Inflaming the diseased brain: a role for tainted melanins.

Inflammation plays a crucial role in neurodegenerative diseases, but the irritants responsible for this response remain largely unknown. This report addressed the hypothesis that hypochlorous acid reacts with dopamine to produce melanic precipitates that promote cerebral inflammation. Spectrophotometric studies demonstrated that nM amounts of HOCl and dopamine react within seconds. A second-order rate constant for the reaction of HOCl and dopamine of 2.5 × 10(4)M(-1)s(-1) was obtained by measuring loss of dopaminergic fluorescence due to HOCl. Gravimetric measurements, electron microscopy, elemental analysis, and a novel use of flow cytometry confirmed that the major product of this reaction is a precipitate with an average diameter of 1.5 µm. Flow cytometry was also used to demonstrate the preferential reaction of HOCl with dopamine rather than albumin. Engulfment of the chlorodopamine particulates by phagocytes in vitro caused these cells to release TNFα and die. Intrastriatal administration of 10(6) particles also increased the content of TNFα in the brain and led to a 50% loss of the dopaminergic neurons in the nigra. These studies indicate that HOCl and dopamine react quickly and preferentially with each other to produce particles that promote inflammation and neuronal death in the brain.

Circulating endothelial microparticles in diabetes mellitus.

BACKGROUND: Endothelial Microparticles (EMPs) are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM) is not known. We studied the cellular adhesion molecule (CAM) profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization. METHODS AND RESULTS: EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/microL) specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process. CONCLUSION: Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.

Prostaglandin D(2) synthase induces apoptosis in pig kidney LLC-PK1 cells.

BACKGROUND: Prostaglandin D(2) synthase (PGD(2)S), a unique member of the lipocalin family, is found at elevated levels in the serum of patients with renal impairment and has recently been implicated as a new biochemical marker of renal insufficiency. The aim of this study was to investigate the apoptotic effects of PGD2S on a pig kidney epithelial cell line (LLC-PK1) and to investigate the effects of prostaglandins and growth factors on this process. METHODS: Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL), annexin V staining, and electron microscopy. RESULTS: A four- to fivefold increase in apoptosis was observed in PGD(2)S-treated cells as compared with controls and the apoptosis appeared to act via caspase-3. A cyclooxygenase-2 inhibitor, anti-PGD(2)S antibody, and selenium all significantly inhibited the apoptosis induced by PGD(2)S; however, none had any effect on the apoptosis induced by the known apoptotic inducer camptothecin. Furthermore, prostaglandins E(1) and E(2), known to induce mitogen-activated protein (MAP) kinase phosphorylation and exhibit cytoprotective effects, both inhibited PGD(2)S-induced apoptosis, while prostaglandin H(2) had no significant effect. Growth factors such as insulin, insulin-like growth factor-1, and platelet-derived growth factor also decreased PGD(2)S-induced apoptosis. In addition, PGD(2)S isolated from human serum seemed slightly more effective at inducing apoptosis than recombinantly expressed protein. CONCLUSIONS: We report on the induction of apoptosis by PGD(2)S in LLC-PK1 pig kidney epithelial cells, and speculate that the accumulation of PGD(2)S in the serum of kidney failure patients may further exacerbate renal problems and is most likely regulated by other prostaglandins and growth factors.

Prostaglandin D2 synthase induces apoptosis in PC12 neuronal cells.

Apoptosis of neuronal cells is a proposed cause of certain neurological disorders. Here, we report on a 5- to 6-fold increase in apoptosis by exposure to prostaglandin D2 synthase (PGD2S) in PC12 neuronal cells. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, and appears to be mediated via caspase-3 activation. Neutralization with anti-PGD2S antibody or pre-treatment with selenium, which inhibits PGD2S enzymatic activity, both significantly inhibited the PGD2S-induced apoptosis, however, neither had any effect on the apoptosis induced by the known neuronal apoptotic inducer, glutamate. In addition, prostaglandins E1, E2, and F2alpha all inhibited the PGD2S-induced apoptosis while prostaglandin H2 had no significant effect. Furthermore, PGD2S isolated from human serum was more effective at inducing apoptosis then recombinantly expressed protein, presumably due to glycosylation. This novel role of PGD2S, as an inducer of apoptosis, may have implications in PC12 differentiation and possibly some neurological disorders.

