Kohei Yamauchi

Co‐expression of TNFα and IL‐1β in human acute pulmonary fibrotic diseases: An immunohistochemical analysis

To clarify the Involvement of TNFα and IL‐1β in the Initiation of fibrotic lung diseases, their localization was examined by Immunohistochemistry. Fibrotic lung diseases observed were classified Into acute and old pulmonary fibrotic changes. The acute fibrotic changes included adult respiratory distress syndrome, acute interstitial pneumonia and idiopathic pulmonary fibrosis with acute exacerbation. Acute pulmonary fibrotic changes histopathologically corresponded to a mixture of the exudative and proliferative phases of diffuse alveolar damage. Both TNFα and IL‐1β were positive in the alveolar macrophages and proliferating type II pneumocytes In acute fibrotic changes. In contrast, positive cells for TNFα and IL‐1β were sparse in the areas of old fibrotic change and in the normal tissue. These findings suggest that TNFα and IL‐1β play an Important role In the Initiation of pulmonary fibrotic responses and in the architectural remodeling, irrespective of the etiology of fibrotic lung diseases.

IL‐5, IL‐8 and GM‐CSF immunostaining of sputum cells in bronchial asthma and chronic bronchitis

Background: Granulocyte‐macrophage colony stimulating factor (GM‐CSF) and inlerleukin (IL)‐5 or IL‐8 have been .suggested to play an important role in the pathogenesis of eosinophilic airway inflammation in bronchial asthma or neutrophilic airway inflammation in chronic bronchitis, respectively, However, GM‐CSF and IL‐8 have biological activities to either eosinophils or neutrophils.

Cell-free synthesis of rat liver δ-aminolevulinate synthase and possible occurrence of processing of the enzyme protein in the course of its translocation from the cytosol into the mitochondrial matrix

S-Aminolevulinic acid (ALA) synthase in liver mitochondria can be increased markedly by the administration of porphyrinogenic drugs such as allylisopropylacetamide (AIA) and 3,5dicarbethoxy1,4-dihydrocollidine to animals, and under these conditions a considerable amount of ALA synthase also accumulates in the liver cytosol fraction [I]. The ALA synthase accumulating in the liver cytosol was shown to be a precursor in transit to the mitochondrial matrix [2]. The cytosolic ALA synthase has also been shown to be a dimer consisting of two identical subunits with min mol. wt 51 000 [ 11. Here we attempted in vitro synthesis of ALA synthase in a reticulocyte lysate system with polysomes isolated from the liver of rats treated or untreated with AIA. The results indicated that functional mRNA for ALA synthase was actually increased in the liver of AIA-treated rat and free polysomes were the major site of ALA synthase synthesis, The ALA synthase synthesized in vitro had the same minimum molecular weight as that of the ALA synthase accumulating in the liver cytosol. On the other hand, the minimum molecular weight of the mitochondrial ALA synthase was found to be -45 000 which is significantly smaller than that of the cytosolic ALA synthase. Possibly the cytosolic ALA synthase is subjected to processing in the course of its translocation into the mitochondrial matrix.

Vascular permeability and airway narrowing during late asthmatic response in dogs treated with metopirone

Recently, we have developed an animal model of late asthmatic response (LAR) by treating naturally sensitized dogs to Ascaris suum antigen with the cortisol-synthesizing inhibitor, Metopirone. By using this animal model, we examined the contribution of edema in the airway wall to the development of LAR. To study whether airway microvascular leakage is increased in association with LAR, we performed antigen challenge in dogs treated with Metopirone. We measured the amount of extravasated Evans blue (EB) dye from the esophagus, trachea, and large and small bronchi 8 hours after the antigen challenge in dogs demonstrating immediate asthmatic response alone (IAR) and in dogs demonstrating both IAR and LAR. Airway responses to A. suum antigen were assessed by changes in respiratory resistance measured with the force oscillation technique at 3 Hz. EB dye extravasation did not increase significantly from that of control in any tissues in IAR (P greater than 0.10), but in LAR, it increased significantly from that of control (p less than 0.01) and IAR (p less than 0.05) in large and small bronchi. Histologic assessment of vascular permeability revealed that Monastral blue-labeled leaking vessels were only in sections from LAR, and leaking vessels were limited to small vessels (10 to 25 microns) in the trachea, large (diameter, greater than 5 mm) and small bronchi (2 to 4 mm in diameter), and bronchiole. The permeability index defined as the ratio of area of small vessels labeled with Monastral blue to that of the total small vessels in the walls was highest in the small bronchi. LAR significantly increased submucosal thickness of the small bronchi (p less than 0.05) compared with that in IAR. Both EB dye extravasation and permeability index in large and small bronchi also significantly increased during IAR within 3 minutes after the antigen challenge (p less than 0.05), but IAR did not alter the submucosal thickness of the small bronchi. These results imply that the increase in vascular permeability and submucosal thickness, especially in small bronchi, may be an important factor in the pathogenesis of LAR.

Induction of granule release by intracellular application of calcium and guanosine-5′-O-(3-thiotriphosphate) in human eosinophils

The roles of Ca and G proteins in granule release from human eosinophils were examined by use of a patch-clamp technique in single cells. The morphologic changes and the release of eosinophil peroxidase (EPO) from single cells were simultaneously observed. In addition, the expression of small molecular weight guanine nucleotide binding protein (small G protein) mRNA (smg p25A [rab 3] and smg p21 [rab 1]) was investigated. The intracellular application of Ca, 10 mumol/L, and guanosine-5-O-(3-thiotriphosphate) (GTP-gamma-S), 100 mumol/L, induced fusion of EPO containing granules with the surface membrane, which was associated with a marked increase in membrane capacitance. Ca alone caused a rapid granule release at an early stage of cell dialysis, but most granules still remained in a cluster. GTP-gamma-S alone caused a gradual degranulation. Northern blot analysis revealed the definite expression of smg p21 mRNA with no appreciable expression of smg p25A. These results provide direct evidence of granule fusion by intracellular application of Ca and GTP-gamma-S. In addition, Ca dependent proteins and G proteins act cooperatively in granule release, and these proteins likely regulate the different processes of degranulation. Furthermore, a protein, encoded by smg p21, may be involved in the granule release process in human eosinophils.

