Article

Tumor-derived macrophage migration inhibitory factor modulates the biology of head and neck cancer cells via neutrophil activation

Department of Otorhinolaryngology, University of Duisburg-Essen, Essen, Germany.
International Journal of Cancer (Impact Factor: 5.09). 08/2011; 129(4):859-69. DOI: 10.1002/ijc.25991
Source: PubMed
ABSTRACT
Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor development and progression are not understood. The objectives of our study were to assess the effects of tumor-derived MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro, MIF modulated functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)-dependent chemotaxis, enhancing neutrophil survival and promoting release of chemokine C-C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9). Further, neutrophils activated with tumor-derived MIF enhanced migratory properties of HNC cells. In conclusion, our data indicate that the effects of tumor-derived MIF on neutrophils represent an additional mechanism by which MIF might contribute to tumor progression.

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Available from: Agnes Bankfalvi, Dec 08, 2014
Tumor-derived macrophage migration inhibitory factor
modulates the biology of head and neck cancer cells via
neutrophil activation
Claudia A. Dumitru
1
, Hossein Gholaman
1
, Sokratis Trellakis
1
, Kirsten Bruderek
1
, Nina Dominas
1
, Xiang Gu
1
,
Agnes Bankfalvi
2
, Theresa L. Whiteside
3
, Stephan Lang
1
*
and Sven Brandau
1
*
1
Department of Otorhinolaryngology, University of Duisburg-Essen, Essen, Germany
2
Department of Pathology and Neuropathology, University of Duisburg-Essen, Essen, Germany
3
University of Pittsburgh Cancer Institute, Pittsburgh, PA
Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the
aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor
development and progression are not understood. The objectives of our study were to assess the effects of tumor-derived
MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we
used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed
expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results
show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the
tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro, MIF modulated
functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)-dependent chemotaxis, enhancing
neutrophil survival and promoting release of chemokine C-C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9).
Further, neutrophils activated with tumor-derived MIF enhanced migratory properties of HNC cells. In conclusion, our data
indicate that the effects of tumor-derived MIF on neutrophils represent an additional mechanism by which MIF might
contribute to tumor progression.
Many solid tumors display inflammatory growth throughout
their progression.
1,2
During this process, regulatory T cells,
myeloid-derived suppressor cells, tumor-associated macro-
phages and tumor-infiltrating lymphocytes are either induced
or ‘‘educated’’ by the tumor, acquiring tumor-promoting
activities. Much less is known about the role of tumor-infil-
trating polymorphonuclear neutrophils, although neutrophils
are the most abundant cell type in the peripheral blood and
many human tumors are infiltrated by these cells.
3
As neu-
trophils release a variety of immunomodulatory mediators it
is likely that intense reciprocal interactions exist between the
tumor and tumor-infiltrating neutrophils.
4
The impact of neutrophils on tumor growth is character-
ized by a striking dichotomy, and previous studies showed
that neutrophils may have both antitumor
5–7
and protumor
8–11
activities, respectively. Recent studies from our group indicated
a potential role of neutrophils in head and neck squamous cell
carcinoma (HNSCC).
12
In vitro, we demonstrated that HNSCC
is able to alter the biology of neutrophils by promoting their
motility, survival and proinflammatory properties. In vivo,
HNSC C patients exhibited considerable systemic changes,
such as increase d neutrophil-to-lymphocyte ratios and high
serum concentrations of CXCL8, chemokine C-C motif
liga nd 4 (CCL4) and CCL5. M ost importantly, we found
that a strong neutrophilic infiltration of the tumor tissue was
associated with poorer survival in HNSCC patients with
advanced diseas e. Taken together, these findings su ggest that
neutrophils may be important mediators in the pathophysi-
ology of HNSCC.
The exact cellular and molecular mechanisms responsible
for modulation of host immune functions during
Key words: macrophage migration inhibitory factor, neutrophils, head
and neck cancer, cancer-related inflammation
Abbreviations: CL: Chemokine C-C Motif Ligand; CXCR:
chemokine CXC motif receptor; HNC: head and neck cancer; IL:
interleukin; ISO-1: (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-
isoxazole acetic acid methyl ester; MIF: macrophage migration
inhibitory factor; MMP: matrix metalloprotease
Additional Supporting Information may be found in the online
version of this article.
*S.L. and S.B. contributed equally to this work.
Grant sponsor: Rudolf Bartling Stiftung (S.L.)
DOI: 10.1002/ijc.25991
History: Received 24 Aug 2010; Accepted 27 Jan 2011; Online 15
Feb 2011
Correspondence to: Sven Brandau, Research Division, Department
of Otorhinolaryngology, University of Duisburg-Essen,
Hufelandstraße 55, 45122 Essen, Germany, Tel.: þ49-201-723-3193,
Fax: þ49-201-723-5196, E-mail: sven.brandau@uk-essen.de
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tumorigenesis and tumor progression in HNSCC and other
types of cancer have been only partially clarified. Selected
soluble inflammatory mediators, such as cytokines, chemo-
kines and metabolites of the arachidonic acid pathway, have
been found to change the function and differentiation of
immune cells.
1
Among these molecules, macrophage migra-
tion inhibitory factor (MIF) is emerging as an important reg-
ulator of inflammation in cancer.
13
MIF was originally identi-
fied as an inhibitor of random macrophage migration in
vitro
14
and is best known for its role in microbial sepsis.
15
MIF is an unusual cytokine, because it seems to have isomer-
ase and oxidoreductase enzymatic activities.
16,17
Subsequent
work identified CD74 as the cellular surface receptor for
MIF.
