The Journal of Immunology
Macrophage Migration Inhibitory Factor Increases
Leukocyte–Endothelial Interactions in Human Endothelial
Cells via Promotion of Expression of Adhesion Molecules
Qiang Cheng,* Sonja J. McKeown,*,1Leilani Santos,* Fernando S. Santiago,†
Levon M. Khachigian,†Eric F. Morand,* and Michael J. Hickey*
Macrophage migration inhibitory factor (MIF) has been shown to promote leukocyte–endothelial cell interactions, although
whether this occurs via an effect on endothelial cell function remains unclear. Therefore, the aims of this study were to examine
the ability of MIF expressed by endothelial cells to promote leukocyte adhesion and to investigate the effect of exogenous MIF on
leukocyte–endothelial interactions. Using small interfering RNA to inhibit HUVEC MIF production, we found that MIF deficiency
reduced the ability of TNF-stimulated HUVECs to support leukocyte rolling and adhesion under flow conditions. These reductions
were associated with decreased expression of E-selectin, ICAM-1, VCAM-1, IL-8, and MCP-1. Inhibition of p38 MAPK had
a similar effect on adhesion molecule expression, and p38 MAPK activation was reduced in MIF-deficient HUVECs, suggesting
that MIF mediated these effects via promotion of p38 MAPK activation. In experiments examining the effect of exogenous MIF,
application of MIF to resting HUVECs failed to induce leukocyte rolling and adhesion, whereas addition of MIF to TNF-treated
HUVECs increased these interactions. This increase was independent of alterations in TNF-induced expression of E-selectin,
VCAM-1, and ICAM-1. However, combined treatment with MIF and TNF induced de novo expression of P-selectin, which
contributed to leukocyte rolling. In summary, these experiments reveal that endothelial cell-expressed MIF and exogenous
MIF promote endothelial adhesive function via different pathways. Endogenous MIF promotes leukocyte recruitment via effects
on endothelial expression of several adhesion molecules and chemokines, whereas exogenous MIF facilitates leukocyte recruitment
induced by TNF by promoting endothelial P-selectin expression.
itating this process via their ability to express cell surface adhe-
sion molecules that mediate interactions with leukocytes in the
bloodstream. Activated endothelial cells express molecules in-
volved in leukocyte rolling, such as P- and E-selectin, leukocyte
adhesion (e.g., VCAM-1 and ICAM-1), as well as chemoattrac-
tants, suchasCCL2(MCP-1)and CXCL8(IL-8),which caninduce
arrest of rolling leukocytes and promote leukocyte emigration from
the vasculature (1, 2). Activation of endothelial cells during in-
flammatory responses is typically induced by proinflammatory
cytokines, such as TNF and IL-1b, released during the response.
The Journal of Immunology, 2010, 185: 1238–1247.
eukocyte recruitment is a fundamental element of the
tissue response to stimuli, such as infection or cellular
damage. Endothelial cells play an essential role in facil-
However, the more recently characterized inflammatory mediator
macrophage migration inhibitory factor (MIF) differs from these
cytokines in that it is present in biologically active concentrations
in plasma in the absence of inflammation (3) and is constitutively
expressed by endothelial cells (4, 5). Despite its constitutive ex-
pression, MIF has been demonstrated to be a multifunctional
proinflammatory molecule (5, 6). Moreover, recent evidence indi-
cates that MIF facilitates the key inflammatory process of leuko-
cyte recruitment during inflammation, although the mechanisms
whereby this occurs remain unclear (7–11).
Emerging evidence suggests a role for endogenous MIF in the
promotion of endothelial adhesion molecule expression (8–10, 12).
We have observed that MIF deficiency results in decreased leu-
kocyte–endothelial cell interactions under inflammatory con-
ditions, as demonstrated using intravital microscopy, and that this
is associated with reduced endothelial expression of VCAM-1 (13,
14). The mechanisms whereby MIF facilitates endothelial adhe-
sion molecule expression are not known. Evidence that MIF influ-
ences activation of the p38 and ERK MAPK pathways suggests
that these pathways contribute to the effects of MIF (15–18).
Endothelial expression of VCAM-1, IL-8, and MCP-1 are facili-
tated by ERK and p38 MAPK activation (19–22). Moreover, MIF
has been shown to contribute to thrombin-induced ERK(1/2)
MAPK activation in endothelial cells and to induce migration
and tube formation via activation of the PI3K/Akt pathway (23,
24). However, it is unknown whether these effects extend to ad-
hesion molecule expression. These observations raise the possibil-
ity that MIF expressed by endothelial cells interacts with these
signaling pathways to affect the expression of adhesion molecules
The proadhesive function of exogenously administered MIF is
also poorly understood. We have observed that exogenous MIF
*Department of Medicine, Monash Medical Centre, Centre for Inflammatory Dis-
eases, Monash University, Clayton, Victoria; and
University of New South Wales, Sydney, New South Wales, Australia
†Centre for Vascular Research,
1Current address: Division of Biology, California Institute of Technology, Pasadena,
Received for publication December 22, 2009. Accepted for publication May 7, 2010.
This work was supported by a program grant from the National Health and Medical
Research Council of Australia (334067), an R01 grant from the National Institutes
of Health (AR51807-01), and a grant-in-aid from the Heart Foundation of Australia.
M.J.H. is a National Health and Medical Research Council Senior Research Fellow.
