transcytosis of il-11 and apical redirection of gp130 is ... · motifs are present in the icd of...
TRANSCRIPT
Article
Transcytosis of IL-11 and
Apical Redirection ofgp130 Is Mediated by IL-11a ReceptorGraphical Abstract
Highlights
d Interleukin-11 receptor localizes on apical and basolateral
sides of polarized cells
d Interleukin 11 receptor forces apical redirection of gp130
d Interleukin 11 receptor has transcytotic activity
Monhasery et al., 2016, Cell Reports 16, 1067–1081July 26, 2016 ª 2016 The Author(s).http://dx.doi.org/10.1016/j.celrep.2016.06.062
Authors
Niloufar Monhasery, Jens Moll,
Carly Cuman, ..., Eva Dimitriadis,
Christoph Garbers, J€urgen Scheller
In Brief
Monhasery et al. show that interleukin 11
(IL-11) signaling via IL-11 receptor:gp130
complexes occurs on both the apical and
basolateral sides of polarized cells. The
transcytotic activity of the IL-11 receptor
allows IL-11 and interleukin-6:soluble
interleukin-6 receptor complexes to be
transported across cellular barriers.
Cell Reports
Article
Transcytosis of IL-11 and Apical Redirectionof gp130 Is Mediated by IL-11a ReceptorNiloufar Monhasery,1 Jens Moll,1 Carly Cuman,2,3 Manuel Franke,1 Larissa Lamertz,1 Rebecca Nitz,1 Boris Gorg,4
Dieter Haussinger,3 Juliane Lokau,5 Doreen M. Floss,1 Roland Piekorz,1 Eva Dimitriadis,2,3 Christoph Garbers,5
and J€urgen Scheller1,*1Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, 40225 D€usseldorf, Germany2Centre for Reproductive Health, The Hudson Institute of Medical Research, Clayton, 3168 VIC, Australia3Department of Molecular and Translational Medicine, Monash University, Clayton, 3168 VIC, Australia4Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine University, 40225 D€usseldorf, Germany5Institute of Biochemistry, Kiel University, Olshausenstrasse 40, 24098 Kiel, Germany
*Correspondence: [email protected]://dx.doi.org/10.1016/j.celrep.2016.06.062
SUMMARY
Interleukin (IL)-11 signaling is involved in variousprocesses, including epithelial intestinal cell regener-ation and embryo implantation. IL-11 signaling isinitiated upon binding of IL-11 to IL-11R1 or IL-11R2, two IL-11a-receptor splice variants, andgp130. Here, we show that IL-11 signaling via IL-11R1/2:gp130 complexes occurs on both the apicaland basolateral sides of polarized cells, whereas IL-6 signaling via IL-6R:gp130 complexes is restrictedto the basolateral side. We show that basolaterallysupplied IL-11 is transported and released to the api-cal extracellular space via transcytosis in an IL-11R1-dependent manner. By contrast, IL-6R and IL-11R2do not promote transcytosis. In addition, we showthat transcytosis of IL-11 is dependent on the intra-cellular domain of IL-11R1 and that synthetic transferof the intracellular domain of IL-11R1 to IL-6R pro-motes transcytosis of IL-6. Our data define IL-11Ras a cytokine receptor with transcytotic activity bywhich IL-11 and IL-6:soluble IL-6R complexes aretransported across cellular barriers.
INTRODUCTION
The interleukin (IL)-6-type family cytokines IL-6 and IL-11 signal
via gp130 homodimers (Garbers et al., 2012). To induce gp130-
receptor complex formation, IL-6 and IL-11 bind to their non-
signaling IL-6- or IL-11-a-receptor chains (IL-6R or IL-11R),
respectively. Accordingly, IL-6R and IL-11R production deter-
mines whether IL-6 and IL-11 can activate a cell. The IL-6R
is mainly expressed on hepatocytes and some leukocytes,
including macrophages, monocytes, neutrophils, and B and
T cells (Chalaris et al., 2011). In IL-6 trans-signaling, IL-6
activates cells lacking membrane-bound IL-6R via complexes
of IL-6 and the soluble IL-6R (sIL-6R) (Taga et al., 1989). IL-
11R is produced by lymphocytes, B cells, macrophages, endo-
CellThis is an open access article und
thelial cells, epithelial cells, hematopoietic cells, osteoclasts,
cardiac myocytes, and cardiac fibroblasts (Putoczki and Ernst,
2010).
IL-6 has both pro- and anti-inflammatory activities. IL-6 in-
duces the acute-phase response (Kopf et al., 1994), Th17 differ-
entiation (Ivanov et al., 2006), and commitment of macrophages
to the relatively anti-inflammatory M2 state (Luig et al., 2015;
Mauer et al., 2014). IL-11 leads to the regeneration of intestinal
epithelial cells helping maintain the barrier function of the intes-
tinal epithelium (Gibson et al., 2010). IL-11 has critical roles
in embryo implantation. IL-11 regulates human endometrial
epithelial cell (HEEC) adhesion (Marwood et al., 2009) and has
been shown to regulate trophoblast adhesion and migration in
humans (Paiva et al., 2007) and placentation in mice (Winship
et al., 2015). Additionally, injection of IL-11 to mice reduces car-
diac fibrosis, thereby attenuating cardiac dysfunction in a
myocardial infarction model by coronary ligation (Obana et al.,
2010). Recently, IL-11 has also been implicated in the develop-
ment of gastric cancer (Putoczki et al., 2013).
Polarized cells are divided into apical and basolateral mem-
brane domains, which are separated by tight junctions assem-
bling a tight crossing barrier for hydrophilic macromolecules,
such as proteins. Major borders are constructed by endo-/
epithelial cells, in particular, at the blood-brain barrier, the intes-
tine, and the placenta. Transcytosis enables the passage of
macromolecules through endo- and epithelial barriers. Here,
cell-surface receptors bind to their ligand on the basolateral
side, become endocytosed, and subsequently become exocy-
tosed on the apical side of the cell. The ligand may be released
from the receptor due to the low concentration of free ligand
on the apical side compared to the basolateral side (Rodri-
guez-Boulan and Powell, 1992; Zegers and Hoekstra, 1998).
Preferential basolateral sorting was described for gp130 and
IL-6R in polarized Madin-Darby canine kidney (MDCK) cells
(Martens et al., 2000). The MDCK line is commonly used as a
general model for polarized epithelial cells. Typically, basolateral
sorting depends on motifs within the intracellular, cytoplasmic
domains of transmembrane proteins containing tyrosine-based
or di-leucine motifs. Di-leucine motifs are also involved in
clathrin-dependent endocytosis (Simons and Ikonen, 1997).
Reports 16, 1067–1081, July 26, 2016 ª 2016 The Author(s). 1067er the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Figure 1. Apical and Basolateral Signaling via IL-11R1 in Polarized MDCK Cells and in Primary Human Epithelial Cells
(A–C) One representative experiment out of three is shown, and the scale bars represent 10 mm. (A) Cell-surface expression of IL-6R in polarizedMDCK cells was
analyzed by confocal microscopy. Left panel: Na+-K+-ATPase served as basolateral staining control. Right panel: DAPI was used to stain the nucleus. (B) Cell-
surface expression of IL-11R1 was analyzed by confocal microscopy. Left panel: Na+-K+-ATPase. Right panel: DAPI. (C) Cell-surface expression of IL-6R (left
panel) and IL-11R1 (right panel) after stimulation with IL-6/IL-11.
(legend continued on next page)
1068 Cell Reports 16, 1067–1081, July 26, 2016
Basolateral sorting motifs were identified in the intracellular do-
mains (ICDs) of IL-6R and gp130. For IL-6R, two discontinuous
motifs were assigned for basolateral sorting: a membrane-prox-
imal tyrosine-based motif (Y408SLG) and a more membrane-
distal di-leucine-type motif (L427I) (Martens et al., 2000). Basolat-
eral sorting of gp130 is mediated by the di-leucine motif, which
also controls internalization of gp130 and IL-6R (Dittrich et al.,
1994, 1996; Doumanov et al., 2006). Consequently, STAT3
phosphorylation in polarized, IL-6R-, and gp130-expressing
MDCK cells was induced by basolateral, but not apical, stimula-
tion with IL-6 and IL-6/sIL-6R.
IL-11R is the closest homolog of IL-6R. Membrane sorting of
IL-11R has not been analyzed to date but is of particular impor-
tance because IL-11R is expressed on polarized epithelial and
endothelial cells (Garbers and Scheller, 2013). In humans, two
differentially spliced IL-11R cDNAs exist (Cherel et al., 1995).
The transcript variant IL-11R1 encodes a transmembrane glyco-
protein containing the extracellular domains (ECDs), a trans-
membrane domain (TMD), and a cytoplasmic region that is
32 amino acid residues long. No tyrosine or typical di-leucine
motifs are present in the ICD of IL-11R1. The transcript variant
IL-11R2 lacks the 32-amino-acid-residue-long cytoplasmic re-
gion. Due to differential splicing, the ICD of IL-11R2 consists of
only four alternative amino acid residues. Whereas IL-11R1 is
widely expressed and found on many polarized cells, IL-11R2
expression is restricted to the thymus, testis, and lymph nodes
(Cherel et al., 1995). Both receptors are, however, comparably
potent to induce IL-11 signaling via gp130 in non-polarized cells
(Lebeau et al., 1997).
