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Biomedical subjects

K Matsushima

Publications and source records attributed to K Matsushima.

At least 19 recordsLinked to original sources
PubMed1992

Tumor necrosis factor alpha and interferon gamma synergistically induce interleukin 8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-kB-like binding sites of the interleukin 8 gene.

Interleukin 8 (IL-8) is a novel cytokine which possesses neutrophil chemotactic and activating activities in addition to chemotactic activity for basophils and T lymphocytes. It has been shown that IL-8 is produced by a variety of human somatic cells including monocytes/macrophages, dermal fibroblasts, vascular endothelial cells, keratinocytes, mesangeal cells, and several types of tumor cell lines. We have examined here whether or not human gastric cancer cell lines produce IL-8 in vitro. The production of IL-8 protein was detected by enzyme-linked immunosorbent assay in the culture supernatants derived from eight of nine human gastric cancer cell lines stimulated with either interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), or TNF alpha plus interferon gamma (IFN gamma). In some of the gastric cancer cell lines such as MKN 45 and KATO, TNF alpha plus IFN gamma synergistically induced the production of IL-8. In MKN 45 cells, synergistic increase of the steady state level of IL-8 mRNA by TNF alpha plus IFN gamma was not inhibited by cycloheximide treatment. Scatchard analysis revealed that IFN gamma changed neither the number nor the affinity constant of TNF alpha binding sites on a gastric cancer cell line, suggesting that the synergism was a post-receptor event. Furthermore, synergistic induction of chloramphenicol acetyltransferase activity by TNF alpha plus IFN gamma was observed in MKN 45 that were transiently transfected with chimeric chloramphenicol acetyltransferase reporter genes driven by the transcriptional regulatory region of human IL-8 gene. Through the mutation of the regulatory region of the IL-8 gene, both AP-1- and NF-kB-like factor binding elements were presumed to be involved in conferring the responsiveness to TNF alpha plus IFN gamma. Moreover, gel retardation analyses revealed that TNF alpha and IFN gamma synergistically induced the binding of NF-kB like as well as AP-1 like proteins bound to these sites. These results indicated that IFN gamma synergistically enhanced TNF alpha-induced IL-8 production in a human gastric cancer cell line through synergistic activation of transcription factors without up-regulating TNF alpha receptor.

Base Sequence↗
PubMed1992

A sensitive enzyme-linked immunosorbent assay for human interleukin-8.

In order to quantify human interleukin-8 (IL-8), which is chemotactic for T cells and basophils as well as neutrophils, we developed an enzyme-linked immunosorbent assay (ELISA). Since binding inhibition tests indicated that three monoclonal antibodies (mAbs; BS-1, WS-4, WS-6) blocked the binding of 125I-labelled IL-8 to neutrophils, we tested an ELISA using these mAbs as primary antibodies, rabbit anti-IL-8 Ab as the secondary antibody, and alkaline phosphatase-labelled goat anti-rabbit Ab as the conjugate. Among the three mAbs tested, WS-4 was the most sensitive with a detection limit of 16 pg/ml. Several other cytokines, including monocyte chemotactic and activating factor (MCAF), which is structurally related to IL-8, showed no cross-reactivity in this system, indicating that this ELISA is specific for IL-8. The coefficients of variation for the intra- and interassays were below 10%. Furthermore, this ELISA also detected natural IL-8 (including both 72 and 77 amino acid forms) produced by cultured human cells and cell lines stimulated with IL-1, suggesting that this system will be useful in the detection of natural IL-8 in various body fluids.

Antibodies, Monoclonal↗
PubMed1992

Cloning of 67-kDa laminin receptor cDNA and gene expression in normal and malignant cell lines of the human lung.

Cell-adhesive protein laminin and its specific receptor play an important role in the processes of cancer proliferation, invasion and metastasis. In the present study, we cloned the cDNAs of the 67-kDa laminin receptor both from a human lung cell line (IMR90) and from a human lung cancer cell line (SBC3), and determined the nucleotide sequences. In comparison with both cDNA sequences of the protein-coding region, three nucleotide differences were found. These differences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67-kDa-laminin receptor mRNA was higher in SBC3 than in IMR90.

Amino Acid Sequence↗
PubMed1992

Increased expression of the 67kDa-laminin receptor gene in human small cell lung cancer.

Gene expression of the precursor of the 67kDa-laminin receptor was examined by Northern analysis using 11 established human lung cancer cell lines and 25 lung cancer tissues obtained by operation. As a result, one transcript, the size of which was 1.2 kb was shown in all cell lines and tissues examined. An increased level of mRNA was demonstrated in cell lines which proliferated rapidly and in small cell lung cancer cell lines. It was also indicated that gene expression of the laminin receptor was up-regulated especially in small cell lung cancer tissue. In this context, 67kDa-laminin receptor appears to be a marker for biological aggressiveness of human lung cancer.

