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Reduced leukocyteendothelial cell interactions in the inflamed microcirculation of macrophage migration inhibitory factordeficient mice.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 9, September 2004, pp 3023–3034
DOI 10.1002/art.20470
© 2004, American College of Rheumatology
Reduced Leukocyte–Endothelial Cell Interactions in
the Inflamed Microcirculation of
Macrophage Migration Inhibitory Factor–Deficient Mice
Julia L. Gregory,1 Michelle T. Leech,1 John R. David,2 Yuan H. Yang,1
April Dacumos,1 and Michael J. Hickey1
space were also reduced significantly in MIFⴚ/ⴚ mice.
In each of these models, the level of P-selectin–
dependent rolling was reduced in MIFⴚ/ⴚ mice. Despite
this, no difference in P-selectin expression was observed
following LPS treatment. However, microvascular shear
forces were elevated in MIFⴚ/ⴚ mice, raising a possible
mechanism to explain the reduced interactions in these
animals.
Conclusion. MIFⴚ/ⴚ mice consistently displayed a
reduction in P-selectin–dependent rolling, suggesting
that MIF exerts proinflammatory effects, in part, via the
promotion of P-selectin–mediated rolling. Together,
these data indicate that MIF promotes interactions
between leukocytes and endothelial cells, thereby enhancing the entry of leukocytes into sites of inflammation.
Objective. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with established roles in a range of inflammatory conditions.
However, it is not known whether MIF influences inflammation via the direct promotion of leukocyte–
endothelial cell interactions. Therefore, the aim of these
experiments was to investigate the ability of MIF to
regulate leukocyte–endothelial cell interactions in the
inflamed microvasculature.
Methods. Intravital microscopy was used to examine postcapillary venules in the cremaster muscle
and synovium of wild-type and MIF ⴚ/ⴚ mice.
Leukocyte–endothelial cell interactions (rolling, adhesion, emigration) were compared under a range of
inflammatory conditions.
Results. In cremasteric postcapillary venules of
MIFⴚ/ⴚ mice, lipopolysaccharide (LPS)–induced leukocyte rolling, adhesion, and emigration were significantly
reduced relative to that in wild-type mice. Similar
responses were observed in response to tumor necrosis
factor ␣ and histamine. Examination of the synovial
microvasculature following exposure to carrageenan
revealed that leukocyte rolling and adhesion in synovial
postcapillary venules and leukocyte entry into the joint
Macrophage migration inhibitory factor (MIF)
was originally identified as an “activity” present in the
supernatant of cultured T cells following activation,
which could prevent the random migration of macrophages in vitro. This observation led to the hypothesis
that MIF could promote the retention of these leukocytes in sites of inflammation (1,2). The functions of this
proinflammatory cytokine are now recognized to extend
well beyond those revealed in these original experiments.
At a cellular level, MIF has well-characterized
activating effects on macrophages, inducing responses
such as cytokine release, increased phagocytic activity,
and augmentation of nitric oxide production (3–6).
Furthermore, there is now a growing body of evidence
that MIF plays a central role in promoting inflammatory
responses. MIF expression is up-regulated in human
rheumatoid arthritis (RA) as well as a number of animal
models of inflammation, including endotoxemia/sepsis,
Supported by a project grant from the National Health and
Medical Research Council of Australia. Dr. Hickey is a National
Health and Medical Research Council R. D. Wright Fellow.
1
Julia L. Gregory, BSc, Michelle T. Leech, MBBS, PhD, Yuan
H. Yang, PhD, April Dacumos, BSc, Michael J. Hickey, PhD: Centre
for Inflammatory Diseases, Monash University, Victoria, Australia;
2
John R. David, MD, PhD: Harvard School of Public Health, Boston,
Massachusetts.
Address correspondence and reprint requests to Michael J.
Hickey, PhD, Centre for Inflammatory Diseases, Monash University
Department of Medicine, Monash Medical Centre, 246 Clayton Road,
Clayton, Victoria 3168, Australia. E-mail: michael.hickey@med.
monash.edu.au.
Submitted for publication December 7, 2003; accepted in
revised form May 12, 2004.
3023
3024
delayed-type hypersensitivity (DTH), and adjuvantinduced arthritis (7–9). In addition, studies using either
MIF blockade or MIF-deficient (MIF⫺/⫺) mice have
repeatedly shown attenuation of inflammatory responses in animal models of arthritis, DTH, endotoxemia, colitis, and experimental allergic encephalomyelitis (EAE) (10–17). A number of potential mechanisms
for these apparent proinflammatory actions have been
identified, including promotion of monocyte Toll-like
receptor 4 (TLR-4) expression, increased macrophage
survival via inhibition of p53, and counterregulation of
the antiinflammatory effects of glucocorticoids (8,18–
20). However, the capacity of MIF to directly regulate
the key inflammatory mechanism common to each of
these models, i.e., leukocyte recruitment, has not been
directly assessed.
For leukocytes to be recruited to sites of inflammation, they must undergo a sequence of interactions
with the endothelium of postcapillary venules. Initially,
leukocytes tether and roll along the endothelial surface
until chemoattractant stimuli cause the rolling leukocytes to adhere to the endothelial surface. Some of these
adherent leukocytes detach and return to the circulating
pool. However, leukocytes that remain adherent are
then able to emigrate into the surrounding tissue (21).
These steps are mediated by adhesion molecules found
on both leukocytes and endothelial cells (22,23). The
potential for MIF to directly promote these interactions
has not been examined, although there is some evidence
to support this possibility. In a model of pulmonary
Pseudomonas aeruginosa infection, MIF⫺/⫺ mice displayed reduced neutrophil accumulation (14). In addition, endotoxemia-induced neutrophil accumulation in
the lung is reduced after treatment with anti-MIF antibody (24). Although these studies suggest that one of the
actions of MIF in inflammatory responses is to promote
leukocyte recruitment to affected tissues, this possibility
has never been directly assessed.
