Autolysosomes and caspase-3 control the biogenesis and release of immunogenic apoptotic exosomes.

Apoptotic exosome-like vesicles (ApoExos) are a novel type of extracellular vesicle that contribute to the propagation of inflammation at sites of vascular injury when released by dying cells. ApoExos are characterized by the presence of the C-terminal perlecan LG3 fragment and 20S proteasome, and they are produced downstream of caspase-3 activation. In the present study, we assessed the relative roles of autophagy and caspase-3-mediated pathways in controlling the biogenesis and secretion of immunogenic ApoExos. Using electron microscopy and confocal immunofluorescence microscopy in serum-starved endothelial cells, we identified large autolysosomes resulting from the fusion of lysosomes, multivesicular bodies, and autophagosomes as a site of ApoExo biogenesis. Inhibition of autophagy with ATG7 siRNA or biochemical inhibitors (wortmannin and bafilomycin) coupled with proteomics analysis showed that autophagy regulated the processing of perlecan into LG3 and its loading onto ApoExos but was dispensable for ApoExo biogenesis. Caspase-3 activation was identified using caspase-3-deficient endothelial cells or caspase inhibitors as a pivotal regulator of fusion events between autolysosomes and the cell membrane, therefore regulating the release of immunogenic ApoExos. Collectively, these findings identified autolysosomes as a site of ApoExo biogenesis and caspase-3 as a crucial regulator of autolysosome cell membrane interactions involved in the secretion of immunogenic ApoExos.

Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles.

Several virulence genes have been identified thus far in the herpes simplex virus 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level. To address this issue, we resorted to flow cytometry (flow virometry), a powerful approach we recently employed to analyze individual viral particles, to identify which tegument proteins vary and directly address if such variability is biologically relevant. We found that the stoichiometry of the UL37, ICP0, and VP11/12 tegument proteins in virions is more stable than the VP16 and VP22 tegument proteins, which varied significantly among viral particles. Most interestingly, viruses sorted for their high VP16 or VP22 content yielded modest but reproducible increases in infectivity compared to their corresponding counterparts containing low VP16 or VP22 content. These findings were corroborated for VP16 in short interfering RNA experiments but proved intriguingly more complex for VP22. An analysis by quantitative Western blotting revealed substantial alterations of virion composition upon manipulation of individual tegument proteins and suggests that VP22 protein levels acted indirectly on viral fitness. These findings reaffirm the interdependence of the virion components and corroborate that viral fitness is influenced not only by the genome of viruses but also by the stoichiometry of proteins within each virion.IMPORTANCE The ability of viruses to spread in animals has been mapped to several viral genes, but other factors are clearly involved, including virion heterogeneity. To directly probe whether the latter influences viral fitness, we analyzed the protein content of individual herpes simplex virus 1 particles using an innovative flow cytometry approach. The data confirm that some viral proteins are incorporated in more controlled amounts, while others vary substantially. Interestingly, this correlates with the VP16 trans-activating viral protein and indirectly with VP22, a second virion component whose modulation profoundly alters virion composition. This reaffirms that not only the presence but also the amount of specific tegument proteins is an important determinant of viral fitness.

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.

EphA4, a receptor tyrosine kinase, is expressed in various pre-, post- and peri-synaptic organelles and implicated in the regulation of morphological and physiological properties of synapses. It regulates synaptic plasticity by acting as a binding partner for glial ephrin-A3 and possibly other pre- or post-synaptic ephrins. Now, its trafficking mechanisms remain unknown. In this study, we examine the association of EphA4 with transport, clathrin-coated and synaptic vesicles using cell fractionation, vesicle immunoisolation and electron microscopy. EphA4 was found in highly purified fractions of clathrin-coated or synaptic vesicles. It was also detected in vesicles immuno-isolated with antibodies anti-synaptophysin, anti-vesicular glutamate transporter or anti-vesicular GABA transporter; demonstrating its presence in synaptic vesicles. However, it was not detected in immuno-isolated piccolo-bassoon transport vesicles. In vivo and in dissociated cultures, EphA4 was localized by immunoelectron microscopy in vesicular glutamate transporter 1-positive terminals of hippocampal neurons. Remarkably, the cell surface immunofluorescence of EphA4 increased markedly in cultured hippocampal neurons following KCl depolarization. These observations indicate that EphA4 is present in subsets of synaptic vesicles, can be externalized during depolarization, and internalized within clathrin-coated vesicles. This trafficking itinerary may serve to regulate the levels of EphA4 in the synaptic plasma membrane and thereby modulate signaling events that contribute to synaptic plasticity.

Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection.

Viral proteins are usually processed by the 'classical' major histocompatibility complex (MHC) class I presentation pathway. Here we showed that although macrophages infected with herpes simplex virus type 1 (HSV-1) initially stimulated CD8(+) T cells by this pathway, a second pathway involving a vacuolar compartment was triggered later during infection. Morphological and functional analyses indicated that distinct forms of autophagy facilitated the presentation of HSV-1 antigens on MHC class I molecules. One form of autophagy involved a previously unknown type of autophagosome that originated from the nuclear envelope. Whereas interferon-gamma stimulated classical MHC class I presentation, fever-like hyperthermia and the pyrogenic cytokine interleukin 1beta activated autophagy and the vacuolar processing of viral peptides. Viral peptides in autophagosomes were further processed by the proteasome, which suggests a complex interaction between the vacuolar and MHC class I presentation pathways.

Low-voltage transmission electron microscopy reveals SV40 viral particles within secretory granules in pancreatic cells.

Novel approach in low voltage transmission electron microscopy (TEM) has revealed the presence of SV40 viral like particles in the secretory zymogen granules and in spherical membrane-bound dense bodies of SV40 infected pancreatic cells. The presence of SV40 antigen in these cellular compartments was confirmed by immunocytochemistry of the VP1 antigen. Visualization of the viral particles was only possible by examining ultrathin tissue sections with low-voltage TEM that significantly enhances imaging contrast. Results indicate that following infection of the cell entry and trafficking of the viral particles are present in unique cellular compartments such as ER, dense bodies, and secretory granules.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats.

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2). ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPKalpha(1), AMPKalpha(2), and AMPKbeta(2) were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding subunit beta(2) were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 mug/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

Transcytosis of gastric leptin through the rat duodenal mucosa.

Leptin is secreted into the gastric juice by epithelial Chief cells and reaches the duodenum in a biologically intact active form. We assessed the possibility that this gastric leptin crosses the intestinal mucosa by transcytosis through enterocytes to reach blood circulation. Endogenous gastric leptin secretion was triggered by cholinergic stimulation. In another set of experiments, recombinant leptin was inserted in vivo into the duodenal lumen. Plasma levels of leptin were assessed by enzyme immunoassay and Western blot, and duodenal tissue was processed for immunocytochemistry. We first observed that leptin was found inside duodenal enterocytes from fed rats but not inside those from fasted ones. Stimulation of gastric secretion by a cholinergic agent led to rapid increases in plasma leptin levels (202 +/- 39%) except when the pylorus was clamped. Insertion of recombinant leptin into the duodenal lumen raised plasma leptin concentrations (558 +/- 34%) quite rapidly, whereas carrier solution without leptin had no effect. The use of FITC-tagged leptin reinforced these results. Light and electron microscopy revealed the cellular compartments involved in its transcytosis, namely, the enterocyte microvilli, the endocytotic vesicles, the Golgi complex, and the basolateral interdigitations. Leptin was also present in the lamina propria, in capillary endothelial cell plasmalemmal vesicles, and in capillary lumina. These results demonstrate that gastric exocrine leptin is internalized by duodenal enterocytes and delivered to the lamina propria and blood circulation. It may thus be able to play important paracrine and endocrine functions for the control of gastric emptying and nutrient absorption.

In situ localization of P-glycoprotein (ABCB1) in human and rat brain.

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.

Internalization and transcytosis of pancreatic enzymes by the intestinal mucosa.

As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation.

Secretion of soluble leptin receptors by exocrine and endocrine cells of the gastric mucosa.

