RESEARCH ARTICLE
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Alarming and Calming: Opposing Roles of S100A8/S100A9
Dimers and Tetramers on Monocytes
Antonella Russo, Hendrik Schürmann, Matthias Brandt, Katja Scholz,
Anna Livia L. Matos, David Grill, Julian Revenstorff, Maximilian Rembrink, Meike von
Wulffen, Lena Fischer-Riepe, Peter J. Hanley, Hans Häcker, Monika Prünster,
Francisco Sánchez-Madrid, Sven Hermann, Luisa Klotz, Volker Gerke, Timo Betz,
Thomas Vogl,* and Johannes Roth
Mechanisms keeping leukocytes distant of local inflammatory processes in a
resting state despite systemic release of inflammatory triggers are a pivotal
requirement for avoidance of overwhelming inflammation but are ill defined.
Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but
extracellular calcium concentrations induce S100A8/S100A9-tetramers
preventing TLR4-binding and limiting their inflammatory activity. So far, only
antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin)
are described. It is demonstrated that extracellular S100A8/S100A9 tetramers
significantly dampen monocyte dynamics as adhesion, migration, and
traction force generation in vitro and immigration of monocytes in a
cutaneous granuloma model and inflammatory activity in a model of irritant
contact dermatitis in vivo. Interestingly, these effects are not mediated by the
well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically
mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the
quaternary structure of these S100-proteins determines distinct and even
antagonistic effects mediated by different receptors. As S100A8/S100A9 are
released primarily as dimers and subsequently associate to tetramers in the
high extracellular calcium milieu, the same molecules promote inflammation
locally (S100-dimer/TLR4) but simultaneously protect the wider environment
from overwhelming inflammation (S100-tetramer/CD69).
A. Russo, K. Scholz, J. Revenstorff, M. Rembrink, M. von Wulffen,
L. Fischer-Riepe, T. Vogl, J. Roth
Institute of Immunology
University of Münster
48149 Münster, Germany
E-mail: vogl@uni-muenster.de
A. Russo, A. L. L. Matos, V. Gerke, T. Betz, J. Roth
Cells in Motion Interfaculty Centre
University of Münster
48149 Münster, Germany
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/advs.202201505
© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
DOI: 10.1002/advs.202201505
1. Introduction
Many molecular pathways activating in-
flammatory mechanisms or promoting cel-
lular dynamics, migration, and recruitment
of leukocytes during inflammation have
been characterized during the last years.
Pathways avoiding spreading of inflamma-
tory activity and keeping leukocytes distant
from the primary site of inflammation in a
resting state; however, are less understood.
The latter, however, is a pivotal requirement
to avoid uncontrolled systemic spreading of
inflammatory activity and to restrict inflam-
matory processes to local sites of infection
or injury.
Monocytes and macrophages are ma-
jor effector cells in almost every inflam-
matory disease. They show pro- as well
as anti-inflammatory properties depending
on their stage of activation or differentia-
tion. Regulated recruitment and differenti-
ation of monocytes and macrophages are
therefore crucial processes for physiolog-
ical inflammatory responses.[1,2] However,
we are only beginning to understand the
H. Schürmann, M. Brandt, T. Betz
Institute of Cell Biology
Centre for Molecular Biology of Inflammation
ZMBE
University of Münster
48149 Münster, Germany
A. L. L. Matos, D. Grill, V. Gerke
Institute of Medical Biochemistry
Centre of Molecular Biology of Inflammation
ZMBE
University of Münster
48149 Münster, Germany
P. J. Hanley
Faculty of Medicine
HMU Health and Medical University Potsdam
14471 Potsdam, Germany
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mechanisms driving recruitment of specific populations such as
monocytes/macrophages to sites of inflammation while simulta-
neously preventing systemic spreading of inflammation.
S100A8 and S100A9, originally described as myeloid related
protein (MRP) 8 and MRP14,[3] are cytosolic calcium-binding
proteins highly expressed in phagocytes such as neutrophils
and monocytes. Both proteins belong to the family of danger-
associated molecular pattern molecules (DAMPs) or alarmins
and are secreted as S100A8/S100A9-heterodimers during the
activation of phagocytes by an unconventional mechanism.[4,5]
Heterodimer-complexes promote inflammatory responses via
Toll-like receptor 4 (TLR4) signaling cascades.[6,7,8] In many clin-
ically relevant disorders, such as rheumatoid arthritis, inflam-
matory bowel diseases or psoriasis, S100A8/S100A9-complexes
are the most abundant alarmins expressed and released at lo-
cal sites of inflammation, and the S100A8/S100A9-complex is
widely used as a biomarker under the brand name “calprotectin”
for monitoring inflammatory disease activity.[9,10,11] In the pres-
ence of extracellular calcium concentrations as found in the sys-
temic circulation in vivo (≈2.09–2.54mmol L−1) or under cell cul-
ture conditions in vitro (≈ 0.89 mmol L−1), two S100A8/S100A9-
dimers form rapidly a S100A8/S100A9-tetramer.[5,8,12,13] S100-
tetramer formation results in steric shielding of the S100 amino
acids responsible for the interaction with TLR4 in the interface
of the tetramer; thus, preventing TLR4 binding of these S100-
proteins.[8] Intracellular tetramer formation has been shown to
modulate microtubule stability but the only function of extra-
cellular S100A8/S100A9-tetramers described so far was an an-
timicrobial activity by chelating bivalent cations as calcium, zinc,
H. Häcker
Department of Pathology
Division of Microbiology and Immunology
University of Utah
Salt Lake City, UT 84112, USA
M. Prünster
BioMedical Center
Walter-Brendel-Centre for Experimental Medicine
Ludwig-Maximilians-University
Planegg-Martinsried, 82152 Munich, Germany
F. Sánchez-Madrid
Immunology Service
Hospital de la Princesa
Universidad Autónoma de Madrid
Instituto Investigación Sanitaria Princesa
Madrid 28006, Spain
F. Sánchez-Madrid
Department of Vascular Biology and Inflammation
Centro Nacional de Investigaciones Cardiovasculares (CNIC)
Madrid 28029, Spain
S. Hermann
European Institute for Molecular Imaging (EIMI)
University of Münster
48149 Münster, Germany
L. Klotz
Department of Neurology with Institute of Translational Neurology
University Hospital Muenster
48149 Muenster, Germany
T. Betz
Third Institute of Physics– Biophysics
Georg August University Göttingen
37077 Göttingen, Germany
manganese or nickel, which are important for the growth of
many pathogens but a functional relevance of the high systemic
levels of this protein complex under noninfectious conditions has
not been described so far.[5,14,15,16]
Here, we demonstrate that monocytes lacking
S100A8/S100A9 exhibit major alterations in their cell dynamics.
Interestingly, these effects are reversed upon addition of extra-
cellular S100A8/S100A9-tetramers but not S100A8/S100A9-
dimers. We show that these phenotypes are not induced by
S100A8/S100A9 binding to TLR4 but are specifically mediated
by S100-tetramers in a CD69-dependent manner. The in vivo
relevance of our novel findings could be verified by a reduced
immigration of monocytes in a cutaneous granuloma model and
diminished inflammation during irritant contact dermatitis in
the presence of S100A8/S100A9-tetramers. Thus, we can finally
demonstrate different functions of dimeric and tetrameric
S100A8/S100A9 complexes which either trigger local inflam-
matory activity via TLR4 (S100-dimers) or systemically dampen
migratory cell dynamics of monocytes via CD69 (S100-tetramers)
depending on their quaternary structure.
2. Results
2.1. Monocyte Migration and Morphology are Dependent on
S100A8/S100A9 Expression
Using the ER-Hoxb8 system,[17] we generated transiently im-
mortalized myeloid progenitor cells of enhanced green flu-
orescent protein (EGFP)-Lifeact wild type (WT) and EGFP-
Lifeact mice crossed with S100A9 knock-out (KO) mice which
were subsequently differentiated to monocytes (day 3). KO of
S100A9 in mice is generally associated with absence of both
S100A9 and S100A8 at the protein level, resulting functionally in
S100A8/S100A9 double KOmice and cells.[18,19] We observed sig-
nificant morphological differences between ER-Hoxb8 WT and
S100A9 KO. While S100A9 KO cells exhibited elongated trailing
ends (uropods) with numerous filopodia-like extensions, which
are probably largely retraction fibers, WT cells had compact mor-
phologies. This altered morphology was confirmed by quantify-
ing the circularity as well as the perimeter of S100A9 KO andWT
cells (Figure 1a).
To ensure that these cellular differences are not caused by the
artificial ER-Hoxb8 expression, we also compared primary bone
marrow derived cells (BMC) obtained from WT and S100A9
knockout mice. BMCs were differentiated to macrophages for
7 days in the presence of macrophage colony-stimulating factor
(M-CSF) and analyzed over time regarding the expression of
surface markers such as Cd11b, Ly6c and F4/80 as well as ex-
pression of S100A9 (Figure S1a, Supporting Information). Bone
marrow derived WT and S100A9 KO monocytes (d3) showed
major differences in their morphology comparable to the results
observed in the ER-Hoxb8 system (Figure S1b, Supporting In-
formation; Figure 1a). We next analyzed the migratory behavior.
