Hisao Tomioka

T cell receptor repertoire of infiltrating T cells in lips of Sjögren's syndrome patients.

Infiltrating T cells around salivary glands in the lips of Sjögrens syndrome (SjS) patients are crucial in the pathogenesis of this disease. To analyze the nature of infiltrating T cells, their T cell receptor repertoire was examined with quantitative polymerase chain reaction. The repertoire of V beta transcripts in lips of SjS was not restricted; however, the V beta 2 and V beta 13 genes were predominantly expressed on the T cells of lip specimens in six and four of seven lips, respectively. Predominance of these genes was specific in lips because no predominant V beta transcripts were found in lips from healthy subjects and PBLs from SjS patients. These results indicated that the V beta 2- and V beta 13-positive T cells expanded specifically and preferentially in SjS lips, thereby suggesting the possible role in triggering the autoimmunity of this disease.

IL-5 but not interferon-gamma (IFN-gamma) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression.

In order to determine regulatory mechanisms of eosinophil apoptosis, we examined the effect of recombinant IL‐5 and interferon‐gamma (IFN‐γ) on eosinophil apoptosis and bcl‐2 expression. rhIL‐5 (2.5u2003ng/ml) significantly inhibited eosinophil apoptosis in 96u2003h in vitro culture compared with medium only‐cultured eosinophils (89.4u2003±u20033.6% versus 31.3u2003±u200312.2% (meanu2003±u2003s.d.); nu2003=u20037, Pu2003<u20030.05). Further, rhIL‐5 significantly increased bcl‐2 protein and mRNA expression on cultured eosinophils. A phosphorothioate antisense oligonucleotide targeted at the ATG translation initiation codon of bcl‐2 (10−5u2003m) could significantly block the supportive effect of rhIL‐5 (0.25u2003ng/ml) for eosinophil survival compared with sense cDNA of bcl‐2 on 96u2003h culture (inhibition rate 28.01u2003±u20034.56% versus 0.07u2003±u20031.73%; nu2003=u20034, Pu2003<u20030.05). In contrast, rhIFN‐γ (100u2003U/ml) significantly inhibited eosinophil apoptosis on 96u2003h in vitro culture (72.7u2003±u200310.5%; nu2003=u20037, Pu2003<u20030.05), but did not significantly up‐regulate bcl‐2 protein and mRNA. These results indicate that IL‐5 has inhibitory effects on eosinophil apoptosis by regulation of bcl‐2 expression.

Human peripheral eosinophils express functional interferon‐gamma receptors (IFN‐γR)

In order to determine whether or not IFN‐γR is associated with regulatory mechanisms on human eosinophil function, we examined the expression of functional IFN‐γR on human peripheral eosinophils. In this study, peripheral blood eosinophils were obtained from seven normal controls and 12 patients (bronchial asthma, nu2003=u20039, and hypereosinophilic syndrome (HES), nu2003=u20033), and the purity of eosinophils was 97.11u2003±u20032. 31%, nu2003=u200319. We first showed that anti‐IFN‐γR α‐chain MoAb reacted with all tested eosinophils of both normal controls and patients by flow cytometry analysis. We also showed expression of mRNA for the α‐chain of IFN‐γR in all purified eosinophils of six individuals. Further, to characterize IFN‐γR on eosinophils, we did binding experiments with 125I‐IFN‐γ on purified peripheral eosinophils. The linear Scatchard plot indicated a single type of high‐affinity binding sites (dissociation constant (Kd)u2003=u20033.89–4.95u2003×u200310−10u2003M, numbers of binding sitesu2003=u2003183–233/cell, nu2003=u20033). To determine whether IFN‐γR on eosinophils is functional, we examined surface eosinophilic cationic protein (ECP) and CD69 induction after IFN‐γR ligation with recombinant human IFN‐γ (rhIFN‐γ) on eosinophils by flow cytometry. rhIFN‐γ stimulation significantly induced both ECP and CD69 expression on the 2–18u2003h‐cultured eosinophils in a dose‐dependent manner. Further, the effects of rhIFN‐γ stimulation were significantly blocked by both a neutralizing anti‐IFN‐γ MoAb and a blocking anti‐IFN‐γR MoAb. These results suggest that human peripheral eosinophils express functional IFN‐γR.

