Seiichi Kitani

Nonreleasing basophils convert to releasing basophils by culturing with IL-3

The extent of basophil histamine release initiated by IgE cross-linking stimuli has been known to vary greatly among donors. Studies on anti-IgE nonreleasing basophils are useful in understanding the IgE-specific control mechanism of mediator release. We attempted to determine (1) whether a mutation of Fc epsilon RI is present in nonreleasing basophils and (2) whether treatment with IL-3 converts anti-IgE nonreleasing basophils to releasing basophils. Basophils were purified from normal human blood and donors were divided into releasers (maximal histamine release > 5%) and nonreleasers (< 5%). The mutation of Fc epsilon RI alpha, beta, and gamma was evaluated by reverse transcriptase-polymerase chain reaction, and the DNA sequence was determined from amplified polymerase chain reaction products. Although antibodies against Fc epsilon RI failed to cause histamine release in anti-IgE nonreleasing basophils, no primary structural change of Fc epsilon RI was observed in nonreleaser basophils. After culturing with IL-3 for 7 days, nonreleasing basophils released histamine in response to anti-IgE, and dose-response curves of anti-IgE were equal in both releasers and nonreleasers. The conversion of nonreleasing basophils to releasing basophils was evident after 3 days of culture with IL-3. These findings indicate that nonreleasing basophils have recoverable defect(s) in the signal transduction pathway after IgE cross-linking.

IgG-Mediated Histamine Release from Canine Mastocytoma-Derived Cells

Background: Recent data suggest that normal tissue mast cells can express functional receptors for IgG under certain conditions. However, little is known about IgG receptor expression and functional consequences in mast cell neoplasms. Methods: In this study, neoplastic mast cells were obtained from a dog with cutaneous mastocytoma (CM-MC) and from a dog with visceral mastocytoma (VI-MC). Both cell populations were characterized morphologically and functionally. Results: Most cells proliferated constantly in suspension without particular supplements. Doubling times of CM-MC and VI-MC were 52.2 and 27.5 h, respectively. Both cell types were sensitive to formalin fixation, did not contain heparin and were tryptase and chymase positive. Electron microscopy showed fine granules with electron-dense content in both cell populations. The total histamine content of CM-MC and VI-MC was 0.25 and 0.10 pg/cell, respectively. Calcium ionophore A23187 and substance P induced dose-dependent histamine release, whereas compound 48/80 had no effect. Most significantly, both cell types, when sensitized with monomeric dog IgG, released histamine upon stimulation by anti-dog IgG. Conclusions: Dog mastocytoma-derived cells may be useful to study the regulation of neoplastic mast cell growth and differentiation, as well as IgG receptor-mediated activation in neoplastic mast cells. Further research is required to clarify the pathophysiological significance of constitutive expression of IgG receptors in neoplastic (canine) mast cells.

Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.

Isolation and characterization of a major allergenic component (gp55) of Aspergillus fumigatus

IgE class antibodies specific for antigens in a water-soluble extract of Aspergillus fumigatus (strain NHL-5759) were analyzed by immunoblotting with sera from patients with allergic bronchopulmonary aspergillosis. All the sera tested were reactive with a major 50 to 60 kd protein in the extract. This allergen, designated gp55, was purified by gel filtration and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was found to be present in the water-soluble extract in the form of a complex composed of approximately eight molecules of gp55. The carbohydrate and phosphate content of the purified antigen were 23.1% and 0.46%, respectively. The molar ratio of mannose to galactose residues was 2.76:1, and the protein was glycosylated predominantly with N-linked oligosaccharides. The serologic activity of the gp55 antigen was abolished by treatment with nonspecific protease (Pronase) but not by treatment with sodium metaperiodate or endoglycosidases. Thus the major antigenic site of the glycoprotein is located within its peptide moiety. The antigen itself displayed no chymotryptic or tryptic activity. The amino acid sequence of the 20 N-terminal residues of the antigen (ATPHEPVFFSWDAGAVTSFP) is different from that of any other protein previously reported.

Tea catechins have dual effect on mast cell degranulation induced by compound 48/80☆

Green tea catechins are emerging as one of the most efficient and safest ingredient in health promoting food. We investigated catechins effects on intracellular ROS generation in mast cell activation and degranulation. Compound 48/80, receptor mimetic basic secretagogues for mast cell, induced ROS generation dose-dependently with bell-shaped degranulation pattern in canine cutaneous mastocytoma cells (CM-MC). When intracellular ROS level was relatively low, catechins decreased both ROS and the degranulation. However, when intracellular ROS level was remarkably high, catechins decreased ROS level but increased the degranulation paradoxically. Gallocatechins showed the stronger effects than non-gallated catechins. Exogenous H(2)O(2) also shows dual effect on degranulation dose-dependently. EGCG shows the dual effect on the tyrosine and threonine phosphorylation depending on the concentration of compound 48/80. Particularly, 60 kDa protein tyrosine-phosphorylated by EGCG with 3 microg/ml of compound 48/80 might be a negative regulator for the degranulation. Taken together, there is an optimal level of ROS for the degranulation, and the catechins have a dual function by controlling ROS level.