In the absence of a chemokine gradient, S100A9 KO monocytes
migrated with a higher speed compared to WT monocytes. In
the presence of the chemoattractant complement C5a; how-
ever, WT monocytes increased their migratory speed toward the
chemotactic stimulus whereas S100A9 KO cells did not show any
additional speed increase (Figure 1b) and migrated slower than
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Figure 1. S100A8/S100A9 deficiency alters morphology and migration capacity of monocytes and depends on small GTPase activities. a) Imaging of
Lifeact WT (n = 20) and Lifeact S100A9 KO monocytes (n = 20). The circularity and perimeter of these two cell types were determined by ImageJ. One-
way ANOVA with Bonferroni post-test analysis. Scale bar = 10 μm. b) 2D migration assay experiment showing the speed of Lifeact WT (n = 66) and
Lifeact S100A9 KO monocytes (n = 66) in a chemokine free and in a complement C5a gradient (20 nm) after 6 h of migration. One-way ANOVA with
Bonferroni post-test analysis. c) Analysis of adhesion properties of untreated and 5 min complement C5a (20 nm) stimulated Lifeact WT and Lifeact
S100A9 KO monocytes after 30 min of adhesion on 50 μg mL−1 fibronectin coated 96 well plate. Quantification was obtained via optical density of
crystal violet stained adherent cells. One-way ANOVA with Bonferroni post-test analysis. d) Left panel, strain energy of Lifeact WT (n = 16) and Lifeact
S100A9 KO monocytes (n = 16) under steady state conditions and after stimulation with complement C5a (20 nm). Mann–Whitney U test. Right panel
shows representative force maps of untreated and complement C5a (20 nm) stimulated Lifeact WT and Lifeact S100A9 KO monocytes. e) Analysis of
transmigrated Lifeact WT and Lifeact S100A9 KO monocytes in a chemokine free and chemokine rich environment (20 nm complement C5a) via a
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the WT cells under this condition. Furthermore, under steady
state conditions,WTmonocytes showed stronger adherence to fi-
bronectin compared to S100A9 KO cells. In WT cells, this strong
adhesion was lost after complement C5a stimulation whereas
S100A9 KO cells did not change their already low basal adhesion
properties (Figure 1c). To analyze whether the adhesion differ-
ences reflect the traction force generation capacities, we studied
the forces of these cells transferred to extracellular matrix struc-
tures using traction force microscopy.[20] EGFP-Lifeact WT and
EGFP-Lifeact S100A9 KOmonocytes were seeded on fibronectin
coated polyacrylamide gels (Young’s modulus: 3 kPa) containing
red fluorescent beads. Analyses of the strain energy showed a
significantly higher deformation of the gel; and hence, larger
force generation by unstimulated WT compared to S100A9 KO
monocytes. After activation of WT cells with complement C5a,
the deformation was significantly reduced. In contrast, the strain
energy of unstimulated S100A9 KO monocytes was already low
in the steady state condition and did not further decrease after
complement C5a stimulation (Figure 1d). In order to assess the
transmigration ability of WT and S100A9 KO monocytes, we
performed two-chamber transmigration assays. Consistent with
the observed elevated chemotactic migration speed, significantly
more S100A9 KO monocytes transmigrated compared to WT
cells. Chemoattractant complement C5a stimulation had no
effect on the transmigration of S100A9 KO cells but significantly
enhanced the transmigration of WT cells (Figure 1e). To rule out
that S100A9 KO monocytes are generally not responsive to com-
plement C5a, we used a fluorescent calcium indicator to monitor
calcium signaling. No differences in complement C5a-induced
calcium signaling could be observed between WT and S100A9
KO monocytes (Figure S2, Supporting Information).
Hence, regarding morphology, migration, and force genera-
tion, the S100A9 KO cells resemble the activated WT, suggesting
that S1008/S100A9 is required to restrain monocyte activation.
2.2. Increased Activities of Small Rho GTPases Alter Cell Shape
and Migration Speed in S100A9 KO Cells
To gain insight into the mechanisms leading to the observed ac-
tivation like phenotype in the S100A9 KO cells, we investigated
intracellular signaling pathways relevant for migration and force
generation. Small GTPases of the Rho family are regulators of
the cytoskeleton, membrane dynamics, and cell shape, switch-
ing from an inactive GDP- to an active GTP-bound form via gua-
nine nucleotide exchange factors (GEFs) and vice versa from a
GTP- to GDP-bound inactive form via GTPase-activating proteins
(GAPs).[21] S100A9 KO cells expressed slightly elevated levels of
total Cdc42. To study more relevant effects on Rho GTPase acti-
vation, we quantified the GTP-bound form of Cdc42, RhoA, and
Rac by immunoprecipitation assays. Under steady state condi-
tions, S100A9 KO cells expressed significantly higher amounts of
active GTP-bound Cdc42 even after normalization to the slightly
elevated total Cdc42 levels. The same was true for RhoA and Rac
compared to WT monocytes. Activation of all GTPases was not
further enhanced after stimulationwith complement C5a, in con-
trast to WT monocytes, which responded to complement C5a
stimulation with significantly increased GTP-bound RhoGTPase
levels (Figure 1f).
Incubation of S100A9 KOmonocytes with specific GTPase in-
hibitors (ZCL28 for Cdc42; Rhosin hydrochloride for RhoA; and
W56 for Rac) for 30 min reduced the GTP-bound amounts of
Cdc42, RhoA, and Rac to those present in WT cells, confirming
the efficacy of these inhibitors in our experimental settings (Fig-
ure S3a, Supporting Information).
Consistent with its importance in cell migration, specific in-
hibition of either RhoA or Rac slightly reduced the migration
speed of S100A9 KO cells, whereas simultaneous inhibition of
both RhoA and Rac reduced their migratory speed to the WT
level, while Cdc42 inhibition had no effect on velocity (Figure 1g).
In contrast to the effects seen on velocity, inhibition of Cdc42
significantly reduced the number of transmigrating S100A9 KO
monocytes (Figure 1h). Inhibition of RhoA or Rac, as well as si-
multaneous inhibition of both Rho GTPases, had no effect on
the transmigration rates (Figure S3b, Supporting Information).
We next analyzed whether the morphological differences seen in
S100A9 KO cells are also dependent of GTPase activities. Accord-
ing to the well-known fact that Cdc42 controls filopodia forma-
tion via formins, inhibition of Cdc42 converted the morphology
of S100A9KO to that seen inWT cells, inducing rounded-upmor-
phologies with only few filopodia-like structures (Figure 1i). Our
data demonstrate that lack of S100A8/S100A9 changes the activ-
ity of Rho GTPases and that the activity of Cdc42 is the dominant
factor responsible for changes in directed transmigration prop-
erties and dysregulation of monocyte morphology in S100A9 KO
cells under steady state conditions. On the other hand, effects of
S100A8/S100A9 on RhoA and Rac activities are the molecular
basis for changes in the migratory speed in S100A9 KO cells.
2.3. STAT3 is a Central Signaling Molecule Affected by
S100A8/S100A9
Previous work has shown that signal transducer and activator of
transcription 3 (STAT3) is involved in cell migration and actin
cytoskeleton reorganization.[22,23] We analyzed the total levels of
STAT1, 3, 5, and 6 and the phosphorylation status of all four STAT
(P-STAT) proteins. While all four STAT proteins were equally
boyden chamber assay. f) Immunoprecipitation assays of Cdc42, RhoA, and Rac. Lifeact WT and Lifeact S100A9 KO monocytes were left untreated or
stimulated for 5 min with the complement C5a (20 nm). Immunoprecipitated GTPases (GTP-bound) and total amounts were analyzed by western blot.
The intensity of the IP-bands was measured via ImageJ. g,) 2D migration assay experiment showing the speed of Lifeact WT (n = 82), Lifeact S100A9
KO monocytes (n = 82), and Lifeact S100A9 KO monocytes treated for 30 min with Rhosin hydrochloride (30 μm; 82 cells), W56 (250 μm; 82 cells),
Rhosin hydrochloride and W56 (82 cells), and ZCL28 (50 μm; 82 cells) after 6 h of migration. One-way ANOVA with Bonferroni post-test analysis. h)
Transmigration of Lifeact WT, Lifeact S100A9 KO, and Lifeact S100A9 KO monocytes treated with ZCL278 (30 min, 50 μm) in a chemokine free set-up,
via a boyden chamber assay. One-way ANOVA with Bonferroni post-test analysis. i) Imaging of Lifeact WT (n = 20), Lifeact S100A9 KO (n = 20), and
Lifeact S100A9 KO monocytes treated for 30 min with ZCL278 (50 μm) (n = 20, Scale bar = 10 μm). Morphological (circularity and perimeter) analysis
was performed using ImageJ. Data were pooled from three independent experiments. For statistical analysis, one-way ANOVA with Bonferroni post-test
analysis was used for all experiments. n.s: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
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expressed in WT- and S100A9 KO monocytes, only S100A9 KO
monocytes had significantly higher levels of P-STAT3 in the un-
stimulated or steady state condition compared toWTmonocytes,
and after stimulation with complement C5a, only WT cells re-
sponded with significantly increased phosphorylation of STAT3
(Figure 2a). For P-STAT1, 5, and 6, we could not detect any differ-
ent bands in the western blot (Figure S4a, Supporting Informa-
tion).
