Masayuki Furukawa

Inhibitory Effect of Macrolides on Interleukin-8 Secretion From Cultured Human Nasal Epithelial Cells

The mechanism of macrolide therapy in chronic sinusitis patients is unclear. The authors studied the effect of macrolides on interleukin (IL)‐8 secretion from cultured human nasal epithelial cells. Epithelial cells harvested from the nasal polyps of patients with chronic sinusitis were primary‐cultured, and secreted IL‐8 in culture media was measured by enzyme immunoassay. The cells secreted considerable amounts of IL‐8 constitutively and in response to lipopolysaccharide. The secretion was significantly inhibited by 10−5 M of erythromycin, clarithromycin, roxithromycin, and josamycin. 10−6 M erythromycin still showed the inhibitory effect, whereas the same concentration of josamycin did not. These results indicate that macrolide antibiotics may act as an immunomodulator to reduce IL‐8 in inflammatory sites and, at least partially, account for the clinically discrepant effects between 14‐ and 16‐membered ring macrolides in long‐term low‐dose therapy for chronic sinusitis.

Infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and ICAM-1 production.

To further understand the early biochemical events that occur in infected surface epithelium, we developed for the first time a model in which a respiratory submucosal gland cell population can be infected with rhinovirus (RV). Viral infection was confirmed by demonstrating with PCR that viral titers in supernatants and lysates from infected cells increased with time. Infection by RV14 upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major RV receptor, on submucosal gland cells, and it increased production of interleukin (IL)-1α, IL-1β, IL-6, IL-8, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor in supernatants. Antibodies to ICAM-1 inhibited RV infection of submucosal gland cells and decreased the production of cytokines after RV infection. Both IL-1α and IL-1β upregulated ICAM-1 mRNA expression and increased susceptibility to RV infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, neutralizing antibodies to IL-1α and IL-1β significantly decreased the viral titers in supernatants and ICAM-1 mRNA expression after RV infection, but a neutralizing antibody to tumor necrosis factor-α was without effect. These findings suggest that respiratory submucosal gland cells play an important role in the initial stages of inflammation and provide useful insights into the pathogenesis of RV infection.To further understand the early biochemical events that occur in infected surface epithelium, we developed for the first time a model in which a respiratory submucosal gland cell population can be infected with rhinovirus (RV). Viral infection was confirmed by demonstrating with PCR that viral titers in supernatants and lysates from infected cells increased with time. Infection by RV14 upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major RV receptor, on submucosal gland cells, and it increased production of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor in supernatants. Antibodies to ICAM-1 inhibited RV infection of submucosal gland cells and decreased the production of cytokines after RV infection. Both IL-1alpha and IL-1beta upregulated ICAM-1 mRNA expression and increased susceptibility to RV infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, neutralizing antibodies to IL-1alpha and IL-1beta significantly decreased the viral titers in supernatants and ICAM-1 mRNA expression after RV infection, but a neutralizing antibody to tumor necrosis factor-alpha was without effect. These findings suggest that respiratory submucosal gland cells play an important role in the initial stages of inflammation and provide useful insights into the pathogenesis of RV infection.

Expression of voltage-dependent chloride channels in the rat cochlea

Voltage-dependent chloride (ClC) channels have not yet been identified in the cochlea. In this study, an approach utilizing the reverse transcription-polymerase chain reaction (RT-PCR) was devised to clone the cDNA of ClC channels. PCR was performed using degenerate primers corresponding to two highly conserved regions of the ClC channels. By Southern hybridization and sequencing studies, the sequences corresponding to ClC-2 and ClC-3 were found in the cochlear lateral wall, while ClC-1 was not detected. These results suggest that ClC-2 and ClC-3 might be involved in Cl- transport in the cochlear lateral wall.

Relative importance of maternal and embryonic microsomal epoxide hydrolase in 7,12-dimethylbenz[a]anthracene-induced developmental toxicity

Microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of epoxide intermediates derived from drugs and environmental chemicals. The response of in vivo (embryo) and in vitro (embryo fibroblast) tests were analyzed using mEH-null and wild-type mice to determine the relative role of maternal and embryonic mEH in the developmental toxicity induced by 7,12-dimethylbenz[a]anthracene (DMBA). Embryos derived from DMBA-treated [50mg/kg, daily from gestational day (GD) 11 to GD 15] dams were analyzed. Although weight (P=0.0009) and crown-rump length (P=0.0003) of wild-type fetuses on GD 18 were significantly lower than those of mEH-null fetuses, respectively, no significant difference was found between mEH-null and heterozygous fetuses of mEH-null dams. Cell viability was decreased to 50% in wild-type mouse embryo fibroblasts (MEFs) treated with 3 microM DMBA, but no significant decrease was found in mEH-null MEFs. DMBA-3,4-diol produced a significant decrease in cell viability and suppressed the proliferation of wild-type MEFs at a 10-fold lower concentration than did DMBA. Although mEH protein was expressed in liver microsomes from wild-type embryos (GD 15), DMBA-3,4-diol was not detected among the DMBA metabolites. However, it was detected in the serum of wild-type pregnant mice treated with DMBA, but not in that of mEH-null mice. These results suggest that maternal mEH plays a major role in DMBA-induced developmental toxicity, and embryonic mEH is less involved in the toxicity.