Role of JAK2 signal transductional pathway in activation and survival of human peripheral eosinophils by interferon-gamma (IFN-γ)

The purpose of this study was to determine whether the JAK pathway is involved in eosinophil activation and survival through IFN‐γ receptor signalling in human peripheral eosinophils. Eosinophils were purified from the blood of six atopic disease patients by anti‐CD16 magnetic bead‐negative selection. IFN‐γ significantly up‐regulated survival and CD69 expression in 24–48u2003h cultured eosinophils. Further, IFN‐γ induced tyrosine phosphorylation of JAK2 in eosinophils, as indicated by Western blot analysis. Finally, the specific JAK2 inhibitor AG‐490 inhibited the tyrosine phosphorylation of JAK2, IFN‐γ‐induced survival and CD69 expression in eosinophils. In conclusion, these results indicate that IFN‐γ induces eosinophil survival and CD69 expression through the activation of JAK2 in peripheral eosinophils, suggesting that JAK2 may play a significant role in eosinophil regulation by IFN‐γ–IFN‐γR interaction.

Substance P-induced granulocyte infiltration in mouse skin : the mast cell-dependent granulocyte infiltration by the N-terminal peptide is enhanced by the activation of vascular endothelial cells by the C-terminal peptide

Previous studies have shown that substance P induces granulocyte infiltration in mouse skin, which is mediated through mast cell degranulation. However, it is not yet known whether the direct effect of substance P on vascular endothclial cells is involved in the granulocyte infiltration in the skin. To solve this issue, we used the N‐terminal peptide substance P1–9 (SP1–9), which is active for mast cells but inactive for vascular endolhelial cells, and the c‐terminal peptide SP6–11, which is active for vascular endothelial cells but inactive for mast cells, since substance P activates both mast cells and vascular endothelial cells. The subcutaneous administration of substance P (10−7−10−5 m) caused granulocyte (neutrophil and eosinophil) infiltration in the skin of BALB/c mice 6 h after the injection. SP1–9 (10−5–10−4 m) also caused granulocyte infiltration of mouse skin which was associated with mast cell degranulation. In contrast. SP6–11 (10−7‐10−4 M), which was found to increase the vascular permeability of endothelial cells in mouse skin, induced no significant granulocyte infiltration nor mast cell degranulation. However, SP6–11 (10−5 10−4 m) enhanced SP1–9 ‐induced granulocyte infiltration in the skin without any significant increase in mast cell degranulation. We conclude that substance P causes granulocyte infiltration in mouse skin through both mast cell degranulation induced by the N‐lerminal peptide of substance P and the activation of vascular endothelial cells induced by the c‐terminal peptide of substance P.

Preventive effects of an antiallergic drug, pemirolast potassium, on restenosis after percutaneous transluminal coronary angioplasty

BACKGROUNDnWe recently confirmed that pemirolast potassium, an antiallergic agent, markedly inhibits migration and proliferation of vascular smooth muscle cells. It has also been reported that pemirolast inhibits intimal hyperplasia in animal experiments.nnnMETHODS AND RESULTSnTo elucidate the preventive effects of pemirolast on restenosis after percutaneous transluminal coronary angioplasty (PTCA), 227 patients were enrolled in this prospective, randomized trial. A total of 205 patients who were compatible with the protocol were analyzed (pemirolast group, 104 patients with 140 lesions; control group, 101 patients with 133 lesions). Patients in the pemirolast group received 20 mg/d of pemirolast from 1 week before PTCA until the time of follow-up angiography (4 months after PTCA). Angiographic restenosis was defined as diameter stenosis >/=50% at follow-up. Restenosis rates were significantly lower in the pemirolast group than in the control group (24.0% vs 46.5% of patients, 18.6% vs 35.3% of lesions, P <.01, respectively). During 8 months of follow-up, there were no coronary events (death, myocardial infarction, coronary artery bypass surgery, or repeated PTCA) in 81.7% of the pemirolast group and in 63.4% of the control group (P =.013).nnnCONCLUSIONSnThis study suggested that pemirolast would be useful in the clinical setting to prevent restenosis after PTCA.

