Related Experiment Videos
The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes
F W Luscinskas1, D E Mark, B Brunkhorst
1Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
Abstract:
The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.
Insights
Monovalent cations like sodium (Na+) and potassium (K+) influence polymorphonuclear leukocyte (PMNL) activation. While both gradients aid depolarization, neither is essential for immune complex activation or superoxide production, except in NH4Cl-prepared cells.
Area of Science:
- Immunology
- Cellular Physiology
Background:
- Polymorphonuclear leukocytes (PMNLs) are crucial immune cells.
- PMNL activation involves transmembrane depolarization and superoxide production.
- Monovalent cation gradients are implicated in cellular signaling.
Purpose of the Study:
- To investigate the role of sodium (Na+) and potassium (K+) gradients in human PMNL activation.
- To determine the impact of these gradients on immune complex-stimulated depolarization and superoxide production.
Main Methods:
- Immune complex stimulation of human PMNLs.
- Monitoring transmembrane potential changes (depolarization).
- Measuring superoxide production.
- Manipulating extracellular Na+ and K+ concentrations and using ionophores/inhibitors (DMA, valinomycin).
Main Results:
- Abolishing the Na+ gradient reduced depolarization by 50%; collapsing both Na+ and K+ gradients reduced it to 11%.
- Neither Na+ nor K+ gradients were required for immune complex-induced activation or respiratory burst activity.
- Enhanced superoxide production occurred without Na+ or K+ gradients or with valinomycin.
- NH4Cl-prepared PMNLs showed an artifactual requirement for an intact Na+ gradient.
Conclusions:
- Both Na+ and K+ gradients contribute to immune complex-induced membrane depolarization in PMNLs.
- Neither gradient is essential for PMNL activation or respiratory burst.
- The NH4Cl lysis technique introduces an artifactual requirement for Na+ gradients in PMNL stimulation.