Motojima S

Eosinophil apoptosis caused by theophylline, glucocorticoids, and macrolides after stimulation with IL-5☆☆☆★★★

BACKGROUND Glucocorticoids have long been used as the most potent drugs in the treatment of bronchial asthma. Data reported recently have led to the proposal that theophylline and macrolides have antiinflammatory effects. OBJECTIVE We examined the abilities of theophylline, glucocorticoids, and macrolides to counteract the prolongation of eosinophil survival caused by IL-5. METHODS Purified guinea pig eosinophils were cultured in the presence or absence of human IL-5 and with or without the aforementioned drugs at various concentrations. The percentage of cells alive after 3 days in culture was determined. RESULTS Aminophylline (AM), methylprednisolone (MP), erythromycin (EM), and clarithromycin (CAM) suppressed the IL-5 induced prolongation of eosinophil survival in a dose-dependent manner. The effects of these drugs on eosinophil survival were significantly greater at low concentrations of IL-5 than at high concentrations of IL-5. When eosinophils were cultured in the presence of IL-5 (1 ng/ml) with physiologic concentrations of MP (10(-6) mol/L), AM (10(-4) mol/L), and either EM or CAM (both 10 micrograms/ml), the effect of IL-5 was almost completely abolished, and the morphologic changes in eosinophils observed by electron microscopy were consistent with apoptosis. DNA extracted from eosinophils cultured with IL-5 and each of the drugs was definitely fragmented. CONCLUSIONS One mechanism of the effectiveness of these drugs is induction of eosinophil apoptosis. Some combination of these drugs may be useful in the treatment of bronchial asthma.

The eosinophilic leukocyte and the pathology of fatal bronchial asthma : Evidence for pathologic heterogeneity

Classically, bronchial asthma is associated with peripheral blood eosinophilia and striking eosinophilia of bronchial tissues and sputum. Evidence exists that eosinophil degranulation commonly occurs during bronchial asthma and eosinophil granule proteins are discharged onto damaged bronchial epithelium. Analyses of 10 patients with fatal asthma showed striking eosinophil participation in eight, whereas two patients showed marked epithelial desquamation in the virtual absence of eosinophils. These results point to the possibility that a pathologic heterogeneity exists in fatal bronchial asthma, with most, but not all, patients showing evidence of eosinophil participation.

Eosinophil peroxidase stimulates the release of granulocyte-macrophage colony-stimulating factor from bronchial epithelial cells

BACKGROUND Asthma is characterized by an accumulation of activated eosinophils in the airway. Eosinophil viability-enhancing activity is present in the sputum of patients with asthma, largely because of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bronchial epithelial cells have been shown to release cytokines including GM-CSF when stimulated with IL-1 beta or tumor necrosis factor-alpha. OBJECTIVE The study was designed to determine whether eosinophil peroxidase (EPO) stimulates the release of GM-CSF from bronchial epithelial cells. METHODS Epithelial cells (BEAS-2B) were cultured in serum free HD-F12 medium in a 24-well tissue culture plate until they became confluent. The cells were then exposed to EPO (5.9 x 10(-8) to 5.9 x 10(-7) mol/L) for 15 minutes, washed twice, and cultured in 1 ml of HD-F12. The supernatants were harvested at 3, 6, or 24 hours, and GM-CSF concentration was measured by ELISA. BEAS-2B cells were also treated with a system comprising EPO (1.9 x 10(-9) to 5.9 x 10(-8) mol/L) + 10(-5) mol/L H2O2 + 10(-4) mol/L Br for 24 hours. RESULTS The GM-CSF concentration in the supernatant pretreated with EPO increased in a time- and concentration-dependent manner compared with control. The release of GM-CSF was not inhibited by catalase but was inhibited by cyclohexamide and by mixing of EPO with heparin, suggesting that the action is due to the cationic property of EPO. When EPO was combined with H2O2 and Br, 5.9 x 10(-9) mol/L EPO + 10(-5) mol/L H2O2 released two times more GM-CSF into the supernatants compared with control. CONCLUSION These results suggest that EPO stimulates epithelial cells to release GM-CSF and forms a self-stimulatory cycle.