Therefore, the aim of this study was to determine
whether MIF promotes leukocyte–endothelial cell interactions during inflammatory responses. To examine this
issue, we used intravital microscopy to observe inflamed
postcapillary venules of the cremaster muscle and synovium in both wild-type and MIF⫺/⫺ mice, allowing the
direct assessment of leukocyte–endothelial cell interactions. In these experiments, MIF⫺/⫺ mice displayed
significant reductions in leukocyte rolling and adhesion,
as well as reduced entry of leukocytes into the site of
inflammation. These observations indicate that one of
the mechanisms whereby MIF may contribute to inflammatory responses is through the promotion of
GREGORY ET AL
leukocyte–endothelial cell interactions in the microcirculation.
MATERIALS AND METHODS
Animals. MIF⫺/⫺ mice were generated via homologous recombination in J1 embryonic stem cells as described
previously (14). They were maintained on a mixed background
of 129/Sv ⫻ C57BL/6. Wild-type mice of the same background
were bred from MIF⫹/⫹ littermates and used as controls.
Antibodies and reagents. Antibodies used for this
study were RB40.34, a monoclonal antibody (mAb) against
murine P-selectin (20 ␮g/mouse; BD Biosciences, San Diego,
CA); RME-1, a mAb against rat and mouse E-selectin (100
␮g/mouse) and RMP-1, a mAb against rat and mouse
P-selectin (100 ␮g/mouse) (both generously provided by Dr.
Andrew Issekutz, Dalhousie University, Halifax, Nova Scotia,
Canada); and 6C7.1, a mAb against murine vascular cell
adhesion molecule 1 (VCAM-1) (90 ␮g/mouse; hybridoma
generously provided by Drs. Dietmar Vestweber and Britta
Engelhardt, Max Planck Institut, Muenster, Germany). The
doses of all function-blocking antibodies used have been shown
to be effective in blocking their specific target molecules in
vivo (25,26). A110-1 (rat anti–keyhole limpet hemocyanin
[anti-KLH]; BD Biosciences) was used as a control mAb in
adhesion molecule expression experiments. RB6-8C5 (anti–
Gr-1), FA-11 (anti-CD68), and KT3 (anti-CD3) were purified
from hybridoma supernatants. Alexa Fluor protein labeling
kits were purchased from Molecular Probes (Eugene, OR).
Tumor necrosis factor ␣ (TNF␣) was purchased from R&D
Systems (through Bioscientific, Gymea, New South Wales,
Australia).
Local administration of lipopolysaccharide (LPS),
TNF␣, and carrageenan. To observe the effects of MIF on the
cremasteric microcirculation, LPS in 200 ␮l of saline was given
via intrascrotal injection, adjacent to the right cremaster
muscle. The cremaster muscle was prepared for intravital
microscopy 4 hours after injection, allowing time for significant
leukocyte recruitment to occur. Pilot experiments using doses
between 1 ng and 100 ng of LPS revealed that 10 ng induced a
consistent increase in leukocyte rolling, adhesion, and emigration without compromising microvascular blood flow.
A similar inflammatory response model was established in wild-type mice using TNF␣. The procedure for
injection and the time course of these experiments were the
same as for the LPS model. Pilot experiments using doses
between 50 ng and 500 ng of TNF␣ revealed that 50 ng of
TNF␣ induced consistent increases in leukocyte recruitment
that were similar to those observed previously for the LPS
model.
Carrageenan administration was used as a model of
joint inflammation, as previously described (27). Briefly, 20 ␮l
of carrageenan (1% in 0.5% carboxymethylcellulose in saline)
was injected into the joint using a 30-gauge needle. Sixty
minutes after injection, the synovial microvasculature was
prepared for intravital microscopy as described below.
Intravital microscopy. Cremaster muscle preparation.
Intravital microscopy of the mouse cremaster muscle was
performed as previously described (28). Briefly, mice were
anesthetized using a combination of 10 mg/kg of xylazine (Troy
LEUKOCYTE INTERACTIONS IN THE MICROCIRCULATION OF MIF⫺/⫺ MICE
Laboratories, Smithfield, New South Wales, Australia) and
200 mg/kg of ketamine hydrochloride (Pfizer, West Ryde, New
South Wales, Australia) administered via intraperitoneal injection. The left jugular vein was cannulated for administration of
additional anesthetic and antibodies. The mouse was then
placed on a thermo-controlled heating pad to regulate the core
temperature to 37°C. The cremaster muscle was dissected free
of surrounding tissues and exteriorized onto an optically clear
viewing pedestal. The muscle was cut longitudinally with a
cautery and held flat against the pedestal by attaching silk
sutures to the edges of the tissue. The muscle was then
superfused with bicarbonate buffered saline (pH 7.4; at 37°C)
and covered with a coverslip that was held in place with
vacuum grease.
An intravital microscope (Axioplan 2 Imaging; Carl
Zeiss Australia, Carnegie, Victoria, Australia) with a 20⫻
objective lens (20⫻/0.40 numerical aperture) and 10⫻ eyepiece was used to observe the cremasteric microcirculation. A
color video camera (Sony SSC-DC50AP; Carl Zeiss Australia)
was used to project the images onto a calibrated monitor (Sony
PVM-20N5E), and the images were recorded for playback
analysis using a videocassette recorder (Panasonic NV-HS950;
Klapp Electronics, Prahran, Victoria, Australia). We selected
1–3 venules (25–40 ␮m in diameter) for recording in each
experiment. Leukocyte rolling flux, rolling velocity, adhesion,
and emigration as well as the venular diameter (Dv) were
quantitated off-line using playback analysis, as previously
described (28,29). The centerline red blood cell (RBC) velocity
(VRBC) in the vessel was measured with an optical Doppler
velocimeter (Microcirculation Research Institute, Texas A&M
University, College Station, TX), and the mean RBC velocity
(Vmean) was determined as the VRBC/1.6. The venular wall
shear rate (␥) was calculated based on the Newtonian definition: ␥ ⫽ 8(Vmean/Dv) (28).
