Aya Nakata

Dynamics of antigen-specific helper T cells at the initiation of airway eosinophilic inflammation

Bronchial asthma is characterized by chronic eosinophilic inflammation of the bronchial mucosa in which Th2 cells play crucial roles. Ovalbumin‐reactive Th2 clones were labeled with a fluorescent‐probe then infused into unprimed mice to elucidate the dynamics of antigen‐specific T cells involved in allergic inflammation. Infiltration of not only labeled antigen‐specific T cells, but alsounlabeled nonspecific CD4+ T cells into the bronchial mucosa following inhaled antigen challenge was detectable under confocal microscopy and flow cytometry. Accordingly, labeled T cells in the spleen were decreased, whereas those in hilar lymph nodes were increased upon antigen challenge. Approximately 45% of antigen‐specific T cells that migrated into the lungs bore CD25, while another early activation marker, CD69, was expressed on 80% of the migrated T cells. Accordingly, antigen challenge to the mice induced in situ proliferation of antigen‐specific T cells as well as bronchial epithelial cells in the lungs. Expression of vascular cell adhesion molecule (VCAM)‐1, but not intercellular adhesion molecule (ICAM)‐1, on the vascular endothelium in the lungs was enhanced following antigen challenge. Nevertheless, treatment with anti‐VCAM‐1 antibody, and also anti‐ICAM‐1 antibody strongly suppressed the accumulation of T cells, suggesting that both VCAM‐1 and ICAM‐1 are essential for antigen‐stimulated T cell mobilization into peripheral tissues. Our current study visualized the kinetics and the mechanism of antigen‐specific T cell migration in response to local challenge with a protein antigen.

Cloned Th cells confer eosinophilic inflammation and bronchial hyperresponsiveness.

Background: The essential role of Th cells and T cell cytokines in eosinophilic inflammation has been established. Methods: To determine whether Th cells are sufficient for the development of airway eosinophilic inflammation, ovalbumin–reactive murine Th clones were established and infused into unprimed mice. Results: Eosinophilic infiltration into the lung was induced upon antigen inhalation in parallel with the rise in bronchoalveolar lavage fluid (BALF) eosinophil peroxidase activity. Neither IgG, IgA, nor IgE antibodies were present in this model. Pathological examination showed swelling and desquamation of epithelial cells, mucous plugs, and goblet cell hyperplasia, all of which well resemble human asthma. Fluorescent probe labeled Th clones migrated into the lung prior to the eosinophil accumulation. Bronchial hyperresponsiveness (BHR) was clearly induced upon antigen inhalation. Anti–IL–5 monoclonal antibody abrogated the responses. Dexamethasone and cyclosporin A suppressed cytokine production by Th cells both in vitro and in vivo, BALF eosinophilia, and BHR. The number of eosinophils recovered in the BALF correlated with the intensity of BHR. Conclusion: The results clearly indicated that monoclonal Th cells are sufficient for the development of both airway eosinophilia and BHR. Agents capable of downregulating IL–5 production seem promising for the treatment of bronchial asthma.

Inhibition of Pulmonary Eosinophilia Does Not Necessarily Prevent the Airway Hyperresponsiveness Induced by Sephadex Beads

Background: The Lewis rat among highly inbred strains exhibits significant airway hyperresponsiveness (AHR) following intravenous administration of Sephadex G-200 (Sephadex). The aim of this study was to investigate the association of Sephadex-induced AHR with changes in airway inflammation in Lewis rats. Methods: A suspension (0.5 mg/ml/rat) of Sephadex was intravenously administered to male Lewis rats on days 0, 2 and 5. Measurement of airway responsiveness to serotonin, bronchoalveolar lavage (BAL) and histological study were performed on day 2–11. Results: Significant AHR induced by Sephadex was recognized on day 2 (p < 0.05), and AHR reached a maximum on day 7 (p < 0.001). In the BAL study, eosinophils increased on day 2 (p < 0.01) with a peak on day 5 (p < 0.05). In the histological study, we found Sephadex beads trapped in small arteries of the lung and granulomatous arteritis on day 2 or later. Pulmonary granulomas, horseshoe-shaped multinuclear giant cells, eosinophils and goblet cell hyperplasia were observed on day 2, and the degree became intense on day 5–7. GCC-AP0341 (10 mg/kg, i.p. × 3) inhibited the recruitment of eosinophils in BAL fluid and in lung tissue, but it did not inhibit AHR. The compound also inhibited pulmonary granulomas and goblet cell hyperplasia. Conclusion: The mechanism of Sephadex-induced AHR may not be directly associated with inflammatory changes such as recruitment of eosinophils, pulmonary granulomas and hyperplasia of goblet cells in rats.

