A Novel Assay to Measure the Calcium Flux in Human Basophils: Effects of Chemokines and Nerve Growth Factor

Abstract

The calcium flux in human basophils was measured by flow cytometry. Peripheral blood mononuclear cells were labeled with anti-CD123 and anti-HLA-DR antibodies, loaded with fluo-3 acetoxymethyl ester (2 µmol/l) in the presence of probenecid (2.5 µmol/l) and Pluronic F-127 (0.02%) for 20 min, and equilibrated with Ca²⁺ (1.8 mmol/l) and Mg²⁺ (1 mmol/l) for 5 min. The levels of intracellular free calcium were monitored as changes in fluorescence. Cross-linking of surface IgE on basophils with anti-IgE antibodies caused effective calcium flux in atopic, but not in healthy, donors. Concentration-dependent responses to monocyte chemoattractant protein 1 (MCP-1), eotaxin, macrophage inflammatory protein 1 alpha (MIP-1α), and C5a (0.3–10 nmol/l) were observed in all subjects, with a rank order of potency of C5a = MCP-1 > eotaxin > MIP-1α. In contrast, the rank order of potency in causing basophil shape change (i.e., increase in forward scatter) was eotaxin > C5a > MCP-1 > MIP-1α. Nerve growth factor (NGF; 15 nmol/l) did not induce calcium flux in basophils, and pretreatment of cells with a low concentration of NGF (0.3 nmol/l), which has previously been shown to prime basophils for mediator release, had no effect on the calcium response to subsequent stimulation with C5a. We conclude that calcium mobilization is differentially involved in signaling to chemoattractants in basophils and that it is correlated with the agonist’s efficacy to induce mediator release. The data also suggest that priming of basophil responses by NGF does not rely on enhanced calcium mobilization.

Identification of Selective Basophil Chemoattractants in Human Nasal Polyps as Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-2

“…In contrast, IGF-1 and IGF-2 did not cause basophil degranulation, measured by CD63 surface expression, or Ca 2ϩ flux. This is consistent with our previous observation that stimulation of Ca 2ϩ flux in basophils is more closely associated with degranulation than shape change responses (34). Significantly, IGF-2 also inhibited basophil apoptosis and enhanced the antiapoptotic effect of IL-3.…”

“…Changes in intracellular calcium levels were analyzed by three-color flow cytometry, as described previously (34). Briefly, PBMC were labeled with FIGURE 4.…”

The Prostaglandin E 2 Receptor EP4 Is Expressed by Human Platelets and Potently Inhibits Platelet Aggregation and Thrombus Formation

“…[27][28][29] Washed platelets were loaded with 5 mol/L of the acetoxymethyl ester of Fluo-3 in the presence of 2.5 mmol/L probenecid at 37°C for 30 minutes and were then resuspended in PBS containing 0.9 mmol/L Ca 2ϩ and 0.5 mmol/L Mg 2ϩ . Changes in intracellular free Ca 2ϩ levels in response to ADP (10 to 1000 nmol/L) were detected as increase in fluorescence intensity of the Ca 2ϩ -sensitive dye Fluo-3 in the FL-1 channel.…”

Interaction of eosinophils with endothelial cells is modulated by prostaglandin EP4 receptors

“…Polymorphonuclear leukocytes (PMNLs, containing eosinophils and neutrophils) were isolated by dextran sedimentation of erythrocytes then by centrifugation on Histopaque gradients as described previously [51,52]. Eosinophils were purified from polymorphonuclear leukocyte using negative magnetic selection with an Ab cocktail (CD2, CD14, CD16, CD19, CD56, and glycophorin A) and colloidal magnetic particles from StemCell Technologies (Vancouver, Canada) [53].…”