18
In a murine model of atherosclerosis, MIF was
recently shown to also interact with chemokine CXC motif
receptor 2 (CXCR2), a chemokine receptor expressed primar-
ily on neutrophils and monocytes.
19
A possible role of MIF in the modulation of myeloid
immune cells in cancer has not been investigated thus far. Our
study evaluates the effects of MIF on neutrophil functions in
head and neck cancer (HNC). We demonstrate that HNC cells
express and release MIF both in vitro and in vivo. Our results
show that MIF triggers neutrophil recruitment via CXCR2,
prolongs survival and upregulates the inflammatory activity of
neutrophils. Furthermore, tumor-derived MIF promotes the
migration of HNC cells via neutrophil-released factors. An
association between intratumoral MIF expression and neutro-
philic infiltration as well as major clinical parameters such as
lymph node metastasis and survival emphasizes the in vivo
role of MIF-mediated events in progression of HNC.
Material and Methods
Cell lines and HNC supernatants
The human hypopharyngeal carcinoma cell line FaDu was
obtained from the American Type Culture Collection
(ATCC, Manassas, VA). The cells were cultured and main-
tained in RPMI-1640 (Invitrogen, Karlsruhe, Germany) sup-
plemented with 10% fetal calf serum (Biochrom, Berlin, Ger-
many), 100 units/mL penicillin and 100 l g/mL streptomycin
(PAA-Laboratories GmbH, Coelbe, Germany). We followed
the guidelines provided by ATCC Technical Bulletin No. 8,
2007 to assess the quality and identity of our cell line. To
obtain FaDu supernatants (FaDu SN), we incubated 2 10
6
cells/mL for 24 hr at 37
C in RPMI-1640 supplemented as
above. The resulting supernatant was centrifuged to remove
cellular debris and stored at 20
C.
To reduce the levels of MIF in FaDu SN, FaDu cells were
transfected with 1 nM validated MIF siRNA (Qiagen, Hilden,
Germany) for 48 hr according to the manufacturer’s instruc-
tions. In parallel, FaDu cells were transfecte d with 1 nM All-
Stars Negative Control siRNA (Qiagen). Supernatants were
collected daily after transfection, and MIF levels were assessed
by ELISA (see below). Supernatants with approximately 70%
reduced MIF expression were used to stimulate neutrophils.
Study subjects
Neutrophils were isolated from the peripheral blood of
healthy volunteers. For double immunofluorescence analysis
of MIF and CD66b, tissues from 25 HNC patients were used.
For analysis of MIF expression by immunohistochemistry
and correlation with clinical parameters, tissue samples were
collected from 91 patients with HNSCC of the oropharynx
and hypopharynx. MIF expression was evaluated and scored
by two independent investigators. The patients were treated
at the Department of Otorhinolaryngology (University of
Duisburg-Essen) between 1995 and 2000 and clinical follow-
up was retrieved. Patient characteristics are shown in Sup-
porting Information Table S1. All experiments were approved
by the local ethics committee, and informed written consent
was obtained from each individual.
Isolation and culture of neutrophils
Diluted blood (1:1, v/v in phosphate buffered saline [PBS])
was subjected to density gradient centrifugation using LSM
1077-lymphocyte separation medium (PAA-Laboratories
GmbH). The mononuclear cell fraction was discarded, and
the neutrophil fraction was collected in a fresh test tube.
Erythrocytes were removed by sedimentation with a solution
containing 1% polyvinyl alcohol and, subsequently, by lysis
with prewarmed Aqua Braun (B. Braun, Melsungen, Ger-
many). The resulting neutrophils were cultured in RPMI-
1640 supplemented as above. The purity of the neutrophil
population after isolation was >98% and the remaining con-
taminating cells were lymphocytes (Supporting Information
Fig. S1).
Antibodies, stimuli and inhibitors
Polyclonal rabbit anti-human MIF and rabbit IgG antibodies
were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Monoclonal mouse anti-human MIF and neutralizing
mouse anti-human CXCR1 and CXCR2 antibodies were from
R&D Systems (Wiesbaden-Nordenstadt, Germany). Mouse
IgG1, mouse IgG2a and biotin-conjugated anti-human CD74
antibodies were from BioLegend (San Diego, CA, USA).
Mouse anti-human CD66b antibodies were obtained from
Immunotech (Marseille Cedex 9, France). All secondary anti-
bodies (Cy3-goat anti-mouse, Fluorescein Isothiocyanate
(FITC)-goat anti-mouse, DyLight 488-donkey anti-rabbit,
DyLight 488-streptavidin) were from Dianova (Hamburg,
Germany). MIF activity inhibitor (S,R)-3-(4-hydroxyphenyl)-
4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) was
obtained from Merck (Darmstadt, Germany). Recombinant
MIF and neutralizing mouse anti-MIF antibodies (NIH-
III.D9) were a kind gift from Dr. J. Bernhagen (University of
Aachen, Germany).
Immunofluorescence analysis of cells and frozen tissues
FaDu cells were fixed and permeabilized using BD Cytofix/
Cytoperm (BD Bioscience s, San Diego, CA). Cells were
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incubated with rabbit anti-MIF or rabbit IgG antibodies and
then with FITC-labeled anti-rabbit antibodies for 1 hr and
for 30 min at room temperature, respectively. For fluores-
cence microscopy, nuclei were visualized by 7-aminoactino-
mycin D (7-AAD) staining according to the protocol pro-
vided by the manufacturer (BD Pharmingen, San Diego, CA).
