MIP-1alpha and MCP-1 Induce Migration of Human Umbilical Cord Blood Cells in Models of Stroke.

Abstract

Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein (MIP-1alpha) are implicated in monocyte infiltration into the central nervous system (CNS) under pathological conditions. We previously showed that in vivo human umbilical cord blood cells (HUCB) migrate toward brain injury after middle cerebral artery occlusion (MCAO). We hypothesized that MCP-1 and MIP-1alpha may participate in the recruitment of HUCB towards the injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), and 24 hours later the production of MCP-1 and MIP-1alpha in the brain was examined with immunohistochemistry, ELISA, and western blotting. The chemotactic effect of MCP-1 and MIP-1alpha, and the expression of MCP-1 receptor CCR2 and MIP-1alpha receptor CCR1, CCR5 on the surface of HUCB were also examined. MCP-1 and MIP-1alpha expression were significantly increased in the ischemic hemisphere of brain, and significantly promoted HUCB cell migration compared to the contralateral side. This cell migration was neutralized with polyclonal antibodies against MCP-1 or MIP-1alpha. Also chemokine receptors were constitutively expressed on the surface of HUCB cells. The data suggested that the increased chemokines in the ischemic area can bind cell surface receptors on HUCB, and induce cell infiltration of systemically delivered HUCB cells into the CNS in vivo.

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Current Neurovascular Research, 2008, 5, 118-124

1567-2026/08 $55.00+.00 ©2008 Bentham Science Publishers Ltd.
MIP-1? and MCP-1 Induce Migration of Human Umbilical Cord Blood
Cells in Models of Stroke
Lixian Jiang1,2,3, Mary Newman1,2,4, Samuel Saporta1-3, Ning Chen1,2, Cyndy Sanberg7,
Paul R. Sanberg1-5,7 and Alison E. Willing1-3,6,*
1Center of Excellence for Aging and Brain Repair, Departments of 2Neurosurgery, 3Pathology and Cell Biology, 4Psy-
chology, 5Psychiatry, 6Molecular Pharmacology & Physiology, University of South Florida, MDC78, 12901 Bruce B.
Downs Blvd, Tampa, FL 33612 and 7Saneron CCEL Therapeutics, Inc., 3802 Spectrum Blvd., Suite 145, Tampa, FL
33612, USA
Abstract: Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein (MIP-1?) are implicated
in monocyte infiltration into the central nervous system (CNS) under pathological conditions. We previously showed that
in vivo human umbilical cord blood cells (HUCB) migrate toward brain injury after middle cerebral artery occlusion
(MCAO). We hypothesized that MCP-1 and MIP-1? may participate in the recruitment of HUCB towards the injury.
Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), and 24 hours later the production of
MCP-1 and MIP-1? in the brain was examined with immunohistochemistry, ELISA, and western blotting. The chemotac-
tic effect of MCP-1 and MIP-1?, and the expression of MCP-1 receptor CCR2 and MIP-1? receptor CCR1, CCR5 on the
surface of HUCB were also examined. MCP-1 and MIP-1? expression were significantly increased in the ischemic hemi-
sphere of brain, and significantly promoted HUCB cell migration compared to the contralateral side. This cell migration
was neutralized with polyclonal antibodies against MCP-1 or MIP-1?. Also chemokine receptors were constitutively ex-
pressed on the surface of HUCB cells. The data suggested that the increased chemokines in the ischemic area can bind cell
surface receptors on HUCB, and induce cell infiltration of systemically delivered HUCB cells into the CNS in vivo.
Key Words: ? chemokines, stroke, human umbilical cord blood, migration, Transplantation.
INTRODUCTION