Morphological and functional changes in the colonic epithelial cells in a rabbit model of colitis.

A rabbit model of TNBS-colitis was used to study the effect of intestinal inflammation on epithelial cell function. Epithelial cells were isolated using a non-enzymatic isolation method without any apparent contamination with infiltrating immune cells. The isolated cells were found to be viable using dye exclusion studies, unidirectional Na+ -fluxes, proliferation assays and morphological studies. The cells, however, showed morphological changes that suggested the presence of increased number of secretory vesicles. This increase correlated well with the increase observed in ion and water secretion as measured by the short-circuit current. Finally, in the colitic tissue the number of PGE2 receptors was greatly reduced with no changes observed in the affinity of PGE2 to its receptor. The reduced number of PGE2 receptors might be due to sensitization of the receptor. In conclusion, we have demonstrated that morphologically and functionally normal epithelial cells can be isolated from the rabbit inflamed distal colon.

Partial characterization of apoptotic factor in Alzheimer plasma.

We have previously demonstrated that a plasma natriuretic factor is present in Alzheimer's disease (AD), but not in multi-infarct dementia (MID) or normal controls (C). We postulated that the natriuretic factor might induce the increased cytosolic calcium reported in AD by inhibiting the sodium-calcium antiporter, thereby activating the apoptotic pathway. To test for a factor in AD plasma that induces apoptosis, we exposed nonconfluent cultured LLC-PK1 cells to plasma from AD, MID, and C for 2 h and performed a terminal transferase-dUTP-nick-end labeling (TUNEL) assay. The plasma from AD increased apoptosis nearly fourfold compared with MID and C. The effect was dose dependent and the peak effect was attained after a 2-h exposure. Additionally, apoptotic morphology was detected by electron microscopy, and internucleosomal DNA cleavage was found. We inhibited apoptosis by removing calcium from the medium, inhibiting protein synthesis with cycloheximide, alternately boiling or freezing and thawing the plasma, and digesting a partially purified fraction with trypsin. Heating AD plasma to 56 degrees C did not deactivate the apoptotic factor. These results demonstrate the presence of an apoptotic factor in the plasma of patients with AD.

Gap junctional communication between murine macrophages and intestinal epithelial cell lines.

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.

In vitro expansion of CD13+CD33+ dendritic cell precursors from multipotent progenitors is regulated by a discrete fas-mediated apoptotic schedule.

We provide new information on how apoptosis regulates the expansion and survival of dendritic cell (DC) elements during in vitro hematopoiesis. Functionally distinct apoptotic schedules were associated with different phases of DC development when multipotent CD34+ progenitor cells were treated with GM-CSF + TNF +/- SCF (c-kit ligand). During early phases of growth, unselected progenitors underwent apoptosis. During intermediate stages, high levels of apoptosis resulted in the preferential selection of DC precursors, as revealed by the massive expansion of DR+CD33+CD13+ cells. Late apoptosis was associated with the death of mature DCs. Apoptotic events surrounding the earlier periods were related to the exogenous addition of TNF-alpha and appeared to be mediated by fas. In contrast, those events associated with terminally differentiated DCs were fas independent because there was no correlation between fas expression and cell death. The bcl-2 protein family appeared to confer resistance to apoptotic death, as revealed by the high levels of bcl-2 and bclxL during peak DC development and in long-term DC cultures. We demonstrate that activation of distinct apoptotic programs regulates DC development and homeostasis. Although suppression of apoptosis may prolong the survival of late DC elements, an earlier apoptotic schedule appears to be required for the selective expansion of DC elements from multipotent progenitors. Our data also provides insight into the mechanism(s) of myeloid lineage selection by cytokines such as TNF-alpha, which may promote both cell death and survival.

Unscheduled apoptosis during acute inflammatory lung injury.

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.

Cellular oxygen toxicity. Oxidant injury without apoptosis.

