Download and Adaptive Immune Responses an Endogenous

Identification of Pancreatic Glycoprotein 2 as
an Endogenous Immunomodulator of Innate
and Adaptive Immune Responses
This information is current as
of May 7, 2017.
Lael Werner, Daniela Paclik, Christina Fritz, Dirk Reinhold,
Dirk Roggenbuck and Andreas Sturm
J Immunol 2012; 189:2774-2783; Prepublished online 13
August 2012;
doi: 10.4049/jimmunol.1103190
http://www.jimmunol.org/content/189/6/2774
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Supplementary
Material
The Journal of Immunology
Identification of Pancreatic Glycoprotein 2 as an Endogenous
Immunomodulator of Innate and Adaptive Immune
Responses
Lael Werner,* Daniela Paclik,* Christina Fritz,† Dirk Reinhold,‡ Dirk Roggenbuck,† and
Andreas Sturm*
I
nflammatory bowel diseases (IBD), consisting of Crohn
disease (CD) and ulcerative colitis (UC), is a chronic condition affecting a steadily rising number of patients worldwide. IBD results in significant social and economic costs, as it is
complex, unpredictable, and incompletely understood.
Diagnosis of IBD and the differentiation between UC and CD
are established by a combination of clinical, laboratory, radiologic,
endoscopic, histopathologic, and serologic markers. The major
serologic markers for IBD are antineutrophil cytoplasmic Abs, antiSaccharomyces cerevisiae Abs, and Abs to outer membrane porin
C of Escherichia coli. However, the significance of these autoantibodies for discriminating between UC and CD is still controversial (1), and thus the need for accurate serologic markers in
IBD remains essentially.
Novel markers discovered in recent years, such as antichitobioside, anti-laminaribioside, and anti-mannobioside, are
Abs directed against glycans (2). As such, glycans are being
recognized as key compounds in mucosal immunology, and gly-
*Division of Hepatology and Gastroenterology, Department of Medicine, Charité–
Campus Virchow Clinic, Medical University of Berlin, 13353 Berlin, Germany;
†
Medipan GmbH, Dahlewitz/Berlin, 15827 Dahlewitz, Germany; and ‡Institute of
Molecular and Clinical Immunology, Otto von Guericke University of Magdeburg,
39106 Magdeburg, Germany
Received for publication November 14, 2011. Accepted for publication July 13,
2012.
This work was supported in part by a grant from the German Federal State of
Brandenburg.
Address correspondence and reprint requests to Prof. Dr. Andreas Sturm at the current
address: Head, Department of Internal Medicine and Gastroenterology, Krankenhaus
Waldfriede, Argentinische Allee 40, 14163 Berlin, Germany. E-mail address: a.sturm@
waldfriede.de
The online version of this article contains supplemental material.
Abbreviations used in this article: ADA, adalimumab; CD, Crohn disease; GP2,
glycoprotein 2; h, human; IBD, inflammatory bowel disease; IBS, irritable bowel
syndrome; IEC, intestinal epithelial cell; IEL, intraepithelial lymphocyte; IFX,
infliximab; LPMC, lamina propria mononuclear cell; PAB, pancreatic autoantibody;
PI, propidium iodide; SA, streptavidin; THP, Tamm–Horsfall protein; Treg, regulatory T cell; UC, ulcerative colitis.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103190
cans are proposed to bridge innate and adaptive immune recognition and response (3). This realization led to a growing interest in
Abs to carbohydrate (glycan)-based Ags, as cellular and humoral
immune responses rely heavily on interactions between glycans
and specific glycan-binding proteins (4). One such glycan-directed,
CD-specific serological marker is pancreatic autoantibody (PAB),
first described more than two decades ago (5). However, its antigenic epitope was unknown until recently.
In 2009, Roggenbuck et al. (6) identified glycoprotein 2 (GP2)
as the major autoantigenic target of a PAB. Anti-GP2 IgG and IgA
have been reported to be novel serologic parameters in CD (7).
Their possible association with disease behavior and activity in
CD is controversial (8). Why GP2 is an autoantibody target in
patients with CD remains also elusive, and whether it plays
a pathophysiological role in the development of the disease or is
an epiphenomenon needs to be determined.
GP2 is a 78-kDa GPI-anchored protein. Initially, GP2 was proposed to be the major zymogen granule membrane in the pancreas
(9), accounting for a large percentage of all of the zymogen (glyco)
proteins. Upon stimulation, GP2 is shed into the pancreatic duct
during exocrine secretion, and then into the intestine (10, 11), for
a possible, yet unknown physiological function.
However, as GP2 knockout mice do not exhibit nutrient malabsorption or predisposition to pancreatitis (12), and GP2 overexpression does not influence secretory processes (13), another,
previously unknown functional and physiological relevance for
GP2 needs to be discerned. Remarkably, apart from the pancreas,
GP2 has been shown to be overexpressed at the site of CD inflammation in the gut, in contrast to UC patients, supporting
a pathophysiological role for this protein in the development of the
disease (7).
The GP2 amino acid sequence exhibits 85% similarity to Tamm–
Horsfall protein (THP; uromodulin) (14), suggesting that they
might possess homologous functions. THP is the most abundant
protein in urine, and autoantibodies to THP have also been reported in urinary tract inflammation (15). Moreover, THP has been
implicated in several inflammatory kidney disorders (16), as well
as in downregulation of both innate and adaptive immunity (17).
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Pancreatic autoantibodies are Crohn disease-specific serologic markers. The function and immunological role of their recently
identified autoantigen, glycoprotein 2 (GP2), are unknown. We therefore investigated the impact of GP2 on modulation of innate
and adaptive immune responses to evaluate its potential therapeutic use in mucosal inflammation. Our data indicate a previously
unknown function for GP2 as an immunomodulator. GP2 was ubiquitously expressed on cells vital to mucosal immune responses.
