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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Published on: August 23, 2013
Inhibition of neutrophil activation by fibrinogen
A A Higazi1, I I Barghouti, S K Ayesh
1Department of Clinical Biochemistry, Hadassah University Hospital Mount Scopus, Jerusalem, Israel.
Abstract:
Physiological levels of human fibrinogen markedly inhibited the chemotactic activity of human neutrophils triggered by zymosan-activated serum (ZAS), C5a, or IL-8 in a Boyden chamber assay. Fibrinogen also slightly inhibited the N-formyl-methionyl leucyl-phenylalanine (FMLP)-induced migration of human neutrophils. Albumin was devoid of the inhibitory activities displayed by fibrinogen in this system. The inhibition of chemotaxis by fibrinogen was dose-dependent and saturable. Fibrinogen placed in the upper compartment of the Boyden chamber produced a larger inhibition than that obtained with fibrinogen placed in the lower compartment. Lysine as well as the lysine analog 6-aminohexanoic acid (AHA) decreased the inhibitory capacity of fibrinogen. In contrast, both arginine and glutamine failed to suppress the fibrinogen-mediated inhibition of neutrophil chemotaxis. AHA counteracts the inhibition of ZAS-induced chemotaxis by anti-CD18 monoclonal antibody, suggesting that lysine binding sites are required for integrin function in chemotaxis. Fibrinogen also inhibited, in a dose-dependent manner, the oxygen consumption of neutrophils activated by opsonized zymosan. Taken together, the present results indicate that fibrinogen modulates neutrophil functions and suggest that in addition to its role in blood coagulation, circulating fibrinogen may be involved in regulation of the inflammatory response.
Insights
Physiological fibrinogen significantly inhibits neutrophil chemotaxis and oxygen consumption. This suggests circulating fibrinogen plays a role in regulating inflammatory responses beyond blood coagulation.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Neutrophils are critical immune cells involved in inflammatory responses.
- Fibrinogen is a key protein in blood coagulation.
- The role of fibrinogen in modulating neutrophil function is not fully understood.
Purpose of the Study:
- To investigate the effect of physiological fibrinogen levels on human neutrophil chemotaxis.
- To explore the impact of fibrinogen on other neutrophil functions, such as oxidative burst.
- To elucidate the mechanisms underlying fibrinogen's modulation of neutrophil behavior.
Main Methods:
- Boyden chamber assay to assess neutrophil chemotaxis induced by various chemoattractants (ZAS, C5a, IL-8, FMLP).
- Dose-response and saturation studies of fibrinogen's inhibitory effects.
- Investigation of the role of lysine binding sites using lysine and 6-aminohexanoic acid (AHA).
- Measurement of neutrophil oxygen consumption upon activation with opsonized zymosan.
Main Results:
- Fibrinogen markedly inhibited neutrophil chemotaxis induced by ZAS, C5a, and IL-8, and slightly inhibited FMLP-induced migration.
- Inhibition was dose-dependent, saturable, and influenced by the location of fibrinogen in the Boyden chamber.
- Lysine and AHA reduced fibrinogen's inhibitory capacity, suggesting involvement of lysine binding sites in integrin-mediated chemotaxis.
- Fibrinogen dose-dependently inhibited neutrophil oxygen consumption.
Conclusions:
- Circulating fibrinogen modulates key neutrophil functions, including chemotaxis and oxidative burst.
- Fibrinogen's inhibitory effects on neutrophil chemotaxis appear to involve lysine binding sites and integrin function.
- Beyond its role in coagulation, fibrinogen may actively regulate inflammatory responses by influencing neutrophil behavior.
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