John H. T. Power

Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.

Remodeling of the conducting airway epithelium is a common finding in the chronically injured lung and has been associated with increased risk for developing lung cancer. Pulmonary neuroendocrine cells and clusters of these cells termed neuroepithelial bodies (NEBs) play a central role in each of these processes. We previously developed an adult mouse model of airway injury and repair in which epithelial regeneration after naphthalene-induced Clara cell ablation occurred preferentially at airway branch points and gave rise to nascent Clara cells. Continued repair was accompanied by NEB hyperplasia. We now provide the following evidence that the NEB microenvironment serves as a source of airway progenitor cells that contribute to focal regeneration of the airway epithelium: 1) nascent Clara cells and NEBs localize to the same spatial domain; 2) within NEB, both Clara cell secretory protein- and calcitonin gene-related peptide-immunopositive cells are proliferative; 3) the NEB microenvironment of both the steady-state and repairing lung includes cells that are dually immunopositive for Clara cell secretory protein and calcitonin gene-related peptide, which were previously identified only within the embryonic lung; and 4) NEBs harbor variant Clara cells deficient in cytochrome P450 2F2-immunoreactive protein. These data suggest that the NEB microenvironment is a reservoir of pollutant-resistant progenitor cells responsive to depletion of an abundant airway progenitor such as the Clara cell.

Multiple-system atrophy: a new α-synuclein disease?

Multiple-system atrophy is a neurodegenerative disease that involves various combinations of parkinsonism, ataxia, corticospinal motor signs, and postural hypotension or urinary incontinence (Shy-Drager syndrome). The pathological hallmark of the disease is the presence of glial and neuronal cytoplasmic inclusions, shown by modified Bielschowsky silver impregnation and, to a limited extent, with antisera to ubiquitin or B-crystallin. We show that glial cytoplasmic inclusions and degenerating neurites in multiple-system atrophy are strongly immunopositive for -synuclein, a presynaptic protein present in brainstem and cortical Lewy bodies and related neurites in idiopathic Parkinson’s disease and dementia with Lewy bodies, and, in a mutated form, in some cases of familial Parkinson’s disease. Antiserum against human -synuclein (residues 111–131) was raised in a rabbit. Paraffin-embedded sections (5 m) and frozen sections (50 m) from different brain regions were immunostained with the -synuclein antibody (1 in 1000–2000 dilution) and the avidin-biotin-peroxidase procedure. Immunostaining was not seen with preadsorbed or preimmune serum or in the absence of the primary antiserum. Brains from people with multiple-system atrophy (n=8) were obtained from the South Australian Brain Bank. The clinical diagnoses before death had included Parkinson’s disease, multiple-system atrophy with olivopontocerebellar involvement, and multiple-system atrophy with autonomic symptoms. The neuropathological diagnosis of multiplesystem atrophy was made by the demonstration of glial cytoplasmic inclusions with the modified Bielschowsky silver stain and immunostaining for ubiquitin and B-crystallin. We assessed the same regions in 12 brains judged to be normal, five from people who had had motor-neuron disease, and ten from people with idiopathic Parkinson’s disease, all also from the brain bank. Sections from brainstem, basal ganglia, cerebellar and cerebral cortices in the multiple-system-atrophy brains all showed strong immunostaining of glial cytoplasmic inclusions, mainly in white matter (figure). Pronounced -synuclein immunoreactivity was also seen in degenerating neurites (figure) and in the cytoplasmic and intranuclear inclusions of neurons in the putamen and pontine nuclei. In normal brains and those from people with motor-neuron disease, no abnormal -synuclein-containing structures were seen; immunostaining for -synuclein was limited to the normal background pattern, which shows the presence of the protein in nerve terminals. In idiopathic Parkinson’s disease, Lewy bodies and degenerating neurites were positive for synuclein, as shown previously, but no immunopositive glial cytoplasmic inclusions were seen. We used confocal microscopy and double-staining for synuclein (1 in 500) and ubiquitin (monoclonal antibody 1510, Chemicon, 1 in 200) to assess the colocalisation of these two markers in glial cytoplasmic inclusions and degenerating neurites in multiple-system atrophy. Small numbers of synuclein-positive degenerating neurites and inclusions were ubiquitin-positive, but -synuclein immunoreactivity was clearly more sensitive than ubiquitin immunoreactivity as a neuropathological marker for the lesions of multiple-system atrophy (figure). The pronounced increase in the number of multiplesystem-atrophy inclusions and neurites shown with the synuclein antibody (compared with the ubiquitin antibody) contrasts with the number in Parkinson’s disease and dementia with Lewy bodies, disorders in which the two antibodies show similar neuropathological abnormalities. Immunoreactivity for -synuclein occurs in plaque-associated neurites in Alzheimer’s disease, but is absent from neuronal and glial inclusions in Alzheimer’s disease and other neurodegenerative disorders. Antibodies to -synuclein