The expression of GP2 was upregulated on activated human T cells, and it was further influenced by pharmaceutical TNF-a
inhibitors. Recombinant GP2 significantly decreased human intestinal epithelial cells, mucosal and peripheral T cell proliferation,
apoptosis, and activation, and it distinctly modulated cytokine secretion. Furthermore, intestinal epithelial cells stimulated with
GP2 potently attracted T cells. In conclusion, we demonstrate a novel role for GP2 in immune regulation that could provide
a platform for new therapeutic interventions in the treatment of Crohn disease. The Journal of Immunology, 2012, 189: 2774–2783.
The Journal of Immunology
Thus, a similar immunological mechanism may be operative in the
mode of action of GP2, and this needs further scrutiny.
Elucidation of the role played by GP2 in immunity, as well as
the pathophysiological involvement of the GP2 Abs in IBD, could
yield great benefit for medical intervention. As defects in T cell
expansion and apoptosis are major events in triggering and perpetuating mucosal inflammation in IBD (18), restricting T cell expansion by GP2 might provide a new therapeutic option to restrict
inflammation. Moreover, anti-GP2 Abs might serve as a diagnostic
tool for recognizing different subtypes of CD.
As an anti-inflammatory function for THP has been described,
we conceived a similar immune modulation for GP2. Thus, based
on the knowledge currently available and our preliminary data, we
hypothesize that anti-GP2 Abs not only differentiate CD from UC
and healthy controls, but also that GP2 itself modulates innate and
adaptive immune responses.
2775
transfection of GP2-DNA–containing baculovirus, as described previously
(7). GP2 (200 mg/ml) was maintained in 50 mM Tris buffer (pH 7.5). To
exclude influence of the GP2 buffer, all of the abovementioned experiments were also performed with Tris buffer alone. No influence was observed by the buffer on any of the parameters (data not shown).
Flow cytometry
Cells were analyzed by flow cytometry using FACSCalibur (Beckman
Coulter, Krefeld, Germany). For propidium iodide (PI; Calbiochem,
Darmstadt, Germany) analysis, cells were fixed and permeabilized with BD
Fix/Perm solution (BD Pharmingen). Apoptosis was detected using annexin
V (BD Pharmingen) and PI. For membrane GP2 quantification, cells were
incubated with anti-GP2 (Sigma-Aldrich, clone HPA016668) for 25 min at
4˚C. Anti-GP2 was preincubated for 24 h with biotinylated Ab (Miltenyi
Biotec) at room temperature. Subsequently, streptavidin (SA)-FITC
(eBioscience, Frankfurt, Germany) was added for 25 min at 4˚C. All
other Abs, including respective IgG controls, were purchased from BD
Pharmingen.
Caspase detection
Materials and Methods
Cell isolation
IEC cell lines
T84 cells were cultured at 37˚C, 5% CO2 and maintained in DMEM/F-12
(Life Technologies) supplemented with 10% FCS, 2.5% penicillinstreptomycin, and 5 ml sodium pyruvate (Lonza, Cologne, Germany).
Cells used were between passages 20 and 30. Cells (2 3 105) were cultured in 24-well tissue culture plates (Corning Glass Works, Corning, NY)
and allowed to grow for 21 d before usage.
Binding of GP2
For binding assays, cells were incubated with biotinylated GP2 for 40 min at
4˚C and subsequent incubation with SA-FITC. Binding was determined by
flow cytometry. SA-FITC alone served as negative control.
MTT assay
Cells (5 3 105) were placed in 96-well plates in triplicates with 100 ml
MTT (4 mg/ml; Sigma-Aldrich), for 3 h at 37˚C. Afterward, plates were
centrifuged and supernatant was removed and then lysed with 100 ml 1 N
HCl/isopropanol. Plates were read in a spectrophotometer at 570 nm.
GP2 PCR
Cells were stimulated as mentioned. For RNA isolation, cells were harvested and dissolved in RNA-Bee (Tel-Test, Friendswood, TX), with
consequent treatment with chloroform, isopropanol, and ethanol. RNA (1
mg) was taken for cDNA production using a kit from Invitrogen (Karlsruhe, Germany). Negative controls were without reverse transcriptase. PCR
amplification was performed with TaqPCR Master mix (Qiagen, Mainz,
Germany) according to the manufacturer’s instructions. Primer sequences
were as follows: GP2, forward, 59-CAATGTGCCTACCCACTGGA-39,
reverse, 59-ATGGCACCCACATACAGCAC-39; b-actin, forward, 59-CTGGACTTCGAGCAAGAGATG-39, reverse, 59-TGAAGGTAGTTTCGTGGATGC-39. After predenaturation at 93˚C for 5 min, 36 cycles were
performed (30 s denaturation at 93˚C, 30 s annealing at 62˚C, and 30 s
elongation at 72˚C), followed by final elongation at 72˚C for 5 min. Five
microliters was loaded onto 2% agarose gel and visualized under UV
light.
Phagocytosis assay
Uptake of FITC-labeled E. coli was performed with a commercial kit
(Vybrant phagocytosis kit; Molecular Probes, Goettingen, Germany).
Briefly, monocytes or T84 cells were incubated with different doses of GP2
for 2 h. Subsequently, GP2 was removed and FITC-labeled E. coli were
added to the cells for 2 h. Next, nonphagocytosed E. coli were removed
and trypan blue was added for 1 min to quench the extracellular probe.
Phagocytosis was measured using a spectrophotometer. Cytochalasin D
and nocodazole were purchased from Sigma-Aldrich.
