MECCANISMO MOLECOLARE DELLA SECREZIONE NELLE CELLULE INFIAMMATORIE

2004/2005I complessi meccanismi di difesa che vengono messi in atto da un organismo in risposta ad un agente infettivo si traducono in uno stato generalmente definito come infiammazione, che rappresenta anche la base del processo di guarigione. Il processo infiammatorio, che viene tipicamente scatenato per arginare un'infezione, è un fenomeno utile, che allo stesso tempo può però rappresentare un potenziale pericolo per la salute. Infatti, se si instaurano le condizioni che conducono ad un'infiammazione cronica (l'agente perturbato re non è eliminato, il processo di riparazione non è ultimato e l'equilibrio interno non è ristabilito) si possono verificare gravi danni tissutali e si possono addirittura creare le condizioni favorevoli alla crescita e alla progressione neoplastica. Per queste ragioni risulta importante capire in dettaglio quali siano i meccanismi che lo regolano, al fine di favorirlo o di ostacolarlo, a seconda della necessità. Il processo secretorio è uno degli eventi di base più importanti per l'innesco dello stato d'infiammazione e per l'attività anti-batterica delle cellule infiammatorie. Lo scarico del contenuto dei granuli secretori dei mastociti nell'ambiente extracellulare ha un notevole potenziale pro-infiammatorio e il killing dei granulociti neutrofili si realizza in un micro-ambiente che imprigiona agenti infettanti, il fagosoma, reso altamente tossico dal rilascio al suo interno, delle sostanze microbicide contenute nei granuli secretori. Appare sempre più chiaro che le modalità di regolazione dell' esocitosi pur potendo essere specifiche da cellula a cellula, seguono uno schema unitario, dal neurone al mastocita. Ad un segnale percepito da specifici recettori, la cellula secretoria costruisce un complesso proteico che aggancia la faccia esterna della membrana granulare alla faccia interna della membrana plasmatica, favorisce la fusione tra le due membrane e quindi lo scarico del contenuto granulare all'esterno. In questa tesi si dimostra nei granulociti neutro fili umani l'espressione della proteina NCS-1 (Neuronal Calcium Sensor-1), già nota nei fenomeni di trasmissione sinaptica, ma ugualmente espressa in cellule neuroendocrine ed ematopoietiche (mastociti). La sua localizzazione sui granuli azurofili dei granulociti neutrofili porta a formulare l'ipotesi di un suo possibile coinvolgimento nella degranulazione calcio-dipendente di questo compartimento granulare Le proteine appartenenti alla famiglia SM (sec-1/ m un c) sono evolutivamente conservate e presenti in ogni compartimento garnulare in cui si verifichi la fusione membranaria. Si dimostra in questa tesi che due proteine della famiglia, Munc 18-2 e Munc 18-3 sono espresse nei granulociti neutrofili, dove svolgono probabilmente un ruolo fondamentale nello scarico dei granuli azurofili. Inoltre, è indagata nei mastociti, la diretta interazione tra la proteina Munc 18-2 e la tubulina, entrambe probabilmente coinvolte nel processo dell'esocitosi composta. In questa tesi, si illustra infine un metodo per l'isolamento dei corpi dell'asbesto, che sono molto probabilmente il risultato della modificazione delle fibre di asbesto o amianto ad opera della fagocitosi e della secrezione di macrofagi e di neutro fili. Si dimostra come tali corpi dell'asbesto, considerati a lungo come inerti, possiedano in realtà una potenziale attività citotossica dovuta ai componenti che formano il loro "coating".XVIII Ciclo1974Versione digitalizzata della tesi di dottorato cartacea

VAMP-8 segregates mast cell–preformed mediator exocytosis from cytokine trafficking pathways

AbstractInflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow–derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8–deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8–deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3–positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways

Role of SpdA in Cell Spreading and Phagocytosis in Dictyostelium.

Dictyostelium discoideum is a widely used model to study molecular mechanisms controlling cell adhesion, cell spreading on a surface, and phagocytosis. In this study we isolated and characterize a new mutant created by insertion of a mutagenic vector in the heretofore uncharacterized spdA gene. SpdA-ins mutant cells produce an altered, slightly shortened version of the SpdA protein. They spread more efficiently than WT cells when allowed to adhere to a glass substrate, and phagocytose particles more efficiently. On the contrary, a functional spdA knockout mutant where a large segment of the gene was deleted phagocytosed less efficiently and spread less efficiently on a substrate. These phenotypes were highly dependent on the cellular density, and were most visible at high cell densities, where secreted quorum-sensing factors inhibiting cell motility, spreading and phagocytosis are most active. These results identify the involvement of SpdA in the control of cell spreading and phagocytosis. The underlying molecular mechanisms, as well as the exact link between SpdA and cell spreading, remain to be established