Here, we compared the apical and basolateral expression
of the human IL-11R transcription variants 1 and 2, IL-11R1
and -R2, which were found on the apical and basolateral mem-
branes of polarized MDCK cells. Synthetic transfer of the ICD
of IL-6R to the ECD of the IL-11R1, and vice versa, resulted in
localization of the IL-11RECD/IL-6RICD chimeric proteins specif-
ically to the basolateral side, whereas the IL-6RECD/IL-11R1ICDchimeric proteins were transferred to both the apical and the
basolateral membranes. Stimulation of polarized IL-11R-ex-
pressing MDCK cells with IL-11 resulted in STAT3 phosphoryla-
tion from the apical and basolateral sides, demonstrating that
IL-11R redirects gp130 to the apical side. Functionally, basolat-
eral treatment of primary HEECs with IL-11 stimulated tropho-
blast spheroid adhesion to the apical epithelial cell surface,
suggesting that IL-11 acted via transcytosis to alter endometrial
epithelial cell adhesion. Finally, IL-11R1, but not IL-11R2 and
IL-6R, acted as a transcytosis receptor, resulting in basolat-
eral-to-apical transport, and released biologically active IL-11
and IL-6:sIL-6R complexes.
(D) Confocal analysis was quantified with ImageJ software. In each group, the a
surface of MDCK-IL-6R and MDCK-11R1 from 20 cells was determined. Data a
experiments.
(E and F) One representative experiment out of two is shown. (E) Western blotting
and basolateral stimulation with IL-6. (F) Western blotting of STAT3/pSTAT3 of
stimulation with IL-11.
(G and H) Polarized primary human endometrial epithelial cells (HEECs) (n = 6)
adhesive capacity. HTR8sv/Neo spheroid adhesion to HEECs was increased aft
presented as percent change from control. IL-11 treatment group data are expr
basolateral, p = 0.0136; n = 6. Original non-normalized data were analyzed by p
RESULTS
Apical/Basolateral Localization of IL-11R1 andBasolateral Localization of IL-6R in Polarized MDCKCellsTo investigate apical/basolateral localization of cytokine recep-
tors, MDCK cells were generated that stably expressed IL-6R
and IL-11R1 (MDCK-IL-6R and MDCK-IL-11R1). MDCK-IL-6R
and MDCK-IL-11R1 cells were polarized, and localization of
IL-6R and IL-11R1 was analyzed by confocal microscopy. As
reported previously (Martens et al., 2000), the IL-6R is predomi-
nantly located on the basolateral membrane (Figure 1A; control
stainings for all applied antibodies are shown in Figures S1A
and S1B). IL-11R1 localized on the apical and basolateral mem-
branes of polarized MDCK-IL-11R1 cells (Figure 1B). Na+-K+-
ATPase staining served as a control for basolateral polarization.
Of note, stimulation with IL-6 and IL-11 had no influence on api-
cal/basolateral localization of IL-6R and IL-11R1 in polarized
MDCK cells (Figure 1C). Quantitative analysis of IL-6R and
IL-11R1 localization in the presence and absence of cytokine
stimulation revealed that cytokine stimulation did not change
the sorting pattern of IL-6R and IL-11R1 (Figure 1D).
Next, we compared the biological activity of polarized and non-
polarizedMDCK-IL-6R andMDCK-IL-11R1 cells in dose-response
experimentswith IL-6 and IL-11, respectively. STAT3phosphoryla-
tion was used as biological readout for IL-6:IL-6R:gp130- and IL-
11:IL-11R1:gp130-receptor complex activation. STAT3 activation
is a general process controlling cellular proliferation, differentiation,
migration, inflammation, and apoptosis (Vogel et al., 2015). IL-6
stimulation from the basolateral, but not from the apical, side
of MDCK-IL-6R cells induced STAT3 phosphorylation in a dose-
dependent manner (Figure 1E). STAT3 phosphorylation in non-
polarized MDCK cells was comparably strong as compared to
basolateral stimulation of polarized MDCK-IL-6R cells (Figure 1E).
MDCK-IL-11R1cells treatedwith IL-11bothapically andbasolater-
ally induced STAT3 phosphorylation in a dose-dependent manner,
demonstrating that apical and basolateral localization of IL-11R1
and gp130 on the membrane were functionally active (Figure 1F).
IL-11 induced STAT3 phosphorylation to a similar extent similarly
in both non-polarized and polarized MDCK cells following apical
and basolateral treatment (Figure 1F).
Basolateral IL-11 Increases HEEC AdhesionHEECs are responsive to IL-11 and endogenously express IL-
11R1 in an apical and basolateral manner (Cork et al., 2002; Mar-
wood et al., 2009). Polarized primary HEECs were treated with
IL-11, either apically or basolaterally, and the effect on human
trophoblast spheroid adhesion to HEECs was measured using
verage of IL-6R or IL-11R1 expression on the apical surface and basolateral
re expressed as mean values ± SD calculated from at least two independent
of STAT3/pSTAT3 of unpolarized and polarized MDCK-IL-6R cells after apical
unpolarized and polarized MDCK-IL-11R1 cells after apical and basolateral
treated with IL-11 (50 ng/ml) apically (G) and basolaterally (H) increased their
er apical or basolateral treatment of the HEECs compared to control. Data are
essed as means ± SEM; control versus apical, p = 0.0157, and control versus
aired t test. *p < 0.05.
Cell Reports 16, 1067–1081, July 26, 2016 1069
a co-culture adhesion assay. Apical treatment of HEECs with IL-
11 (50 ng/ml) significantly increased trophoblast HTR8sv/Neo
spheroid-cell adhesion to HEECs by 44.6% ± 25.0%, compared
to control (p < 0.05) (Figure 1G). Similarly, HEECs treated with IL-
11 basolaterally significantly increased HTR8sv/Neo spheroid-
cell adhesion to HEECs by 18.0% ± 5.6%, compared to control
(p < 0.05) (Figure 1H), suggesting biological active apical and
basolateral expression of endogenous IL-11R and gp130.
Apical Sorting of IL-11R1/2 and IL-6RDICD Forced theTypically Basolaterally Restricted gp130 to the ApicalMembrane and Enabled Apical IL-11 and IL-6 Trans-signalingGp130 is sorted to the basolateral side of polarized cells (Douma-
nov et al., 2006). Heterologous gp130 was visualized in polarized
MDCK-IL-6R and -IL-11R1 cells by confocal microscopy. Gp130
was found exclusively on the basolateral membrane in polarized
MDCK-IL-6R cells (Figures 2A and 2B) but, surprisingly, also at
the apical membrane in polarized MDCK-IL-11R1 cells (Figures
2C and 2D). In trans-signaling, IL-6:sIL-6R complexes induce
STAT3 phosphorylation solely via gp130 without the need of
membrane-bound IL-6R. Hyper-IL-6 (HIL-6) is a fusion protein
of IL-6 and sIL-6R, specifically inducing IL-6 trans-signaling
(Fischer et al., 1997). In polarizedMDCK-IL-6R cells, basolateral,
but not apical, application of HIL-6 induced STAT3 phosphoryla-
tion (Figure 2E). We detected, however, little background STAT3
phosphorylation, which might be caused by limited leakage of
HIL-6. These results support our finding that gp130 was not
expressed on the apical side in polarized MDCK-IL-6R cells.
Interestingly, basolaterally supplied, but also apically supplied,
HIL-6 to polarized MDCK-IL-11R1 cells led to sustained STAT3
phosphorylation (Figure 2E). Deletion of the ICD of the IL-6R
(IL-6RDICD) comprising the basolateral sorting signal (Figure 2F)
resulted in predominantly apical localization of IL-6RDICD. In
line, apical stimulation of polarized MDCK-IL-6RDICD cells with
IL-6 stimulated STAT3 phosphorylation, suggesting that gp130
was also redirected, whereas STAT3 phosphorylation from the
basolateral side was greatly reduced (Figures 2G and 2H).
Whereas the transcript variant IL-11R1 contains a 32-amino-
acid-residue-long cytoplasmic region, the transcript variant IL-
11R2encodes the four alternative intracellular amino-acid residues
LGLW (Figure 2I). In polarizedMDCKcells, IL-11R2was also found
on the basolateral side and, to a lesser extent, the apical side (Fig-
ure2J).Accordingly, apical IL-11stimulationofpolarizedMDCK-IL-
11R2 cells was much weaker but clearly detectable, as compared
to basolateral stimulation and apical stimulation of MDCK-IL-11R1
cells with IL-11 (Figures 2K and 2L).
Taken together, our data demonstrated that apical sorting of
the IL-11R1/2 and IL-6RDICD forced gp130 to the apical mem-
brane, thereby enabling apical gp130 signaling. Our data sug-
gest that the ICD of the IL-11R1 was crucial for efficient apical
sorting of IL-11R1, as compared to IL-11R2.