Address correspondence and reprint requests to Dr. Michael J. Hickey, Department of
Medicine, Centre for Inflammatory Diseases, Monash University, Monash Medical
Centre, 246Clayton Road,Clayton,Victoria 3168,Australia.E-mail address: michael.
The online version of this article contains supplemental material.
Abbreviations used in this paper: IP-10, IFN-g inducible protein-10; MFI, mean
fluorescence intensity; MIF, macrophage migration inhibitory factor; NC, negative
control; RT-PCR, real-time PCR; Sc, scrambled control; siRNA, small interfering
promotes monocyte recruitment invivo, in part via the induction of
endothelial release of CCL2/MCP-1 (25), but without increasing
VCAM-1 expression. In vitro experiments have shown that expo-
sure to MIF renders endothelial cells capable of supporting leuko-
cyte adhesion, although the mechanisms underlying this response
have not been clearly defined (12, 26). Lin et al. (27) reported that
MIF induces ICAM-1 expression in an endothelial cell line. More-
over, MIF has been found to increase VCAM-1 in mononuclear
leukocytes (12), although a similar effect on endothelial cells has
not been reported. Several studies have linked MIF exposure to
induction of chemokine expression (25, 28, 29). However, whether
MIF has these effects in primary human endothelial cells remains
Therefore, the aim of this study was to examine the ability of
MIF, either endogenously expressed by endothelial cells or applied
exogenously, to promote leukocyte–endothelial cell interactions,
and to regulate endothelial adhesion molecule expression and
chemokine production. These experiments demonstrate that MIF
expressed by endothelial cells promotes expression of several
adhesion molecules and chemokines, whereas exogenous MIF
specifically promotes P-selectin expression.
Materials and Methods
Proteins, Abs, and chemicals
MN). Recombinant human MIF was produced using an Escherichia coli
expression system and provided by Cortical (Melbourne, Victoria, Aus-
tralia). PE-labeled mouse anti-human E-selectin mAb, PE-labeled mouse
VCAM-1 mAb were all purchased from BD Biosciences (San Diego,
CA). Polyclonal anti-human P-selectin Ab was provided by Dr. R. Andrews
(Australian Centre for Blood Diseases, Monash University, Prahran, Victo-
ria, Australia). The function-blocking anti–E-selectin mAb (BBIG-E4) was
purchased from R&D Systems. PD98059 (ERK inhibitor) and SB203580
(p38 inhibitor) were purchased from Alexis Biochemicals (Plymouth Meet-
ing, PA), and Bay 11-7082 (NF-kB inhibitor) was obtained from Calbio-
chem (Darmstadt, Germany).
FITC-labeled mouse anti-human
HUVECs were isolated from umbilical veins using 0.1% collagenase II
(Worthington Biochemical, Lakewood, NJ) and cultured in 0.2% gelatin-
coated culture flasks with M199 medium (Invitrogen, Carlsbad, CA) con-
taining 20% FCS, endothelium mitogen (50 mg/ml), 0.1 mg/ml heparin,
2 mM L-glutamine, 1 U/ml penicillin, and 0.1mg/ml streptomycin, as de-
scribed previously (30). Primary HUVECs were allowed to grow for 3–5 d
to reach 100% confluence. Cells were subsequently passaged once before
use in experiments. To activate HUVECs, cells were treated with TNF
(33 or 1000 pg/ml) and/or MIF (100 ng/ml) for 4 h in serum-free medium
(M199 containing 2 mM L-glutamine, 1 U/ml penicillin, and 0.1 mg/ml
streptomycin). For the NF-kB and MAPK inhibition experiments, cells
were pretreated with the following inhibitors: ERK inhibitor (PD98059,
20 mM); p38 inhibitor (SB203580, 10 mM); and NF-kB inhibitor (Bay 11-
7082, 2 mM) for 1 h before being exposed to TNF in the presence of the
same inhibitor for 4 h.
MIF silencing via small interfering RNA
For MIF silencing, a human MIF-specific small interfering RNA (siRNA)
(sense, 59-CCUUCUGGUGGGGAGAAAUtt-39; antisense, 39-ttGGAAG-
ACCACCCCUCUUUA 59) was used. Control cells were treated with the
following nontargeting scrambled control (Sc) siRNA (sense, 59-CACUC-
Gtt-39). Both siRNAs were purchased from Ambion (Austin, TX). siRNA
was transfected into HUVECs using the Lonza HUVEC Nucleofector Kit
(Lonza, Basel, Switzerland), according to the manufacturer’s instructions.
105cells wereseededontofibronectin(RocheDiagnostics, CastleHill,New
South Wales, Australia)-coated 3.5-cm culture dishes (Corning, Lowell,
MA) at 1 3 106cells/ml. Cells were then cultured for 2 d before being used
for other experiments. To assess cell viability after these treatments, cells
were exposed to propidium iodide (2.5 mg/ml, 5 min) and assessed using
fluorescence microscopy. Cells that stained positive with propidium iodide
were considered nonviable.
Quantitation of mRNA expression by real-time PCR
Total mRNA was extracted with the RNeasy mini kit (Qiagen, Cologne,
Germany). RNA was then treated with TURBO DNA-free kit (Applied
Biosystems, Foster City, CA) to degrade contaminating genomic DNA.
