Toshiaki Takaishi

Identification of basophils by immunohistochemistry in the airways of post-mortem cases of fatal asthma.

There is increasing evidence for the role of basophils in the pathogenesis of bronchial asthma. To examine the presence of basophils in the airways of patients with fatal asthma by immunohistochemistry, we stained lung tissues from four post‐mortem cases who had died from severe asthmatic attacks and four controls with a monoclonal antibody raised against tryptase (AA‐1) and anti‐IgE. Mast cells and basophils were identified in the bronchioles as A A‐1‐ and anti‐IgE‐positive cells, and anti‐IgE‐posilive cells, respectively. Airway mast cells were found beneath the basemenl membrane, near blood vessels in the submucosa, and adjacent to the submucosal glands, and scattered throughout the muscle bundles. There was a significant increase of mast cells in the asthma group compared with the control group (203.5 ± 84.6/mm2, mean ± s. d. vs 37.7 ± 8.7/mm2, P < 0.05, n= 4). In contrast, basophils were observed in the airway lumen, in the bronchial epithelium and in the submucosa. The number of basophils in the bronchioles was 81.8 ± 55.5/mm2 (n= 4); however, basophils were not found at all in the airways of the control group. Although eosinophils, B lymphocytes and macro‐phages bear low affinity IgE receptors and could react with anti‐IgE, the location of these cells in the close sections did not correspond closely with basophils. The presence of basophils in lung tissues obtained from fatal asthma patients supports the view that basophils play a role in the pathogenesis of bronchial asthma.

Airway Basophil and Mast Cell Density in Patients with Bronchial Asthma: Relationship to Bronchial Hyperresponsiveness

We investigated the role of basophils and mast cells in the pathogenesis of bronchial asthma. Eight asthmatics (6 atopic, 2 nonatopic) and 6 control subjects were enrolled in this study. Bronchial responsiveness to acetylcholine (PC20ACh) was measured in asthmatics and endobronchial biopsy from right upper lobe bronchus was performed on the same day. Basophils and mast cells in the airways were identified by immunohistochemistry using a monoclonal antibody against tryptase and anti-IgE. The number of basophils of asthmatics was 52.2 +/- 12.5/mm(2). In contrast, no basophils were found in the airways of control subjects. There was a significant increase of number of mast cells in the asthma group compared to the control group (168.6 +/- 32.6 vs. 22.3 +/- 6.1, p<0.01). There was an inverse correlation between airway basophil and mast cell numbers and PC20ACh (r=-0.82, r=0.72, p<0.05). These findings suggest a possible role for basophils and mast cells in the pathophysiology of asthma.

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.

Glucocorticoids inhibit chemokine generation by human eosinophils

Recent identification of eosinophils as a cellular source of various cytokines suggests that eosinophil-derived cytokines contribute to allergic inflammation through either an autocrine or a paracrine fashion. The profound inhibitory effects of glucocorticoids (GCCs) on the production of various cytokines have been well recognized, however, there has been no definitive evidence that GCCs in fact inhibit cytokine generation by eosinophils. To verify the inhibitory ability of GCCs on eosinophil cytokine generation, we studied the effect of GCCs by determination of IL-8 and monocyte chemoattractant protein-1 (MCP-1) as parameters. Dexamethasone (DEX) inhibited both generation and secretion of IL-8 in a dose-dependent fashion. DEX also dampened formyl-methionyl-leucyl-phenylalanine-or ionomycin-induced eosinophil IL-8 production. Furthermore, MCP-1 production was also inhibited by DEX. The slope and the shape of the dose-response curve of DEX were similar irrespective of either the input stimuli or the output cytokines; half-maximal inhibition was observed at 10(-8) mol/L, and nearly complete abolishment was observed at 10(-7) mol/L. The competitive polymerase chain reaction for IL-8 mRNA and semiquantitative polymerase chain reaction for MCP-1 mRNA revealed that the inhibition occurred at a level of pretranslation. These results indicate that the beneficial effect of GCCs in allergic inflammation might be related, at least in part, to a direct effect of the drugs on eosinophil cytokine synthesis.

Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells

Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.

Hemopoietic Growth Factors Regulate the Survival of Human Basophils in vitro

Human basophils were purified from normal peripheral blood, using density gradient followed by negative panning selection. We tested the effects of hemopoietic growth factors on the survival of these basophils in vitro. In the absence of exogenous factors, basophils (purity greater than 90%) decreased in number rapidly. At day 7 only 11% of the cells remained alive in cultures; less than 1% of cells survived at day 14. Interleukin (IL)-3 maintained numbers of viable cells; cell viability was 67% at day 7 and 45% at day 14. Granulocyte-macrophage (GM)-colony-stimulating factor (CSF) exhibited slight effect on the survival; 33% of cells remained at day 7. Other growth factors including granulocyte (G)-CSF, macrophage (M)-CSF, and IL-4 had no significant effect on the survival of basophils at all. Morphological and functional characterization of cells maintained by IL-3 revealed that they belonged to the basophil lineage. These observations indicate that normal basophils possess functional receptors for IL-3 and GM-CSF and that both factors modulate immediate- and delayed-type hypersensitivity reactions by prolonging the life span of basophils.