Leptin is a hormone secreted by the gastric mucosa into the lumen of the stomach. It is present in its intact form in the intestine where it regulates nutrient absorption and intestinal mucosa integrity. We have identified the binding protein that protects leptin from the harsh conditions of the gastric juice. Immunoprecipitations and Western blot analyses demonstrated that leptin is present in the gastric mucosa and the gastric juice, bound to a protein corresponding to the extracellular domain of the leptin receptor. In the absence of this soluble receptor, leptin is rapidly degraded. Immunocytochemistry on rat gastric mucosa identified the cells and intracellular compartments involved in secretion of this complex. Leptin receptor extracellular domain and leptin are present along the rough endoplasmic reticulum-Golgi-granules secretory pathways and form a complex in the secretory granules of Chief and specific endocrine cells. The long-form membrane leptin receptor OB-Rb, the protease activator furin, and proprotein convertase 7 were found in Chief cell granules but not in those of endocrine cells. The shedding of the receptor occurs in the immature granules. It is concluded that in the immature secretory granules of Chief cells, furin activates proprotein convertase 7 that, in turn, cleaves the extracellular portion of membrane-bound leptin receptors. Leptin bound to its soluble receptor forms a complex that is resistant to the gastric juice. Endocrine cells, on the other hand, generate a soluble leptin receptor by mechanisms different from those of the exocrine cells.

Endocrine and exocrine secretion of leptin by the gastric mucosa.

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum-Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.

Evaluation of pancreatic amylase mRNA upon cholinergic stimulation of secretion.

The primary function of the exocrine pancreas consists of the synthesis and secretion of several digestive enzymes. It is well established that amylase secretion by rat pancreatic tissue or by isolated acinar cells in culture can be stimulated by the cholinergic agonist carbachol. However, the effect of this secretagogue on enzyme synthesis remains unclear. Some studies demonstrated increases in rates of synthesis, whereas others reported increases in secretion with or without decreases in synthesis. We have evaluated changes in pancreatic amylase mRNA and total RNA after a single injection of carbachol and under fasting conditions. Two approaches in molecular morphology were applied on rat pancreatic tissue: in situ hybridization and RNase A-gold. Both revealed decreases in RNA labeling at the level of the rough endoplasmic reticulum (RER) 5 min after stimulation of secretion and after fasting. Gradual recovery was registered 15 and 30 min after stimulation of secretion. Northern blotting confirmed drastic decreases in amylase mRNA 5 min after stimulation and after fasting. The combination of such different approaches has demonstrated drastic decreases in RNA at the RER level, reflecting declines in rates of synthesis at the translational level under all conditions tested.

Redistribution of integrins in tubular epithelial cells during diabetic glycogen nephrosis.

BACKGROUND/AIMS: Even though many aspects of glycogen nephrosis in diabetes have already been studied, adhesion interactions between the glycogen-accumulating clear cells and the tubular basement membranes have not been addressed. As integrins play key roles in cell-to-matrix interactions, we investigated the expression and distribution of alpha3-, alphaV-, beta1- and beta3-integrin subunits in renal tissues from streptozotocin-induced hyperglycemic rats (3 months old) and their age-matched controls as well as from streptozotocin-injected normoglycemic animals. METHODS: The levels and distribution of integrins were studied by immunocytochemistry and Western blot analysis. RESULTS: Immunoblotting analysis of fractions enriched in glycogen-accumulating clear cells demonstrated enhanced expression of alpha3, alphaV and beta1 subunits while expression of beta3 did not differ from controls. The most striking cytochemical result was the redistribution of the alpha3-, alphaV-, and the beta1-integrin subunits to the apical plasma membrane of these cells. This was found by light and electron microscopy. CONCLUSION: Our results suggest that the altered expression and distribution of integrins in clear cells of diabetic animals must have defined roles in the development of the renal tubulopathy.

Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures.

We investigated the cellular/subcellular localization and functional expression of P-glycoprotein, an ATP-dependent membrane-associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT-PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P-glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P-glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin-1 polyclonal H-97 antibody demonstrated that P-glycoprotein is physically associated with caveolin-1 in both cell culture systems. The accumulation of [(3)H]digoxin (an established P-glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P-glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P-glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.