Then, cDNA was generated using random primers (7.5 mg/ml) and the
SuperScript III reverse transcriptase (Invitrogen). The gene-specific primer
sequences used for real-time PCR (RT-PCR) were as follows: MIF (59-
CCGGACAGGGTCTACATCAACTATTAC-39 and 59-TAGGCGAAGG-
TGGAGTTGTTCC-39), 18s (59-GCAATTATTCCCCATGAACG-39 and
59-TGTACAAAGGGCAGGGACTT-39) (31), and P-selectin (59-GCCA-
AGCAGGACCATTGACTAT-39 and 59-AGCCACCGCTCCACCAA-39).
RT-PCR wasperformedusing Power SybrGreen PCR Master Mix(Applied
Wales, Australia). The level of target gene expression was normalized
against 18S rRNA expression, and the results are expressed as the number
of mRNA copies per 10618S rRNA copies (32).
A total of 0.25 3 105cultured HUVECs were lysed in cell lysis buffer
(Cell Signaling Technology, Danvers, MA) containing proteinase inhib-
itors (Roche Diagnostics), and lysates were stored at 270˚C until use. The
MIF ELISA was performed using commercially available paired Abs
(R&D Systems) following manufacturer’s protocols, with a detection limit
of 16 pg/ml. Briefly, ELISA plates were coated with mouse anti-MIF Ab
overnight at 4˚C. Plates were then blocked with PBS containing 1% BSA
and 5% sucrose for 2 h at room temperature and then washed with wash
buffer (0.05% Tween 20 in PBS). Cell lysates were diluted in Tris buffer
(0.1% BSA and 0.05% Tween 20 in pH 7.3 Tris, 1/200) and incubated
overnight at 4˚C. Plates were washed with wash buffer and then incubated
sequentially with biotinylated goat anti-human MIF and streptavidin-HRP
(Millipore, Billerica, MA) for 2 h each at room temperature. After wash-
ing, 100 ml tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO)
was added and incubated for 5–10 min. The reaction was stopped with
0.5 M H2SO4. Plates were then analyzed at 450 nm. The total protein
concentrations of all lysate samples were determined using BCA Protein
assay kit (Pierce, Rockford, IL). Results are presented as picograms MIF
protein per milligram total protein.
Flow chamber assay
The whole-blood flow chamber assay was performed using a parallel plate
flow chamber (Glycotech, Gaithersburg, MD) at a shear rate of 150 s21(33,
34). Blood was collected from healthy volunteers in lithium heparin blood
collection tubes and stored on ice until use. Fifteen minutes before use,
blood was diluted 1/10 in HBSS and incubated at 37˚C. The flow chamber
was placed into the HUVEC culture dish and observed using a Axiovert
200 microscope (Carl Zeiss, North Ryde, New South Wales, Australia).
The diluted blood was then pulled into the flow chamber and allowed to
perfuse over the HUVEC monolayer for 5 min using a syringe pump
(Harvard Apparatus, Holliston, MA). Subsequently, blood was replaced
with HBSS to clear the field, and 8–12 random fields (0.23 mm2/field)
were recorded for 10 s each using a video camera (Sony SSC-DC50AP,
Carl Zeiss) and VCR. Videos were then analyzed, and the number of
rolling and adherent (defined as cells that remained stationary for the entire
10 s of recording period) leukocytes were quantified. Data for both param-
eters are expressed as cells per square millimeter.
Quantitation of endothelial adhesion molecule expression
flow cytometry. Briefly, HUVECs were first trypsinized from the culture
PE-labeled anti-human E-selectin, FITC-labeled anti-human VCAM-1, or
PE-labeled mouse anti-human ICAM-1 at room temperature for 20 min.
Cells were then washed, fixed in paraformaldehyde and analyzed using
a MoFlo Analyzer (Beckman Coulter, Brea, CA). Unless otherwise stated,
that of a sample stained with an appropriate isotype control Ab.
Quantitation of HUVEC cytokine and chemokine production
The concentrations of cytokines and chemokines in HUVEC culture super-
natants were measured using the Cytometric Bead Array Human In-
flammatory Cytokines Kit and Human Chemokine Kit (both from BD
Biosciences), according to the manufacturer’s instructions. These kits allow
The Journal of Immunology1239
simultaneous measurement of six cytokines (IL-8, IL-1b, IL-6, IL-10, TNF
inducible protein-10 (IP-10)] in each sample. All bead samples were ana-
lyzed by a FACSCanto II Cell Analyzer (BD Biosciences), and the results
were analyzed using FCAP Array software (version 1.0.1; Soft Flow, St.
Louis Park, MN). Cytokine/chemokine concentrations were determined by
reference to the cytokine/chemokine standards.
Quantitation of MAPK phosphorylation
MAPK (p38 and ERK) phosphorylation was measured using BD Cyto-
metric Bead Array Flex Set assay (BD Biosciences). Cells were lysed with
cell lysis buffer (Cell Signaling Master Buffer kit; BD Biosciences) con-
taining proteinase inhibitor (Roche Diagnostics). Samples were then ana-
lyzed by a FACSCanto II Cell Analyzer (BD Biosciences), according to the
manufacturer’s instructions. All flow cytometric results were analyzed
using FCAP Array software (version 1.0.1, Soft Flow). Concentrations of
phosphorylated MAPKs were determined using MAPK standards provided
by the array kits. Total protein concentrations of all lysate samples were
determined using a BCA protein assay kit (Pierce). Phosphorylation results
were then normalized to the protein concentration and presented as
phosphorylated protein (U) per milligram total protein.