All forms of aerobic life are faced with the threat of oxidation from molecular oxygen (O2) and have evolved antioxidant defenses to cope with this potential problem. However, cellular antioxidants can become overwhelmed by oxidative insults, including supraphysiologic concentrations of O2 (hyperoxia). Oxidative cell injury involves the modification of cellular macromolecules by reactive oxygen intermediates (ROI), often leading to cell death. O2 therapy, which is a widely used component of life-saving intensive care, can cause lung injury. It is generally thought that hyperoxia injures cells by virtue of the accumulation of toxic levels of ROI, including H2O2 and the superoxide anion (O2-), which are not adequately scavenged by endogenous antioxidant defenses. These oxidants are cytotoxic and have been shown to kill cells via apoptosis, or programmed cell death. If hyperoxia-induced cell death is a result of increased ROI, then O2 toxicity should kill cells via apoptosis. We studied cultured epithelial cells in 95% O2 and assayed apoptosis using a DNA-binding fluorescent dye, in situ end-labeling of DNA, and electron microscopy. Using all approaches we found that hyperoxia kills cells via necrosis, not apoptosis. In contrast, lethal concentrations of either H2O2 or O2- cause apoptosis. Paradoxically, apoptosis is a prominent event in the lungs of animals injured by breathing 100% O2. These data indicate that O2 toxicity is somewhat distinct from other forms of oxidative injury and suggest that apoptosis in vivo is not a direct effect of O2.

Morphological factors influencing transepithelial conductance in a rabbit model of ileitis.

BACKGROUND & AIMS: Infection of rabbits with coccidia (Eimeria magna) causes chronic ileal inflammation and diarrhea. Inflamed ileum also shows decreased transmural conductance. The aim of this study was to characterize morphological factors known to affect paracellular permeability that may alter transmural conductance in inflamed ileum. METHODS: Ileal mucosa was mounted in Ussing chambers for study of [3H]mannitol and [3H]inulin fluxes. Light and electron microscopy were used for morphometric studies. Alterations in the zonula occludens of epithelial cells were evaluated in freeze-fracture replicas. RESULTS: Inflamed ileum showed diminished paracellular fluxes. Inoculated rabbits showed marked lymphoplasmocytic infiltration and villus blunting in ileum. Villus linear junctional density was unaffected. However, total villus apical surface area per square centimeter of tissue was reduced in inflamed ileum, causing a diminished total villus linear junctional pathway per square centimeter of apical surface. Villus zonula occludens strand number was reduced in inflamed ileum, whereas the frequency of both villus and crypt lateral surface extrajunctional strands increased. CONCLUSIONS: Chronic inflammation exerts a profound effect on ileal paracellular permeability. Morphological data suggest that this effect may be caused in part by alterations in inflamed ileal mucosal structure and tight junctional organization and density, particularly on villi.

Effects of serosal hypertonicity on water permeability in toad urinary bladder.

We studied in toad urinary bladder the effects of serosal hypertonicity on tissue water permeability, granular cell luminal membrane water permeability, and granular cell luminal membrane particle aggregates and compared them with effects of antidiuretic hormone (ADH). In tissues challenged by a hypertonic (447 mosmol/kgH2O) serosal bath, luminal membrane aggregates were structurally similar to those caused by ADH. The tissue water permeability increase induced by serosal hypertonicity was much less than that caused by a maximally stimulating concentration of ADH on tissue in isotonic serosal baths with approximately the same transmural gradient. The difference is explained not only by a reduced incidence of luminal membrane aggregates but also by an increased resistance to water movement at a postluminal membrane site. Measurements of luminal membrane water permeability showed a close correlation with luminal membrane aggregate frequency, indicating that the calculated permeability of an individual aggregate was a constant. Thus the relation of luminal membrane aggregates to tissue osmotic permeability is modified by serosal hypertonicity. Morphological examination of these tissues suggested that luminal membrane aggregates may be less stable in the absence of hormone. This was evident by the proportionally greater number of structures interpreted as aggregates captured in the process of disassembly ("patches"). Membrane depressions containing intramembrane particles ("craters") were also observed. They corresponded in terms of frequency and size to coated pits as seen in thin sections.