Expression of adhesion molecules in nonallergic chronic sinusitis

Endothelial and epithelial adhesion molecules are important in the recruitment of leukocytes to inflammatory sites. To determine the relationship between recruited leukocytes and adhesion molecules in the paranasal sinus mucosa of nonallergic chronic sinusitis, we surgically obtained mucosa from 16 patients and identified the expression of intercellular adhesion molecules(ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), E‐selectin, and P‐selectin by immunohistochemistry. Neutrophils were significantly dominant in the nasal discharge as compared with eosinophils. The degree of neutrophil infiltration in the paranasal sinus mucosa was prominent in both intraepithelial and subepithelial areas as compared with the lamina propria. In each tissue site, the degree of infiltration of neutrophils was similar to that of eosinophils. These findings suggest that the tissue neutrophils actively and rapidly migrated into the lumen. All the adhesion molecules except VCAM‐1 were expressed in the vascular endothelial cells. On the other hand, the surface epithelial cells showed the expression of only ICAM‐1. The expression of ICAM‐1 on the endothelial cells correlated with the degree of neutrophil infiltration in the mucosa. The eosinophil infiltration was not dependent on any adhesion molecules examined here. It was concluded that ICAM‐1 expression in the mucosa may be involved in neutrophil recruitment and may contribute to the establishment of the inflammatory cell distribution in the paranasal sinus of nonallergic chronic sinusitis.

Expression of the Voltage-Dependent Chloride Channel ClC-3 in Human Nasal Tissue

In the present study, ClC-3, one of the voltage-dependent chloride channels, was identified in human nasal tissue. In situ hybridization and immunohistochemical investigations demonstrated the localization of ClC-3 in the serous acini and ductal portions of submucosal nasal glands, which are the primary source of nasal secretion. Our data suggest that this channel contributes to nasal secretion via chloride transport. Its dysfunction might lead to abnormal nasal secretion in such pathological states as sinusitis.

Na+,K+-ATPase activity in the cochlear lateral wall of the gerbil

We examined Na+,K(+)-ATPase activity in four regions of the gerbil cochlear lateral wall by measuring intracellular Na+ concentration ([Na+]i). The application of 1 mM ouabain and removal of external K+ caused a most striking increase in [Na+]i of the suprastrial fibrocytes, followed by the stria vascularis and the type II fibrocytes, but no detectable response in the type I fibrocytes. These results may imply the potency of the Na+,K(+)-ATPase activity. The high values of the resting [Na+]i in these cells under perilymph-like solution returned to the [Na+]i within physiological values after exposure to low-Na+ conditions similar to endolymph. Our results are interpreted in the light of recent morphological findings with regard to the roles in the secretion of K+ into endolymph of several cell types within the lateral wall.

Quantitative uronic acid analysis of the otoconial membrane of the guinea pig

Histochemical and biochemical studies have shown that the otoconial membrane, as well as the tectorial membrane and the cupula, contains glycoproteins and proteoglycans. However, uronic acids, which are essential elements of glycosaminoglycans (GAGs), have not so far been directly detected or quantitatively measured in inner ear samples. In the present study, we quantitatively analyzed the glucuronic acid (GlcA) content of the otoconial membrane of the guinea pig by methanolysis combined with reversed-phase high-performance liquid chromatography (RP-HPLC). The utricular otoconial membrane was treated with thermolysin to separate the supernatant (OM-sup; gelatinous layer) and precipitate (OM-ppt; otoconia). The samples were then subjected to methanolysis followed by brief TFA hydrolysis, incubated with 1-phenyl-3-methyl-5-pyrazolone for labeling, and then analyzed by RP-HPLC coupled with the detection of UV absorbance at 245 nm. The GlcA contents of OM-sup and OM-ppt were 2.1 and 5.8 pmol/100 ng protein, respectively. Based on this result, GlcA-containing GAGs estimated as (GlcA+monosulfo-N-acetylhexosamine)n would be 1.0% of protein in OM-sup and 2.61% of protein in OM-ppt, indicating that this type of glycoconjugate is a minor component in both fractions. It is, accordingly, presumed that both the gelatinous layer and otoconia of the otoconial membrane have totally different properties from those of the tectorial membrane and cartilaginous matrix, which contain abundant GAGs.