Assessment of P-selectin expression by immunofluorescence
HUVECs were seeded and grown on 35-mm glass-bottom dishes for 2 d to
reach confluence before being exposed to TNF (33 pg/ml) with or without
formaldehyde for 10 min and incubated with sheep serum (diluted 1/10) for
40 min at room temperature. Subsequently, P-selectin was detected using
a two-layer staining protocol with rabbit anti-human P-selectin Ab (5 mg/
BSA (35). Monolayers were then mounted with Vectashield mounting me-
confocal microscope, using a 340 1.0 NA oil immersion lens.
Significance was assessed by Student t tests, and a value of p , 0.05 was
considered to be significant. Where appropriate, such as in the comparison
of differently treated cells isolated from the same cord, paired analysis was
MIF siRNA inhibits MIF expression in HUVECs
To suppress MIF expression in HUVECs, an siRNA approach was
used. Forty-eight hours posttransfection with MIF-specific siRNA,
HUVECs showed a .95% reduction in MIF mRNA, compared
with levels detected in the control siRNA-treated cells (Fig. 1A).
Similarly, MIF siRNA resulted in a .90% decrease in intracellular
MIF protein relative to control-treated cells (Fig. 1B), indicating
that MIF siRNA transfection successfully suppressed MIF expres-
sion in HUVECs. MIF siRNA-transfected cells showed normal
cobblestone morphology (Fig. 1C), indicating that the depletion
of MIFdoes notmarkedly alter cell physiology.This was supported
by the finding that cells treated with either control (Sc) or MIF
siRNA retained the ability to exclude propidium iodide, an indica-
tor of cell viability (data not shown).
MIF-deficient HUVECs support fewer leukocyte–endothelial
To determine the effect of MIF depletion on the ability of endo-
thelial cells to support interactions with leukocytes under flow
conditions, a whole-blood flow chamber assay was used. In the ab-
were seen on transfected HUVECs (Fig. 1D, 1E), indicating that
siRNA transfection alone did not result in cell activation. TNF
(33 pg/ml; 4 h) exposure induced significant increases in rolling
ever, MIF-deficient HUVECs supported significantly fewer rolling
and adhesive interactions in response to TNF (Fig. 1D, 1E)
compared with the control siRNA-treated cells, with rolling being
reduced by 30% and adhesion by 26%. To determine whether this
ability applied under conditions of maximal TNF stimulation, we
also examined cells treated with TNF at 1000 pg/ml. Under these
conditions, MIF depletion via siRNA did not reduce rolling and
adhesive interactions (data not shown), indicating that the adhe-
sion-enhancing abilities of endogenous MIF are operative at lower
TNF concentrations. All subsequent experiments assessing the
using TNF at 33 pg/ml.
cell interactions. HUVECs were transfected with Sc siRNA or MIF siRNA.
MIF mRNA (A; n = 5) and intracellular MIF protein (B; n = 4) were
measured 48 h posttransfection using RT-PCR and ELISA, respectively. C,
Morphology of monolayers of either control (Sc)-treated cells or MIF
siRNA-treated cells (original magnification 3400). The ability of control
(Sc) cells and MIF siRNA-transfected cells to support rolling and adhesion
of human leukocytes was assessed using a flow chamber system in the
absence of treatment (n = 4) or following TNF treatment (33 ng/ml; 4 h;
n = 7). The number of rolling (D) and adherent (E) leukocytes were quan-
tified. pp , 0.05; ppp , 0.01, for comparison of TNF-treated control (Sc)
and MIF siRNA-treated cells.
MIF-deficient HUVECs support fewer leukocyte–endothelial
1240 MIF AND ENDOTHELIAL CELL ADHESIVE FUNCTION
MIF-deficient HUVECs show reduced expression of E-selectin,
VCAM-1, and ICAM-1
To investigate the possibility that endogenous endothelial MIF
influences leukocyte rolling and adhesion through modulating ad-
hesion molecule expression, cell surface expression of E-selectin,
VCAM-1, and ICAM-1 was measured. TNF significantlyincreased
the expression of E-selectin in control siRNA-transfected cells.
However, in MIF-deficient HUVECs, TNF-induced E-selectin ex-
pression was significantly reduced (by 51%) relative to that in
TNF-treated controlcells (Fig.2A).HUVECsexpressedsubstantial
levels of VCAM-1 and ICAM-1 in the absence of TNF stimulation.
In MIF-deficient HUVECs, constitutive VCAM-1 and ICAM-1
expression was significantly reduced by 28 and 36%, respectively
(Fig. 2B, 2C). In addition, TNF increased the expression of
VCAM-1 and ICAM-1, although in MIF-deficient HUVECs, these
increases were significantly attenuated (VCAM-1, 31% reduction;
ICAM-1, 28% reduction) (Fig. 2B, 2C).
MIF deficiency decreases HUVEC production of IL-6, IL-8,
TNF stimulation of HUVECs results in the expression of in-
flammatory mediators, including chemokines, which promote
leukocyte arrest. The role of endogenous endothelial cell MIF in
modulating these responses is unknown. Therefore, we next de-
termined the impact of endogenous MIF on cytokines and chemo-
at detectable levels in culture supernatants, whereas IL-1b, IL-10,
not shown). Basal release of IL-6, IL-8, and MCP-1 was all signif-
icantly reduced in MIF-deficient HUVECs by 55, 31, and 49%,
respectively (Fig. 3). Similarly, TNF-induced production of each
of IL-6, IL-8, and MCP-1 was also significantly decreased in
MIF-deficient HUVECs, with the following percentage reductions
relative to TNF-treated control cells: IL-6, 20%; IL-8, 17%; and
MCP-1, 19% (Fig. 3).