In accordance with the western blot results, S100A9 KO
monocytes showed a significantly higher ratio of nuclear-to-
cytoplasmic P-STAT3 as compared to WT cells, which was re-
verted after Cryptotanshinone (STAT3 inhibitor) treatment (Fig-
ure S4 c,d, Supporting Information). Treatment with Cryptotan-
shinone also reduced the amount of active Cdc42, RhoA, and Rac
in S100A9 KO monocytes to the levels found in WT cells (Fig-
ure S4d, Supporting Information). Moreover, blocking STAT3
phosphorylation restored morphology (Figure 2b) as well as
transmigration rates (Figure 2c) and migration speeds (Fig-
ure 2d) of S100A9 KO cells to WT levels. Knock-out of STAT3
alone did not alter wild-type morphology and STAT3 KO on the
S100A9 KO background even restored the wild-type morphology.
Only WT monocytes were able to respond to C5a (Figure S4e,
Supporting Information).
2.4. Extracellular S100A8/S100A9-Tetramers Control Cell
Dynamics of Monocytes
Next, we investigated whether the observed effects on cell dynam-
ics described above were due to intracellular S100A8/S100A9
or S100A8/S100A9 proteins that following secretion, could act
in an autocrine manner. During differentiation of monocytes to
macrophages, the cells constitutively released S100A8/S100A9-
dimers which rapidly formed S100A8/S100A9-tetramers due to
the high calcium ion concentrations present in the cell culture
medium.[8] We performed a S100A8/S100A9-ELISA which
detects both S100A8/S100A9-dimers and S100A8/S100A9-
tetramers to quantify the amounts of spontaneous secretion
of S100A8/S100A9 in unstimulated WT and S100A9KO
monocytes. As expected, no S100A8/S100A9 could be de-
tected in S100A9 KO monocytes, whereas for WT cells, we
detected ≈350 ng mL−1 S100A8/S100A9 in the supernatant
(Figure 2e). To analyze the effect of the extracellular alarmin,
we added S100A8/S100A9-tetramers, S100A8-homodimers, and
S100A8/S100A9-N70A mutated heterodimers (deletion of the
high affinity calcium-binding site in S100A9) to S100A9 KO
monocytes. S100A8-homodimers and S100A8/S100A9-N70A
mutated heterodimers are unable to form tetramers and; are
therefore, constitutively active triggers of TLR4.[8] Interestingly,
addition of wildtype S100A8/S100A9-complexes, which are able
to form tetramers, significantly reduced the phosphorylation
state of STAT3 (Figure 2f) and its nuclear translocation (Fig-
ure S5a, Supporting Information) in S100A9 KO monocytes to
a level similar to WT cells, whereas both forms of constitutively
active dimers had no effect. For WT monocytes we could not
observe additional effects after stimulation with tetramers or
dimers (data not shown).
We therefore used S100A8-homodimers and S100A8/S100A9-
tetramers in the following experiments because all S100-dimers
show a similar activation of TLR4[8] and purification of mu-
tated S100A8/S100A9-N70A heterodimers needs a complex and
time-consuming de-/renaturation protocol. The morphological
changes of S100A9 KOmonocytes were also completely reversed
upon tetramer stimulation, while no effects could be observed for
the pro-inflammatory active homodimer (Figure 2g). Moreover,
in tetramer-stimulated S100A9 KO cells, the amount of active
GTP-bound Rho-GTPases was reduced to the level of WT cells
(Figure S5b, Supporting Information). Next, we investigated the
cellular dynamics of S100A9 KO cells treated with S100-dimers
or S100-tetramers using transmigration assays (Figure 2h), 2D
migration (Figure 2i), and adhesion assays (Figure 2j) as well
as traction force microscopy (Figure 2k). S100A9 KO monocytes
behaved similar to unstimulated WT monocytes after treatment
with S100-tetramers but showed no changes after application of
S100-dimers.
2.5. Extracellular S100A8/S100A9-Tetramers Control STAT3
Activation Via Expression of SOCS3
The family of suppressor of cytokine signaling (SOCS) pro-
teins are feedback inhibitors of the Janus kinase (JAK) and
signal transducer and activator of transcription (STAT) signal-
ing pathway; especially SOCS3 is a known inhibitor of STAT3
phosphorylation.[24] We analyzed SOCS3 expression via west-
ern blot and observed higher SOCS3 levels in WT compared to
S100A9 KOmonocytes. Again, S100-tetramer stimulation signif-
icantly increased SOCS3 expression in S100A9 KO monocytes,
while S100-dimers had no effect (Figure 3a).
We next addressed the question of which receptor may me-
diate the effects of S100-tetramers on cell dynamics in mono-
cytes. First, we compared the morphology of ER-Hoxb8 mono-
cytes lacking two well studied S100A8/S100A9-receptors, TLR4
or receptor for advanced glycation end products (RAGE), with
WT controls. As clearly shown in Figure 3b, TLR4 KO as well
as RAGE KO monocytes showed the same morphology, circu-
larity, and perimeter as WT cells. In addition, the amount of P-
STAT3 in TLR4 KO and RAGE KOmonocytes was similar to WT
monocytes and significantly lower compared to S100A9KOs (Fig-
ure 3c). Accordingly, only the S100A9 KO monocytes presented
higher P-STAT3 intensity in the nucleus compared to WT, TLR4
KO, and RAGE KO monocytes, as indicated by immunofluores-
cence staining (Figure S6, Supporting Information).
These data exclude the involvement of TLR4 and RAGE in the
effects described above and implicate the involvement of another
receptor in this newmechanism, which is in accordance with the
fact that S100-dimers do not correct the S100A9 KO phenotype.
2.6. CD69 is a Specific Receptor for S100A8/S100A9 Tetramers
on Monocytes
CD69 is a so-called early activationmarker present on hematopoi-
etic stem cells and many different immune cells and is known to
regulate the phosphorylation of STAT3 via SOCS3.[25] Therefore,
CD69 constituted a candidate receptor for S100A8/S100A9
tetramers, in particular, because an interaction between
S100A8/S100A9 and CD69 has been reported before in T
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Figure 2. Increased P-STAT3 activity in S100A8/S100A9 deficient EGFP-Lifeact ER-Hoxb8monocytes and extracellular S100A8/S100A9-tetramers rescues
the S100A9 KO to WT monocytes. a) Immunoblot analysis of P-STAT3 and total STAT3 of Lifeact WT and Lifeact S100A9 KO monocytes in the presence
and absence of complement C5a (20 nm, 5 min). Quantification of STAT3 activation corresponds to the ratio of P-STAT3 on total STAT3 levels after
normalization to their GAPDH bands. b) Imaging of Lifeact WT (n = 20), Lifeact S100A9 KO (n = 20), and Lifeact S100A9 KO monocytes treated for
30 min with 5 μm of Crypto. (n = 20). Circularity and perimeter were analyzed via ImageJ software. Scale bar = 10 μm. c) Transmigration of Lifeact WT,
Lifeact S100A9 KO, as well as Lifeact S100A9 KO monocytes treated for 30 min with Crypto (5 μm) was analyzed in a chemokine free environment. d)
2D migration assay showing the speed of Lifeact WT (n = 77), Lifeact S100A9 KO (n = 77), and Lifeact S100A9 KO monocytes treated for 30 min with
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cells.[25] We first analyzed the expression of CD69 inWT, S100A9
KO, and in CD69 KO monocytes. No differences could be de-
tected in the expression levels of CD69 between WT and S100A9
KO monocytes and as expected, lack of CD69 expression was
confirmed in CD69 KO monocytes (Figure 4a).
Next, we analyzed the spontaneous secretion of
S100A8/S100A9 in WT, S100A9 KO, and CD69 KO mono-
cytes; there was no difference in the amount of secreted
S100A8/S100A9 between WT and CD69 KO monocytes, indi-
cating that a cellular uptake mechanism by CD69 is no major
pathway to influence extracellular S100A8/S100A9 concentra-
tions. In addition, as expected, no secretion by S100A9 KO
monocytes was observed (Figure 4b). Interestingly, CD69 KO
monocytes presented a phenotype comparable to S100A9 KO
monocytes, showing a significant reduction in expression of
SOCS3, which could not be up-regulated by further stimulation
with either S100-tetramers or S100-dimers (Figure 4c, upper
panel). Accordingly, high levels of P-STAT3 could be detected
in CD69 KO monocytes, which were not downregulated by
additional S100-tetramers or S100-dimers (Figure 4c, lower
panel). Moreover, changes in morphology with lower circularity
and larger perimeters (Figure 4d), lower adhesion (Figure 4e),
higher transmigration rates (Figure 4f), and migration speeds
(Figure 4g) could be detected for CD69 KO cells, comparable to
S100A9 KO cells, and no recovery was seen upon S100-dimer or
S100-tetramer stimulation.