IL-6R and IL-11R1 Have Comparable Cell-Surface Half-Lives and Are Internalized by Clathrin-DependentEndocytosisNext, we compared the cell-surface half-life of IL-6R and IL-
11R1. For this analysis, we chose the suspension cell lines Ba/
1070 Cell Reports 16, 1067–1081, July 26, 2016
F3-gp130-IL-6R and Ba/F3-gp130-IL-11R1. These cells are
ideal tools for determining the cell-surface half-life by flow cy-
tometry (Garbers et al., 2014). Cell-surface-expressed IL-6R
and IL-11R1 were labeled with IL-6R and IL-11R1 antibodies,
respectively. Labeling was conducted at 4�C, and non-bound
IL-6R/IL-11R antibodies were washed away. Cells were shifted
back to 37�C for 0, 60, 120, 180, 240, 300, and 360 min to allow
IL-6R and IL-11R1 internalization. After the indicated time points,
cells were stained with secondary detection antibodies at 4�C to
quantify the remaining cell-surface expression of IL-6R and IL-
11R1 by flow cytometry. During the flow cytometry pulse-chase
experiment, the time-dependent reduction of cell-surface IL-6R
and IL-11R1 expression was analyzed, indicating a cell-surface
half-life of about 120 min for IL-6R and IL-11R1 (Figures 3A–
3D). The internalization rate of IL-6R determined in our experi-
ments was in good agreement with the previously determined
internalization half-life of about 2–3 hr (Gerhartz et al., 1994).
Dynasore and Pitstop2 were used to compare the internal-
ization route of IL-6R and IL-11R1. During clathrin-mediated
endocytosis, Dynamin promotes the formation of clathrin-
coated vesicles, but Dynamin also affects other routes such as
CLIC/GEEC-type endocytosis and phagocytosis (Doherty and
McMahon, 2009). Dynasore is a potent inhibitor of Dynamin (Ma-
cia et al., 2006). Pitstop2 preferentially inhibits clathrin-indepen-
dent endocytosis (Dutta et al., 2012). Dynasore, but not PitStop2,
prolonged the cell-surface expression of IL-6R and IL-11R1 (Fig-
ures 3A–3D, middle and lower panels). Analysis of IL-6R and
IL-11R1 in polarized MDCK cells by confocal microscopy sup-
ported our data from flow cytometry, because Dynasore, but
not PitStop2, decelerated the internalization of IL-6R and IL-
11R1 (Figures 4A and 4B). Dynamin-1-K44A is a dominant-nega-
tive variant of Dynamin-1, which is essential for clathrin-coated
vesicle formation in endocytosis (Al-Hasani et al., 1998). Overex-
pression of Dynamin-1-K44A, but not wild-type Dynamin-1, or
mock transfection resulted in a clearly delayed internalization
of cell-surface IL-11R1 (Figures 4C–4F). Finally, using super-res-
olution structured illumination microscopy (SR-SIM), we demon-
strated that clathrin heavy chain (CHC) in endocytosed pits was
in close contact to IL-6R and IL-11R and that clathrin-coated en-
dosomes contain IL-6R and IL-11R (indicated by white arrows in
Figures 4G and 4H and quantified in Figure 4I). Here, also IL-6R-
and IL-11R-containing vesicles were detected, which were not
co-stained by CHC, likely representing exocytotic rather than
endocytotic vesicles. In conclusion, our data demonstrated
that IL-6R and IL-11R1 were internalized with comparable ki-
netics via clathrin-mediated endocytosis.
Apical Localization of IL-11R1 and Apical/BasolateralLocalization of IL-6R in Polarized MDCK Cells WereAchieved by Exchange of ICDsChimeric IL-6R/IL-11R1 proteins were generated to analyze
whether the basolateral and apical/basolateral localization pat-
terns of IL-6R and IL-11R1 can be swapped by transfer of the
TMD and/or ICD. IL-6RcbECD/IL-11R1S,TMD,ICD contains the three
ECDs of the IL-6R (cytokine-binding ECD; cbECD), whereas the
stalk region (or S), the TMD, and the ICD are derived from the IL-
11R1. In IL-6RECD/IL-11R1ICD, only the ICD of the IL-6R was
replaced by the 32-amino-acid-residue-long ICD of IL-11R1. In
Figure 2. IL-11R1 Redirects gp130 to the Apical Membrane of Polarized MDCK Cells
(A–D) In (A) and (B), cell-surface expression of gp130 and IL-6R in polarizedMDCK cells was analyzed by confocal microscopy. (C and D) Cell-surface expression
of gp130 and IL-11R1 was analyzed by confocal microscopy. Scale bars represent 10 mm. In (A)–(C), one representative experiment out of three is shown.
(E) Western blotting of STAT3/pSTAT3 in polarized MDCK, MDCK-IL-6R, and MDCK-IL-11R1 cells after basolateral and apical stimulation with HIL-6.
(F) Schematic illustration of IL-6R and IL-6RDICD proteins. D1–D3, domains 1–3, S, stalk; TMD: transmembrane domain; ICD, intracellular domain.
(G) Western blotting of STAT3/pSTAT3 in polarized MDCK, MDCK-IL-6R, and MDCK-IL-6RDICD cells after basolateral and apical stimulation with IL-6.
(H) Quantification of three western blots from (G).
(I) Schematic illustration of IL-11R1 and IL-11R2 proteins. D1–D3, domains 1–3; S, stalk; TMD, transmembrane domain; ICD, intracellular domain.
(J) Cell-surface expression of IL-11R2 was analyzed by confocal microscopy. Na+-K+-ATPase and DAPI staining served as controls. Scale bars represent 10 mm.
(K) Western blotting of STAT3/pSTAT3 of polarized MDCK-IL-11R1 and MDCK-IL-11R2 cells stimulated apically or basolaterally supplied IL-11.
(L) Quantification of three western blots from (K).
In (E), (G), and (K), one representative experiment out of three is shown. Data are expressed as mean values ± SD calculated from at least two independent
experiments. ***p < 0.001.
Cell Reports 16, 1067–1081, July 26, 2016 1071
(legend on next page)
1072 Cell Reports 16, 1067–1081, July 26, 2016
IL-11R1cbECD/IL-6RS,TMD,ICD, the three ECDs of the IL-11R were
genetically fused to the S, the TMD, and the ICD of the IL-6R. In
IL-11R1ECD/IL-6RICD, only the ICD of the IL-11R1 was replaced
by the ICD of the IL-6R (Figure 5A). MDCK cells were stably
transduced with cDNAs of the chimeric variants, and their
expression was verified by western blotting (Figure 5B). Next,
polarized MDCK cells stably expressing the chimeras were
analyzed for apical/basolateral receptor localization by confocal
microscopy. Transfer of the IL-11R1ICD to the IL-6RECD in IL-
6RECD/IL-11R1ICD converted the basolateral localization of the
IL-6R to a IL-11R1-type apical/basolateral localization (Figures
5C and 5D), whereas no role for basolateral sorting was assigned
to the transmembrane and stalk regions of IL-6R included in IL-
6RcbECD/IL-11R1S,TMD,ICD (Figure 5C). Transfer of the IL-6RICD to
the IL-11R1ECD in IL-11R1ECD/IL-6RICD was sufficient to achieve
strict basolateral localization (Figures 5E and 5F), and no role
for basolateral sorting was assigned to the transmembrane
and stalk regions, which were included in IL-11R1cbECD/IL-
6RS,TMD,ICD (Figure 5E).
We also determined STAT3 phosphorylation capacity of the
receptor chimeras in polarized and non-polarizedMDCK cells af-
ter stimulation with IL-6 and IL-11, demonstrating that all chi-
meras were biologically active (Figure 5G). The IL-6-responsive
IL-6RcbECD/IL-11R1S,TMD,ICD and IL-6RECD/IL-11R1ICD chimeras,
which were equally localized to the apical and basolateral mem-
branes induced IL-6-dependent STAT3 phosphorylation from
both sides (Figure 5G). The IL-11-responsive IL-11R1cbECD/IL-
6RS,TMD,ICD and IL-11R1ECD/IL-6RICD chimeras, which were
sorted to the basolateral membrane induced IL-11-dependent
STAT3 phosphorylation specifically from the basolateral side
(Figure 5G). Thus, our data showed that transfer of the ICD of
IL-11R1 forced IL-6R also to the apical/basolateral membrane.
IL-11R1 and gp130 Are Transcytosis Receptors for IL-6-Type CytokinesTranscytosis is a mechanism to transport macromolecules
through cellular barriers, including the blood-brain barrier,
placenta, and intestinal epithelium. Polarized MDCK cells have
been widely used as a cellular model system to analyze trans-
cytosis of macromolecules such as immunglobulin G (IgG)
via neonatal Fc receptors (FcRn) and immunoglobulin A (IgA)
via polymeric immunoglobulin receptor (pIgR) (Jerdeva et al.,
2010). Polarized MDCK cells were incubated for 24 hr with baso-
laterally supplied IL-11 (50 ng/ml). After 0, 1, and 24 hr, IL-11
was quantified by ELISA on the basolateral and apical sides,
as well as in cellular lysates. After 24 hr, a concentration of about
10 ng/ml IL-11 was detected on the apical side of MDCK-IL-
Figure 3. Cell-Surface Half-Life of IL-6R and IL-11R1
(A) Cell-surface half-life of IL-6R was determined in Ba/F3-gp130-IL-6R cells in t
cytometry. Color code: red, 0 min; blue, 60 min; dark purple, 120 min; green, 18
without IL-6R.