Histamine-induced rolling. To assess the role of MIF in
regulating histamine-induced rolling, the cremaster muscle of
an untreated mouse was exteriorized. Thirty minutes was
allowed for the initial elevated level of rolling to abate. At this
point, when rolling was equivalent in wild-type and MIF⫺/⫺
mice, histamine (100 ␮M in superfusion buffer) was superfused
over to the cremaster muscle continuously for 60 minutes, and
changes in rolling during the 60-minute period were assessed.
This concentration of histamine has been shown previously to
cause a rapid, P-selectin–dependent increase in leukocyte
rolling in rat mesenteric postcapillary venules (30).
Knee joint preparation. The murine synovial microcirculation of the knee joint was examined as previously described
(31). Briefly, the mouse was anesthetized, placed on a heating
pad as described above, and a catheter was inserted into the
tail vein to allow administration of additional anesthetic and
fluorescent markers. The left hind limb was immobilized with
the knee joint in slight flexion. The skin overlying the knee
joint was incised and resected, and the patellar tendon was
mobilized and divided distally. The patellar tendon and knee
joint were carefully removed, exposing the intraarticular synovial tissue. The exposed tissue was flushed with 5 ml of sterile
saline, then immersed in a saline-filled chamber, and covered
with a coverslip.
The synovial microvasculature was examined microscopically using a 20⫻ immersion lens (20⫻/0.50 WI). To
visualize leukocytes, mice were injected intravenously with 50
3025
␮l of 0.05% rhodamine 6G (Sigma-Aldrich, St. Louis, MO)
immediately prior to microscopy, as described previously (32).
Rhodamine 6G–associated fluorescence was visualized by epiillumination at 510–560 nm, using a 590-nm emission filter.
Microscopic images of the synovial microcirculation were
projected onto a monitor using a low-light video camera
(Dage-MTI IR-1000; Sci-Tech, Preston South, Victoria, Australia), and rolling and adhesion parameters were analyzed as
for the cremaster muscle.
Quantitation of MIF messenger RNA (mRNA) via
real-time polymerase chain reaction (PCR). Real-time PCR
was used to measure MIF mRNA in the cremaster muscle.
RNA was extracted from tissue samples via homogenization in
TRIzol (Gibco BRL, Grand Island, NY). Total RNA (1 ␮g)
was reverse transcribed using Superscript II reverse transcriptase (Gibco BRL) and oligo(dT)12-18. PCR amplification was
performed on a LightCycler (Roche Diagnostics, Castle Hill,
New South Wales, Australia) using SYBR Green I, as previously described (33). Murine MIF and ␤-actin PCR products
were used as assay standards. Amplification (40 cycles) was
carried out in a total volume of 10 ␮l containing 1 ␮l of dNTP
mixture, Taq polymerase, SYBR Green I dye, and the following primers at 3 pM: MIF 5⬘-TGACTTTTAGCGGCACGAAC-3⬘ and 5⬘-GACTCAAGCGAAGGTGGAAC-3⬘, and
␤-actin 5⬘-TGTCCCTGTATGCCTCTGGT-3⬘ and 5⬘-GATGTCACGCACGATTTCC-3⬘.
Melting curve analysis and agarose gel electrophoresis
were performed at the end of each PCR reaction. The level of
MIF mRNA expression was quantitated and expressed relative
to ␤-actin expression. The results are presented as the ratio of
mRNA expression in LPS-treated mice to that in untreated
animals.
Quantitation of P-selectin expression in the cremaster
muscle. P-selectin expression in the cremaster muscles of
LPS-treated wild-type and MIF⫺/⫺ mice was quantitated as
previously described (29). Briefly, Alexa 488–conjugated
RMP-1 (detected via epiillumination at 450–490 nm, with a
515-nm emission filter) was used to label P-selectin, and Alexa
594–conjugated anti-KLH mAb (detected using a 530–585-nm
excitation filter, with a 615-nm emission filter) was used as a
nonspecific control IgG. Images were visualized using a SIT
video camera (Dage-MTI VE-1000; Sci-Tech) on predefined
gain and black-level settings, and recorded for playback analysis using a video recorder. Four hours after LPS treatment,
recordings were made of background fluorescence in the
cremaster muscle at each of the excitation wavelengths, and
these data were then subtracted from subsequent intensity
readings.
Mice then received 100 ␮g of Alexa 488–labeled
RMP-1 and 20 ␮g of Alexa 594–labeled anti-KLH via intravenous injection. Antibodies were allowed to circulate for 5
minutes, and then the mouse was exsanguinated. P-selectin
expression in the exsanguinated muscle was then quantitated
exactly as previously described (29). Data were expressed as 1)
millimeters of positive vessels per square-millimeter of tissue
area and 2) the mean relative fluorescence intensity.
Flow cytometric analysis of leukocyte P-selectin–
binding activity. Whole blood was isolated from wild-type and
MIF⫺/⫺ mice, and RBCs were lysed with ammonium chloride.
Aliquots (100 ␮l) of the resultant leukocyte suspension were
incubated with 5 ␮g of P-selectin IgG fusion protein (BD
3026
GREGORY ET AL
Table 1. Leukocyte rolling flux, velocity, adhesion, and emigration in untreated versus LPS-treated
wild-type mice 5 hours after injection*
Mouse group
Rolling flux,
cells/minute
Rolling velocity,
␮m/second
Adhesion,
cells/100 ␮m
Emigration,
cells/field
Untreated (n ⫽ 6)
LPS-treated (n ⫽ 6)
48.4 ⫾ 8.9
89.6 ⫾ 12.4†
63.9 ⫾ 7.2
36.6 ⫾ 11.9†
3.0 ⫾ 0.3
34.2 ⫾ 7.2†
11.7 ⫾ 2.7
35.5 ⫾ 6.3†
* Values are the mean ⫾ SEM. LPS ⫽ lipopolysaccharide (10 ng).