The above data suggested that CD69 could function as a spe-
cific surface receptor for S100A8/S100A9 tetramers on mono-
cytes. To directly assess this possibility, we performed direct
binding experiments with purified components by employing
the quartz crystal microbalance with dissipation (QCM-D) tech-
nique. QCM-D measures shift in the resonance frequency of a
quartz crystal that under certain conditions is proportional to
the adsorbed mass; and thus, can be used to measure protein–
protein interactions that result in the change of mass adsorbed
to a sensor chip attached to the quartz crystal.[27] To mimic a
condition where CD69 is anchored in a biological membrane,
we first established a solid-supported bilayer (SLB) consisting of
phosphatidylcholine (PC) and DGS-Ni-NTA on the surface of the
sensor chip. This allowed the association of the His-tagged ex-
tracellular domain of CD69, and thereby, the anchoring of this
domain in a lipid bilayer. Next, soluble S100-dimers or S100-
tetramers were perfused over the chip to assess potential bind-
ing by a shift in quartz crystal resonance frequency. Figure 4h-(2)
(left and right panel) shows that recombinant His-tagged CD69
bound strongly to the Ni-containing SLB as evidenced by the de-
crease in resonance frequency. Addition of S100-tetramers led to
a further decrease in frequency indicative of an additional mass
adsorption and thus direct binding (Figure 4h-(3) [left panel]).
Interestingly, no such binding to the CD69 receptor was seen
for S100A8/S100A9-N70A-dimers (Figure 4h-(3) [right panel]). At
the end of each measurement, imidazole was added to reach to-
tal desorption of the receptor from the lipid bilayer. Thus, S100-
tetramers but not S100-dimers could interact directly with the
extracellular domain of CD69. To ensure that no S100 protein
bonded directly to the lipid bilayer, we performed the same exper-
iment with and without CD69 receptor (Figure S7a,b, Supporting
Information).
For correct CD69 insertion into the membrane, we performed
the same experiment with a well-known binding partner for
CD69 which is galectin-1 (Figure S7c, Supporting Information).
Finally, via a titration ELISA, we determined the dissociation con-
stant of the receptor CD69 and S100A8/S100A9-tetramer which
was in the low nanomolar range (Figure 4i).[26]
2.7. S100A8/S100A9-Tetramers Restrict the Immune Response in
a Cutaneous Granuloma Model and a Model of Irritative Contact
Dermatitis In Vivo
To confirm the biological relevance of our data obtained in vitro,
we used a cutaneous granuloma model, in which two biogel
“plugs” were injected into the right (addition of lipopolysaccha-
ride (LPS) plus S100A8/S100A9-tetramer) and the left flank (ad-
dition of LPS only) of the mice. The addition of LPS to both plugs
served as a defined inflammatory trigger. After i.v. application of
5 × 106 DID stained ER-HoxB8-derived monocytes, mice were
imaged at times indicated in Figure 5. In vivo fluorescence re-
flectance imaging (FRI) showed a significant decrease of cellular
infiltration into tetramer/LPS plugs compared to LPS plugs 24 h
after injection of the cells (ratio tetramer-LPS/LPS: 0.83+/− 0.11,
p < 0.004; three independent experiments were performed using
three mice per experiment each, Figure 5a). In addition, we per-
formed amousemodel of irritative contact dermatitis (ICD). ICD
was induced in S100A9 ko mice with and without application of
S100A8/S100A9 tetramers (100 μg iv). Ear swelling was signifi-
cantly lower in S100A9 KO mice recieving S100-tetramers com-
pared to controls as determined by CT scan analysis (Figure 5b).
Crypto (5 μm; n = 77) in the absence of a chemokine gradient after 6 h of migration. e) S100A8/S100A9 enzyme-linked immunosorbent assay (ELISA)
of the supernatant of untreated Lifeact WT and Lifeact S100A9 KO monocytes. f) Immunoblot analysis of P-STAT3 and STAT3 in Lifeact WT and Lifeact
S100A9 KO monocytes as well as Lifeact S100A9 KO cells treated with S100A8/S100A9-tetramer (250 ng mL−1), S100A8/S100A8-homodimer (250 ng
mL−1), or S100A8/S100A9-N70A-heterodimer (250 ng mL−1) for 3 days of differentiation. g) Imaging of Lifeact WT (n = 20), Lifeact S100A9 KO (n =
20), and Lifeact S100A9 KO monocytes treated with S100A8/S100A9-tetramer (250 ng mL−1; n = 20) or S100A8/S100A8-homodimer (250 ng mL−1; n =
20) for 3 days. Scale bar = 10 μm. Circularity and perimeter were analyzed via ImageJ software. h) Transmigration rates of Lifeact WT, Lifeact S100A9 KO,
and Lifeact S100A9 KO monocytes treated with S100A8/S100A9-tetramer (250 ng mL−1) or S100A8/S100A8-homodimer (250 ng mL−1) were analyzed
via a boyden chamber system in a chemokine free condition. i) 2D migration experiment showing the speed of Lifeact WT (n = 77), Lifeact S100A9 KO
(n = 77), and Lifeact S100A9 KO monocytes treated for 3 days with S100A8/S100A9-tetramer (250 ng mL−1; n = 77) or S100A8/S100A8-homodimer
(250 ng mL−1; n = 77) after 6 h of migration. j) Adhesion properties of Lifeact WT, Lifeact S100A9 KO monocytes, and Lifeact S100A9 KO cells treated
with S100A8/S100A9-tetramer (250 ng mL−1) or S100A8/S100A8-homodimer (250 ng mL−1). K) Left panel, traction force microscopy of Lifeact WT (n
= 16), Lifeact S100A9 KO monocytes (n = 16), and Lifeact S100A9 KO monocytes (n = 16) treated for 3 days with S100A8/S100A9-tetramer (250 ng
mL−1; n = 19) or S100A8/S100A8-homodimer (250 ng mL−1; n = 15). Right panel shows the representative force maps. Data were pooled from three
independent experiments. For statistical analysis, one-way ANOVA with Bonferroni post-test analysis was used for all experiments. n.s: not significant,
* P < 0.05, ** P < 0.01, and *** P < 0.001.
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Figure 3. TLR4 and RAGE are not involved in S100-tetramer specific effects on monocytes. a) Immunoblot analysis of SOCS3 in Lifeact WT, Lifeact
S100A9 KO, as well as Lifeact S100A9 KO monocytes stimulated with either S100A8/S100A9-tetramer (250 ng mL−1) or S100A8/S100A8-homodimer
(250 ng mL−1) or S100A8/S100A9-heterodimer (250 ng mL−1). b) Morphological analysis of Lifeact WT (n = 20) and Lifeact S100A9 KO (n = 20) and
Alexa Fluor 488-phalloidin labeled TLR-4 KO (n = 20) and RAGE KO monocytes (n = 20). Scale bar = 10 μm. c) Immunoblot analysis of P-STAT3 and
total STAT3 in Lifeact WT and Lifeact S100A9 KO as well as TLR4 KO and RAGE KO monocytes. Data are pooled from three independent experiments.
For statistical analysis, one-way ANOVA with Bonferroni post-test analysis was used. n.s: not significant, ** P < 0.01, and *** P < 0.001.
