Keisuke Masuyama

A newly identified MAGE‐3‐derived epitope recognized by HLA‐A24‐restricted cytotoxic T lymphocytes

Five MAGE‐3‐derived peptides carrying an HLA‐A24‐binding motif were synthesized. Binding capacity of these peptides was analyzed by an HLA‐class‐I stabilization assay. Two of the 5 peptides bound to HLA‐A*2402 molecule with high affinity, and 3 peptides with low affinity. Peripheral‐blood mononuclear cells (PBMC) depleted of CD4+T cells were stimulated with the peptides to determine whether these peptides would induce cytotoxic T lymphocytes (CTL) from PBMCs obtained from 7 healthy HLA‐A*2402+ donors. Peptide M3‐p97 (TFPDLESEF; corresponding to amino‐acid residues 97–105 of MAGE‐3), with high binding capacity to the HLA‐A*2402 molecule, elicited the peptide‐specific and HLA‐A24‐restricted CD8+CTL lines in 2 of the 7 donors, while none of the 4 other peptides induced CTL specific for the corresponding peptide in any of the donors. CTL lines induced by stimulation with peptide M3‐p97 exhibited cytolytic activities against HLA‐A*2402 transfectant cell lines (C1R‐A*2402) in the presence of peptide M3‐p97, but not in unloaded or irrelevant peptide‐pulsed C1R‐A*2402 cells. The CTL lines and a cloned CD8+CTL isolated from one of the bulk populations by limiting dilution could lyse MAGE‐3+/HLA‐A*2402+ squamous‐cell‐carcinoma(SCC) lines but neither MAGE‐3−/HLA‐A*2402+ nor MAGE‐3+/HLA‐A*2402− SCC lines, indicating that M3‐p97 can be naturally processed and presented on the tumor‐cell surface in association with HLA‐A*2402 molecules. Combined with the 4 currently reported CTL epitopes derived from MAGE‐3 and presented by HLA‐A1, HLA‐A2, HLA‐A24 or HLA‐B44, identification of this CTL epitope presented by the HLA‐A*2402 molecule will extend the application of MAGE‐3‐derived peptides for immunotherapy for cancer patients. Int. J. Cancer 81:387–394, 1999.

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Histamine application to the nasal mucosa induces release of calcitonin gene-related peptide and substance P from peripheral terminals of trigeminal ganglion: A morphological study in the guinea pig

Short-term effects of application of histamine to the nasal mucosa on trigeminal ganglion neurons containing calcitonin gene-related peptide (CGRP) and substance P (SP) were examined in guinea pig. Immunoreactivities to CGRP and SP in these neurons were decreased 30 min after the histamine application. The decreases were most marked at 1-3 h after application, after which the immunoreactivities began to increase, reaching the base line by 6 h after the application. The immunoreactivities to CGRP and SP in the nerve endings of nasal mucosa were not decreased. The expression of mRNAs for both peptides in the soma of trigeminal neurons was unchanged. The histamine application to the nasal mucosa may cause release of CGRP and SP from terminals of peripheral processes of trigeminal ganglion neurons, and enhance axonal transport of these peptides, but does not affect their biosynthesis in the soma of trigeminal ganglion neurons.

Immunoregulatory Effect of Substance P in Human Eosinophil Migratory Function

Substance P (SP) is a tachykinin involved in the regulation of inflammatory processes. To investigate a modulatory role of the neuropeptide SP in allergic inflammation, we studied its priming effect on human eosinophil chemotaxis and kinetic responses towards platelet activating factor (PAF) and recombinant human interleukin 5 (rhIL-5). Blood was obtained from normal subjects and eosinophils were separated by Percoll discontinuous density gradient centrifugation. High purification was obtained by negative selection procedure (CD16-beads) and the experiments were performed in a 48-well microchemotaxis Boyden chamber. In the present study we demonstrate a potent synergistic effect of 1OOnM dose of SP on the migratory function of human eosinophils stimulated by PAF and rhIL- 5. This synergism was chemotaxis specific and was abolished by NK-1 receptor antagonist (FK888). The results suggest that neurogenic stimuli may play a significant role in eosinophil infiltration via its priming effect on the cell.