Adenocarcinoma↗
PubMed1992

A comparative study of the neutrophil stimulatory activity in vitro and pro-inflammatory properties in vivo of 72 amino acid and 77 amino acid IL-8.

IL-8, a potent neutrophil-activating protein, can be produced by many cell types including monocytes, lymphocytes, fibroblasts, neutrophils, and endothelial cells. Depending on the cell source, the N-terminal amino acid sequence of IL-8 displays heterogeneity that has been shown to confer differences in its neutrophil stimulatory activity in vitro. Despite these observations the relative potency of different IL-8 molecules in vivo is unknown. To address this question we have investigated the biologic activity of the two predominant forms of IL-8, the 72 and the 77 amino acid proteins, in vitro and in vivo. In vitro, human rIL-8(72) and human rIL-8(77) dose dependently induced adherence of rabbit peritoneal neutrophils and human neutrophils to laminin-coated plates and elevated cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2 loaded neutrophils. In these in vitro assays human rIL-8(72) was more potent than human rIL-8(77) while inducing comparable responses to human rC5a. With respect to enhancing [Ca2+]i, neutrophils desensitized to human rIL-8(72) failed to respond to human rIL-8(77). However, neutrophils fully desensitized to human rIL-8(77) could exhibit a partial response to human rIL-8(72). Further, human rIL-8(72) was approximately 10-fold more effective than human rIL-8(77) in displacing human [125I]rIL-8(72) from rabbit peritoneal neutrophils in a receptor-binding assay. In vivo, intradermally administered human rIL-8(72) and human rIL-8(77) induced 111In-neutrophil accumulation and edema formation in rabbit skin. In contrast to the in vitro studies, the two forms of IL-8 gave identical responses in vivo although they were less potent than human rC5a. Our results demonstrate that, in vitro, human rIL-8(72) is more potent than human rIL-8(77) in stimulating neutrophils. It may be that IL-8)72) has a greater affinity and/or efficacy for the neutrophil IL-8 cell-surface receptors. One possibility for the observation that both forms of IL-8 are equipotent in inducing inflammatory responses in vivo is that the extended form is proteolytically cleaved to the more biologically active IL-8(72).

Animals↗
PubMed1992

Interleukin-8 is constitutively and commonly produced by various human carcinoma cell lines.

We examined the production of interleukin-8 and interleukin-6 by 30 human carcinoma cell lines. Serum levels of interleukin-8 were measured in 14 patients with hepatocellular carcinoma by enzyme-linked immunosorbent assay and Northern blotting. Furthermore, serum interleukin-8 was also investigated in a nude mouse bearing a tumor of the HuH7 hepatoma cell line producing interleukin-8. Of the 30 cell lines, 29 (96.7%) constitutively produced interleukin-8, and 19 of the 29 (65.5%) were high producers (> 1 ng/ml culture supernatant). Among the high producers, 4 cell lines released both interleukin-8 and interleukin-6. Interleukin-6 was constitutively produced by 17 of the 30 (56.7%) cell lines, 4 of which (23.5%) were high producers (> 1 ng/ml). By Northern blot analysis, mRNAs of interleukin-8 and interleukin-6 were detected in producing cell lines. Of 14 patients with hepatocellular carcinoma 4 (28.5%) showed increased levels of serum interleukin-8. Furthermore, inoculation of the HuH7 hepatoma cell line which produced the highest amount of interleukin-8 into a nude mouse resulted in tumor production accompanied by an elevated level of human interleukin-8 (646 pg/ml) in the peripheral blood. Thus, interleukin-8 is constitutively and commonly produced by various carcinoma cell lines. The production of interleukin-8 by carcinoma cells may be related to the elevation of serum interleukin-8 in patients with hepatocellular carcinoma. Finally, these cell lines may be valuable for studying the relationship between interleukin-8 and cancer.

Animals↗
PubMed1992

Human polymorphonuclear leucocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/18 complex.