(HUCB) decreases neural damage in rodents subjected to
middle cerebral artery occlusion (MCAO), and provides bet-
ter behavioral and neurological recovery in a rodent model of
stroke compared to direct intracranial delivery (Willing et
al., 2003). While an additional advantage of the systemic
route is the ease of delivery, how these cells migrate into the
brain to produce their effects is not clear, since the brain is
insulated by the blood brain barrier (BBB) and has immune
privilege. One possibility is that BBB is disrupted after the
ischemic insult and granulocytes, macrophages, and other
inflammatory cells leak into the ischemic brain passively. A
second possibility is that stroke up-regulated chemoattrac-
tants, which promote HUCB cell migration into the brain.
One of the potential chemoattractants is monocyte chemoat-
tractant protein-1 (MCP-1). MCP-1 has been detected in
injured brain after stroke (Babock et al., 2003), predomi-
nantly in astrocytes (Che et al., 2001). Other studies found
that hypoxia-ischemia increases MCP-1 expression in multi-
ple cell types around the site of injury in neonatal rodent

Intravenous delivery of human umbilical cord blood cells
*Address correspondence to this author at the Center of Excellence for
Aging & Brain Repair, MDC78, University of South Florida, 12901 Bruce
B. Downs Blvd, Tampa FL 33612, USA; Tel: 813-974-7812; Fax: 813-974-
3078; E-mail: awilling@health.usf.edu
Received: December 27, 07, Revised: April 2, 08, Accepted: April 4, 08
brain (Ivacko et al., 1997). Following MCP-1 expression,
leukocytes could be found in the lesioned hippocampus. In
addition, mutant mice with a CCR2 (MCP-1 receptor) deficit
had neither T cells nor macrophage infiltration in the dener-
vated hippocampus, suggesting a critical role for MCP-1 and
its receptor CCR2 in leukocyte migration (Babock et al.,
2003).

ficking of lymphoid and mononuclear cell into the CNS is
macrophage inflammatory protein (MIP-1?). It is up regu-
lated as early as 3 to 6 hr post stroke in the ipsilateral hemi-
sphere to the stroke, whereas the level of MIP-1? in the con-
tralateral hemisphere was similar to control levels (Kim et
al., 1995a; Takami et al., 1997). MIP-1? mRNA was also
found present in an immortalized microglia cell line, cortical
astrocytes and monocytes in culture (Murphy et al., 1995). In
vivo, MIP-1? expression was correlated with mononuclear
cell infiltration (Glabinski et al., 1998). Further, extracts
from ischemic brain induced human bone marrow stromal
cell migration in culture, and this cell migration could be
blocked by an MIP-1? antibody, suggesting a chemo-
attractant effect of MIP-1? (Wang et al., 2002).
In this study we examined whether MIP-1? and MCP-1
expression increased in the damaged hemisphere after stroke
and if the presence of these chemokines in extracts of stroke
brain would induce HUCB migration in an ex vivo assay
system.
Another potential chemoattractant implicated in the traf-

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Chemokine Induced Migration of Cord Blood in Stroke Current Neurovascular Research, 2008, Vol. 5, No. 2 119
MATERIALS AND METHODS
Middle Cerebral Artery Occlusion (MCAO)

the MCAO or normal groups as previously described
(Willing et al., 2003). Briefly, animals were anesthetized
with isoflurane (2-5% in 2 L/min O2). The right common,
external and internal carotid arteries were isolated and an
embolus inserted retrogradely through the external carotid,
into the internal carotid, past the base of the skull to the ori-
gin of the MCA (approximately 25mm from insertion). The
filament was permanently anchored in place and the incision
closed. All animals were euthanized 24h post surgery, and
the brains were harvested. Some of the brains were fixed
with 4% paraformaldehyde in PBS, and the others were im-
mediately frozen in liquid nitrogen.
Twenty Sprague Dawley rats were randomly assigned to
Neural Cell Hypoxia Culture

fetal rat brain at embryonic day (E) 17 as described (Mackay,
2001; Rothenberg et al., 1999), and grown separately in cul-
ture to confluency. Before hypoxia, the gas-tight chamber
(Hornung et al., 2000; Wang et al., 2002) was flushed with
5% CO2 and 95% N2 and the plates washed with low glucose
color free DMEM medium. For the hypoxia group, medium
was changed to 1 ml hypoxia pre-treated, low glucose, color
free DMEM and cultures placed in the gas-tight chamber.
The cultures were exposed to hypoxia (5% CO2 and 95%N2)
at 37oC for 2 hours (Lombardi et al., 2003). The control
group was treated with low glucose color free DMEM under
normoxic conditions for 2 hour. The media were harvested,
and cells prepared for western blotting.
Neurons, astrocytes and microglia were isolated from
Immunohistochemistry Staining
MIP-1? (or MCP-1) Double Labeling

