(Stroke. 1995;26:661-666.)
© 1995 American Heart Association, Inc.
Articles |
Monocyte Chemoattractant Protein–1 Messenger RNA Expression in Rat Ischemic Cortex
From the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.
Correspondence to Giora Z. Feuerstein, MD, Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, PO Box 1539, King of Prussia, PA 19406.
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Abstract |
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Background and Purpose Previously we demonstrated that focal cerebral ischemia results in an increased expression of several cytokines/chemokines that precede the infiltration of leukocytes into the ischemic cortex after focal stroke induced by occlusion of the middle cerebral artery (MCAO). Monocyte chemoattractant protein–1 (MCP-1) is a potent chemoattractant specific for monocytes. The aim of the present study was to examine whether MCP-1 messenger RNA (mRNA) is expressed in ischemic brain tissue after MCAO.
Methods The expression of MCP-1 mRNA in the ischemic cortex was first identified by means of a sensitive reverse transcription and polymerase chain reaction technique. The time course of expression of MCP-1 mRNA in the ischemic and nonischemic cerebral cortex after both permanent MCAO and temporary MCAO (160 minutes) with reperfusion was then examined by means of Northern blot analysis.
Results Almost no expression of MCP-1 mRNA was found in the sham-operated or nonischemic (contralateral) cortex. A significant increase in MCP-1 mRNA expression in the ischemic cortex was observed after either permanent or temporary MCAO. MCP-1 mRNA was elevated at 6 hours (4.4-fold increase over sham; n=4), reached its highest expression from 12 hours to 2 days (22.7-fold at the peak level; P<.01), and remained elevated up to 5 days (5.6-fold; P<.01) after permanent MCAO. The profile of MCP-1 mRNA expression in the ischemic cortex after MCAO with reperfusion was similar to that of permanent MCAO except that MCP-1 mRNA was increased earlier (ie, 12.5-fold increase at 3 hours; n=4; P<.01). Also, MCP-1 mRNA expression in the ischemic cortex after permanent MCAO was significantly greater in hypertensive rats than in two normotensive rats (n=4; P<.05).
Conclusions The demonstration of induced MCP-1 mRNA expression early after focal ischemia suggests that MCP-1 may represent a locally expressed monocyte chemoattractant that plays an important role in monocyte infiltration into ischemic tissue and therefore may contribute to the tissue injury in ischemic stroke. Further studies must concentrate on identifying the induced expression of MCP-1 and its cellular localization in the ischemic brain when the appropriate antibodies become available.
Key Words: cerebral ischemia, focal • cytokines • proteins • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocyte chemoattractant protein–1 (MCP-1) was initially discovered as a rapidly induced gene in platelet-derived growth factor (PDGF)–stimulated murine fibroblasts and designated as JE.1 2 The mature MCP-1 secreted product contains 76 amino acids and belongs to the chemokine ß superfamily (CC chemokines). Similar to other family members, MCP-1 functions as a chemoattractant specific for monocytes and basophils but not neutrophils.3 MCP-1 also induces adhesion molecule expression and cytokine production in monocytes.4 5 MCP-1 is secreted by many cell types in response to lipopolysaccharide, interleukin (IL)-1, IL-4, tumor necrosis factor–
(TNF-),
interferon gamma, and PDGF.5 Permanent occlusion of the middle cerebral artery (PMCAO) and temporary occlusion of the middle cerebral artery (TMCAO) with reperfusion in rats are the most commonly used animal models for ischemic stroke6 and resemble the focal stroke that occurs in humans as a result of thromboembolic events. One well-characterized consequence of cerebral ischemia relevant to humans7 is the inflammatory reaction that occurs in the ischemic brain. This reaction involves the infiltration of leukocytes, which is led by polymorphonuclear cells and followed by mononuclear cells (for review, see Reference 88 ). Systematic histological studies of the development of cerebral ischemia in spontaneously hypertensive rats (SHR) after both PMCAO and TMCAO have been described in detail.9 10 After focal ischemia, monocyte infiltration and macrophage activation occur just after the initial neutrophil recruitment. Specifically, monocytes or macrophages can be identified in ischemic cortical tissue by 2 to 3 days after PMCAO or 1 day after TMCAO.9 10 After 5 days of MCAO, macrophages are the predominant inflammatory cells located in the infarct zone and appear to play a significant role in phagocytosis of necrotic tissue.