IgG1 Antibodies to House Dust Mite (Dermatophagoides farinae) and Late Asthmatic Response

Thirteen asthmatic patients sensitive to mite were challenged by inhalation of an extract of mites (Dermatophagoides farinae). Seven showed dual bronchial reactions and 5 showed isolated immediate responses. No patient showed an isolated late reaction. Six of seven patients with dual reaction had higher IgG1 antibodies than the 5 patients with isolated immediate reaction when examined before the challenge. A similar result was obtained in terms of levels of immune complex. IgE, IgG4 and total IgG antibodies were not predictive for late reaction. These results suggest that there is a close correlation of the presence of high IgG1 antibodies with a propensity to develop late asthmatic responses. The meaning of this observation is discussed.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A2-induced degranulation in mast cells

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of β-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G(M1)), di-sialoganglioside (G(D1a)) and tri-sialoganglioside (G(T1b)). In contrast, honeybee venom-derived phospholipase A(2) induced the net degranulation directly without cytotoxicity, which was not inhibited by G(M1), G(D1a) and G(T1b). For analysis of distribution of Gα(q) and Gα(i) protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα(q) and Gα(i) at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A(2)-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A(2)-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

Global environmental pollutant substance vanadium activates mast cells and basophils at the late phase in the presence of hydrogen peroxide.

Vanadium is contained in fossil fuel such as coal, oil and sand oil and released in the air through the combustion. We studied the allergic and inflammatory potentials of vanadium as the factor of a recent increase of allergic disease. Vanadium oxide (V(2)O(5)) and orthovanadate (Na(3)VO(4)) released histamine from human basophils, rat mast cells and rat basophilic leukemia (RBL-2H3) cells in the presence of hydrogen peroxide (H(2)O(2)). Activation of RBL-2H3 cells by vanadium/H(2)O(2) was accompanied by leukotriene synthesis, increases of [Ca(2+)](i), multiple protein tyrosine phosphorylations and remarkable morphological changes. Pharmacological study suggests that vanadium/H(2)O(2) activates mast cells at the late phase, bypassing the early signaling components. Thus, vanadium can amplify the allergy in the presence of H(2)O(2) at the inflammation site in the global environment of industrial age.

Pharmacologic Study of Basophil Histamine Release Induced by Monocyte Chemotactic Protein-1 with Kinase Inhibitors

Monocyte chemotactic protein-1 (MCP-1)/monocyte chemotactic activating factor has a potent histamine-releasing activity for basophils and is a major component of IgE-independent histamine-releasing factors (HRF). In this study, we examined the effect of a panel of kinase inhibitors on MCP-1-induced histamine release from human basophils to characterize the signaling pathway used by this chemokine. Genistein (3 micrograms/ml), an inhibitor of tyrosine kinase, inhibited MCP-1-induced histamine release by 44%. Wortmannin is a specific inhibitor of phosphatidylinositol 3 kinase (PI-3 kinase). It blocked MCP-1-induced histamine release with an IC50 of 3.3 x 10(-8) M indicating a role of PI-3 kinase in this reaction. KT5926, an inhibitor of myosin light chain kinase, also inhibited histamine release in response to MCP-1 with an IC50 of 10(-6) M. Staurosporine, a potent inhibitor of protein kinase C, although being not specific, augmented MCP-1-induced histamine release by 31.9% at 10(-6) M. These results indicate the possible involvement of a series of kinases, including PI-3 kinase, in the signal transduction pathway used by MCP-1.

IgG-Mediated Signal Transduction in Canine Mastocytoma-Derived Cells

Background: We have reported canine cutaneous mastocytoma-derived cells named CM-MC sensitized with monomeric IgG released histamine upon anti-IgG stimulation. However, IgG or IgE-mediated signal transduction in the cells remains to be examined. Methods: Monomeric IgG-binding to cells was measured by flow cytometry using FITC-anti-IgG. IgG-mediated protein tyrosine phosphorylation was studied by Western blotting using anti-phosphotyrosine antibody. We monitored the intracellular Ca²⁺ concentration ([Ca²⁺]_i) when IgG-primed cells were activated with anti-canine IgG. Release of Ca²⁺ from intracellular stores was analyzed with thapsigargin in the absence of extracellular Ca²⁺. The Ca²⁺ entry via store-operated Ca²⁺ channel from the external environment was characterized using Ba²⁺, Ni²⁺ and EGTA. Cells sensitized with canine serum abundant in IgG and IgE or heat-inactivated serum were activated by anti-canine IgG or anti-canine IgE. The effect of extracellular Ca²⁺ and reaction time on IgG-mediated histamine release was examined. Staurosporine and ER-27319 were used to clarify the IgG-mediated protein tyrosine phosphorylation. Results: Abundant IgG-binding sites on the cell were detected by FACS analysis. Anti-IgG induced rapid protein tyrosine phosphorylation and [Ca²⁺]_i elevation. When extracellular Ca²⁺ was excluded by EGTA, a mild and transient increase in [Ca²⁺]_i was observed, indicating the release of Ca²⁺ from anti-IgG-sensitive intracellular Ca²⁺ stores. The constant Ba²⁺ entry from external environment proved the Ca²⁺ influx occurred mainly via a store-operated Ca²⁺ channel which was inhibited by Ni²⁺ and EGTA. Canine serum-sensitized cells showed a rapid and sustained increase in [Ca²⁺]_i upon both anti-IgG and anti-IgE stimulation. The [Ca²⁺]_i elevation induced by anti-IgE was decreased in the cells sensitized with heat-inactivated serum. Histamine release from CM-MCs was absolutely dependent on extracellular Ca²⁺, and reached equilibrium within 5 min. Staurosporine inhibited the tyrosine phosphorylation of 38-, 65-, 70-, 80-kD proteins. ER-27319 inhibited the tyrosine phosphorylation of 38- and 70-kD proteins. Staurosporine also inhibited IgG-mediated [Ca²⁺]_i elevation and histamine release in a dose-dependent manner. Conclusions: Canine cutaneous mastocytoma-derived (CM-MC) cells were activated by both IgG- and IgE-mediated mechanisms. IgG-mediated protein tyrosine phosphorylation and Ca²⁺ influx were similar to those mediated by IgE. CM-MC cells are useful for the study of allergic inflammation caused by IgG-dependent mechanisms.