 mice
Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105,
Israel
Received for publication May 10, 2011. Accepted for publication April 25, 2012.
This work was supported by Juvenile Diabetes Research Foundation Grant 2-2007-10
and Israel Science Foundation Grant 1027/07.
Address correspondence and reprint requests to Dr. Eli C. Lewis, Ben-Gurion University of the Negev, Faculty of Health Sciences, POB 151, Beer-Sheva 84105, Israel.
E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AAT, a-1 antitrypsin; BMDC, bone marrowderived DC; CT, control; DC, dendritic cell; DLN, draining lymph node; GITR,
glucocorticoid-inducible TNFR; mDC, mature DC; MHCII, MHC class II; smDC,
semimature DC; Treg, T regulatory; WT, wild-type.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101340
exhibit reduced numbers of protective CD4 +CD25 + Foxp3positive Treg cells (9), and human DC deficiencies exhibit a
sharp loss in Treg cell numbers (10).
Unlike mDC, semimature DC (smDC) present intermediate
levels of CD40, CD80, CD86, and MHCII (reviewed in Ref. 11);
smDC also produce high levels of IL-10 (12, 13). Whereas this
profile might appear to render the cells less than optimal APCs,
their ability to migrate to the DLN is suggested to be preserved
(5). Specifically, like mDC, smDC require surface CCR7 to migrate to the DLN (14). Once migration is achieved, smDC have
been shown to mediate auto- and alloimmune tolerance by the
preferential expansion of Ag-specific IL-2–dependent CD4+
CD25+Foxp3+ Treg cells (8, 15–18). In fact, in the context of a
tolerizing DC phenotype, an intact IL-2 activation pathway has
been shown to be essential for Treg differentiation (19, 20). In
addition to the prototypical CD4+CD25+Foxp3+ phenotype, Foxp3
appears to drive CD39 expression, an ectonucleotidase that hydrolyzes extracellular ATP and whose catalytic activity is
strongly enhanced by TCR ligation; CD39 expression has been
shown to be specifically relevant to Treg protection in the context
of transplantation immunology (21). Similarly, expression of
glucocorticoid-inducible TNFR (GITR) has been shown to occur
in protective Treg cells (22). Induction of smDC has to date been
experimentally achieved by DC stimulation under diminished inflammatory environments, such that are achieved by added vitamin D (23, 24), IL-4 (18), estrogen (25), MyD88 inhibitors (26),
and IL-10 (27).
Our group has reported that a-1 antitrypsin (AAT), a naturally
occurring anti-inflammatory glycoprotein, promotes smDC in the
presence of LPS and also during pancreatic islet allograft transplantation (28). AAT also facilitates Treg expansion in the NOD
mouse model, protecting the mice from autoimmune diabetes
(29). The tolerogenic effects of AAT have been shown to be
consistent using gene-derived human AAT without the use of
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Tolerogenic IL-10–positive CCR7-positive dendritic cells (DC) promote T regulatory (Treg) cell differentiation upon CCR7dependent migration to draining lymph nodes (DLN). Indeed, in human DC deficiencies, Treg levels are low. a-1 antitrypsin
(AAT) has been shown to reduce inflammatory markers, promote a semimature LPS-induced DC phenotype, facilitate Treg
expansion, and protect pancreatic islets from alloimmune and autoimmune responses in mice. However, the mechanism behind
these activities of AAT is poorly understood. In this study, we examine interactions among DC, CD4+ T cells, and AAT in vitro and
in vivo. IL-1b/IFN-g–mediated DC maturation and effect on Treg development were examined using OT-II cells and human AAT
(0.5 mg/ml). CCL19/21-dependent migration of isolated DC and resident islet DC was assessed, and CCR7 surface levels were
examined. Migration toward DLN was evaluated by FITC skin painting, transgenic GFP skin tissue grafting, and footpad DC
injection. AAT-treated stimulated DC displayed reduced MHC class II, CD40, CD86, and IL-6, but produced more IL-10 and
maintained inducible CCR7. Upon exposure of CD4+ T cells to OVA-loaded AAT-treated DC, 2.7-fold more Foxp3+ Treg cells were
obtained. AAT-treated cells displayed enhanced chemokine-dependent migration and low surface CD40. Under AAT treatment (60
mg/kg), DLN contained twice more fluorescence after FITC skin painting and twice more donor DC after footpad injection,
whereas migrating DC expressed less CD40, MHC class II, and CD86. Intracellular DC IL-10 was 2-fold higher in the AAT group.
Taken together, these results suggest that inducible functional CCR7 is maintained during AAT-mediated anti-inflammatory
conditions. Further studies are required to elucidate the mechanism behind the favorable tolerogenic activities of AAT. The
Journal of Immunology, 2012, 189: 000–000.
2
injected clinical-grade plasma-derived human AAT (30). In addition, AAT has recently been shown to promote IL-10–producing
DC in three animal models for graft-versus-host disease (31) and
to promote Treg cells in experimental autoimmune encephalomyelitis (32).