thick. The sections were blocked with 10% goat serum, 0.3%
Triton X100 in PBS for 1 hour, and then incubated with pri-
mary antibody cocktail at 4oC overnight. The cocktail con-
sisted of goat anti MIP-1? (Santa Cruz; 1:100) or rabbit anti
MCP-1 (Novus; 1:100) with either mouse anti OX-42
(CD11b/c) (Abcam; 1:100), mouse anti TuJ1 (Chemicon;
1:400) or chicken anti GFAP (Chemicon; 1:100). After
washing, the sections were incubated with a secondary anti-
body cocktail of rhodamine-conjugated goat anti-rabbit IgG
(Molecular Probes; 1:500) and FITC-conjugated goat anti-
mouse IgG (Molecular Probes; 1:500). The sections were
mounted and examined with a confocal microscope.
The fixed brains were sectioned on a cryostat at 30?m
Chemokine Receptor Immunolabeling

washed and then air dried. The slides were stained with pri-
mary and secondary antibody as described above. The pri-
mary antibodies used were goat anti-human CCR5 (Ca-
pralogics Inc, 1:200), rabbit anti- human CCR1, and mouse
anti-human CCR2 (Abcam Inc, 1:200). Secondary antibodies
were FITC-conjugated goat anti-mouse IgG or rhodamine-
conjugated goat anti-rabbit IgG (Molecular Probes; 1:500)
(Molecular Probes; 1:500).
HUCB smears were fixed with 4% paraformaldehyde,
Western Blotting

to MCAO) and non-stroke side (contralateral to MCAO) and
the tissue lysed. An equivalent amount of protein was loaded
on SDS-12% polyacrylamide gel and transferred to nitrocel-
lulose paper. The membranes were immunoblotted with anti-
rat MIP-1? (Chemicon) followed by horseradish peroxidase-
conjugated secondary antibody. After the final wash, mem-
branes were probed using enhanced chemiluminescence dye
(ECL, Amersham Pharmacia Biotech, Piscataway, NJ) and
autoradiographed. Neural cells from culture were treated
similarly.
The brains were cut sagitally into stroke side (ipsilateral
MCP-1 ELISA Assay

stroked side. Tissues were weighed, placed in clear DMEM
(Gibco, 150mg/ml), homogenized and centrifuged at 2000g
for 20 minutes. The supernatants were collected, filtered and
adjusted to the same protein concentration. The ELISA assay
was performed according to manufacturer’s protocol (Amer-
sham Bioscience), and the concentration of chemokine was
determined on a plate reader at absorbance of 450nm and
550nm.
The brains were cut sagitally into stroked side and non-
Cell Migration Assay

ples (300?L) with or without MCP-1 or MIP-1? antibodies
(1:100) were pipetted into the bottom wells of a 96-well
plate. Freshly thawed HUCB cells were directly pipetted into
the top well at a concentration of 100,000 cells per 60 ?L.
The migration chamber was incubated at 37oC with 5% CO2
from 4 hours to 24 hours. The top well plate was then re-
moved and the bottom plate was centrifuged, and 200?l me-
dia removed. The number of migrated cells was determined
with Cell Titer-Glo Luminescent Cell Viability Assay,
(Promega) according to the manufacturer’s protocol. The
plate was read in a plate reader. The migration assays were
performed twice. Each sample, control and standard was
performed in triplicate.
The standard (chemokine protein) or tissue extract sam-
RESULTS
Presence of MIP-1? and MCP-1 in the Stroked Brain