(B) Quantification of (A). T, time.
(C) Cell-surface half-life of IL-6R was determined in Ba/F3-gp130-IL-11R1 cells in
cytometry. Color code: red, 0 min; blue, 60 min; dark purple, 120 min; green, 18
without IL-11R.
(D) Quantification of (C).
In (A) and (C), one representative experiment out of three is shown. Data are
experiments.
11R1 cells, whereas no IL-11 was detectable on the apical side
of untransfected and MDCK-IL-11R2 cells. Whereas almost no
IL-11 was detected in lysates of untransfected MDCK cells,
comparable amounts of IL-11 were detected in lysates of
MDCK-IL-11R1 and -IL-11R2 cells, demonstrating that endocy-
tosis of IL-11 was dependent on IL-11R expression (Figure 6A).
About 20% of total basolaterally supplied IL-11 was transported
to the apical side by transcytosis within 24 hr of cultivation. A
24-hr incubation time overall reduced total basolateral IL-11 in
untransfected MDCKs, most likely due to the instability of IL-
11. The decreased basolateral IL-11 was, however, much stron-
ger in MDCK-IL-11R1 cells compared to untransfected MDCK
cells. We also observed a larger decrease of basolateral IL-11
in MDCK-IL-11R1 cells compared to MDCK-IL-11R2 cells. This
difference reflected the amount of IL-11 detected on the apical
side of MDCK-IL-11R1 cells (Figure 6A). Apical IL-11R1, but
not IL-11R2, led to the internalization of apical IL-11. Transcyto-
sis of IL-11 from the apical to the basolateral side of MCDK-IL-
11R1 cells was not observed (Figure 6B).
Non-polarized MDCK-IL-11R1 cells were stimulated with api-
cal supernatants from untransfected MDCK, MDCK-IL-11R1,
and MDCK-IL-11R2 cells obtained after 24-hr incubation with
basolaterally supplied IL-11. Only IL-11 containing apical super-
natants from MDCK-IL-11R1 cells induced STAT3 phosphoryla-
tion of non-polarized MDCK-IL-11R2 cells, demonstrating that
transcytosed IL-11 was biologically active (Figure 6C). As a
control, stimulation of non-polarized MDCK-IL-11R1 cells with
10 ng/ml recombinant IL-11 induced sustained STAT3-phos-
phorylation compared to unstimulated cells (Figure 6C).
Next, we analyzed whether IL-6 was also transcytosed by IL-
6R, IL-6RDICD, or IL-6RECD/IL-11R1ICD. As expected from our
confocal microscopy data, IL-6 was not trancytosed by polar-
ized MDCK and IL-6R-expressing MDCK cells (Figure 6D).
Even though IL-6RDICD was expressed on both apical and ba-
solateral sides, IL-6RDICD did not mediate transcytosis of IL-6
(Figure 6D). Interestingly, the fusion receptor protein IL-6RECD/
IL-11R1ICD transcytosed IL-6 (Figure 6D). Basolateral IL-6 was
endocytosed by IL-6R but not IL-6RDICD, supporting our finding
that most IL-6RDICD localized on the apical membrane (Fig-
ure 6D). Non-polarized MDCK-IL-6R cells were stimulated with
apical supernatants from untransfected MDCK and MDCK-IL-
6R, MDCK-IL-6RDICD, or MDCK-IL-6RECD/IL-11R1ICD cells
obtained after 24-hr incubation with basolaterally supplied
IL-6. As expected from the ELISA quantification, only IL-6-con-
taining apical supernatants from IL-6RECD/IL-11R1ICD-express-
ing MDCK cells induced STAT3 phosphorylation of non-polar-
ized MDCK-IL-6R cells (Figure 6E).
he presence and absence of Dynasore (100 mM) and Pitstop2 (30 mM) by flow
0 min; yellow, 240 min; pink, 300 min; light blue, 360 min; black, control cells
the presence and absence of Dynasore (100 mM) and Pitstop2 (30 mM) by flow
0 min; yellow, 240 min; pink, 300 min; light blue, 360 min; black, control cells
expressed as mean values ± SD calculated from at least two independent
Cell Reports 16, 1067–1081, July 26, 2016 1073
(legend on next page)
1074 Cell Reports 16, 1067–1081, July 26, 2016
Next, we asked whether IL-11R was crucially needed for cyto-
kine transcytosis or whether simple redirection of gp130 by
IL-11R was sufficient. Therefore, we analyzed transcytosis of
HIL-6, consisting of IL-6 fused to sIL-6R. HIL-6 specifically binds
to gp130 without the need of IL-11R. If IL-11R redirects endog-
enous gp130 from the basolateral to the apical membrane, bind-
ing of HIL-6 to endogenous gp130 on the basolateral side would
result in apical transcytosis of HIL-6. Polarized MDCK cells were
incubated for 24 hr with basolaterally supplied HIL-6 (50 ng/ml).
After 0 and 24 hr, HIL-6 was quantified by ELISA on the apical
side. After 24 hr, a concentration of about 1.5 ng/ml HIL-6 was
detected on the apical side of MDCK-IL-11R1 and MDCK-IL-
6RECD/IL-11RICD cells. No HIL-6 was detectable on the apical
side of untransfected MDCK cells and MDCK-IL-11R2, MDCK-
IL-6R and MDCK-IL-6RDICD cells (Figure 6F), demonstrating
that gp130 was able to transcytose IL-6:sIL-6R complexes in
an IL-11R-dependent manner.
Finally, we independently verified the biological activity of
transcytosed cytokines in proliferation assays using Ba/F3-
gp130-IL-11R1 and Ba/F3-gp130-IL-6R, where proliferation
is dependent on IL-11, IL-6, or IL-6:sIL-6R (HIL-6), respec-
tively. MDCK, MDCK-IL-11R1, MDCK-IL-11R2, and MDCK-IL-
11R1ECD/IL-6RICD cells were stimulated from the basolateral
side with IL-11 (50 ng/ml) and HIL-6 (50 ng/ml) for 24 hr. The api-
cal and basolateral media were collected and separately incu-
bated with Ba/F3-gp130-IL-11R1 cells for 48 hr. Basolaterally,
IL-11 and HIL-6 induced the proliferation of Ba/F3-gp130-IL-
11R1 cells. However, only apical media from MDCK-IL-11R1
cells containing transcytosed IL-11 and HIL-6 induced the
proliferation of Ba/F3-gp130-IL-11R cells (Figure 6G). Accord-
ingly, MDCK, MDCK-IL-6R, MDCK-IL-6RDICD, and MDCK-IL-
6RECD/IL-11RICD cells were stimulated basolaterally with IL-6
(50 ng/ml) and HIL-6 (50 ng/ml) for 24 hr. Again, basolateral
and apical media were collected and incubated for 48 hr with
Ba/F3-gp130-IL-6R. Basolateral treatment with IL-6 and HIL-6
induced the proliferation of Ba/F3-gp130-IL-6R cells, only apical
media from MDCK-IL-6RECD/IL-11RICD cells containing trans-
cytosed IL-6 and HIL-6 induced the proliferation of Ba/F3-
gp130-IL-6R cells (Figure 6H). Our results confirmed that the
transcytosed cytokines were biologically active and induced
cellular proliferation.
In conclusion, our results defined IL-11R1 as a transcytosis
receptor and assigned the ICD as the transcytosis regulatory
unit within the IL-11R1. Moreover, in the presence of IL-11R1,
endogenous gp130 served as a general transcytosis receptor
for IL-6:sIL-6R complexes.
Figure 4. IL-6R and IL-11R1 Co-localize with CHC
(A) Internalization of IL-6R was analyzed in HeLa cells in the presence and absen
(B) Internalization of IL-11R1 was analyzed in HeLa cells in the presence and ab
(C) Internalization of IL-11R1 was analyzed in HeLa cells by confocal microscop
(D) Internalization of IL-11R1 was analyzed in HeLa cells overexpressing Dynam
(E) Internalization of IL-11R1 was analyzed in HeLa cells overexpressing the dom
(F and G) Localization of IL-6R and IL-11R1 and clathrin heavy chain (CHC) in He
(SR-SIM; ELYRA PS.1 microscope). The scale bars in the overview images repre
(H and I) Super-resolution structured illumination microscopy (SR-SIM) images
co-localizated with CHC in HeLa cells from ten spots were determined.
In (A)–(E), the scale bars represent 10 mm. In (A)–(G), one representative experime
calculated from at least two independent experiments. ***p < 0.001.
