Vol. 275, Issue 6, H2300-H2307, December 1998
Myocardial ischemia-reperfusion injury in CD18- and
ICAM-1-deficient mice
Anthony J.
Palazzo1,
Steven P.
Jones1,
Wesley G.
Girod1,
Donald C.
Anderson2,
D. Neil
Granger1, and
David J.
Lefer1
1 Department of Molecular and
Cellular Physiology, Louisiana State University Medical Center,
Shreveport, Louisiana 71130-3392; and
2 Discovery Research, Pharmacia
and Upjohn, Kalamazoo, Michigan 49001
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ABSTRACT |
Previous studies have demonstrated that
circulating neutrophils (PMNs) contribute to the pathophysiology of
myocardial ischemia-reperfusion (MI/R) injury. PMN-endothelial
cell interactions are highly regulated by adhesive interactions between
PMN CD11/CD18 and coronary endothelial cell intercellular adhesion
molecule-1 (ICAM-1). We investigated the effects of MI/R in wild-type,
CD18-, and ICAM-1-deficient (
/) mice. Wild-type
(n = 6), CD18 /
(n = 6), and ICAM-1 / (n = 6) mice were subjected to 30 min
of myocardial ischemia and 120 min of reperfusion to determine
the extent of PMN infiltration and myocardial cell necrosis. Myocardial
infarction (% of the area at risk) was 45.1 ± 5.9 in wild-type
mouse hearts. In contrast, the extent of myocardial infarction was
significantly (P < 0.05) reduced in
the CD18 (19.3 ± 5.1%)- and ICAM-1 (17.9 ± 3.2%)-deficient mice. Similarly, PMN infiltration into the ischemic-reperfused myocardium was attenuated by 54% in the CD18 / mice and
by 32% in ICAM-1 / mice compared with wild-type hearts.
Deficiency in either CD18 or ICAM-1 expression results in a marked
reduction in PMN accumulation and myocardial necrosis after acute MI/R.
neutrophil; infarction; mouse; intercelleular adhesion molecule-1
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INTRODUCTION |
NUMEROUS EXPERIMENTAL studies have focused on the role
of polymorphonuclear leukocytes (PMNs) in the inflammatory response associated with coronary artery occlusion and reperfusion in animal models. Early investigations suggested that PMNs modulate coronary endothelial cell injury (26, 30), myocardial necrosis (6, 20), and
reductions in coronary blood flow that occur after myocardial
ischemia and reperfusion (MI/R; see Ref. 4). Recently, clinical
investigations have provided some preliminary evidence that PMNs and
endothelial cell adhesion molecules are activated in the coronary
circulation under conditions of unstable angina (5, 23), coronary
vasospasm (14, 16), and acute myocardial infarction (24) in humans.
There is an abundance of experimental evidence suggesting that
CD18-intercellular adhesion molecule-1 (ICAM-1) interactions are vital
for PMN-mediated myocardial reperfusion injury. The involvement of
ICAM-1 in ischemic-reperfused myocardial tissue has been previously
demonstrated by Kukielka et al. (17) and by Youker et al. (39). In
addition, it has also been shown that PMN CD18 participates in the firm
adhesion of activated PMNs to cardiac myoctes via interactions with
ICAM-1, resulting in myocyte injury and necrosis (8, 34). Previous
investigations (1, 2, 19, 22, 31) utilizing animal models have also
reported cardioprotective effects with monoclonal antibodies (MAbs)
directed against the PMN
2-integrin complex CD11/CD18.
Similarly, immunoneutralization of the endothelial ligand for
CD11/CD18, ICAM-1, has also been shown to reduce the injury produced by
MI/R (10, 18, 21, 38). However, some studies have reported that
anti-CD18 MAbs fail to attenuate MI/R injury (27, 36). Thus the precise
role of CD18 and ICAM-1 in the pathogenesis of myocardial reperfusion injury remains unclear.
Despite the wealth of information obtained in studies of MI/R injury
with MAbs directed against CD18 and ICAM-1, the use of these agents can
be somewhat problematic for a number of reasons, including species
cross-reactivity, antibody dosage, timing of administration, and
half-life. Recently, a number of gene-targeted animals have been
developed in which the genes encoding either CD18 (37) or ICAM-1 (32)
have been disrupted. As a result, animals are now available in which
the level of PMN CD18 or endothelial ICAM-1 expression is nearly
abolished and in which the inflammatory response is markedly blunted.
