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N Mukaida

Publications and source records attributed to N Mukaida.

At least 181 records · Page 10Linked to original sources
PubMed1991

Regulation of human interleukin 8 gene expression and binding of several other members of the intercrine family to receptors for interleukin-8.

IL-8 is produced by a wide variety of cells in response to polyclonal mitogens and cytokines. Northern blotting analysis revealed that IL-1, TNF and PMA could induce rapid expression of IL-8 mRNA in the absence of new protein synthesis. Nuclear run-off assays using different cell types demonstrated that IL-8 mRNA expression could at least be partly due to the activation of transcription. Cloning and determination of the entire sequence of IL-8 genomic DNA enabled us to explore the functional significance of the 5'-flanking enhancer region of the IL-8 gene by employing CAT assays. The results indicated that the region spanning from -94 to -71 bp is minimally sufficient for conferring responsiveness to IL-1, TNF and PMA. Further analysis using point-mutations revealed that this region consisted of two distinct cis-elements; one being the potential binding site for NFkB-like and the other for a C/EBP-like factor. These results suggested that all three stimuli, IL-1/TNF/PMA, modulate the identical combination of nuclear factors possibly by phosphorylation. We previously reported that these three stimuli activated the same serine protein kinase which phosphorylates identical 65 kDa and 74 kDa cytosol proteins in human PBMC. This IL-1/TNF/PMA-activated protein kinase is distinct from protein kinase A, protein kinase C or casein kinase in substrate specificity; in Ca and phospholipid dependency; in cyclic nucleotide dependency; and sensitivity to protein kinase inhibitors. Taken collectively, IL-1/TNF/PMA may activate a common serine protein kinase and this protein kinase may in turn directly or indirectly modulate several nuclear factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Properties of the novel proinflammatory supergene "intercrine" cytokine family.

A family consisting of at least ten distinct novel 8-10 kd cytokines has been identified over the past 12 years. These cytokines exhibit from 20 to 45% homology in amino acid sequence, are probably all basic heparin-binding polypeptides, and have proinflammatory and reparative activities. The cDNA for these cytokines are characterized by conserved single open reading frames, typical signal sequences in the 5' region, and AT rich sequences in the 3' untranslated regions. Those human cytokines known as interleukin 8, platelet factor 4, beta thromboglobulin, IP-10 and melanoma growth stimulating factor or GRO can be assigned to a subfamily based on their location on chromosome 4 and unique structural features, whereas the second subset consisting of LD78, ACT-2, I-309, RANTES, and macrophage chemotactic and activating factor (MCAF) are all closely linked on human chromosome 17. In this review we have summarized and discussed the available information concerning the regulation and structure of the genes, the structure and biochemical properties of the polypeptide products, their receptors, signal transduction, cell sources, and in vitro as well as in vivo activities of these cytokines.

Amino Acid Sequence↗
PubMed1991

IL-1 and TNF-alpha induction of IL-8 and monocyte chemotactic and activating factor (MCAF) mRNA expression in a human astrocytoma cell line.

In order to elucidate the role of inflammatory cytokines in the central nervous system (CNS), we examined whether IL and TNF-alpha induce cells in the CNS to produce two newly identified leucocyte chemo-attractants, IL-8 and monocyte chemotactic and activating factor (MCAF). Several human astrocytoma and glioblastoma cell lines expressed high levels of IL-8 and MCAF mRNA in vitro upon stimulation with IL-1 and TNF-alpha. In particular, an astrocytoma cell line U373MG subclone responded markedly to IL-1 with high expression levels of IL-8 and MCAF mRNA as well as IL-6 mRNA. Both IL-8 and MCAF mRNA expression depended on the dose of IL-1 and appeared as early as 30 min to 1 hr after IL-1 stimulation, confirming that these are early inducible genes. The production of IL-8 and MCAF in the U373MG cell culture supernatants was confirmed by a competitive radioimmunoassay (RIA) as well as chemotactic activities on human neutrophils and monocytes. IL-1-induced IL-8 and MCAF mRNA expression appeared to occur at least at the transcriptional level as revealed by a nuclear run-off assay. Moreover, IL-1 treatment increased the half-life of IL-8 and MCAF mRNA markedly, suggesting that increased mRNA stability was also responsible for the enhanced gene transcription. These data suggest that IL-1 and TNF-alpha induce astrocytes to produce IL-8 and MCAF transcriptionally and post-transcriptionally, both of which may be responsible for leucocytosis seen in inflammation of the CNS.