DISCUSSION
The sorting of proteins to the apical or basolateral surface in
polarized cells such as epithelial cells, neurons, hepatocytes,
or migratory cell types is essential for the maintenance of the
cellular architecture and overall physiological function at the
cellular and (inter-) organ level. Cell polarity is executed by a
complex regulation of selective targeting of newly synthesized
proteins to their respective membrane domains, i.e., apical or
basolateral. Here, we compared apical/basolateral sorting of
the two differentially spliced isoforms IL-11R1 and IL-11R2
with the IL.6R. Although both IL-11Rs were found on apical
and basolateral membranes, only IL-11R1 acts as a transcytosis
receptor (Figure 7). Moreover, we showed that the membrane
half-life of IL-11R1 and IL-6R was about 120 min and that both
receptors were internalized by clathrin-mediated endocytosis.
Previously, it was shown that the cytoplasmic domain of IL-6R
was dispensable for endocytosis because internalization of IL-
6R was mediated via gp130 and depended on a cytoplasmic
di-leucine internalization motif of the signal transducer (Dittrich
et al., 1994, 1996). A recent study however, showed that cla-
thrin-mediated internalization of IL-6R is independent of gp130
and results in lysosomal degradation rather than recycling of
IL-6R (Fujimoto et al., 2015).
Expression of IL-11R2 is restricted to cells of thymus, testis,
and lymph nodes (Putoczki and Ernst, 2010). IL-11R1, however,
is widely expressed and found in the lung, liver, thymus, spleen,
kidney, intestinal epithelial cells, bone marrow, and uterus.
Moreover, immunohistochemical staining of IL-11R in human
endometrium demonstrates both apical and basolateral localiza-
tion of polarized epithelial cells in the mid-secretory phase of the
menstrual cycle (Cork et al., 2002), suggesting a role in endome-
trial receptivity and embryo implantation. While IL-11 is less
important for the hematopoietic system, it has potent anti-
apoptotic and anti-necrotic properties on, e.g., the intestinal
mucosa (Putoczki and Ernst, 2010). Consequently, widespread
IL-11 signaling is important in intestinal epithelial cell homeosta-
sis and mucosal protection, thrombopoiesis, embryogenesis,
(gastric) tumor development, immunomodulation, hematopoie-
sis, macrophage and osteoclast differentiation, and promo-
tion of stem cell development (Putoczki and Ernst, 2010). Intes-
tinal wound healing depends on polarized intestinal epithelial
cells and hematopoietic-cell-derived IL-11, which is severely
impaired in IL-11R1-deficient mice. Administration of IL-11 in
mice is protective in several disease states, among them colitis,
renal ischemia and reperfusion injury, experimental autoimmune
ce of Dynasore (100 mM) and Pitstop2 (30 mM) by confocal microscopy.
sence of Dynasore (100 mM) and Pitstop2 (30 mM) by confocal microscopy.
y.
in-1 by confocal microscopy.
inant-negative Dynamin-1-K44A mutant by confocal microscopy.
La cells was analyzed by Super-resolution structured illumination microscopy
sent 5 mm; in the magnified left and right images, scale bars represent 0.5 mm.
were quantified. In each group, the vesicles containing IL-11R1 and IL-6R
nt out of two is shown. In (F) and (I), data are expressed as mean values ± SD
Cell Reports 16, 1067–1081, July 26, 2016 1075
Figure 5. The ICD of IL-11R1 Mediates Apical Sorting
(A) Schematic illustration of IL-6R/IL-11R1 chimeric proteins. cbECD, extracellular domain containing the three extracellular domains D1–D3; ECD, extracellular
domain including transmembrane domain; S, stalk; TMD, transmembrane domain; ICD, intracellular domain.
(B) Western blotting of IL-6R, IL-11R1, and the indicated IL-6R/IL-11R1 chimeric proteins in MDCK cells.
(C–F) Cell-surface expression of IL-6R/IL-11R1 chimeric proteins—(C) IL-6RcbECD/IL-11R1S/TMD/ICD; (D) IL-6RECD/IL-11R1ICD; (E) IL-11R1cbECD/IL-6RS/TMD/ICD;
and (F) IL-11R1ECD/IL-6RICD)—in polarized MDCK cells was analyzed by confocal microscopy. Na+-K+-ATPase served as control. In (C)–(F), the scale bars
represent 10 mm.
(G) Western blotting of STAT3/pSTAT3 of polarized MDCK cells expressing the chimeric IL-6R/IL-11R1 proteins stimulated with apically or basolaterally supplied
IL-6 and IL-11 from (C)–(F). One representative experiment out of three is shown.
encephalomyelitis, acute liver injury, and cardiac fibrosis after
myocardial infarction (Gurfein et al., 2009; Lee et al., 2012; Nish-
ina et al., 2012; Obana et al., 2010; Qiu et al., 1996). By contrast,
in gastric tumors, IL-11R1-deficient mice displayed a delayed
onset and reduced overall tumor formation, demonstrating that
it drives tumorigenesis (Putoczki et al., 2013). While it is clear
1076 Cell Reports 16, 1067–1081, July 26, 2016
that IL-6 and IL-11 have overlapping functions, the full extent
by which each display unique biological activities is unknown.
A simplified view suggests that IL-6 is more important for im-
mune functions, whereas IL-11 regulates polarized epithelial
proliferation and regeneration. While apical and basolateral
secretion of cytokines by polarized epithelial cells, including
Figure 6. IL-11R1 Is a Cytokine Transcytosis Receptor
(A) Basolateral stimulation of polarized MDCK, MDCK-IL-11R1, and MDCK-IL-11R2 cells with 50 ng/ml IL-11 for 24 hr. IL-11 was quantified after the indicated
time points on the apical and basolateral sides and in cell lysates by ELISA.
(B) Apical stimulation of polarized MDCK, MDCK-IL-11R1, and MDCK-IL-11R2 cells with 50 ng/ml IL-11 for 24 hr. IL-11 was quantified after the indicated time
points on the apical and basolateral sides and in cell lysates by ELISA.
(C) Western blotting of STAT3/pSTAT3 of non-polarized MDCK-IL-11R2 cells stimulated with apical MDCK-supernatants from (A) (24-hr time point).
(D) Apical stimulation of polarizedMDCK,MDCK-IL-6R, MDCK-IL-6RDICD, andMDCK-IL-6RECD/IL-11R1ICD cells with 50 ng/ml IL-6 for 24 hr. IL-6 was quantified
after the indicated time points on the apical and basolateral sides by ELISA.
(E) Western blotting of STAT3/pSTAT3 of non-polarized MDCK-IL-11R2 cells stimulated with apical MDCK supernatants from (D) (24-hr time point).
(F) Apical stimulation of polarizedMDCK,MDCK-IL-6R,MDCK-IL-6RDICD,MDCK-IL-11R1,MDCK-IL-11R2, andMDCK-IL-6RECD/IL-11R1ICD cells with 50 ng/ml
Hyper-IL-6 (HIL-6) for 24 hr. HIL-6 was quantified after the indicated time points on the apical side by ELISA.
(G and H) Ba/F3-gp130-IL-11R (G) and Ba/F3-gp130-IL-6R (H) cells were stimulated for 48 hr with apical and basolateral supernatants from the indicated MDCK
cells stimulated for 24 hr with HIL-6 (50 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml) or left untreated. One representative experiment out of two is shown.
In (A), (B), (D), and (F)–(H), data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.
IL-6 and IL-8, has been described previously, there is no infor-
mation on IL-11.
Basolateral sorting of the IL-6R is triggered by two basolateral
sorting motifs (Martens et al., 2000), and deletion of the IL-6R
ICD results in unpolarized localization of IL-6R. Unpolarized api-
cal/basolateral localization is rather uncommon, because most
receptors show a strong preference for basolateral localization
or, in some cases, for apical localization (Cao et al., 2012).
Cell Reports 16, 1067–1081, July 26, 2016 1077
Figure 7. Apical/Basolateral Sorting and
Mode of Activation in Polarized MDCK Cells
Expressing IL-6R, IL-6RDICD, IL-11R1, and
IL-11R2
Left: IL-6R-expressing cell. Middle left: IL-6RDICD-
expressing cell. Middle right: IL-11R1-expressing
cell. Right: IL-11R2-expressing cell.
Both IL-11R protein variants were, however, found on basolat-
eral and apical membranes. We demonstrated that the basolat-
erally sorted gp130 was transported along with IL-11R1 and IL-
11R2 to the apical side, which enabled IL-11 signaling from both
the basolateral and apical membranes. In this respect, IL-11
signaling was different from IL-6 classic and trans-signaling,
which was induced exclusively from the basolateral membrane.
Redirection of gp130 was suggested previously for heterodi-
meric gp130/LIFR (leukemia inhibitory factor receptor) signaling.