In addition, the techniques required for the study of MI/R in mice have
been described recently (13, 15, 25). We investigated the effects of
acute coronary artery ischemia and reperfusion on myocardial
infarct development in CD18-deficient and ICAM-1-deficient (/)
mice. Moreover, we also determined the extent of PMN infiltration in
the ischemic-reperfused myocardium in these gene-targeted mice to
further our understanding of the role of PMNs in the pathophysiology of
myocardial reperfusion injury.
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MATERIALS AND METHODS |
Transgenic mice. Male wild-type
(C57BL/6), CD11/CD18-deficient, and ICAM-1-deficient mice (C57BL/6J
background) were utilized for the experimental studies. The CD11/CD18
and ICAM-1 transgenic mice have been previously described and well
characterized (3, 32, 37). The wild-type mice (C57BL/6) were purchased
from Jackson Laboratories (Bar Harbor, ME), and the gene-targeted mice were prepared and provided by Pharmacia Upjohn Laboratories (Kalamazoo, MI). All experimental procedures complied with the
Guide for the Care and Use of Laboratory
Animals [DHHS Publication No. (NIH) 86-23,
revised 1985. Animal Resources Program, DRR/NIH, Bethesda, MD
20205] approved by the Council of the American Physiological Society and with federal and state regulations. All experimental procedures were approved by the Louisiana State University Medical Center Animal Care and Use Committee.
Myocardial ICAM-1 expression.
Radiolabeled ICAM-1 and control MAbs were prepared using the Iodo-Gen
(Sigma) method as previously described (11). Wild-type
(n = 5), CD18 /
(n = 5), and ICAM-1 /
(n = 5) mice were anesthetized (100 mg/kg pentobarbital sodium ip) and instrumented with carotid artery and
jugular vein catheters. Radiolabeled antibodies (volume titrated to 200 µl with 0.9% NaCl) were injected. Monoclonal radiolabeled
(125I) antibody directed against
ICAM-1 (YN-1; Bayer Laboratories, West Haven, CT) and a nonbinding
radiolabeled (131I) antibody
(P-23; Pharmacia-Upjohn) were slowly administered through the jugular
vein catheter. After 5 min of circulation (20 min of reperfusion), a
50-µl plasma sample was drawn. The animal was then perfused with 15 ml of warm (pH 7.4), heparinized bicarbonate-buffered saline, while
being exsanguinated, to flush the excess ICAM-1 MAb and nonbinding
control antibody. The heart was then excised and measured for
radioisotopic activity. Cardiac radioactivity was measured using an
automatic gamma counter (1480 Wizard; Wallac) to determine constitutive
ICAM-1 expression. Additionally, the following items were also measured
using the gamma counter: the 50-µl plasma sample, syringe,
intravenous catheter, and 2 liters of the original antibody mixture.
The gamma counts of these items were factored into the determination of
ICAM-1 expression using the following equation:
[(125I
cpm/g)/(125I cpm injected)] [(131I
cpm/g)/(131I cpm injected)],
where cpm is counts per minute.
MI/R surgical procedures. Animals were
initially anesthetized with pentobarbital sodium (100 mg/kg ip) before
any surgical procedure. Anesthesia was maintained via supplemental
doses of pentobarbital sodium (30 mg/kg ip) as needed. Mice were
secured to the operating table by taping the extremities. A 4-0 silk
ligature was placed behind the upper incisors and pulled tautly to
extend the neck. A midline incision was made from the xiphoid process to the submentum. The salivary glands were separated from the midline
to allow access to the trachea. A tracheotomy was then performed, and a
section of polyethylene-90 tubing was inserted into the animal's
trachea and connected via a loose junction to a Harvard respirator
(model 683 rodent respirator; Harvard Apparatus). The respirator's
tidal volume was set at 1.2 ml/min, and the rate was set at 120 strokes/min and was supplemented with 100% oxygen. The right carotid
artery was then cannulated with polyethylene-10 tubing to monitor mean
arterial pressure. The arterial cannula was connected to a blood
pressure transducer and BP-1 (World Precision Instruments) blood
pressure monitor.
