Extracellular vesicles, news about their role in immune cells: physiology, pathology and diseases.

Two types of extracellular vesicles (EVs), exosomes and ectosomes, are generated and released by all cells, including immune cells. The two EVs appear different in many properties: size, mechanism and site of assembly, composition of their membranes and luminal cargoes, sites and processes of release. In functional terms, however, these differences are minor. Moreover, their binding to and effects on target cells appear similar, thus the two types are considered distinct only in a few cases, otherwise they are presented together as EVs. The EV physiology of the various immune cells differs as expected from their differential properties. Some properties, however, are common: EV release, taking place already at rest, is greatly increased upon cell stimulation; extracellular navigation occurs adjacent and at distance from the releasing cells; binding to and uptake by target cells are specific. EVs received from other immune or distinct cells govern many functions in target cells. Immune diseases in which EVs play multiple, often opposite (aggression and protection) effects, are numerous; inflammatory diseases; pathologies of various tissues; and brain diseases, such as multiple sclerosis. EVs also have effects on interactive immune and cancer cells. These effects are often distinct, promoting cytotoxicity or proliferation, the latter together with metastasis and angiogenesis. Diagnoses depend on the identification of EV biomarkers; therapies on various mechanisms such as (1) removal of aggression-inducing EVs; (2) EV manipulations specific for single targets, with insertion of surface peptides or luminal miRNAs; and (3) removal or re-expression of molecules from target cells.

Differential repression by the transcription factor REST/NRSF of the various Ca2+ signalling mechanisms in pheochromocytoma PC12 cells.

Expression of the nerve cell phenotype is orchestrated by the REST/NRSF transcription repressor, working on hundreds of genes recognized at a specific regulatory binding sequence. Most PC12 clones, the most frequently employed neuronal model, maintain low levels of REST; however a few, defective of neurosecretion, express high levels. To investigate the role of REST in Ca2+ signalling we studied the [Ca2+](i) changes in single cells of four clones, two wild-type and two defective, pre-treated for 5 days with NGF. We focused on Ca2+ influxes induced by depolarization and ATP. Only a subpopulation ( approximately 15%) of the defective, high REST cells responded to depolarization (Ca(V) expression approximately 10%). The ATP-induced intracellular Ca2+ release was little changed, whereas influx via ionotropic P2X receptors was decreased, in agreement with the decreased expression of P2X2 receptors. The percentage of defective cells expressing store-operated calcium entry (SOCE) following ATP stimulation was also lower. The responses of the defective clones were little affected by their differentiated state. In conclusion, our results revealed important new aspects of REST control of Ca2+ homeostasis, of potential physiological importance. The mechanisms of this control remain to be investigated.

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation.

AIMS/HYPOTHESIS: We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. METHODS: The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. RESULTS: Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin +/- high glucose release of chromogranin B predominated. Weak, Ca(2+)-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca(2+) sensitivity of the specific granules. CONCLUSIONS/INTERPRETATION: The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.

Triggering receptor expressed in myeloid cells 2 (TREM2) trafficking in microglial cells: continuous shuttling to and from the plasma membrane regulated by cell stimulation.

Cell biology of triggering receptor expressed in myeloid cells 2, a receptor expressed in brain cells (microglia and possibly neurons and oligodendrocytes) which is responsible for a neurological and psychiatric genetic disease, polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy otherwise called the Nasu-Hakola disease, is still largely unknown. Using immortalized mouse N9 microglial cells we demonstrate that triggering receptor expressed in myeloid cells 2 is mostly distributed intracellularly in two pools: a deposit in the Golgi complex and a population of exocytic vesicles, distinct from endosomes and lysosomes, which is continuously translocated to, and recycled from the cell surface. Results with ionomycin and gamma-interferon, showing rapid and slow increases, respectively, of triggering receptor expressed in myeloid cells 2 surface density, documented that the exocytosis of the receptor-rich vesicles is regulated. Pulse labeling in the cold of surface triggering receptor expressed in myeloid cells 2 with its antibody (or Fab fragment) followed by chase at 37 degrees C showed internalization, with recovery of the antibody in endosomes and lysosomes. However, part of the receptor/antibody complex, internalized for up to 30 min chase, was recycled to the cell surface within 2 min of ionomycin stimulation, together with a fraction of the total biotinylated surface protein chased in parallel. The internalized receptor appears therefore to get access to exocytic organelles distinct from lysosomes which may resemble the exocytic vesicles of resting cells. These results document that, in microglial cells, the surface density of the triggering receptor expressed in myeloid cells 2 and thus, presumably, the response to its activation, is continuously adapted and can be greatly increased, even at rapid rate, as a function of cell activation.

Surface wound healing: a new, general function of eukaryotic cells.

The ability to repair surface wounds is a property, necessary for long-term survival, expressed to various extents by all eukaryotic cell types except erythrocytes. The process is based on the rapid Ca(2+)-induced exocytosis of various types of specific organelles, such as lysosomes and enlargeosomes, that decreases surface tension and makes possible the spontaneous fusion of lipid monolayers at the lesion edges. The recognized importance of the process in physiology and in several cases of pathology is discussed.