after MCAO using immunolabeling. No positive staining
was found in the contralateral hemisphere. Double immuno-
chemistry staining revealed that both MCP-1 and MIP-1?
were found in neurons (Fig. (1)), astrocytes (Fig. (2)), and
some microglia (Fig. (3)). MIP-1? expression was verified
with western blotting (Fig. (4A)) from extracts of stroked
brain. Further, in enriched cultures, MIP-1? was expressed in
cultured astrocytes and neurons. After hypoxia, MIP-1? in-
creased in neurons compared to neurons only exposed to
normoxia conditions (Fig. (4B)). MCP-1 expression was
verified with ELISA. It was significantly increased on the
stroked side of the brain compared to the contralateral side
after stroke (p<0.05) (Fig. (5A)). In enriched cultures, hy-
poxia induced MCP-1 expression particularly in microglia
and astrocytes (p<0.05) (Fig. (5B)).
Both MCP-1 and MIP-1? were found in the brain 24 hour

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120 Current Neurovascular Research, 2008, Vol. 5, No. 2
Jiang et al.



















Fig. (1). MCP-1and MIP-1? expression in neurons after stroke. Stroked rat brain was immunolabelled with MCP-1 (A, red) and then la-
beled with an antibody to TuJ1 to identify immature neurons (B, green). (C) MCP-1 was found in neurons on the ipsilateral (stroked) side of
the brain. (D) MIP-1? (red), (E) TuJ1 and (F) MIP-1? positive neurons (TuJ1, yellow, arrows).

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Fig. (2). MCP-1and MIP-1? expression in astrocytes after stroke. Stroked rat brain was immunolabelled with MCP-1 (A, C, red) or MIP-
1? (D, F, red), and then double labeled with antibody to GFAP (astrocytes, B, E, green). Both MCP-1 and MIP-1? could be found in astro-
cytes of the infarcted area (merged C, F). Inset in C showing that not all GFAP+ astrocytes expressed MCP-1.
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Chemokine Induced Migration of Cord Blood in Stroke
?
Current Neurovascular Research, 2008, Vol. 5, No. 2 121
?

















Fig. (3). MCP-1and MIP-1? expression in microglia after stroke. Stroked rat brain was immunolabelled with MCP-1 (A, C, red) or MIP-
1? (D, F, red), and then double labeled with antibody to OX-6 (microglia, B, E, green). Both MCP-1 and MIP-1? could be found in microglia
of the infarcted area (merged C, F).


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Fig. (4). Western blot analysis of MIP-1? protein expression in
the brain after MCAO. (A) MIP-1? was present within the ipsilat-
eral (Ipsi, stroked) hemisphere and not on the contralateral side of
the brain (Contra). PC = positive control. (B) MIP-1? was ex-
pressed in enriched cultures of neurons, astrocytes and microglia
under hypoxic (H) and normoxic (N) conditions.
Chemotactic Effect of MCP-1 and MIP-1? on HUCB Cell
Migration

receptor CCR2 were all expressed on the cell surface of
HUCB cells (Fig. (6)) suggesting that HUCB cells could
respond to expression of these chemokines. Indeed, MIP-1?
induced migration in vitro, especially at the lowest dose, 30
ng/ml (p<0.05; Fig. (7A)). HUCB cell migration to MCP-1
reached its maximum at a concentration 600 ng/ml although
MIP-1? receptor CCR1 and CCR5 as well as MCP-1



















Fig. (5). Protein expression in MCP-1 brain after MCAO tissue
and in enriched neural cell cultures. (A) MCP-1 expression in-
creased on the stroked side of the brain as determined with ELISA.
(B) After enriched cultures were exposed to hypoxia, MCP-1 con-
centration in the media of microglia and astrocyte cultures was
significantly higher than in neuronal medium (*, p<0.05).
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122 Current Neurovascular Research, 2008, Vol. 5, No. 2
Jiang et al.