After an equilibration period of 10 min, a thoracotomy was performed.
With the use of an electrocautery (model-100; Geiger Instrument), an
incision was made to the left of the sternum. Retraction stitches
(4-0 silk) were used on each side of the thoracotomy to aid in
visualization of the heart. The pericardial sac was then removed.
Ligation of the left anterior descending (LAD) coronary artery was
performed using a 7-0 silk suture attached to a BV-1 needle
(Ethicon). A small piece of polyethylene tubing was used to secure the
ligature without damaging the artery. The retraction sutures were
removed, and the chest wall was approximated and covered with parafilm
wax paper to prevent desiccation.
MI/R infarct protocols. A diagram of
the MI/R protocols is presented in Fig. 1,
A and
B. In initial experiments, wild-type (n = 6), CD18-deficient
(n = 6), and ICAM-1-deficient
(n = 6) mice were subjected to 30 min
of LAD coronary artery occlusion and 120 min of reperfusion. In
additional experiments (Fig. 1B), wild-type (n = 5), CD18-deficient
(n = 6), and ICAM-1-deficient (n = 6) mice were subjected to 60 min
of LAD coronary artery occlusion and 120 min of reperfusion.
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Fig. 1.
Mouse myocardial ischemia (MI)-reperfusion experimental
protocols. After a 20-min equilibration period, mice were subjected to
either 30 min (A) or 60 min (B) of left anterior
descending (LAD) coronary artery ischemia followed by 120 min
of reperfusion. TTC, 1.0% 2,3,5-triphenyltetrazolium chloride.
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Determination of area at risk and infarct
size. At the conclusion of the 2-h period of
reperfusion, the LAD coronary artery was religated with 7-0 silk
suture. Evans blue dye (1.5 ml of 1.0%; Sigma) was retrogradely
injected into the carotid artery catheter to delineate the in vivo area
at risk.
At the end of the protocol, the heart was excised and fixed in 1.5%
solution of SeaPlaque agarose gel (FMC BioProducts). After the gel
solidified, the heart was sectioned perpendicular to the long axis in
1-mm portions using a McIlwain tissue chopper (Brinkmann Instruments).
The 1-mm sections were placed in individual wells of a six-well cell
culture plate (Cell Wells; Corning Glass Works) with the basal side
exposed. Each slice was then counterstained with 3.0 ml of 1.0%
2,3,5-triphenyltetrazolium chloride (Sigma) solution for 5 min at
37°C. The right ventricle was excised, and each slice was weighed
and visualized under an Olympus SZ4045 (Olympus America) dissecting
microscope equipped with a Sony charge-coupled device Iris color video
camera (Sony Electronics). The left ventricular area, area at risk, and
area of infarction for each slice were then determined by computer
planimetry using NIH Image (v1.57) software. The size of the myocardial
infarction was determined by the following previously described
equation (25): weight of infarction = (A1 × Wt1) (A2 × Wt2) (A3 × Wt3) (A4 × Wt4) (A5 × Wt5), where
A is percent area of infarction by
planimetry from subscripted numbers
1-5 representing sections. Wt is the weight of the
same-numbered sections.
Hematology of peripheral blood.
Leukocyte, PMN, and platelet counts were performed by LabCorp (BioVet
Division). Whole blood samples were obtained from the carotid artery
and collected into pediatric (
1 ml) lavender-topped (EDTA-containing)
Microtainers (Becton-Dickinson).
Myocardial histology. Routine
histological staining was performed on multiple sections of
midventricular cardiac sections to determine the extent of PMN
infiltration. Wild-type (n = 3), CD18
/ (n = 3), and ICAM-1
/ (n = 3) hearts were
subjected to 30 min of myocardial ischemia and 120 min of
reperfusion and stained as described above. The hearts were stored
overnight in 4.0% paraformaldehyde at 4°C. The tissue was cut into
sections and dehydrated using graded acetone washes at 4°C. Tissue
sections were embedded in plastic (Immunobed; Polysciences), and
4-µm-thick sections were cut and transferred to Vecatabond-coated
slides (Vector Laboratories). The slides were soaked in 95% ethanol
for 10 min to remove some of the plastic embedding and to allow the tissue to stain. After the 10-min ethanol wash, the tissue sections were stained with either Gill no. 3 hematoxylin solution for 10 min
(Sigma) or Giemsa stain for 3 min (Sigma). The slides were then
observed microscopically, and the number of PMNs was counted per field
of view. For each of the three hearts examined the number of PMNs was
counted in 4 fields for a total of 12 fields.