Detection of interleukin-5 messenger RNA and interleukin-5 protein in bronchial biopsies from asthma by nonradioactive in situ hybridization and immunohistochemistry☆☆☆★★★

Recently direct evidence for the role of interleukin-5 (IL-5) in eosinophilic inflammation in the airways of persons with asthma has been provided by an in situ hybridization study that used radioisotope-labeled IL-5 complementary RNA probes. Radioisotope-labeled probes, although sensitive, require autoradiographic detection, which is time-consuming. In the most recent study we attempted to detect IL-5 messenger RNA in the bronchial biopsy specimens from patients with asthma using nonradioactive in situ hybridization, which gives rapid results. Bronchial biopsy specimens were obtained from eight patients with asthma and seven diseased control subjects. IL-5 complementary DNA probes were labeled with digoxigenin-deoxyuridine triphosphate and hybridized to permeabilized sections. Hybridization signals were visualized by an immunohistochemistry technique. Positive hybridization signals were observed in six of the eight biopsy specimens from patients with asthma. Pretreatment with ribonuclease or hybridization with an unrelated probe produced negative results. Immunohistochemical staining of serial sections with a monoclonal antibody to IL-5 revealed that a few cells within the mucosa positively stained, suggesting active synthesis of IL-5. Biopsy results from the seven diseased control subjects did not show any hybridization signal. These results confirm and extend previous observations of IL-5 messenger RNA expression in the airways of patients with asthma, and suggest that digoxigenin-labeled IL-5 complementary DNA probes would be a powerful research tool.

151 In vitro model of eosinophil migration into the site of acute allergic reactions

PEROXIDE GENERATION BY EOSINOPHIS IN ALLERGIC RHINITIS. H.Oqasawara,M.D.,S.Yoshimura, M.D. and T.Kumoi,M.D.,Nishinomiya,Japan We previously showed that H202 which could be produced by eosinophils or neutrophils from peripheral blood itself induced histamine release ,and that low concentration of H202 augmented anti-IgE or antigen mediated histamine release, while high concentration of H202 which were not cytotoxic inhibited anti-IgE or antigen mediated histamine release. The function of eosinophils in nasal secretions or peripheral blood in house dust nasal allergic patients was studied. Nasal secretion were collected, soon the patient were challenged with house dust, and 5,15 and 30 minutes later respectively,additional nasal secretion were collected. Leukocytes were isolated using five different concentrations(50,56,64,68 and 72%)of Percoll solution from heparinized venous blood. After nasal challenge,the population of eosinophils was increased with various times elapsed to 47,64,65 and 73%,the concentration of H202 in nasal secretion was B&739,728 and 697 pmol/g,the activity of peroxidase was 0.90,0.65,1.67 and 0.91 microgram/g respectively, and the activity of arylsulfatase was not change during this time. Eosinophi chemotactic activity was seen in 5 minute nasal secretion. The generation of H202 increased with time elapsed;stimulation with zymosan enhanced the H202 generation. The rate of phagocytosis for zymosan in eosinophils was not changed. In the high IgE group, oxidative metabolic function in eosinophils from peripheral blood was higher than in the low IgE group. Eosinophil chemotactic activity seemed to stimulate oxidative metabolic function. H202 plays a significant role in vivo was suggested.

Inhibitory effect of an anti-allergic drug (Ibudilast) on eosinophil infiltration, changes of epithelial tissue and eosinophil microstructure in antigen-induced airway responses of passively sensitized guinea pigs.

We evaluated whether or not eosinophil infiltration induced by an antigen (ovalbumin) inhalation caused the damage of the bronchial epithelium. The inhibitory effects of the anti-allergic drug, Ibudilast (Ibd), on eosinophil infiltration, eosinophil activation and epithelial tissue damage were also examined. Fourteen days pretreatment of Ibd inhibited eosinophil infiltration in the epithelial and subepithelial tissue 6, 24 hours after an antigen inhalation. A significant correlation was noted between change of the electron densities of the eosinophil specific granules and the damage of tracheal epithelium 6 hours after an antigen inhalation. Changes of the electron densities of the eosinophil specific granules suggest the activation of the cells.The observation in this study shows that infiltrated eosinophils were activated and contributed to the damage of the bronchial epithelium by releasing toxic granules proteins and may explain the mechanism that Ibd exerts bronchial hypersensitivity.