Human eosinophil peroxidase induces surface alteration, killing and lysis of Mycobacterium tuberculosis

The antimycobacterial role of eosinophil peroxidase (EPO), one of the most abundant granule proteins in human eosinophils, was investigated. Our data indicate that purified EPO shows significant inhibitory activity towards Mycobacterium tuberculosis H37Rv. On a molar basis, this activity was similar to that exhibited by neutrophil myeloperoxidase (MPO) and was both dose and time dependent. In contrast to the activity of MPO, which requires H(2)O(2), EPO also exhibited anti-M. tuberculosis activity in the absence of exogenously added peroxide. Morphological evidence confirmed that the mechanism of action of EPO against mycobacteria differs from that of MPO. While MPO kills M. tuberculosis H37Rv exclusively in the presence of hydrogen peroxide, it does not induce morphological changes in the pathogen. In contrast, EPO-treated bacteria frequently had cell wall lesions and eventually underwent lysis, either in the presence or in the absence of H(2)O(2)

WT and <i>spdA-ins</i> cells have similar sizes.

<p>(A) Cell size was analyzed by electric current exclusion using a CASY 1 cell counter. (B) The packed cell volume of a known number of cells was determined in graded tubes. (C) The amount of protein per cell was determined using a Lowry assay. For each experiment, the average and SEM of three independent experiments is indicated. No significant differences were seen between WT and <i>spdA-ins</i> cells.</p

The cellular amounts of SibA, Phg1 and Talin are similar in WT cells and in <i>spdA-ins</i> cells.

<p>To determine the cellular amount of SibA, Pgh1A or Talin, cellular proteins were separated by electrophoresis and specific proteins revealed with antibodies against SibA (A), Talin (B) or Phg1A (C). The intensity of the signal was quantified and expressed in arbitrary units (a.u.). The average and SEM of four independent experiments are represented. The amounts of SibA, Phg1a and Talin were similar in WT cells and in <i>spdA-ins</i> cells.</p

Role of SpdA in the regulation of phagocytosis by cell density.

<p>(A) Cells were grown to the indicated densities, and allowed to phagocytose fluorescent latex beads for 20 minutes. Phagocytosis was measured by flow cytometry. The results of three independent experiments were pooled in this figure. (B) In the experiment described in A, phagocytosis in mutant cells was directly compared to phagocytosis by WT cells grown at the same density. While both mutant cells phagocytosed like WT cells at low cell density, marked differences appeared when cellular density increased.</p

The actin organization is not significantly altered in <i>spdA-ins</i> cells.

<p>Cells were allowed to adhere to a glass coverslip for 10 min in HL5. After fixation filamentous actin was labeled with fluorescent phalloidin. The contact area between cells and their substrate was visualized by confocal microscopy, and did not reveal gross alterations of actin organization in <i>spdA-ins</i> cells. When cells were incubated in phosphate buffer (PB), formation of filopodia was induced in both WT and <i>spdA-ins</i> cells.</p

Characterization of <i>spdA-ins</i> mutant cells.

<p>(A) <i>SpdA-ins</i> mutant cells were originally created by the random insertion of a REMI mutagenic vector (pSC) in the coding sequence of gene DDB_G0287845 (position 2635). (B) To quantify growth of <i>Dictyostelium</i> on bacteria, we applied 10'000, 1'000, 100 or 10 <i>Dictyostelium</i> cells on a lawn of <i>K</i>. <i>pneumoniae</i> or <i>M</i>. <i>luteus</i> bacteria (black). WT cells created a phagocytic plaque (white). <i>SpdA</i> mutant cells grew as efficiently as WT cells on a lawn of <i>K</i>. <i>pneumoniae</i> but less efficiently in the presence of <i>M</i>. <i>luteus</i>. (C) Growth of <i>Dictyostelium</i> mutant strains in the presence of different bacterial species.</p

<i>SpdA-ins</i> cells adhere more efficiently than WT cells to their substrate.

<p>(A) Side view of a cell attached to its substrate and exposed to a flow of medium. The adhesion of the cell to its substrate can be assessed by determining the speed of a flow of medium that is necessary to detach the cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160376#pone.0160376.ref019" target="_blank">19</a>]. The strength applied by the flow of medium on the cell is σh², and its mechanical moment (σh³) is balanced by the adhesive force (F). Inspired from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160376#pone.0160376.ref018" target="_blank">18</a>]. (B) Percentage of detached cells as a function of the applied shear stress. At a low flow (between 0 and 0.5 Pa), <i>spdA-ins</i> cells detached less readily than WT cells from the substrate. At higher flow (>0.5 Pa) no significant difference can be seen between WT cells and <i>spdA-ins</i> cells. Data from three independent experiments is represented in this graph. A decrease in cell detachment can be the result of an increase in the adhesion force (F) or of a decrease in h (i.e. of a more efficient cell spreading).</p