Role of α4 Integrin and VCAM-1 in CD18-Independent Neutrophil Migration Across Mouse Cardiac Endothelium
Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. Although leukocyte β2 integrins (CD18) play a critical role, significant neutrophil emigration persists when CD18 is neutralized or absent. This study examined the role of leukocyte β1 integrin (α4) and its endothelial ligand VCAM-1 in CD18-independent neutrophil migration across cardiac endothelium. In a mouse model of myocardial ischemia and reperfusion, we show that compared with wild-type mice, neutrophil infiltration efficiency was reduced by 50% in CD18-null mice; in both types of mice, myocardial VCAM-1 staining increased after reperfusion. In wild-type mice, antibodies against CD18, ICAM-1 (an endothelial ligand for CD18), or VCAM-1 given 30 minutes before ischemia did not block neutrophil emigration at 3 hours reperfusion. Although anti-VCAM-1 attenuated neutrophil emigration by 90% in CD18-null mice, it did not diminish myocardial injury. To determine if CD18-independent neutrophil emigration was a tissue-specific response, we used isolated peripheral blood neutrophils from wild-type or CD18-null mice and showed neutrophil migration across lipopolysaccharide-activated cultured cardiac endothelium is CD18-independent, whereas migration across endothelium obtained from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against α4 integrin or VCAM-1. We conclude tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.
Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. The process begins when flowing neutrophils leave the blood stream by tethering, rolling, and finally arresting on the inflamed endothelial surface. Firm adhesion (arrest) is mediated largely by leukocyte β2 integrins (CD18) and endothelial ICAM-1 (CD54), a ligand for CD11a/CD18 and CD11b/CD18.1 Two major lines of evidence support an important role for CD18 and ICAM-1 in neutrophil infiltration of the postischemic myocardium. First, in cat2,3⇓ and dog4,5⇓ models of myocardial ischemia and reperfusion injury, blocking antibodies against CD18 or ICAM-1 reduce neutrophil infiltration by 60% to 90%. Second, in mouse models of myocardial injury, targeted deletions of CD18 or ICAM-1 show a 30% to 50% reduction in neutrophil emigration after ischemia and reperfusion.6,7⇓ Because significant levels of neutrophil emigration persist when CD18 or ICAM-1 are neutralized or absent, the possibility that a CD18-independent mechanism for neutrophil emigration exists in the heart.
The observation that significant neutrophil emigration persists in ischemic-reperfused myocardium when CD18 is neutralized or absent contrasts with many published studies of inflammation at various tissue sites.8–11⇓⇓⇓ Although Doerschuk et al12 found certain stimuli would induce CD18-independent emigration of neutrophils in pulmonary tissue, ischemia/reperfusion typically induces CD18-dependent migration.13–16⇓⇓⇓ However, baseline expression and cytokine-induced changes in endothelial adhesion molecule expression vary widely between tissues. Compared with other tissues, mouse heart has high levels of ICAM-1 and VCAM-1 (CD106), both of which increase markedly after lipopolysaccharide (LPS) stimulation.17 Leukocyte receptors for VCAM-1 include the β1 integrins, VLA-4 (CD49d/CD29 aka α4β1), and α9β1. Circulating human neutrophils express low levels of α4 and high levels of α9,18,19⇓ but α4 levels appear to increase during transendothelial migration.20 Whether α9β1 is expressed on mouse neutrophils is unknown. However, α4 integrin is expressed at low levels on mouse neutrophils and increases as much as 8-fold after neutrophil emigration.21,22⇓ Hence, adhesive interactions between neutrophil α4 integrin and endothelial VCAM-1 may be partially responsible for CD18-independent neutrophil trafficking into the injured myocardium.
The purpose of this study was to determine the role of α4 integrin and VCAM-1 in CD18-independent neutrophil migration across heart endothelium. In a mouse model of myocardial ischemia and reperfusion, we assessed neutrophil infiltration in wild-type and CD18-deficient (CD18-null) mice and examined the role of VCAM-1 using antibody blockade. In addition, we developed a novel in vitro model for assessing neutrophil trafficking across mouse endothelial monolayers using endothelium derived from heart or inferior vena cava that allowed greater experimental control and quantification.
Materials and Methods
Eight- to 30-week-old C57BL/6 wild-type mice (Harlan Biosciences, Houston, Tex) and CD18-null mice (backcrossed with C57BL/6 mice for >8 generations) were used in this study. CD18-null mice were generated as described previously.23 Neutrophils from these mice do not have CD11a or CD11b on their cell surface.11 All experimental protocols were reviewed and approved by the Office of Research at Baylor College of Medicine.