Role of MAPKs in regulation of inflammatory response in
The MAPK and NF-kB signaling pathways provide a potential link
between endogenous MIF and endothelial adhesion molecule ex-
pression. Also, previous studies have shown NF-kB signaling to
expression (36). Therefore, we next investigated the involvement
of the NF-kB pathway, and p38 and ERK MAPK, in endothelial
activation in response to TNF by analyzing the effect of specific
selectin, VCAM-1, and ICAM-1. Expression of E-selectin (A), VCAM-1
(B), and ICAM-1 (C) were measured in control (Sc) siRNA-treated and
MIF siRNA-treated HUVECs, in the absence of treatment (n = 5), or
following treatment with TNF (33 ng/ml; 4 h; n = 5), using flow cytometry.
Data represent mean 6 SEM of MFI following subtraction of MFI of cells
stained with appropriate isotype control Ab. pp , 0.05; ppp , 0.01, for
comparison of similarly-treated control (Sc) and MIF siRNA-treated cells.
MIF-deficient HUVECs show reduced expression of E-
and MCP-1. Production of IL-6 (A), IL-8 (B), and MCP-1 (C) were mea-
sured in supernatants of control (Sc) siRNA-treated and MIF siRNA-
treated HUVECs, in the absence of treatment (n = 5), or following treat-
ment with TNF (33 ng/ml; 4 h; n = 10), via Cytometric Bead Array kits.
Data are presented as mean 6 SEM. pp , 0.05; ppp , 0.01, for compar-
ison of similarly-treated control (Sc) and MIF siRNA-treated cells.
MIF deficiency decreases HUVEC production of IL-6, IL-8,
The Journal of Immunology 1241
inhibitors on adhesion molecule expression. Inhibition of the NF-
kB pathway completely inhibited the TNF-induced increase in ex-
p38 MAPK also significantly reduced TNF-induced expression
of the adhesion molecules E-selectin (34% reduction relative to
TNF/DMSO-treated cells), VCAM-1 (38% reduction), and ICAM-1
(16% reduction) (Fig. 4), although to a lesser extent than inhibition
of NF-kB. In contrast, inhibition of ERK had no effect on TNF-
induced adhesion molecule expression.Thesefindings are consistent
with a mechanism for TNF-induced adhesion molecule expression
in which NF-kB activity is required, whereas p38 MAPK plays
a facilitatory role.
MIF deficiency reduces TNF-induced p38 phosphorylation in
Given the established ability of MIF to promote TNF-induced
MAPK activation (15), the observation of a role for p38 in pro-
moting adhesion molecule expression in response to TNF raised
a potential mechanism to explain the inhibitory effect of MIF
siRNA on this response. Therefore, in the next series of experi-
ments, we compared TNF-induced MAPK phosphorylation in
control and MIF siRNA-transfected cells. In control-transfected
HUVECs, TNF caused an increase in p38 phosphorylation of .6-
fold but failed to alter ERK phosphorylation significantly (Table I).
In MIF-deficient HUVECs, p38 phosphorylation was significantly
reduced relative to control-transfected HUVECs, both in untreated
5A). In contrast, MIF deficiency had no effect on ERK activation
(Fig. 5B). These results suggest a role for MIF in facilitation of
TNF-induced p38 activation in endothelial cells.
Lack of effect of exogenous MIF on HUVEC expression
of E-selectin, VCAM-1, and ICAM-1
Exogenous MIF has been observed to promote leukocyte adhesion
both invitro and invivo, although its effects on endothelial adhesion
molecule expression remain poorly understood (12, 25, 26). There-
fore, the aim of the next series of experiments was to investigate the
role of exogenous MIF in promoting adhesion molecule expression
and leukocyte–endothelial cell interactions in vitro. HUVECs were
exposed to MIFat a range of concentrations that induce responses in
of E-selectin, VCAM-1, and ICAM-1 induced by TNF. To compare the
roles of MAPK and NF-kB in TNF-induced adhesion molecule expression,
HUVECs were pretreated with either the NF-kB inhibitor (Bay 11-7082,
2 mM; n = 4), p38 inhibitor (SB203580, 10 mM; n = 4), or the ERK inhibi-
tor (PD98059, 20 mM; n = 4) for 1 h before being exposed to TNF (33 ng/
ml; 4 h) in the presence of the same inhibitor. Comparison was made with
either untreated (NC) cells (n = 4–7) or cells treated with TNF and vehicle
(DMSO; n = 7). Expression of E-selectin (A), VCAM-1 (B), and ICAM-1
(C) were measured by flow cytometry. Data represent mean 6 SEM of MFI
following subtraction of MFI of cells stained with appropriate isotype con-
trol Ab. Proteins targeted by specific inhibitors are indicated on the graph.
Data are shown as mean 6 SEM. pp , 0.05; ppp , 0.01, relative to TNF
+DMSO-treated cells. NC, negative control.
