In Vitro Activation of Neutrophils by TNF-α, a Mediator of the Sepsis Syndrome

Document Type

Article

Publication Date

10-1-1996

Description

Purpose: Polymorphonuclear leukocytes (PMNs) have generally been considered incapable of modulating specific immune responses. However, recent studies from several laboratories demonstrate their ability to express cytokines, inflammatory mediators and surface molecules critical to a directed inflammatory response. We therefore studied alterations in the levels of the surface markers ICAM-1, LFA-1α, CD-69 and HLA-DR (crucial for adaptive immunity) on PMNs induced by activation in vitro with TNF-α, a major mediator of gram negative sepsis. Methods: PMNs were isolated from a healthy volunteer using the 1-Step Polymorphs isolation method and were activated with lipopolysaccharide (LPS;10μg/ml) or TNF-α (10 U/ml) for 0, 3, 6, 9, 24 and 48 hours. The PMNs were then stained with FITC or PE conjugated monoclonal antibodies to HLA-DR, ICAM-1 (CD54), LFA-1α (CD11a), FcγRIII (CD16), and CD69 and analyzed by flow cytometry. Results: The neutrophil preparation contained 0-3% lymphocytes, but over 99% of PMNs were gated for analysis. In media control group increased incubation time decreased CD11a expression, but increased CD16, HLA-DR, ICAM-1 and CD69 expression, both in absolute number and density. In TNF-α treated PMN culture, CD11a expression decreased, but CD16, HLA-DR, ICAM-1 and CD69 expression increased when compared to media control. These results were compatible to those of LPS treated positive control group. Conclusions: The data suggests that TNF-α downregulates CD11a, while upregulating CD16, HLA-DR, ICAM-1 and CD69 expression on neutrophils. Clinical implications: The expression of HLA-DR antigen on PMNs suggests a more specific role for these cells in adaptive immune response and in the defense mechanism against infectious agents.

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