The present study demonstrates that tumour necrosis factor (TNF) and FMLP, but not IL-1 or IL-8, enhanced the adherence of polymorphonuclear neutrophil (PMN) to fibronectin, an extracellular matrix protein. The adherence induced by FMLP was very rapid, within 5 min while the induction of adherence by TNF was much slower, reaching maximum at 60 min. TNF also enhanced an adhesion of PMN to other extracellular matrix proteins, such as laminin, collagen IV and gelatin II, but not to human serum albumin. Anti-CD18 MoAb completely inhibited the binding of TNF-stimulated PMN to fibronectin and partially inhibited the binding to laminin. Further investigation showed that adhesion of TNF-stimulated PMN to fibronectin and laminin was inhibited by anti-CD11b MoAb and to a lesser extent by CD11a MoAb. In contrast to TNF-stimulated PMN the binding of unstimulated PMN to fibronectin and laminin was only inhibited by anti-CD11a MoAb. Anti-CD11c had no effect on PMN adherence. These results suggest that unstimulated PMN adhere to extracellular proteins through the CD11a/18, while TNF-stimulated PMN adhere through the CD11b/18. These results suggest that TNF secreted at the site of inflammation may enhance the interaction of PMN with the extravascular environment through the CD11b/18 complex.

Antigens, CD↗
PubMed1992

[Acute hepatitis A (HA) presenting findings of meningoencephalitis].

A 39-year-old man, who had high grade fever and headache for 4 days was admitted to our hospital because of generalized seizure and disturbance of consciousness. He was pyrexial, but not icteric. Neurological examination revealed disorientation, nuchal rigidity and bilateral Babinski reflexes. Laboratory test results included the following: GOT 1,740 U/l, GPT 2,800 U/l, bilirubin 1.2 mg/dl, serum IgM-HA antibody cut-off index 6.8. CSF was clear, with 10 leukocytes/mm3 and protein level of 108 mg/dl. Head CT and MRI revealed no abnormality. An EEG demonstrated diffuse slowing. During the following 2 days, he had increased obtundation and labored breathing. In the second week of hospitalization his neurological conditions and liver function test results improved. A diagnosis of HA was confirmed by a finding of serum IgM-HA antibody. The neurological findings, CSF findings and clinical course indicated acute meningoencephalitis in association with HA. To our knowledge, there have been only 4 previous case reports of meningoencephalitis associated with serologically confirmed HA infection. HA virus infection might pass unnoticed, as many cases of HA infection remain anicteric or subclinical. Therefore, HA virus should also be considered as one of the etiological agents in meningoencephalitis.

Acute Disease↗
PubMed1992

[Function, molecular structure and gene expression of interleukin-1].

Recent cloning of human and murine IL-1 receptor (IL-1R) has revealed that there are at least two type of IL-1R: type I IL-1R is detected on T cells and fibroblasts and consists of 552 AAs with a cytoplasmic domain of 213 AAs, while type II is detected on B cells and monocytic cell lines and consists of 398 AAs with a short stretch intracytoplasmic domain of 29 AAs. Extracytoplasmic portion of IL-1R has some homology with vaccinia virus B15 Ag or fibroblast protein ST-2, while cytoplasmic portion has considerable similarity with Drosophila toll gene. By transfecting murine type I IL-1R cDNA into a human Jurkat cell line, structural and functional potion required for the IL-1 signal transduction is determined. At least broad portion of cytoplasmic domain including 364-474 AAs from N-terminus are found to be essential, while PKC acceptor site (Ser-431 and Ser-509), and PKA acceptor site (Ser-528) are not essential for the IL-8 gene expression.

Amino Acid Sequence↗
PubMed1992

[Function, molecular structure and gene expression of IL-8].

Interleukin-8 (IL-8) is a potent chemotactic factor for neutrophils and T lymphocytes. Various reagents such as lectins, mitogens, IL-1, TNF, induce IL-8 production in a wide range of cells and tissues. The IL-8 gene is known to be activated by AP-1, NF-kB like factor and C/EBP like factor, but the relative importance of these transcriptional factors varies from cell to cell. Two types of human IL-8 receptor cDNA have recently been cloned. Both are G-protein coupled receptors and the amino acid sequences are highly homologous. Other members of the IL-8 family, such as GRO/MGSA and MIP2, bind to IL-8 receptors, and the receptors of other chemoattractants such as fMLP and C5a, show high homology to the IL-8 receptors.

Amino Acid Sequence↗
PubMed1992

[Two sibling patients with late-onset familial amyloidotic polyneuropathy and atypical clinical manifestations].

Brothers (case 1 and case 2) had familial amyloidotic polyneuropathy type 1 (FAP type 1) confirmed with sural nerve biopsy and DNA analysis. Both patients were unique in that their ages at onset were 56 and 52, and that their only manifestation was sensori-motor polyneuropathy, without clinically apparent autonomic involvements such as orthostatic hypotension, sweat dysfunction and sphincter dysfunction, or severe organ involvement such as gastrointestinal features and myocardial involvement after the onset. They are also unique in that their parents were healthy. The initial manifestation was sensori-motor polyneuropathy starting in the lower extremities. These atypical manifestations made the diagnosis of FAP type 1 difficult in the present cases. Based on reports in the literature and the present cases, there might be a tendency that in patients with late-onset FAP type 1 the clinical manifestations are generally mild and autonomic involvement and organ disturbance are absent or mild. In the etiological diagnosis of polyneuropathy, FAP type 1 should be considered especially in steadily progressive patients.