Synaptic vesicles: is kissing a matter of competence?

The "kiss-and-run" model of exocytosis and endocytosis predicts that synaptic vesicles can undergo fast and efficient recycling, after fusion with the plasmalemma, without intermixing of membranes. Evidence is mounting from several new experimental approaches that kiss-and-run occurs at synapses. Distinct vesicle pools, which initially were identified in morphological terms, are now being characterized in biochemical and functional terms. In addition, at least two functional recycling pathways, operating on different time scales (from milliseconds to tens of seconds), have been shown to coexist in the same synaptic system, and the two pathways appear to be differentially regulated. Taken together, these data suggest that kiss-and-run operates in parallel with the classical, coated-vesicle recycling. Here, we review recent evidence for kiss-and-run recycling and discuss whether it is a distinct process, dependent on the molecular organization of the fusing vesicle. We propose that vesicles undergo a process of "competence maturation". According to this view, the specific molecular make-up of the vesicles, their location and their interactions with nerve terminal proteins might determine not only the differential availability of the vesicles for fusion and neurotransmitter release but also the recycling path that they will follow.

Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins.

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.

Ultra rapid calcium events in electrically stimulated frog nerve terminals.

Fast calcium events occurring in cytoplasmic organelles after a single electrical stimulus were investigated by electron spectroscopic imaging (an electron microscope technique that reveals total calcium with high sensitivity and spatial resolution) in quick frozen presynaptic terminals of the frog neuromuscular junction. In resting preparations synaptic vesicles showed a prominent calcium signal whereas mitochondria were mostly negative and only some of the cisternae of the endoplasmic reticulum were clearly positive. In preparations quick frozen 10 ms after the application to the nerve of a single, supramaximal electric stimulus, no obvious change was observed in synaptic vesicles, while calcium levels rose to high values in the endoplasmic reticulum cisternae and in the matrix of mitochondria. Voltage-induced influx of Ca(2+) within synaptic terminals appears therefore to induce an extremely rapid uptake into selected organelles. The possible physiological role of this response is discussed.

CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity.

Astrocytes actively participate in synaptic integration by releasing transmitter (glutamate) via a calcium-regulated, exocytosis-like process. Here we show that this process follows activation of the receptor CXCR4 by the chemokine stromal cell-derived factor 1 (SDF-1). An extraordinary feature of the ensuing signaling cascade is the rapid extracellular release of tumor necrosis factor-alpha (TNFalpha). Autocrine/paracrine TNFalpha-dependent signaling leading to prostaglandin (PG) formation not only controls glutamate release and astrocyte communication, but also causes their derangement when activated microglia cooperate to dramatically enhance release of the cytokine in response to CXCR4 stimulation. We demonstrate that altered glial communication has direct neuropathological consequences and that agents interfering with CXCR4-dependent astrocyte-microglia signaling prevent neuronal apoptosis induced by the HIV-1 coat glycoprotein, gp120IIIB. Our results identify a new pathway for glia-glia and glia-neuron communication that is relevant to both normal brain function and neurodegenerative diseases.

Total calcium ultrastructure: advances in excitable cells.

This is a review which is written on the basis of a cell calcium lecture delivered on 22 July 2000 at the European Research Meeting 'Calcium as a molecule of cellular integration'.

Nitric oxide inhibits tumor necrosis factor-alpha-induced apoptosis by reducing the generation of ceramide.

Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-alpha (TNF-alpha) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-alpha, however, ceramide potentiated, whereas NO inhibited, TNF-alpha-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-alpha-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.

Requirement of pyk2 for the activation of the MAP kinase cascade induced by Ca(2+) (but not by PKC or G protein) in PC12 cells.

The role of the Ca(2+)-activated tyrosine kinase, Pyk2, in the pleiotropic coupling of nerve cell stimulation to the MAP kinase cascade still remains undefined. Using a panel of PC12 clones, one of which was defective in Pyk2, we demonstrate (1) that the MAP kinase response induced by a [Ca(2+)](i) rise (following application of the Ca(2+) ionophore, ionomycin) is inappreciable in the defective clone and is re-established after Pyk2 transfection; and (2) that the responses to both protein kinase C and P(2y2) receptor activation occur normally even in the defective cells. We conclude that Pyk2 is the key mediator in the pathway activated by Ca(2+) but has minor roles with the other types of stimulation.

Neurosecretory cells without neurosecretion: evidence of an independently regulated trait of the cell phenotype.

Neurosecretion competence is a fundamental property that enables differentiated neurones and professional neurosecretory cells to store neurotransmitters and hormones in specialized organelles, the synaptic-like vesicles and dense granules, and to release them by regulated exocytosis. In our laboratory, the study of rat phaeochromocytoma (PC12) clones that fail to express the above organelles or any other components involved in neurosecretion, whilst maintaining most of the general markers of the parental population, has served to demonstrate that this trait is controlled independently from the rest of the phenotype. The present review focuses on recent advances in elucidating the molecular mechanisms governing neurosecretion competence. Moreover, the opportunities that such neurosecretion-defective PC12 clones offer for the investigation of new aspects of regulated exocytosis and the localization of its components are summarized.