Neutrophil Transendothelial Migration In Vivo
A murine model of myocardial ischemia/reperfusion injury was used to assess the role of adhesion molecules on neutrophil transendothelial migration as described previously.24,25⇓ Briefly, mice were anesthetized, ventilated, and the chest opened to expose the left ventricle. Using a 1-mm piece of PE10 tubing and a surgical tie, the left anterior descending (LAD) branch of the left coronary was occluded and myocardial ischemia assessed by left ventricular blanching and electrocardiographic (ECG) S-T segment elevation. After 30 minutes, the ligature was removed and blood flow restored for 3 or 24 hours. Sham-operated mice underwent an identical procedure but did not undergo coronary occlusion. To examine the effect of antibody blockade on neutrophil emigration, blocking antibodies (4 mg/kg body weight in 0.3 mL saline) against VCAM-1 and ICAM-1 (MK2.7 and YN1, respectively; American Type Culture Collection [ATCC], Rockville, Md) or control antibody against human HLA-DR5 (SFR-DR5; ATCC, Rockville, Md) were injected into the jugular vein 30 minutes before occlusion.
To assess neutrophil influx, hearts were excised and fixed with 10% zinc-buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin or immunostained for neutrophils using a rat anti-mouse neutrophil monoclonal antibody (7/4; Serotec Inc, Raleigh, NC) followed by anti-rat IgG biotinylated monoclonal antibody (mAb) (Vectastain, ABC Kit Peroxidase Rat IgG PK-4004, Vector Laboratories, Burlingame, Calif). Morphometric measurements were made using a Zeiss Axioscop light microscope and image analysis software (Zeiss Image; Zeiss, Oberkochen, Germany). For immunofluorescent detection of adhesion molecules, sections were labeled with rat mAbs against ICAM-1 (YN1; ATCC, Rockville, Md) or VCAM-1 (429(MVCAM.A); BD Pharmingen, Franklin Lakes, NJ). Rat anti-HLA-DR5 (SFR3-DR5; ATCC, Rockville, Md) was used as a control. Goat anti-rat IgG conjugated to Alexa 594 (Molecular Probes, Eugene, Oreg) was used for secondary detection. To assess area at risk and infarct size, hearts were removed and perfused with 1% Evans blue through the aorta, sectioned, stained with 1.5% 2,3,5-tripheyltetrazolium chloride, and subjected to microscopic image analysis as previously described.24,25⇓
Isolation and Culture of Mouse Endothelial Cells
Inferior vena cava or heart obtained from anesthetized newborn mice was digested with collagenase A (400 U/mL) at 37°C for 45 minutes and then labeled with anti-ICAM-2 (10 μg/mL; BD Pharmingen) followed by secondary labeling with goat anti-rat IgG conjugated to ferritin (20 μg/mL; Miltenyi Biotech Inc, Auburn, Calif). ICAM-2-positive endothelial cells were immunomagnetically isolated according to manufacturer’s instructions (Miltenyi-Biotech Inc). Endothelial cells were seeded in 35-mm Petri dishes and incubated with D-MEM containing D-valine, 20% FBS, 5% human platelet-poor plasma, 5% heparin, 50 μg/mL endothelial growth supplement, 1% Pen/Strep, and 1% Fungizone. Confluent monolayers were passed onto glass coverslips coated with glutaraldehyde-crosslinked gelatin26 or 96-well polystyrene plates coated with adsorbed gelatin.
Characterization of Mouse Endothelial Cells
Endothelial cell surface adhesion molecule expression was evaluated using an enzyme-linked immunosorbent assay (ELISA) as previously described.27 Briefly, endothelial cells were seeded in 96-well Corning plastic plates. At 4 days after confluence, monolayers were treated with or without LPS (30 ng/mL) for 4 hours, fixed in PBS containing 0.25% paraformaldehyde, and labeled in triplicate with 10 μg/mL antibody against ICAM-1 (YN1; ATCC, Rockville, Md), ICAM-2,VCAM-1, PECAM-1, E-selectin, P-selectin, and VE-cadherin (3C4, 429(MVCAM.A), MEC 13.3, 10E9.6, RB40.34 BD, and 11D4.1, respectively; Pharmingen). Rat mAb M170 (anti-human CD11b; eBioscience, San Diego, Calif) was used as a control. Binding was assessed by secondary detection with alkaline phosphatase conjugated to goat anti-rat IgG (Sigma, St Louis, Mo). Plates were read at 405 nm by an automatic microplate reader (Cambridge Technology, Inc).