Age Factors↗
PubMed1992

Molecular analysis of the inhibition of interleukin-8 production by dexamethasone in a human fibrosarcoma cell line.

In order to analyse the effects of glucocorticoids on interleukin-8 (IL-8) production more precisely, we examined the effects of dexamethasone on IL-8 production at the molecular level in a human fibrosarcoma cell line, 8387, which IL-1 induces to express IL-8 messenger RNA (mRNA) and to secrete IL-8. Over a wide dose range, dexamethasone inhibited IL-8 production induced by IL-1 alpha stimulation. Northern blotting analysis showed that dexamethasone also inhibited the IL-8 mRNA accumulation in a similar dose-related manner. Nuclear run-off assay revealed that dexamethasone decreased the transcription of the IL-8 gene and the degree of inhibition of transcription correlated well with the inhibition of IL-8 production, suggesting that the action of glucocorticoids is mainly at the transcriptional level. Furthermore, transfection with chloramphenicol acetyl transferase (CAT) expression vectors inserted with the 5'-deleted IL-8 gene demonstrated that the 5'-flanking region which contains the glucocorticoid response element (GRE) was mainly involved in the dexamethasone-induced repression of the IL-8 gene. These data suggest that the inhibition of the IL-8 gene transcription by glucocorticoids occurs through the interaction of the glucocorticoid receptor complex with GRE in the 5'-flanking region of the IL-8 gene.

Blotting, Northern↗
PubMed1991

Stimulation and priming of human neutrophils by interleukin-8: cooperation with tumor necrosis factor and colony-stimulating factors.

Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time-courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM-CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.

Adult↗
PubMed1991

Inhibition of IL-8-induced W3/25+ (CD4+) T lymphocyte recruitment into subcutaneous tissues of rats by selective depletion of in vivo neutrophils with a monoclonal antibody.

IL-8 recruits both neutrophils and lymphocytes in vitro and in vivo. To elucidate the mechanisms of lymphocyte recruitment in vivo by IL-8, we examined the role of neutrophil infiltration through selective depletion of circulating neutrophils using a mAb, RP-3. Selective depletion of neutrophils inhibited the IL-8 induced in vivo migration of the W3/25+ (CD4+) T cell subset but did not inhibit that of the MRC-OX8+ (CD8+) subset. These results suggest that CD4+ T cell migration into IL-8-injected s.c. tissues depends on the prior infiltration of neutrophils chemoattracted directly by IL-8.

Animals↗
PubMed1991

The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine.

Recombinant monocyte-chemotactic and activating factor (rMCAF; alternative acronyms MCP-1, TDCF, human JE) induced migration of human monocytes across polycarbonate or nitrocellulose filters. Maximal induction of migration was observed at a concentration of 10 ng/ml (10(-9) M). Checkerboard analysis revealed that rMCAF elicited true gradient-dependent chemotactic migration, although a gradient independent chemokinetic effect was observed at low concentrations (1-5 ng/ml). rMCAF caused a rapid (less than 5 s) and transient (approximately 1.5 min) increase of free cytosolic Ca2+ ions, as assessed by the fura-2 probe. No Ca2+ increase was detected in neutrophils or lymphocytes stimulated by rMCAF. Studies conducted in the absence of extracellular Ca2+ or in the presence of Ni2+ (an inhibitor of Ca2+ influx) suggested that the increase of intracellular Ca2+ induced by rMCAF is dependent on the influx of extracellular Ca2+ through plasma membrane channels. Bordetella pertussis toxin inhibited the intracellular Ca2+ elevation and chemotaxis caused by rMCAF. The possible involvement of Ca(2+)-dependent protein kinases in rMCAF signaling pathway(s) was explored using inhibitors. Inhibitors of GMP-dependent kinase and myosin L chain kinase had no effect on rMCAF-induced monocyte migration. In contrast, protein kinase C/cAMP-dependent kinase inhibitors (such as, C-I, H-7, HA-1004, KT5720, and Staurosporine) markedly decreased rMCAF induced chemotaxis suggesting the involvement of a serine/threonine protein kinase, possibly protein kinase C, in rMCAF signaling pathway.

Calcium↗