The chemotactic mediators involved in monocyte recruitment and
activation after brain ischemia are not clear. Recently we demonstrated
that cytokines (TNF-, IL-1ß, IL-6) and chemokines
(cytokine-induced neutrophil chemoattractant [CINC]/KC) are
upregulated early in the ischemic brain after both
PMCAO11 12 13 14 and TMCAO with reperfusion.15
Since TNF- and IL-1ß are known to stimulate MCP-1 gene expression
in vitro,16 17 we postulated that MCP-1 might also be
induced in focal ischemia and could play a role in monocyte recruitment
under these conditions. Therefore, in the present study we
evaluated the time course of MCP-1 messenger RNA (mRNA) expression
after both PMCAO and TMCAO with reperfusion in rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Focal Brain Ischemia
Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Bethesda, Md: Office of Science and Health Reports, Division of Research Resources/National Institutes of Health; 1985. US Dept of Health, Education, and Welfare [Dept of Health and Human Services] publication [NIH] 85-23). Procedures in which laboratory animals were used were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.
Cerebral focal ischemia or sham surgery was carried out in male SHR (Taconic Farms, Germantown, NY) and in three normotensive rat strains (Fisher-344, Wistar-Kyoto, or Sprague-Dawley; Charles River, Danvers, Mass) (age, 18 weeks; weight, 250 to 330 g) by PMCAO or TMCAO, as described in detail previously.18 19 20 Briefly, for PMCAO, the middle cerebral artery (MCA) was permanently occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein by means of electrocoagulation (Force 2 Electrosurgical Generator, Valley Lab Inc). For TMCAO with reperfusion, the MCA was lifted from the brain surface to occlude blood flow for 160 minutes and then reperfused, as described in detail previously.19 20 In sham-operated rats the dura was opened over the MCA, but the artery was not occluded. Rats were overdosed with pentobarbital, and forebrains were removed and dissected at various times after PMCAO or after MCAO with reperfusion and at 12 hours after sham surgery. The ischemic cortex (ie, the cortex ipsilateral to surgery) was dissected from the ipsilateral frontoparietal cortical hemisphere; the contralateral cortex was dissected as the nonischemic control from the same rat.18 20 The cortical samples were immediately frozen in liquid nitrogen and stored at -80°C.
Reverse Transcription and Polymerase Chain Reaction
Total cellular RNA was prepared from cortical samples by means
of the acid guanidinium thiocyanate, phenol, and chloroform extraction
procedure.21 To evaluate the expression of MCP-1 mRNA in
the ischemic cortex, we initially used a highly sensitive reverse
transcription and polymerase chain reaction (RT/PCR) technique. Two
amplifying primers for rat MCP-1 mRNA (5' sense primer,
5'-CAGGTCTCTGTCACGCTTCT-3', from bases 4 to 23; 3' antisense primer,
5'-AGTATTCATGGAAGGGAATAG-3', from bases 510 to 530)22 and
the amplifying primer pairs for rat ribosomal protein L32 (rpL32) (5'
sense primer, 5'-GTGAAGCCCAAGATCGTC-3', from bases 28 to 45; 3'
antisense primer, 5'-GAACACAAAACAGGCACAC-3', from bases 422 to
440)23 were synthesized according to the published
sequences. RNA isolated from the cortical samples at 1 hour, 6 hours,
12 hours, and 5 days after PMCAO was reverse transcribed with RNase
H- reverse transcriptase (GIBCO BRL) for 60 minutes at
37°C in the presence of oligo(dT)12-18 primer (GIBCO BRL)
according to the manufacturer's specification. After phenol/chloroform
extraction and ethanol precipitation, the RT products (0.1 µg RNA per
sample) were subjected to coamplification in the presence of primers
for MCP-1 and rpL32 at conditions described in detail
previously.24 25 In this study rpL32 was used as an
internal control for the coamplification to normalize any difference of
the samples because a constant expression of rpL32 mRNA in the cortical
samples after MCAO has been observed.26
The identities of the amplified complementary DNA (cDNA) for MCP-1 (527 base pairs) and rpL32 (412 base pairs) were confirmed by DNA sequencing. These DNA fragments were also used as probes for Northern analysis, as described below.
Northern Hybridization Analysis
RNA samples (40 µg per lane) were electrophoresed through
formaldehyde-agarose slab gels27 and transferred to
GeneScreen Plus membranes (Du Pont–New England Nuclear). For Northern
blot analysis, MCP-1 cDNA or rpL32 cDNA was uniformly labeled with
[-32P]dATP (3000 Ci/mmol, Amersham Corp) with the use
of a random-priming DNA labeling kit (Boehringer Mannheim).