The pulmonary consequences of a deep breath

We used the isolated rat lung perfused with Krebs bicarbonate and 4.5% albumin, to examine the effect of a transient increase in peak inspired pressure (PIP). The lung was ventilated with 5% CO2 in O2 at a Vr of 2.5 ml, an f of 60 min-1 and an end expired pressure of 2 cm H2O. After 30 min we increased the PIP from 9 to 18 cm H2O for one breath; following a further 30 sec of normal ventilation we lavaged the lung. The large breath increased the amount of alveolar surfactant phospholipids (PLalv) (control: 7.0 +/- 0.73 (11); large breath: 8.3 +/- 1.33 (14), mean +/- SD in mg . g dry lung-1), and decreased the percentage of PLalv associated with tubular myelin (control: 30.2 +/- 3.49% (9); deep breath: 25.4 +/- 2.99% (9). In rats that had received 20 microCi . kg-1 of [methyl-3H]choline chloride 3 h previously, there was also an increase in the tritium in PLalv expressed as a percent of that in tissue (control: 4.4 +/- 0.77% (5); deep breath: 5.7 +/- 1.0% (7). The deep breath also resulted in an increase in oxygen diffusing capacity. We conclude, that a single deep breath results in the opening of atelectatic alveoli, the release of pulmonary surfactant and possibly also the transfer of PLalv from the tubular myelin to the monomolecular phase.

Peroxiredoxin 6 in human brain: molecular forms, cellular distribution and association with Alzheimer’s disease pathology

Peroxiredoxin 6 is an antioxidant enzyme and is the 1-cys member of the peroxiredoxin family. Using two-dimensional electrophoresis and Western blotting, we have shown for the first time that, in human control and brain tissue of patient’s with Alzheimer’s disease (AD), this enzyme exists as three major and five minor forms with pIs from 5.3 to 6.1. Using specific cellular markers, we have shown that peroxiredoxin 6 is present in astrocytes with very low levels in neurons, but not detectable in microglia or oligodendrocytes. In control brains, there was a very low level of peroxiredoxin 6 staining in astrocytes that was confined to a “halo” around the nucleus. In AD, there were marked increases in the number and staining intensity of peroxiredoxin 6 positive astrocytes in both gray and white matter in the midfrontal cortex, cingulate, hippocampus and amygdala. Confocal microscopy using antibodies to Aβ peptide, tau and peroxiredoxin 6 showed that peroxiredoxin 6 positive astrocytes are closely involved with diffuse plaques and to a lesser extent with neuritic plaques, suggesting that plaques are producing reactive oxygen species. There appeared to be little astrocytic response to tau containing neurons. Although peroxiredoxin 6 positive astrocytes were seen to make multiple contacts with tau positive neurons, there was no intraneuronal colocalization. In brain tissue of patients with AD, many blood vessels exhibited peroxiredoxin 6 staining that appeared to be due to the astrocytic foot processes. These results suggest that oxidative stress conditions exist in AD and that peroxiredoxin 6 is an important antioxidant enzyme in human brain defenses.