Effect of inhibition of NF-kB and MAPKs on the expression
TNF-induced p38 and ERK MAPK activation in endothelial
MAPK ProteinUntreated (U/mg)a
14.94 6 2.1
0.87 6 0.08
95.37 6 11.8
1.66 6 0.15
HUVECs were either untreated or treated with TNF (33 ng/ml, 30 min), then
MAPK phosphorylation was determined as described in Materials and Methods.
aData are shown as units of phosphorylated MAPK protein per milligram total
protein; n = 4 for all groups; p values represent results of comparison of untreated and
TNF-treated cells via paired t test.
assess the role of MIF in promoting activation of p38 MAPK, HUVECs
transfected with either control (Sc) siRNA or MIF siRNA were treated with
or without TNF (33 ng/ml; 30 min) prior to cell lysis. Phosphorylation of p38
(A) and ERK (B) MAPKs were measured using a BD Cytometric Bead Array
Flex Set assay. Data are shown asphosphorylationunits permilligram protein
(U/mg) and represent mean 6 SEM of four to five individual cultures. pp ,
0.05 for comparison of control (Sc) and MIF siRNA-treated cells.
1242MIF AND ENDOTHELIAL CELL ADHESIVE FUNCTION
other cell types (25, 26). As shown in Fig. 6, MIF failed to induce
molecule expression in the same cells in response to LPS confirmed
their ability to respond to other inflammatory stimuli (Fig. 6). These
data suggest that, in contrast to the facilitatory role of endogenous
MIF, exogenous MIF is not able to independently induce the expres-
sion of these adhesion molecules in HUVECs.
Exogenous MIF increases TNF-induced leukocyte rolling and
MIF is present in the circulation and extracellular space during
inflammatory responses in which endothelial activation is initiated
by other mediators. Therefore, we next examined the ability of
exogenous MIF to increase leukocyte–endothelial interactions
induced by TNF. Treatment of HUVECs with MIF alone did not
induce leukocyte rolling or adhesion (Fig. 7), consistent with the
lack of effect of exogenous of MIF on adhesion molecule ex-
pression. However, in cells treated with TNF (33 pg/ml), the ad-
dition of MIF resulted in significantly increased leukocyte rolling
and adhesion relative to cells treated with TNF alone (Fig. 7).
Increasing the concentration of TNF to 1000 pg/ml eliminated this
effect of MIF (data not shown). Therefore, subsequent experi-
ments were performed at the lower TNF concentration.
MIF does not increase TNF-induced expression of E-selectin,
VCAM-1, ICAM-1, and chemokines
To determinewhether exogenous MIF increased TNF-induced roll-
ing and adhesion via effects on adhesion molecule expression, the
expression of any of these adhesion molecules above that induced
by TNFalone (Supplemental Fig. 1). Similar results were found for
IL-6, IL-8, and MCP-1 in that MIF treatment of HUVECs did not
increase basal secretion of these mediators or alter the level of
their release following TNF stimulation (Supplemental Fig. 2).
MIF increases TNF-induced leukocyte rolling by inducing
endothelial P-selectin expression
An alternative candidate adhesion molecule to explain the in-
creased leukocyte–endothelial interactions in HUVECs cotreated
with TNF and MIF is P-selectin (35, 37). Although TNF does not
induce P-selectin expression by HUVECs (38), the role of MIF in
the regulation of P-selectin expression in HUVECs is unknown.
We therefore assessed the possibility that cotreatment with MIF
and TNF-induced endothelial P-selectin expression. Measurement
of P-selectin mRNA revealed that neither MIF nor TNF alone
increased P-selectin mRNA expression. However, cotreatment
with MIF and TNF significantly increased P-selectin mRNA ex-
pression in HUVECs compared with either treatment alone
(Fig. 8A). This treatment also resulted in increased expression of
P-selectin on the cell surface. As shown in Fig. 8B, surface ex-
pression of P-selectin was negligible following treatment with
either MIF or TNF, whereas robust P-selectin expression was
detected when cells were cotreated with TNF and MIF. Finally,
we tested the functional contribution of E- and P-selectin to TNF-
ber assay. In cells treated with TNF alone, inhibition of E-selectin
E-selectin, VCAM-1, and ICAM-1 on HUVECs. HUVECs were treated
with LPS (as positive control; 20 ng/ml; 4 h) or MIF (50, 100, or 200 ng/
ml; 4 h) and expression of E-selectin (A; n = 4), VCAM-1 (B; n = 3), and
ICAM-1 (C; n = 3) was assessed via flow cytometry. For E-selectin and
ICAM-1, data are shown as MFI, whereas VCAM-1 results are shown as
percentage of positive cells, as determined by comparison with an appro-
priate isotype control Ab.
Exogenous MIF does not induce surface expression of
and adhesion. To assess the ability of exogenous MIF to increase leuko-
cyte–endothelial cell interactions induced by TNF, HUVECs were treated
with TNF (33 ng/ml; 4 h), MIF (100 ng/ml; 4 h), or TNF and MIF (33 and
100 ng/ml; 4 h) in combination. Subsequently, leukocyte rolling (A) and
adhesion (B) were assessed in a whole-blood flow chamber assay. Data are
shown as mean 6 SEM of rolling or adherent cells per square millimeter.
Data for untreated (NC) cells are also shown; n = 4–8 individual cultures;
pp , 0.05 for comparison of TNF versus TNF+MIF-treated cells. NC,
Exogenous MIF increases TNF-induced leukocyte rolling
The Journal of Immunology 1243
caused a significant reduction in rolling, an effect that was not
further reduced with a function-blocking anti–P-selectin Ab
(Fig. 8C). In contrast, when HUVECs were cotreated with TNF
and MIF, inhibition of P-selectin resulted in a significant additional
reduction in leukocyte rolling relative to that induced by inhibition
in P-selectin detected on cells cotreated with TNF and MIF con-
tributed to the increased adhesive interactions observed under
The proinflammatory function of MIF has been demonstrated in
numerous forms of inflammation affecting a wide range of tissues
(5, 6). Although MIF has been shown to modulate a wide range of
inflammatory processes, several studies have shown that reducing
MIF function, either by Ab neutralization or gene deletion, results
in reduced leukocyte recruitment to sites of inflammation (8, 11,
39–41) and suggest this as a major mechanism underlying the
proinflammatory effects of MIF. Effects of MIF on leukocyte re-
cruitment, as demonstrated by findings of reduced leukocyte–
endothelial cellinteractions inMIF2/2mice (14), couldbe mediated
either through altering responses of leukocytes or endothelial cells.