Isolation of Peripheral Blood Mouse Neutrophils
To optimize the isolation of mouse neutrophils, we used a method described by Williams et al28 for use in the rat and later modified by Sugawara and colleagues for mice.29 Briefly, using an anesthetized mouse, a PE10 tube (fitted to a PE50 tube that is attached to a 23-gauge needle connected to a 3-way stopcock) is inserted into the jugular vein. Blood (0.5 mL) is drawn into a heparinized syringe, the stopcock turned, and 0.5 mL of Hespan (6% hetastarch) is infused into the animal. The infusion/extraction cycle is repeated until the mouse dies, yielding approximately 7 mL blood/hetastarch, which is then layered on a NIM-2 two-component step gradient (Cardinal Associates, Inc) and centrifuged for 30 minutes at 1000g. The lower neutrophil-rich band is collected and contaminating red cells removed by hypotonic lysis. Using this method, the number of neutrophils recovered from one mouse is typically 1.5×106 and the purity is 95%, a significant improvement over what can be obtained from blood (without Hespan) recovered by cardiac puncture (1.5 to 3×105 neutrophils and 75% purity).
Flow Cytometric Characterization of Neutrophils
Flow cytometry (FACSCalibur; BD) of whole blood or isolated neutrophils was performed using FITC- and PE-tagged antibodies to L-selectin (BD Pharmingen), α4, or CD11b (Caltag). Rat anti-mouse neutrophil antibody clone 7/4 was used to positively identity neutrophils in whole blood.
Neutrophil Transendothelial Migration In Vitro
Neutrophil transmigration was analyzed using modified Muntz adhesion chambers.26 Mouse endothelial cells were cultured on 5-mm glass coverslips, placed in Muntz chambers, and filled with PBS containing 20 000 mouse neutrophils (chamber volume=7 μL). This yielded a neutrophil to endothelial cell ratio of 2:1, identical to that used in our human neutrophil transmigration studies.26,30⇓
To study effects of antibody blockade on CD18 (GAME-46; BD Pharmingen) or α4 (PS/2; Serotec Inc) integrin function, neutrophils were premixed with 10 μg/mL of antibody for 20 minutes at room temperature before injection. To study effects of blocking antibodies on endothelial adhesion molecule function, monolayers were preincubated with anti-VCAM-1 (429(MVCAM.A) from BD Pharmingen or MK2.7 from ATCC) or anti-ICAM-1 (YN1; ATCC) antibody for 20 minutes at room temperature before beginning the assay.
Assessment of CD18 Dependency in Neutrophil Trafficking in the Myocardium
To determine the CD18 independency of neutrophil emigration in our model myocardial injury, we assessed neutrophil emigration after 30 minutes ischemia and 3 hours reperfusion. Figure 1 shows large numbers of extravascular neutrophils infiltrated the myocardium of wild-type and CD18-null mice. Transverse sections of heart (base, middle, and apex) revealed neutrophil emigration was confined to the middle and apical regions. Quantitative comparisons between wild-type and CD18-null mice for each region showed similar numbers of extravasated neutrophils present after 3 or 24 hours reperfusion (Table 1). At face value, these data suggest in CD18-null mice, neutrophil emigration into the reperfused myocardium is entirely CD18-independent. However, in comparison to wild-type mice, the number of circulating neutrophils in CD18-null mice can be 5 to 40 times higher because of increased levels of granulocyte-colony stimulating factor and interleukin (IL)-17.31 Although the relationship between circulating neutrophil levels and magnitude of the emigration response is complex and not linear,12 increased neutrophil recruitment at sites of tissue injury or infection is frequently accompanied by increased circulating neutrophil counts.11,32⇓ Although some argue that neutrophil levels have little to do with neutrophil recruitment,33–35⇓⇓ a common practice among investigators working with mice deficient in adhesion molecules is to normalize neutrophil emigration by the circulating neutrophil counts. As shown in Table 2, CD18-null mice had higher baseline numbers of circulating white blood cells (WBCs) compared with wild-type mice (2.7-fold, P<0.05); this was primarily due to increased numbers of neutrophils (8.4-fold, P<0.05). In both CD18-null and wild-type mice, WBC and neutrophil counts were not affected by 30 minutes of ischemia and 3 hours of reperfusion. Because neutrophil levels potentially influence neutrophil recruitment, we adjusted for differences in neutrophil counts between wild-type and CD18-null mice and an emigration ratio [(neutrophils/mm2 of ventricle area)/WBC counts after 3 hours reperfusion)×100%] was calculated.36 In the absence of CD18, there was a 69% and 44% decrease in the neutrophil emigration ratio for the middle and apical regions of the injured myocardium, respectively, yielding a combined average reduction of 50%.