3. Discussion
The results reported in this study provide evidence that S100-
dimers and S100-tetramers exhibit distinct active and even
antagonistic functions in regulating the cellular dynamics
of monocytes. Our data clearly demonstrate that extracel-
lular S100A8/S100A9 tetramer formation represents a cell
autonomous mechanism keeping monocytes in a quiescent
state. This finding was surprising because it has been generally
accepted that the alarmin S100A8/S100A9 triggers or amplifies
inflammatory responses via binding to TLR4.[6,7,14,28] Moreover,
S100A8/S100A9 complexes have been described to bind to
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Figure 4. S100A8/S100A9-tetramers signal via the early activation marker CD69. a) Fluorescence activated cell sorting (FACS) analysis of CD69 surface
expression in Lifeact WT, Lifeact S100A9 KO, and CD69 KO monocytes. b) S100A8/S100A9 ELISA of Lifeact WT, Lifeact S100A9 KO, and CD69 KO
monocytes supernatant. c) Immunoblot of SOCS3 in the upper panel and P-STAT3/STAT3 in the lower panel. LifeactWT, Lifeact S100A9 KO, CD69 KO, and
CD69 KOmonocytes treated with S100A8/S100A9-tetramer (250 ng mL−1) or S100A8/S100A8-homodimer (250 ng mL−1). d) Morphological changes in
circularity and perimeter of untreated Lifeact WT (n= 20), Lifeact S100A9 KO (n= 20), CD69 KO, and CD69 KOmonocytes treated with S100A8/S100A9-
tetramer (250 ng mL−1; n = 20) or S100A8/S100A8-homodimer (250 ng mL−1; n = 20). Scale bar = 10 μm. Circularity and perimeter were analyzed via
ImageJ software. Untreated Lifeact WT, Lifeact S100A9 KO, CD69 KO, and CD69 KOmonocytes treated with S100A8/S100A9-tetramer (250 ng mL−1) or
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several other receptors, for example, TLR4, RAGE, CD33, CD68,
CD69, CD85j, EMMPRIN (CD147), or MCAM, but none of
these published interactions could explain a specific effect of the
S100A8/S100A9-tetramers.[25,29–35] In the presence of high extra-
cellular calcium-ion concentrations alone or in combination with
zinc-, nickel-, or manganese-ions, the S100A8/S100A9-dimers
form S100A8/S100A9-tetramers, which mask the TLR4 binding
site in the tetramer interface and block binding to TLR4.[8,15,16]
The tetramers diffuse into the systemic circulation, where they
can be found in relatively high concentrations.[5,36] Our study
now demonstrates a novel relevant biological function for these
highly abundant S100-tetramers in the absence of infectious
S100A8/S100A8-homodimer (250 ng mL−1) were analyzed for: e) adhesion properties, f) transmigration abilities, g) 2D migration experiment showing
the speed of Lifeact WT (n = 77), Lifeact S100A9 KO (n = 77), CD69 KO monocytes, and CD69 KO monocytes treated with S100A8/S100A9-tetramer (n
= 77) or S100A8/S100A8-homodimer (n = 77) in the absence of a chemokine gradient after 6 h of migration. h) Quartz Crystal microbalance analysis,
black lines show the resonance frequency, and the orange lines the dissipation shifts of the quartz sensors during measurement over time. 4h-(1))
formation of the lipid bilayer. 4h-(2)) CD69 receptor (250 nm) adsorption onto the lipid bilayer. 4h-(3)) S100A8/S100A9-tetramer (1 μg mL−1) adsorption
adds mass without changing the dissipation, indicating that the lipid film is not perturbed (left graph). 4h-(3)) S100A8/S100A9-N70A-heterodimer (1 μg
mL−1) adsorption does not add mass (right graph). 4h-(4)) The recovery of the frequency baseline upon Imidazole chelation indicates total desorption
of the receptor from the lipid bilayer. i) Estimation of the binding constant of CD69 to S100-tetramer. The constant was calculated using the equation
of a 1:1 binding model as described by Eble.[26] Each dot represents the mean value of three independent experiments. For statistical analysis, one-way
ANOVA with Bonferroni post-test analysis was used for (a–g). n.s.: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Significance on capped line
is the same for all conditions referred to as the WT or S100A9 KO control, respectively.
Figure 5. S100A8/S100A9-tetramers dampen the immune response in inflammatory disease models. a) Time series of in vivo fluorescence reflectance
imaging (FRI) in a cutaneous granuloma model. Images of two representative mice show recruitment of i.v. injected DID-labeled monocytes to the
biogel plugs (dashed circles) over time. At 24 h p.i., the LPS+/S100-tetramer+ plugs (* label) in the right flank show a decreased cellular signal as
compared to the LPS+ plug without S100-tetramer in the left flank. Three independent experiments were performed using three mice per experiment
each and the figure images of two representative mice are shown. Injection of S100-tetramers significantly reduced immigration of monocytes (ratio
tetramer-LPS/LPS: 0.83 +/- 0.11, p<0.004). One-way ANOVA with Bonferroni post-test was used. b) Computed tomography (CT) scans of healthy (0 h)
and inflamed (4 h) ears in irritant contact dermatitis (ICD). Images of two representative mice before and 4 h after ICD induction with and without
tetramer application (100 μg, iv) prior induction of ICD are shown. The control mouse (upper CT pictures) shows increase of ear swelling over time
which is diminished after tetramer application (lower CT pictures). The lower graph shows that the percentage of ear volume increases after 4 h of ICD
induction. At 4 h p.i., S100A9 KO mice plus tetramer application show statistically significant less increase in ear volumes compared to mice without
tetramer. Both ears per mouse were analyzed in three independent experiments using two, two, and three mice per experiment each and for statistical
analysis, t-test was used, * P < 0.05.
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conditions. The specific interaction of these S100A8/S100A9-
tetramers but not dimers with CD69 controls the constitutive
migratory capacities of monocytes. Tetramers induced by dif-
ferent bivalent cations as calcium or calcium/manganese or
calcium/nickel may differ in their structure and consequently,
also in their function regarding binding to CD69. However,
known S100A8/S100A9 tetrameric 3D-structures in the pres-
ence of different cations are almost identical; so, we expect no
functional consequences on the S100-tetramer–CD69 interaction
by binding of different cations.[16,37,38] The analysis of wildtype
S100A8/S100A8 heterodimers is not possible because their life
time is too limited after release by activated phagocytes in vivo or
under cell culture conditions in vitro due to the rapid calcium-
induced tetramer formation. We therefore use two independent
controls for dimer-induced effects: S100A8 homodimers[6,7]
and S100A8/S100A9N70A heterodimers. The latter does not
form tetramers due to a mutation in the calcium binding site
which however does not induce significant structural changes or
block inflammatory activities.[39,8] We confirmed the biological
relevance of our in vitro findings in vivo using a cutaneous
granuloma model and a model of irritative contact dermatitis.
The granuloma model allows a direct quantitative comparison of
the effect of S100A8/S100A9 tetramers on leukocyte recruitment
triggered by a defined inflammatory stimulus within the same
mouse. In the ICD model, the degree in ear swelling represents
the response of mainly phagocytes to a non-specific irritative trig-
ger in a complex inflammatory scenario. In both inflammatory
in vivo settings, S100A8/S100A9-tetramers significantly reduce
the response of immune cells even to strong inflammatory
stimuli such as LPS or croton oil.
Binding of S100A8/S100A9-tetramers to CD69 blocks STAT3
signaling via activation of SOCS3, which finally results in the con-
trol of small GTPase activities leading to a resting state in mono-
cytes. GTPases may activate STAT3[22] or STAT3may activate GT-
Pase activity.[23] Our data of STAT3 KO cells and STAT3/S100A9
double KO cells and use of the STAT3 inhibitor Cryptotanshinone
point to the latter mechanism. Signaling via other members of
the STAT family is not relevant in S100A8/S100A9-tetramer me-
diated effects. SOCS3 is a known inhibitor of STAT3 phospho-
rylation and regulator of activation in monocytes[24] and STAT3
has been described to promote migration in different cell types
through activation of different Rho GTPases.[22,23,40,41] It is well
known that different GTPases exhibit specific effects in mono-
cytes and macrophages. Rac and Cdc42 promote actin polymer-
ization and generation of lamellipodial membrane protrusions
at the front end of migrating cells, whereas RhoA, known to
induce the formation of actin stress fibers, mediates the re-
traction of membrane protrusions and the trailing end of the
cell.[21] Accordingly, our pharmacological inhibition experiments
also revealed specific effects for the individual GTPases. Uncon-
trolled activation of the Rho GTPase Cdc42 is the key pathway
for increased filopodia formation and enhanced transmigration
of S100A9 KO monocytes, whereas activation of RhoA and Rac
in S100A9 KO monocytes is responsible for the enhanced basal
velocity in accordance to the main functions described for these
two GTPases.[42,43]
Although CD69 was initially described as an early activation
marker of lymphocytes, the receptor is also expressed on innate
immune cells and also exhibits anti-inflammatory effects.[44,45]
For example, analysis of CD69 KO mice demonstrated a reg-
ulatory role of CD69 in attenuating inflammation and the im-
mune response in arthritis, in pathogen clearance or tumor
immunity.[46] These effects have been largely attributed to lym-
phocytes but our data point to the fact that regulatory mecha-
nisms induced by S100-tetramers in monocytes could be a so
far neglected pathway of CD69-mediated regulatory effects on in-
flammation. In addition, S100-tetramer binding to CD69 could
affect signaling lipid sphingosine 1-phosphate (S1P) pathway,
which also may participate in the control of an appropriate im-
mune response.[47,48,49]
S100A8 and S100A9 form intracellular dimers which are re-
leased at sites of inflammation.[39,50] High extracellular calcium
concentrations; however, induce a shift from pro-inflammatory
S100-dimers binding to TLR4 to regulatory S100-tetramers with
a high binding affinity to CD69 which are the predominant form
in the systemic circulation.[51] In fact, in a previous study, we
already showed in our model of granulomatous inflammation
induced by parallel injection of two subcutaneous biogel pel-
lets (one containing LPS as inflammatory stimulus, the other
PBS as control), a significantly higher abundance of S100-dimers
in the inflammatory LPS-pellet compared to the control pellet
within the same mouse.[8] Thus, the same molecules drive in-
flammation locally but prevent spreading of inflammatory ac-
tivity throughout the body. Several mechanisms have been de-
scribed which may contribute to the stabilization of the dimeric
form of S100A8/S100A9. Low pH values, which is a common
finding at sites of inflammation, shift the equilibrium to S100-
dimers; and thus, promote inflammatory processes.[8] Accord-
ingly, analysis of local inflammatory exudates with a high abun-
dance of S100A8/A9 shows indeed conditions which favor a shift
from tetramers to dimers, that is, low calcium and lower pH.[52,53]
In addition, reactive oxygen molecules released by phagocytes at
sites of inflammation may lead to oxidation of S100A9 which
has been described to inhibit tetramer formation as well.[54] As
tetramer formation is also induced by zinc ions, our findingsmay
also present a novel mechanism explaining why zinc deficiency
promotes inflammatory processes and tissue damage.[55,56] The
high systemic concentrations of S100A8/S100A9 tetramers are
thus not simply an inactive byproduct as supposed so far but
an important buffer protecting from harmful spreading of in-
flammatory processes which may be of high relevance because
S100A8 and S100A9 are the most abundant alarmins in for ex-
ample, infections, autoimmune, or cardiovascular diseases.[5,57]
However, the relevance of this mechanism in specific inflam-
matory diseases is currently difficult to assess because reliable
methods to quantify the different complex forms are still lacking.