† P ⬍ 0.05 versus untreated wild-type mice.
Biosciences) for 30 minutes at 37°C. Cells were then washed
and treated for 25 minutes with fluorescein isothiocyanate–
conjugated anti-human IgG (Chemicon Australia, Boronia,
Victoria, Australia) that had been preincubated with 5%
mouse serum. The percentage of cells displaying P-selectin–
binding activity and the mean fluorescence intensity of positive
cells were determined by analysis in a MoFlo flow cytometer
(Cytomation, Fort Collins, CO). These parameters were assessed for granulocytic and mononuclear cell populations,
which were identified using forward and side scatter properties.
Identification of infiltrated leukocytes via immunohistochemistry. LPS-treated leukocytes present in cremaster muscles were identified using 3-layer immunoperoxidase staining
as previously described (34). Following fixation in
paraformaldehyde–lysine–periodate, cryostat sections (6 ␮m)
of cremaster muscles were prepared and stained for neutrophils, macrophages, and T cells using mAb RB6-8C5 (anti–Gr1), mAb anti–FA-11 (anti-CD68), and mAb KT3 (anti-CD3),
respectively (34). The numbers of each of these cell types were
assessed on a scale of 0–4⫹.
Statistical analysis. Data are presented as the mean ⫾
SEM. Student’s t-tests were performed to compare experimental groups. P values less than 0.05 were considered significant.
(Figure 1A). In addition, leukocyte rolling velocity 4
hours after LPS administration was significantly higher
in MIF⫺/⫺ mice (mean ⫾ SEM 56.4 ⫾ 10.7 ␮m/second)
RESULTS
Reduction of LPS-induced leukocyte recruitment
in MIFⴚ/ⴚ mice. Since LPS is one of the best characterized stimuli of MIF release, we first examined leukocyte
trafficking induced by this mediator. Local treatment of
the cremaster muscle with LPS (10 ng for 4–5 hours)
induced a characteristic pattern of increased leukocyte
rolling flux, decreased rolling velocity, and increased
adhesion and emigration in wild-type mice (Table 1),
findings that are consistent with previous observations
(35). LPS treatment was also associated with a 1.6-fold
increase in MIF mRNA in the cremaster muscle of
wild-type mice, as assessed by real-time PCR (average
data from 4 mice) (data not shown). We then used this
model to compare leukocyte trafficking in wild-type and
MIF⫺/⫺ mice.
In LPS-treated MIF⫺/⫺ mice, leukocyte rolling
flux was reduced by ⬃50% relative to that in similarly
treated wild-type mice 4–5 hours after LPS treatment
Figure 1. Leukocyte rolling flux (A), adhesion (B), and emigration (C) in
cremasteric postcapillary venules of lipopolysaccharide (LPS)–treated (10
ng) wild-type (n ⫽ 6) and MIF⫺/⫺ (n ⫽ 6) mice at 4, 4.5, and 5 hours after
LPS treatment. ⴱ ⫽ P ⬍ 0.05 versus LPS-treated wild-type mice.
LEUKOCYTE INTERACTIONS IN THE MICROCIRCULATION OF MIF⫺/⫺ MICE
3027
Table 2. Hemodynamic parameters and circulating leukocyte counts in LPS-treated and TNF␣-treated wild-type and MIF⫺/⫺
mice 4–5 hours after treatment*
Shear rate, seconds⫺1
Venule
diameter, ␮m
4 hours
4.5 hours
5 hours
Leukocyte count,
⫻10⫺6/ml
30 ⫾ 1.6
28 ⫾ 1.0
350 ⫾ 57
668 ⫾ 109†
425 ⫾ 110
613 ⫾ 104
449 ⫾ 119
601 ⫾ 105
5.8 ⫾ 1.3
7.2 ⫾ 0.5
28 ⫾ 1.0
29 ⫾ 1.0
526 ⫾ 37
688 ⫾ 51†
486 ⫾ 34
645 ⫾ 63†
470 ⫾ 60
634 ⫾ 62†
5.7 ⫾ 1.1
12.3 ⫾ 1.6†
LPS-treated mice
Wild-type (n ⫽ 6)
MIF⫺/⫺ (n ⫽ 6)
TNF␣-treated mice
Wild-type (n ⫽ 6)
MIF⫺/⫺ (n ⫽ 6)
* Values are the mean ⫾ SEM. LPS ⫽ lipopolysaccharide (10 ng); TNF␣ ⫽ tumor necrosis factor ␣ (50 ng).
† P ⬍ 0.05 versus wild-type mice receiving the same treatment.
than in wild-type mice (26.3 ⫾ 3.6 ␮m/second) (P ⬍
0.012). Leukocyte adhesion was also significantly reduced in MIF⫺/⫺ mice at 4 and 5 hours (Figure 1B).
Moreover, 5 hours after LPS treatment, the number of
emigrated cells in MIF⫺/⫺ mice was significantly reduced relative to that in similarly treated wild-type mice
(Figure 1C). These reductions were not due to differences in the number of circulating leukocytes, since this
value did not differ significantly between the strains
(Table 2). However, the normal reduction in microvascular shear rate induced by LPS was absent in MIF⫺/⫺
mice, such that 4 hours after LPS dosing, the shear rate
was significantly elevated in MIF⫺/⫺ mice relative to that
in wild-type mice (Table 2). This difference was no
longer significant at 4.5 and 5 hours post-LPS.