The possibility that MIF affects endothelial cell adhesive function
directly is supported by in vitro studies showing that MIF treatment
of endothelial cells promotes leukocyte adhesion (13, 26), although
we have specifically examined the ability of MIF to modulate en-
dothelial adhesive function. These experiments revealed that defi-
ciency of MIF in HUVECs was associated with reductions in TNF-
induced expression of adhesion molecules and chemokines, with
concomitant reductions in TNF-induced leukocyte–endothelial cell
of reduced TNF-induced rolling and adhesive interactions in post-
capillary venules of MIF2/2mice following TNF treatment (14). In
the current study, MIF deficiency also reduced basal endothelial cell
expression of VCAM-1, ICAM-1, IL-6, IL-8, and MCP-1, suggest-
ing that the presence of MIFcontributes toconstitutive expression of
minimal effect on nonactivated HUVECs, addition of MIF to TNF-
stimulated endothelial cells increased leukocyte rolling and adhe-
sion, in part via the induction of P-selectin expression. Taken to-
gether, these data indicate that MIF has important roles in
promoting endothelial adhesive function, both when expressed by
endothelial cells and when applied to cells undergoing stimulation
by other inflammatory mediators. However, the current observations
suggest the similar effects of endogenous and exogenous MIF are
achieved via different cellular pathways.
The present data indicate that reduction of MIF expression in
HUVECs decreases p38 MAPK activation in response to TNF,
resulting in reduced expression of molecules associated with leu-
kocyte adhesion. This is in agreement with recent studies that in-
MIF has been found to facilitate MAPK activation in response to
diverse stimuli, such as LPS, cytokines, or TCR activation (15, 16,
on permissive inhibition by MIF of MAPK phosphatase 1 (also
MIF (100 ng/ml), or TNF and MIF (33 and 100 ng/ml; 4 h) in combination and P-selectin mRNA levels (A) assessed by RT-PCR (A; n = 5). Data for
untreated cells (NC) are also shown. pp , 0.05 for comparison of TNF versus TNF+MIF. B, Immunohistochemical assessment of surface P-selectin
expression in HUVECs under the same experimental conditions (P-selectin shown in green; DAPI-stained nuclei shown in blue; representative images from
one of three individual experiments; original magnification 3800). C and D, Comparison of functional roles of E- and P-selectin in mediating leukocyte
rolling on HUVECs treated with either TNF alone (C) or TNF+MIF (D). C, Leukocyte rolling data for cells treated with TNF alone before and after
addition of either anti–P-selectin, anti–E-selectin, or both Abs in combination. D, Leukocyte rolling data for cells treated with TNF+MIF, presented in the
same format as C. Data show the number of rolling leukocytes (cells/mm2) in the individual treatments normalized to data from monolayers prior to
addition of anti-selectin Abs. n = 3–5. ##p , 0.01 for comparison of rolling following blockade of E-selectin alone versus no blockade controls; ppp , 0.01
for comparison of rolling following blockade of E-selectin alone versus combined blockade of E- and P-selectin. NC, negative control.
MIF increases TNF-induced leukocyte rolling by inducing endothelial P-selectin expression. HUVECs were treated with TNF (33 ng/ml),
1244 MIF AND ENDOTHELIAL CELL ADHESIVE FUNCTION
known as DUSP1), a critical negative regulator of MAPK phos-
phorylation (16, 43). In contrast to these effects of MIF on MAPK
activation, effects of MIF on NF-kB activation are less well
established. For example, in other cell types, MIF does not di-
rectly induce NF-kB activity and is not required for cytokine-
induced NF-kB nuclear translocation or DNA binding (15, 44).
In contrast, NF-kB activation is considered essential for TNF-
induced adhesion molecule expression in HUVECs (36). In the
current study, we found that although TNF-induced endothelial
expression of adhesion molecules was prevented by inhibition of
the NF-kB pathway, inhibition of p38 also attenuated this induction,
albeit to a lesser degree. In addition, in parallel with the inhibitory
effect of MIF depletion on TNF-induced endothelial adhesive inter-
actions and adhesion molecule expression, MIF depletion also re-
duced TNF-induced endothelial cell p38 MAPK phosphorylation.
Notably, the magnitude of inhibitory effects of MIF depletion was
comparable to that of p38 MAPK inhibition. Taken together, these
findings are consistent with a mechanism whereby endogenous MIF
promotes TNF-induced p38 activation, which further supports in-
creased expression of adhesion molecules and chemokines.
role of MAPKs, such as p38, in endothelial cell adhesion molecule
expression (45–47), there is evidence that endothelial cell NF-kB
activity can be enhanced via the actions of MAPKs (22, 23). For
example, MIF and ERK MAPK have each been shown to amplify
thrombin-induced NF-kB transcriptional activity in endothelial
cells (22, 23). Also, p38 inhibition has been shown to attenuate
NF-kB DNA binding in stimulated HUVECs (22, 23). Amplifying
effects of MAPK on NF-kB transcriptional activity are well de-
scribed in other contexts (48). This is believed to occur via effects
of MAPK on mitogen- and stress-activated protein kinases, which
phosphorylate NF-kB p65, enhancing the ability of the NF-kB
heterodimer to interact with other transcriptional proteins. These
observations, together with the present data, support the contention
that p38 MAPK has the capacity to contribute to NF-kB–associated
endothelial adhesion molecule expression, at least under some
activating conditions, and that this is facilitated by MIF.