Environmental factors regulating dimer/tetramer ratios at local
sites of inflammation may differ significantly under specific in-
flammatory conditions, tissues, exudates, granulomas, or even
abscesses. Quantitative data about local calcium and zinc con-
centrations, pH, or even ROS production are astonishingly rare
for most inflammatory diseases. Differences in these factors may
explain some organ specific impact on the beneficial or destruc-
tive consequences of inflammation. Future approaches need to
address the question of under which inflammatory conditions
environmental factors in tissues such as ions, pH or ROS de-
crease to retard the formation of S100 tetramers, and therebymay
promote unwanted inflammation.
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Figure 6. Alarming and calming: Functions of S100A8/S100A9 dimers
and tetramers on monocytes. High release of S100A8/S100A9-dimers at
local sites of inflammation leads to the well-known activation of mono-
cytes via binding to TLR4 (left cell: alarming). However, calcium-induced
S100A8/S100A9-tetramer formation, which occurs rapidly in the extracel-
lular space, blocks the TLR4-binding site restricting their inflammatory ac-
tivities. S100A8/S100A9-tetramers keepmonocytes in a steady state condi-
tion via a CD69 receptor mediated mechanism (right cell: calming). Thus,
due to their calcium-induced quaternary structure, S100A8 and S100A9
show local and systemic antagonistic functions due to interactions with
different receptors.
Changes in a quaternary structure have often been described
to regulate the activity of proteins, in particular, enzymatic ac-
tivities, protein–protein interactions, or the assembly of multi-
protein complexes. However, in this case, we have the unique fea-
ture of a quaternary structure not simply switching the activity of
protein complexes “on” or “off” but rather inducing a shift from
binding and activating a pro-inflammatory receptor (TLR4) to an
opposite regulatory function mediated specifically by a different
receptor (CD69) (Figure 6), a situation which to our knowledge
has not been described so far.
4. Experimental Section
Animals: S100A9-/-mice (9–15 weeks old, C57BL/6 background) were
bred and housed in individually ventilated cages. Of note, S100A9-/- mice
are double deficient for S100A9 and S100A8 at the protein level.[8,19] The
animal housing conditions and animal experiments were approved by the
local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz
Nordrhein-Westfalen register no. 81-02.04.2018.A103). For generation of
ER-Hoxb8 cells male C57BL/6 wild-type, S100A9 knockout, CD69 knock-
out, TLR4 knockout, and RAGE knockout as well as EGFP-Lifeact wild-
type and EGFP-Lifeact S100A9 knockout mice were maintained in specific
pathogen free conditions at the animal facility of the Institute of Immunol-
ogy Muenster.
Isolation of Murine Bone Marrow Cells: Bone marrow cells (BMC) were
isolated from femur and tibia by flushing with PBS containing 1% FCS
(PBS/FCS). After lysis of erythrocytes and washing in PBS/FCS, cells were
differentiated for up to 7 days in RPMI1640 medium supplemented with
10% FCS, 1% Pen/Strep, 1% L-Glutamine, and 20% supernatant of M-CSF
expressing L-929 cells (Lonza).
Cells: Generation of ER-Hoxb8 cell lines were made as previously
described (Wang et al., 2006). Cells were cultured in RPMI1640 Medium
(Merck Millipore) supplemented with 10% FCS (Pan Biotech), 1%
Pen/Strep (Biochrom), 1% L-Glutamine (Biochrom), 2 μg recombinant
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Immuno-
tools), and 1 μm 𝛽-Estradiol (Sigma–Aldrich). For differentiation to
monocytes, cells were washed twice in PBS containing 1% FCS to remove
𝛽-Estradiol. Cells were differentiated for up to 7 days in RPMI1640medium
supplemented with 10% FCS, 1% Pen/Strep, 1% L-Glutamine, and 20%
supernatant of M-CSF expressing L-929 cells (Lonza). Of note, the final
calcium concentration in the differentiationmediumwas≈0.89mmol L−1.
Preparation of S100 Proteins: Murine S100A8, S100A9, and S100A9-
N70A proteins were prepared according to the protocol for the human
S100 proteins. Briefly, murine S100A8, S100A9, and S100A9-N70A pro-
teins were cloned in pET1120 vector, expressed in E.coli BL21 bacteria and
purified from inclusion bodies. S100 containing inclusion bodies were pu-
rified and solubilized in 8 m urea in the presence of 10 mm DTT. Renatura-
tion was performed by stepwise increase of the pH from 2 to 4.7 and 7.4
followed by two chromatography runs (Äcta-FPLC, anion exchange, and
size exclusion chromatography). Pure fractions of the S100 proteins were
collected, dialized inHBS, and frozen in aliquots before use. Heterodimers
were prepared by mixing equal amounts of S100A8 and S100A9 or S100A8
and S100A9-N70A in the presence of 1 mm DTT followed by a denatura-
tion step at pH 2. Renaturation was again achieved by stepwise increase
of the pH to 7.4. Due to that, heterodimers were much more stable com-
pared to homodimers; the formation of heterodimers is preferred. Pos-
sible residual contaminating homodimers in the preparations were sep-
arated by anion exchange chromatography runs (Äcta FPLC). Fractions
containing only heterodimers were collected, aliquoted, and frozen before
use. Tetramers were induced by adding calcium (1 mm). The identity of all
preparations was tested by western blot and functional activity measure-
ments and only those batches were used which were entirely free of any
LPS contaminations.[6,7,8,55]
Cell Culture: Cells were stimulated or treated with different in-
hibitors as indicated in the figures. As stimuli, the complement C5a
(20 nm; R&D) as well as purified S100A8/S100A8-homodimer (250 ng
mL−1), S100A8/S100A9-heterodimer (250 ng mL−1), and the mutated
S100A8/S100A9-N70A-heterodimer (250 ng mL−1) were used. For the
inhibition of the Rho GTPases, RhoA, Rac, and Cdc42, the cells were
treated with the specificGEF inhibitors Rhosin hydrochloride (30 μm),W56
(250 μm), and ZCL278 (50 μm) for 30min, respectively (Tocris). Cryptotan-
shinone was used to inhibit the phosphorylation of STAT3 at a concentra-
tion of 5 μm for 30 min (Sigma–Aldrich).
Adhesion Assay: 2 × 105 cells were seeded on 50 μg mL−1 fibronectin
(Sigma–Aldrich) coated 96 well plates and allowed to adhere for 30 min at
37 °C under 5% CO2. The optical density of the adhering cells was mea-
sured at 560 nm wavelength after having fixed the adherent cells with 2%
glutaraldehyde (Roth) for 10 min, stained with 0.5 % crystal violet, and
lysed with 10 % acetic acid (Roth). Quantification was obtained via optical
density of crystal violet stained adherent cells.
Transmigration Assay: A boyden chamber assay was used to investi-
gate the transmigration properties of the cells. The two chambers were
separated by a porous membrane (5 μm pore size, ThermoFisher Scien-
tific). The lower chamber was filled either with medium or medium con-
taining the chemoattractant (complement C5a, 20 nm) and in the upper
chamber, 1× 106 cells were seeded. After six hours at 37 °C under 5%CO2
the transmigrated cells in the lower chamber were harvested and counted
with a cell counter (Schärfe System).