Intramembrane particle structures in epithelial cells of the toad urinary bladder: a quantitative freeze-fracture study.

Freeze-fracture electron microscopy reveals intramembrane particle arrays in basal membranes of granular epithelial cells as well as both upper and lower plasma membranes of the underlying basal cells in the toad urinary bladder. These particle arrays are morphologically indistinguishable from the luminal membrane aggregates which are known to be associated with antidiuretic hormone (ADH)-stimulated water transport. In both granular and basal cells particle arrays are frequently located in and/or around the openings of vesicular and/or tubular structures fused to the plasma membranes, suggesting that they may be transferred from the cytoplasm by membrane fusion. Quantification of cytoplasmic aggrephores in control granular cells shows that they can be numerous and as close to the basolateral membrane as they are with the luminal membrane, to which they are known to fuse and deliver aggregates upon ADH stimulation. Aggrephore-like tubules were also found in the basal cells. Particle array densities were quantified for 6 pairs of control and ADH-stimulated hemibladders. At least 1440 microns 2 area of plasma membrane for each membrane domain was examined. Results indicate that the presence of these particle arrays in granular and basal cell membranes is highly variable and that exposure to ADH does not cause a statistically significant increase in their frequency.

Increase in blood-brain barrier permeability by altitude decompression.

Previous studies indicated that exposure to compression-decompression increases blood-brain barrier (BBB) permeability to vital dyes and antibiotics. This report concerns functional and ultrastructural BBB changes induced by altitude decompression. A 2% trypan blue solution was intravenously injected (4 ml.kg-1) into 29 experimental and 19 control rabbits. Some animals also received horseradish peroxidase. The experimental animals were subjected to 30,000 ft (4.3 psi) for 45 min. Controls were kept at ground level. The animals were sacrificed 90 min postinjection. Gross and microscopic examination and spectrophotometric dye determination revealed significantly greater tracer penetration in experimental brains (mean dye concentration 27.06 +/- 4.42 micrograms.g-1) than in controls (4.52 +/- 1.52 micrograms.g-1). No sex differences were noted. Electron microscopy suggested that the increased BBB permeability was due to transendothelial vesicular transport and, occasionally, to penetration through interendothelial junctions. These observations may have relevance to pharmacotherapy in space and at high altitudes and to the pathogenesis of altitude decompression sickness.

Prevention of cholesterol-induced gallstones by hyodeoxycholic acid in the prairie dog.

Prairie dogs of both sexes were fed a semisynthetic diet containing 0.35% cholesterol for a period of 8 weeks. This lithogenic diet induced cholesterol gallstones in ten "lithogenic control animals", five males and five females. Three animals maintained with a high glucose, fat-free diet did not develop gallstones although the cholesterol saturation of their bile approached unity. The formation of gallstones was prevented in four out of five males and all five females fed the lithogenic diet plus 0.1% hyodeoxycholic acid (30 mg per kg body weight per day). The biles of the prairie dogs receiving hyodeoxycholic acid were abnormally colored, cloudy, and highly saturated with cholesterol but contained neither cholesterol crystals nor gallstones (with the exception of one male). Feeding the relatively hydrophilic bile acid, hyodeoxycholic acid, was associated with an increase in hepatic microsomal HMG-CoA reductase activity. Cholesterol 7 alpha-hydroxylase, on the other hand, was inhibited by the administered bile acid. The dietary hyodeoxycholic acid was transformed, in part, to 3 alpha, 6 beta-dihydroxy-5-beta-cholanoic acid and hyocholic acid. It is concluded that hyodeoxycholic acid and its metabolites did not prevent the induced cholelithiasis by causing a decrease in the concentration of biliary cholesterol. Instead, this hydrophilic bile acid apparently increases the amount of cholesterol in the bile, probably in the form of a liquid crystalline mesophase. Hyodeoxycholic acid apparently prevents gallstones by preventing the nucleation and aggregation of cholesterol crystals. The lithogenic diet induced moderate to marked bile duct proliferation together with portal fibrosis and inflammatory infiltration. The addition of hyodeoxycholic acid to the lithogenic diet reduced all of the portal tract changes.