Assessment of ICAM-1 and VCAM-1 Dependency in Neutrophil Trafficking in the Myocardium
Having confirmed neutrophil emigration has a significant CD18-independent component, we wished to assess the expression patterns of endothelial ICAM-1 (a ligand for the leukocyte β2 integrins CD11a/CD18 and CD11b/CD18) and VCAM-1 (a ligand for the leukocyte β1 integrin CD49d/CD29). Using immunofluorescence microscopy, ICAM-1 and VCAM-1 expression patterns in myocardium of wild-type mice were indistinguishable from those in CD18-null animals. In control wild-type mice, ICAM-1 staining was found throughout the heart microvasculature (Figure 2A) with far fewer vessels staining positively for VCAM-1 (Figure 2B). After 30 minutes of ischemia and 24 hours of reperfusion, but not 3 hours of reperfusion, there was a marked increase in the number of VCAM-1-positive vessels, but the extent of staining was always less than that for ICAM-1 (compare Figures 2C and 2D). Similar expression patterns were found in myocardium of CD18-null mice, and again, an increase in the number of vessels staining positively for VCAM-1 was only appreciated after 24 hours reperfusion (Figures 2E and 2F).
To determine the functional contribution of ICAM-1 and VCAM-1 to neutrophil emigration, we assessed neutrophil emigration into injured (30 minutes ischemia/3 hours reperfusion) myocardium in the presence of blocking antibodies against ICAM-1 and VCAM-1. Figure 3 shows in wild-type mice, neither anti-ICAM-1 nor anti-VCAM-1 were effective at reducing neutrophil emigration. As expected, in CD18-null mice, anti-ICAM-1 had no effect (because neutrophils lack the CD18 receptor). Conversely, anti-VCAM-1 reduced neutrophil emigration by 55%. Anti-VCAM-1 antibody had no effect on peripheral neutrophil counts (7.3±0.6×103 neutrophils/μL blood before anti-VCAM-1 injection and 8.6±0.3×103 neutrophils/μL blood after anti-VCAM-1 injection; mean±SD, n=3). Expressing the data as a neutrophil emigration ratio, to adjust for elevated neutrophil counts found in CD18-null mice (see previous section in Results), suggests anti-VCAM-1 decreased neutrophil emigration by 90%. Despite this decrease, no reductions in infarct size or area at risk were observed. A comparison between CD18-null mice receiving control antibody or anti-VCAM-1 revealed that 24 hours after reperfusion, there was no significant difference in percentage of left ventricle that was infarcted (14±0.1% and 13±0.1%, respectively; mean±SD, n=6), infarct size expressed as a percentage of area at risk (26±0.1% and 27±0.1%, respectively), or area at risk expressed as a percentage of total left ventricle (52±0.2% and 50±0.2%, respectively).
Finally, VCAM-1 dependency for neutrophil emigration in CD18-null mice was not due to compensatory changes in α4 integrin (a leukocyte ligand for endothelial VCAM-1) expression on the neutrophil. By FACScan, baseline mean fluorescence intensity values for α4 integrin expression were similar on circulating wild-type and CD18-null neutrophils (0.7±1.1 [SD] and 1.9±2.1, respectively, n=3) and this expression, although more variable in the wild-type mice, was not significantly increased by 30 minutes of ischemia and 3 hours reperfusion (11.0±15.1 and 0.8±0.8, respectively, n=3).
Assessment of CD18 Dependency on Neutrophil Trafficking In Vitro
Because neutrophil emigration during ischemia/reperfusion injury is a complex process involving multiple inflammatory mediators (eg, C5a, LTB4, IL-8, PAF), it is difficult to test the hypothesis that CD18-independent neutrophil migration across heart endothelium is tissue-specific. To overcome this limitation, we developed an in vitro model of neutrophil trafficking where we examined the ability of freshly isolated mouse neutrophils to migrate across endothelial monolayers derived from 2 tissue sources (vena cava and heart) and activated by an identical stimulus, LPS (30 ng/mL, 4 hours). As shown by cell surface ELISA (Figure 4), endothelium derived from vena cava or heart express the expected and characteristic array of endothelial adhesion molecules (ICAM-1, ICAM-2, VCAM-1, PECAM-1, and VE-cadherin). After LPS stimulation, VCAM-1 expression increased and there was de novo surface expression of P-selectin and E-selectin. Although similar results were obtained with tumor necrosis factor-α (TNF-α; 100 or 1000 U/mL), we restricted our study to LPS because it consistently induced greater levels of neutrophil adhesion and migration (data not shown).
