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ically signals cellular responses to bacterial lipopolysaccharide (LPS) in conjunction with accessory molecules.8 Activation of TLR4 is linked to expression of proinflammatory
cytokines and activation of nuclear factor-B signaling pathways in several cell types.7,9 TLR4 is expressed by cells of
myeloid lineage, which are central to innate immune responses, but TLR4 is also expressed in tissues without a
recognized immune function, notably the heart and vasculature.10,11 Consistent with its role as a receptor for LPS, cardiac
expression of TLR4 is essential for LPS-induced LV dysfunction and myocardial expression of tumor necrosis factor ,
interleukin (IL)-1, and inducible NO synthase.12,13 Expression of tumor necrosis factor , IL-1, and adhesion molecules
vascular cellular adhesion molecule 1 and intracellular adhesion molecule 1 on coronary endothelial cells exposed to LPS
is similarly dependent on TLR4.14 These studies support a
role for TLR4 in cardiovascular inflammation and dysfunction during bacterial sepsis, but there is no evidence that
TLR4 participates in myocardial inflammatory responses
Received January 14, 2003; de novo received July 14, 2003; revision received October 2, 2003; accepted October 6, 2003.
From the Department of Cardiovascular Medicine (J.O., C.B., M.P.); Pulmonary and Critical Care Division (X.L.); Anesthesiology, Perioperative and
Pain Medicine (T.B.), Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Womens Hospital, Boston; Department of Pathology
(L.K.), Harvard Medical School, Boston; and Genzyme Corporation (R.A.K.), Framingham, Mass.
Correspondence to Todd Bourcier, PhD, Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Therapeutics and
Reperfusion Injury, Brigham & Womens Hospital, Medical Research Building #502, Boston, MA 02115. E-mail tbourcier@rics.bwh.harvard.edu
2004 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000112575.66565.84
784
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by guest on May 4, 2015
Oyama et al
Methods
Experimental Animals
The murine strains C57 BL/10 ScCr and C3H/HeJ do not express
functional TLR4 because of naturally occurring mutations in the
TLR4 gene.8 C57 BL/10 ScCr mice were obtained from Dr Peter
Tobias of Scripps Research Institute, La Jolla, Calif. The C57 BL/10
ScSn, C3H/HeJ, and C3H/OuJ mice were purchased from Jackson
Laboratories, Bar Harbor, Maine. Animals were housed in accordance with the Harvard Medical Area Standing Committee on
Animals in a specific pathogen-free environment.
785
hexadecyltrimethyl ammonium bromide, homogenized, and centrifuged for 15 minutes at 14 000 rpm at 4C. The supernatants were
mixed 1:9 (vol/vol) with 50 mmol/L PBS (pH 6.0) containing 0.2
mg/mL o-dianisidine and 0.0006% hydrogen peroxide; the absorbance change was measured at 30 and 90 seconds at a wavelength of
460 nm. Murine myocardial myeloperoxidase (MPO) activity was
normalized using human MPO as a standard.
Immunohistochemistry
Hearts were fixed in methyl Carnoys solution at 4C for 5 hours and
then placed in 70% ethanol overnight. Hearts were paraffin embedded and cut into 10-m sections. Sections were stained with
hematoxylin and eosin to assess morphology and evidence of injury.
Histological stains included anti-mouse CD45 antibody (Ly-5;
1:1000, Pharmingen), the neutrophil-specific marker GR-1 (Ly-6g;
1:1000, Pharmingen), and goat anti-rat complement-3 (C3, 1:500,
Cappel, ICN Pharmaceuticals). Secondary antibodies consisted of
biotinylated anti-mouse or anti-goat antibodies (diluted 1:200, Vector Laboratories), followed by incubation with horseradish peroxidase coupled streptavidin (ABC reagent, Vector Laboratories) and
development with 3,3-diaminobenzidine substrate. Specificity of
the primary antibodies was checked by using species and isotypematched nonimmune antibodies. Sections were counterstained with
hematoxylin and mounted for light microscopy. Histological analysis
was performed on hearts from 3 mice from each strain.
Thioglycollate-Induced Peritonitis
Mice were given a 2-mL intraperitoneal injection of 4% Brewers
thioglycollate medium (Difco No. 0236-17-7). After 3 hours, mice
were anesthetized with pentobarbital, and abdominal infiltrates were
collected with 3 washes with PBS. Cells were centrifuged and
resuspended in 1.0 mL PBS, and total cell numbers were determined
with a hemocytometer. Ratios of granulocyte to monocyte populations were determined from the forward and side-scatter characteristics on flow cytometry.
Serum Endotoxin
Levels of serum endotoxin were measured with a chromogenic
Limulus amebocyte lysate assay according to the manufacturers
directions (BioWhittaker, QCL-1000). Sensitivity of the assay is 0.1
EU/mL.
Statistics
Data are expressed as meanSEM of n observations. Comparisons
of data between Sn versus Cr and between HeJ versus OuJ were
made using a 2-sample t test assuming equal variances. Differences
were considered statistically significant when P0.05.