Statistical analyses. All values in
text and in Figs. 1-5 are presented as means ± SE of
n independent experiments. The infarct size, area at risk, left ventricle size, leukocyte counts, and hemodynamic data were analyzed with an analysis of variance coupled with post hoc analysis (Scheffé's test for significance). All statistics were calculated with StatView 4.5 (Abacus Concepts). Statistical significance was set at P < 0.05.
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RESULTS |
Exclusion criteria. A total of six
wild-type, six CD18 /, and six ICAM-1 /
mice entered the 30-min ischemia and 120-min reperfusion
infarct size determination protocol. All mice in these three groups
survived the protocol and were included for data analysis. Conversely,
~56% (5 out of 9) of the wild-type mice, 86% (6 out of 7) of the
CD18 / mice, and 100% (6 out of 6) of the ICAM-1
/ mice survived the 60-min ischemia and 120-min
reperfusion protocol and were included for data analysis. The mice that
did not survive this protocol died between 30 and 60 min of reperfusion.
Myocardial ICAM-1 expression. Basal
ICAM-1 expression (µg MAb/g tissue) in wild-type, CD18
/, and ICAM-1 / hearts is presented in
Table 1. Hearts from wild-type mice
expressed significantly (P < 0.05)
higher levels of ICAM-1 (1.79 ± 0.24 µg MAb/g tissue) than CD18
/ (0.92 ± 0.06 µg MAb/g tissue) and ICAM-1
/ (0.02 ± 0.01 µg MAb/g tissue) hearts. ICAM-1
expression in the ICAM-1 / hearts was significantly lower
than both the wild-type and CD18 / hearts.
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Table 1.
Constitutive expression of ICAM-1 in wild-type, CD18
/, and ICAM-1 / mouse hearts using a
dual radiolabeled monoclonal antibody technique
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Hematology data. The circulating
levels of leukocytes, PMNs, and platelets for the wild-type, CD18
/, and ICAM-1 / mice under baseline
conditions are presented in Table 2. Total
leukocyte counts were significantly (P < 0.01) higher in the CD18 / mice (6,367 ± 953 cells/µl blood) compared with the wild-type mice (2,660 ± 320 cells/µl blood). However, circulating leukocyte counts in ICAM-1
/ mice (4,250 ± 189 cells/µl blood) were not
significantly different from either wild-type or CD18 /
mice. The number of circulating PMNs was similar in the wild-type (960 ± 169 cells/µl blood), CD18 / (1,267 ± 211 cells/µl blood), and ICAM-1 / (983 ± 114 cells/µl
blood) mice. Furthermore, circulating platelet counts were also similar
in the wild-type and transgenic animals. Baseline platelet counts were
9,640 ± 507, 10,750 ± 774, and 10,900 ± 308 cells/µl
blood in the wild-type, CD18 /, and ICAM-1
/ mice, respectively.
Hemodynamics. Mean arterial blood
pressures (MABP) were recorded for all animals in each experimental
group throughout the MI/R experimental protocol. MABP data for the
30-min ischemia and 120-min reperfusion experiments are
presented in Table 3. There were no
significant group differences in MABP observed at any time throughout
the experimental protocol. MABP data for the 60-min
ischemia/120-min reperfusion group are presented in Table 4. MABP in the CD18 / group
was significantly different compared with the ICAM-1 /
group at 60 min of reperfusion. No other significant differences were
observed at any times during the experimental protocol.
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Table 3.
Mean arterial blood pressures in wild-type, CD18-deficient, and
ICAM-1-deficient mice subjected to 30 min of coronary artery
ischemia and 120 min of reperfusion
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Table 4.
