Effect of heparin anticoagulation on neutrophil adhesion molecules and release of IL8: C3 is not essential.
OBJECTIVE
To examine the role of heparin in modulating neutrophil activation and release of cytokine.


BACKGROUND
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
El Habbal
M H
MH
Cardiothoracic Unit, Institute of Child Health, London, UK.
Smith
L
L
Elliott
M J
MJ
Strobel
S
S
D000293:Adolescent; D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D003176:Complement C3 / Q000172:deficiency / Q000502:physiology*; D015938:Complement Membrane Attack Complex / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D016209:Interleukin-8 / Q000097:blood / Q000378:metabolism*; D019041:L-Selectin / Q000097:blood / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000097:blood / Q000378:metabolism; D008297:Male; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D011479:Protamines / Q000494:pharmacology
D000925:Anticoagulants; D015815:Cell Adhesion Molecules; D002951:Citrates; D003176:Complement C3; D015938:Complement Membrane Attack Complex; D016209:Interleukin-8; D016177:Macrophage-1 Antigen; D011479:Protamines; D019041:L-Selectin; D019343:Citric Acid; D006493:Heparin; D004492:Edetic Acid