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Membrane fluidity changes accompanying phagocytosis in normal and in chronic granulomatous disease polymorphonuclear
Abstract:
We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by-products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.
Insights
Polymorphonuclear leukocytes (PMN) membrane fluidity remains unchanged in chronic granulomatous disease (CGD) patients at rest. During phagocytosis, reactive oxygen species, particularly hydrogen peroxide (H2O2), alter PMN membrane biophysical properties.
Area of Science:
- Biophysics
- Cell Biology
- Immunology
Background:
- Polymorphonuclear leukocytes (PMN) are crucial immune cells involved in phagocytosis.
- Membrane fluidity plays a role in cellular functions, including phagocytosis.
- Chronic granulomatous disease (CGD) is a genetic disorder affecting phagocyte function.
Purpose of the Study:
- To investigate changes in PMN membrane fluidity during phagocytosis.
- To determine the role of reactive oxygen species (ROS) in modulating PMN membrane fluidity.
- To compare membrane fluidity between normal and CGD PMN during phagocytosis.
Main Methods:
- Electron spin resonance (ESR) spectroscopy using a spin probe (5DS) to assess membrane fluidity via the order parameter (S).
- Incubation of PMN from normal subjects and CGD patients with opsonized zymosan.
- Treatment of PMN with ROS scavengers (catalase, superoxide dismutase, sodium benzoate) and glucose oxidase.
Main Results:
- Resting PMN from normal and CGD subjects showed no significant difference in membrane fluidity (S values).
- Normal PMN exhibited a significant increase in membrane order (decreased fluidity) upon zymosan stimulation, while CGD PMN did not.
- ROS scavengers and catalase treatment in normal PMN mimicked the resting state, indicating H2O2's role in fluidity changes.
- Glucose oxidase-induced H2O2 production in CGD PMN led to altered membrane fluidity.
Conclusions:
- Reduced oxygen (O2) by-products, especially hydrogen peroxide (H2O2), significantly alter PMN membrane biophysical properties during phagocytosis.
- CGD, characterized by a defect in ROS production, shows impaired changes in membrane fluidity during phagocytosis.
- These findings highlight the critical role of ROS in modulating immune cell membrane dynamics.