Western Blot: Cells were lysed with a Mammalian Protein Extraction
Reagent lysis buffer (ThermoFisher Scientific) containing a protease
inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Santa
Cruz). Proteins were separated according to the protein size by a 10%
or 15% SDS-PAGE and transferred onto a nitrocellulose membrane
(ThermoFisher Scientific). Membranes were blocked for 1 h at room
temperature in TBST containing 10% milk powder (Roth) followed by
overnight incubation with primary antibodies diluted in TBST containing
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5% bovine serum albumin (Roth) at 4 °C. For detection, horseradish
peroxidase-conjugated secondary antibody (Cell Signaling) diluted in
TBST containing 5% milk powder was used in combination with en-
hanced chemiluminescence (ECL) solution to detect the bands via a
Chemidoc XRS+ (BioRad). For statistical analysis, the intensity of the
bands was measured via ImageJ and the quantification of STAT3 acti-
vation corresponded to the ratio of P-STAT3 on total STAT3 levels after
normalization to their GAPDH bands and the quantification of SOCS3
corresponded to the ratio of SOCS3 levels after normalization to GAPDH
levels.
Immunofluorescence: EGFP-Lifeact ER-Hoxb8 WT and EGFP-Lifeact
ER-Hoxb8 S100A9 KO monocytes were differentiated on coverslips for 3
days and afterward treated as indicated in the figures. Cells were fixed with
4% of PFA for 30 min, permeabilized with 0.1% of TritonX for 10 min, and
incubated overnight with the primary antibody P-STAT3. Cells were washed
and incubated with a secondary antibody conjugated to Cy3 for 30min and
subsequently, the coverslips were mounted on a slide. For statistical anal-
ysis, the intensity of the P-STAT3 signal was measured via ImageJ in the
nucleus and normalized to the signal in the cytoplasm.
Immunoprecipitation: EGFP-Lifeact ER-Hoxb8 WT and EGFP-Lifeact
ER-Hoxb8 S100A9 KO monocytes were treated and analyzed for the GTP-
bound RhoA, Rac, and Cdc42 according to the manufacturer’s protocol
(Cytoskeleton, Inc.). Immunoprecipitated GTPases (GTP-bound) and to-
tal amounts were analyzed by western blot. The intensity of the bands was
quantified using ImageJ and the GTPase activities = relative intensities
shown in the lower bar graphs corresponded to the ratio of GTP bound
GTPase (IP:GTPase) to total GTPase and for total GTPases, a GAPDH blot
was performed as a loading control.
Flow Cytometry: Surface expression of antigens was performed after
washing of cells with PBS/FCS. Cells were incubated for 30 min in 4 °C
with fluorescence-labeled antibodies. After washing, cells were measured
or fixed in 2%PFA at 4 °C for 20min prior to intracellular staining. PFA fixed
cells were permeabilized (BD Bioscience) at 4 °C for 20 min. Intracellular
staining was performed with the desired antibodies at 4 °C for 30 min.
After washing, cells were measured on a Navios flow cytometer (Beckman
Coulter). Flow cytometer and data were analyzed using FlowJo software
(Tree Star).
ELISA: Concentrations of S100A8/S100A9 in culture medium were
quantified by a sandwich ELISA system established in our laboratory as
described previously.[58]
Quartz Crystal Microbalance Dissipation (QCM-D): 1,2-dioleoyl-sn-
glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[(N-(5-
amino-1- carboxypentyl)iminodiacetic acid)succinyl] Ni salt (DGS-Ni-
NTA) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL).
All other chemical/solvents were purchased from Merck, Carl Roth,
or AppliChem (Darmstadt, Germany). For the experiments, recombi-
nant mouse CD69 (250 nm; R&D) as well as purified S100A8/S100A9-
heterodimer (1 μg mL−1), and the mutated S100A8/S100A9-N70A-
heterodimer (1 μg mL−1) were used.
Liposome Preparation: To generate small unilamellar vesicles (SUV), the
respective lipids were dissolved in chloroform/methanol (1:1 v/v) and
mixed to obtain the composition DOPC/DGS-Ni-NTA of 96:4. The organic
solvents were then removed under a stream of nitrogen above the lipid
gel–fluid phase transition temperature. Residual traces of the organic sol-
vent were evaporated in high vacuum for 1 h at the same temperature and
lipid films were stored at 4 °C until use. Dry lipid films were resuspended in
citrate buffer [10 mm trisodium citrate (Merck, Darmstadt, Germany) and
150 mm NaCl, pH = 4.6 (at RT)] at 60 °C and kept for 30 min with steps
of vortex mixing every 5 min resulting in the formation of multilamellar
vesicles (MLVs). To obtain SUVs, the MLVs were extruded 21 times using
a polycarbonate membrane with a pore size of 50 nm (Avanti Polar Lipids
Inc., Alabaster, AL).
Supported Lipid Bilayer (SLBs): QCM-DQuartz sensors (QSX 303, 50 nm
SiO2, 4.95 MHz) were cleaned in 2% (w/v) SDS and hydrophilized by a
10-min O2-plasma treatment (Harrick Plasma, Ithaca, NY). Subsequently,
surfaces were rinsed with ultra-pure water and dried in a stream of nitro-
gen. To prepare the supported bilayer membrane on the quartz sensor by
vesicle rupture, SUVs (0.5 mg mL−1, 50 nm) were applied in the QCM-
D loop-flow mode. Following membrane formation, the citrate buffer was
exchanged to a HBS buffer (10 mm HEPES and 150 mm NaCl, pH of 7.4
[at RT], containing CaCl2, EGTA or imidazole, as indicated) prior to the
protein solution.
QCM-D Measurements: Quartz crystal microbalance measurements
with dissipation (QCM-D) were performed on a Q-Sense E4 QCM-D An-
alyzer (Q-Sense, Gothenburg, Sweden) equipped with four temperature-
controlled flow cells in a parallel configuration. Flow cells were connected
to a peristaltic pump (Ismatec IPC, Glattbrugg, Switzerland) employing a
flow rate of 80.4 μLmin−1. Binding analysis was performed at 20 °C inHBS
Buffer supplemented with 250 μm CaCl2. Frequency and dissipation shifts
(7th overtone) of the quartz sensor (QSX 303, 50 nm SiO2, 4.95 MHz)
were monitored and considered for data evaluation. Calculations were car-
ried out using OriginPro v. 9.1 (OriginLab Corp., Northampton, MA).
Titration ELISA: A 10 nm S100A8/S100A9-tetramer concentration
(HBS plus 250 μm CaCl2) was coated to the bottom of a 96-well plate. Af-
ter three washing steps, unspecific binding sites were blocked by 0.25%
BSA/HBS plus 250 μm CaCl2. Afterward, His-tagged-CD69 (R&D) was
added at increasing concentrations as indicated in the figure. After 1 h,
unbound CD69 was removed by three washing steps and an anti-His-
antibody (10 μg mL−1, Thermo Fisher) was added in excess to quantify
bound CD69. The plate was again washed for three times followed by
the addition of a secondary horseradish peroxidase (HRP) coupled anti-
body (Cell Signaling). Afterward, a peroxidase substrate solution (TMB;
GE Healthcare) was dispensed to the plate and after 15 min of incubation,
the stop solution H2SO4 (2N) was added to each well. The absorbance at
540 nm was measured using an ELISA reader (Anthos). A non-linear re-
gression analysis was performed to calculate the binding constant of CD69
to S100-tetramer assuming a 1:1 binding model of one S100A8/S100A9-
tetramer binding one CD69-dimer.[26]
2D Migration Assay: EGFP-Lifeact ER-Hoxb8 monocytes were seeded
into a narrow channel of a fibronectin coated μ-slide chemotaxis chamber
(Ibidi) which is connected with two reservoirs. After 30 min, the chemo-
taxis chamber was filled with RPMI 1640 (bicarbonate) medium plus 10%
FCS and 1% Pen/Strep. The reservoirs were filled with medium contain-
ing 0.003% Patent Blue V (Sigma–Aldrich) (blue dye) either with or with-
out complement C5a (20 nm). The observation area was imaged with
an inverted Axio Observer.Z1 epifluorescence microscope (Zeiss) with
10×/0.25 objective. Images were acquired every minute for up to 6 h and
analyzed with ImageJ (National Institutes of Health) using amanual track-
ing plugin and the chemotaxis and migration tool from Ibidi.
Spinning Disk Microscopy: EGFP-Lifeact ER-Hoxb8 cells were seeded
into a narrow channel of a fibronectin coated μ-slide chamber (Ibidi) for
30 min. ER-Hoxb8 WT, S100A9 knockout, TLR4 knockout, RAGE knockout
and CD69 knockoutmonocytes were seeded on coverslips, fixed (4%PFA),
and labeled with Alexa Fluor 488-phalloidin (ThermoFisher Scientific). Im-
ages were taken with an inverted Nikon Eclipse Ti-e equipped with a Spin-
ning Disk module (Yokogawa, W1). The circularity and perimeter of the
cells were determined by ImageJ.