Results
Reduced Infarct Size in TLR4-Deficient Mice
After Myocardial Ischemia-Reperfusion
Myocardial ischemia-reperfusion was performed on C57/
BL10 ScCr mice, which lack expression of TLR4, and on
C57/BL10 ScSn (Sn) mice, which express TLR4 normally.
After 1 hour of ligation of the LAD and 24 hours of
reperfusion, the extent of myocardial infarction was measured. The LV area affected by LAD ligation, referred to as
the area at risk (AAR), was similar between Sn and Cr mice
(515% versus 535%, respectively; PNS; Figure 1).
Despite sustaining equal areas at risk, TLR4-deficient Cr
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Circulation
mice had significantly smaller myocardial infarctions compared with Sn mice, whether normalized to the area at risk or
to the LV area (Figure 1).
In addition to a lack of TLR4 expression, Cr mice harbor a
point mutation in the IL-12 receptor resulting in impaired
microbial-induced production of interferon-, a characteristic
not shared by other TLR4-deficient murine strains.18 Serum
levels of IL-12 are also reported to increase after myocardial
infarction in humans.19 Thus, part of the myocardial protection observed in Cr hearts may be linked to defective IL-12 as
well as TLR4 signaling. We determined that serum IL-12
levels in Cr mice were similar before and after 1 hour and 24
hours of MIR (Cr before MIR, 19.611 pg/mL; Cr after
MIR, 23.91.9 pg/mL). In addition, IFN-, the primary
response gene induced by IL-12, was not detected in serum
from Cr or Sn mice after MIR (2 pg/mL). Although not
exclusionary, these data suggest that IL-12/IL-12R signaling
is not activated during the course of our experiments. To
additionally address this concern, we performed MIR on
C3H/HeJ (HeJ) mice, which harbor a distinct point mutation
in the conserved TIR signaling domain of TLR4. As shown in
Figure 1, HeJ mice also sustained smaller myocardial infarctions compared with the control strain C3H/OuJ (OuJ). Thus,
both Cr and HeJ mice that harbor distinct TLR4 mutations
sustained less myocardial damage after ischemia reperfusion,
as measured by infarct size, than did mice expressing functional TLR4.
As depicted in the Table, body weights did not differ
significantly between the murine strains tested. The TLR4defective Cr mice showed an increased survival rate compared with the Sn control mice of 71.4% versus 56.8%,
respectively. However, there was not a similar increase in
survival of TLR4-defective HeJ compared with OuJ control
mice.
Body Weight and Survival Rate for Experimental Mice After 1
h/24 h Myocardial Ischemia/Reperfusion
Murine Strain
No.
Body Weight, g
% Survival
Sn
44
270.41
57
Cr
35
250.31
71
OuJ
12
240.61
58
HeJ
12
250.31
58
Oyama et al
787
Discussion
Figure 2. A, Histochemical sections of hearts from TLR4deficient (Cr) or wild-type control (Sn) mice after 1-hour/24-hour
MIR. Sections were stained with hematoxylin and eosin (A and
B), CD45 Ab (brown, C through F), or neutrophil-specific marker
GR-1 Ab (G and H). Results are representative of 3 mice from
each strain. B, MPO activity in heart homogenates from Cr
(n6) and Sn (n6) mice after 1-hour/24-hour MIR. MPO values
for sham-operated hearts were Cr, 0.370.12; and Sn,
0.520.085 U/100 mg (P0.38, NS). Units of myocardial MPO
activity are expressed as units per 100 mg tissue weight and
are meanSEM. *Significant difference by Students t test at
P0.05.
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Circulation
Figure 5. Complement-3 (C3) deposition on representative histochemical sections of hearts from TLR4-deficient (Cr, A) or
wild-type control (Sn, B through D) after 1-hour/24-hour MIR.
Areas stained positive for C3 are depicted by arrows in A and
B. Lower bar graph depicts quantification of C3 deposition as a
percentage of the LV area from Cr and Sn mice. Values represent meanSEM from 3 observations per group. *Significant
difference by Students t test at P0.05.
Conclusions
In summary, 2 distinct TLR4-deficient murine strains were
protected from myocardial infarction caused by ischemiareperfusion injury. Inflammatory pathways linked to MIRinduced infarction were reduced in TLR4-deficient Cr mice,
including PMN infiltration, oxidative stress, and complement
activation. This study demonstrates that in addition to its role
in innate immune responses, TLR4 serves a proinflammatory
function in a murine model of MIR injury.
Acknowledgments
This study was supported by grants from the National Heart, Lung,
and Blood Institute (R01 HL63927 to Dr Kelly) and the American
Heart Association (SDG 0130323N to Dr Bourcier); a Banyu
Fellowship in Cardiovascular Sciences (to Dr Oyama); and the
Medical Research Council of Canada (to Dr Blais). Special thanks to
Dr Gregory Stahl for critically reading this manuscript.
Oyama et al
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