To assess the requirement for CD18 in neutrophil trafficking across mouse endothelial monolayers, we used isolated neutrophils obtained from wild-type and CD18-null mice. Neutrophil adhesion and migration were quantitated using an inverted microscope and phase contrast optics as previously described.26 Figure 5 shows that after endothelial activation with LPS, CD18-null neutrophils migrated across heart endothelium but not endothelium derived from vena cava. These results are consistent with our in vivo finding that a significant fraction of neutrophil emigration in the heart is CD18-independent and support the idea that CD18 independency is due to a tissue-specific difference (heart versus vena cava).
Assessment of VCAM-1 and VLA-4 Dependency on Neutrophil Trafficking In Vitro
We wished to determine if CD18-independent neutrophil migration across heart endothelium was dependent on endothelial VCAM-1 and the leukocyte β1 integrin receptor α4 (VLA-4). Figure 6 shows that CD18-null neutrophil migration, but not adhesion, was significantly reduced (90%) in the presence of anti-α4 or anti-VCAM-1 antibodies. By comparison, wild-type neutrophil adhesion and migration were unaffected by these antibodies. It is worth noting that in vivo, anti-VCAM-1 also blocked 90% of neutrophil emigration (net migration after adjusting for circulating neutrophil counts) in CD18-null mice. This suggests that the neutrophil emigration ratio calculation is appropriate for estimating neutrophil emigration efficiency in vivo. In summary, these results show migration of CD18-deficient neutrophils across heart endothelium in vitro is largely dependent on endothelial VCAM-1 and neutrophil α4 integrin. VCAM-1 dependency is consistent with our in vivo observations on neutrophil trafficking in the reperfused myocardium, and it potentially explains why neutrophils continue to migrate into the injured myocardium of CD18-null mice.
We show for the first time that antibody blockade of VCAM-1 significantly attenuates CD18-independent neutrophil emigration in a CD18-null mouse model of myocardial ischemia and reperfusion injury. Our in vitro model of neutrophil trafficking provides the first evidence that CD18-independent neutrophil migration across heart endothelium is a tissue-, rather than stimulus-, specific response. The data show clearly that neutrophil migration across LPS-activated cardiac endothelium is CD18-independent, whereas migration across LPS-activated endothelium derived from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, CD18-independent neutrophil migration on cardiac endothelial monolayers is completely inhibited by antibodies against α4 integrin or VCAM-1. Collectively, these data support the conclusion that tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.
Using CD18-null mice, not only did we confirm previous studies showing that neutrophil emigration persists in the ischemic/reperfused myocardium,6,7⇓ but we extended these observations by demonstrating for the first time that CD18-independent emigration in CD18-null mice is largely dependent on VCAM-1. This conclusion is supported by the following evidence: (1) histologically, the injured myocardium of wild-type and CD18-null mice contain similar numbers of emigrated neutrophils. However, CD18-null mice have elevated circulating numbers of neutrophils, which may enhance delivery of neutrophils to the injured myocardium. Using a neutrophil emigration ratio to adjust for this difference (see Results), CD18-null mice show a 50% reduction in neutrophil emigration efficiency after 3 hours reperfusion. (2) In CD18-null mice, neutrophil emigration efficiency is further reduced (90%) after VCAM-1 antibody blockade. Despite this additional decrement in reperfusion-induced neutrophil accumulation, VCAM-1 blockade does not reduce infarct size or area at risk after 24 hours reperfusion. This likely relates to the observation that, although migration efficiency is inhibited by 90%, the actual number of neutrophils infiltrating the injured myocardium after VCAM-1 blockade is reduced by only 55% (see Figure 3).