LIFR is expressed in an unpolarized fashion in polarized MDCK
cells, albeit an apical expression that was not shown directly
(Buk et al., 2004). Glycosylphosphatidylinositol (GPI)-anchored
CNTFR (ciliary neurotrophic factor receptor) is the a-receptor
for ciliary neurotrophic factor (CNTF)-induced signaling via
CNTF:CNTFR:gp130:LIFR complexes. CNTFR was specifically
found on the apical side of polarized MDCK cells (Buk et al.,
2004), because GPI anchorage of membrane proteins promotes
their apical sorting (Simons and Ikonen, 1997). In accordance
with our findings, CNTFR renders polarized MDCK cells respon-
sive to CNTF from the apical side via CNTF:CNTFR:gp130:LIFR
complexes (Buk et al., 2004), suggesting that CNTFR and LIFR
forced the apical localization of gp130. Deletion of the ICD in
IL-6RDICD also renders polarized MDCK cells responsive to
IL-6 classic signaling at the apical side. A key question is, how
does IL11R force the redirection of gp130 to the apical mem-
brane? Our data suggest that apical redirection of gp130 was
mediated by the ICD of the cytokine a-receptors. We have
previously demonstrated that gp130 exists in preformed homo-
dimers in the absence of IL-6 (Tenhumberg et al., 2006). Future
experiments will test whether IL-6R and IL-11R are also included
in preformed complexes tomediate cellular localization of gp130
in polarized cells.
IL-11R1 and IL-11R2 only differ in the composition of their ICD
(Cherel et al., 1995). IL-11R1 contains 32 amino acid residues,
whereas the transcription variant IL-11R2 contains the four alter-
1078 Cell Reports 16, 1067–1081, July 26, 2016
native intracellular amino acid residues
LGLW. No tyrosine- or di-leucine basolat-
eral sorting motifs were identified in
the ICDs of the IL-11R1 and IL-11R2.
Moreover, general sequence-based api-
cal sortingmotifs aremissing. Apical sort-
ing motifs were localized to the ECD,
membrane domain, and ICDs. Many api-
cal sorting signals contain extracellular
post-translational modifications, such as
O- or N-linked glycosylation (Ihrke et al.,
2001; Kinlough et al., 2011; Lisanti et al.,
1989; Naim et al., 1999; Potter et al.,
2006), or GPI-membrane anchors that
mediate their association with cholesterol/sphingolipid-enriched
lipid raft microdomains, as in the case of CNTFR (Buk et al.,
2004; Simons and Ikonen, 1997). Only in some cases are apical
sorting motifs defined by extra- and intracellular amino acid
sequences within the target protein, as observed for megalin
and P2Y2 receptor (Qi et al., 2005; Takeda et al., 2003). It is
still unclear how these different classes of apical proteins are
sorted into distinct trans-Golgi-network (TGN)-derived transport
carriers.
Our data suggest that the membrane distribution of IL-11R2
and IL-6RDICD, which both lack significant ICDs in polarized
cells, is executed in a non-regulated, stochastic manner. This
is clearly opposite to the IL-11R1 isoform. Our results demon-
strated that IL-11R1 bound IL-11 on the basolateral side and
transported and released biologically active IL-11 to the apical
side, thereby defining IL-11R1 as transcytosis receptor within
the IL-6R family. By contrast, IL-11R2 and IL-6RDICD did not
mediate transcytosis. Transcytosis of IL-11 was, however, unidi-
rectional, since no IL-11 was transported from the apical to
the basolateral side. Lastly, IL-6R could be transformed into an
IL-6 transcytosis receptor by swapping the ICD of IL-11R to
IL-6R. Our data demonstrated that the ICD of IL-11R1 was
responsible for transcytosis by successive sorting of IL-11R1
from the basolateral membrane to the apical membrane.
Gp130 can serve as a transcytosis receptor for IL-6 trans-
signaling mediated by IL-6 and sIL-6R in cells expressing
IL-11R1. We propose that additional IL-6-type cytokines that
do not require direct binding to IL-11R, such as leukemia inhib-
itory factor (LIF), oncostatin M (OSM), and clathrin light chain
(CLC), may be transcytosed by gp130 due to IL-11R-induced
redirection.
The cell localization pattern of IL-11R1 and IL-11R2 suggests
that transcytosis is not needed for IL-11R2-mediated function,
because only IL-11R1 is primarily expressed on polarized cells.
Functionally, we demonstrated that basolateral stimulation of
polarized primary HEECs stimulated the adhesion of human
trophoblast cell line spheroids to the apical surface of the endo-
metrial epithelial cells. This suggests the IL-11 acts via transcy-
tosis to regulate human blastocyst attachment to endometrial
surface epithelial cells and the very early stages of human em-
bryo implantation. IL-11 is known to regulate human endometrial
epithelial adhesive capacity via exogenous apical stimulation
(Marwood et al., 2009); however, this is the first study to demon-
strate that apical treatment of the cells alters their adhesion.
Although IL-11R1-deficient mice demonstrated the overall
importance of IL-11 signaling in tissue regeneration and tumor
development, at this point, it is not clear what the significance
is of the transcytosis of IL11 in these processes. IL-11 transcyto-
sis may be required for efficient organ-wide distribution of IL-11
during regeneration or delivery across cellular barriers, such as
the blood-brain barrier. It was suggested that polarized cytokine
expression is needed for directed migration and activation of im-
mune cells by, e.g., IL-8 and IL-6 (Chow et al., 2010; Healy et al.,
2015; Rossi et al., 2013). In this scenario, migrating immune cells
would only be activated after they reached the site of infection.
Subsequently, regeneration triggered by transcytosed IL-11
might be specifically and locally induced at sites of infection
from the apical side. Selective deletion of the ICDof IL-11R in vivo
will, however, reveal the role in regeneration tumor development,
and endometrial remodeling in preparation for embryo implanta-
tion is fulfilled by polarized expression of IL-11R1 and transcyto-
sis of IL-11.
EXPERIMENTAL PROCEDURES
Cell Culture
MDCK (CCL-34TM) cells were from ATCC, and HeLa cells (ACC-57) were from
DSMZ. Ba/F3-gp130 cells (Gearing et al., 1987) were cultured with 10 ng/ml
HIL-6 (Fischer et al., 1997), Ba/F3-gp130-IL-6R cells were cultured with
10 ng/ml IL-6, and Ba/F3-gp130-IL-11R cells were cultured with 20 ng/ml IL-
11. Stably transfected MDCK cells were selected with 600 mg/ml G418 or
2 mg/ml puromycin. All stable cell lines were selected from single clones. Ba/
F3-gp130 and HeLa cells were cultured in high-glucose DMEM/10% fetal
calf serum (FCS), and MDCK cells were cultured in low-glucose DMEM/10%
FCS (GIBCO, Life Technologies) at 37�C with 5% CO2.
Polarization and Stimulation of MDCK Cells
MDCK cells were seeded at 104 cells per six-well plate in Transwell filter plates
with transparent polyethylene terephthalate (PET) membrane (pore size,
0.4 mm, Corning Life Sciences) and grown for 4–5 days to form a polarized
monolayer. Cells were washed twice with PBS, subsequently starved for
2 hr in serum-free DMEM, and exposed for 30 min with IL-6 (10 ng/ml), IL-11
(20 ng/ml), or HIL-6 (10 ng/ml).
Immunofluorescence Staining of Polarized Cells
Polarized MDCK cells were fixed in 4% paraformaldehyde (PFA) for 20 min at
room temperature (RT), washed once with PBS, and permeabilized in PBS
with 0.25% Triton X-100 for 5 min. After washing with PBS, cells were
blocked for 1 hr in PBS with 1% BSA and 0.25% Triton X-100 for 1 hr,
and proteins were stained at 4�C overnight: IL-6R (4–11 mAb [monoclonal
antibodies]), IL-11R (N20), and gp130 (ab87969), CHC (D3C6), Na+-K+-
ATPase (H-300). The secondary antibodies Alexa Fluor 546 goat anti-mouse
IgG (immunoglobulin G) and Alexa Fluor 488 goat anti-rabbit IgG were
applied for 1 hr at RT. Transwell filters were washed three times with PBS,
and the membrane was mounted onto microscopy slides with ProLong
Gold Antifade reagent containing DAPI (Invitrogen). Analyses were performed
with a Leica TCS SP2/AOBS microscope equipped with an HCX PL APO
633 immersion objective (Leica Microsystems). The images were taken us-
ing an LSM 510-Meta microscope (Zeiss) at excitation wavelengths of 488
and 546 nm. SR-SIM was performed using the ELYRA PS.1 microscope
(Zeiss).
Transcytosis
Polarized MDCK cells were washed with PBS and starved for 2 hr at 37�C.50 ng/ml IL-6, IL-11, or HIL-6 was added basolaterally or apically. Cytokine
concentration was quantified by ELISA (R&D Systems IL-11 ELISA kit for IL-
11, Novex IL-6 Elisa kit from Life technology for IL-6 and ELISA for IL-6R as
described; Chalaris et al., 2007).
Human Ethics Statement
Written informed consent was obtained from each patient, and the study was
approved by theMonash Health Research and Ethics Committee (#09317B) at
the Monash Medical Centre, Melbourne, Australia.
