Cardiac derived neutrophil chemotactic factors; preliminary biochemical characterization

Abstract

The purpose of this study is to investigate whether isolated hearts perfused with cardioplegic solution release inflammatory mediators such as neutrophil chemotactic factors (NCF). Three conditions were tested, including: (1) perfusion of rabbit hearts with crystalloid cardioplegic solution (4°C) saturated with air (95% oxygen) and containing dextrose (i.e. complete system), (2) perfusion of rabbit hearts with non-oxygenated cardioplegic solution, containing dextrose (i.e. minus oxygen system), and (3) perfusion of hearts with cold cardioplegic solution saturated with air in the absence of dextrose (i.e. minus dextrose system). At various time intervals (5 min, 1, 2, 3 and 4 h) samples of circulated perfusate were removed and assayed for the presence of NCF using modified Boyden chambers. Rabbit peritoneal neutrophils were the indicator cells. The standard chemoatractant, f-Met-Leu-Phe (f-MLP) was the positive control. High levels of neutrophil chemotactic activity were detected in perfusate of all above described hearts perfused for 4 h (i.e. 194 ± 22% of f-MLP control—complete system, 126 ± 13%—minus oxygen and 136 ± 10%-minus dextrose). Histological evaluation of these hearts showed evidence of global ischemia. We also detected significant levels of NCF in effluent of hearts perfused for 5 min, 1, 2 and 3 h. Similar to perfused hearts, isolated rabbit hearts incubated for 4 h with non-oxygenated cardioplegic solution (in presence and absence of dextrose) released high levels of NCF (132 ± 18%-intact heart; and 100 ± 6% myocardial segments). Standard checkerboard analysis revealed that the observed activity released from these hearts is chemotatic. Preliminary biochemical characterization of chemotactic factors generated from intact hearts showed that the activity is protease sensitive (76% inhibition— P < 0.001) and exhibits a molecular weight of greater than 300 kDa by Amicon ultrafiltration. The activity was not extractable by organic solvents, suggesting the chemoattractants are not leukotriene dependent. These studies indicate that isolation of the heart from its blood supply results in the release of a tissue derived chemotactic factor and that oxygenation and the addition of dextrose to the perfusing fluid does not reduce the level of the detected factor. These results also suggest that cardiac derived NCF are different from previously known low molecular weight factors such as C5a, C3a, leukotriene B 4 and interleukin 1.