Immunohistochemical analysis of the cremaster
muscle indicated that in wild-type mice, the infiltrate
consisted predominantly of Gr-1⫹ cells (neutrophils)
and FA-11⫹ cells (monocyte/macrophages), with occasional KT3⫹ cells (lymphocytes) (Table 3). Although
total leukocyte numbers were lower in the cremaster
muscles of MIF⫺/⫺ mice, the leukocyte subset proportions were similar in both groups.
Reduction of exteriorization-induced leukocyte
rolling in MIF ⴚ/ⴚ mice. To determine whether
leukocyte–endothelial cell interactions were altered in
MIF⫺/⫺ mice in the absence of exogenous mediators of
inflammation, we compared interactions in untreated
wild-type and MIF⫺/⫺ mice. Initially after tissue exteriorization, leukocyte rolling was significantly reduced in
MIF⫺/⫺ mice relative to that in wild-type mice (Figure
2A). At the same time point, rolling velocity was significantly increased (P ⬍ 0.02) in MIF⫺/⫺ mice (65.8 ⫾ 9.4
␮m/second) compared with wild-type mice (38.3 ⫾ 6.6
␮m/second) (Figure 2B). However, 30 minutes after
exteriorization, leukocyte rolling flux in wild-type mice
was reduced by ⬃40% and leukocyte rolling velocity was
increased, such that both parameters were no longer
statistically significantly different from those in MIF⫺/⫺
mice.
Leukocyte adhesion and emigration were consistently low in both groups and were not significantly
different. Microvascular shear rate did not differ between the groups throughout the observation period
(data not shown).
Reduction in TNF␣-induced leukocyte rolling
and adhesion in MIFⴚ/ⴚ mice. To determine if the
reduction in interactions observed in MIF⫺/⫺ mice was
restricted to LPS or whether they also occurred in
response to other inflammatory mediators, we examined
leukocyte recruitment induced by TNF␣. In wild-type
mice, TNF␣ (50 ng) induced a significant reduction in
Table 3. Immunohistochemical assessment of infiltrated leukocytes in cremaster muscles of wild-type
and MIF⫺/⫺ mice 4 hours after lipopolysaccharide treatment*
Wild-type mice
Individual scores
Group mean
MIF⫺/⫺ mice
Individual scores
Group mean
Gr-1 positive
FA-11 positive
KT3 positive
2⫹, 3⫹, 1⫹
2
3⫹, 2⫹, 2⫹
2.3
1⫹, 1⫹, 1⫹
1
1⫹, 1⫹, 1⫹, 1⫹
1
1⫹, 2⫹, 1⫹, 1⫹
1.3
1⫹, 1⫹
1
* The numbers of neutrophils (Gr-1 positive), monocyte/macrophages (FA-11 positive), and lymphocytes
(KT3 positive) were assessed on a scale of 0–4⫹.
3028
GREGORY ET AL
Figure 2. Leukocyte rolling flux (A) and rolling velocity (B) in cremasteric postcapillary venules of untreated wild-type (n ⫽ 6) and
MIF⫺/⫺ (n ⫽ 6) mice at 0, 30, and 60 minutes after tissue exteriorization. ⴱ ⫽ P ⬍ 0.05 versus untreated wild-type mice.
rolling velocity and significant increases in adhesion and
emigration, as previously described (36). Four hours
after TNF␣ treatment, leukocyte rolling flux did not
differ significantly between wild-type and MIF⫺/⫺ mice;
however, by 4.5 and 5 hours, leukocyte rolling flux in
MIF⫺/⫺ mice was significantly reduced relative to that in
wild-type mice (Figure 3A). In addition, the characteristic reduction in leukocyte rolling velocity induced by
TNF␣ was significantly attenuated in MIF⫺/⫺ mice at all
time points from 4 to 5 hours (at 4 hours, 17.2 ⫾ 2.6
␮m/second in wild-type mice versus 38.4 ⫾ 3.5 ␮m/
second in MIF⫺/⫺ mice; P ⬍ 0.001). Levels of leukocyte
adhesion were also significantly decreased in MIF⫺/⫺
mice relative to those in wild-type mice (Figure 3B).
As was seen in LPS-treated mice, microvascular
shear rates following TNF␣ treatment were significantly
higher in MIF⫺/⫺ mice than in wild-type mice (Table 2).
In contrast, TNF␣-induced leukocyte emigration did not
differ significantly between the 2 groups at 5 hours after
TNF␣ administration (Figure 3C).
Figure 3. Leukocyte rolling flux (A), adhesion (B), and emigration
(C) in cremasteric postcapillary venules of tumor necrosis factor ␣
(TNF␣)–treated (50 ng) wild-type (n ⫽ 6) and MIF⫺/⫺ (n ⫽ 6) mice
at 4, 4.5, and 5 hours after initiation of TNF␣ treatment. ⴱ ⫽ P ⬍ 0.05
versus TNF␣-treated wild-type mice.
Roles of endothelial selectins in LPS-induced
leukocyte rolling. The decreased leukocyte rolling observed in LPS-treated MIF⫺/⫺ mice raised the possibility
of altered usage of the molecules responsible for rolling
under these conditions. Therefore, we next determined
which endothelial adhesion molecules were responsible
for rolling in LPS-treated wild-type mice. In LPS-treated
wild-type mice, P-selectin blockade eliminated ⬎95% of
the leukocyte rolling. Similarly, in MIF⫺/⫺ mice, anti–Pselectin treatment virtually abolished rolling (Figure 4).
