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Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils.

ABSTRACT: Platelet-activating factor (PAF) desensitizes as well as stimulates its various target cells, We find that human polymorphonuclear neutrophils (PMN) exposed to PAF became maximally unresponsive to a second PAF challenge within 15-90 s in assays of Ca2+ mobilization and degranulation. The cells regained full PAF-sensitivity over the ensuing 20-40 min. These effects correlated with changes in PAF receptor availability. PMN treated with PAF, washed in regular buffer and assayed for PAF binding exhibited falls (maximal in 15 s), followed by rises (reaching control levels by 60 min), in the number of high-affinity PAF receptors. However, tracking studies showed that [3H]PAF accumulated on the cell surface for approximately 2 min before being internalized. Regular-buffer washes did not remove this superficial PAF, whereas a washing regimen using excess albumin to adsorb PAF removed 99% of the surface compound. PMN washed by the latter regimen after PAF exposure lost PAF receptors relatively slowly (maximal at approximately 5 min), but the ultimate extent of this loss and the rate at which receptor expression normalized were similar to those of cells washed in regular buffer. Neither cycloheximide nor actinomycin D influenced the course of the receptor changes, but two protein kinase C (PKC) blockers, staurosporine and 1-(5-isoquinolinesulphonyl)piperazine, inhibited the receptor-receptor-depleting actions of PAF. Indeed, a phorbol diester activator of PKC also caused PMN to decrease high-affinity PAF receptor numbers, and the two PKC blockers antagonized this action at concentrations that inhibited PAF-induced PAF receptor losses. We conclude that: (a) PAF induces PMN to down-regulate and then to re-express PAF receptors independently of protein synthesis; (b) these changes are likely to underlie the later stages and reversal of desensitization; (c) the onset (t < or = 2 min) of desensitization, however, precedes receptor down-regulation and must be due to receptor uncoupling from transductional elements; and (d) down-regulation of receptors for PAF appears to be mediated by PKC and/or elements inhibited by PKC blockers.

SUBMITTER: O'Flaherty JT

PROVIDER: S-EPMC1132104 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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Project description:During the past two decades, studies describing the chemistry and biology of PAF have been extensive. This potent phosphoacylglycerol exhibits a wide variety of physiological and pathophysiological effects in various cells and tissues. PAF acts, through specific receptors and a variety of signal transduction systems, to elicit diverse biochemical responses. Several important future directions can be enumerated for the characterization of PAF receptors and their attendant signalling mechanisms. The recent cloning and sequence analysis of the gene for the PAF receptor will allow a number of important experimental approaches for characterizing the structure and analysing the function of the various domains of the receptor. Using molecular genetic and immunological technologies, questions relating to whether there is receptor heterogeneity, the precise mechanism(s) for the regulation of the PAF receptor, and the molecular details of the signalling mechanisms in which the PAF receptor is involved can be explored. Another area of major significance is the examination of the relationship between the signalling response(s) evoked by PAF binding to its receptor and signalling mechanisms activated by a myriad of other mediators, cytokines and growth factors. A very exciting recent development in which PAF receptors undoubtedly play a role is in the regulation of the function of various cellular adhesion molecules. Finally, there remain many incompletely characterized physiological and pathophysiological situations in which PAF and its receptor play a crucial signalling role. Our laboratory has been active in the elucidation of several tissue responses in which PAF exhibits major autocoid signalling responses, e.g. hepatic injury and inflammation, acute and chronic pancreatitis, and cerebral stimulation and/or trauma. As new experimental strategies are developed for characterizing the fine structure of the molecular mechanisms involved in tissue injury and inflammation, the essential role of PAF as a primary signalling molecule will be affirmed. Doubtless the next 20 years of experimental activity will be even more interesting and productive than the past two decades.