Cells were analyzed by flow cytometry with a BD FACSCanto
flow cytometer (BD, Heidelberg, Germany) or mounted in
Fluoprep (bioMerieux, Marcy l’Etoile, France) and analyzed
by fluorescence microscopy with a Zeiss Axioscope 2 (Zeiss,
Jena, Germany).
Frozen sections of HNC tissue were fixed with BD Cyto-
fix/Cytoperm and incubated with rabbit anti-MIF and mouse
anti-CD66b antibodies overnight at 4
C. The sections were
then stained with Cy3-goat anti-mouse and DyLight 488-
donkey anti-rabbit for 30 min at room temperature. Nuclei
were visualized by DRAQ5 staining according to the manu-
facturer’s protocol (eBiosciences, San Diego, CA). Samples
were mounted in Fluoprep and analyzed by fluorescence mi-
croscopy with a Zeiss Axioscope 2.
Colorimetric immunohistochemistry
For MIF, immunohistochemical staining was performed with
an automated staining device (Dako Autostainer; DakoCyto-
mation, Hamburg, Germany) using mouse anti-human MIF
or mouse IgG1 antibodies. Secondary and tertiary immunore-
actions were performed with a commercially available anti-
mouse IgG detection kit (En-Vision; DakoCytomation).
Flow cytometric analysis of surface antigens
Cells were incubated with FITC-conjugated anti-CD66b, bio-
tin-conjugated anti-CD74, mouse anti-CXCR1, mouse anti-
CXCR2 or mouse IgG2a antibodies for 30 min at 4
C. Where
necessary, secondary reaction s were performed for 20 min at
4
C. Cells were fixed in a 1.5% paraformaldehyde solution
and analyzed by flow cytometry with a BD FACSCanto II
flow cytometer.
Chemotaxis assay
Directed migration (chemotaxis) of neutrophils was assessed
using 3 lm cell culture inserts together with 24-well compan-
ion plates (Becton Dickinson, Franklin Lakes, NJ) as previ-
ously described .
12
Data are expressed as chem otactic index,
calculated as the number of cells migrating in response to
stimulus divided by the number of cells migrating in
response to culture medium.
Quantification of cytokines/chemokines in culture
supernatants
MIF levels in FaDu SN and levels of CCL4 released by neu-
trophils were analyzed by ELISA (assay sensitivity 10 pg/mL)
according to the protocols provided by the manufacturer
(R&D Systems, Abington, England). A Synergy 2 microplate
reader (BioTek, Bad Friedrichshall, Germany) was used to
determine sample absorbance at 450 nM.
Multiplex screening assays for HNC supernatants were
performed using Human Cytokines Group I 18-plex and
Group II 5-plex (Bio-Rad Life Sciences, Mu¨nchen, Germany),
according to the protocol provided by the manufacturer.
Data were analyzed using the Bio-Plex Manager software ver-
sion 4.1.1 (Bio-Rad Laboratories).
Gelatin zymography
The release of matrix metalloproteases (MMPs) by neutro-
phils was analyzed according to the method of Kleiner and
Stetler-Stevenson.
20
Briefly, 10
6
cells/mL were stimulated as
indicated for 1 hr at 37
C. Supernatants were collected and
incubated with Zymogram sample buffer at a final concentra-
tion of 80 mM Tris pH 6.8, 1% SDS, 4% glycerol and 0.006%
bromphenol blue. Proteins were separated by Sodium Do-
decyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-
PAGE) containing 0.2% gelatin. Samples were renatured in
2.5% Triton-X-100 for 1 hr at room temperature, and the en-
zymatic reaction was allowed to proceed for 16 hr at 37
Cin
a buffer containing 50 mM Tris pH 7.5, 200 mM NaCl, 5
mM CaCl
2
and 1% Triton-X-100. To visualize digested
bands, the gel was incubated with 0.5% Coomassie blue, 30%
methanol and 10% acetic acid for 2 hr at room temperature
followed by multiple destaining steps in a solution containing
30% methanol and 10% acetic acid.
Apoptosis assays
Neutrophils (10
6
cells/mL) were stimulated as indicated and
were stained with phycoerythrin-conjugated Annexin-V and
7-AAD according to the manufacturer’s instructions (BD
Pharmingen). Quantification was performed with a BD
FACSCanto II flow cytometer.
Wound healing (scratch) assays
FaDu cells were seeded in 24-well plates containing 1-mm
wide ‘‘wound’’-forming culture inserts, which were removed
when a confluent monolayer was formed. Cells were stimu-
lated as indicated and fixed/stained with a solution contain-
ing 2% formaldehyde, 30% ethanol, 60 mM NaCl and 0.7%
crystal violet. Wounds were visualized with a Zeiss Axioscope
2 microscope at 25 magnifica tion. Quantification of wound
closure was performed with the ImageJ software.
Collagen adhesion assays
96-well black microplates (FIA black plate, Greiner Bio-One)
were coated with 20 lg/mL collagen VI (Sigma) overnight at
4
C. FaDu cells were dissociated nonenzymatically using
accutase (PAA-Laboratories GmbH) and were labeled with
the fluorescent dye DiIC
12
(3) according to the protocol pro-
vided by the manufacturer (BD Biosciences, MA). FaDu cells
were stimulated as indicated for 2 hr at 37
C and then
seeded on the collagen-coated plates at a concentration of 5
10
4
cells/well. After an incubation period of 30 min at
37
C, plates were washed with PBS and the fluorescence of
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the remaining attached FaDu cells wa s detected with a Syn-
ergy 2 microplate reader.