Most circulating AAT is synthesized during steady-state conditions by the liver, and its levels rise up to 6-fold during acute-phase
responses. In addition to exerting anti-inflammatory effects, AAT is
a potent inhibitor of multiple inflammatory and immune-activating
serine proteases and exhibits tissue-protective properties (33).
Nevertheless, the tolerogenic activities of AAT are still under investigation.
In the current study, we focus on the DC as one of the cellular
targets of AAT that holds a tolerogenic potential. Specifically, we
examine the ability of AAT to facilitate Treg-promoting, IL-10–
producing, and readily migrating smDC.
Materials and Methods
Mice
Generation of DC
Splenic DC were enriched using magnetic bead enrichment kit (Miltenyi
Biotec). Bone marrow-derived DC (BMDC) were generated, as described
(36). Briefly, cells that were released by flushing of mouse femur and tibia
with PBS were cultured (2 3 106 per well) in 10 ml RPMI 1640 containing
50 U/ml penicillin, 50 mg/ml streptomycin, 10% FBS (all Biological Industries), and 2-ME 50 mM (Sigma-Aldrich), and supplemented with rGMCSF (10 ng/ml; ProSpec). On day 3, 10 ml medium containing 10 ng/ml
GM-CSF was added. On day 6, half of the medium was replaced by
medium containing 10 ng/ml GM-CSF. On day 9, .95% of the cells were
CD11c+ according to FACS staining.
In vitro and in vivo DC maturation and migration experiments
According to each experimental setting, unless otherwise specified, BMDC
and splenic DC were pretreated overnight with AAT (0.5 mg/ml; Aralast,
Baxter, Westlake Village, CA) and then stimulated with murine rIL-1b and
murine rIFN-g (5 ng/ml each; R&D Systems) for 24 h. Also, systemic
AAT was introduced i.p. at 60 mg/kg throughout the study.
OVA-induced CD4+ T cell differentiation. BMDC were cultured with AAT,
and then OVA was added (10 mg/ml; GenScript, produced by bacteria-free
solid-phase peptide-automated synthesis). Twenty-four hours later, spleenderived OT-II CD4+ T cells (EasySep enrichment kit; STEMCELL Technologies) from Foxp3-GFP3OT-II mice were added. Medium was replaced
after 3 d, and cells were analyzed after another 3 d by FACS. Supernatant
were sampled for cytokine levels.
Agarose-plate DC migration. Culture dishes were precoated with poly-Llysine (0.01%; Sigma-Aldrich) and then covered by agarose (10%, prepared in RPMI 1640 supplemented with 10% FCS; Biological Industries).
Recombinant CCL19 and CCL21 (30 ng/ml each; Biological Industries)
or PBS were placed in agarose indents adjacent to wells containing BMDC
(5 3 106 per well). Plates were incubated at 37˚C overnight, and migration
distance was evaluated by microscope digital image analysis (Nikon
Eclipse TS-100).
Islet cell emigration. Islets were isolated, as described (28). Freshly isolated
islets were cultured in 48-well plates (50 islets per well), and cell emigration was determined by imaging and manual removal of islets and
transfer of adherent cells by cell scraper to an automatic cell counter
(Countess; Invitrogen) or FACS analysis.
FITC skin painting. FITC (15 ml; Sigma-Aldrich) was dissolved in acetone
and DMSO (1:1 ratio; Sigma-Aldrich) and freshly applied onto a 2-cm2–
shaved area of recipient mouse abdominal skin. Inguinal lymph node
single-cell suspensions were prepared 24 h later and analyzed by fluorometer (TECAN; SpectraFluor Plus).
Footpad-injected DC. BMDC were injected to the footpad of recipient mice
(2 3 106 cells per animal). Twenty-four hours prior to cell injection,
footpads were preconditioned by IL-1b and IFN-g (200 ml, 250 mg/ml
each), and AAT was administered systemically. Inguinal lymph nodes were
harvested 24 h later and analyzed by FACS. In related experiments, spleen
were harvested on day 7 for evaluation of Treg population. In addition,
OVA was loaded on BMDC as in the in vitro settings, and cells were introduced into footpads of OT II mice; spleen were harvested on day 7 for
evaluation of Treg population.
Graft-derived DC migration. Skin was removed from ventral sides of GFP
transgenic donor mouse ears, as described (37), and then placed s.c. at the
hypogastric abdominal area of recipient mice. Inguinal lymph nodes were
collected in separate experiments for RT-PCR and FACS analysis. In related experiments, skin grafting was performed into DTR-CD11c+ mice
with or without preconditioning using sublethal doses of diphtheria toxin
(Sigma-Aldrich; 100 ng/injection), as described (38); spleens were harvested on day 7 for evaluation of Treg population.
