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Biomedical subjects

P Rovere

Publications and source records attributed to P Rovere.

51 records · Page 3Linked to original sources
PubMed1997

Processing of engulfed apoptotic bodies yields T cell epitopes.

Programmed death via apoptosis is the metazoan physiologic mode of cell death. Apoptotic cells are recognized by scavenger phagocytes via a number of membrane receptors and engulfed. Thereafter, little is known of their fate, or that of phagocytes. Here, we have traced apoptotic cells upon their engulfment by macrophages. After 3 h, apoptotic cells were contained in discrete well-defined vacuoles. Upon overnight chase, several small vesicles, possibly originating from the fragmentation of original vacuoles, were evident all over the macrophage body. Furthermore, Ags were diffused in the cytosol of some cells, which raises the possibility that epitopes from engulfed apoptotic cells may associate with macrophage MHC class I molecules and be recognized by T lymphocytes. Indeed, Ag-specific CTLs recognize and specifically lyse syngeneic macrophages upon phagocytosis of MHC class I-positive or -negative apoptotic cells, provided that they contain the relevant Ags. Synthesis and membrane expression of class I molecules by macrophages, together with functional transporters associated with Ag presentation, were necessary for recognition and lysis. The indirect presentation of epitopes from engulfed apoptotic cells by scavenger Ag-presenting phagocytes may, in the absence of "danger" signals, have implications for the establishment of central and peripheral self-tolerance.

Animals↗
PubMed1997

Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells.

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.

Animals↗
PubMed1997

Autocrine nitric oxide modulates CD95-induced apoptosis in gammadelta T lymphocytes.

Gammadelta T lymphocytes play an important early role in the defense against pathogens. Their function is terminated by acquisition of susceptibility to CD95-triggered apoptosis. Here we show that the regulation of this process depends on the activity of the endothelial NO synthase expressed by gammadelta T lymphocytes, which is modulated in an activation-dependent way. The effects of nitric oxide thus generated, mediated via cGMP generation, are exerted at at least two sites along the CD95 signaling cascade: one at, or upstream, and the other downstream of ceramide generation. At either site, nitric oxide/cGMP action is sufficient for protection from apoptosis. The effect of NO is selective for apoptosis induced by CD95 cross-linking, since it does not affect apoptotic program triggered by other stimuli. The evidence here reported demonstrates a new physiological role for nitric oxide, acting as a survival factor for T lymphocytes.

Apoptosis↗
PubMed1997

Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.

The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

Animals↗
PubMed1997

Quantitative cytometry of MHC class I digestion from living cells.

Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the beta2-microglobulin (beta2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). beta2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, "immature" MHC molecules.

Animals↗
PubMed1997

Dendritic cells from mice lacking the invariant chain express high levels of membrane MHC class II molecules in vivo.

We investigated in H-2k mice bearing a genetically disrupted invariant chain (Ii) gene, the MHC class II expression and antigen presentation ability of dendritic cells (DC) freshly purified from the spleen (SpDC) or derived from bone marrow precursors (BMDC) upon treatment with GM-CSF. In the absence of Ii, class II alpha/beta heterodimers are expressed on the DC membranes to a similar extent than in control mice, in contrast to splenic B cells. Class II molecules immunoprecipitated from the plasma membrane of Ii deficient DC are compact indicating that the dimers are stabilized by antigenic peptides. Furthermore DC from Ii mutant mice are able to present to CD4+ T lymphocytes, epitopes derived from the processing of the hen egg lysozyme (HEL) that normally require expression of the Ii molecule for presentation by B cells. All together, our results show that the antigen processing machinary of DC provides peptides that can reach class II molecules and stabilize their conformation in the absence of Ii mediated targeting of class II complexes.

Animals↗
PubMed1997

Differential mRNA expression in untreated and TNF-alpha elicited murine dendritic cells precursors.

We have compared the pattern of gene expression in long term cultured precursor dendritic cells (DC), either untreated (immature) or cultured for two days in the presence of recombinant murine (rm)-TNF alpha (mature). The hybridization signature of complex cDNA probes prepared from total RNA extracted from immature and mature DC were analyzed using a mouse thymic cDNA library, gridded on high density filters. For each clone spotted on the filters, we have measured using an imaging plate device the hybridization signals of the complex probe obtained from immature or mature DC. Comparative analysis of these values allowed us to identify differentially expressed gene products. Our goal is to identify a new set of genes induced or repressed during DC maturation elicited by rmTNF alpha treatment.

Animals↗
PubMed1997

Histological and structural study of the adhesive tissue in knee fibroarthrosis: a clinical-pathological correlation.

In fibroarthrosis of the knee, it is still unknown if joint range of motion is affected by anatomopathological differences in adhesive tissue, such as tissue maturity, location, and quantity. A retrospective study of 78 patients who underwent arthoscopic arthrolysis was performed to determine a correlation between location of adhesions and preoperative range of motion (ROM). In another 17 patients, a histological and structural evaluation of adhesive tissue was performed. Based on vascularization, number and shape of cells, and collagen fiber orientation, the adhesive tissue was classified into three groups: low, medium, and high maturity. Preoperative joint ROM and the time of onset of joint stiffness was correlated with the degree of adhesion tissue maturity. A strong and statistically significant correlation between the location of adhesions and joint restriction was found. However, histological and structural evaluation showed no correlation between the degree of tissue maturity, the time of onset of joint stiffness, and the amount of joint ROM.

