Emigrated Neutrophils Regulate Ventricular Contractility via α ₄ Integrin

Abstract

Abstract-We have previously shown that CD18 and ␣ 4 integrin were important in the adherence of emigrated neutrophils to cardiac myocytes. Whether either of these molecules is important in myocyte dysfunction is unclear. In this study, we measured contractility as an index of myocyte function. Control contractility was compared with shortening response in myocytes exposed to neutrophils in the presence and absence of anti-CD18 or anti-␣ 4 antibodies. Control unloaded cell shortening, expressed as a percentage of resting cell length, measured 10.06Ϯ1.16% (nϭ10) at 5 minutes. Circulating neutrophils caused a 35% reduction in cell shortening, an event prevented by anti-CD18, but not by anti-␣ 4 antibody. When emigrated neutrophils were added to the myocytes, a profound reduction (50%) in unloaded cell shortening was noted. A significant increase in CD18 and ␣ 4 integrin was found on emigrated neutrophils. Addition of anti-CD18 antibody did not protect the myocyte from the emigrated neutrophils, whereas the addition of an anti-␣ 4 antibody significantly reduced neutrophil-induced cell shortening, despite some neutrophils still adhering to the myocytes. Furthermore, emigrated neutrophils were able to cause myocytes to go into contracture within 5 minutes in the presence of neutrophils with or without anti-CD18 antibody. In addition to the impairment in unloaded cell shortening, at later times (10 minutes), neutrophils also caused a 40% reduction in the rate of contraction and relaxation. The addition of either anti-CD18 or anti-␣ 4 antibody protected the myocytes from these changes. The data suggest that immunosuppression of CD18 on emigrated neutrophils was only partially effective in reducing myocyte dysfunction. In contrast, immunosuppression of the ␣ 4 integrin alone was sufficient to dramatically reduce all parameters of cell dysfunction measured in this study. (Circ Res. 1999;84:1245-1251.)Key Words: myocyte Ⅲ emigrated neutrophil Ⅲ ischemia/reperfusion Ⅲ contractility Ⅲ ␣ 4 integrin N eutrophils have been implicated as having a direct role in myocardial injury during inflammation, [1][2][3][4] given that depletion of neutrophils from the circulation has been found to reduce myocardial injury after ischemia-reperfusion. [5][6][7] It is thought that after neutrophils infiltrate myocardium, 8 -11 they release cytotoxic factors such as oxygen free radicals, proteases, and arachidonic acid metabolites. 3,8,12 Targeting these molecules also reduced the extent of myocardial injury after ischemia-reperfusion. [13][14][15][16] In earlier studies, a very important observation was that firm adhesion between myocytes and neutrophils was required for both the release of these toxic mediators 17,18 and the subsequent injury. 17,19 Detailed reports have proposed that (1) the engagement of CD18 (the ␤ 2 integrin responsible for firm adhesion) was essential for neutrophils to release cytotoxic molecules 2,18,20,21 and (2) the tight seal between the neutrophil and myocyte may exclude plasma, which contains important antioxidant...

Role of α 4 Integrin and VCAM-1 in CD18-Independent Neutrophil Migration Across Mouse Cardiac Endothelium

“…21,22 Although circulating human and mouse neutrophils express very low levels of ␣ 4 , current evidence suggests transendothelial migration triggers ␣ 4 upregulation. 20 -22,37 Our study supports and extends this concept and suggests that ␣ 4 integrin activation begins when the neutrophil makes first contact with the endothelial surface.…”

“…However, ␣ 4 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 ␣ 4 integrin and endothelial VCAM-1 may be partially responsible for CD18-independent neutrophil trafficking into the injured myocardium.…”

Selective Impairment of Nuclear Factor-κB-Dependent Gene Transcription in Adult Cardiomyocytes

“…12,13 In brief, 8-week-old male mice were anesthetized (92:7 mg/kg ketamine/xylazine) and sacrificed by cervical dislocation. Hearts were rapidly removed and placed into Tyrode buffer (140 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L Na 2 HPO 4 , 5 mmol/L HEPES, 10 mmol/L glucose, and 1 mmol/L MgCl 2 , pH 7.4), containing 1 mmol/L CaCl 2 at 4°C and then cannulated via the aorta for retrograde perfusion of the coronary arteries.…”

Immune Cell Toll-Like Receptor 4 Is Required for Cardiac Myocyte Impairment During Endotoxemia

“…Ventricular myocytes were isolated as previously described 19 and, unless otherwise stated, all chemicals were purchased from SigmaAldrich. Briefly, 6-to 16-week-old male septic (LPS, Escherichia coli serotype 0127:B8, 10 mg/kg intraperitoneally, 4 hours), wildtype (C57Bl/6), TLR4-deficient, and TLR4 chimeric mice were anesthetized with methoxyflurane (Metafane; Janssen Pharmaceuticals).…”