Bronchial response to methacholine and histamine in monkeys with beta adrenergic blockade

The possibility has been investigated that propranolol administration could alter bronchial reactivity to methacholine and histamine in monkeys (Macaca fuscata and Macaca fascicularis). The impedance of the total respiratory system was measured by the forced 3-HZ oscillation method through an endotracheal tube. Methacholine and histamine dose-dependently increased the impedance in monkeys irrespective of the route of administration (inhalation of aerosol or intravenous injection). Propranolol treatment increased the bronchial response to intravenously injected methacholine and caused no significant change in the bronchial response to aerosolized methacholine. No marked difference was observed in the bronchial response to histamine due to treatment with propranolol regardless of whether administered by intravenous injection or aerosol challenge.

Pharmacological effect of KC-404 on leukotriene release from human leukocytes induced by ionophore A23187.

Release of leukotriene (LT) B4, LTC4 and histamine from human peripheral mixed leukocytes stimulated with calcium ionophore A23187 was evaluated. The amounts of LTB4 and LTC4 in supernatants were analyzed by high performance liquid chromatography and histamine was measured by an automated fluorometric technique. A time-course study demonstrated the characteristic patterns of the release of these mediators. LTB4 and LTC4 were more slowly released than histamine and the release of these LTs was inhibited by KC-404, a novel anti-asthmatic drug which has no effect on histamine release.

HMT regulates histamine-induced Cl- secretion across the canine tracheal epithelium.

Although histamine N-methyltransferase (HMT), the primary enzyme responsible for the inactivation of histamine, has been shown to exist in the airway epithelium, it is still unknown whether this enzyme regulates ion transport across the airway epithelium. Using an Ussing chamber, we examined the effect of a HMT inhibitor, SKF 91488, on potential difference (PD) and short circuit current (SCC) in epithelial membranes from the posterior portion of canine trachea. SKF 91488 itself did not significantly alter PD or SCC values. Pretreatment with SKF 91488 significantly augmented PD and SCC induced by histamine. Amiloride did not significantly alter the augmentation by SKF 91488 in histamine-induced PD and SCC rises. These findings indicate that HMT regulates Cl- secretion across airway epithelium.

HMT regulates histamine-induced glycoconjugate secretion from human airways in vitro

To determine whether histamine N-methyltransferase (HMT) regulates mucus glycoprotein (MGP) secretion from airways, we examined the effect of an HMT inhibitor, SKF 91488, on MGP secretion from human airways in vitro. MGP secretion from human airway explants (with epithelium) and isolated submucosal glands was estimated by measuring trichloroacetic acid (TCA) precipitable glycoconjugates using secretory indices. Histamine induced significant MGP secretion from both explants and isolated glands. Pretreatment with SKF 91488 significantly inhibited histamine-induced secretion from explants, while it did not alter significantly the secretion from isolated glands. H1-blocker significantly reversed the inhibition by SKF 91488 of the secretion from explants, while H2-blocker abolished histamine-induced secretion from both explants and isolated glands. Prostaglandin E2 (PGE2) significantly inhibited histamine-induced secretion from isolated glands. The inhibitory action of SKF 91488 in airway explants was blocked by indomethacin and was significantly reduced by a prostanoid EP4 receptor antagonist (AH23848B). These findings suggest that HMT regulates MGP secretion from human airway submucosal glands through an interaction with epithelial cells which involves the release of PGE2.

Opsonized zymosan decreases cytoplasmic motility of alveolar macrophages in dogs

To examine the mechanisms of changes in alveolar macrophage (AM) activities caused by phagocytic stimulus, we studied the effect of opsonized zymosan (OZ) on cytoplasmic motility (CM) of AM from dog lungs in vitro. Four days after the instillation of ferrimagnetic particles (Fe3O4, 3 mg/kg) into the lower lobe bronchus, AM were harvested by broncho-alveolar lavage. AM were adhered to the bottom of plastic vials (10(6) cells of AM per each vial). Remanent field strength (RFS) from the AM containing Fe3O4 particles was measured immediately after magnetization. RFS decreased with time due to particle rotation (relaxation), which is related to cytoplasmic motility of AM. OZ (1-500 micrograms) decreased lambda 0 (the relaxation rate for the first min) in a concentration-dependent fashion. Neither BW755C (10(-5) M), indomethacin (10(-6) M), leupeptin (10(-5) M), bestatin (10(-5) M), nor superoxide dismutase (1000 U/ml) inhibited OZ (500 micrograms)-induced inhibitory effects on lambda 0, suggesting that cyclooxygenase and lipoxygenase products, serine, thiol enzymes, aminopeptidase and superoxide anion wer not responsible for OZ-induced effects. OZ (500 micrograms) significantly increased the intracellular concentration of Ca2+ (P less than 0.01). Likewise, OZ (500 micrograms)-induced effects on lambda 0 of AM were significantly inhibited by replacement of the medium with a Ca2+ free solution (P less than 0.01). These results imply that opsonized zymosan inhibits cytoplasmic motility of AM via external calcium influx.