Fig. (6). MIP-1? receptor CCR1 (A), CCR5 (B) and MCP-1 recep-
tor CCR2 (C) were present on the surface of HUCB cells (x100).
this was not significantly different from the lower doses. In
contrast, the 400 ng/ml concentration was significantly dif-
ferent from the 30 ng/ml, 50 ng/ml and 100 ng/ml doses,
respectively (* p<0.05; Fig. (7B)).
Effect of Brain Tissue Extract on HUCB Cell Migration

HUCB cells to migrate across a membrane than extract of
the non-stroked side did (p<0.05; Fig. (8A & 8B)). Antibod-
ies to both MIP-1? (Fig. (8A)) and MCP-1 (Fig. (8B)) anti-
body depressed migration toward non-stroked values
(p<0.05).
Tissue extract from the stroked brain induced more
DISCUSSION

the brain 24 hours after MCAO as determined with immuno-
histochemistry and western blotting. Our results are consis-
tent with previous studies that reported MIP-1? expression
as early as 1 hour after onset of the MCAO and which
peaked at 4-6 hour post surgery in the injured hemispheres
(Takami et al., 1997; Kim et al., 1995). MIP-1? has also been
found to increase after other brain injuries such as ischemia
(Takami et al., 1997), stab wound (Ghirnikar et al., 1996),
hypoxia, olfactory target ablation (Thomas et al., 2002).
MIP-1? was observed on the ipsilateral injured side of

lating monocytes. In the CNS, the inducible MIP-1? could
exist in astrocytes, microglia, endothelial cells or neurons.
Controversy exists, with some groups finding MIP-1? in the
astrocytes (Kim et al., 1995b; Miyamoto and Kim, 1999)
while others report that it is only produced by Mac-1
mRNA-positive cells including microglia /macrophages
(Takami et al., 1997). Babcock et al. (2003) observed that
the MIP-1? was expressed by glial cells and could direct
leukocytes to the CNS after axonal injury (Che et al., 2001).
Other investigators indicated MIP-1? was expressed by neu-
ronal cells in culture (Miyamoto and Kim, 1999). Immu-
nostaining shows that MIP-1? can be in all three neural cells,
but western blotting shows MIP-1? was mainly produced by
astrocytes and neurons. The inconsistency between these
studies most likely reflects differences between animal mod-
els and cell culture techniques.
Similar to MIP-1?, MCP-1 was also found in the ische-
mic brain around the infarct. ELISA assay revealed that the
amount of MCP-1 on the stroked side was 7 fold greater than
the contralateral side. MCP-1 was produced by neurons, as-
trocytes and microglial cells. This is consistent with previous
work showing that MCP-1 was detected as early as 3 to 6 hr
post stroke in the ipsilateral hemisphere (Babock et al.,
2003) and peaked at 2 -3 days post MCAO (Che et al.,
2001). When we examined cultures enriched for neurons,
astrocytes or microglia, MCP-1 was mainly expressed in
microglia and astrocytes after hypoxia. Using immunochem-
istry double staining, Che et al. (2001) found the majority of
MCP-1 positive cells were astrocytes. This is consistent with
work in the cortico-striatal slice (Noraberg et al., 1999).
The majority of MIP-1? in the body is produced by circu-

after stroke is not known. Recent studies revealed that the
production of MCP-1 and MIP-1? was regulated by pro- and
anti - inflammatory cytokines induced by the ischemic insult.
However, what leads to MCP-1 and MIP-1? upregulation