Reagents
Wound healing assay
Where mentioned, the following reagents were added: CD28 (Ancell,
Bayport, MN), anti-CD3 (clone OKT3; provided by Janssen-Cilag, Neuss,
Germany), anti-CD2 (clones T112 and T113; provided by Dr. Ellis Reinherz,
Boston, MA), and LPS (Sigma-Aldrich). The TNF-a inhibitors adalimumab
(ADA; Abbott, Wiesbaden, Germany) and infliximab (IFX; Essex Pharma,
Munich, Germany) were used at concentrations of 10 mg/ml.
T84 cells or rat IEC6 epithelial cells were grown to confluency on 60 mm
tissue culture plates (BD Pharmingen). On the day of the experiment, cells
were cultured with starvation medium (0.1% FCS) for 6 h. Afterward,
a scratch was made as previously described (22) and 25 or 50 mg/ml GP2
was added for 16 h. Images were taken with a Nikon D70 camera. Cells
migrating over the scratch were counted manually.
GP2 stimulation
Transwell assay
The recombinant GP2 used in this study was either purchased from Diarect
(Freiburg, Germany) or expressed in Spodoptera frugiperda 9 cells by
In vitro migration of cells was assessed using Costar Transwells (Costar/
Corning, Corning, NY). Filters were precoated with fibronectin (Milli-
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PBMCs from healthy volunteers were isolated using Ficoll-Hypaque (GE
Healthcare, Otelfingen, Switzerland) as previously described (19, 20). Cells
were cultured in RPMI 1640 (Life Technologies, Darmstadt, Germany)
containing 10% FCS (Biochrom, Berlin, Germany) and 2.5% penicillinstreptomycin (Biochrom). Depletion of monocytes and regulatory T cells
(Tregs; CD4+CD25+CD127dim/2 cells) from PBMCs was performed by
negative selection using magnetic cell sorting kits (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instruction.
Approximately 95% of the isolated Tregs were CD4+CD25+. From those,
80–90% were confirmed by flow cytometry to be Foxp3+ (data not shown).
Lamina propria mononuclear cells (LPMCs) were isolated from mucosa
of patients undergoing colectomy owing to noninflammatory disorders as
previously described (19). Briefly, minced mucosa was digested in DTT
(Sigma-Aldrich, Taufkirchen, Germany), two dispase (Life Technologies)
cycles, and collagenase (Roche Applied Science, Mannheim, Germany)
plus DNase (Worthington Biochemical, Lakewood, NJ). Forty percent
Percoll (GE Healthcare) was used to separate LPMCs. Separation of human intestinal epithelial cells (hIECs) and intraepithelial lymphocytes
(IELs) was performed on supernatant collected after the two dispase
cycles. A Percoll gradient of 100–60/40–30/0% was performed in a 15-ml
tube for 30 min, with break-off. hIECs were collected from the 0–30%
interface and IELs from the 30–40% interface.
Organ cultures were also performed as previously described (21). Fresh
mucosal biopsies (10–20 mg) were placed on a stainless steel grid in a center
well organ culture dish (BD Pharmingen, San Diego, CA) containing 700 ml
RPMI 1640, either with or without GP2. Mucosal surface was placed facing
downward. After 24 h, supernatants were collected for future ELISA assays.
Weight of biopsy was divided from the final cytokine concentration to
standardize results. Sera were separated by centrifugation for 10 min, 3000
rpm, 4˚C. Where human cells, sera, or tissues were used, signed, informed
consent was obtained from each subject and the experimental protocol was
approved by the Local Ethics Committees of the Charité.
Caspase 3-FITC (BD Pharmingen) was determined using BD Fix/Perm solution. Activation of caspase-8 and -9 was determined using a CaspGLOW
staining kit (MBL International, Woburn, MA) according to the manufacturer’s instructions.
2776
GP2 IS A NOVEL IMMUNOREGULATOR OF INNATE/ADAPTIVE IMMUNITY
pore, Schwalbach, Germany) for 1 h at 37˚C. Subsequently, cells (2–5 3
105) in 100 ml PBS/0.1% BSA were placed in the upper chamber. The
lower chamber contained 600 ml RPMI 1640 alone, RPMI 1640 with GP2,
or T84 cells pretreated with GP2. Cells were allowed to migrate for 3 h at
37˚C. Afterward, cells that transmigrated to the lower well were collected
and counted. The percentage of migrating cells was calculated by flow
cytometry or a counting grid. For flow cytometry, cells were acquired by
FACS for a fixed period of time (90 s). Percentage migration was calculated as the number of migrating cells divided by the total number of cells
placed in the upper wells, counted in the same time period, and multiplied
by 100.
ELISA
ELISA kits for anti-GP2 (Generic Assays, Dahlewitz, Germany) were
performed on sera. Sera were obtained from healthy volunteers or patients
suffering from CD, UC, irritable bowel syndrome (IBS), or pancreatitis.
Patients’ demographics, as well as medications taken, are detailed in
Supplemental Table I. In mentioned experiments, supernatants were collected and ELISAs for b-defensin-2 (PeproTech, Hamburg, Germany),
CXCL8, IL6, TSLP, TNF-a (R&D, Minneapolis, MN), IL-17, and IFN-g
(both from eBioscience) were performed according to the manufacturers’
instructions. For TGF-b1 ELISA (BD Pharmingen) supernatants were
activated for 1 h with 1 N HCl and then neutralized with 1 N NaOH.
Data are means 6 SEM. Statistical analysis for significant differences was
performed by using ANOVA, with the Student t test for parametric samples
(GraphPad Prism version 4; GraphPad Software, San Diego, CA).
