Actin S-Nitrosylation Inhibits Neutrophil β2 Integrin Function

Abstract

The focus of this work was to elucidate the mechanism for inhibition of neutrophil ␤ 2 integrin adhesion molecules by hyperoxia. Results demonstrate that exposure to high oxygen partial pressures increases synthesis of reactive species derived from type 2 nitric-oxide synthase and myeloperoxidase, leading to excessive S-nitrosylation of ␤-actin and possibly profilin. Hyperoxia causes S-nitrosylation of the four cysteine moieties closest to the carboxyl-terminal end of actin, which results in formation of short actin filaments. This alters actin polymerization, network formation, and intracellular distribution, as well as inhibits ␤ 2 integrin clustering. If neutrophils are exposed to ultraviolet light to reverse S-nitrosylation, or are incubated with N-formyl-methionyl-leucine-phenylalanine to trigger "insideout" activation, the effects of hyperoxia are reversed. We conclude that cytoskeletal changes triggered by hyperoxia inhibit ␤ 2 integrin-dependent neutrophil adhesion.

Factors Associated with Nitric Oxide-mediated β2 Integrin Inhibition of Neutrophils

“…Fibrinogen-coated Plate Adherence-Preparation and use of fibrinogen-coated plates to measure ␤ 2 integrin-specific neutrophil adherence in calcein AM-loaded cells were as previously described (24,26). Suspensions of 25,000 cells in 100 l of PBS were added to plate wells, and at the end of a 10-min incubation, wells were washed twice with 4 ml of PBS to remove nonadherent cells.…”

“…Subjects were healthy adults who had taken no medications for at least 4 days prior to phlebotomy. Blood was acquired in heparinized tubes, and neutrophils were isolated as previously described (24,31).…”

“…Lysates were centrifuged at 2000 ϫ g for 10 min, and supernatant was recovered and made 0.4% CHAPS using the 10% stock solution. The biotin switch assay was carried out following published methods (24).…”

“…With regard to ␤ 2 integrin regulation, S-nitrosylation of cytoplasmic actin (SNO-actin) was shown to increase formation of Triton-soluble actin filaments, leading to alterations in the cytoskeletal network that inhibit ␤ 2 integrin-dependent adherence-associated with exposure to high pressure oxygen (24). Reactive nitrogen species are generated because nitric-oxide synthase type 2 (NOS-2 or iNOS) activation occurs (25).…”

“…Reactive nitrogen species are generated because nitric-oxide synthase type 2 (NOS-2 or iNOS) activation occurs (25). Agents capable of causing S-nitrosylation are generated when ⅐ NO synthesized by iNOS reacts with reactive oxygen species (ROS) that are synthesized by myeloperoxidase that is concurrently activated by the hyperoxic exposure (24). An autocatalytic cycle occurs because vasodilator stimulated phosphoprotein (VASP) exhibits high affinity for S-nitrosylated Triton-soluble filamentous actin (sF-actin) (26).…”