Adoptive Immunotherapy for Head and Neck Cancer with Killer Cells Induced by Stimulation with Autologous or Allogeneic Tumour Cells and Recombinant Interleukin-2

Peripheral blood lymphocytes drawn by leukapheresis using Haemonetics V50 were mixed and cultured with autologous or allogeneic tumour cell line to activate killer cells by tumour antigenic stimulation, and further with recombinant interleukin-2 (rIL-2). Killer cells were intra-arterially infused, as a primary therapy, in 5 patients with maxillary and one with lingual cancer (squamous cell carcinoma). Effects on reduction of primary tumour size were significantly high without any severe side effects. The effects were interpreted mainly by direct day-by-day observation of the site, findings of CT and histology. Histological findings of the tissue obtained by surgical operation performed after adoptive immunotherapy were remarkable changes, such as infiltration of lymphoid cells around the cancer nets, degeneration of cancer cells, infiltration of scavenger macrophages (giant cells) and so on. The results suggested that adoptive immunotherapy by the killer cells can be a powerful treatment to bring the cancer under control, in with combination of other therapies.

Human eotaxin induces eosinophil-derived neurotoxin release from normal human eosinophils

Background: Eosinophil granule proteins deposition at the site of allergic inflammation contributes to the late-phase reaction of hypersensitivity diseases. In the present communication, we describe the effect of human eotaxin on normal human eosinophil exocytosis measured as degranulation of eosinophil-derived neurotoxin (EDN). Methods: Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Purified eosinophils were stimulated with various concentrations of eotaxin and the amount of EDN released was analysed by radioimmunoassay. Flow cytometry was used to examine the surface expression of adhesion molecules on eosinophils. Results: Eotaxin significantly induced EDN release in a dose-dependent manner. The potency of eotaxin in this effect was equal to that of RANTES, and comparable to that of platelet-activating factor. Eotaxin-induced EDN release was blocked by cytochalasin B in a dose-dependent manner. The surface expression of CD11a, CD11b, CD18 and VLA-4 adhesion molecules on normal human eosinophils were not modulated by eotaxin stimulation. Conclusions: These results indicate that eotaxin may play an important role not only as a selective chemotaxin for the cell type but also as a secretagogue. Furthermore, they demonstrate a degranulation mechanism(s) involving cytoskeletal changes which is probably independent of the quantitative expression of adhesion molecules.

Mechanisms Involved in Activation of Human Eosinophil Exocytosis by Substance P: An in Vitro Model of Sensory Neuroimmunomodulation

Substance P (SP), a tachykinin with a wide range of biological activities including a priming effect on human eosinophil chemotaxis, was investigated for its influence on eosinophil cytotoxic function measured as degranulation of eosinophil-derived neurotoxin (EDN). Peripheral blood was obtained from healthy volunteers and the degranulation assays were performed using radioimmunoassay (RIA). SP and its C-terminal elicited EDN release in a time-dependent mode at a narrow range of doses with optimal activity of 10(-6) M. FK888 (NK-1 receptor antagonist) inhibited EDN release stimulated by SP in dose dependency, also a complete inhibition was observed when eosinophils were preincubated with 1000 ng/ml pertussis toxin (PTX). Pre-exposure of eosinophils to staurosporine resulted in blockage of SP-induced EDN release in a dose-dependent mode. On the other hand, SP at 10(-7) M and 10(-8) M primed eosinophils to suboptimal dose (10(-8) M) of Platelet activating factor (PAF) resulting into significant enhancement of EDN release. SP(4-11) fragment showed a similar activity while SP(1-4) fragment was not active. SP priming of eosinophils was not affected by Ca2+ depletion, however, it caused a change in the pattern of the intracellular calcium influx against the suboptimal dose of PAF. These results suggest that SP i) may induced human eosinophil matrix protein degranulation through a receptor mediated mechanism coupled to PTX sensitive G protein(s) with the probability of linkage to phospholipase C activation, and, ii) primes human eosinophils for an exalted inflammatory response through a Ca2+ independent pathway.