Proliferation assay
FaDu cells were seeded in 96-well black microplates at a den-
sity of 2.5 10
4
cells/well. Cells were stimulated as indicated
and labeled with DiIC
12
(3) poststimulation. Fluorescence was
detected with a Synergy 2 microplate reader.
Statistical analysis
For the in vitro studies, data are presented as means and stand-
ard deviations (S.D.) and statistical analysis was performed
with the two-tailed paired Student’s t-test. Analysis of tumoral
MIF expression versus the degree of neutrophilic infiltration
was performed by cross-tabulation and v
2
significance testing.
Clinical data were analyzed by nonparametric exact tests (Krus-
kal–Wallis). Correlations were assessed using Spearman’s rank
correlation coefficient. Survival curves were plotted according
to the Kaplan–Meier method and significance was tested using
Cox regression analysis. The level of significance was set at p
0.05. The statistical calculations were performed with SPSS Ver-
sion 16 software (SPSS, Chicago, IL).
Results
We have recently demonstrated that HNC cells activate neu-
trophils in vitro and that high neutrophilic infiltration of tu-
mor tissue correlates with poor survival in HNC patients.
12
Here, we investigate the tumor-derived factors that mediat e
neutrophil activation and the potential feedback effect of neu-
trophil activation on the biology of tumor cells.
Cytokine/chemokine content of HNC cell supernatants
We initially performed multiplex screening arrays to deter-
mine the identity of soluble factors present in HNC cell SN
that might be responsible for neutrophil activation. Among
the 22 cytokines/chemokines tested, interleukin (IL)-6, IL-8
and MIF were the most abundant (Supporting Information
Table S2). While the role of IL-6 and IL-8 in carcinoma–neu-
trophil interactions has been partially clarified,
12,21,22
almost
nothing is known about the role of tumor-derived MIF in
this process. Therefore, we next evaluated MIF expression in
HNC cells. MIF was found to be strongly expressed in FaDu
cells, as indicated by fluorescence microscopy (Fig. 1a) and
flow cytometry (Fig. 1b). Furthermore, FaDu cells released
MIF already after 6 hr of culture, and MIF concentrations
increased to approximately 2000 pg/mL after 24 hr of culture
Figure 1. HNC cells express and release MIF in vitro.(a) FaDu cells were grown on glass coverslips overnight. After fixation and
permeabilization, cells were stained with FITC-coupled anti-MIF antibodies or isotype control. Nuclei were counterstained with 7-AAD, and
the samples were analyzed by fluorescence microscopy. The results are representative for two independent experiments. (b) FaDu cells
were fixed, permeabilized and stained as described in (a). The flow cytometry results are representative for three independent experiments.
(c) FaDu cells were cultured for up to 24 hr, and SNs were collected over time. MIF levels were determined by ELISA. Data are means 6
S.D. of three independent experiments.
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(Fig. 1c). As significant levels of MIF were detected in 24 hr
FaDu SN, all further studies were performed using these SN.
Inhibition of tumor-derived MIF
To investigate whether the in vitro effects of HNC cell SN on
neutrophils
12
were mediated by MIF, we stimulated neutro-
phils with FaDu SN in the presence of ISO-1, a MIF specific
inhibitor
23
or Dimethylsulfoxide (DMSO) control. Initial
titrations were performe d to determine the nontoxic concen-
tration of ISO-1 and DMSO on neutrophils and to exclude a
potential effect of this inhibitor on the basal responses of
neutrophils (data not shown). The results showed that the in-
hibition of HNC-derived MIF downregula ted chemotaxis
(Fig. 2a) and enhanced apoptosis of neutrophils (Fig. 2b).
Furthermore, the release of proinflammatory factors (CCL4
and MMP9) by neutrophils stimulated with FaDu SN was
significantly downregulated in the presence of ISO-1 (Figs. 2c
and 2d). FaDu SN alone contained no CCL4 or MMP9 (data
not shown), which demonstrates that these proinflammatory
factors are released by neutrophils on activation. To confirm
these findings, we stimulated neutrophils with supernatants
obtained from MIF siRNA-transfected FaDu cells or with
FaDu SN containing MIF neutralizing antibodies. The results
showed that inhibition of tumor-derived MIF by siRNA or
neutralizing antibodies significantly downregulated chemo-
taxis (Figs. 3a and 3b) and release of CCL4 by neutrophils
(Figs. 3c and 3d), in a manner comparable to ISO-1. These
data strongly indicate that MIF produced by the tumor is re-
sponsible for modulation of neutrophil functions.
Receptors mediating neutrophil recruitment by MIF
To elucidate the re ceptors involved in neutrophil recruitment
by MIF, we first determined the expression of CD74 and
CXCR2 on neutrophils, as these receptors were shown to be
involved in MIF-induced chemotaxis.
