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Peripheral blood eosinophils from normal healthy subjects were purified on a Percoll gradient and were incubated with H3(32)PO4. 32P-labeled eosinophils were stimulated by IL-5 under various conditions. After the reaction was stopped. SDS/PAGE and autoradiography were performed to determine the incorporation of 32P into proteins. 32P incorporation into the bands with molecular weights of 52 kDa and 65 kDa was obtained. 32P incorporation into the band of 52 kDa was presumed to be non-specific. 32P incorporation into the band of 65 kDa increased when it was incubated with 1-3 U/ml IL-5. IL-5-induced 32P incorporation into the band of 65 kDa was dependent on the temperature of the reaction system and the incubation time with IL-5. However, IL-5-stimulated eosinophils generated no significant superoxide anion as measured by MCLA-dependent chemiluminescence method.

Peripheral blood eosinophils from normal subjects and patients with atopic dermatitis were isolated on a Percoll gradient and incubated with [gamma 32P] ATP in the presence of Mg2+. After the reaction was stopped, SDS/PAGE was performed and autoradiography was used to determine the incorporation of 32P into the proteins in the eosinophils. Both normodense and hypodense eosinophils showed 32P incorporation into the proteins at the molecular weights of 65 kDa & 66.2 kDa. These reactions were dependent upon Mg2+ concentration, and maximal response was observed at concentrations of 2-6 mM MgCl2. 32P incorporation into the bands was dependent upon the reaction time and temperature, and maximal response was observed at 20 degrees C. 32P incorporation into the proteins was inhibited by Ca2+ in a dose-dependent manner. Cyclic GMP at concentrations of 5 x 10(-8) to 5 x 10(-6) M enhanced the 32P incorporation. These data suggest the possible presence of protein kinase in human peripheral blood eosinophils.

Peripheral blood eosinophils from normal healthy subjects were isolated on a Percoll gradient and were incubated with [32P] orthophosphoric acid. 32P-labeled eosinophils were washed and stimulated with GM-CSF under different conditions. After reaction was stopped, SDS/PAG electrophoresis was performed along with autoradiographs to determine the incorporation of 32P into proteins. GM-CSF-stimulated eosinophils produced an increase of 32P incorporation into the bands in the 31 kDa and 35 kDa areas after 30 sec and 1 min of stimulation. The increase of 32P incorporation was dependent on Mg2+ concentration and temperature, suggesting that proteins in the eosinophils with apparent molecular weights of 31 kDa and 35 kDa can be phosphorylated with the stimulation of GM-CSF.

Vasoactive intestinal peptide (VIP) is suspected to be a neurotransmitter of nonadrenergic and noncholinergic inhibitory nerves in human respiratory tracts. Although VIP affects T lymphocytes through its specific receptor, the effects of VIP on inflammatory cells except T lymphocytes are obscure. We investigated the effects of VIP on superoxide anion (O2-) generation from five kinds of human cells; neutrophils, eosinophils and mononuclear cells isolated from peripheral blood, alveolar macrophages obtained from the bronchoalveolar lavage, and human monocyte cell line, U937, the capacity of which for O2- generation was induced by interferon-gamma. O2- generation from human cells stimulated by 10(-5) M fMLP was measured by the cypridina luciferin analog, MCLA, -dependent chemiluminescence method. VIP inhibited O2- generation from fMLP-stimulated neutrophils, mononuclear cells and U937 in a dose-dependent manner. 3 x 10(-6) M VIP inhibited O2- generation from fMLP-stimulated eosinophils and alveolar macrophages significantly. These results indicate that VIP might inhibit the activation of inflammatory cells and act as an antiinflammatory agent.

Peripheral blood eosinophils from the patients with atopic dermatitis were isolated on a Percoll gradient and incubated with H3(32)PO4. After stopping the reaction, SDS/PAG electrophoresis was performed and autoradiographs were prepared to determine the incorporation of 32P into proteins. Eosinophils developed at least 14 protein bands below 66.2 K by SDS/PAG electrophoresis and the differences of the staining patterns between hypodense and normodense eosinophils were observed. In the autoradiographs 5 distinct radioactive bands were observed below 31 K. 32P incorporation into the bands of hypodense eosinophils were stronger than that of normodense eosinophils, suggesting possible involvement of protein phosphorylation in the activation process of eosinophils.

We measured the expression of the IgE Fc receptor, Fc epsilon RII, on peripheral blood monocytes isolated from asthmatic patients and normal subjects by laser flow cytometry. After peripheral blood mononuclear cells were incubated with monoclonal antibodies, H107, which recognize Fc epsilon RII, FITC-labeled second antibodies were reacted with them. The cells were then incubated with PE-labeled Leu M3 monoclonal antibodies and the ratios of H107 positive monocytes were measured by two color analysis. The ratio of H107 positive monocytes in atopic asthmatic patients was significantly greater than the ratio in normal subjects. But the ratio was not higher in 9 out of the 14 atopic asthmatic patients. This indicated that atopic asthmatic patients are divided into two groups by the expression of Fc epsilon RII on monocytes. Total serum IgE level was not related to the ratio of H107 positive monocytes.