Results
Anti-GP2 levels are significantly elevated in CD
Initially, we confirmed GP2 as an autoantigenic target by assessing
anti-GP2 IgG and IgA levels in IBD patients and different control
groups. As shown (Fig. 1), Abs to GP2, of both IgG and IgA
isoforms, are significantly elevated in CD compared with healthy
controls, UC, and patients with pancreatitis or IBS. We were unable to observe any correlations of anti-GP2 levels with medications taken (Supplemental Table I).
GP2 is distinctly expressed on various cell types and is
regulated on T cells
Because anti-GP2 Abs are elevated in CD, we next investigated
expression profiles of the respective Ag GP2 on key cells involved
in mucosal inflammation (epithelial, monocytes, T cells, B cells)
using a commercial Ab. The specificity of the Ab was verified by
preincubating cells with serum of rabbits hyperimmunized with
recombinant GP2 (Supplemental Fig. 1). These sera contain Abs
against GP2 and thus block GP2 epitopes reactive with the commercial anti-GP2. Preincubation of cells with these hyperimmune
sera dose-dependently reduced the binding of anti-GP2, thus
proving the specificity of the commercial Ab.
FIGURE 1. Abs against GP2 are increased in CD. Sera were collected from
healthy volunteers (n = 10) and CD (n = 16),
UC (n = 13), pancreatitis (n = 5), and IBS
(n = 5) patients. Anti-GP2 IgG (A) and IgA
(B) ELISAs confirmed elevated levels in CD,
in comparison with all other groups examined. *p # 0.002.
GP2 regulates epithelial cell phenotype and function
Having shown that GP2 is expressed and regulated on cells
critically involved in mucosal immunity, we went on to evaluate
the effect of GP2 itself on central mechanisms of innate and
adaptive immunity. Epithelial cells are the first cells to encounter
pancreas-originating GP2 in the gastrointestinal lumen (10).
Moreover, they represent the first line of defense and are central
elements of innate immunity (23). To elucidate the influence of
GP2 on epithelial cells, we incubated hIECs or epithelial cell line
T84 with recombinant GP2 for 24 and 48 h, after which cells and
supernatant were acquired for the different experiments. As the
pattern by which GP2 influenced epithelial function was similar
at these two time points, results shown are after 48 h GP2
stimulation.
Examination of expression of key receptors in IEC function using
flow cytometry showed, to our knowledge, for the first time that
GP2 differentially modulates key molecules involved in epithelial
cell function (Fig. 3A). A decreased percentage of cells expressed
E-cadherin and an increased percentage expressed a4 integrin
(CD49d). GP2 also increased the percentage of cells expressing
molecules used in Ag presentation (HLA-DR and CD40). Moreover, MICA, which presents Ag to gd T cells, was also upregulated
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Statistical analysis
Further evidence for the specificity of the Ab was deduced by
preincubation of the Ab with recombinant GP2 itself for 24 h at
4˚C. Staining of cells (T cells, monocytes, and epithelial cells;
Supplemental Fig. 2) with the preincubated protein-Ab mixture
negated expression of surface GP2. Additionally, a further negative control was conducted using isotype-matched IgG control,
which confirmed no unspecific binding as observed by lack of
staining (data not shown).
As shown, GP2 expression was detected to varying degrees on all
cells investigated (Fig. 2A). Approximately a third of peripheral
blood and lamina propria T cells expressed GP2. A high percentage of B cells (80.5 6 4.2%) and monocytes (92.9 6 7.1%)
also expressed GP2. Also, about three quarters of primary hIECs
expressed GP2, in contrast to two epithelial cell lines (Caco-2 and
T84) that exhibited only modest GP2 expression (12.5 6 7.2 and
18.8 6 2.1%, respectively). Our control groups also exhibited
variable GP2 expressions. Percentage of pancreatic (PANC1,
ASPC1, and DANG), eosophageal (KYSE180), and neuronal cell
lines (Sy5y) expressing GP2 ranged from 8 to 46% (Supplemental
Fig. 1). As the aim of our study was to investigate the biological
relevance of GP2, we next investigated whether GP2 expression
can be regulated on T cells. As depicted in Fig. 2B and 2C, GP2
expression was significantly upregulated after 48 h T cell activation via the TCR, both at the RNA as well as the protein expression levels.
The Journal of Immunology
2777
by GP2, suggesting that GP2 has a function in bridging epithelialto-T cell responses. In contrast to the demonstrated capability of
GP2 to modulate other epithelial cell receptors, TLR2 and TLR4
expression were not altered by GP2 (data not shown).
When we examined modulation of apoptosis and proliferation by
GP2 on epithelial cells using annexin V/PI staining and MTT incorporation, respectively, we observed that GP2 dose-dependently
decreased apoptosis and proliferation of both hIECs and T84 (Fig.
3B, 3C). We also determined the influence of GP2 on epithelial
migration over a lesion using the wound-healing assay with two
different epithelial cell lines. Interestingly, but confirming the
differences between cell migration and cell proliferation (24), cell
migration over the wound edge was not influenced by GP2 coculture (Fig. 3D). In inflammatory responses, such as IBD, CXCL8 is
upregulated and serves as a potent chemoattractant. Using ELISA,
we also revealed that GP2 dose-dependently decreases CXCL8
secretion from epithelial cells (Fig. 3E). However, b-defensin-2
secretion, which is dysregulated in CD (25), and TSLP were unaffected by GP2. IL-6 was not detected in T84 cells (data not
shown).