For the calcium measurements, EGFP-Lifeact ER-Hoxb8 cells were
seeded into a narrow channel of a fibronectin coated μ-slide chamber
(Ibidi) to let adhere for 30 min. The calcium dye Cal590AM (Biomol) was
dissolved in Pluronic F-127/DMSO (Invitrogen). 4 μL of this solution was
mixed with 4mL RPMI 1640 (bicarbonate) and supplemented with 0.5mm
Probenecid (Santa Cruz). The reservoirs connected to the channel were
filled with the dye mix and after 20 min at 37 °C under 5% CO2, cells were
washed with medium and imaged. Time-lapse imaging was performed
using an UltraVIEW Vox 3D live cell imaging system (Perkin Elmer) cou-
pled to a Nikon Eclipse Ti inverted microscope. The system incorporated
a Yokogawa (Japan) CSU-X1 spinning disk scanner, a Hamamatsu (Japan)
C9100-50 EM-CCD camera (1000 × 1000 pixels), and Volocity software.
Cells were imaged every 10 s for up to 6 min and after the first minute
of imaging complement C5a (20 nm) was added. The temperature was
maintained at 37 °C using an Okolab all-in-one stage incubator (Okolab,
Ottavianio) and images were taken via a Nikon 60x/1.49 oil immersion
objective. The Ca2+ intensity signal was measured over time via ImageJ.
Traction Force Microscopy (TFM): Glass bottom dishes were pre-
treated with 200 μL APTMS washed and incubated for 30 min with 0.5%
Adv. Sci. 2022, 9, 2201505 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH2201505 (13 of 15)
www.advancedsciencenews.com www.advancedscience.com
glutaraldehyde. A pre-mix of 500 μL acrylamide plus 4 μL acrylic acid
and 250 μL bisacrylamide was prepared. To 75 μL of the pre-mix, 415 μL
65% PBS, 10 μL polystyrene beads (100 nm; Micromod), 1.5 μL TEMED
(Sigma–Aldrich), and 5 μL APS (Roth) were added. 5 μL of this gel mix was
pipetted onto the glass bottom dish, covered with a coverslip. After solidi-
fication, the gel was covered with 65% PBS and the coverslip was removed.
100 μL of 50 μg EDC (Roth) dissolved in 1 mL NHS/NaCl stock solution
was pipetted on top of the gel for 15 min. After washing the gels with PBS,
they were incubated with 50 μg mL−1 fibronectin (Sigma–Aldrich) for 1
h at 37 °C under 5% CO2. Day 3 differentiated cells treated as indicated
in the figures were seeded on the gels and after 30 min, the gels were
flushed with RPMI 1640 (bicarbonate) and imaged. TFM was performed
at 37 °C with an inverted Nikon Eclipse Ti-e microscope equipped with
a spinning disk unit (Yokogawa, W1), 60×/1.2 objective and 488 nm and
561 nm lasers. A z-stack of 5 μm (plane distance 0.33 μm) was taken every
5 s for 15 min, employing the perfect focus system (Nikon). After 15 min,
10% SDS was added and a null force image was taken. The traction force
analysis was performed with a custom written MATLAB program.[20]
Cutaneous Granuloma Model (CG): The cutaneous granuloma model
was performed as previously described.[50] Briefly, mice where shaved
and 200 μL of Biogel (P-100, Bio Rad, Hercules, USA) was injected as a
“plug” subcutaneously at both flanks of the mice. Both biogels contained
1 μg/200 μL lipopolysaccharide (LPS from Salmonella enterica serotype
enteritidis, Sigma–Aldrich, Munich, Germany) as inflammatory trigger.
100 μg/200 μL S100A8/S100A9 tetramers were added only to the right plug
(seen from the dorsal surface). FRI measurements were performed at day
one after injection of DID labeled ER-HoxB8 cells at baseline: 3, 6, and
24 h.
Eliciting of Irritant Contact Dermatitis (ICD): ICD was induced by ap-
plication of 3% croton oil in olive oil-acetone (1:4) to the dorsal surface
of both ears of mice (7 per group). S100A9 deficient mice were chosen
for these experiments which allowed to control potentially confounding
effects of endogenously released S100A8/S100A9. S100A9ko mice were
divided into two groups and prior to induction, one group received addi-
tional 100 μg of S100A8/S100A9 tetramers intravenously. Prior to t = 0 h
and 4 h after ICD induction, CT scans were performed to evaluate the ear
volumes.
Labeling: ER-HoxB8 cells differentiated for 3 days toward monocytes
were harvested and 1 × 106 cells per mL were labeled with 5 μmol/l 1,1-
Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DID)/EtOH (Ther-
moFisher Scientific, Waltham, USA) for 5 min and washed three times.
Labeling efficiency was checked by flow cytometry, and viability controlled
by trypan blue or 7AAD staining. For in vivo FRI experiments, 5 × 106 la-
beled cells/200 μL PBS were injected (i.v.) into the lateral tail vein.[50]
FRI Measurements: In vivo imaging of labeled ER-HoxB8 cells by FRI
in the CG model was carried out using the IVIS Spectrum system (epi-
illumination) by PerkinElmer, Waltham, USA (605 nm excitation/680 nm
emission for DID). Identical illumination settings (exposure time: auto-
matic, lamp voltage = 21 V, f-stop = 2, field of view = B [6.6 cm], and bin-
ning = 2) were used for all experiments. Mice were kept under anesthesia
(DRÄGER Isofluran Vapor, Lübeck, Germany) and kept warm during the
imaging process. Fluorescence images were analyzed by using in-house
softwareMEDgical. Fluorescence emission wasmeasured by fluorescence
emission radiance per incident excitation irradiance (radiant efficiency) in
p/s/cm2/sr/μW/cm2. Grayscale photoimages and fluorescence color im-
ages were overlaid.
Computed Tomography (CT) Scanning: Animals were anesthetized
with isoflurane and placed on a heat controlled multimodal scanning bed
and transferred to the computed tomography scanner (Inveon, Siemens
Medical Solutions, USA) and a medium resolution (25 μm) computed
tomography acquisition was performed for each mouse. Computed to-
mography was reconstructed into a volume data set with a voxel size of
0.09×0.09×0.09mm3. An inhouse-developed softwareMEDgical was used
to segment and quantify the volumes of the outer ear.
Quantification and Statistical Analysis: Statistical analysis was per-
formed with Graph Pad Prism Software using the appropriate tests ac-
cording to normal or non-normal data distribution: unpaired t-test (Fig-
ures 1e, 4a, and 5b), Mann-Whitney U test (Figures 1d and 2k), and
Wilcoxon Signed Rank Test. Three independent experiments were per-
formed using three mice per experiment each (Figure 5a) and One-way
ANOVA with Bonferroni post-test (for all other figures). P values were de-
rived from one-way ANOVA with Bonferroni post-test analysis unless oth-
erwise noted. Error bars denote the SD. Statistical significance was defined
as p < 0.05. All quantitative data shown using representative data were re-
peated in at least three independent replications.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
The authors thank Heike Hater, Heike Berheide, Andrea Stadtbäumer, and
Ina Halbig for technical help. The authors also thank Nina Knubel for the
graphical abstract. A.R. is a fellowship holder and A.L.L.M. is a member
of CiM-IMPRS, the joint graduate school of the Cells in Motion Interfac-
ulty Centre, University of Münster, Germany, and of the International Max
Planck Research School –Molecular Biomedicine,Münster, Germany. This
work was funded by grants of the Interdisciplinary Center of Clinical Re-
search at the University of Münster (Vo2/011/19, Ro2/023/19; T.V. and
J.R.), the German Research Foundation CRC 1009 B8, B9 and Z2 (T.V. and
J.R.), CRC TRR332 B5 and C7 (T.V. and J.R.), CRU 342 P3 and P5 (T.V. and
J.R.) and RO 1190/14-1 (J.R.).
Open access funding enabled and organized by Projekt DEAL.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
Designed and performed experiments, analyzed results, and wrote the
manuscript: A.R. Performed experiments and provided inputs on the
manuscript: H.S.,M.B., A.L.L.M., D.G.,M.v.W., L.F.-R., P.J.H., and S.H. Pro-
vided access to the Hoxb8 vector, analyzed data, and provided inputs on
themanuscript: H.H. Provided EGFP-LifeactWT and EGFP-Lifeact S100A9
knockout mice and inputs on the manuscript: M.S. Provided access to the
CD69 knockout mice and input on themanuscript: F.S.-M. Performed revi-
sion experiments: K.S., J.R., M.R., and L.K. Designed and supervised exper-
iments, discussed data, and provided input on the manuscript: V.G. and
T.B. Conceived and supervised the study, designed experiments, discussed
data, and wrote the manuscript: T.V. and J.R.
Data Availability Statement
The data that support the findings of this study are available from the cor-
responding author upon reasonable request.
Keywords
alarmin, calprotectin, CD69, MRP8/MRP14, migration, S100A8/S100A9
tetramer
Received: March 15, 2022
Revised: September 30, 2022
Published online: October 30, 2022
Adv. Sci. 2022, 9, 2201505 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH2201505 (14 of 15)
www.advancedsciencenews.com www.advancedscience.com
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