Adult↗
PubMed1996

CD95 engagement releases calcium from intracellular stores of long term activated, apoptosis-prone gammadelta T cells.

Engagement of the CD95 (Apo-1, Fas) membrane receptor is known to induce apoptosis in a variety of sensitive cells, even in the absence of extracellular Ca2+. The signal transduction events implicated in this pathway are poorly understood. We have recently characterized normal human Vgamma9/Vdelta2+ T cell clones that maintain similar levels of CD95 membrane expression throughout the culture. Here we show that 3 wk of culture after in vitro restimulation are necessary for the cells both to die and to acquire the ability to mobilize intracellular Ca2+ upon CD95 ligation. Buffering of intracellular Ca2+ by accumulation of the chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N1,N-tetraacetic acid protects from CD95-triggered apoptosis, suggesting that the two phenomena are causally related. As intracellular Ca2+ release by inhibition of endoplasmic reticulum ATPases induces apoptosis in both recently and long term activated gamma delta cells, the molecular regulation of activation-dependent apoptosis is likely to involve events upstream of CD95-dependent Ca2+ release. The CD95-triggered increase in the intracellular Ca2+ concentration depends on depletion of the same intracellular Ca2+ stores mobilized by ligation of the TCR, and Ca2+ release does not depend on inositol 1,4,5-trisphosphate generation.

Apoptosis↗
PubMed1996

Feedback modulation of ligand-engaged alpha L/beta 2 leukocyte integrin (LFA-1) by cyclic AMP-dependent protein kinase.

Rapid interconversion between a firmly adherent and a nonadherent, circulating phenotype is a distinctive feature of mature leukocytes and is thought to be essential for efficient immune surveillance. Leukocyte adhesion is a finely regulated process controlled in part by reversible, activation-dependent up-regulation of beta 1- and beta 2- integrin function. To investigate the molecular basis of such reversibility in human T lymphocytes, we developed a model of alpha L/beta 2 (LFA-1)-dependent adhesion that uses a heterologous cell line expressing human intercellular adhesion molecule-1 as a selected ligand. We show here that intracellular cAMP elevation, followed by cAMP-dependent kinase activation, promotes T cell deadhesion by disassembling the actin-based cytoskeleton, thus dissociating LFA-1 from cytoskeletal anchoring proteins that normally connect the adhesion receptor to F-actin in lymphocytes engaged in intercellular adhesion. Cells costimulated via the CD3 and LFA-1 receptors by specific Abs or by binding to intercellular adhesion molecule-1 display gradual and persistent intracellular cAMP elevations due to the synergistic induction of a protein kinase C-dependent adenylyl cyclase isoform. On the basis of these findings, we propose a feedback model for short term regulation of leukocyte integrins, involving sequential, integrin-dependent activation of the protein kinase C and adenylyl cyclase/cAMP-dependent kinase enzymatic pathways and leading to disengagement of the adhesion receptor from its specific ligand.

Actins↗
PubMed1996

CD31/PECAM-1-driven chemokine-independent transmigration of human T lymphocytes.

We assessed the relative contribution of CD31/PECAM-1 (platelet-endothelial cell adhesion molecule-1) to T lymphocyte transmigration by the use of transfected murine fibroblasts stably expressing either the human CD31/PECAM-1 or the intercellular adhesion molecule-1 (CD54/ICAM-1). Unlike CD54/ICAM-1, CD31/PECAM-1 supported migration of activated T cells in the absence of chemokines: most of the migrating lymphocytes were CD31+ and displayed a phenotype corresponding to the naive subpopulation (LFA-1 dull and CD45RA+). Migration of activated T lymphocytes through CD54/ICAM-1+ transfected monolayers could be induced by creating a chemotactic gradient with the chemokine monocyte chemotactic protein-1, and the migrating cells mainly displayed a memory phenotype (LFA-1 bright CD45RO+) under these conditions. Furthermore, we found that in transfected cells CD54/ICAM-1 is uniformly distributed along the apical surface of the cells, while CD31/PECAM-1 is concentrated at the intercellular junctions, suggesting the existence of a haptotactic gradient (i.e. a gradient of substrate- or cell-bound molecules) responsible for T cell migration. This was also confirmed by the finding that monolayers of murine fibroblasts transfected with a CD31/PECAM-1 mutant lacking the cytoplasmic domain (CD31/PECAM-1-delta cyto), which has a reduced tendency to localize at cell-cell contact areas, supported efficient adhesion but were unable to induce migration of activated T cells unless a chemotactic gradient was created. We propose that in lymphocytes, homophilic CD31/PECAM-1 adhesion may be primarily involved in transmigration of naive T cells and that its role is complementary to that of CD54/ICAM-1.

3T3 Cells↗