Phagocytotic potential of GP2
Previously, GP2 was shown to assist IECs in bacterial handling and
uptake (26). Therefore, we wanted to confirm this function in our
experimental settings. When we examined the capacity of epithelial cells to phagocytose E. coli as described in Materials and
Methods, we observed that GP2 did not influence the phagocytosis
of E. coli (Fig. 4A) by T84 cells. Interestingly, isolated human
monocytes incubated with increasing doses of GP2 exhibited
significantly increased uptake of E. coli compared with cells
cultured in the absence of GP2, confirming a unique effect of GP2
on monocyte phagocytosis. Further experiments revealed that
neither cytochalasin D nor nocodazole altered GP2-mediated E.
coli phagocytosis by monocytes (data not shown).
Chemoattractant potential of GP2
As epithelial cells attract cells to sites of inflammation, and as Ag
presentation receptors were upregulated in response to GP2, we
examined the ability of GP2 to chemoattract T cells, a critical step
in mucosal inflammation. To do so we performed Transwell migration assays and determined how many PBMCs migrated toward
GP2-stimulated epithelial cells. Migration was determined using
flow cytometry for a fixed time of 90 s, and only gated mononuclear
cells on forward versus side scatter were counted. As depicted in
Fig. 4B, GP2 significantly increased mononuclear cell migration
toward GP2-treated T84 cells. CXCL8-treated T84 cells served as
positive control. Importantly, PBMCs did not migrate toward GP2
alone (data not shown).
GP2 binds directly to epithelial and T cells
Because our results so far suggest that GP2 modulates key cell
functions, we sought to delineate whether GP2 can bind directly to
these cells, suggesting a possible mode of action for GP2. To
investigate this important question, we incubated PBMCs with
biotinylated GP2 for 40 min at 4˚C, followed by secondary SAFITC incubation. As shown (Fig. 4C), GP2 bound to unstimulated
T cells to some extent, but CD3 stimulation of the cells greatly
increased this attachment. Furthermore, GP2 was also shown to be
bind potently to the T84 IEC cell line.
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FIGURE 2. GP2 is expressed on diverse cells and is regulated on T cells. (A) Using flow cytometry, GP2 was found to be expressed on all cell populations examined (full line). SA-FITC alone served as negative control (dashed line). Representative of n = 3–10. Additional cell populations and validation of specificity of the Abs are included as Supplemental Fig. 1. Expression of GP2 is upregulated on CD3-stimulated PBMCs, both on the membrane
(B) (flow cytometric costaining with CD3-PerCP; n = 6; *p # 0.05 versus unstimulated) and at the mRNA level (C) (representative of n = 3).
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GP2 IS A NOVEL IMMUNOREGULATOR OF INNATE/ADAPTIVE IMMUNITY
GP2 modulates T cell activation, proliferation, apoptosis, and
cytokine secretion
Having discovered the effect of GP2 on critical cells of innate
immunity, we went on to explore the influence of GP2 on the
adaptive immune system. We isolated PBMCs from healthy volunteers, as well as LPMCs and IELs, and stimulated with anti-CD3
or anti-CD2, respectively. Flow cytometric analysis revealed that in
all T cell populations examined, GP2 significantly and dosedependently decreased activation as determined by CD25 expression (Fig. 5A). Of note, CD69 expression was not influenced
by GP2. Moreover, annexin V/PI staining uncovered that apoptosis is decreased by as much as 25–50% upon GP2 stimulation
in PBMCs, LPMCs, and IELs in comparison with CD3-stimulated
cells (Fig. 5B). Further exploring underlying apoptosis pathways,
we found that GP2 decreased activation of caspase-3 and -8, but
not caspase-9 (Fig. 5C).
Proliferation of CD3-activated PBMCs was also significantly
inhibited by GP2 as verified by PI staining (Fig. 5D). However, in
LPMCs and IELs we were unable to induce or observe any proliferation, using both PI staining and MTT incorporation.
Similar to epithelial cells, GP2 coculture of activated PBMCs did
not influence TLR2 and TLR4 expression on CD3+ cells, compared
with cells stimulated in the absence of GP2 (data not shown).
Importantly, as outlined in Materials and Methods, all experiments were performed with two different sources of recombinant
GP2, showing identical patterns of effects.
We also examined the influence of GP2 on cytokine secretion
from the different cell populations. Using ELISA, we observed that
GP2 decreased secretion of the proinflammatory TNF-a and IL-17
(Fig. 6A, left panel) from CD3-stimulated PBMCs, but increased
regulatory IL-6 (Fig. 6A, left panel) and TGF-b1 release (Fig. 6A,
right panel on a different scale). A similar pattern was observed in
CD2-stimulated IELs and LPMCs (Fig. 6B, 6C); that is, GP2
coculture decreased proinflammatory (TNF-a and IL-17) and increased regulatory cytokine (TGF-b1) release compared with cells
cultured in the absence of GP2. In contrast to PBMCs, however,
IL-6 was not detected in the mucosal cells, and concentrations of
both TNF-a and IL-17 were considerably lower than in peripheral
cells.
Finally, to gain an insight into the influence of GP2 in the more
complex mucosal milieu, we examined cytokine secretion in response to GP2 from freshly resected mucosal specimens, using
organ culture assay. A slightly different representation of cytokine
secretion emerged (Fig. 6D). Proinflammatory CXCL8 secretion
decreased and regulatory TGF-b1 increased in response to GP2.
However, in contrast to isolated cells, IL-6 secretion in response
to GP2 was decreased, and the cytokines TNF-a, IL-17, and
b-defensin-2 were not observed. All the data mentioned above
regarding the influence of GP2 on T cell function and phenotype
were examined both 24 and 48 h after stimulation. As there were
no differences in the pattern by which GP2 influenced T cell
function, data shown are for 48 h after GP2 stimulation.