In contrast, blockade of either E-selectin or VCAM-1 in
LPS-treated wild-type mice did not alter leukocyte roll-
LEUKOCYTE INTERACTIONS IN THE MICROCIRCULATION OF MIF⫺/⫺ MICE
Figure 4. Role and expression of P-selectin in lipopolysaccharide
(LPS)–treated wild-type and MIF⫺/⫺ mice 4 hours after LPS (10 ng)
treatment. A, Effects of P-selectin blockade on leukocyte rolling flux in
cremasteric postcapillary venules of LPS-treated wild-type (n ⫽ 6) and
MIF⫺/⫺ (n ⫽ 6) mice. ⴱ ⫽ P ⬍ 0.05 versus LPS-treated MIF⫺/⫺ mice.
mAb ⫽ monoclonal antibody. B and C, LPS-induced P-selectin
expression in cremasteric postcapillary venules in wild-type (WT; n ⫽
7) and MIF⫺/⫺ (n ⫽ 7) mice. The average length of vessels positive for
P-selectin expression is shown in B; the mean intensity of P-selectin
expression within individual postcapillary venules is shown in C.
ing flux or velocity significantly (data not shown), indicating that these adhesion molecules played no role in
mediating rolling in this low-dose LPS model. Together,
these findings indicate that in this model of LPS treatment, P-selectin is the predominant mediator of rolling
in both wild-type and MIF⫺/⫺ mice and that the reduced
leukocyte rolling observed in MIF⫺/⫺ mice is due to a
reduction in P-selectin–dependent interactions.
To determine if the reduction in P-selectin–
dependent interactions in MIF⫺/⫺ mice was due to an
alteration in endothelial P-selectin expression, we next
3029
compared LPS-induced P-selectin expression in wildtype and MIF⫺/⫺ mice, using fluorochrome-conjugated
mAb. It was previously established that local LPS induces up-regulation of P-selectin in the cremaster muscle (35). Comparison of P-selectin expression in wildtype and MIF⫺/⫺ mice 4 hours after LPS treatment
revealed that the number of vessels expressing P-selectin
and the level of P-selectin expression within individual
venules did not differ significantly between the 2 types of
mice (Figures 4B and C).
It is also conceivable that the reduced rolling
reflected an altered ability of circulating leukocytes from
MIF⫺/⫺ mice to bind P-selectin. Therefore, we assessed
P-selectin binding activity in leukocytes from each
mouse strain. Examination of both the granulocyte
compartment and the mononuclear leukocytes revealed
that there was no difference in P-selectin binding activity
between leukocytes from wild-type and MIF⫺/⫺ mice
(data not shown).
MIF and rapid expression of P-selectin. In murine endothelial cells, mediators such as TNF␣ and LPS
induce expression of P-selectin in a protein synthesis–
dependent manner (37). However, mediators such as
histamine are capable of inducing P-selectin expression
via an alternate pathway, such that preformed P-selectin
is rapidly (within 5 minutes) translocated to the endothelial surface (30). In the present study, the observation
that the initial increase in leukocyte rolling seen in the
cremaster muscle of naive wild-type mice immediately
after exteriorization (known to be P-selectin–dependent
[25]) was absent in MIF⫺/⫺ mice raised the possibility
that MIF may promote this rapid translocation of
P-selectin. Therefore, we compared histamine-induced
leukocyte rolling in cremasteric postcapillary venules in
wild-type and MIF⫺/⫺ mice (Figure 5).
In wild-type mice, histamine (100 ␮M) induced a
rapid increase in leukocyte rolling, peaking 5 minutes
after initial exposure, before partially abating to a level
of ⬃80 cells/minute. At the peak of the rolling response,
treatment with anti–P-selectin mAb eliminated rolling
(data not shown), indicating that this response was
mediated exclusively by P-selectin. Histamine-treated
MIF⫺/⫺ mice showed a similar initial profile of response,
with rolling peaking at the same time and the same level
as in wild-type mice. However, in contrast to wild-type
mice, in MIF⫺/⫺ mice, rolling rapidly returned to basal
levels (⬃40 cells/minute), such that 20 minutes after
initiation of histamine superfusion, rolling in MIF⫺/⫺
mice was significantly lower than that in wild-type mice.
This significant difference persisted for the remainder of
the 60-minute histamine treatment (Figure 5).
3030
Figure 5. Effect of histamine (100 ␮M) on leukocyte rolling flux in
cremasteric postcapillary venules of wild-type (n ⫽ 6) and MIF⫺/⫺
(n ⫽ 6) mice. Histamine was superfused over the cremaster muscle
continuously from time 0 to 60 minutes. ⴱⴱ ⫽ P ⬍ 0.05 versus the
–5-minute time point (before histamine treatment) for each group of
mice; ⌿ ⫽ P ⬍ 0.05 versus wild-type mice at the corresponding time
point.
Reduction of synovial leukocyte trafficking in
MIFⴚ/ⴚ mice. Given the proinflammatory role of MIF in
experimental arthritis and human RA, we were also
interested in examining by intravital microscopy the role
of MIF in regulating leukocyte trafficking in the synovial
microvasculature. In untreated mice, leukocyte rolling
was detected in synovial venules, consistent with previous observations (31). Use of anti–P-selectin mAb revealed that this constitutive rolling was entirely dependent on P-selectin (data not shown). Furthermore,
similar to the observations in the cremaster muscle, the
level of leukocyte rolling flux observed in synovial postcapillary venules immediately upon exteriorization was
significantly reduced in MIF⫺/⫺ mice (Figure 6). Minimal leukocyte adhesion (⬍2 cells/100 ␮m) was observed
in untreated mice of either strain.
We then examined the effects of carrageenan.