Fig. (7). Chemokines dose dependently induced HUCB cell migration. (A) MIP-1? at a dose of 30 ng/ml produced optimal HUCB migra-
tion after 12 hour incubation, compared to all other MIP-1? concentrations. (B) HUCB cell migration reached its maximum at a concentra-
tion of 600 ng/ml. The 400 ng/ml concentration was significantly different from the 30 ng/ml, 50 ng/ml and 100 ng/ml doses, respectively (*
p<0.05).
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Chemokine Induced Migration of Cord Blood in Stroke Current Neurovascular Research, 2008, Vol. 5, No. 2 123
Interleukin (IL)-1? production increased shortly after the
onset of the stroke (Minami et al., 1991); following that,
MCP-1 and MIP-1? production increased. Further, dex-
amethasone and IL-10 (both anti-inflammatory) significantly
reduced MCP-1 expression. However, in knockout mice
where interleukin-1 converting enzyme (ICE), a cysteine
protease that cleaves inactive pro-IL-1? to generate mature
IL-1?, was removed, MCP-1 expression was similar to wild
type animals when animals underwent severe hypoxia. With
a less severe hypoxic incident, ICE-/- did attenuate MCP-1
expression compared to wild type animals (Xu et al., 2001).
The inductive MCP-1 expression is regulated by ligands that
trigger nuclear factor kappa B (NF-kappa B) DNA binding.
High doses of IL-6 treatment remained without effect
(Thibeault et al., 2001). The early expression of IL-1? could
activate the NF-?B promoter to induce expression. The func-
tional NF-?B then binds the promoter of MIP-1?, and in-
duces MIP-1? production. When IL-1? expression is
blocked, MIP-1? expression is inhibited (Guo et al., 2004).

blood cell migration into the brain. In a stab wound model,
MIP-1? induced T cell and neutrophil infiltration into the
brain 3 days after surgery; monocytes/ macrophages were
present in the injured area 12 days post injury (Ghirnikar et
al., 1996). In a human blood-brain-barrier model, MIP-1?
expression increased shortly after amyloid-beta stimulation,
and was followed by monocyte migration from the blood
side to the brain side (Fiala et al., 1998). Further, in MIP-1?
knock out animals, there is a decrease in CD8?? dendritic
cell migration into the CNS after infection with mouse hepa-
titis virus (MHV) (Trifilo and Lane, 2004). In addition, both
MCP-1 and MIP-1? are involved in inflammatory cell re-
cruitment in other tissues (Cook et al., 1995; Domachowske
et al., 2000) after infection. MCP-1 and MIP-1? increase in
hippocampus after entorhinodentate lesions prior to T cell
and macrophage migration into the denervated hippocampus.
When CCR2 (MCP-1 receptor) knockout animals were used,
the cell migration was quenched, demonstrating a critical
role for the CCR2 ligand MCP-1/CCL2 in leukocyte migra-
The production of MCP-1 and MIP-1? could in turn elicit
tion. Cellular infiltration was not altered by a mutation to
CCR5 (a receptor of MIP-1? and RANTES/CCL5) (Che et
al., 2001). The deletion of MCP-1 may also result in up-
regulation of other cytokines (Ferreira et al., 2005), suggest-
ing a key role of MCP-1 in a cytokine network.
These findings suggested that MCP-1 and MIP-1? may
be responsible for HUCB cell infiltration into the CNS after
intravenous administration in stroke animals. However, it
can be argued that a large enough difference in species ho-
mology could result in no HUCB response to rodent
chemokine signals. This is unlikely since our interface mi-
gration system did induce HUCB migration, and exhibited a
dose effect. This could be due to the conservation of both
MCP-1 and MIP-1? ligands and their receptors across spe-
cies (Nibbs et al., 1998; Rutledge et al., 1995). In addition,
we found constitutive expression of MCP-1 and MIP-1?
receptors on HUCB.

ischemia begins with IL-1? secretion at the injury site, which
initiates MCP-1 and MIP-1? secretion in activated astro-
cytes, microglia and some neurons. The accumulated MCP-1
and MIP-1? form a concentration gradient and when com-
bined with the receptor at the surface of HUCB cells, induce
migration of systemically administered HUCB cells.
In conclusion, we hypothesize that HUCB migration after
ACKNOWLEDGEMENTS

Association grant (#0355183B to AEW). The HUCB cells
were provided by Saneron CCEL Therapeutics, Inc (SCT).
MN, SS and AEW are consultants to SCT. CS is an em-
ployee of SCT. PRS is co -founder of SCT. AEW, MN and
PRS are inventors on cord blood related patents.
This work was supported in part by the American Heart
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Fig. (8). Effect of MIP-1? and MCP-1 on HUCB migration to brain tissue extract. (A) Extracts from the stroked side of the brain at-
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