Project description:This data series contains spotted oligo microarray data from 10 different experiments using Agilent Rat v2 microarrays. This data is being made public in support of Fillon S et al. Journal of Immunology, (2006). Proinflammatory bacterial components are at least partially responsible for causing the clinical features of sepsis, a syndrome that causes >100,000 deaths each year in the US (1). In the case of Gram positive infection, a key bacterial element recognized by the innate immune system is the cell wall, a complex network of peptidoglycan covalently linked to teichoic acids, proteins and lipoproteins. The current model of innate immune recognition of Gram positive bacteria suggests bacterial cell wall interacts with host recognition proteins, such as toll-like receptors (TLR) and Nod proteins. We describe an additional recognition system mediated by the platelet activating factor receptor (PAFr) and directed to the pathogen associated molecular pattern (PAMP) phosphorylcholine that results in uptake of bacterial components into host cells. Intravascular choline-containing cell walls bound to endothelial cells and caused rapid lethality in wild type, Tlr2-/- and Nod2-/- mice, but not in Pafr-/- mice. Cell wall exited the vasculature into the heart and brain, accumulating within endothelial cells, cardiomyocytes and neurons in a PAFr-dependent way. Physiological consequences of the cell wall/PAFr interaction were cell specific, being noninflammatory in endothelial cells and neurons, but causing rapid loss of cardiomyocyte contractility that contributed to death. Thus, PAFr shepherds phosphorylcholine-containing bacterial components such as cell wall into host cells from where the response ranges from quiescence to severe pathophysiology. Keywords: Competitive hybridizations The ten experiments in this series comprise of four distinct experiments, two of which were performed as biological triplicates and two as biological duplicates. The table below describes the overall design in detail: File Name Experiment 16011868017643v41_GEO_format.txt RBCEC Replicate 1 16011868017644v41_GEO_format.txt RBCEC Replicate 2 251186821865v41_GEO_format.txt Neuron Replicate 1 16011868021377v41_GEO_format.txt Neuron Replicate 2 251186821690v41_GEO_format.txt CW/Lyt44 Replicate 1 251186821691v41_GEO_format.txt CW/Lyt44 Replicate 2 251186821692v41_GEO_format.txt CW/Lyt44 Replicate 1 251186821693v41_GEO_format.txt CW+TNF/Lyt44+TNF Replicate 1 251186821694v41_GEO_format.txt CW+TNF/Lyt44+TNF Replicate 2 251186829677v41_GEO_format.txt CW+TNF/Lyt44+TNF Replicate 3

Project description:Inflammation is an essential process of the host defense against infections, illness, or tissue injuries. However, unregulated inflammation has been associated with chronic inflammatory autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, and atherosclerosis. Polymorphonuclear neutrophils (PMNL) are amongst the first immune cells involved in the acute inflammatory response used to fight bacterial infections. Once activated, PMNL releases inflammatory mediators, enzymes and large quantities of microparticles in the extracellular milieu to recruit various immune cells required to fight the invading pathogens. Recent evidence also shows that platelets (PLTs), well known for their coagulation proprieties, are also implicated in the body’s inflammatory response. Interestingly, activated PLTs can release fully functional mitochondria in the extracellular milieu. Known as the powerhouse of the cell, the mitochondria share similar characteristics with bacteria. Therefore, we hypothesize that PLTs-derived mitochondria present in the extracellular milieu, acting in a similar way as bacteria, induce a sterile inflammatory response that involves the PMNL. The objective of this study was to investigate the sterile inflammatory response of PMNL caused by the exposure to PLTs-derived extracellular mitochondria. Blood was obtained from healthy consenting donors, then PMNL and PLTs-derived mitochondria were isolated and purified. Following the co-incubation of PMNL with various physiological doses of PLTs-derived mitochondria, a characterization of the interaction between PMNL and PLTs-derived mitochondria and an investigation of the inflammatory proprieties of PMNL were performed using flow cytometry, transmission electron microscopy, and high-resolution respirometry. While the transcriptomic analysis indicated that several mitochondrial genes had increased expression, no mitochondrial-dependent increase in oxygen consumption was observed by high-resolution respirometry experiments. However, our data show that PLTs-derived mitochondria significantly induce, in a dose-dependent manner, the release of PMNL microparticles. This study provides new insight into the role of PLTs-derived mitochondria in the context of sterile inflammation.

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Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils.

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