Figure 7 illustrates that carrageenan induced significant
increases in leukocyte rolling and adhesion in wild-type
mice. Comparison of carrageenan-induced rolling flux
and adhesion in wild-type and MIF⫺/⫺ mice revealed
that these interactions were significantly reduced in
MIF⫺/⫺ mice (Figures 8A and C). In addition, the
leukocyte count in synovial lavage fluid was significantly
reduced in MIF⫺/⫺ mice compared with wild-type mice
(Figure 8D), indicating that the absence of MIF also
resulted in a reduction in leukocyte egress from the
synovial microvasculature. In both strains of mice,
carrageenan-induced leukocyte rolling in the synovial
microvasculature was abolished with an anti–P-selectin
mAb (Figure 8B). Together, these findings illustrate that
GREGORY ET AL
Figure 6. Leukocyte rolling flux in synovial postcapillary venules of
untreated wild-type (n ⫽ 7) and MIF⫺/⫺ (n ⫽ 8) mice. ⴱ ⫽ P ⬍ 0.05
versus wild-type mice.
the ability of MIF to promote leukocyte recruitment
extends to the synovial microvasculature and that, as
seen in the LPS model, the absence of MIF was associated with a reduction in P-selectin–mediated rolling.
DISCUSSION
There is a growing body of evidence that MIF
contributes to the development of inflammatory responses induced by a wide range of stimuli (3,8,10,13).
However, whether MIF regulates one of the most critical
aspects of inflammatory responses, leukocyte recruitment, remains unknown. Therefore, in order to examine
this issue, we used intravital microscopy to directly assess
leukocyte–endothelial cell interactions in the microvasculature of wild-type and MIF⫺/⫺ mice. In the cremasteric microvasculature, the absence of MIF was associated with significant reductions in leukocyte rolling and
adhesion induced by LPS and TNF␣, as well as LPSinduced leukocyte emigration. Similar findings were
observed in the synovial microvasculature in response to
carrageenan, demonstrating that this putative function
of MIF is also active in the setting of joint inflammation.
MIF⫺/⫺ mice also displayed reductions in the leukocyte
rolling induced by mild inflammatory stimuli such as
tissue exteriorization and histamine treatment. Together, these findings reveal a previously undescribed
function of MIF in the regulation of leukocyte–
endothelial cell interactions and leukocyte entry into
inflamed tissues and demonstrate an additional mechanism whereby MIF may promote inflammatory responses.
Data from previous studies have raised the possibility that MIF directly promotes leukocyte recruitment in inflammation. MIF immunoneutralization has
LEUKOCYTE INTERACTIONS IN THE MICROCIRCULATION OF MIF⫺/⫺ MICE
3031
Figure 8. Leukocyte rolling flux (A and B) and adhesion (C) in
synovial vessels and leukocyte numbers in the joint space as assessed by
joint lavage (D) in carrageenan-treated wild-type (n ⫽ 6) and MIF⫺/⫺
(n ⫽ 6) mice. The effect of P-selectin blockade on synovial leukocyte
rolling in separate groups of carrageenan-treated wild-type (n ⫽ 4)
and MIF⫺/⫺ (n ⫽ 5) mice is shown in B. ⴱ ⫽ P ⬍ 0.05 versus wild-type
mice. mAb ⫽ monoclonal antibody.
Figure 7. Intravital microscopic images of synovial postcapillary
venules (broken lines indicate margins) of wild-type mice, demonstrating interacting rhodamine 6G–labeled leukocytes. A, Low level of
leukocyte–endothelial cell interactions in an untreated knee. B,
Marked increase in the number of leukocytes interacting with the
venular endothelium 75 minutes after injection of carrageenan into the
joint. Arrows show adherent leukocytes, with the remainder undergoing rolling interactions. C, Rolling flux and D, adhesion of leukocytes
in synovial vessels after carrageenan (Carrag) treatment in wild-type
mice compared with saline-treated wild-type mice. ⴱ ⫽ P ⬍ 0.05 versus
saline-treated wild-type mice.
been shown to reduce leukocyte numbers in inflamed
tissues in models of DTH, arthritis, colitis, and EAE and
in the lung in response to endotoxemia (11,16,17,24,38).
However, whether these reductions in leukocyte accumulation were due to an ability of MIF to promote the
entry of leukocytes into these sites has not been directly
assessed. Indeed, an alternative explanation for these
observations is that MIF promotes the survival of leukocytes that are present in inflammatory lesions. This
possibility is supported by the finding that MIF can
inhibit p53-dependent apoptosis (20,39). In the present
study, however, the potential for apoptosis to affect the
number of leukocytes present in tissues was minimized
by the use of acute models of leukocyte recruitment.
Over the time courses studied, it is unlikely that the
effects of MIF on apoptosis or proliferation could
explain the observed differences in the numbers of
recruited leukocytes. An alternative hypothesis supported by the present observations is that MIF promotes
leukocyte recruitment via effects on leukocyte–
endothelial cell interactions.
One possible explanation for the reduced interactions in MIF⫺/⫺ mice in some of the models examined
here relates to effects on microvascular shear rate. The
propensity for a leukocyte to undergo interactions with
the endothelial surface is determined by the balance
between proadhesive forces and the dispersive effects of
the flowing blood, as evaluated by the microvascular
shear rate (40). In both the LPS-treated and the TNF␣treated mice, the microvascular shear rate was signifi-
3032
cantly higher in MIF⫺/⫺ mice at various points between
4 and 5 hours after treatment. This correlated with
reduced rolling and adhesive interactions within postcapillary venules in each of these models. It is possible
that one of the myriad effects of MIF is to interact with
an as-yet-unidentified pathway that controls the microvascular shear rate. The resultant reduction in this
parameter then enhances the ability of leukocytes in the
mainstream of the blood flow to undergo interactions
with the endothelial surface. However, the observation
that in histamine-treated mice, leukocyte rolling is reduced in MIF⫺/⫺ mice in the absence of an alteration in
the microvascular shear rate indicates that this is not the
sole mechanism whereby MIF promotes leukocyte–
endothelial cell interactions.