19
The results indicate
that neutrophils do not express CD74 (Fig. 4a) but, as
expected, have a high expression of CXCR2 (Fig. 4b). Next,
we allowed the neutrophils to migrate toward recombinant
MIF in the presence or absence of neutralizing antibodies
against CXCR2 or CXCR1. Initial titration of these antibodies
indicated that 1 lg/mL anti-CXCR1 and 5 lg/mL anti-
CXCR2 are required to maximally bind and thus, block the
respective receptors on neutrophils (data not shown). The
results demonstrate that MIF-induced neutrophil recruitment
was downregulated upon inhibition of CXCR2 (Fig. 4c). In
contrast, neutralizing antibodies against CXCR1 had no effect
Figure 2. HNC-derived MIF modulates neutrophil chemotaxis, survival and release of proinflammatory factors. (a) Neutrophils were allowed
to migrate toward FaDu SN (with or without ISO-1) in a transwell culture system. Migrated cells were counted after 3 hr, and the
chemotactic index was determined. Data are means 6 S.D. of three independent experiments. (b) Neutrophils were stimulated with FaDu
SN (with or without ISO-1) for 24 hr at 37
C. Cells were stained with PE-conjugated annexin-V and 7-AAD and analyzed by flow cytometry.
Data are means 6 S.D. of four independent experiments. (c) Neutrophils were stimulated with FaDu SN (with or without ISO-1) for 24 hr at
37
C. Neutrophil SNs were collected, and the concentration of CCL4 was determined by ELISA. CCL4 levels in the FaDu SN-stimulated
samples were set at 100%. Data are means 6 S.D. of four independent experiments. (d). Neutrophils were stimulated with FaDu
supernatants (with or without ISO-1) for 1 hr at 37
C. Neutrophil SN was collected, and MMP9 levels were determined by gelatine
zymography. The results of three independent experiments are shown.
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on MIF-induced neutrophil recruitment (Fig. 4c). These
results are in accordance with previous findings
19
and con-
firm that the main chemotactic receptor for MIF in neutro-
phils is CXCR2.
Feedback effects of neutrophils exposed to
tumor-derived MIF
In the next part of the study, we considered the possibility that
neutrophils stimulated by tumor-derived MIF produced factors
which exerted feedback effects on tumor cells. To address this
hypothesis, we used the experimental design outlined in Figure
5a. Neutrophils were stimulated with supernatant from FaDu
cells in the presence or absence of MIF inhibitor ISO-1. After
24 hr of neutrophil activation (priming) the supernatant of
those ‘‘FaDu-primed’’ neutrophils was used to stimulate FaDu
cells. Wound healing (scratch assay), adhesion to collagen and
proliferation of FaDu tumor cells were determined.
Interestingly, the closure of the scratch was augmented,
when the assay was performed in the presence of the super-
natant obtained from ‘‘FaDu-primed’’ neutrophils (Fig. 5b).
When the supernatant of ‘‘FaDu-primed’’ neutrophils was
generated under conditions of MIF inhibition, this effect was
abolished (Fig. 5b). Similar effects were observed with respect
to collagen adhesion of the tumor cells. Adhesion of tumor
cells was enhanced in the presence of the supernatant
obtained from ‘‘FaDu-primed’’ neutrophils, and again this
effect was abolished upon inhibition of MIF (Fig. 5c). In con-
trast, proliferation of HNC cells did not seem to be affected
by HNC-induced neutrophil factors (Fig. 5d). These experi-
ments indicated that HNC-derived MIF is responsible for the
Figure 3. Inhibition of HNC-derived MIF by siRNA or neutralizing antibodies blocks neutrophil chemotaxis and release of CCL4. (a)
Neutrophils were allowed to migrate toward supernatants obtained from FaDu cells transfected with 1 nM MIF siRNA or negative control
siRNA (mock) in a transwell culture system. Migrated neutrophils were counted after 3 hr, and the chemotactic index was determined. Data
are means 6 S.D. of three independent experiments. (b) Neutrophils were allowed to migrate toward FaDu SN in the presence of 10 lg/mL
anti-MIF neutralizing antibodies (NIH-III.D9) or isotype control. Migrated cells were counted after 3 hr, and the chemotactic index was
determined. Data are means 6 S.D. of three independent experiments. (c) Neutrophils were stimulated with supernatants obtained from
FaDu cells transfected with 1 nM MIF siRNA or negative control siRNA (mock) for 24 hr at 37
C. Neutrophil SN was collected, and the
concentration of CCL4 was determined by ELISA. CCL4 levels in neutrophil cultures stimulated with mock siRNA FaDu SN were set at 100%.
Data are means 6 S.D. of three independent experiments. (d) Neutrophils were stimulated with FaDu SN in the presence of 10 lg/mL anti-
MIF neutralizing antibodies (NIH-III.D9) or isotype control antibodies for 24 hr at 37
C. Neutrophil SN was collected, and the concentration
of CCL4 was determined by ELISA. CCL4 levels in the neutrophil cultures stimulated with FaDu SN containing isotype control antibodies
were set at 100%. Data are means 6 S.D. of three independent experiments.
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regulation of neutrophil functions which, in turn, modulate
functions of the tumor cells.
MIF expression in the tumor and clinical outcome
of HNC patients
Previous studies demonstrated that high numbers of tumor-
associated neutrophils correlate with poor clinical outcome in
HNC patients.
12
Here, we performed exploratory studies to
determine whether the degree of neutrophilic infiltration
might correlate with the expression of MIF by the tumor cells.
To this end, frozen sections from 25 HNC tissues were cos-
tained against MIF and CD66b (neutrophil marker). The
expression of MIF and CD66b was independently scored as
‘‘weak’’, ‘‘medium’’ and ‘‘strong’’ (examples are shown in Fig.
6a) and plotted for each patient in a graph (Fig. 6b). Cross-
tabulation analysis indicated that the numbers of CD66b-posi-
tive cells significantly correlated (p ¼ 0.002, v
2
) with the in-
tensity of MIF expression in the tumor cells (Fig. 6b). Thus,
our data suggest that there is an association between HNC-
derived MIF and neutrophils in vivo. To elucidate the poten-
tial role of tumor-derived MIF–neutrophil interactions in
HNC pathophysiology, MIF expression was immunohisto-
chemically determined in a cohort of 91 patients with HNC.