Rat peritoneal mast cells purified on a Percoll gradient were challenged with substance P and effect of phospholipase A2 inhibitor ONO-RS-082 on substance P-induced histamine release from the cells were investigated. Substance P at the concentration of 10(-5) M caused a significant histamine release and the amount of histamine release reached to its submaximum at 1 min after the challenge and then slowly increased. ONO-RS-082 inhibited the substance P-induced histamine release in a concentration-dependent manner at the concentration from 10(-6) to 10(-4) M, suggesting that phospholipase A2 may play some roles in the process of substance P-induced histamine release from rat peritoneal mast cells.

Guinea pigs were actively sensitized by intraperitoneally administered ovalbumin and challenged by intravenous injections of ovalbumin. Respiratory resistance and dynamic compliance were measured by pulmonary mechanics analyzer Buxco Model 6. Changes in resistance (delta R) and compliance (delta C) induced by intravenously administered histamine were investigated before and after the challenge. Responses to the histamine in sensitized guinea pigs were also compared with those in naive guinea pigs. The following results were obtained. 1) A significant increase in delta R by histamine administration was observed following the antigen challenge. delta C by histamine administration showed a tendency to increase after the challenge. 2) A significant increase of delta R by 7.2 micrograms/kg histamine administration was observed in actively sensitized guinea pigs. However, delta C by the histamine administration did not change in the sensitized animals.

Rat peritoneal mast cells obtained by Percoll gradient were incubated with different concentrations of naturally occurring aliphatic polyamines, spermine and spermidine, at 0.1-10 mM and the amount of histamine release into the supernatant solutions was measured. The addition of each polyamine to the suspensions of the mast cells caused a histamine release in a dose-dependent manner. The histamine release from the cells incubated with each polyamine was rapid and the amount of the histamine release into the supernatant solutions reached maximum at one minute with the incubations.

Peptide histidine isoleucine (PHI) as well as vasoactive intestinal peptide (VIP) is considered to be a neurotransmitter of non-adrenergic and non-cholinergic inhibitory nerves in the respiratory tract. We tried to measure PHI immunoreactivity in rat respiratory tracts to evaluate the movement of PHI. A high titer of PHI antiserum which did not react with VIP was obtained from a rabbit immunized with rat PHI, and rat PHI radioimmunoassay was established with this antiserum. Tracheas, extrapulmonary bronchi and lungs were dissected from rats sacrificed by microwave irradiation. They were homogenized with 0.5 N acetic acid and centrifuged at 35,000 X g. Lyophilized supernates were used as samples for the radioimmunoassay. PHI immunoreactivity per g wet weight tissue was 5.12 +/- 1.35 (mean +/- SD) pmol in the tracheas, 1.66 +/- 1.15 pmol in the extrapulmonary bronchi and 0.12 +/- 0.06 pmol in the lungs. PHI immunoreactivity occurred more in the central airways than in the peripheral ones. VIP immunoreactivity in rat respiratory tracts, assayed simultaneously by radioimmunoassay using commercialized VIP antiserum, was as strong as the of PHI, suggesting that PHI and VIP may be produced from the same precursor at the ratio of 1:1 in rat respiratory tracts.

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine to the granules caused an increase in DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines on DPI synthesis in the rat mast cell granules was dose-dependent and maximal effects were observed at 1 mM spermine and 10 mM spermidine respectively. When the effect of 1 mM spermine on 32P incorporation into DPI in rat mast cell granules was investigated serially, 32P incorporation into DPI in rat mast cell granules incubated with spermine for 15 min was enhanced significantly (p less than 0.05) compared with that in the granules in the absence of spermine.

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. DPI synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HC1 and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of PMA to the granules caused an increase of DPI synthesis, which can be catalysed by PI kinase. Neither an inactive phorbol ester, 4-alpha-phorbol-12, 13-didecanoate, nor dimethyl sulfoxide (DMSO) used as a solvent for PMA had any effect. The effect of PMA in the DPI synthesis was dose-dependent and maximal effects were observed at 10-100 ng/ml. Dose-response curves of the effects of PMA in DPI synthesis in the granules corresponded to those of other biochemical effects of PMA in rat mast cells, such as mediator release mediated through the activation of protein kinase C. These results suggest that PMA may directly affect PI kinase or indirectly regulate its activity in rat mast cell granules.

To evaluate some of the pathologic roles of prostaglandins (PGs) in bronchial asthma, plasma PGs levels in bronchial asthmatic patients were measured by the 9-anthryldiazomethane-HPLC method. Plasma TXB2 levels from stable bronchial asthmatic patients were higher than those from healthy subjects. Plasma PGF2 alpha levels were more elevated in atopic patients than in non-atopic ones. Plasma PGF2 alpha levels were more elevated in patients who suffered from attacks than in those who did not and plasma 6-keto PGF1 alpha levels were lower in patients who suffered from attacks than in those who did not. There was no significant difference in plasma TXB2 and PGs levels between mild and moderate asthmatic subjects. In the patients with mild attacks, aminophylline injection showed no significant effect on plasma TXB2 and PGs levels before and after the injection. These results suggest some of the roles of PGs in the pathogenesis of bronchial asthma.