Tregs mediate the immunosuppressive effect of GP2
Our results so far assign a previously unknown, immunosuppressive effect of GP2 on T cell function. We were now intrigued to
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FIGURE 3. GP2 modulates epithelial phenotype and function. hIECs (n = 3) or T84 cells (n = 3–5) were incubated with the mentioned concentrations of
GP2 for 48 h. (A) Expression of cell surface molecules was determined using flow cytometry. (B) Decreased apoptosis by GP2 was determined using
annexin V/PI staining. (C) Decreased proliferation by GP2 was determined using the MTT assay. (D) Migration of T84 (n = 2) or IEC6 (n = 3) over a scratch
lesion in the presence of GP2 was examined by the wound healing assay. (E) Influence of GP2 on cytokine secretion was detected by ELISA. *p # 0.05,
**p # 0.01 versus 0 mg/ml GP2.
The Journal of Immunology
2779
elucidate the role of specific cell populations in the response to
GP2. Tregs are a specialized subpopulation of T cells that suppress
the activation of the immune system and might be responsible for
GP2’s effect. Therefore, we used the same experimental conditions as described above and examined the response of isolated
CD3+ cells, Tregs, or monocytes to GP2. Interestingly, we
detected no influence by GP2 on activation, proliferation, apoptosis, or cytokine secretion on either isolated T cells, Tregs, or
monocytes (data not shown). Moreover, when we repeated these
experiments on the PBMC fraction depleted from Tregs, GP2 was
also unable to influence activation, proliferation, or apoptosis of
the T cells (Fig. 7A, gray columns). However, when we then
stimulated Treg-depleted PBMCs for 3 h in the presence of GP2,
subsequently washed away the GP2, and then returned the autologous Tregs for a further 48 h, the effect of GP2 on T cell
activation, proliferation, or apoptosis was restored as shown
above (Fig. 7A, black columns).
TNF-a inhibitors regulate GP2 expression in PBMCs and IECs
Finally, owing to the immunomodulatory properties exerted by GP2,
we questioned whether GP2 is involved in the mode of action
of pharmaceutical TNF-a inhibitors, potent inhibitors of mucosal
inflammation. To this end, PBMCs or Caco2 cells were stimulated
with CD3 Abs or LPS, respectively, either with or without 10 mg/
ml IFX or ADA. After 48 h, cells were collected for GP2 surface
expression using flow cytometry or mRNA levels of GP2 using
PCR. In PBMCs (Fig. 8A), CD3 stimulation increased expression
of GP2 at both the RNA and protein levels. Addition of either IFX
or ADA increased transcription of GP2 RNA in T cells, but decreased surface expression of GP2. Incubation of Caco2 cells (Fig.
8B) with either IFX or ADA increased GP2 at both the RNA and
surface levels. Moreover, addition of LPS to these TNF-a inhibitortreated IECs further increased GP2 RNA expression but decreased
GP2 expression. In light of our intriguing, novel results we further
inquired into the relative effect of GP2 and TNF-a inhibitors on
T cell activation and survival either alone or in combination (Fig.
8C). As shown, CD3 stimulation increased both activation and
apoptosis. Again, addition of GP2 decreased T cell activation and
apoptosis. IFX decreased activation, but did not influence apoptosis. Combination of IFX with GP2 on CD3-stimulated PBMCs
further decreased T cell activation, without influencing apoptosis.
These experiments suggest that GP2 itself can indeed partake
a role in downmodulating immune response, further enhancing the
anti-inflammatory effect of TNF-a inhibitors.
Discussion
The major zymogen granule membrane glycoprotein, GP2, is secreted by pancreatic cells and is suggested to be involved in the
“opsonization” of FimH+ in the intestine. Furthermore, GP2 was
demonstrated to be a specific receptor on M cells facilitating
translocation of FimH+ bacteria across the intestinal epithelium.
Interestingly, GP2 has recently been identified as the main
autoantigenic target recognized by the CD-specific PABs. IBD
patients have a higher prevalence of PABs and consequently antiGP2 Abs compared with controls (27–30), assuming a potential
role for GP2 in mucosal immunology. An immunosuppressive
effect has been reported in the mid-1980s for the urinary homolog
of GP2, THP (31, 32). Thus, as the role(s) of GP2 in central
mechanisms of the mucosal immune system are not defined, we
hypothesized a similar mode of influence by GP2.
Downloaded from http://www.jimmunol.org/ by guest on May 7, 2017
FIGURE 4. GP2 influences phagocytosis and chemotaxis, possibly via direct binding to cells. (A) Phagocytosis of T84 cells (n = 4) or monocytes (n = 3)
was determined using fluorescently labeled E. coli particles. Results are calculated as percentage effect in comparison with cells incubated with E. coli
without GP2. (B) Chemotaxis was performed using Transwell plates (n = 4). Confluent T84 cells were pretreated for 1 h with the mentioned substances.
Afterward, 5 3 105 PBMCs were placed in Transwell inserts and placed above the T84 cells for a further 3 h. Migrating cells were collected from the lower
chamber and enumerated for 90 s in a flow cytometer. (C) Binding of GP2 to cells was evaluated by means of biotinylated GP2. Cells were incubated for 40
min at 37˚C, with subsequent staining with SA-FITC. Negative controls were performed with SA-FITC incubation alone (gray-shaded areas). Representative histograms are from three experiments in each group. *p # 0.05, **p # 0.01 versus 0 mg/ml GP2.
2780
GP2 IS A NOVEL IMMUNOREGULATOR OF INNATE/ADAPTIVE IMMUNITY
Indeed, two different recombinant GP2s elicited a largely
downregulated immune response, revealing a novel immuno-
modulatory role for GP2 in modulating both innate and adaptive
immune responses.