Notwithstanding these effects of endogenous endothelial cell
MIF, exogenous MIF has also been found to induce leukocyte
adhesion and recruitment under flow conditions, both in vitro and
in vivo (25, 26). As potential mechanisms underlying this re-
sponse, MIF has been observed to induce endothelial ICAM-1
expression in an endothelial-derived cell line (EA.hy926), and
we previously observed MIF-dependent induction of MCP-1 re-
lease from murine microvascular endothelial cells (25, 27). In
contrast, in the current study, exogenous application of MIF alone
had no effect on expression of adhesion molecules or chemokines
in HUVECs and failed to induce rolling and adhesion of leuko-
cytes in a whole-blood flow chamber assay. The reasons underly-
ing these differences in reported responses to MIF treatment are
unclear, although it is conceivable that they stem from functional
differences in endothelial cells from different vascular sites. In
vivo studies have established that endothelial cells from arterial
and venous sources have differing capacities in terms of adhesion
molecule expression (49–52). Studies in which MIF treatment was
found to induce mononuclear leukocyte arrest were performed
using aortic endothelial cells, whereas the present studies used
endothelial cells derived from umbilical veins (26). Similarly,
our previous experiments showing MIF-induced MCP-1 release
were performed using pulmonary microvascular endothelial cells
(25). To clarify this issue and fully understand the effects of MIF
in the vasculature, it may be necessary to directly compare the
effects of MIF on endothelial cells from arterial, venous, and mi-
In contrast to the effects of exogenous MIF alone, coadminis-
tration of MIF to TNF-treated cells resulted in an increase in TNF-
induced leukocyte–endothelial interactions. This indicated that ex-
ogenous MIF can facilitate the proinflammatory effects of TNF
and LPS on leukocyte recruitment. Given the presence of extracel-
lular MIF in normal plasma, this finding is likely to be relevant to
the observation that MIF facilitates in vivo leukocyte–endothelial
cell interactions induced by TNF or LPS (13, 14). However, the
finding that this effect was not associated with increased HUVEC
expression of E-selectin, ICAM-1, VCAM-1, or various chemo-
kines indicated the involvement of an alternative pathway for this
effect, which we found to be induction of P selectin expression.
Although it is known that TNF is not able to induce P-selectin
in HUVECs (38), the present data show that the addition of
MIF results in the induction of P-selectin expression by TNF-
stimulated HUVECs. Moreover, P-selectin was expressed at func-
tional levels, as demonstrated by the ability of anti–P-selectin to
inhibit E-selectin–independent rolling on cells stimulated with TNF
plus MIF, but not on cells stimulated with TNF alone. Notably,
MIF-dependent induction of P-selectin occurred in the absence of
induction of E-selectin, ICAM-1, and VCAM-1, suggesting that
MIF has a previously unidentified specific role in promotion of ex-
conditions. Although this has not been shown before in human
cells, we previously observed that histamine-induced P-selectin–
dependent leukocyte rolling in murine postcapillary venules was
attenuated in MIF2/2mice (14). This experiment was the first to
suggest a link between MIF and P-selectin expression. It is notewor-
thy that, in both the human and murine systems, MIF alone was in-
sufficient to induce P-selectin expression and was only active in the
context of an existing inflammatory stimulus (e.g., TNF or hista-
mine). These findings are consistent with a process whereby exoge-
induced by additional mediators.
immune disorders continues to grow, so does the concept that MIF
may be a valid therapeutic target (6, 53). Although it is established
data suggest that one of the effects of MIF inhibition during in-
flammation would be reduction in endothelial expression of adhe-
sion molecules and chemokines and concomitant reduction in
sites. In combination with other potential effects of MIF inhibition,
this could result in a highly effective anti-inflammatory strategy.
In conclusion, the current study demonstrates that both endog-
enous and exogenous MIF enhance the ability of endothelial cells to
support interactions with leukocytes under flow conditions. Endog-
enous MIF was required for basal and TNF-induced adhesion mole-
cule and chemokine expression and in vitro leukocyte–endothelial
important facilitator of cell activation entrained by proinflammatory
stimuli. In addition, exogenous MIF increased the level of leukocyte
rolling and adhesion induced by TNF via amplification of P-selectin
expression. Given that MIF is constitutively expressed by human
endothelial cells and is also present at high levels in human blood
in the absence of inflammation, these observations identify multiple
pathways whereby MIF may contribute to the promotion of inflam-
matory leukocyte recruitment.
We thank Dr. Robert Andrews (Australian Centre for Blood Diseases, Mon-
ash University) for the polyclonal anti-human P-selectin Ab, and Joanne
Mockler and Prof. Euan Wallace (Department of Obstetrics and Gynecol-
ogy, Monash University) for assistance with umbilical cord collections.
The Journal of Immunology1245
E.F.M. is a consultant to Cortical, a biotechnology company involved in the
development of anti-MIF therapies. The remaining authors have no financial
conflicts of interests.
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