HEEC-Trophoblast Spheroid Adhesion Assay
Primary HEECs were grown to confluence in 24-well Transwell filter plates with
amembrane pore size of 0.4 mm (Corning Life Sciences). Prior to the addition of
HEECs, the wells were overlaid with Matrigel (Corning) (1:10) for polarization of
the endometrial epithelial cells. The cells were washed with PBS and serum
starved for 12 hr. Recombinant human IL-11 (50 ng/ml) or diluent control (a
kind donation from Genetics Institute) was added to the basolateral or apical
media for 24 hr. Spheroids were formed using a first-trimester-derived tropho-
blast cell line, HTR8sv/Neo (2,000 cells per spheroid), in a CELLSTAR
U-shaped 96-well suspension culture plate (Greiner Bio-One International)
and incubated at 37�C for 48 hr (Krishnan et al., 2013). Spheroids (approxi-
mately ten per well) were transferred into the 96-well plate containing treated
HEECs. Spheroid number was determined visually prior to incubation at 37�Cfor 2 hr. Co-culture wells were washed gently, with 150 ml serum-free DMEM/
F12 media (GIBCO), and the remaining spheroids were counted to determine
the number of adhered spheroids; attachment was expressed as a percentage
of the original spheroid number, as previously described (Cuman et al., 2015;
Krishnan et al., 2013).
Statistical Analysis
Data are expressed asmean values ±SD calculated from at least two indepen-
dent experiments. Statistical analysis was performed by using Student‘s t test.
A p value of <0.05 (*) was defined as statistically significant (**p < 0.01; ***p <
0.001).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and one figure and can be found with this article online at http://dx.doi.org/
10.1016/j.celrep.2016.06.062.
AUTHOR CONTRIBUTIONS
J.S. and N.M. designed the study. N.M., J.M., B.G., C.C., E.D., and M.F. per-
formed experiments. R.N., J.L., D.M.F., D.H., C.G., and E.D. provided mate-
rials and discussed the data. N.M., R.P., E.D., and J.S. contributed to drafting
the manuscript.
ACKNOWLEDGMENTS
We thank Hadi Al-Hasani (German Diabetes Center, Leibniz Center for Dia-
betes Research, Heinrich-Heine-University) for the generous gift of plasmids
encoding Dynamin-1 and Dynamin-1 K44A mutant.
This work was funded by grants from the Deutsche Forschungsgemein-
schaft (DFG SCHE907/3-1 and SFB974 A12 to J.S. and GA2048/1-1 and
SFB877 project A10 to C.G.), the National Health andMedical Research Coun-
cil (NHMRC) of Australia Senior Research Fellowship (ID 1019826 to E.D.), and
the Victorian Infrastructure Support Program and Australian Government
NHMRC IRIISS (to E.D.).
Cell Reports 16, 1067–1081, July 26, 2016 1079
Received: November 11, 2015
Revised: April 8, 2016
Accepted: June 14, 2016
Published: July 14, 2016
REFERENCES
Al-Hasani, H., Hinck, C.S., and Cushman, S.W. (1998). Endocytosis of the
glucose transporter GLUT4 is mediated by the GTPase dynamin. J. Biol.
Chem. 273, 17504–17510.
Buk, D.M., Waibel, M., Braig, C., Martens, A.S., Heinrich, P.C., and Graeve, L.
(2004). Polarity and lipid raft association of the components of the ciliary neu-
rotrophic factor receptor complex in Madin-Darby canine kidney cells. J. Cell
Sci. 117, 2063–2075.
Cao, X., Surma, M.A., and Simons, K. (2012). Polarized sorting and trafficking
in epithelial cells. Cell Res. 22, 793–805.
Chalaris, A., Rabe, B., Paliga, K., Lange, H., Laskay, T., Fielding, C.A., Jones,
S.A., Rose-John, S., and Scheller, J. (2007). Apoptosis is a natural stimulus of
IL6R shedding and contributes to the proinflammatory trans-signaling function
of neutrophils. Blood 110, 1748–1755.
Chalaris, A., Garbers, C., Rabe, B., Rose-John, S., and Scheller, J. (2011). The
soluble Interleukin 6 receptor: generation and role in inflammation and cancer.
Eur. J. Cell Biol. 90, 484–494.
Cherel, M., Sorel, M., Lebeau, B., Dubois, S., Moreau, J.F., Bataille, R., Min-
vielle, S., and Jacques, Y. (1995). Molecular cloning of two isoforms of a recep-
tor for the human hematopoietic cytokine interleukin-11. Blood 86, 2534–2540.
Chow, A.W., Liang, J.F., Wong, J.S., Fu, Y., Tang, N.L., and Ko, W.H. (2010).
Polarized secretion of interleukin (IL)-6 and IL-8 by human airway epithelia
16HBE14o- cells in response to cationic polypeptide challenge. PLoS ONE
5, e12091.
Cork, B.A., Tuckerman, E.M., Li, T.C., and Laird, S.M. (2002). Expression of
interleukin (IL)-11 receptor by the human endometrium in vivo and effects of
IL-11, IL-6 and LIF on the production of MMP and cytokines by human endo-
metrial cells in vitro. Mol. Hum. Reprod. 8, 841–848.
Cuman, C., Van Sinderen, M., Gantier, M.P., Rainczuk, K., Sorby, K., Rom-
bauts, L., Osianlis, T., and Dimitriadis, E. (2015). Human blastocyst secreted
microRNA regulate endometrial epithelial cell adhesion. EBioMedicine 2,
1528–1535.
Dittrich, E., Rose-John, S., Gerhartz, C., M€ullberg, J., Stoyan, T., Yasukawa,
K., Heinrich, P.C., and Graeve, L. (1994). Identification of a region within the
cytoplasmic domain of the interleukin-6 (IL-6) signal transducer gp130 impor-
tant for ligand-induced endocytosis of the IL-6 receptor. J. Biol. Chem. 269,
19014–19020.
Dittrich, E., Haft, C.R., Muys, L., Heinrich, P.C., and Graeve, L. (1996). A di-
leucine motif and an upstream serine in the interleukin-6 (IL-6) signal trans-
ducer gp130 mediate ligand-induced endocytosis and down-regulation of
the IL-6 receptor. J. Biol. Chem. 271, 5487–5494.
Doherty, G.J., and McMahon, H.T. (2009). Mechanisms of endocytosis. Annu.
Rev. Biochem. 78, 857–902.
Doumanov, J.A., Daubrawa, M., Unden, H., and Graeve, L. (2006). Identifica-
tion of a basolateral sorting signal within the cytoplasmic domain of the inter-
leukin-6 signal transducer gp130. Cell. Signal. 18, 1140–1146.
Dutta, D.,Williamson, C.D., Cole, N.B., and Donaldson, J.G. (2012). Pitstop 2 is
a potent inhibitor of clathrin-independent endocytosis. PLoS ONE 7, e45799.
Fischer, M., Goldschmitt, J., Peschel, C., Brakenhoff, J.P., Kallen, K.J.,
Wollmer, A., Grotzinger, J., and Rose-John, S. (1997). I. A designer cytokine
with high activity on human hematopoietic progenitor cells. Nat. Biotechnol.
15, 142–145.
Fujimoto, K., Ida, H., Hirota, Y., Ishigai, M., Amano, J., and Tanaka, Y. (2015).
Intracellular dynamics and fate of a humanized anti-interleukin-6 receptor
monoclonal antibody, tocilizumab. Mol. Pharmacol. 88, 660–675.
Garbers, C., and Scheller, J. (2013). Interleukin-6 and interleukin-11: same
same but different. Biol. Chem. 394, 1145–1161.
1080 Cell Reports 16, 1067–1081, July 26, 2016
Garbers, C., Hermanns, H.M., Schaper, F., M€uller-Newen, G., Grotzinger, J.,
Rose-John, S., and Scheller, J. (2012). Plasticity and cross-talk of interleukin
6-type cytokines. Cytokine Growth Factor Rev. 23, 85–97.
Garbers, C., Monhasery, N., Aparicio-Siegmund, S., Lokau, J., Baran, P.,
Nowell, M.A., Jones, S.A., Rose-John, S., and Scheller, J. (2014). The inter-
leukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145
confers increased proteolytic conversion rates by ADAM proteases. Biochim.
Biophys. Acta 1842, 1485–1494.
Gearing, D.P., Gough, N.M., King, J.A., Hilton, D.J., Nicola, N.A., Simpson,
R.J., Nice, E.C., Kelso, A., and Metcalf, D. (1987). Molecular cloning and
expression of cDNA encoding a murine myeloid leukaemia inhibitory factor
(LIF). EMBO J. 6, 3995–4002.
Gerhartz, C., Dittrich, E., Stoyan, T., Rose-John, S., Yasukawa, K., Heinrich,
P.C., and Graeve, L. (1994). Biosynthesis and half-life of the interleukin-6
receptor and its signal transducer gp130. Eur. J. Biochem. 223, 265–274.
Gibson, D.L., Montero, M., Ropeleski, M.J., Bergstrom, K.S., Ma, C., Ghosh,
S., Merkens, H., Huang, J., Mansson, L.E., Sham, H.P., et al. (2010). Inter-
leukin-11 reduces TLR4-induced colitis in TLR2-deficient mice and restores
intestinal STAT3 signaling. Gastroenterology 139, 1277–1288.
Gurfein, B.T., Zhang, Y., Lopez, C.B., Argaw, A.T., Zameer, A., Moran, T.M., and
John, G.R. (2009). IL-11 regulates autoimmune demyelination. J. Immunol. 183,
4229–4240.