Transwell migration assay. BMDC (5 3 105 cells per 5-mm Transwell
inserts; Costar) were submerged in culture medium. After overnight incubation, cell migration to the bottom chamber was determined by an
automatic cell counter (Countess; Invitrogen).
RT-PCR and cytokine analysis
Total RNA was extracted using PerfectPure RNA Tissue Kit (5 PRIME), and
reverse transcription was followed using Verso cDNA kit (Thermo Scientific). Mouse b-actin forward, 59-GGTCTCAAACATGATCTGGG-39 and
reverse, 59-GGGTCAGAAGGATTCCTATG-39; CD86 forward, 59-TCCAGAACTTACGGAAGCACCCACG-39 and reverse, 59-CAGGTTCACTGAAGTTGGCGATCAC-39; CCR7 forward, 59- GAGGAAAAGGATGTCTGCCACG-39 and reverse, 59-GGCTCTCCTTGTCATTTTCCAG-39; CCL21
forward, 59- GAGGAAAAGGATGTCTGCCACG-39 and reverse, 59-GGCTCTCCTTGTCATTTTCCAG-39; and CCL19 forward, 59- CCTTCCGCTACCTTCTTAATG-39 and reverse, 59-CTTCTGGTCCTTGGTTTCCTG-39. Equal amounts of total RNA were used to generate cDNA for all the
samples. Prior to gene expression analysis, b-actin levels were compared
between the samples and had displayed no significant differences. For
semiquantitative PCR, each sample was amplified by at least two different
numbers of cycles to ensure that amplification was in the exponential phase
of PCR. Gene expression profile was analyzed by densitometry using NIH
ImageJ (http://rsbweb.nih.gov/ij/) and normalized to b-actin. Quantitative
real-time PCR was performed to confirm results obtained in the latter
technique (data not shown). Cell culture supernatants were analyzed for IL1b, IL-2, IL-6, and IL-10 using Q-Plex mouse cytokine chemiluminescencebased ELISA on Q-View imager (Quansys Biosciences, Logan, UT) and
quantified using Quansys Q-View software (Quansys Biosciences).
FACS analysis
Analysis was performed using Cytomics FC 500 Beckman Coulter, BD
Canto II, and BD FACSCalibur (each experiment was repeated using at least
two different FACS instruments). Data were analyzed by FlowJo (version
7.6.3; Tree Star, Ashland, OR). Islet-derived DC migration required detection of rare populations and was analyzed using MACSQuant FACS
analyzer (Miltenyi Biotec). After washing with FACS buffer (PBS containing 1% BSA, 0.1% sodium azide, and 2 mM EDTA [pH 7.4]), cells (1 3
106 per sample) were incubated with FcgRII/III blocker (BioLegend) and
then stained with the following Abs: anti-CD86 FITC, anti-MHCII PE, antiCD39 PE, anti-CD11c allophycocyanin, and anti-Foxp3 allophycocyanin
(from eBioscience, according to manufacturer’s guidelines); anti-CD4 Pacific Blue, anti-GITR PE, anti-CD3 FITC, anti-CD40 PE, and anti-CD25
allophycocyanin-Cy7 (from BioLegend, according to manufacturer’s
guidelines); and anti-CD8 V500 from BD Biosciences, according to manufacturer’s guidelines. Intracellular IL-10 staining was performed according
to BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD GolgiStop). Briefly, stimulated cells were incubated with GolgiStop for 5 h, then
harvested and stained for surface molecules, and washed twice with FACS
staining buffer and resuspended in fixation/permeabilization solution. Cells
were then washed and stained with anti–IL-10 PE. Fluorescent intensity
,103 was defined negative, as obtained from unstained samples and isotype
controls (CT) (data not shown). Fluorescent intensity in the range of 102–
103 was defined as high for CD40 and CD86 (CD40high and CD86high), and
in the range of 102–104 was defined as high for MHCII (MHCIIhigh).
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Wild-type (WT) C57BL/6, BALB/c, and chicken b-actin promoter GFP
transgenic mice (C57BL/6 background) were obtained from Harlan Laboratories. Foxp3-GFP knock-in mice that express GFP under the promoter
for Foxp3 were provided by T. Strom (Harvard Institutes of Medicine,
Boston, MA; transgenic animals described in Ref. 34), and OVA-specific
OT-II TCR transgenic mice (OT-II) were provided by S. Jung [Weizmann
Institute of Science; mice originally generated by Barnden et al. (35)], both
C57BL/6 background. Male F1 from Foxp3-GFP3OT-II mice were used
for OVA-responsive CD4+ T cell experiments (34). All animals were males
between the ages of 7 and 10 wk. Experiments were approved by the BenGurion University of the Negev Animal Care and Use Committee, and
animals were bred and maintained in accordance with the guidelines of the
Care and Use of Laboratory Animals published by the Israeli Ministry of
Health.
a-1 ANTITRYPSIN PROMOTES MIGRATING SEMIMATURE DC
The Journal of Immunology
Statistical analysis
Results are expressed as mean 6 SEM. Two-way ANOVA, followed by
Student t test, was used to assess differences among groups. A p value
,0.05 was considered significant.