Astrocytoma↗
PubMed1990

Cooperative interaction of nuclear factor-kappa B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines.

A novel cytokine, interleukin-8 (IL-8), may play major roles in the inflammatory process by recruiting neutrophils and T cells into inflammatory sites. The production of this cytokine is not constitutive and is induced in a variety of cell types by stimulation with mitogens and cytokines. Among cytokines, only IL-1 and tumor necrosis factor (TNF) can induce IL-8 gene expression at the transcriptional level. Transfection of a human fibrosarcoma cell line with chloramphenicol acetyltransferase expression plasmids linked to a 5'-flanking deletion mutants of the IL-8 gene demonstrated that the nucleotides between -94 and -71 base pairs from the start of the first exon are essential and sufficient for the IL-8 induction by either IL-1, TNF, or phorbol 12-myristate 13-acetate. This sequence is composed of two cis-elements; one is the potential binding site for a nuclear factor-kappa B-like factor and the other for a cis-regulatory enhancer binding protein-like factor. Mutations in either elements abolished IL-1, TNF, and phorbol 12-myristate 13-acetate responsiveness. This report provides the first evidence that cooperation between two distinct cis-elements may be required for induction of gene expression by either IL-1 or TNF.

Base Sequence↗
PubMed1990

Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily.

Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8, suggested gene symbol IL8) is a cytokine that chemoattracts and activates neutrophils. Using a panel of human-rodent cell hybrids that preferentially segregate human chromosomes and in situ hybridization, the MDNCF/IL-8 gene was placed on the human gene map at position 4q12-q21. This is the same location where at least three other members (platelet factor 4, melanoma growth stimulatory activity, and interferon-gamma induced factor) of the platelet factor 4 gene super-family reside. In addition, a restriction fragment length polymorphism was identified using MDNCF as a probe in screening genomic DNA digested with HindIII from unrelated individuals.

Chemotactic Factors↗
PubMed1990

IL-1-induced production of IL-2 and IFN-gamma in subclones of human T-cell derived leukaemia HSB.2 cells: regulation by phytohaemagglutinin-mediated (poly)phosphoinositide breakdown and cyclic AMP.

A human T-leukaemic cell line, HSB.2-C5B2, which produces high levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) when stimulated with phytohaemagglutinin (PHA) plus IL-1, was recloned to obtain spontaneous variants in IL-2 production in response to the stimuli. In these subclones, the ability of one clone to produce IL-2 correlated well with that to produce IFN-gamma. Three C5B2 subclones: clone no. 28, a high IL-2 producer, clone no. 61, an intermediate IL-2 producer, and clone no. 40, a non-producer, were selected and examined for differences in signal transduction mechanisms. Since the three subclones were shown to express about the same number of IL-1 binding sites with similar affinities, the loss of ability to produce IL-2 was not due to decreased cell-surface receptor or changes in receptor property. In support of this, IL-1 induced expression of the IL-2 receptor (Tac/p55 antigen) to the same extent on the three subclones. The levels of conventional intracellular second messengers were compared and it was revealed that loss of responsiveness was closely related to the subclones' degree of (poly)phosphoinositide (PI) turnover, protein kinase C (PKC) activation and cyclic AMP formation in response to PHA. Moreover, resting intracellular cyclic AMP concentrations were found to be increased in subclones with attenuated IL-2 production. These results indicate that the variation of IL-1-induced production of IL-2 and IFN-gamma in this T-cell line is attributed to the difference in the PHA-mediated signal transduction pathway and, presumably, to the different regulation of intracellular cyclic AMP.

Cell Line↗
PubMed1989

Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8.

To identify possible regulatory sites of the gene expression, we cloned the genomic DNA of human monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8), whose mRNA is induced by IL-1 or TNF and determined its entire nucleotide sequence. The results show that the MDNCF/IL-8 gene consists of 4 exons and 3 introns with a single "CAT"- and "TATA"-like structure. The 5'-flanking region of MDNCF/IL-8 gene shows no overall sequence similarity with that of other cytokine and acute phase reactant genes whose production is also affected by IL-1 and TNF. The 5' flanking region, however, contains potential binding sites for several nuclear factors including activation factor-1, activation factor-2, IFN regulatory factor-1, and hepatocyte nuclear factor-1. In addition, the glucocorticoid responsive element and heat shock element were located in the 5'-flanking region. Inasmuch as PMA induces MDNCF/IL-8 mRNA accumulation in human PBMC and a glucocorticoid inhibits the induction of MDNCF/IL-8 mRNA by LPS, the expression of this gene is probably regulated by interaction of these nuclear factors with the 5'-flanking DNA.

