Kazuaki Tabe

The evidence of platelet activation in bronchial asthma

The possible involvement of platelets in bronchial asthma was investigated under three different conditions: (1) chronic asthma, (2) bronchial provocation inhaling house dust mite (HDM), and (3) status asthmaticus. Plasma levels of beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), and in part, platelet-activating factor (PAF) were measured. Approximately one third of the subjects with symptomatic or asymptomatic chronic asthma showed an increased level of beta-TG or PF4. Statistically significant differences occurred in beta-TG and PF4 levels only between healthy controls and symptomatic subjects. Five out of six subjects showed no elevation of beta-TG and PF4 during immediate asthmatic response. In two out of nine subjects with status asthmaticus, beta-TG or PF4 was elevated, and statistically significant correlations occurred between the initial level of PAF and that of beta-TG or PF4. Those results suggest that the platelet activation in the circulation is sometimes provoked in asthma, but plasma level of alpha-granule-derived proteins does not reflect the intensity or severity of asthma, and that PAF is likely to be a mediator responsible for the platelet activation.

Eosinophil Transmigration across VCAM-1-Expressing Endothelial Cells Is Upregulated by Antigen-Stimulated Mononuclear Cells

Background: Adhesion to and transmigration across endothelial cells expressing vascular cell adhesion molecule 1 (VCAM-1) may be key steps in the development of selective eosinophil accumulation at the allergic inflammation sites. There is evidence that cytokines/chemokines produced by CD4+ T cells play a prominent role in these processes. Objective: The objective of this study was to evaluate whether eosinophil migration across human pulmonary microvascular endothelial cells (HPMEC) expressing VCAM-1 is modulated by the supernatants of antigen-stimulated mononuclear cells obtained from atopic asthmatics. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from Dermatophagoides farinae(Df)-sensitive asthmatic subjects and cultured for 96 h at 37°C in the presence or absence of 1 µg/ml Df antigen. Eosinophils were isolated from blood of healthy subjects and placed on the HPMEC monolayers cultured on a transwell filter (3-µm pore size) stimulated with IL-4 plus TNF-α (both at 100 pM, 24 h) The supernatants of PBMC were then applied to the lower compartment and the transmigration of eosinophils was examined. Results: The supernatants of PBMC stimulated with Df significantly enhanced the eosinophil transmigration across VCAM-1-expressing HPMEC (% migration: 7.6 ± 0.6 by the supernatants of PBMC cultured without Df vs. 12.3 ± 1.2 by the PBMC cultured with Df, p < 0.01, n = 8). The enhanced migration, but not spontaneous migration, was blocked by the anti-α4 integrin antibody. Moreover, the enhanced transmigration was blocked by anti-CCR3 antibody. Conclusion: The antigen-stimulated PBMC from atopic asthmatics produce an activity to induce eosinophil migration across VCAM-1-expressing endothelial cells. This activity appears to involve CCR3 as an essential molecule.

Effect of Immunotherapy on the Production of Eosinophil Adhesion-Inducing Activity from Mononuclear Cells in House-Dust-Mite-Sensitive Bronchial Asthma

An initial step of eosinophil (EOS) accumulation in the sites of allergic inflammation is the adhesion to endothelial cells. There is increasing evidence that immunotherapy (IT) modulates the production of cytokines from mononuclear cells and hence attenuates allergic inflammation. To examine whether IT modifies the production of factor(s) which induce EOS adhesion, peripheral blood mononuclear cells (PBMC) from house-dust-mite-sensitive asthmatics treated with or without IT were cultured for 96 h in the presence or absence of 1/µg/ml Dermatophagoides farinae antigen. EOS were isolated from the peripheral blood of healthy subjects. EOS adhesion-inducing activity (EAIA) in the PBMC culture supernatants was examined by the ability to modify EOS adhesion to paraformaldehyde-fixed human umbilical vein endothelial cells (HUVEC) which were stimulated with IL-4 plus TNF-α. In asthmatics without IT, the addition of D. farinae antigen significantly promoted the production of EAIA from PBMC (EOS adhesion: 22.4±13.1% by medium control vs. 30.5±18.9% by D. farinae, n=10, p=0.023). This enhancing effect was blocked by an anti-β2-integrin antibody. In contrast, the addition of D. farinae did not modulate EAIA production from PBMC in asthmatics treated with IT (23.1±10.3% vs. 21.5±12.3%, n=10, p=NS). These results suggest that IT induces the inhibition of antigen-dependent production of EAIA from PBMC. This may contribute to the inhibitory effect of IT on eosinophil recruitment in allergic inflammation.