MIF expression levels were evaluated and correlated with dif-
ferent clinical and pathologic al parameters. Figure 6c shows
examples of ‘‘weak’’ (32 patients), ‘‘medium’’ (31 patients) and
‘‘strong’’ (28 patients) expression of MIF in the tumors.
Because MIF was present at substantial levels even in the ‘‘me-
dium’’ group, we combined the patients with ‘‘medium’’ and
‘‘strong’’ MIF into one group. Statistical analysis (Kruskal–
Wallis and Spearman’s rank) showed that MIF expression in
the tumor significantly correlated with lymph node metastasis
(N): p ¼ 0.020 (for Kruskal–Wallis), and p ¼ 0.045, Rho ¼
0.211 (for Spearman), respectively. As shown in Figure 6d, the
percentage of patients with medium/strong MIF expression
increased with higher N-stage, while the percentage of patients
with weak MIF expression decreased or was absent in the N3
group (Fig. 6 d). Furthermore, Cox regression analysis
demonstrates that MIF expression also associates with 5-year
overall survival, as patients with tumors exhibiting medium/
strong MIF express ion had significantly decreased survival
Figure 4. MIF induces neutrophil recruitment via CXCR2. (a) Flow cytometric analysis of neutrophils (upper panel) or monocytes (lower
panel) stained with biotin-coupled anti-human CD74 antibodies. The results indicate that, in contrast to monocytes, neutrophils do not
express CD74. The results are representative for three independent experiments. (b) Flow cytometric analysis of neutrophils stained with
anti-CXCR1, anti-CXCR2 or isotype control antibodies. The results show that neutrophils highly express CXCR2 and CXCR1 on the cell
surface. The results are representative for two independent experiments. (c) Neutrophils were incubated with neutralizing anti-CXCR2, anti-
CXCR1 or isotype control antibodies and allowed to migrate toward 10 ng/mL of recombinant MIF in a transwell system. Migrated cells
were counted after 3 hr, and the chemotactic index was determined. Data are means 6 S.D. of five independent experiments.
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rates (p ¼ 0.013) compared to the patients with tumors
expressing weak MIF levels (Fig. 6e).
Discussion
Tumors are known to produce cytokines that can regulate
the functions of immune cells found in situ as well as in the
systemic circulation of patients.
24
Here we present evidence
that HNC-derived MIF mo dulates crucial functions of neu-
trophils, which respond to MIF stimulation with increased
CXCR2-dependent chemotaxis, prolonged survival and
release of proinflammatory factors. Furthermore, neutrophils
stimulated by tumor-derived MIF appear to increase the
Figure 5. MIF-induced neutrophil factors modulate the migratory properties of HNC cells. (a) Neutrophils were incubated with FaDu SN in
the presence or absence of ISO-1 (50 lM). The resulting supernatants (FaDu/neutrophil SN) were used to stimulate FaDu cells and
‘‘wound’’-healing, adhesion to collagen and proliferation were assessed as below. (b) FaDu cells were grown to confluence in the presence
of ‘‘wound’’-forming culture inserts. Stimulation was performed as indicated in (a). Cells were fixed and stained with a solution containing
crystal violet. ‘‘Wounds’’ were visualized by light microscopy at 25-fold magnification. Wound closure was quantified with the ImageJ
software. Data are means 6 S.D. of three independent experiments (left). A representative result is shown on the right-hand side of the
figure. (c) DiIC
12
(3)-labeled FaDu cells were stimulated as indicated in (a) for 2 hr at 37
C. Cells were allowed to adhere on collagen-coated
black microplates for 30 min at 37
C. Fluorescence was quantified for each sample, and adhesion was expressed as percentage of the
control sample. Data are means 6 S.D. of three independent experiments, each performed in triplicate. (d) FaDu cells were stimulated as
indicated in (a) for 36 hr at 37
C. Cells were labeled with the fluorescent dye DiIC
12
(3), and fluorescence was quantified for each sample.
Proliferation was expressed as a percentage of the control sample (FaDu SN only). Data are means 6 S.D. of two independent experiments,
each performed in triplicate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Tumor Immunology
866 MIF modulates neutrophil biology in HNC
Int. J. Cancer: 129, 859–869 (2011)
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migratory properties of tumor cells via a feedback mecha-
nism. In vivo, we observe an association between tumoral
MIF expression and the numbers of tumor-infiltrating neu-
trophils. Importantly, immunohistochemical analysis of HNC
tissues in 91 patients shows that the MIF expression levels in
the primary tumor correlates with the presence of lymph
node metastasis and with poor survival of the patients.
Expression of MIF by the tumor together with the presence
of neutrophils in tumor tissues prompted the question of
whether HNC-derived MIF plays a role in the recruitment of
these cells to the tumor tissue. Although MIF was initially
described as ‘‘macrophage migration inhibitory factor,’’
14
newer evidence has indicated that MIF can exert opposite
effects on cellular motility. For instance, it has been reported
that the migration of lung adenocarcinoma cells is modulated
by MIF.
25
Recently, Bernhagen et al.
19
demonstrated that MIF
promotes the atherogenic recruitment of monocytes and T
cells. Here, we showed that the directed in vitro migration of
neutrophils is upregulated by HNC-derived MIF. The mecha-
nism(s) responsible for MIF-mediated regulation of cellular
Figure 6. Expression of MIF by the tumor is proportional to the degree of neutrophilic infiltration and is associated with lymph node
metastasis and poor survival in HNC patients. (a) Frozen sections of HNC tissues were double stained against MIF (green) and CD66b (red).