Morphofunctional studies of the glomerular wall in mice lacking entactin-1.

The architecture of the basement membranes is essential for proper function. This architecture is based on interactions among its components, which assemble in a complex network. Entactin-1 appears to be the mastermind of this assembling. In entactin-1-null transgenic mice, immunocytochemistry established the absence of entactin-1 in the glomerular basement membrane, and morphological thickening of this membrane was demonstrated. This prompted us to investigate the organization of other components of the glomerular basement membrane in the transgenic animals. The distribution of type IV collagen and laminin remained unchanged, whereas that of anionic charges was significantly altered. We also evaluated the impact of the absence of entactin-1 on cell relays by studying the alpha(3)- and the alpha(v)-integrins along the endothelial and epithelial glomerular cell plasma membranes. Only the density of alpha(v) was found to be increased. Finally, the filtration properties of the glomerular wall were evaluated by revealing endogenous albumin distribution across the basement membrane. This was altered in transgenic animals, suggesting changes in permselectivity properties. Entactin-1 appears to be an essential component in basement membranes because its absence appears to modify the molecular organization leading to alterations in functional properties.

Apoptosis of tubular epithelial cells in glycogen nephrosis during diabetes.

The important problem of the fate of glycogen-accumulating clear cells in glycogen nephrosis is still unsettled. In this study, we examine whether apoptosis plays a relevant role in the development of diabetic glycogen nephrosis and explore the involvement of the Fas/Fas-L system and the activation of the caspase cascade. Diabetes was induced in rats by streptozotocin injection. Glycogen-accumulating clear cells were identified in renal tissues of hyperglycemic rats. They were found to be concentrated in the thick ascending limbs and distal tubules. Large cellular glycogen accumulations were confirmed by biochemical assays and enzyme-gold cytochemistry. Clear cells displayed apoptotic features such as Annexin V binding, nuclear TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling), and the simultaneous occurrence of Fas, Annexin V, and TUNEL positivity. Western blot analysis demonstrated enhanced expression of Fas receptor/ligand and the activation of the caspase cascade in these cells because cleaved forms of the caspase-3, -8, and -9 were detected. Furthermore, active caspase-3 was located in nuclei by immunoelectron microscopy. Our results indicate that epithelial cells in thick ascending limbs and distal tubules that develop glycogen nephrosis in response to hyperglycemia undergo Fas/Fas-L mediated cell death. Thus, apoptosis could be playing a significant role in renal epithelial cell deletion during diabetes.

Circulating bile salt-dependent lipase originates from the pancreas via intestinal transcytosis.

BACKGROUND & AIMS: Bile salt-dependent lipase (BSDL) has been detected in human blood, where it is assumed to play a substantial role in atherosclerosis. The origin of this circulating enzyme is unknown. The aim of this study was to show that blood BSDL originates from pancreatic exocrine secretions via a transcytotic motion across the intestinal epithelium. METHODS: Fluorescein isothiocyanate- or [(125)I]-labeled human pancreatic BSDL was instilled into the lumen of intestinal loops of the rat, and combined biochemical and immunocytochemical investigations were performed in intestinal cells and in the blood of these animals. RESULTS: In vivo pancreatic BSDL is internalized by duodenal enterocytes. The pancreatic enzyme was associated with microvilli and present in endocytic vesicles and Golgi apparatus as well as along the basolateral membrane of enterocytes. It was also detected in intestinal interstitial spaces. Radiolabeled pancreatic BSDL internalized by the duodenal epithelium is the one further detected in circulation. The radiolabeled protein was immunoprecipitated from plasma and had a 100-kilodalton molecular mass compatible with native pancreatic BSDL. In blood, BSDL was mainly associated with low-density lipoproteins. CONCLUSIONS: These in vivo data show that BSDL, normally present in blood, originates from exocrine pancreatic secretion and support the pathophysiologic relevance of BSDL transcytosis through the intestinal mucosa cell lining. Based on this, the implication of circulating BSDL in atherosclerosis merits careful attention.