Effect of heparin anticoagulation on neutrophil adhesion molecules and release of IL8: C3 is not essential.

Abstract

To examine the role of heparin in modulating neutrophil activation and release of cytokine. Up-regulation of CD11b, down-regulation of L-selectin on neutrophil cell surface and release of IL8 occur in response to extracorporeal circulation (ECC) and were proposed to cause leakage of the capillaries in patients. In a series of experiments, we examined the effect of heparin (4 U/ml) comparing it with ethylenediamine tetra-acetate (EDTA, 1.5 mg/ml) and citrate mixture (100 microliters/ml), heparin dose-response, IL8 (human recombinant IL8) dose-response and protamine (80 micrograms/ml) neutralisation of heparin (4 U/ml) using donor blood (total of 38). The role of complement component type 3 (C3) was tested. Neutrophils from a patient with complete C3 deficiency were stimulated by using heparin and cobra venom factor (10 micrograms/ml) and compared with controls (n = 5). CD11b and L-selectin expressions were assayed immediately and serially up to 120 min using immune fluorescence and flow cytometry. Serum concentrations of IL8 were determined by using enzyme-linked immunosorbent assay. The medians of up-regulation of CD11b were 540.2 (range 235.2-653.3) for heparin vs. 186.5 (55.7-207.1) for EDTA and 192.5 (69.2-263.8) for citrate mixture, P < 0.01. The medians of down-regulation of L-selectin were 79 (32-192) for heparin vs. 18.4 (0-188) for EDTA and 36.2 (7.4-135) for citrate mixture, P < 0.05. Up-regulation of CD11b, down-regulation of L-s and release of IL8 were inversely related to heparin concentration (r = 0.87, P < 0.05). Serum concentration of IL8 had a direct relationship to the changes in CD11b and L-selectin expression (r = 0.92). Heparin-protamine complex was less stimulant to expression of CD11b and L-selectin than heparin or protamine (P < 0.05). In blood samples from C3-deficient patients, heparin and cobra venom factor caused up-regulation of CD11b and down-regulation of L-selectin similar to that of controls (P > 0.05). Heparin stimulates up-regulation of neutrophil adhesion molecules CD11b, down-regulation of L-selectin and release of IL8. These effects are inversely related to heparin concentration and are independent of C3 activation. IL8 has a direct relationship to activation of neutrophil adhesion molecules. Increasing heparin dosage reduces neutrophil activation and may reduce the morbidity of patients.