DRAQ5 (blue pseudocolor) was used to visualize nuclei. The intensity of MIF staining in tumor tissue and the numbers of CD66b-positive
cells were scored separately. Shown are examples of ‘‘weak,’’ ‘‘medium’’ and ‘‘strong’’ stainings for MIF and CD66b. (b) The intensity of
MIF expression by the tumor and the degree of CD66b-positive cells infiltration were plotted for each patient. Statistical analysis was
performed by cross-tabulation and v
2
testing. The level of significance was set at p 0.05. (c) Paraffin-embedded sections of HNC tissue
were stained with antibodies against MIF. The intensity of MIF staining in tumor tissue was scored as indicated. (d) HNC patients were
divided in groups according to the intensity of MIF expression and the stage of lymph node metastasis (N stage). Association between MIF
expression and N stage was evaluated using Kruskal–Wallis test and Spearman’s rank correlation coefficient. The level of significance was
set at p 0.05. (e) Kaplan–Meier survival curves were plotted for patients with weak versus patients with medium/strong MIF expression
in the tumor. Statistical significance was determined by Cox regression analysis with the level of significance set at p 0.05.
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Int. J. Cancer: 129, 859–869 (2011)
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motility have not yet been fully elucidated. MIF may promote
cellular motility via activation of Rac1 and stabilization of lipid
rafts.
25
It has been recently reported that MIF may also act as a
noncognate ligand for CXC receptors.
19
MIF has been found to
compete with CXCR2 and CXCR4 ligands and to bind directly
to CXCR2. Here, we demonstrate that MIF induces neutrophil
recruitment in a CXCR2-dependent manner. The mechanisms
of MIF-mediated chemotaxis through CXCR2 was proposed to
involve formation of a complex between CXCR2 and CD74
which, subsequently, amplified MIF signaling via activation of
CD44/Src kinases.
19
However, as we clearly show in our study,
neutrophils lack CD74. Therefore, it remains to be determined
whether MIF-induced neutrophil chemotaxis relies solely on
CXCR2 or migh t require additional coreceptors in these cells.
Recently, it has been shown that recombinant MIF can in-
hibit the intrinsic (mitochondria initiated) apoptotic pathway
and delay the spontaneous apoptosis of neutrophils.
26
In our
study, we observed a constant but modest effect of HNC-
derived MIF on neutrophil survival (approximately 15%),
which suggests that other tumor-derived factors are mainly
responsible for the strong anti-apoptotic effect of the HNC
supernatants. For instance, IL-8 and IL-6, both of which are
present in considerable amounts in FaDu SN can prolong the
lifespan of neutrophils.
27,28
Similarly, growth factors such as
epidermal growth factor, fibroblast growth factor or vascular
endothelial growth factor are known to activate the Phospha-
tidylinositol-3-Kinase (PI3K) pathway,
29
consequently inhibi-
ting apoptosis. Therefore, it is plausible that because of the
presence of other prosurvival mediators in tumor-conditioned
medium, the inhibition of MIF alone would not result in dra-
matic effects on neutrophil apoptosis.
MIF is best known for its proinflammatory functions.
Numerous studies, particularly on infectious diseases, have
provided evidence that MIF directly or indirectly promotes
the production or expression of proinflammatory molecules,
such as tumor necrosis factor, interferon gamma, IL-6 and
IL-8, nitric oxide or products of the arachidonic acid path-
way.
30
Our study demonstrates that HNC-derived MIF elicits
increased inflammatory responses from neutrophils by induc-
ing release of CCL4 and MMP9. Although the significance of
CCL4 production for the tumor environment is not com-
pletely elucidated, this chemokine could play a protumoral
role, because the expression of CCL4 has been found to be
increased in gastric and colon cancer
31,32
and has been linked
to the biochemical recurrence of prostate cancer.
33
In con-
trast to CCL4, the role of MMPs in cancer has been exten-
sively investigated. Many studies demonstrated that MMPs,
in particular MMP9, are involved at several stages of cancer
progression including neoangiogenesis and migration/metas-
tasis of the tumor cells.
34,35
An association between tumor
angiogenesis and neutrophils has been previously suggested.
For instance, an influx of neutrophils was shown to foster tu-
mor angiogenesis, whereas their depletion reduced angiogene-
sis in various animal tumor models.
36,37
In hepatocellular
carcinoma, tumo r-associated neutrophils were shown to be a
major source of MMP9 and were positively correlated with
angiogenesis progression.
38
In our study, we address the
interaction between neutrophils and tumor cells and demon-
strate that upon stimulation with tumor-derived MIF, neutro-
phils release factors which promote cell-matrix adhesion and
migration of HNC cells. Interestingly, we also find a signi fi-
cant association between the levels of tumor-derived MIF
and lymph node metastasis as well as overall survival in
HNC patients. It would be, therefore, tempting to speculate
that the clinical outcome of patients with HNC may be, at
least partially, a consequence of altered neutrophil functions
in response to tumor-derived MIF.
In summary, we have demonstrated that HNC-derived MIF
modulates functions of neutrophils and, by feedback mecha-
nisms, also the biology of the HNC cells. Thus, MIF seems to
play a complex role in the tumor microenvironment by regu-
lating not only the tumor cells, as shown by most of the previ-
ously published studies,
39–41
but also the tumor-associated neu-
trophils and perhaps other tumor infiltrating immune cells.
Ultimately, these changes may result in the amplification of
inflammatory processes in the tumor microenvironment and
may lead to a more aggressive tumor cell phenotype.