Evidence that cyclosporin A and dexamethasone inhibit allergic airway eosinophilic inflammation via suppression of interleukin-5 synthesis by T cells

1 We have recently demonstrated that airway eosinophilic inflammation can be transferred to unprimed mice by infusing interleukin (IL)‐5‐producing T cell clones. Using that murine model, we performed this study to delineate the mechanism of cyclosporin A and dexamethasone to inhibit allergic airway eosinophilic inflammation. 2 The ovalbumin‐reactive murine T cell clones, FJ17, produced IL‐2, IL‐4 and IL‐5 upon stimulation with relevant antigen. In FJ17‐transferred mice, messenger RNA (mRNA) of IL‐2 and IL‐5 expressed in the lungs, the number of eosinophils in bronchoalveolar lavage fluid (BALF) was increased and the bronchial responsiveness to acetylcholine was enhanced after antigen provocation. 3 Cyclosporin A (10, 100 ng ml−1) and dexamethasone (10, 100 ng ml−1 suppressed the production of IL‐5 as well as IL‐2 and IL‐4 by FJ17 in vitro. 4 Subcutaneously administered cyclosporin A (30 mg kg−1) and dexamethasone (10 mg kg−1) inhibited antigen‐induced mRNA expression of IL‐2 and IL‐5, increase of BALF eosinophils and bronchial hyperresponsiveness of FJ17‐transferred mice in vivo. The number of BALF eosinophils was correlated with the bronchial responsiveness to acetylcholine (r = 0.672). 5 The results clearly indicated that the suppression of IL‐5 synthesis by T cells is involved in the effects of cyclosporin A and dexamethasone to inhibit allergic airway eosinophilic inflammation.

Facilitatory role of somatostatin via muscarinic cholinergic system in the generation of long-term potentiation in the rat dentate gyrus in vivo

We investigated whether somatostain modulates the generation of long-term potentiation (LTP) in rat perforant path-dentate gyrus synapse in vivo. When somatostatin was injected intracerebroventricularly (i.c.v.) 20 min prior to the tetanus, the intensity of LTP increased dose dependently. Synaptic potential evoked by a low-frequency test stimulation, however, was not altered by somatostatin. We next tested whether the LTP-augmenting effect of somatostain is mediated by cholinergic activation, because somatostatin was demonstrated to promote acetylcholine release in rat hippocampal slice. Pirenzepine (50 nmol/rat), a muscarinic M1 receptor antagonist, did not affect the tetanus-induced LTP by itself. But when it was co-applicated with the somatostatin (50 ng/rat) 20 min before tetanus, it completely abolished the LTP-augmenting effect of somatostatin. Then we examined the effect of octreotide, a potent agonist specifically binding to somatostatin receptor subtypes 2 and 4, on the generation of LTP. Octreotide (500 ng/rat) also facilitated the intensity of LTP. These results suggest that somatostatin facilitates the generation of perforant path-dentate gyrus granule cell LTP by activating the muscarinic cholinergic receptor and the effect of somatostatin is induced, at least partly, by somatostatin receptor subtypes 2 and 4 in vivo.