That VCAM-1 and CD18 play important roles in neutrophil emigration is consistent with recent radiolabeled antibody data showing mouse heart has high constitutive levels of vascular VCAM-1 and ICAM-1.17 Using immunofluorescence microscopy, we extend these observations by documenting that basal endothelial ICAM-1 expression is relatively uniform throughout the heart microvasculature, whereas VCAM-1 expression is patchy. After ischemia/reperfusion injury, VCAM-1 expression increases as evidenced by a greater number of vessels showing a positive staining reaction. This latter observation agrees with radiolabeled antibody data from heart showing that after 5 hours, inflammatory mediators (LPS and TNF-α) increase VCAM-1 and ICAM-1 expression 2-fold.17 Taken together, these data show that inflamed heart tissue expresses significant levels of 2 critical endothelial ligands, ICAM-1 (for supporting CD18-dependent neutrophil emigration) and VCAM-1 (for supporting CD18-independent neutrophil emigration).
There is mounting evidence from in vivo models of inflammation that molecular mechanisms regulating leukocyte extravasation exhibit both tissue and stimulus specificity. Tissue-specificity is illustrated by the following: in the absence of CD18, neutrophil emigration in skin after TNF-α stimulation is reduced by 95%.36 Conversely, in ischemic/reperfused heart, targeted genetic deletions of CD18 reduce neutrophil emigration by only 50% (present study, after adjusting for increased numbers of circulating neutrophils in CD18-null mice). Stimulus specificity is illustrated by studies in the lung showing that neutrophil emigration in response to LPS is largely CD18-dependent, yet in response to Streptococcus pneumoniae it is CD18-independent.9,12⇓
Using a novel in vitro model of neutrophil trafficking, we provide new data showing that CD18-independent neutrophil migration across heart endothelium is a tissue-specific process. Specificity is controlled at the level of the endothelium and is independent of other tissue factors (eg, myocytes, fibroblasts, and mast cells). This conclusion is based on the observation that CD18-null neutrophils fail to migrate across LPS-activated mouse endothelial monolayers derived from inferior vena cava, but migrate normally across endothelium derived from heart. In vivo, ischemia/reperfusion injury is a complex process involving numerous inflammatory mediators. Our in vitro model with its single stimulus (LPS) avoids this complexity and allows for direct assessment of neutrophil adhesion and migration across activated mouse endothelial cells. The concordant observations that neutrophil migration across heart endothelium is CD18-independent both in vitro and in vivo suggest that this in vitro model is useful for assessing the question of tissue specificity due to endothelial heterogeneity.
Additional observations made with the in vitro model show that CD18-independent migration across heart endothelium was dependent not only on endothelial VCAM-1, but also on leukocyte α4 integrin. Antibody neutralization of VCAM-1 or α4 integrin inhibited transendothelial migration of CD18-null neutrophils by >90%. It is worth noting that LPS-activated endothelial cells derived from vena cava also express VCAM-1 yet neutrophil migration across these cells is completely CD18-dependent. Perhaps VCAM-1 levels are too low on vena cava endothelial cells to support neutrophil transmigration. Support for this notion comes from cell surface ELISA profiles showing that VCAM-1:ICAM-1 ratios are consistently greater on heart endothelium compared with vena cava endothelium (see Figure 4).
Recent studies have clearly defined a role for α4 integrin in neutrophil adhesion and injury to cardiac myocytes.21,22⇓ Although circulating human and mouse neutrophils express very low levels of α4, current evidence suggests transendothelial migration triggers α4 upregulation.20–22,37⇓⇓⇓ Our study supports and extends this concept and suggests that α4 integrin activation begins when the neutrophil makes first contact with the endothelial surface. In evidence is the ability of anti-α4 blocking antibodies to neutralize CD18-independent neutrophil migration (but not adhesion) across LPS-activated heart endothelial cells. Finally, our experimental results obtained using CD18-null mice show that CD18-independent neutrophil migration in the heart is a tissue-specific characteristic that is controlled at the level of the endothelium and critically dependent on endothelial VCAM-1. Conceivably, activation and engagement of α4 integrin with VCAM-1 at the onset of neutrophil transendothelial migration may ready the neutrophil for subsequent α4 integrin-dependent adhesion and injury to cardiac myocytes.
This work was supported by grants from American Lung Association (ALA-RG068N to A.R.B.) and National Institutes of Health (AI-46773 to A.R.B. and HL-42550 to A.R.B., L.H.M., and C.M.B.). The authors thank Celetta Callaway, Elizabeth Priest, Anyssa Aldape, and Cederick Scott for skilled and dedicated technical help.
Original received November 6, 2001; revision received February 14, 2002; accepted February 14, 2002.
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