Computer-assisted instructions for patients with bronchial asthma

We produced computer-assisted instruction (CAI) software for bronchial asthma patients (asthma educational system with computer-assisted instruction; ASTCAI) to assist in self-management and avoid asthmatic attacks and death. ASTCAI is a question-and-answer program operating in a multimedia environment, and was evaluated from questionnaires which 33 patients were asked. Thirty-two patients could perform ASTCAI without any assistance. The responses of 31 patients (94%) indicated that they had no difficulty with manipulation, and 29 patients (88%) stated that the program was beneficial to control of their asthma. Elderly patients (over 65) required more time than younger adults. Emergency visits or admissions of at least 1 year after the first CAI trial decreased in eight out of 26 patients, while only two patients deteriorated compared to the previous year. Our results show that CAI is feasible for most patients, and through active self-learning CAI can improve motivation for self-management as well as supplement the physicians instructions.

Comparison of substance P-induced and compound 48/80-induced neutrophil infiltrations in mouse skin

It has recently been shown that substance P induces neutrophil infiltration in the skin, which is mediated through mast cell degranulation. Since substance P activates both skin mast cells and vascular endothelial cells, we compared the potencies of substance P and a mast cell-degranulating agent, compound 48/80, which is inactive for vascular endothelial cells, in inducing neutrophil infiltration in mouse skin. We also examined the effect of the C-terminal peptide of substance P, SP6-11, which is active for vascular endothelial cells, on compound 48/80-induced neutrophil infiltration in the skin. Subcutaneous administrations of substance P (10(-7) to 10(-5) M; 0.1 ml) and compound 48/80 (0.5-50 micrograms/ml) induced neutrophil infiltrations and mast cell degranulations in mouse skin in a concentration-dependent fashion. Moreover, substance P induced more neutrophil infiltrations than compound 48/80 in terms of the magnitude of mast cell degranulations. SP6-11 (10(-6) to 10(-4) M) induced no significant neutrophil infiltration or mast cell degranulation, but increased the vascular permeability of endothelial cells in the skin. Furthermore, SP6-11 enhanced compound 48/80-induced neutrophil infiltration without any increase in mast cell degranulation. Our results indicate that, in addition to mast cell degranulation, the activation of vascular endothelial cells is involved in substance P-induced neutrophil infiltration in the skin.

Short-term administration of anti-L3T4 MoAb prevents diabetes in NOD mice.

We treated 2‐week‐old and 8‐week‐old non‐obese diabetic (NOD) mice with 1 mg of anti‐L3T4 MoAb weekly for 4 weeks. This short‐term treatment of anti‐L3T4 MoAb prevented the development of overt diabetes in NOD mice, in both groups, even after cessation of the therapy. However, there were overt mononuclear cell infiltrations in the majority of islets, and no appreciable differences in the degree of insulitis between treated and control mice. There were also no significant differences in the percentage of L3T4+ T cells expressing Vβ5, Vβ8 and Vβ11 antigens between the treated and the control group. In contrast, most of male NOD mice injected with 200 mg/kg of cyclophosphamide did not become diabetic when the spleen cells from the MoAb‐treated female NOD mice were transferred to these animals 48 h before the cyclophosphamide injection. Thus, the tolerance induced by the short‐term administration of anti‐L3T4 MoAb to NOD mice may not be due to clonal deletion, but rather to newly generated suppressor cells in the animals.

Inhibitory Effect of Pemirolast, a Novel Antiallergic Drug, on Leukotriene C4 and Granule Protein Release from Human Eosinophils

To determine whether pemirolast, a new antiallergic drug, inhibits the activation of eosinophils, we investigated the effect of pemirolast on the release of leukotriene C4 (LTC4) and eosinophil cationic protein (ECP) from human eosinophils. Calcium ionophore A23187 caused both LTC4 and ECP release from human eosinophils, whereas PAF and FMLP induced only ECP release from the eosinophils. Pemirolast (10(-6) to 10(-3) M) inhibited A23187-induced LTC4 release from the eosinophils in a dose-dependent fashion with 77% inhibition at 10(-3) M. Pemirolast (10(-5) to 10(-3) M) inhibited A23187-induced ECP release from the eosinophils in a dose-dependent fashion with 42% inhibition at 10(-3) M. Pemirolast (10(-4) and 10(-3) M) also inhibited PAF-induced and FMLP-induced ECP release from the eosinophils. We conclude that pemirolast prevents the activation of human eosinophils to inhibit LTC4 and ECP release. These results suggest that pemirolast might be useful in controlling allergic diseases by inhibiting eosinophil activation.