Changes in membrane potential of human granulocytes antecede the metabolic responses to surface stimulation

Abstract

Human granulocytes (polymorphonuclear leukocytes) exposed to surface stimuli [e.g., immune complexes, concanavalin A (Con A)] generate°2--, undergo a respiratory burst, and secrete lysosomal enzymes. To study the earliest reaction of ligands with surface receptors of granulocytes, purified cells were exposed to bovine serum albumin-anti-albumin complexes (Fc receptors) MATERIALS AND METHODS Preparation of PMN Suspensions. PMN suspensions containing 98 + 1% PMN were prepared from heparinized venous blood (10 units of heparin per ml of blood) obtained from healthy adult donors. Standard techniques (Hypaque/Ficoll gradients) were used (19) followed by dextran sedimentation and hypotonic lysis. The cells were suspended in Krebs-Ringer pH 7.45 phosphate buffer (KRP; Na+, 149 mM; K+, 4.8 mM;Ca2+, 1.3 mM; Mg2+, 1.2 mM; P043-, 15.6 mM; Cl-, 140 mM).This buffer was used throughout unless otherwise indicated. In buffers from which divalent cations were omitted, an equal volume of 0.9% NaCl was substituted. The high-K+ buffer was prepared by substituting KCl for NaCl and potassium phosphate buffer for the sodium phosphate buffer.TPMP+ Distribution Measurements. Uptake of TPMP+ was measured by a minor modification of the method of Schuldiner and Kaback (13). PMNs were incubated in KRP at 370 in the presence of 100MuM [H3]TPMP+ (120 Ci/mol), in a final volume of 100 gl. Incubations were carried out in 5-ml glass culture tubes treated with Siliclad (Clay-Adams) in order to minimize cell adhesion to the tube. The reaction was terminated by the addition of 3 ml of ice-cold 0.9% saline (<1 sec) followed by rapid filtration on a 0.5-,m-pore-diameter Celotate filter (Millipore). This was followed by a further rinse with 3 ml of ice-cold saline. The number of PMNs per assay (usually 4 X 106) was chosen to ensure that the entire filtration and washing procedure could be carried out in less than 20 sec. The Abbreviations: PMN, polymorphonuclear leukocyte; KRP, KrebsRinger phosphate buffer; TPMP+, triphenylmethyl phosphonium ion; Con A, concanavalin A; Ab/Ag, immune complex of bovine serum albumin and anti-bovine serum albumin IgG; Alt', membrane potential; PNa+, Na+ permeability; PK+, K+ permeability; EGTA, ethylene glycol-bis(fl-aminoethyl ether)-N,N'-tetraacetic acid.

Effect of a Radical Scavenger "Water Soluble Protein" from Broad Beans (Vicia faba) on Antioxidative Enzyme Activity in Cellular Aging.

“…On the other hand, treatment with WSP or HC caused 10-70% decreases in SOD activity, except for the WSP treatments for 4wk in young cells and for 6wk in old cells, and HC treatments for 6wk in young cells and 6wk in old cells. It can be assumed from these changes in SOD activity that WSP or HC treatment brings about a reduction in oxidative stress since the generation of superoxide radicals in cultured cells is inhibited by WSP (15)or HC (11).…”

“…Fulder (10) also reported that the increase in density resulted from a faster growth rate. In addition, it was shown that HC inhibited superoxide generation (11).…”

Interferon-γ Corrects the Respiratory Burst Defect In Vitro in Monocyte-Derived Macrophages from Glycogen Storage Disease Type 1b Patients

“…The generation of 0 2 -by MDM was measured on the 9th d of culture in the well plate as superoxide dismutaseinhibitable cytochrome c reduction by endpoint analysis (33). DNA assay.…”

In Vitro and In Vivo Effects of Granulocyte Colony-Stimulating Factor on Neutrophils in Glycogen Storage Disease Type IB: Granulocyte Colony-Stimulating Factor Therapy Corrects the Neutropenia and the Defects in Respiratory Burst Activity and Ca2+ Mobilization

“…The generation of 0 2 -by neutrophils and monocytes was measured as superoxide dismutase-inhibitable cytochrome c reduction by a continuous recording spectrophotometric method (34). Cells were preincubated for 5 min at 37°C before addition of stimulus.…”