Cellular glutathione peroxidase in human brain: cellular distribution, and its potential role in the degradation of Lewy bodies in Parkinson’s disease and dementia with Lewy bodies

Glutathione peroxidase (GPx-1) is regarded as one of the mammalian cell’s main antioxidant enzymes inactivating hydrogen peroxide and protecting against oxidative stress. Using control, Parkinson’s disease (PD), and dementia with Lewy bodies tissue (DLB) we have shown that GPx-1 is a 21-kD protein under reducing conditions in all tissues examined but is not in high abundance in human brain. Using immunohistochemistry we have mapped the cellular distribution of GPx-1 and have shown it to be in highest levels in microglia and with lower levels in neurons. Only a trace amount was detectable in astrocytes using immunofluorescence and GPx-1 was not detectable in oligodendrocytes. GPx-1 positive microglia were hypertrophied and more abundant in PD and DLB tissues and were seen to be making multiple contacts with neurons. In some cases neurons containing Lewy bodies were surrounded by microglia. Unstructured Lewy bodies were enveloped with a layer of GPx-1 that was partially colocalized with α-synuclein whereas concentric Lewy bodies had discrete deposits of GPx-1 around the periphery which appeared to be involved in the degradation of the Lewy bodies. These results suggest that abnormal α-synuclein as found in Lewy bodies produce hydrogen peroxide and these neurons are capable of directing antioxidant enzymes to regions of oxidative stress. These results also suggest that GPx-1 positive microglia are involved in neuroprotection in PD and DLB and that GPx-1 is an important antioxidant enzyme in neuronal defences.

Cyclic stretch induces both apoptosis and secretion in rat alveolar type II cells.

We examined the effects of short‐term cyclic stretch on both phosphatidylcholine (PC) secretion and apoptosis in primary cultures of rat alveolar type II cells. A 22% cyclic stretch (3 cycles/min) was applied to type II cells cultured on silastic membranes using a Flexercell strain unit. This induced, after a lag period of about 1 h, a small, but significant release of [3H]PC from prelabelled cells. In addition, stretch increased nuclear condensation, the generation of oligosomal DNA fragments and the activation of caspases. Similar responses were triggered by sorbitol‐induced osmotic shock, but not by the secretagogue ATP. We conclude that stretch can induce both apoptosis and PC secretion in alveolar type II cells and propose that these diverse responses occur within the lung as a consequence of normal respiratory distortion of the alveolar epithelium.

Body Temperature Alters the Lipid Composition of Pulmonary Surfactant in the Lizard Ctenophorus nuchalis

In any 24-h period the body temperature (Tb) of the central Australian agamid lizard, Ctenophorus nuchalis, may vary from 13 to 45 degrees C; the mean preferred Tb is 37 degrees C. We have analyzed surfactant-type lipids in lizards that underwent rapid changes in Tb from 37 degrees C to 14, 19, 27, or 44 degrees C. Lipids were extracted from lung lavage and lamellar body fractions, and phospholipids and cholesterol components were measured. There was no change in either the total amount or relative proportions of the different classes of phospholipids, but cooling increased the cholesterol content of lavage. An increase in the cholesterol: phospholipid ratio was evident within 2 h of cooling to 19 degrees C and was maintained for at least 48 h. The ratio increased from 8% at 37 degrees C, to 15% after 4 h at 19 degrees C, and 18% after 4 h at 14 degrees C. Possibly the increase in cholesterol promotes fluidity and absorption of surfactant within the alveoli of lizards with low Tb. Cold lizards collapse their lungs during prolonged periods of apnea and the surfactant may prevent the epithelial walls from adhering.

Nonselenium Glutathione Peroxidase in Human Brain: Elevated Levels in Parkinson's Disease and Dementia with Lewy Bodies

Nonselenium glutathione peroxidase (NSGP) is a new member of the antioxidant family. Using antibodies to recombinant NSGP we have examined the distribution of this enzyme in normal, Parkinsons disease (PD), and dementia with Lewy body disease (DLB) brains. We have also co-localized this enzyme with alpha-synuclein as a marker for Lewy bodies. In normal brains there was a very low level of NSGP staining in astrocytes. In PD and DLB there were increases in the number and staining intensity of NSGP-positive astrocytes in both gray and white matter. Cell counting of NSGP cells in PD and DLB frontal and cingulated cortices indicated there was 10 to 15 times more positive cells in gray matter and three times more positive cells in white matter than in control cortices. Some neurons were positive for both alpha-synuclein and NSGP in PD and DLB, and double staining indicated that NSGP neurons contained either diffuse cytoplasmic alpha-synuclein deposits or Lewy bodies. In concentric Lewy bodies, alpha-synuclein staining was peripheral whereas NSGP staining was confined to the central core. Immunoprecipitation indicated there was direct interaction between alpha-synuclein and NSGP. These results suggest oxidative stress conditions exist in PD and DLB and that certain cells have responded by up-regulating this novel antioxidant enzyme.

