Abstract
BackgroundPolymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods.ResultsTo identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis.ConclusionOur data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.
Highlights
Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli
Resolution of plasma membrane-enriched fractions from resting PMN The light membrane fraction recovered from a two-step Percoll density gradient separation of cavitated resting PMN [13], the γ fraction, is enriched for plasma membrane vesicles (PMV) and contains secretory vesicles (SV), a labile intracellular compartment whose membranes contain several functionally important proteins [17]
PMN isolated by sequential dextran sedimentation and differential density centrifugation on Hypaque-Ficoll generate 1.01 ± 0.21 nmoles superoxide anion/106 PMN/10 min (n = 9), whereas PMN stimulated with 100 ng/ml of phorbol myristate acetate produce 78.47 ± 2.48 nmoles superoxide anion/106 PMN/10 min (n = 9)
Summary
Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. The NADPH oxidase is a multicomponent enzyme complex that is unassembled and inactive in the resting PMN, with essential components segregated in distinct cellular compartments (i.e. membrane vs cytoplasm) in the unstimulated cell. Antimicrobial proteins, and hydrolytic enzymes act independently and cooperate synergistically to create an environment within the phagosome that is extremely inhospitable to the ingested microbe. Both oxidase assembly and degranulation represent agonistdependent redistribution of prefabricated biological elements, a strategy of cellular response that is especially tailored to the physiologic responsibilities of PMN within the context of innate immunity and distinctly different from one dependent on transcriptional control of the production of reactive molecules [12]
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