Mean arterial blood pressures in wild-type, CD18-deficient, and
ICAM-1-deficient mice subjected to 60 min of coronary artery
ischemia and 120 min of reperfusion
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Myocardial area at risk and infarct
size. Representative photomicrographs of area at risk
and infarct staining for wild-type, CD18 /, and ICAM-1
/ mice after 30 min of LAD coronary artery occlusion and
120 min of reperfusion are presented in Fig.
2, A and
B. Despite similar-sized areas at
risk, wild-type hearts suffered a significantly larger area of
infarction after MI/R compared with both CD18 (Fig.
2A)- and ICAM-1 (Fig.
2B)-deficient mouse hearts. Summary
data for area at risk and infarct size are shown in Fig.
3. All groups of animals experienced
similar-sized areas at risk per left ventricle after coronary artery
occlusion. The areas at risk per left ventricle were 50.4 ± 3.2, 49.8 ± 4.8, and 54.8 ± 5.2% in wild-type, CD18
/, and ICAM-1 / mice, respectively. Infarct
size per area at risk was 45.1 ± 5.9% in wild-type mice and
19.3 ± 5.1% of the area at risk in the CD18
/ mice (P < 0.05). Furthermore, infarct size per area at risk was also
significantly (P < 0.05) reduced in
ICAM-1 / hearts.
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Fig. 2.
A: representative photomicrographs
(magnification ×25) of midventricular myocardial tissue from
wild-type and CD18-deficient (KO) mouse hearts after 30 min of
ischemia and 120 min of reperfusion. Degree of myocardial
necrosis was significantly reduced in the CD18 KO hearts compared with
wild-type hearts. KO, knockout. B:
representative photomicrographs (magnification ×25) of
midventricular myocardium from wild-type and intercellular adhesion
molecule-1 (ICAM-1)-deficient (KO) hearts subjected to 30 min of
coronary artery ischemia followed by 120 min of reperfusion.
Myocardial cell injury was dramatically attenuated in the ICAM-1 KO
mouse hearts compared with wild-type control hearts.
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Fig. 3.
Myocardial area-at-risk (AAR)/left ventricle (LV), infarct (Inf)/AAR,
and Inf/LV data for wild-type, CD18 /, and ICAM-1
/ mouse hearts subjected to 30 min of myocardial
ischemia and 120 min of reperfusion. Data are expressed as a
percentage of the AAR or LV. Myocardial Inf size was significantly
(* P < 0.05) reduced in the
CD18- and ICAM-1-deficient mice. /, deficient.
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Myocardial PMN accumulation. Graphic
interpretation of PMN counts within the ischemic-reperfused myocardium
after 30 min of myocardial ischemia and 120 min of reperfusion
is presented in Fig. 4. PMN accumulation in
ischemic-reperfused myocardium was 41.1 ± 2.6 PMNs/field in
wild-type hearts at 120 min of reperfusion. In contrast, the degree of
PMN infiltration was 19.1 ± 0.9 and 28.0 ± 1.7 PMNs/field in
the CD18-deficient and ICAM-1-deficient mice, respectively
(P < 0.01 and
P < 0.05 vs. wild type).
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Fig. 4.
Myocardial neutrophil (PMN) accumulation within the
ischemic-reperfused myocardium after 30 min of ischemia and
120 min of reperfusion in wild-type (n = 3), CD18-deficient (n = 3), and
ICAM-1-deficient (n = 3) mouse hearts.
A total of 12 fields was examined in each heart, and the number of
PMNs/field was counted. Myocardial PMN infiltration was significantly
ameliorated in the CD18 / and ICAM-1 /
hearts compared with wild-type hearts.
* P < 0.05 and
** P < 0.01.
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Myocardial infarct size after prolonged
ischemia. The results obtained from experiments
involving a longer duration of myocardial ischemia (i.e., 60 min) and reperfusion are presented in Fig. 5. The size of the area at risk relative to
the left ventricle was 49.5 ± 4.6% in wild-type mice. This is
similar to that observed in the CD18-deficient mice (50.4 ± 6.8%)
and in the ICAM-1-deficient mice (56.7 ± 8%). There were no
significant group differences in the size of the area at risk. In
addition, the area of infarction per area at risk was also similar in
the three groups. Infarct size per area at risk was 57.2 ± 4.9% in
wild-type, 62.8 ± 2.7% in CD18 /, and 50.3 ± 6.9% in ICAM-1 / hearts
(P = not significant between groups).