Acknowledgements
We are grateful to Dr. Ju¨rgen Bernhagen (Department of Biochemistry, Uni-
versity of Aachen) for providing recombinant MIF and NIH-III.D9 antibod-
ies. We thank Petra Altenhoff and Anne-Marie Heider (Department of
Otorhinolaryngology, University of Duisburg-Essen) for excellent technical
support.
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  • Source
    • "Both cell types secrete cytokines and chemokines that recruit each other and enhance each other's proinflammatory activities, thus enhancing resolution of inflammation [155] (seeTable 3 for details of some cytokines and chemokines in the microenvironment). Macrophages secrete the macrophage migration inhibitory factor (MIF) to enhance neutrophil survival and secretion of MMP-9, in the context of both cancer [156] and autoimmunity [157]. The manner by which neutrophils die profoundly affects macrophage polarization, and, therefore, the subsequent course of disease. "
    [Show abstract] [Hide abstract] ABSTRACT: Cancer and autoimmune diseases are fundamentally different pathological conditions. In cancer, the immune response is suppressed and unable to eradicate the transformed self-cells, while in autoimmune diseases it is hyperactivated against a self-antigen, leading to tissue injury. Yet, mechanistically, similarities in the triggering of the immune responses can be observed. In this review, we highlight some parallel aspects of the microenvironment in cancer and autoimmune diseases, especially hypoxia, and the role of macrophages, neutrophils, and their interaction. Macrophages, owing to their plastic mode of activation, can generate a pro- or antitumoral microenvironment. Similarly, in autoimmune diseases, macrophages tip the Th1/Th2 balance via various effector cytokines. The contribution of neutrophils, an additional plastic innate immune cell population, to the microenvironment and disease progression is recently gaining more prominence in both cancer and autoimmune diseases, as they can secrete cytokines, chemokines, and reactive oxygen species (ROS), as well as acquire an enhanced ability to produce neutrophil extracellular traps (NETs) that are now considered important initiators of autoimmune diseases. Understanding the contribution of macrophages and neutrophils to the cancerous or autoimmune microenvironment, as well as the role their interaction and cooperation play, may help identify new targets and improve therapeutic strategies.
    Full-text · Article · Feb 2016 · Mediators of Inflammation
  • Source
    • "In the presence of bacterial infection for example, inflammation is normally triggered , and the subsequent release of MIF allows for prolonged inflammation through the release of other pro-inflammatory cytokines like TNF-α, IL-1β, IL-6, IL-8, IL-2, and IFN-γ [22,44]. In the context of tumor biology, MIF has been shown to induce the infiltration of several cell types into tumors, including myeloid derived suppressor cells (MDSC)[13] , neutro- phils [45], and mast cells [46] , as well as to affect the polarization of tumor associated macro- phages [47]. The tumor microenvironment becomes immunocompromised, reducing host antitumor responses. "
    [Show abstract] [Hide abstract] ABSTRACT: The macrophage migration inhibitory factor (MIF) has been increasingly implicated in cancer development and progression by promoting inflammation, angiogenesis, tumor cell survival and immune suppression. MIF is overexpressed in a variety of solid tumor types in part due to its responsiveness to hypoxia inducible factor (HIF) driven transcriptional activation. MIF secretion, however, is a poorly understood process owing to the fact that MIF is a leaderless polypeptide that follows a non-classical secretory pathway. Better understanding of MIF processing and release could have therapeutic implications. Here, we have discovered that ionizing radiation (IR) and other DNA damaging stresses can induce robust MIF secretion in several cancer cell lines. MIF secretion by IR appears independent of ABCA1, a cholesterol efflux pump that has been implicated previously in MIF secretion. However, MIF secretion is robustly induced by oxidative stress. Importantly, MIF secretion can be observed both in cell culture models as well as in tumors in mice in vivo. Rapid depletion of MIF from tumor cells observed immunohistochemically is coincident with elevated circulating MIF detected in the blood sera of irradiated mice. Given the robust tumor promoting activities of MIF, our results suggest that an innate host response to genotoxic stress may mitigate the beneficial effects of cancer therapy, and that MIF inhibition may improve therapeutic responses.
    Full-text · Article · Jan 2016 · PLoS ONE
  • Source
    • "These could be the result of a more sustained immune response to the tumour. However, tumour-associated macrophages and neutrophils have also been shown to play a role in creating an immune-suppressive tumour microenvironment in many cancers including head and neck cancer [18, 19]. It is now believed that immunosurveillance is the first of a three-step process referred to as cancer immunoediting, in which abnormal cells are first eliminated, as indicated above, but with the possibility that clonal evolution results in cells able to survive immune attack and, following immune evasion, begin aberrantly proliferating [17]. "
    [Show abstract] [Hide abstract] ABSTRACT: Oral squamous cell carcinoma (OSCC) is a prevalent cancer with poor prognosis. Most OSCC progresses via a non-malignant stage called dysplasia. Effective treatment of dysplasia prior to potential malignant transformation is an unmet clinical need. To identify markers of early disease, we performed RNA sequencing of 19 matched HPV negative patient trios: normal oral mucosa, dysplasia and associated OSCC. We performed differential gene expression, principal component and correlated gene network analysis using these data. We found differences in the immune cell signatures present at different disease stages and were able to distinguish early events in pathogenesis, such as upregulation of many HOX genes, from later events, such as down-regulation of adherens junctions. We herein highlight novel coding and non-coding candidates for involvement in oral dysplasia development and malignant transformation, and speculate on how our findings may guide further translational research into the treatment of oral dysplasia.
    Full-text · Article · Oct 2015 · Oncotarget
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