Results
AAT promotes tolerogenic smDC
Although the appearance of LPS-induced smDC in the presence
of AAT has been established (28, 31), we sought to examine the
effect of AAT on DC stimulated by rIL-1b/IFN-g (Fig. 1A–C).
Splenic DC were incubated overnight in the presence of AAT (0.5
mg/ml), and then stimulated with IL-1b/IFN-g. For FACS analysis, 24-h cultures were examined, and CD11c+ cells were identified to determine the surface levels of MHCII, CD40, and CD86.
Fig. 1A depicts representative FACS images from each condition
and analyte. As shown, stimulated cells displayed elevated levels
3
of all three surface markers, whereas stimulated cells that were
added AAT displayed reduced surface levels of all three markers
to different extents; by setting the stimulated amplitude for each
marker at 100%, we calculate that stimulation in the presence of
AAT allowed MHCII to reach 72.9 6 8.8% of stimulation levels
(p = 0.048), CD40 16.6 6 0.55% of stimulated levels (p = 0.052),
and CD86 35.4 6 2.05% of stimulated levels (p = 0.039). The
kinetics of CD86 expression was examined by RT-PCR. As shown
in Fig. 1B, CD86 transcript levels increased in stimulated cells by
up to 1.14 6 0.03-fold from CT in the absence or presence of AAT
in the first 5 h after stimulation. However, CD86 transcript levels
decreased to nonstimulated values at 9 and 24 h after stimulation
in the presence of AAT.
Release of IL-6 and IL-10 was determined 72 h after stimulation
(Fig. 1C). As shown, stimulated cells displayed an increase in IL-6
and IL-10, albeit without significant difference from CT. However,
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FIGURE 1. Facilitation of smDC and a tolerogenic environment. (A–C) In vitro stimulation of DC with IL-1b/IFN-g (5 ng/ml each) in the presence of
AAT (0.5 mg/ml). (A) FACS analysis at 24 h. Spleen-derived magnetic bead-enriched CD11c+ cells (1 3 106/well in triplicates) were either untreated (CT)
or cytokine stimulated, in the absence or presence of AAT. Representative dot plots; shown are percentage events out of CD11c+ in separate gates for high
(upper gate) and low (bottom gate) expression levels of surface MHCII, CD40, and CD86. Representative experiment of five independent experiments. (B)
RT-PCR. Gene expression kinetics for CD86, as analyzed in 5 3 105 cells/well in multiples of seven at 0, 5, 8, and 24 h after exposure to cytokine
stimulation. Results are shown as fold from untreated (CT) cells. Mean 6 SEM, *p , 0.05 between conditions in each time point. (C) Cytokine release at
24 h. Cultured DC (5 3 105 per well in triplicates) were allowed to respond to IL-1b/IFN-g for 24 h, after which supernatants were analyzed for IL-6 and
IL-10. Mean 6 SEM, *p , 0.05. Representative data from three independent experiments. (D–F) Generation of Treg cells in mixed cultures of BMDC and
OVA-reactive CD4+ T cells. BMDC (4 3 104 cells per round-bottom 96-plate wells in multiples of 12) were either untreated or preincubated with AAT, and
then OVA Ag (10 mg/ml) was added overnight and washed. Splenic bead-enriched CD4+ T cells from OT II mice were added to the culture (4 3 105 cells/
well) and allowed to interact with DC for 3 and 6 d. (D) Supernatant cytokines. IL-1b, IL-6, IL-2, and IL-10 were measured; day of supernatant collection is
depicted. Mean 6 SEM, *p , 0.05, **p , 0.01. (E) FACS for surface CD40. Left, Representative overlay of CD11c+ cells out of the coculture of DC and
CD4+ T cells under the various treatment protocols; right, representative bar graph from three independent experiments. Mean 6 SEM, **p , 0.01. (F)
FACS for surface CD4 and CD25, and depiction of Foxp3+ cells. Representative FACS contour images as obtained by collecting gated data from nonCD11c+ cells. Center, Cells positive for Foxp3 (using the transgenic Foxp3-driven GFP signal) out of CD4+CD25+ cells. Representative histogram and bar
graph from pooled results (right). Representative experiment of five independent experiments. Mean 6 SEM, **p , 0.01.
4
the population of CD3+CD4+CD82Foxp3+CD39+ cells in the
spleen increased 1.72 6 0.26-fold in the presence of AAT. Similarly, when OVA-loaded BDMC were introduced to OT II mice
(Supplemental Fig. 2), CD3+CD4+CD25+GITR+ cell population
size increased 2.71 6 0.69-fold in the AAT-treated group. Furthermore, the requirement of DC for the process of AAT-induced
Treg elaboration gained support by the use of DTR-CD11c mice;
as shown in Supplemental Fig. 3, skin allograft recipient animals
exhibited a larger Treg population size when treated systemically
with AAT, whereas animals that were pretreated with DC-depleting doses of diphtheria toxin exhibited a lack of AAT-mediated
increment in Treg population size.