Similarly, no significant differences were observed in the data
collected for infarct size expressed as a percentage of the left
ventricle.
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Fig. 5.
Myocardial AAR/LV, Inf/AAR, and Inf/LV data for wild-type, CD18
/, and ICAM-1 / mouse hearts subjected to
60 min of myocardial ischemia and 120 min of reperfusion. Data
are expressed as a percentage of the AAR or LV. Myocardial AAR and
myocardial Inf size were similar in all 3 study groups.
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DISCUSSION |
The present study clearly demonstrates a significant reduction of
myocardial infarction in CD18- and ICAM-1-deficient mice compared with
wild-type mice after acute coronary artery occlusion and reperfusion.
Coronary endothelial cell ICAM-1 levels were measured in
ICAM-1-deficient mouse hearts and was reduced by >90% compared with
wild-type hearts. Myocardial necrosis within the area at risk was
reduced by 57 and 60% in the CD18 and ICAM-1 null mice, respectively.
It is unlikely that the observed reductions in infarct size are a
result of differences in the hemodynamic status of the gene-targeted
mice because MABPs were similar in all groups throughout the
experimental protocol. In addition, the number of circulating PMNs was
similar in the wild-type and both strains of gene-targeted mice,
suggesting that the infarct size reduction observed was not due to
diminished levels of circulating PMNs. Moreover, the degree of PMN
infiltration into the ischemic-reperfused myocardial tissue was also
attenuated in the CD18 and ICAM-1 knockout mice. Taken together, the
results of the present study confirm earlier reports demonstrating
beneficial effects of anti-CD18 (1, 2, 19, 22) and anti-ICAM-1 MAbs
(10, 18, 21, 38, 40) in myocardial reperfusion injury.
Inhibition of CD18-ICAM-1 interactions after reperfusion of the
ischemic myocardium has been investigated extensively as a possible
therapy in patients for a number of reasons. Elegant in vitro studies
have clearly demonstrated that PMN adhesion to vascular endothelium or
cardiac myocytes is highly regulated by the interplay between CD18 and
ICAM-1. For example, the adhesion and transmigration of human PMNs has
been shown to be highly dependent on the cooperative interaction among
LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and ICAM-1 (34,
35). Moreover, hydrogen peroxide treatment of isolated
canine vessels clearly promotes PMN adherence, which involves PMN CD18
binding to endothelial cell ICAM-1 (9). In addition, the locomotion of
human PMNs also involves recruitment of CD11b/CD18 to the PMN surface
followed by firm adhesion, as described previously (12). It is also
well appreciated that PMN-mediated myocardial cell injury involves
CD18-ICAM-1 interactions (7, 8, 33) in vitro.
The roles of CD18 and ICAM-1 in the regulation of PMN-endothelial cell
interactions in vivo have also been investigated. Firm adhesion of
leukocytes in the microcirculation occurs via a CD18 mechanism at low
shear rates and has been shown to be more prevalent in postcapillary
venules compared with arterioles (28). In addition, induction of ICAM-1
mRNA has been reported in ischemic-reperfused myocardium as early as 1 h after reperfusion and increasing mRNA levels for 24 h after
reperfusion (17). Moreover, ICAM-1 mRNA staining has also
been demonstrated in the ischemic viable "border zone" of the
ischemic-reperfused myocardium within the first few hours of
reperfusion (39). ICAM-1 mRNA is typically only observed in
ischemic-reperfused tissues at early times after reperfusion but is
observed in nonischemic tissues by 24 h of reperfusion (17, 39). All of
these data provide strong support for the involvement of ICAM-1 in the
recruitment of circulating PMNs into the myocardium after
ischemia and reperfusion.