Hybridization was carried out overnight with 1x106
cpm/mL of probe at 42°C in 5x SSPE (750 mmol/L NaCl, 50 mmol/L
NaH2PO4 [pH 7.6], 5 mmol/L
ethylenediaminetetraacetic acid [EDTA]), 50% formamide, 5x
Denhardt's solution, 2% sodium dodecyl sulfate (SDS), and 200 µg/mL
boiled salmon sperm DNA. The membranes were washed in 2x SSPE, 2% SDS
at 65°C for 1 to 2 hours with a change for every 30 minutes, then
autoradiographed at -80°C with a Cronex Lightning Plus intensifying
screen for various times, depending on the signal intensity. A probe
was stripped from the membranes by heating at 95°C in a solution
containing 10 mmol/L tris(hydroxymethyl)aminomethane (pH 7.5), 1 mmol/L
EDTA (pH 8.0), and 1% SDS for 20 minutes before rehybridization with
the other probe.
PhosphorImager (Molecular Dynamics) was used to quantitate the band intensities of the Northern blots, and IMAGEQUANT-TM software version 3.0 (Molecular Dynamics) was used to analyze the results. The relative mRNA was calculated by normalizing to the rpL32 signal in each sample.24 26
Statistical Analysis
Statistical evaluation was performed on four complete sets of
cortical samples at various times with the use of both Student's
t test and one-way ANOVA followed by a post hoc t
test. Since the two methods provided identical results, we illustrated
the data according to Student's t test. The results are
expressed as mean±SE. Significance was accepted for P<.05
by comparing the relative mRNA levels in the ischemic cortex with the
sham-operated cortex.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Semiquantitative RT/PCR (with rpL32 as internal control for the coamplification) was initially used to detect the expression of MCP-1 mRNA in the ischemic cortex at selected time points (see "Materials and Methods") after PMCAO. Marked increase of MCP-1 mRNA expression in the ischemic cortex was observed (data not shown). Northern analysis was then used to measure the MCP-1 mRNA expression in the extended time course after MCAO. Fig 1A
illustrates a
representative Northern blot for MCP-1 mRNA expression in the
ipsilateral (ischemic) and contralateral (nonischemic) cortical samples
after PMCAO. Quantitative Northern blot data (n=4), after normalizing
to an rpL32 probe, are illustrated graphically in Fig 1B. A low level
of MCP-1 mRNA was detected in sham-operated animals, and almost no
signal was detected in the cortical samples from normal or
contralateral cortical samples. The absence of MCP-1 mRNA expression in
normal or nonischemic cortex was also confirmed even when the highly
sensitive RT/PCR technique was used (data not shown). MCP-1 mRNA began
to be induced in the ischemic cortex after 6 hours (4.4-fold increase
compared with sham; n=4), reached its largest expression level between
12 hours and 2 days (22.7-fold increase at peak level, ie, at 24 hours;
P<.01), and remained elevated up to 5 days (5.6-fold;
P<.01) after PMCAO (Fig 1).
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MCP-1 mRNA expression in the ischemic cortex after TMCAO (160 minutes)
with reperfusion exhibited a temporal profile similar to that of PMCAO.
Fig 2A shows a representative Northern blot for
the increase in MCP-1 mRNA levels after TMCAO with reperfusion.
Quantitative data (n=4) are depicted in Fig 2B. MCP-1 mRNA after TMCAO
with reperfusion was upregulated somewhat earlier, ie, beginning at 3
hours after reperfusion (12.5-fold increase compared with sham; n=4;
P<.01), and the elevated MCP-1 mRNA returned to baseline
earlier, ie, there was no significant increase at day 5 (4.3-fold
increase) (Fig 2).
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MCP-1 mRNA expression in the ischemic cortex was relatively higher in
SHR compared with three normotensive rat strains (Fisher-344,
Wistar-Kyoto, and Sprague-Dawley) at 12 hours after PMCAO. A
representative comparative Northern blot for MCP-1 mRNA
expression in these rat strains is shown in Fig 3A, and
the quantitative data (n=4) are illustrated in Fig 3B. Northern blot
analysis revealed that although MCAO induced a high level of MCP-1
mRNA expression in the ipsilateral (ischemic) cortex of all four rat
strains (P<.01), the MCP-1 mRNA expression follows an
order, ie, SHR>Fisher-344>Sprague-Dawley>Wistar-Kyoto
(Fig 3). The elevated MCP-1 mRNA level was significantly lower in
Wistar-Kyoto rats (0.36-fold of the mean value; P<.01) and
Sprague-Dawley rats (0.5-fold; P<.05) than in
SHR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Focal brain ischemia produces an inflammatory reaction that is characterized by the infiltration and accumulation of leukocytes. This phenomenon has been demonstrated repeatedly in animal models of stroke9 10 18 20 28 29 and has also been observed in humans.7 Of these inflammatory cells present in the ischemic tissue, monocytes/macrophages are the predominant cells present after early polymorphonuclear cell infiltration into the ischemic lesions has occurred.9 10 The chemotactic mediators involved in neutrophil infiltration after MCAO are thought to include CINC/KC, a chemokine specific for neutrophils, as demonstrated previously.11 However, the chemokines that mediate monocyte/macrophage recruitment after brain ischemia are not known. The present study represents the first demonstration that MCP-1 (a chemotactic cytokine for monocytes) mRNA expression is induced in response to cerebral ischemia. The increased MCP-1 mRNA expression clearly precedes the influx of monocytes/macrophages in both focal ischemia models.9 10 If MCP-1 synthesis parallels the induced mRNA expression, MCP-1 could be the initiating factor in this more chronic inflammatory process. However, additional studies will be necessary to demonstrate the time course for translated MCP-1 under these conditions when appropriate antibodies are available.