Amino Acid Sequence↗
PubMed1989

Interleukin 1 regulation of interleukin 2 and interferon-gamma gene activation in a human leukemic HSB.2 subclone.

The role of interleukin 1 (IL1) in causing IL2 and interferon-gamma (IFN-gamma) production and their associated gene activation has been studied in a human leukemic HSB.2 subclone. One of the subclones, HSB.2-C5B2 clone #28, which produced only low levels of IL2 and IFN-gamma when stimulated with phytohemagglutinin (PHA) or with a combination of phorbol-myristate-acetate (PMA) and Ca2+ ionophore, ionomycin, showed marked IL2 and IFN-gamma production in the presence of IL1. The augmentation by IL1 was observed in both dot and Northern blot analysis, indicating that IL1 regulates lymphokine genes at the pretranslational level. The kinetics of IL2 and IFN-gamma production were essentially similar for both lymphokines except that there was a faster accumulation of IFN-gamma mRNA than IL2 mRNA. In contrast to the IL2 and IFN-gamma gene activation, IL2 receptor (Tac/p55 antigen) expression was induced directly by IL1 itself as with PMA in this subclone. In these studies, IL1 action was not replaced by the drugs raising intracellular cyclic AMP (cAMP). Taken collectively, these experiments support the notion that IL1 does not trigger IL2 gene activation per se, but amplifies the preactivated lymphokine genes initiated by PMA and ionomycin, whereas IL1 can activate the IL2 receptor gene without any other known signal requirements.

Blotting, Northern↗
PubMed1988

The role of protein kinase C activation in signal transmission by interleukin 2.

Role of protein kinase C (PKC) in interleukin (IL) 2-induced proliferation was investigated by utilizing two murine IL 2-dependent cell lines, CT6 and CTLL-2 cell lines. CT6 cells showed a marked proliferative response to phorbol 12-myristate 13-acetate (PMA), while CTLL-2 did not. PMA induced PKC translocation from cytosol to membrane only in a PMA-responsive cell line. IL 2 failed to stimulate PKC translocation in both cell lines. H-7, a potent and specific PKC inhibitor, however, inhibited the proliferation of both cell lines induced by IL 2. Taken collectively, IL 2 may induce PKC activation without its translocation.

Animals↗
PubMed1988

Establishment of a highly sensitive enzyme-linked immunosorbent assay for interleukin-1 alpha employing a fluorogenic substrate.

We have previously established a non-competitive solid-phase enzyme-linked immunosorbent assay (ELISA) specific for interleukin-1 alpha (IL-1 alpha) using a combination of polyclonal antibody as the immobilized antibody, biotinylated monoclonal antibody as the second antibody and avidin-peroxidase. The level of detection of that ELISA was 200-500 pg/ml. In order to improve its sensitivity, we have used streptavidin-beta-D-galactosidase and the fluorogenic substance 4-methylumbelliferyl-D-galactopyranoside as enzyme substrate. With this system IL-1 alpha could be detected at concentrations as low as 10-50 pg/ml, which was about 10-20 times more sensitive than conventional mouse thymocyte co-stimulator assays. Furthermore, the assay system was specific for IL-1 alpha in that neither IL-1 beta nor interleukin-2 (IL-2) interfered.

Antibodies, Monoclonal↗
PubMed1987

Signal requirement for interleukin-1-dependent interleukin 2 production by a human leukemia-derived HSB.2 subclone.

We have previously established subclones from human leukemia-derived HSB.2 cell line that produced high levels of interleukin (IL) 2 when stimulated with phytohemagglutinin (PHA) and IL-1. Herein, we investigated the signal requirement for IL-2 production, particularly concerning the role of IL-1 in this system. PHA but not IL-1 rendered marked protein kinase C (PKC) activation and IL-2 production induced by PHA plus IL-1 was totally abrogated by a potent PKC inhibitor, H-7. Concomitantly, PHA alone caused marked Ca2+ influx, whereas IL-1 neither induced Ca2+ influx nor augmented PHA-induced Ca2+ influx. As expected, a signal delivered by PHA could be substituted by phorbol 12-myristate 13-acetate (PMA) and ionomycin while IL-1 was still indispensable, indicating that at least three signals, i.e., those delivered by IL-1 as well as PKC activation and Ca2+ influx were required for optimal IL-2 production. Kinetic study indicated that while PMA and ionomycin should be added at the initiation of culture, delayed addition of IL-1 up to 4 hr later induced even higher levels of IL-2 production, demonstrating the requirement for IL-1 after PKC activation and Ca2+ influx. In this system, it was revealed that IL-1 was not involved in PKC activation, Ca2+ influx, and breakdown of phosphatidylinositols. Whereas PMA, ionomycin, and IL-1 stimulated high levels of IL-2 production, those combinations of signals did not induce breakdown of phosphatidylinositols. It should be noted that IL-2 production induced by these three signals seemed to bypass hydrolysis of phosphatidylinositols in contrast to PHA plus IL-1 stimulation that was accompanied with a marked breakdown of phosphatidylinositols.