Blockade of membrane transport and disassembly of the Golgi complex by expression of syntaxin 1A in neurosecretion-incompetent cells: prevention by rbSEC1.

The t-SNAREs syntaxin1A and SNAP-25, i.e. the members of the complex involved in regulated exocytosis at synapses and neurosecretory cells, are delivered to their physiological site, the plasma membrane, when transfected into neurosecretion-competent cells, such as PC12 and AtT20. In contrast, when transfection is made into cells incompetent for neurosecretion, such as those of a defective PC12 clone and the NRK fibroblasts, which have no endogenous expression of these t-SNAREs, syntaxin1A (but neither two other syntaxin family members nor SNAP-25) remains stuck in the Golgi-TGN area with profound consequences to the cell: blockade of both membrane (SNAP-25, GAT-1) and secretory (chromogranin B) protein transport to the cell surface; progressive disassembly of the Golgi complex and TGN; ultimate disappearance of the latter structures, with intermixing of their markers (mannosidase II; TGN-38) with those of the endoplasmic reticulum (calreticulin) and with syntaxin1A itself. When, however, syntaxin 1A is transfected together with rbSec1, a protein known to participate in neurosecretory exocytosis via its dynamic interaction with the t-SNARE, neither the blockade nor the alterations of the Golgi complex take place. Our results demonstrate that syntaxin1A, in addition to its role in exocytosis at the cell surface, possesses a specific potential to interfere with intracellular membrane transport and that its interaction with rbSec1 is instrumental to its physiological function not only at the plasma membrane but also within the cell. At the latter site, the rbSec1-induced conversion of syntaxin1A into a form that can be transported and protects the cell from the development of severe structural and membrane traffic alterations.

The p75(NTR)-induced apoptotic program develops through a ceramide-caspase pathway negatively regulated by nitric oxide.

SK-N-BE neuroblastoma cell clones transfected with p75(NTR) and lacking Trk neurotrophin receptors, previously reported to undergo extensive spontaneous apoptosis and to be protected by nerve growth factor (NGF) (Bunone, G., Mariotti, A., Compagni, A., Morandi, E., and Della Valle, G. (1997) Oncogene 14, 1463-1470), are shown to exhibit (i) increased levels of the pro-apoptotic lipid metabolite ceramide and (ii) high activity of caspases, the proteases of the cell death cascade. In the p75(NTR)-expressing cells, these parameters were partially normalized by prolonged NGF treatment, which, in addition, decreased apoptosis, similar to caspase blockers. Conversely, exogenous ceramide increased caspase activity and apoptosis in both wild-type and p75(NTR)-expressing cells. A new p75(NTR)-expressing clone characterized by low spontaneous apoptosis exhibited high endogenous ceramide and low caspase levels. A marked difference between the apoptotic and resistant clones concerned the very low and high activities of nitric-oxide (NO) synthase, respectively. Protection from apoptosis by NO was confirmed by results with the NO donor S-nitrosoacetylpenicillamine and the NO-trapping agent hemoglobin. We conclude that the p75(NTR) receptor, while free of NGF, triggers a cascade leading to apoptosis; the cascade includes generation of ceramide and increased caspase activity; and the protective role of NO occurs at step(s) in between the latter events.

Multiple and diverse forms of regulated exocytosis in wild-type and defective PC12 cells.

Regulated exocytosis triggered by the photolysis of a caged Ca2+ compound, DM-nitrophen, was investigated by patch-clamp capacitance measurements in two clones of PC12, the first wild-type and the second (PC12-27) defective of both types of classical secretory vesicles together with the neuronal-type receptors for the attachment proteins of the N-ethylmaleimide-sensitive fusion protein, the so called SNAREs. Moreover, the electrophysiological data were correlated with the ultrastructure of resting quick-frozen-freeze-dried cells of the two clones. Wild-type PC12 exhibited two-component capacitance responses, time constants of 30-100 ms and >10 s, that previous studies had suggested to reflect primarily the fusion of the small and large secretory vesicles, each contributing cell surface increases of approximately 10%. Both of these components were largely and specifically inhibited whether cells previously were microinjected with tetanus toxin light chain. In the defective clone, large responses also were recorded ( approximately 19% surface expansion; time constant, approximately 1 s) that, in contrast to those of the wild-type, were entirely resistant to the toxin. Although secretory organelles, i.e., large vesicles and also profiles of small vesicles, were abundant at the cell periphery and often docked to the plasmalemma of resting wild-type PC12, in the defective clone, no superficial accumulation of vesicles was observed. Our coordinate structural and functional results have revealed diversities between the two classical forms of regulated secretion in wild-type PC12 and have provided evidence of a toxin-insensitive form of Ca2+-induced exocytosis, prominent in the defective clone, that may play an important role(s) in cellular physiology.