The decrease in rolling and adhesive interactions
in MIF⫺/⫺ mice is suggestive of decreases in adhesion
molecule expression and/or function. There are few
existing data on the ability of MIF to regulate adhesion
molecule expression. MIF blockade has been observed
to result in a reduction in ICAM-1 expression in a model
of gastric inflammation and in a reduction in ICAM-1
and VCAM-1 expression in experimental glomerulonephritis, providing indirect evidence of a role for MIF in
supporting adhesion molecule expression (41,42). Analysis of the direct effects of MIF has shown that MIF can
induce small increases in the expression of ICAM-1 by
keratinocytes and dendritic cells in vitro (43). Furthermore, recombinant human MIF has been observed to
induce the expression of ICAM-1 by endothelial cells
(44). These experiments raise the possibility that MIF
can directly mediate the up-regulation of other endothelial adhesion molecules.
Given that all of the leukocyte rolling in the
LPS-treated mice was P-selectin–dependent, we examined the effect of the absence of MIF on P-selectin
expression in vivo following LPS treatment. These experiments showed that the level of P-selectin expression
in postcapillary venules did not differ between wild-type
and MIF⫺/⫺ mice. Similarly, leukocytes from wild-type
and MIF⫺/⫺ mice displayed comparable abilities to bind
murine P-selectin, suggesting that alterations in leukocyte function were not responsible for these observations. These findings indicated that alterations in endothelial P-selectin expression or leukocyte selectin ligand
function were not responsible for the reduced interactions observed in the present study.
Another possible explanation for the reduction in
leukocyte–endothelial cell interactions in MIF⫺/⫺ mice
is the absence of the MIF-mediated counterregulation of
the antiinflammatory effects of glucocorticoids. MIF is
GREGORY ET AL
unique among proinflammatory cytokines in being a
homeostatic regulator of the effects of glucocorticoids
(18). This has been illustrated both in vitro, where MIF
has been shown to override the glucocorticoid-mediated
inhibition of cytokine release by LPS-stimulated human
monocytes, and in vivo, by the observation that MIF can
reverse the antiinflammatory effects of glucocorticoids
during murine antigen-induced arthritis (16,18). These
studies have led to the hypothesis that MIF and glucocorticoids reciprocally regulate inflammatory responses. It is increasingly well accepted that constitutively expressed glucocorticoids serve to limit a number
of inflammatory processes. This ability extends to the
regulation of rolling and adhesion induced by inflammatory stimuli such as LPS and histamine (45,46). In light
of these findings, it is plausible that the reduced
leukocyte–endothelial cell interactions observed in
MIF⫺/⫺ mice result from unopposed activity of glucocorticoids in these animals.
It has previously been shown that MIF deficiency
is associated with a reduction in macrophage expression
of the cellular receptor for LPS, TLR-4 (19). Indeed,
this has been raised as a plausible mechanism for the
reduced susceptibility of MIF⫺/⫺ mice to endotoxemiainduced mortality. It is conceivable that this mechanism
was also responsible for the reduction in LPS-induced
leukocyte recruitment observed in the present study.
This prompted us to examine models of leukocyte
recruitment induced by the alternative stimuli, TNF␣
and carrageenan. In both the TNF␣ and carrageenan
models, it was noteworthy that rolling and adhesion were
reduced in the absence of MIF, which is consistent with
the observations from LPS treatment. Similarly, in the
carrageenan model, leukocyte emigration was reduced
in MIF⫺/⫺ mice. Given that neither of these stimuli is
thought to utilize TLR-4, this indicates that mechanisms
distinct from a reduction in TLR-4 expression are responsible for the modifications in leukocyte–endothelial
cell interactions in MIF⫺/⫺ mice.
It is noteworthy that in response to TNF␣, leukocyte rolling does not differ between wild-type and
MIF⫺/⫺ mice 4 hours after stimulation, although subsequently, rolling decreases more rapidly in MIF⫺/⫺ mice.
It is conceivable that this stems from differential alterations in adhesion molecule expression within this time
frame. Indeed, in response to some mediators, MIF may
not be required for leukocyte rolling flux to reach peak
levels. However, it may have an important function in
preventing re-internalization of adhesion molecules
from the endothelial surface, thereby prolonging expression. This contention is supported by the observations
LEUKOCYTE INTERACTIONS IN THE MICROCIRCULATION OF MIF⫺/⫺ MICE
from the histamine model, in which the peak level of
P-selectin–dependent rolling did not differ between the
2 strains, but rolling dissipated significantly more rapidly
in MIF⫺/⫺ mice. Moreover, in contrast to the LPS
model, no difference in TNF␣-induced leukocyte emigration was detected. During inflammatory responses,
adherent leukocytes respond to chemotactic cues that
cause them to exit the vasculature and enter tissues. It is
probable that the chemotactic mediators that promote
leukocyte emigration in response to local LPS administration differ from those at work in response to TNF␣.
The present data suggest that MIF promotes the chemotactic mechanisms induced by LPS, but not those
invoked by TNF␣.
We have previously reported elevated expression
of MIF in serum, synovial fluid, and cultured synoviocytes from RA patients, and have described an association between disease activity in RA, synovial cellularity,
and synovial MIF expression (8,9). In the present study,
the absence of MIF is associated with a reduction in
leukocyte–endothelial cell interactions in inflamed
cremasteric and synovial venules. These findings illustrate a novel mechanism whereby MIF may promote
inappropriate inflammatory responses such as those
which occur in RA.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
ACKNOWLEDGMENTS
The authors would like to acknowledge the kind assistance of Dr. Andrew Issekutz (Dalhousie University, Halifax,
Nova Scotia, Canada) for providing the RMP-1 and RME-1
antibodies and Drs. Dietmar Vestweber and Britta Engelhardt
(Max Planck Institut, Muenster, Germany) for providing the
6C7.1 antibody.
18.
19.
20.
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