FIGURE 6. GP2 downregulates inflammatory and upregulates regulatory cytokine secretion. PBMCs (A) (n = 6), LPMCs (B) (n = 4), IELs (C) (n = 3),
and organ cultures (D) (n = 3) were stimulated as described in Materials and Methods and incubated with the mentioned doses of GP2. After 48 h,
supernatants were collected and examined for cytokine secretion using ELISA. *p # 0.05, **p # 0.01 versus 0 mg/ml GP2.
Downloaded from http://www.jimmunol.org/ by guest on May 7, 2017
FIGURE 5. GP2 inhibits activation, apoptosis, and proliferation of T cells. CD3-stimulated PBMCs (n = 6) or CD2-stimulated LPMCs (n = 3) and IELs
(n = 3) were incubated for 48 h at the mentioned GP2 concentration. Afterward, cells were acquired for activation (A) (gated on CD3+CD25+ cells from
total PBMCs), apoptosis (B) (annexin V/PI staining), or proliferation (C) (PI staining). (D) Caspase activation was determined using intracellular staining as
described in Materials and Methods. *p # 0.05, **p # 0.01 versus 0 mg/ml GP2.
The Journal of Immunology
Our findings showing that Abs to GP2 are significantly elevated
in CD, but not in UC, confirm its previously reported potential for
the differential serological diagnosis in IBD (27–30). Furthermore,
since pancreatitis and IBS patients appear to lack Abs to GP2, our
data seem to support the assumption that anti-GP2 autoantibodies
do not pertain to inflammation in general, but to CD specifically.
As we speculated that GP2 not only serves as an autoantigen but
is also a constituent of immune cells, we investigated whether GP2
is expressed on key cells of the innate and adaptive immune
systems. Indeed, GP2 expression was detected on human epithelial
cells as well as T cells and monocytes. Moreover, GP2 expression
was regulated by T cell stimulation, upon TCR ligation, at the RNA
as well as at protein expression levels. Our findings appear to be in
contrast to a previous report that GP2 is solely expressed on M cells
in the intestinal epithelium (26), which could be explained by
a different epitope specificity of the anti-GP2 Abs employed. It is
noteworthy that using mRNA transcripts that hybridize to GP2
cDNA, GP2-like homologs have been reported to exist in a variety
of epithelial tissues known to contain regulated secretory processes (14). Thus, as the Abs we used were polyclonal, our data
could pertain to these homologs. Although scarce reports exist
regarding the expression of GP2 in nonpancreatic cell populations
(14), our intriguing finding of GP2 expression on several cell
populations investigated raised the issue of the specificity of the
Ab. We were able to prove the specificity of the Ab by blocking its
binding site using polyclonal rabbit Abs against recombinant GP2,
as well as by preincubating the Abs with recombinant GP2, thus
confirming the broad GP2 expression.
As we hypothesized that GP2 possesses its own biological
function, we next investigated whether and how recombinant GP2
affects primary human immune cell phenotype and function. The
mucosal epithelium of the alimentary tract not only constitutes a key
element of the mucosal barrier, but its cells also serve as important
regulators of the innate immune system. Remarkably, GP2 increased
the percentage of IECs expressing HLA-DR, CD40, and MICA,
which are critically involved in Ag presentation toward the
underlying adaptive immune system, signifying that GP2 bridges
epithelial-to-T cell crosstalk. However, GP2 did not modulate cell
restitution and it decreased epithelial cell proliferation and apoptosis. This suggests an arresting influence by GP2, revealing that
the function of GP2 is beyond its previously known actions. Even
more, as we provide evidence that GP2 dose-dependently decreased
the proinflammatory chemokine CXCL8, we suggest an antiinflammatory role for GP2 in the mucosal immune system.
GP2 binds to E. coli and appears to support defense mechanisms
against potentially pathogenic bacteria (33). It also serves as an
uptake receptor for commensal and pathogenic bacteria by M cells,
but not other epithelial cells (34). In our experiments, we confirmed the inability of GP2 to promote phagocytosis by IECs,
which are not M cells. Moreover, we showed that GP2 promoted
E. coli phagocytosis by monocytes, indicating that GP2 has broader
prophagocytotic ability than previously assumed. Note that we
were unable to inhibit GP2-mediated phagocytosis by monocytes
either with cytochalasin D or with nocodazole, implying that this
mechanism is independent of a disruption of microfilament function (35).
THP, the renal homolog of GP2, was recently reported to increase
transepithelial migration of neutrophils (36). We show that GP2
increased mononuclear cell migration toward GP2-treated epithelial cells. However, as mononuclear cells did not migrate toward
GP2 alone, GP2 is not in itself a chemoattractant but induces this
feature in epithelial cells.
In the lamina propria, the adaptive immune system initiates and
fosters inflammation, especially IBD. In CD, T cells exhibit increased activation, proliferation, and cell cycling compared with
healthy controls (18). Therefore, limiting T cell activation is an
effective therapeutic approach to limit mucosal inflammation (37).
By showing that GP2 potently restricted T cell activation and
proliferation, our data suggest its potential therapeutic use to limit
mucosal inflammation. Moreover, as GP2 also decreased the secretion of the proinflammatory cytokines TNF-a and IL-17 from
intraepithelial, lamina propria, and peripheral blood T cells, its
anti-inflammatory influence was further supported.
We also present data that GP2 decreased T cell apoptosis upon
stimulation via caspase-3– and caspase-8–dependent pathways. In
CD, T cell apoptosis is already decreased (38), indicating that this
process is perhaps not desired in this type of IBD. However, in
UC, T cell apoptosis is elevated (39), implying that these patients
might benefit from this effect.