AAT-treated DC readily migrate
Because DC typically express CCR7 and migrate under inflammatory conditions, the concern was raised that AAT-treated DC
might fail to migrate. We therefore sought to examine whether the
addition of AAT to DC allows for chemokine-directed migration
in vitro and in vivo. Using an agarose-based migration assay (Fig.
2A), AAT-pretreated cytokine-stimulated BMDC were allowed to
migrate overnight through an agarose gel prepared in RPMI 1640,
on the surface of poly-L-lysine–coated plates, toward neighbor
wells that contained rCCL19/CCL21. As shown, CT untreated
cells demonstrated steady-state migration to an average distance of
187.3 mm. Cytokine-stimulated cells, however, migrated to a distance 1.18-fold greater than CT. Unexpectedly, in the presence of
AAT, cells migrated to a distance 1.977-fold greater than CT.
In light of the ability of AAT to promote tolerance in islet
transplantation models and in the NOD mouse, islet-resident DC
were next examined (Fig. 2B). The phenomenon of APC emigration from cultured islets has been described (40). As shown in
representative images, a migrating mass of cells can be observed
extending away from cultured primary pancreatic islets (representative micrographs and pooled automatic cell-counter results),
with a clear increase in cell migration upon added cytokine
stimulation. However, in the presence of AAT, a 2.8-fold further
increase in the number of emigrating cells was observed. Cell
count was consistent with FACS analysis results, which depict
FIGURE 2. DC migration in vitro. (A) Agarose migration assay. BMDC (1 3 106 per well) were pretreated with AAT (0.5 mg/ml) and then stimulated
overnight with IL-1b/IFN-g (5 ng/ml each). CCL19/CCL21 (30 ng/ml each) added in the well next to BMDC. Migration distance was determined after
24 h. Data pooled from five independent experiments. Mean 6 SEM, **p , 0.01. (B) Primary islet resident cell migration assay. Freshly isolated pancreatic
islets (50 per well in multiples of six) were pretreated with AAT (0.5 mg/ml) and then stimulated overnight with IL-1b/IFN-g (5 ng/ml each). Wells were
added to rCCL19/CCL21 (30 ng/ml each), and islet resident cells were allowed to migrate on the surface of the wells for 24 h. Photomicrographs of
representative fields containing 1–3 islets (original magnification 340, unstained). Arrows, adherent and migrating cells. Right, Automatic cell counter
results obtained from scraped adherent cells. The cells were then examined by FACS analysis for the surface markers. (C) CD11c (representative overlay
and pooled results) and (D) CD40 (representative dot plot). Insets, Percentage of high and low CD40 expression. Representative experiment of four
independent experiments. Mean 6 SEM, *p , 0.05. CT, Untreated cells.
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in the presence of AAT, IL-6 release was decreased 2.5-fold from
stimulated levels, and IL-10 release was increased 2.7-fold from
stimulated levels.
To assess the ability of AAT-treated DC to promote Treg differentiation, a mixed culture of DC and T lymphocytes was studied
(Fig. 1D–F). BMDC obtained from WT mice were cultured
overnight in the presence of AAT. Cells were then incubated with
synthetic OVA Ag for 24 h. Splenic Foxp3-GFP3OT-II OVAreactive CD4+ T cells were added to washed DC. Supernatant
content was examined on days 3 and 6 (Fig. 1D). As shown, the
presence of OVA resulted in an increase in the release of IL-1b
and IL-2, and, to a lesser extent, IL-6 (day 3), in accordance with
reports on cytokine release by exposure to OVA (39). However, in
the presence of AAT, IL-1b and IL-6 levels were significantly
reduced from stimulated levels. Unexpectedly, IL-2 release was
further increased by added AAT. Whereas IL-10 levels were below
detection on day 3, day 6 supernatants contained greater levels of
IL-10 in cocultures that contained AAT-pretreated (OVA-treated)
DC and (OT-II OVA-reactive) CD4+ T cells.