Exogenous Nitric Oxide Regulates the Degranulation of Human Basophils and Rat Peritoneal Mast Cells

This study was designed to investigate whether anti-IgE-induced or ionophore A23187-induced histamine release from human basophils is regulated by exogenous nitric oxide (NO), and to assess some similarities between the effect of NO on basophils and that on rat peritoneal mast cells (RPMC). The NO donor, sodium nitroprusside (SNP), inhibited A23187-induced histamine release from crude human basophils and crude RPMC in a dose-dependent fashion. This downregulation was still observed when SNP was washed out just before the cell stimulation, indicating that the effect of SNP was irreversible. The downregulation disappeared in both purified cell populations after the removal of contaminating cells. However, when purified cells were preincubated with SNP in the presence of 5 mM N-acetylcysteine (NAC), increasing the bioavailability of NO, the downregulation was recovered. The presence of NAC significantly augmented the downregulation of SNP on A23187-induced histamine release from both crude cell populations. In contrast, SNP had no effect on anti-IgE-induced histamine release from either crude or purified basophil preparation in the absence of NAC, and SNP plus NAC inhibited anti-IgE-induced histamine release from both cell preparations. The same results were obtained with crude and purified RPMC preparations under the same conditions. These results show that SNP similarly downregulated exocytosis of basophils and RPMC, and acquired the potent effect in the presence of NAC, indicating that exogenous NO plays a part in the regulation of basophil and mast cell activation.

Haemopoietic growth factors induce human basophil migration in vitro

Accumulation of basophils in inflammatory sites is an important aspect of the late‐phase allergic reaction involving skin and upper and lower airways, suggesting the existence of mechanisms for basophil migration. Because haemopoietic growth factors have been shown to stimulate various functions of human basophils, we tested the ability of haemopoietic growth factors to migrate basophils in vitro. Both IL‐3 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) induced migration of purified normal basophils (purity c. 80%) in a dose‐dependent fashion at picomolar concentrations, while granulocyte (G)‐CSF, macrophage (M)‐CSF, and IL‐4 had no effect at all. Chequerboard analyses indicate that migratory activity of both factors are chemokinetic. These results suggest that local production of both factors during allergic reactions might potentially play an initial role in the recruitment of basophils from the circulation to sites of inflammatory reactions.

IgG4 Antibodies in Patients with House-Dust-Mite-Sensitive Bronchial Asthma: Relationship with Antigen-Specific Immunotherapy

Sera from 40 patients with house-dust-mite (Dermatophagoides farinae)-sensitive bronchial asthma were evaluated by solid-phase radioimmunoassay for their mite-specific IgG4 antibody levels. Asthmatic patients undergoing specific immunotherapy possessed a significantly higher mean value of IgG4 antibodies than normal controls and asthmatics without immunotherapy (p less than 0.01 and p less than 0.05, respectively). Moreover, evaluation of 11 patients before and after immunotherapy showed that IgG4 antibodies tend to increase during immunotherapy. The clinical significance of the IgG4 subclass as blocking antibodies in immediate allergic reactions is also briefly discussed.

Effect of cytokines on mediator release from human dispersed lung mast cells

To evaluate the contribution of human lung mast cells (HLMC) to allergic inflammation, we investigated whether or not cytokines, including stem‐cell factor (SCF), monocyte chemotactic and activating factor (MCAF), and RANTES, activate HLMC. SCF induced histamine release from dispersed HLMC in a dose‐dependent fashion (P<0.01). The release was 7.8 ± 1.0% at 500 ng/ml SCF (n= 9). This response was also observed in chopped lung tissue. HLMC from which surface IgE molecules had been removed by treatment with lactic acid responded to SCF, while these cells lost their response to anti‐IgE. The process was relatively rapid and reached a maximum in 5 min. This response required extracellular calcium, and it was observed at 37°C, but not at 4°C or 20°. A brief preincubation (10 min) with lower concentrations of SCF, which were ineffective in releasing histamine, enhanced anti‐IgE‐induced histamine release (P<0.05), while its enhancing effect was lost by the longer preincubation (30 min). SCF did not prime basophils to enhance stimulated‐histamine release. Interleukin (IL)‐1α, IL‐1β, IL‐3, IL‐4, IL‐5, granulocyte/macrophage‐colony stimulating factor (GM‐CSF), MCAF, and RANTES neither induced histamine release nor enhanced the release stimulated by anti‐IgE after a 10‐ or 30‐min preincubation. The combination of IL‐3 and IL‐4 showed no effect on histamine release from HLMC. Leukotriene (LT)C4/D4/E4 production by SCF was negligible, as compared with anti‐IgE‐induced LT production. SCF at 1.5 ng/ml augmented anti‐IgE‐induced LT generation significantly (536+ 117 pg/105 mast cells and 1569 ± 258 pg/105 mast cells; P<0.01). These results provide further evidence that numerous aspects of the phenotype of mast cells and basophils are heterogeneous, including structure, relevant secretagogues, and pharmacologic control.