Eosinophil-Adhesion-Inducing Activity Produced by Antigen-Stimulated Mononuclear Cells Involves GM-CSF

Background: The initial step of eosinophil accumulation in allergic inflammation is adhesion of circulating eosinophils to vascular endothelial cells (EC). There is evidence that the adhesive property of circulating eosinophils is upregulated following antigen exposure. Although the exact mechanism remains to be established, cytokine(s) produced by antigen-stimulated mononuclear cells is (are) likely key factor(s). Objective: The objective of this study was to examine the factor(s) responsible for eosinophil adhesion and migration induced by the antigen-stimulated mononuclear cells obtained from atopic asthmatics. Methods: Peripheral blood mononuclear cells (PBMC) isolated from house-dust-mite-sensitive bronchial asthmatics were cultured for 96 h in the presence or absence of 1 µg/ml Dermatophagoides farinae (Df) antigen. Eosinophils were isolated from peripheral blood of healthy subjects. Eosinophil-adhesion-inducing activity in the culture supernatants of PBMC was examined by the ability to modify the adhesion of eosinophils to human pulmonary microvascular endothelial cells (HPMEC) in the presence or absence of anti-cytokine/chemokine antibodies. Eosinophil migration induced by the supernatants was also examined. Results: Eosinophil adhesion to HPMEC was significantly augmented by the supernatants of Df-stimulated PBMC, which was significantly inhibited by anti-GM-CSF, but not by anti-IL-5, anti-RANTES, or isotype-matched controls. On the other hand, eosinophil migration induced by the supernatants was inhibited by anti-GM-CSF and partly by anti-RANTES. Conclusion: Both eosinophil adhesion and migration induced by the antigen-stimulated PBMC involve GM-CSF. In contrast, RANTES is involved only in the eosinophil migration. These molecules may participate in the development of eosinophil accumulation at the allergic inflammation sites.

Immunotherapy attenuates eosinophil transendothelial migration induced by the supernatants of antigen-stimulated mononuclear cells from atopic asthmatics.

Background: Eosinophil transendothelilal migration across vascular endothelial cells is an initial step of eosinophil accumulation in allergic inflammation. There is increasing evidence that specific immunotherapy (SIT) modulates the production of inflammatory molecules from mononuclear cells. Objective: The present study was undertaken to examine whether SIT modifies eosinophil transendothelial migration induced by the supernatants of antigen-stimulated mononuclear cells from atopic asthmatics. Methods:Dermatophagoides farinae(Df)-sensitive mild persistent asthmatics were divided into a SIT-treated group and a control group. Peripheral blood mononuclear cells (PBMC) were isolated before and after SIT using the rush protocol, and cultured for 96 h at 37°C in the presence or absence of Df antigen. Eosinophils were isolated from the blood of healthy subjects, and put on transwell filters coated with pulmonary microvascular endothelial cell monolayers stimulated with IL-4 plus TNF-α. The supernatants of PBMC were applied to the lower compartment and the transmigration of eosinophils was examined. Results:Df stimulation of PBMC resulted in an augmentation of eosinophil transendothelial migration. This enhancement was abrogated following SIT. In the control group, the antigen-induced effect on eosinophil transmigration did not show an interval change. Conclusion: SIT attenuates eosinophil transendothelial migration induced by antigen-stimulated mononuclear cells.