According to FACS analysis (Fig. 1E), a significantly lower
population size of CD40+ cells was observed in the presence of
AAT, compared with OVA-treated cells. Also, as shown in Fig. 1F,
in two representative FACS images (left), T cells that were cultured in the presence of OVA/AAT-treated DC exhibited reduced
population proportions of CD4+CD25+ T cells (from 16.93 6
1.12% to 8.8 6 1.0% out of CD11c2 cells). Of the CD4+CD25+
cells, we determined the percentage of CD4+CD25+Foxp3+ cells
(center, representative overlay; right, pooled results). The Foxp3
signal in these experiments is derived from transgenic GFP under
Foxp3 promoter, which was confirmed in a separate transplant
experiment to overlap with direct intracellular Foxp3 staining
(data not shown). As illustrated in Fig. 1F, the proportion of Foxp3expressing cells out of cells gated for CD4+CD25+ displayed a
significant increase in the AAT-treated group (from 2.88 6 0.53%
to 7.9 6 0.83%, respectively). In related in vivo experiments, we
examined the changes in Treg population size using cultured
BMDC that were preincubated with AAT and then added IL-1b/
IFN-g (Supplemental Fig. 1). As shown, once injected to animals,
a-1 ANTITRYPSIN PROMOTES MIGRATING SEMIMATURE DC
The Journal of Immunology
cells out of total CD11c+ cells, representing a 2 6 0.12-fold increase in the proportion of donor-derived DC.
In the tissue-grafting model (Fig. 3C), recipient mice were divided into untreated and systemically pretreated AAT mice. Skin
tissue from GFP transgenic mice was grafted to recipient animals,
and 16 h later inguinal lymph nodes were collected and analyzed
by FACS for CD11c+GFP+ donor-derived cells. The proportion of
graft-derived CD11c+GFP+ cells that migrated to DLN was 2.28fold greater on average in the AAT-treated recipient mice (n = 9)
compared with untreated graft recipients (n = 7) (p = 0.0003).
Moreover, as shown in Fig. 3D, surface levels of MHCII, CD40,
and CD86 on donor-derived GFP+CD11c+ cells were consistently
lower in the AAT-treated group (2.64 6 0.11-, 2.12 6 0.07-, and
4.16 6 0.16-fold reduction in markers, respectively).
Unlike the downregulation of maturation markers observed in
migrating donor cells in the AAT group, intracellular DC IL-10
exhibited a positive response to AAT. As shown in Fig. 3E, in
the AAT-treated group IL-10 levels increased 2 6 0.07-fold in
the total population of CD11c+ cells (left, representative contour
FACS images; center, pooled results), and were similarly elevated
in migrating donor-derived GFP+ cells (Fig. 3E, right).
smDC elicited by AAT and cytokine stimulation maintain
inducible levels of CCR7, and migrate in a CCR7-dependent
manner
Because inducible CCR7 expression appears to require inflammation to promote DC migration, we sought to examine whether
the anti-inflammatory environment exerted by AAT allows for
CCR7 expression and for CCR7-dependent migration. BMDC were
cultured in the presence of AAT 24 h before stimulation with IL-1b/
FIGURE 3. smDC migration in vivo. (A) FITC skin painting. Mice were treated systemically with AAT prior to abdominal FITC painting. Sixteen hours
later, cell suspension was prepared from DLN and total fluorescence was examined. CT set at 100%. Representative graph from three independent
experiments. Mean 6 SEM, *p , 0.05. (B) Footpad-injected DC. BMDC from GFP transgenic mice were pretreated with AAT (0.5 mg/ml) and then
stimulated overnight with IL-1b/IFN-g (5 ng/ml each). Recipient mice were preconditioned accordingly. A total of 2 3 106 cells/footpad was introduced,
and 24 h later, the proportion of GFP-positive donor cells out of CD11c+ cells in inguinal DLN was determined. Mean 6 SEM, **p , 0.01. Representative
results from three independent experiments. (C–E) Skin tissue graft model. Mice were pretreated with systemic AAT (n = 9) or untreated (CT, n = 7).
Syngeneic donor tissue from GFP transgenic mice was grafted. (C) FACS analysis of CD11c+GFP+ inguinal DLN content at 24 h. Results representative
of two independent experiments. (D) Donor cell surface markers (left, representative FACS overlay; right, bar graph representing five independent
experiments). Mean 6 SEM, *p , 0.05, **p , 0.01, ***p , 0.001. (E) Intracellular IL-10 levels in migrating DC (left, representative images and pooled
results from total CD11c+ cells; right, donor GFP+CD11c+ cells). Data represent two independent experiments, mean 6 SEM, *p , 0.05, **p , 0.01.
CT, Untreated cells (n 5 3 per group).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
changes in CD11c+ cell population size (Fig. 2C, representative
overlay on the left and a graph of pooled results on the right).
Being that the migrating CD11c+ cells are considered a rare
population in islets, FACS analysis required MACSQuant FACS
analyzer (Miltenyi Biotec), which further allowed the identification of CD40 on the surface of the minute population of migrating
CD11c+ cells (Fig. 2D). As shown, in stimulated cells the population of CD40+CD11c+ migrating cells was elevated 7.76 6 3.4fold from CT, but only 1.43 6 0.62-fold in the presence of AAT
(representative dot plots). Of note, the FACS analysis incorporates
all adherent cells in the plate and thus includes also nonmigrating
adherent cells.