It is of interest that the temporal expression of MCP-1 mRNA follows
that of TNF- and IL-1ß mRNA expression after
PMCAO12 13 or TMCAO with reperfusion.15 Since
TNF- and IL-1ß are known to be able to induce MCP-1 mRNA
expression in a number of cell lines in vitro,5 16 it is
possible that MCP-1 mRNA is induced by these two earlier expressed
inflammatory cytokines after focal stroke. Additional time-course
studies also revealed that the induced expression of chemokines for
neutrophils (eg, CINC/KC) precedes that for monocytes (eg, MCP-1),
which is well in accord with the temporal profile for neutrophil and
monocyte/macrophage infiltration in this focal stroke
model.9 10
It is also of interest to note that the elevated level of MCP-1 mRNA
expression in SHR is higher than that of normotensive rats (Fig 3
).
Increased mRNA expression in SHR has also been demonstrated for
TNF-, IL-1ß, and intercellular adhesion
molecule–113 14 26 and parallels the increase in
neurological deficits and the degree of cortical infarction that occur
when hypertensive rats are compared with normotensive strains after
focal ischemia.19 Hypertensive rats are known to carry an
increased risk factor for stroke and are more vulnerable to focal
stroke than normotensive rats.6 This appears to be due to
increased release of prothrombotic and inflammatory mediators from
brain endothelium in the higher-risk, hypertensive
animals.30 31 The data presented in this
report also demonstrate that increased MCP-1 mRNA expression after
focal ischemia, although greater in hypertensive rats, is not specific
to the SHR strain, because significant postischemia expression was also
observed in the three normotensive rat strains (Fig 3). Whether the
degree of MCP-1 protein level is reflected by the degree of MCP-1 mRNA
expression induced in these rat strains after focal ischemia will need
to be determined in the future.
The cellular sources of MCP-1 mRNA in the ischemic cortex are not known. Different cellular sources could include monocytes/macrophages, fibroblasts, B lymphocytes, endothelial cells, smooth muscle cells, and glioblastoma cells, which are all able to express MCP-1.4 5 It is important to note that normal brain tissue does not express MCP-1, as reflected in the contralateral cortical samples. After MCAO, MCP-1 mRNA expression begins at about the same time as neutrophils begin to enter the ischemic tissue and increased glial activation and cell death are observed.9 10 The presence of mononuclear cells in infarcted tissue begins after 1 to 3 days and peaks at 5 days after ischemia, which is delayed relative to MCP-1 mRNA expression. Therefore, the cellular sources of MCP-1 mRNA expression can be infiltrating leukocytes and/or cells endogenous to the brain. Evidence for the source of MCP-1 mRNA expression will require in situ hybridization studies.
The MCP-1 receptor has recently been cloned from human monocytes and is almost nondetectable in normal brain tissue.32 Therefore, it is possible that induced MCP-1 in focal stroke may be specific for attracting circulating monocytes into ischemic tissue. However, it will be interesting to determine whether brain microglia can be activated by MCP-1 to transform into macrophages after MCAO. The potential importance of cytokines in leukocyte infiltration into the ischemic brain has been described previously.33 34 Also, the potentially positive and negative effects of brain- and blood-derived macrophages have been discussed in detail.35 36 37
In summary, the present study describes the temporal induction profile for MCP-1 mRNA in the ischemic cortex after focal ischemia. The early induction of MCP-1 mRNA expression in ischemic brain tissue suggests that this chemokine may be responsible for the recruitment of circulating monocytes into the ischemic tissue and/or for the activation of local microglia to become macrophages after focal stroke. However, future studies must demonstrate that the induced expression of MCP-1 mRNA is translated in the ischemic lesion. This will be possible when the appropriate antibodies to this peptide become available.
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Acknowledgments |
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The authors wish to acknowledge the excellent technical assistance of Raymond F. White in generating the focal ischemic animals.
Received August 24, 1994; revision received November 7, 1994; accepted December 29, 1994.
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References |
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