Osmotic stress induces both secretion and apoptosis in rat alveolar type II cells

The aim of this study was to analyze the effects of osmotic shock and secretagogues such as ATP and 12-O-tetradecanoylphorbol 13-acetate (TPA) on various intracellular signaling pathways in primary cultures of alveolar type II cells and examine their potential role in regulating events such as secretion and apoptosis in these cells. Sorbitol-induced osmotic stress caused the sustained release of [3H]phosphatidylcholine ([3H]PC) from primary cultures of rat alveolar type II cells prelabeled with [3H]choline chloride. This release was not dependent on protein kinase C because downregulation of the major protein kinase C isoforms (alpha, betaII, delta, and eta) expressed in alveolar type II cells had no effect on [3H]PC secretion. Sorbitol, as well as the known secretagogues TPA and ATP, activated extracellular signal-regulated kinase. Although an inhibitor of the extracellular signal-regulated kinase cascade, PD-98059, blocked this activation, it had no effect on the release of [3H]PC. Sorbitol and ultraviolet C radiation, but not TPA or ATP, were also found to activate both p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, both sorbitol and ultraviolet C radiation induced apoptosis in alveolar type II cells as demonstrated by Hoechst 33258 staining of the condensed nuclei, the generation of DNA ladders, and the activation of caspases. The data indicate that multiple signaling pathways are activated by traditional secretagogues such as TPA and ATP and by cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.The aim of this study was to analyze the effects of osmotic shock and secretagogues such as ATP and 12- O-tetradecanoylphorbol 13-acetate (TPA) on various intracellular signaling pathways in primary cultures of alveolar type II cells and examine their potential role in regulating events such as secretion and apoptosis in these cells. Sorbitol-induced osmotic stress caused the sustained release of [3H]phosphatidylcholine ([3H]PC) from primary cultures of rat alveolar type II cells prelabeled with [3H]choline chloride. This release was not dependent on protein kinase C because downregulation of the major protein kinase C isoforms (α, βII, δ, and η) expressed in alveolar type II cells had no effect on [3H]PC secretion. Sorbitol, as well as the known secretagogues TPA and ATP, activated extracellular signal-regulated kinase. Although an inhibitor of the extracellular signal-regulated kinase cascade, PD-98059, blocked this activation, it had no effect on the release of [3H]PC. Sorbitol and ultraviolet C radiation, but not TPA or ATP, were also found to activate both p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, both sorbitol and ultraviolet C radiation induced apoptosis in alveolar type II cells as demonstrated by Hoechst 33258 staining of the condensed nuclei, the generation of DNA ladders, and the activation of caspases. The data indicate that multiple signaling pathways are activated by traditional secretagogues such as TPA and ATP and by cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.

IMMUNOHISTOCHEMICAL LOCALIZATION AND CHARACTERIZATION OF A RAT CLARA CELL 26-kDa PROTEIN (CC26) WITH SIMILARITIES TO GLUTATHIONE PEROXIDASE AND PHOSPHOLIPASE A2

We have purified and partially sequenced a 26-kDa protein isolated from rat lung lavage. Two-dimensional electrophoresis and Western blotting using an antibody we have raised to this protein indicated that CC26 has 3 isoforms with pIs between 4.9 and 5.5 and is neither a component of surfactant nor present in plasma. The first 10 amino acids of the N-terminal of all 3 isoforms were identical. The first 25 amino acids of the N-terminal sequence were identical to a rat acidic calcium-independent phospholipase A2 and one amino acid different from a mouse nonselenium glutathione peroxidase. Light immunohistochemistry showed a strong reaction with the airway hypophase from the trachea down to the terminal bronchioles, but not in the alveolus. Immunohistochemistry at the electron microscopy level showed that CC26 was localized to the dense secretory bodies and endoplasmic reticulum of the Clara cell and secretory granules of tracheal nonciliated serous and goblet cells. Heavily labeled Clara cell dense secretory bodies were observed in the process of being exocytosed. Biochemical and further sequence analysis will be required to determine if this protein is either a nonselenium glutathione peroxidase or a calcium-independent phospholipase A2.