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Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial
Abstract:
Human neutrophils were isolated from peripheral blood by four methods: 1) Ficoll-Hypaque gradients and erythrocyte lysis, 2) plasma-Percoll gradients, 3) a "lipopolysaccharide (LPS)-free" method yielding 85% neutrophils, and 4) by centrifugation of cells prepared by Method 3 through a plasma-Percoll gradient to produce pure neutrophils. The use of the Ficoll-Hypaque method resulted in spontaneous change of cell shape, enhanced formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated release of superoxide anion, increased release of lysosomal enzymes upon subsequent FMLP stimulation, and reduced chemotactic responsiveness, by comparison with the other methods. These effects were not due to erythrocyte lysis by NH4C1 but were reproduced by exposure of neutrophils prepared by the "LPS-free" method or the use of plasma-Percoll gradients to 10-100 ng/ml LPS. Neutrophil change of shape and stimulated O-2 production were particularly sensitive markers of these effects. The effects of trace concentrations of LPS in the modulation of neutrophil function may have relevance to the pathophysiology of endotoxemia and its resultant tissue injury.
Insights
The Ficoll-Hypaque method for isolating human neutrophils alters their function due to trace lipopolysaccharide (LPS). This impacts neutrophil responses, highlighting LPS sensitivity in endotoxemia research.
Area of Science:
- Immunology
- Cell Biology
Background:
- Human neutrophil isolation is critical for studying immune responses.
- Standard isolation methods may inadvertently affect neutrophil function.
Purpose of the Study:
- To compare four methods for isolating human neutrophils.
- To identify how neutrophil isolation techniques influence neutrophil function and responsiveness.
Main Methods:
- Isolation of human neutrophils using Ficoll-Hypaque gradients, plasma-Percoll gradients, an LPS-free method, and a combination method.
- Assessment of neutrophil shape change, superoxide anion release stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP), lysosomal enzyme release, and chemotactic responsiveness.
- Exposure of neutrophils to varying concentrations of lipopolysaccharide (LPS).
Main Results:
- The Ficoll-Hypaque method induced spontaneous cell shape change and altered FMLP-stimulated responses (superoxide anion and lysosomal enzyme release), alongside reduced chemotaxis.
- These functional changes were attributed to trace lipopolysaccharide (LPS) contamination, not erythrocyte lysis.
- Neutrophil shape change and stimulated superoxide production were sensitive indicators of LPS effects.
Conclusions:
- Trace LPS concentrations significantly modulate neutrophil function.
- The Ficoll-Hypaque method is unsuitable for functional studies due to LPS-induced alterations.
- Findings have implications for understanding endotoxemia pathophysiology and associated tissue injury.