Hiroaki Inamura

Expression of the Interleukin-8 Receptors CXCR1 and CXCR2 on Cord-Blood-Derived Cultured Human Mast Cells

Background: An increase in mast cell number at sites of inflamed tissues has been observed. However, the expression of CXC chemokine receptors on human mast cells is poorly understood. Methods: Cultured human mast cells were raised from human umbilical cord blood cells in the presence of stem cell factor and interleukin-6 (IL-6). The expression of surface chemokine receptors on the mast cells was analyzed by flow cytometry and that of mRNA was examined by the method of reverse transcriptase-polymerase chain reaction (RT-PCR). As functional assays for the receptors, mast cell migration was examined by a microchemotaxis assay and changes in the cytosolic free intracellular calcium concentrations ([Ca2+]i) was measured using fura-2-loaded mast cells, respectively. Results: Expression of IL-8 receptors CXCR1 and CXCR2 was demonstrated by flow cytometry and of both mRNA by RT-PCR; however, CC chemokine receptors including CCR3 were not expressed on cord-blood-derived cultured human mast cells. IL-8 and its homologues showed chemotactic activity toward them in a dose-dependent manner, and IL-8 induced a dose-dependent rapid and transient increase in [Ca2+]i in the mast cells. Conclusions: Our results suggest the surface expression of functional CXCR1 and CXCR2 on cord-blood-derived cultured human mast cells.

Quantitative Analysis of Mast Cells in Benign and Malignant Breast Lesions

Background: It has been reported that the number of mast cells was significantly greater in malignant breast carcinomas than in benign breast lesions. This was due to tryptase-containing mast cells while tryptase, chymase-containing mast cells had no effect. However, analysis of mast cells in breast carcinomas and benign breast lesions based on their histological findings remains to be elucidated. Methods: Using immunohistochemical methods morphological examinations of mast cells were undertaken in benign and malignant breast tissues from 51 patients (30 benign, 21 malignant), which were formalin-fixed and paraffin-embedded. In the study with malignant breast tissues, samples of malignant tissues and adjacent healthy tissues were obtained from a single patient, and the number of mast cells was compared. Results: Among benign breast tissues, the number of mast cells in intracanalicular fibroadenoma was significantly lower than that in pericanalicular fibroadenoma as well as that in mastopathy. The number of mast cells was significantly greater in malignant lesions than that in benign lesions. The number of mast cells in scirrhous carcinoma and that in solid-tubular carcinoma were significantly increased compared with that in adjacent healthy tissues. In addition, the number of mast cells in scirrhous carcinoma was highest among breast carcinomas, and significantly greater than that in papillotubular carcinoma. Conclusion: We were the first to find the significant lower number of mast cells in intracanalicular breast fibroadenoma when compared with that in pericanalicular fibroadenoma as well as that in mastopathy. Moreover, the number of mast cells in scirrhous carcinoma was significantly greater than that in papillotubular carcinoma.

Thromboxane A2 receptor +795T>C and chemoattractant receptor-homologous molecule expressed on Th2 cells -466T>C gene polymorphisms in patients with aspirin-exacerbated respiratory disease.

It is well known that aspirin-exacerbated respiratory disease (AERD) is more common in women than in men, however, whether gene polymorphisms of the thromboxane A2 receptor (TBXA2R) and chemoattractant receptor-homologous molecules expressed on Th2 cells (CRTH2) are associated with the susceptibility of AERD remains unknown. In this study, we examined the gene polymorphisms in a Japanese population. DNA specimens were obtained from the following three groups: 96 patients with AERD, 500 patients with aspirin-tolerant asthma (ATA) and 100 normal controls. The target DNA sequence of each gene was amplified, and an allelic discrimination assay for single nucleotide polymorphisms relating to expression of each gene was carried out. The frequencies of the CC/CT genotype of TBXA2R +795T>C were higher than those of the TT genotype in AERD patients compared to ATA patients (P=0.015). In female AERD patients, but not in males, frequencies of the CC/CT genotype were higher than those of the TT genotype of TBXA2R +795T>C compared to female ATA patients (P=0.013). Also, frequencies of the TT genotype of CRTH2 -466T>C were higher than those of the CC/CT genotype in AERD patients compared to ATA patients (P=0.034). In female AERD patients, but not in male, frequencies of the TT genotype were higher than those of the CC/CT genotype of CRTH2 -466T>C in AERD patients compared to female ATA patients (P=0.046). Based on our investigations, no significant relationship was found between the genotype and the clinical characteristics according to these gene polymorphisms in AERD patients. Our results suggest that an association between the TBXA2R and CRTH2 gene polymorphisms with AERD may exist in the Japanese population.

Arg16Gly β2-Adrenergic Receptor Gene Polymorphism in Japanese Patients with Aspirin-Exacerbated Respiratory Disease

Background: There has been no report that investigated β2-adrenergic receptor (ADRB2) gene polymorphism in patients with aspirin-exacerbated respiratory disease (AERD). Methods: DNA in the specimens in three groups of study subjects classified patients with AERD, patients with aspirin-tolerant asthma (ATA) and normal controls was extracted, and the target DNA sequence of the ADRB2 was amplified using a set of primers to generate an amplicon of 219 bp in length. Allelic discrimination assay for single nucleotide polymorphisms relating to the ADRB2 gene expression was carried out by using a previously described single nucleotide polymorphism detective system, sequence-specific thermal-elution chromatography. Results: The frequency of the Gly variant allele in patients with AERD was significantly lower than that in patients with ATA (p = 0.007), and the odds ratio (OR) of AERD to ATA associated with wild-type ArgArg homozygote was 3.300. Frequencies of wild-type ArgArg homozygote are significantly higher than those of variant-type ArgGly/GlyGly genotype in patients with AERD compared with those with ATA (p < 0.001, OR = 3.153). In patients with AERD, frequencies of wild-type ArgArg homozygote in both female and male patients are significantly higher than those of variant-type ArgGly/GlyGly genotype in male patients compared with those with ATA (p < 0.001, OR = 5.128 and p = 0.007, OR = 4.367, respectively). Also, in patients with AERD, frequencies of wild-type ArgArg homozygote in female patients are significantly higher than those of variant-type ArgGly/GlyGly genotype in female patients compared with those with ATA (p = 0.002, OR = 2.825). Conclusions: We were the first to analyze Arg16Gly ADRB2 gene polymorphism in Japanese patients with AERD, and showed that Arg16Gly ADRB2 gene polymorphism in Japanese patients with AERD is different from that in the patients with ATA.

Incorporation of radioactive phosphate from adenosine triphosphate into phosphatidylinositol-4-phosphate in human peripheral blood hypodense eosinophils is less than in the normodense eosinophils.

Peripheral blood eosinophils from patients with atopic dermatitis and normal healthy controls were isolated on a Percoll gradient and incubated with [gamma 32P]ATP in the presence of Mg2+. After stopping the reaction, lipid extraction was performed with acidic medium and phospholipids were separated by thin-layer chromatography on 1% (w/v) oxalic acid-impregnated silica gel plates. In the autoradiographs considerable amounts of the radioactivity were found to be incorporated into phosphatidylinositol-4-phosphate. 32P incorporation into phosphatidylinositol-4-phosphate in hypodense eosinophils from atopic dermatitis patients was less than in normodense eosinophils from the same patients and controls.