Healy, L.L., Cronin, J.G., and Sheldon, I.M. (2015). Polarized epithelial cells
secrete interleukin 6 apically in the bovine endometrium. Biol. Reprod. 92, 151.
Ihrke, G., Bruns, J.R., Luzio, J.P., and Weisz, O.A. (2001). Competing sorting
signals guide endolyn along a novel route to lysosomes in MDCK cells.
EMBO J. 20, 6256–6264.
Ivanov, I.I., McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille,
J.J., Cua, D.J., and Littman, D.R. (2006). The orphan nuclear receptor
RORgammat directs the differentiation program of proinflammatory IL-17+
T helper cells. Cell 126, 1121–1133.
Jerdeva, G.V., Tesar, D.B., Huey-Tubman, K.E., Ladinsky, M.S., Fraser, S.E.,
and Bjorkman, P.J. (2010). Comparison of FcRn- and pIgR-mediated transport
in MDCK cells by fluorescence confocal microscopy. Traffic 11, 1205–1220.
Kinlough, C.L., Poland, P.A., Gendler, S.J., Mattila, P.E., Mo, D., Weisz, O.A.,
and Hughey, R.P. (2011). Core-glycosylated mucin-like repeats from MUC1
are an apical targeting signal. J. Biol. Chem. 286, 39072–39081.
Kopf, M., Baumann, H., Freer, G., Freudenberg, M., Lamers, M., Kishimoto, T.,
Zinkernagel, R., Bluethmann, H., and Kohler, G. (1994). Impaired immune and
acute-phase responses in interleukin-6-deficient mice. Nature 368, 339–342.
Krishnan, T., Winship, A., Sonderegger, S., Menkhorst, E., Horne, A.W.,
Brown, J., Zhang, J.G., Nicola, N.A., Tong, S., and Dimitriadis, E. (2013). The
role of leukemia inhibitory factor in tubal ectopic pregnancy. Placenta 34,
1014–1019.
Lebeau, B., Montero Julian, F.A., Wijdenes, J., M€uller-Newen, G., Dahmen, H.,
Cherel, M., Heinrich, P.C., Brailly, H., Hallet, M.M., Godard, A., et al. (1997).
Reconstitution of two isoforms of the human interleukin-11 receptor and com-
parison of their functional properties. FEBS Lett. 407, 141–147.
Lee, H.T., Park, S.W., Kim, M., Ham, A., Anderson, L.J., Brown, K.M., D’Agati,
V.D., and Cox, G.N. (2012). Interleukin-11 protects against renal ischemia and
reperfusion injury. Am. J. Physiol. Renal Physiol. 303, F1216–F1224.
Lisanti, M.P., Caras, I.W., Davitz, M.A., and Rodriguez-Boulan, E. (1989). A gly-
cophospholipid membrane anchor acts as an apical targeting signal in polar-
ized epithelial cells. J. Cell Biol. 109, 2145–2156.
Luig, M., Kluger, M.A., Goerke, B., Meyer, M., Nosko, A., Yan, I., Scheller, J.,
Mittr€ucker, H.W., Rose-John, S., Stahl, R.A., et al. (2015). Inflammation-
induced IL-6 functions as a natural brake on macrophages and limits GN.
J. Am. Soc. Nephrol. 26, 1597–1607.
Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., and Kirchhausen,
T. (2006). Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10,
839–850.
Martens, A.S., Bode, J.G., Heinrich, P.C., and Graeve, L. (2000). The cyto-
plasmic domain of the interleukin-6 receptor gp80 mediates its basolateral
sorting in polarized madin-darby canine kidney cells. J. Cell Sci. 113, 3593–
3602.
Marwood, M., Visser, K., Salamonsen, L.A., and Dimitriadis, E. (2009). Inter-
leukin-11 and leukemia inhibitory factor regulate the adhesion of endometrial
epithelial cells: implications in fertility regulation. Endocrinology 150, 2915–
2923.
Mauer, J., Chaurasia, B., Goldau, J., Vogt, M.C., Ruud, J., Nguyen, K.D., The-
urich, S., Hausen, A.C., Schmitz, J., Bronneke, H.S., et al. (2014). Signaling by
IL-6 promotes alternative activation of macrophages to limit endotoxemia and
obesity-associated resistance to insulin. Nat. Immunol. 15, 423–430.
Naim, H.Y., Joberty, G., Alfalah, M., and Jacob, R. (1999). Temporal associa-
tion of the N- and O-linked glycosylation events and their implication in the
polarized sorting of intestinal brush border sucrase-isomaltase, aminopepti-
dase N, and dipeptidyl peptidase IV. J. Biol. Chem. 274, 17961–17967.
Nishina, T., Komazawa-Sakon, S., Yanaka, S., Piao, X., Zheng, D.M., Piao,
J.H., Kojima, Y., Yamashina, S., Sano, E., Putoczki, T., et al. (2012). Inter-
leukin-11 links oxidative stress and compensatory proliferation. Sci. Signal.
5, ra5.
Obana, M., Maeda, M., Takeda, K., Hayama, A., Mohri, T., Yamashita, T., Na-
kaoka, Y., Komuro, I., Takeda, K., Matsumiya, G., et al. (2010). Therapeutic
activation of signal transducer and activator of transcription 3 by interleukin-
11 ameliorates cardiac fibrosis after myocardial infarction. Circulation 121,
684–691.
Paiva, P., Salamonsen, L.A., Manuelpillai, U., Walker, C., Tapia, A., Wallace,
E.M., and Dimitriadis, E. (2007). Interleukin-11 promotes migration, but not
proliferation, of human trophoblast cells, implying a role in placentation. Endo-
crinology 148, 5566–5572.
Potter, B.A., Hughey, R.P., andWeisz, O.A. (2006). Role of N- and O-glycans in
polarized biosynthetic sorting. Am. J. Physiol. Cell Physiol. 290, C1–C10.
Putoczki, T., and Ernst, M. (2010). More than a sidekick: the IL-6 family cyto-
kine IL-11 links inflammation to cancer. J. Leukoc. Biol. 88, 1109–1117.
Putoczki, T.L., Thiem, S., Loving, A., Busuttil, R.A., Wilson, N.J., Ziegler, P.K.,
Nguyen, P.M., Preaudet, A., Farid, R., Edwards, K.M., et al. (2013). Interleukin-
11 is the dominant IL-6 family cytokine during gastrointestinal tumorigenesis
and can be targeted therapeutically. Cancer Cell 24, 257–271.
Qi, A.D., Wolff, S.C., and Nicholas, R.A. (2005). The apical targeting signal of
the P2Y2 receptor is located in its first extracellular loop. J. Biol. Chem. 280,
29169–29175.
Qiu, B.S., Pfeiffer, C.J., and Keith, J.C., Jr. (1996). Protection by recombinant
human interleukin-11 against experimental TNB-induced colitis in rats. Dig.
Dis. Sci. 41, 1625–1630.
Rodriguez-Boulan, E., and Powell, S.K. (1992). Polarity of epithelial and
neuronal cells. Annu. Rev. Cell Biol. 8, 395–427.
Rossi, O., Karczewski, J., Stolte, E.H., Brummer, R.J., van Nieuwenhoven,
M.A., Meijerink, M., van Neerven, J.R., van Ijzendoorn, S.C., van Baarlen, P.,
and Wells, J.M. (2013). Vectorial secretion of interleukin-8 mediates autocrine
signalling in intestinal epithelial cells via apically located CXCR1. BMC Res.
Notes 6, 431.
Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature
387, 569–572.
Taga, T., Hibi, M., Hirata, Y., Yamasaki, K., Yasukawa, K., Matsuda, T., Hirano,
T., and Kishimoto, T. (1989). Interleukin-6 triggers the association of its recep-
tor with a possible signal transducer, gp130. Cell 58, 573–581.
Takeda, T., Yamazaki, H., and Farquhar, M.G. (2003). Identification of an apical
sorting determinant in the cytoplasmic tail of megalin. Am. J. Physiol. Cell
Physiol. 284, C1105–C1113.
Tenhumberg, S., Schuster, B., Zhu, L., Kovaleva, M., Scheller, J., Kallen, K.J.,
and Rose-John, S. (2006). gp130 dimerization in the absence of ligand:
preformed cytokine receptor complexes. Biochem. Biophys. Res. Commun.
346, 649–657.
Vogel, T.P., Milner, J.D., and Cooper, M.A. (2015). The ying and yang of STAT3
in human disease. J. Clin. Immunol. 35, 615–623.
Winship, A.L., Koga, K., Menkhorst, E., Van Sinderen, M., Rainczuk, K., Nagai,
M., Cuman, C., Yap, J., Zhang, J.G., Simmons, D., et al. (2015). Interleukin-11
alters placentation and causes preeclampsia features in mice. Proc. Natl.
Acad. Sci. USA 112, 15928–15933.
Zegers, M.M., and Hoekstra, D. (1998). Mechanisms and functional features of
polarized membrane traffic in epithelial and hepatic cells. Biochem. J. 336,
257–269.
Cell Reports 16, 1067–1081, July 26, 2016 1081