These in vitro findings prompted us to examine whether the
observed trend in enhanced smDC migration by AAT is reproducible in vivo. FITC skin painting model allows evaluation of DC
migration from skin to DLN by analysis of carryover fluorescence.
The population of cells in this model found in the DLN has been
shown to be CD11c+ (3, 41). As shown in Fig. 3A, fluorescence in
DLN increased 2.1 6 0.3-fold in AAT-treated mice compared with
untreated mice (CT, p = 0.03). DC footpad injection and skin
tissue grafting were next performed (Fig. 3B, C–E, respectively).
BMDC from GFP transgenic mice were added AAT overnight and
then stimulated by IL-1b/IFN-g (Fig. 3B). Twenty-four hours after
cytokine stimulation, 2 3 106 cells were injected into the rear
footpads of WT mice. As shown, after 24 h from cell injection,
DLN contained 10.59 6 0.34% donor-derived CD11c+GFP+ cells
out of total CD11c+ cells in mice that received untreated cells
(CT), and 26.88 6 1.22% in mice that received cytokinestimulated cells. DLN from mice that were injected with AATtreated stimulated cells contained 54.11 6 1.3% CD11c+GFP+
5
6
DC migration in stimulated AAT-treated wells, indicating that
AAT drives DC migration in a CCR7-dependent manner.
Discussion
Tolerogenic DC and CD4+CD25+Foxp3+ Treg cells hold the capacity to influence allogeneic and autoimmune immune responses
(17). Typically, DC that are tolerogenic exhibit reduced surface
levels of inducible CD40, CD86, and MHCII; release less inflammatory cytokines; and secrete more IL-10 (11), thus termed
semimature. In a previous report of ours, we demonstrated that the
anti-inflammatory acute-phase circulating protein, AAT, promotes
islet allograft tolerance, as well as smDC (28). Consistent outcomes were generated in the NOD mouse (29), in experimental
autoimmune encephalomyelitis (32), and in three animal models
for graft-versus-host disease (31). Uniformly, treatment with human AAT results in an elevated population size of CD4+CD25+
Foxp3+ Treg cells in the lymph nodes and also at local antigenic
sites. Importantly, other groups as well as ours have established
that AAT does not interfere with IL-2–mediated T cell responses,
thus presumably allowing Treg expansion (28, 29).
In the current study, we revisited the capacity of AAT to generate
smDC by extending the stimuli to include IL-1b and IFN-g.
Consistent with our previous report, we observed favorable
changes in cell surface markers, less proinflammatory cytokine
release, and more IL-10 production. When cocultured with T cells,
smDC increased the proportion of Treg cells. It was interesting
to observe that IL-2 was increased in mixed cultures of AATpretreated DC and T cells. At this point in the study, at least
two major questions remained to be addressed, as follows: 1) can
these smDC migrate toward lymph nodes, and 2) do they convert
T cells into Treg cells?
As shown, AAT allowed in vivo migration of smDC, resulting in
the accumulation of smDC in DLN in multiple experimental setups.
AAT promoted DC migration in an agarose plate toward a chemokine gradient, and also away from stimulated cultured islets.
AAT facilitated DC migration in skin FITC-painting model and in
FIGURE 4. smDC CCR7 expression and CCR7-dependent migration. (A and B) In vitro CCR7 expression. BMDC (2 3 106 in multiples of six) were
pretreated with AAT (0.5 mg/ml) and then stimulated with IL-1b/IFN-g for 4, 12, and 24 h (5 ng/ml each). (A) RT-PCR for CCR7 at 4 h, normalized to
b-actin, shown as fold-change from CT. Mean 6 SEM, representative experiment of two independent repeats, *p , 0.05. (B) FACS analysis for CCR7
surface levels at 12 and 24 h. Representative experiment of 10 independent repeats. Mean 6 SEM, *p , 0.05. (C) Islet-derived DC. Islets were incubated as
in Fig. 2 and then analyzed by FACS for CD11c+CCR7+ migrating cells (representative overlay and pooled results). (D) In vivo expression of CCL19. Mice
were pretreated with AAT and then grafted with allogeneic skin (n = 6 per group). Four hours later, inguinal DLN was analyzed by RT-PCR for CCL19 and
normalized to b-actin. **p , 0.01. (E) CCR7-dependent AAT-facilitated Transwell migration. BMDC (0.5 3 106) were cultured on a Transwell insert in
wells containing CCL19/CCL21 (30 ng/ml each) with or without neutralizing anti-CCR7 Ab. Cells were pretreated with AAT (0.5 mg/ml) and then stimulated
with IL-1b/IFN-g (5 ng/ml each). Migrating cells were determined in the bottom compartment after 24 h by automatic cell counter. Mean 6 SEM.
CT, Untreated cells; Tx, grafted mice.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
IFN-g. Cells were then analyzed for CCR7 expression by RT-PCR
after 4 h and by FACS after 12 and 24 h (Fig. 4A, 4B). As shown,
relative CCR7 mRNA transcript levels displayed a positive response upon inflammatory stimulation, and instead of being reduced by AAT, appeared to have maintained their inducible levels.