Cyclic AMP suppresses interleukin‐5 synthesis by human helper T cells via the downregulation of the calcium mobilization pathway

To delineate the mechanism by which cyclic AMP (cAMP) suppresses interleukin (IL)‐5 synthesis, the effects of prostaglandin (PG) E2, forskolin, dibutyryl (db)‐cAMP and the Ca2+ ionophore, ionomycin on cytokine synthesis, proliferation and CD25 expression of human T cells were investigated. Further studies were performed by measurement of the intracellular concentrations of cyclic AMP ([cAMP]i) and Ca2+ ([Ca2+]i) and by electrophoretic mobility shift analysis (EMSA). PGE2, forskolin and db‐cAMP suppressed IL‐5 production by human T cell line following T cell receptor (TCR)‐stimulation. PGE2 suppressed TCR‐induced messenger RNA (mRNA) expression of IL‐2, IL‐4 and IL‐5, as well as proliferation and CD25 expression. Cyclic AMP‐mediated suppression of cytokine synthesis, proliferation and CD25 expression in human T cells were attenuated by ionomycin. [cAMP]i was increased by PGE2 and forskolin. PGE2 suppressed the TCR‐induced biphasic increase in [Ca2+]i. EMSA revealed that four specific protein‐DNA binding complexes related to NF‐AT were detected at the IL‐5 promoter sequence located from −119 to −90 relative to the transcription initiation site. The slowest migrating complex induced by TCR stimulation was enhanced by PGE2 and further upregulated by ionomycin. Another binding which did not compete with cold AP‐1 oligonucleotides, was constitutively present and was unaffected by PGE2 but enhanced by ionomycin. The suppressive effect of cyclic AMP on human IL‐5 synthesis is mediated by interference with intracellular Ca2+ mobilization but distinct from the NF‐AT‐related pathway.

IL-5-Producing T Cells that Induce Airway Eosinophilia and Hyperresponsiveness Are Suppressed by Dexamethasone and Cyclosporin A in Mice

We have recently demonstrated that airway eosinophilic inflammation can be transferred to unprimed mice by infusion of IL-5-producing T cell clones. In this study, we investigated the effects of dexamethasone and cyclosporin A on the airway eosinophilic inflammation in mice transferred with T cell clones. An ovalbumin-reactive T cell clone, KW29, produced IL-5 as well as IL-2 and IL-4 upon stimulation with relevant antigen. Dexamethasone and cyclosporin A dose-dependently suppressed the production of these cytokines in vitro. The number of eosinophils recovered in the bronchoalveolar lavage fluid and the airway responsiveness to acetylcholine were increased in KW29-transferred mice after antigen provocation. Both responses were dose-dependently suppressed by the administration of dexamethasone or cyclosporin A in vivo. We concluded that airway eosinophilic inflammation can be controlled by agents capable of downregulating IL-5 production in T cells.

Synthesis and biological activities of 1-pyridylisoquinoline and 1-pyridyldihydroisoquinoline derivatives as PDE4 inhibitors.

A novel series of 1-pyridylisoquinoline and 1-pyridyldihydroisoquinoline derivatives has been prepared. These compounds showed potent PDE4 inhibitory activities and a broad margin between the K(i) value of the rolipram binding affinity and the IC(50) value of PDE4 inhibition. They also exhibited potent inhibitory activities toward LPS-induced TNF-alpha production in mice.

Antigen-Induced Airway Migration of a T Helper 2 Clone

Background: The crucial role of T helper (Th) 2 cells in allergic eosinophilic inflammation has been established. Methods: To determine the dynamics of Th2 cells in airway eosinophilic inflammation, ovalbumin-reactive murine Th2 clones were infused into unprimed mice. Results: The infused T cells were activated and migrated into the bronchial submucosa upon provocation with the relevant inhaled antigen. The T cell migration was followed by massive eosinophilic infiltration, edema, swelling of epithelial cells in the lung and a significant increase in bronchial responsiveness to acetylcholine. Conclusion: Clonal Th2 cells have the potential to accumulate and induce eosinophilic inflammation in the lung upon antigenic stimulation.