Expression of the MAGE-1, -2, -3, -4, and -6 Genes in Non-Squamous Cell Carcinoma Lesions of the Head and Neck

The messenger RNA level of several MAGE genes, some of which have been proven to encode tumor rejection antigens recognized by cytotoxic T lymphocytes, were examined in 41 benign and malignant lesions of the head and neck region. By a reverse transcription-polymerase chain reaction assay and Southern blot hybridization, MAGE-1, -2, -3, -4, and -6 genes were expressed in 25%, 41.7%, 33.3%, 8.3% and 33.3% of 12 non-squamous cell carcinomas, respectively. These tumors consisted of 6 papillary adenocarcinomas, 3 adenoid cystic carcinomas, 2 adenocarcinomas, and 1 mucoepidermoid tumor. Of 7 non-Hodgkins lymphomas, one case from the oropharynx and 2 from the nasopharynx expressed for the MAGE-1 and MAGE-2 genes, respectively. In contrast, none of 12 benign tumors expressed any of these MAGE genes. Interestingly, of 10 other lesions including hyperplasia, keratosis, and ulcer, one histologically diagnosed as dysplasia expressed the MAGE-2, -3, -4, and -6 genes. These results suggest that the MAGE genes may be expressed in malignant tumors and precancerous lesions but not in benign tumors. In addition, non-squamous cell carcinomas may be suitable targets for specific immunotherapy against MAGE gene products.

Inhibition of Human Eosinophil Chemotaxis in Vitro by the Anti-Allergic Agent Emedastine Difumarate

Emedastine difumarate (emedastine), an anti-allergic agent with anti-histaminic properties, was studied for its effect on human eosinophil chemotaxis induced by platelet activating factor (PAF). Peripheral blood eosinophils (98% purity) were obtained from healthy donors and chemotaxis assay were performed in microchemotaxis chambers. Emedastine showed a significant inhibitory effect on 10(-6) M PAF-induced eosinophil chemotaxis, in dose dependent fashion, at concentrations from 10(-6) to 10(-8) M. Conversely, no inhibitory effect was observed on human neutrophil chemotaxis. Pretreatment of eosinophils with Pyrilamine did not affect PAF-induced eosinophil chemotaxis. Thus emedastine appears to possess a potent and selective inhibitory effect on eosinophils chemotaxis, an action which is probably unrelated to its anti-histamine properties.

Modulation of Normal Human Eosinophil Chemotaxis in vitro by Herbimycin A, Erbstatin and Pervanadate

Background: The mediators involved in eosinophil accumulation in diseases such as allergy continue to be an area of interest, even though little is known regarding the signaling involved in the human cell type recruitment. In the present study, we demonstrate a novel modulatory role of tyrosine kinase and tyrosine phosphatase activities on normal human eosinophil chemotaxis induced by different groups of chemoattractant. Methods: Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Chemotactic activities against platelet-activating factor (PAF), vasoactive intestinal peptide (VIP) and eotaxin were assessed using a 48-well microchemotaxis chamber assay. Purified eosinophils were pretreated with herbimycin A, erbastatin or pervanadate to examine the role of tyrosine kinase in chemoattractant signaling. Results: Pretreatment of eosinophils with the tyrosine kinase inhibitors herbimycin A and erbstatin significantly blocked chemotaxis induced by eotaxin whilst both inhibitors augmented chemotaxis induced by VIP; however, they had no effect on PAF-induced chemotaxis. On the other hand, pretreatment of eosinophils with the phosphotyrosine phosphatase inhibitor pervanadate resulted in augmentation of eotaxin-induced chemotaxis and inhibition of VIP-induced chemotaxis, but it had no effect on PAF-induced chemotaxis. Conclusions: These results suggest that protein kinase plays a modulatory role in eosinophil chemotaxis induced by various chemoattractants.