Cyclical binding, processing, and functional interactions of neutrophils with leukotriene B₄

Abstract

Leukotriene (LT) B4 activates human polymorphonuclear neutrophils (PMN) by binding to plasmalemmal receptors. It stimulates PMN to raise cytosolic calcium and degranulate. Both responses end within 15-30 sec. However, in less than 15 sec, LTB4-treated PMN lose the ability to respond further to LTB4; decrease the affinity and number of high affinity receptors available for binding LTB4; sequester LTB4 in plasmalemma-associated sites that are inaccessible to a releasing buffer regimen; and begin internalizing LTB4. Over the next 90 min, the cells increasingly internalize LTB4 and convert it to less potent metabolites; release the metabolites; recover LTB4 binding sites; and become fully sensitive to LTB4. Contrastingly, during the entire 90 min incubation with LTB4. PMN retained the capacity to bind and respond normally to a second stimulus, platelet-activating factor. We therefore suggest the following model. LTB4 receptors, when ligand-bound, initiate function but rapidly lose this capacity as they lower their ligand binding affinity and sequester, internalize, or otherwise uncouple from transducing elements. These LTB4 receptor changes contribute to terminating PMN responses and producing a stimulus-selective state of desensitization. During the desensitization period, PMN progressively process and metabolize LTB4. This removes LTB4 from the environment, thereby allowing PMN to recover functional receptors for and sensitivity to the ligand.

Receptors for the 5-Oxo Class of Eicosanoids in Neutrophils

“…Suspensions were placed on ice for 30 min, incubated (4°C) with label for 0 -120 min, layered on 0.4 ml of silicone oil, and centrifuged (12,000 ϫ g for 1 min at 4°C). Isolated supernatant fluids and pellets were counted for radioactivity (32). Results are given as the percentage of total recovered radioactivity in pellets.…”

“…For membrane binding assays, plasma membranes (see below) were incubated (37°C) with radiolabel in 250 l of Hanks' buffer and passed through GF/C filters. Filters were washed with 5 ml of Hanks' buffer (no CaCl 2 or MgCl 2 ; 4°C), air-dried, and counted for radioactivity (32).…”

“…Cells were washed three times, taken up in 8 ml of relaxation buffer, subjected to N 2 -cavitation, and treated with 2 mM EDTA, 50 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. Cavitates were freed of whole cells and nuclei by centrifugation and resolved on Percoll gradients (32 (18)) were pooled. Pools had Ͻ5% of total gradient markers for cytosol (lactate dehydrogenase), endoplasmic reticulum (NADPH-dependent cytochrome c reductase), primary granules (␤-glucuronidase and myeloperoxidase), and secondary granules (lysozyme-and vitamin B-12-binding protein) but ϳ50% of gradient activity for Golgi (UDP-galactose:N-acetylglucosamine galactosyltransferase) and 100% of gradient activity for secretory vesicles (latent alkaline phosphatase (18)).…”

5-Oxo-eicosanoids and Hematopoietic Cytokines Cooperate in Stimulating Neutrophil Function and the Mitogen-activated Protein Kinase Pathway

“…2-4 ϫ 10 8 PMN in 5 ml of Hanks' buffer were challenged at 37°C for 20 min, transferred to 4°C cavitation buffer (5 ml), and further processed at 4°C. Cells were subjected to N 2 cavitation, freed of nuclei by low speed centrifugation, normalized for protein and LDH, and centrifuged through Percoll discontinuous gradients to obtain 19 fractions that were enriched with markers for cytosol (LDH, fractions 1-3), endoplasmic reticulum (NADPH-dependent cytochrome c reductase, fractions 1-7), plasmalemma (surface alkaline phosphatase and [ [12][13][14], and primary granules (␤-glucuronidase, fractions 16 -18), as detailed elsewhere (43). Alkaline phosphatase was assayed by incubating (30 min; 37°C) 60 l of a Percoll gradient fraction in 640 l of a pH 10 solution of 50 mM 2-amino-2-methylpropanol, 0.4% Triton X-100, 14 mM MgCl 2 , and 1.6 mg of p-nitrophenol phosphate.…”

“…GM-CSF (200 pM), G-CSF (10 nM), and LTB 4 (10 nM), but not 500 nM 15-oxoETE, had these same actions. Hence, 5-oxoETE mimics FMLP, PAF, and LTB 4 (42)(43)(44) in translocating secretory vesicles to the cell surface. We also examined unfractionated cavitates.…”