T cells are the backbones of the mucosal adaptive immune system,
and they consist of several phenotypical distinct cell types. Regulatory
CD4+CD25+ T cells are effective in the prevention and downregulation of experimental colitis and mediate mucosal inflammation
(40). In contrast to its potent effect on CD3-stimulated PBMCs, GP2
had no effect on isolated Tregs, regardless of their stimulation status.
Also, GP2 was ineffective on Tregs-depleted PMBCs. However,
indicating the important role of GP2 in Treg function, GP2 was able
to downmodulate activation, proliferation, apoptosis, and cytokine
secretion when the autologous Treg fraction was reinstated.
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FIGURE 7. Depletion of Tregs from PBMCs negates the immunosuppressive influence of GP2. PBMCs were depleted from Tregs (n = 4).
Activation, apoptosis, and proliferation were evaluated as mentioned
above. Reintroduction of Tregs (black bar, PBMC-Treg+Treg) to depleted
PBMCs previously treated with GP2 led to decreased cell activation (flow
cytometric CD3+CD25+ expression), apoptosis (annexinV/PI staining), and
proliferation (PI staining). *p # 0.01 versus 0 mg/ml GP2.
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GP2 IS A NOVEL IMMUNOREGULATOR OF INNATE/ADAPTIVE IMMUNITY
Importantly, although apoptosis and proliferation are considered
to be inversely regulated, we observed their simultaneous downmodulation by GP2. This phenomenon of coupled cell death and
cycling is a hallmark of activation-induced cell death (41, 42),
comparable to our in vitro anti-CD3 stimulations. Moreover, as
caspase-8 is implicated in this regulation of apoptosis and proliferation (41, 43), our data are further supported, as we observed
involvement of caspase-8 in GP2-mediated responses.
Having identified a novel role for GP2 in immunity, we were
finally wondering whether and how IBD therapeutics influence
GP2 expression. TNF-a inhibitors, such as IFX or ADA, inhibit
T cell and epithelial proliferation by inducing apoptosis and thus
limit mucosal inflammation. Interestingly, both TNF-a inhibitors
increased GP2 gene transcription in T cells but decreased its
surface expression. It can be speculated that this seemingly opposite effect indicates an increased endocytosis of GP2. However,
blocking endocytosis did not prevent GP2 downregulation by
TNF-a inhibitors (data not shown).
Thus, the different regulation of GP2 at the mRNA level in
comparison with the membrane level suggests either an intracellular role for GP2, or more probable secretion of GP2 from the
T cells, as it acts as a secreted protein. These ideas need to be
investigated in further studies, and ongoing studies in our laboratory will elaborate this observation.
Indicating a specificity of this process, in cells of the innate
immune system, such as epithelial cells, addition of IFX or ADA
increased both gene and surface GP2 expression.
Our novel data showing additive value of TNF-a inhibitors and
GP2 in downmodulating immune response hint to the fact that
GP2 could be used for future therapeutic medications.
Although it has been known for .20 y that in CD PAB levels are
increased (5), it was not assumed until recently that their main
autoantigenic target GP2 might have a biological function in the
intestine. To our knowledge, our study revealed for the first time that
this autoantigen has a physiological role in the intestinal immune
system. Furthermore, it was recently shown that GP2 binds to
scavenger receptor expressed on endothelial cells (44). Despite our
data regarding the binding of GP2 to epithelial and activated T cells,
whether GP2 exerts its biological function in the innate and adaptive
immune systems via those receptors needs to be further investigated.
THP was reported to influence chemotaxis of polymorphonuclear leukocytes (45), as well as neutrophils (36), and it fosters
bacterial phagocytosis (46, 47). In this study we provide clear
evidence that THP and GP2 share functional and immunological
resemblances. To explore whether GP2 and THP share structural
similarities as well, we generated theoretically calculated threedimensional structures of GP2 and THP based on their amino acid
sequences (Supplemental Fig. 3). Although both proteins exhibit
homology of 53% in protein–protein alignment (using amino acid
sequences of P55259 = GP2_Human, and P07911 = Urom_Human, with BLASTP 2.2.25), and they share some distinctive
structural segments, such as a single a helix and a protruding C
terminus, it was difficult to deduce other homologies. However,
whether these structural differences indicate an unknown physi-
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FIGURE 8. TNF-a inhibitors modulate expression of GP2. PBMCs (A) (n = 3) were stimulated with anti-CD3 and Caco2 cells (B) (n = 3) with 10
mg/ml LPS. Cells were incubated either with or without 10 mg/ml IFX or ADA for 48 h. RNA levels were determined using PCR. b-actin served as
control. GP2 surface expression was determined using flow cytometry, and representative histograms are shown (n = 2–4). (C) PBMCs (n = 3) were
stimulated with anti-CD3 and incubated with GP2, IFX, or their combination. Apoptosis and activation were assessed as described above. *p # 0.01
versus CD3-stimulated.
The Journal of Immunology
ological or functional difference remains to be elucidated further
using protein crystallization and isoform-dependent studies.
In summary, our study revealed that GP2 not only acts as an Ag
for PABs, but more importantly and, to our knowledge, for the first
time, it has its own potent immunoregulatory functions. Our data
showed that GP2 consistently reduced innate and adaptive immune responses at several levels, thus identifying a potential use
for GP2 as a novel anti-inflammatory agent in IBD.
2783
20.
21.
22.
Disclosures
23.
D.R. has a management role and is a shareholder of GA Generic Assays
GmbH and Medipan GmbH. C.F. is an employee of GA Generic Assays
GmbH and Medipan GmbH. Both companies are diagnostic manufacturers. The other authors have no financial conflicts of interest. None of
the sponsors had an influence on any experimental setting or preparation of
the manuscript.
24.
25.
26.
27.
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45.
46.
47.
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