Additional experiments have been performed to determine if ICAM-1
expressed on coronary endothelial cells and cardiac myocytes actually
contributes to the development of myocardial infarcts. Immunoneutralization of either CD18 (1, 2, 19, 22, 31) or ICAM-1 (10,
18, 21, 38, 40) has been shown in a number of laboratories to protect
ischemic-reperfused myocardium. However, some laboratories have
reported that anti-CD18 MAbs do not reduce myocardial cell injury after
ischemia and reperfusion (27, 36). It is now well appreciated
that studies employing MAbs directed against cell adhesion molecules
are complicated by a number of pharmacological and immunological
considerations, including the dosage and timing of antibody as well as
species cross-reactivity and specificity of the MAb that is employed.
We utilized gene-targeted mice in which the genes for either CD18 or
ICAM-1 had been disrupted, resulting in a chronic and marked diminution
in expression of these proteins to avoid the problems associated with
MAbs. Thus the results obtained with these mice clearly demonstrate
that deficiency of either CD18 or ICAM-1 protects ischemic-reperfused murine myocardium.
Despite the profound myocardial protective effects observed in the
present study with chronic deficiency of CD18 or ICAM-1, there is a
major limitation for the use of anti-inflammatory agents during the
process of myocardial infarction in humans. There is an abundance of
data suggesting that activated PMNs contribute to the development of
myocardial cell injury after ischemia and reperfusion, but it
should also be noted that PMNs play a vital role in myocardial healing
and remodeling after infarction (5). Therefore, prolonged suppression
of PMN-dependent inflammation may actually promote myocardial injury at
later times after reperfusion as reported in a previous clinical trial
of glucocorticoid therapy (29). It has become increasingly clear that
immunomodulation of the ischemic-reperfused myocardium is complex.
In addition, it should also be pointed out that, when the ischemic
insult was increased from 30 to 60 min in duration, we failed to
observe any cardioprotection in the CD18-deficient and ICAM-1-deficient
mice. This may be due to the fact that ischemic injury is so severe
after 60 min of coronary ischemia in the mouse that most of the
myocardium within the ischemic zone is necrotic before reperfusion and
therefore not amenable to reperfusion therapy. Alternatively,
reperfusion injury after 60 min of ischemia may be independent
of leukocyte-endothelial cell adhesion molecules in the mouse. Similar
results were observed in a previous study (15) of P-selectin-deficient
mice performed in our laboratory. We observed a marked reduction in
infarct size in the P-selectin / mice after 30 min of
ischemia and 120 min of reperfusion. However, no differences in
infarct size were observed between wild-type and P-selectin
/ mice after 60 min of ischemia and 120 min of reperfusion.
In summary, we have demonstrated a significant reduction in myocardial
necrosis and PMN infiltration in CD18- and ICAM-1-deficient mice after
acute coronary artery ischemia and reperfusion. These data
provide strong evidence that both of these cell adhesion molecules
contribute to myocardial cell injury in the reperfused myocardium. Thus
it is likely that CD18 and ICAM-1 modulate firm adhesive interactions
between PMNs and endothelial cells and adhesive interactions between
PMNs and cardiac myocytes in the ischemic-reperfused myocardium. Our
results serve to confirm and extend the results of earlier studies
demonstrating cardioprotective actions of MAbs that immunoneutralize
either CD18 or ICAM-1. Additional studies are necessary to determine
the effects of CD18 and/or ICAM-1 deficiency after myocardial
ischemia and prolonged reperfusion. Studies utilizing longer
periods of reperfusion will determine if the protection observed in the
knockout mice actually reduces the ultimate extent of myocardial
infarction and if prolonged deficiency of these pivotal adhesion
proteins interferes with myocardial healing.
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ACKNOWLEDGEMENTS |
We acknowledge DeRoyal Industries (Surgical Division; Powell, TN)
and Ethicon Surgical (Somerville, NJ) for generous donations of
surgical supplies.
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FOOTNOTES |
This research was supported by Grant JDF 195065 from the Juvenile
Diabetes Foundation (D. J. Lefer) and by National Institute of Diabetes
and Digestive and Kidney Diseases Grant PO1 DK-43785 (D. N. Granger).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: D. J. Lefer, Dept. of Molecular and
Cellular Physiology, LSU Medical Center, 1501 Kings Highway,
Shreveport, LA 71130.
Received 7 April 1998; accepted in final form 27 August 1998.
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T. So,
A. DiPaula,
T. Aversano,
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