Greater changes were observed in CCR7 surface levels (Fig. 4B);
AAT-treated cells exhibited a 3.7-fold increase in CCR7 levels at
12 h compared with CT. At 24 h, cytokine stimulation resulted in
a 5.3-fold rise in CCR7 surface levels, and a consistent rise in
AAT-treated cells (5.5-fold greater than CT). Thus, surface CCR7
is upregulated earlier in DC that are inflamed and exposed to AAT.
Surface CCR7 was also evaluated in CD11c+ cells that emigrated from cultured islets (Fig. 4C, extended readouts from the
experiment described in Fig. 2B). As shown, CCR7 surface levels
were consistently greater in resident CD11c+ cells that emigrated
from islets in the presence of AAT. Of note, unlike in Fig. 2C, the
FACS analysis in this study incorporates only cells that migrated
through the pores of the Transwell and are thus predominantly
CCR7 positive.
To assess whether inflammation-triggered lymph node-derived
ligands for CCR7 are consistent with an active CCR7 pathway
in the presence of AAT in vivo, the expression of CCL19 was
examined in DLN from tissue-grafted mice (Fig. 4D). As shown,
CCL19 exhibited an increase in DLN transcript levels after tissue
transplantation, and maintained values in the AAT-treated group
that are comparable to the stimulated group. CCL21 did not respond to transplantation at this time point and was unchanged in
the presence of AAT (data not shown); these constitutive expression levels are consistent with reports regarding posttranscriptional proteolytic regulation of CCL21 (6).
The functional role of CCR7 in the migratory effects of AAT on
DC was examined using neutralizing anti-CCR7 Ab in a Transwell
migration assay (Fig. 4E). As shown, cells readily migrated to the
bottom compartment in stimulated conditions, and also in stimulated wells that were added AAT. However, when CCR7 was
blocked by neutralizing Ab, there was a significant reduction in
a-1 ANTITRYPSIN PROMOTES MIGRATING SEMIMATURE DC
The Journal of Immunology
attributes (47, 48). However, we found no evidence for increased
DC viability in the presence of AAT according to live-dead cell
staining and trypan blue exclusion (data not shown). In addition,
because neutralizing anti-CCR7 Ab blocked AAT-related DC
migration, it appears that the large cell numbers at the end of
migration assays are indeed CCR7 dependent and not affected by
enhanced viability. It would be of interest to investigate whether
CCR72/2 mice that have been shown to fail to evolve Foxp3+
Treg cells (9) can achieve Foxp3+ Treg expansion under systemic
AAT treatment.
Our findings support the notion that AAT affects both host and
donor CD11c+ cells in a manner that achieves semimature migrating tolerogenic cells. However, further studies are required
to elucidate the potential use of AAT for ex vivo graft preconditioning, or, conversely, for manipulating host DC toward
immune adaptation.
A diverse variety of agents has been shown to promote smDC,
most of which inhibit nuclear translocation of members of the
NF-kB family of transcription factors (reviewed in Ref. 49). With
this regard, the reports by Churg et al. (50) and Tawara et al. (31),
in which AAT reduces NF-kB translocation to the nucleus, provide a possible mechanism for the elaboration of smDC by AAT.
In addition, IL-1b has been shown to break Treg-mediated tolerance
(51); because AAT induces the endogenous antogonist for IL-1b,
IL-1R antagonist (52), and also reduces IL-1b and IL-6 levels (53,
54), it appears that AAT promotes a tolerogenic profile of cytokines.
Whether the mechanism behind these changes is restricted to the
ability of AAT to inhibit proteases or whether it is serine protease
independent, is a topic of recent interest (33, 48, 55, 56).
In conclusion, the current study describes a particular aspect of
AAT-mediated immune tolerance, namely, the DC as a cellular
target of immune modulation. In addition, we describe the unexpected observation that inducible surface CCR7 is maintained
during AAT-mediated anti-inflammatory conditions, and that
modulated DC migrate despite the lack of an inflammatory cytokine profile. Consistent with the availability of AAT as augmentation therapy for individuals with genetic AAT deficiency, its
favorable safety profile (57), and its islet-protective attributes in
the context of unwarranted immune responses (28, 29), the rapid
testing of weekly administration of i.v. human AAT for youngsters
with recently diagnosed autoimmune type 1 diabetes is currently
investigated in three clinical trials (National Institutes of Health
clinical trial registry codes NCT01304537, NCT01319331, and
NCT01183468). Nevertheless, further studies are required to fully
elucidate the mechanism behind the favorable tolerogenic activities of AAT.
Acknowledgments
We thank Valeria Frishman for excellent technical assistance and Zhana
Moshayev for generous support in FACS studies.
Disclosures
The authors have no financial conflicts of interest.
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