Calcium↗
PubMed1987

Preparation and characterization of polyclonal and monoclonal antibodies against human interleukin 1 alpha (IL 1 alpha).

Polyclonal and monoclonal anti-human IL 1 alpha antibodies (Ab) have been established. These Ab neutralized human recombinant IL 1 alpha (rIL 1 alpha) activity effectively, but did not interfere with human rIL 1 beta, murine rIL 1 alpha, or human rIL 2 activity. Fifty percent of rIL 1 alpha activity (25 U/ml, or 2.5 ng/ml) was neutralized by less than 0.06 microgram/ml of rabbit anti-IL 1 alpha Ab (R-38.3G) and by less than 0.13 microgram/ml of monoclonal Ab (clone 28(3B1], respectively. In other experiments, 10 micrograms/ml of rabbit anti-IL 1 alpha Ab could effectively neutralize 50% of 2000 U of rIL 1 alpha activity, and the same amount of monoclonal Ab neutralized 50% of 500 U/ml of rIL 1 alpha activity. Not only IL 1 alpha activity in the thymocyte costimulator assay, but also IL 1-dependent IL 2 production by a human leukemic cell line, HSB.2 subclone, were blocked by these polyclonal or monoclonal Ab. In addition, pI 4.9 IL 1 activity produced by the myelomonocytic cell line THP-1 and by the Epstein-Barr virus-transformed B cell lines, were neutralized by these Ab, suggesting that these cell lines also produce IL 1 alpha. The specificity of these polyclonal and monoclonal Ab was further confirmed by immunochemical method (Western blotting), in which anti-IL 1 alpha Ab reacted with rIL 1 alpha in a specific manner. Furthermore, an enzyme-linked immunosorbent assay system has been developed that can detect low levels of IL 1 alpha activity (less than 0.3 ng/ml or less than 3 U/ml), which is still less sensitive than thymocyte comitogenic assay and considerably less sensitive than the D10 assay. Finally, anti-IL 1 alpha Ab-conjugated affinity columns were prepared, by which IL 1 alpha activity, but not IL 1 beta activity, was specifically adsorbed and eluted effectively.

Antibodies, Monoclonal↗
PubMed1987

Differentiation-inducing factor for a human leukemic cell line produced by colony-stimulating factor producing human lung cancer tissue.

We have found that medium conditioned by a colony-stimulating factor producing tumor derived from a granulocytosis case with lung cancer contained a factor to differentiate a human promyelocytic leukemic cell line (HL-60) to macrophage-like cells that were butyrate esterase-positive and had phagocytosing activity and membrane Fc receptors. This differentiation-inducing factor was not active for a human myeloblastic cell line (KG-1), and was separated from a colony-stimulating factor by its molecular weight and isoelectric point. The conditioned medium did not contain a detectable amount of gamma interferon when tested by bioassay as well as by radioimmunoassay. This is the first report that a human lung cancer tissue produces not only a colony-stimulating factor, but also a differentiation-inducing factor. The conditioned medium is considered to be a good source of differentiation-inducing factor.

Animals↗
PubMed1986

A radioimmunoassay that sandwiches human interleukin-2 between radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line.

Two monoclonal antibodies were raised against human interleukin-2 (IL-2) produced by E. coli harboring recombinant complemental DNA. Both antibodies did not neutralize its activity, nor did they inhibit the binding of IL-2 to the receptor on target cells. Taking advantage of the ability of monoclonal antibodies to detect IL-2 that had bound to the receptor, a radioimmunoassay was developed that sandwiched IL-2 between the radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line infected with human T cell leukemia virus Type I. The assay had the advantage of detecting only IL-2 with the ability to bind to the receptor, and displayed a linear dose-response relationship over concentrations ranging from 5 to 100 ng/ml.

Antibodies, Monoclonal↗