Search PubMed⌕ Search

Biomedical subjects

S K Dower

Publications and source records attributed to S K Dower.

At least 73 records · Page 4Linked to original sources
PubMed1993

Comparison of IL-1 alpha effectiveness in activating murine pre-B and T cell lines.

Interleukin-1 (IL-1) is a potent agent that induces a wide range of biological effects. The action of IL-1 is mediated by surface IL-1 receptors (IL-1R). Two types of IL-1 receptors have been identified in lymphocytes. In this study we examined activity of IL-1 alpha in two murine lymphocyte lines that express different types of IL-1 receptors. The T lymphoid cell line EL-4 6.1 C10 expresses type I IL-1R that mediates IL-1 alpha-induced IL-2 gene expression and secretion of IL-2. The pre-B lymphoid cell line 70Z/3 was previously shown to express type II IL-1R and responds to IL-1 alpha by expressing immunoglobulin kappa light chain mRNA and increased levels of surface IgM. We found that IL-1 alpha was as potent in inducing IgM expression in 70Z/3 cells as it was in inducing IL-2 secretion in EL-4 6.1 C10 cells. Likewise, the IL-1 alpha concentration sufficient to trigger kappa light chain gene expression in 70Z/3 cells was similar to the concentration of IL-1 alpha sufficient to trigger IL-2 gene expression in EL-4 6.1 C10. In both cell lines, IL-1 alpha activated NF-kappa B-like DNA-binding activity but in EL-4 6.1 C10 cells the IL-1 alpha concentration sufficient to induce NF-kappa B response was 1000-fold lower than in 70Z/3 cells. Monoclonal antibody, mAb M15, to the type I IL-1R blocked IL-1-induced responses in EL-4 6.1 C10 cells. Surprisingly mAb M15 also blocked IL-1 action in 70Z/3 cells, even though these cells predominantly express type II IL-1R. 15% of the total IL-1 binding sites in 70Z/3 cells were recognized by mAb M15. Human IL-1 receptor antagonist (IL-1ra), which binds to the natural murine type I but not the type II IL-1R, blocked IL-1 alpha responses in EL-4 6.1 C10 and 70Z/3 cells. Although at low levels, Northern blot analysis confirmed that 70Z/3 cells express low levels of type I IL-1R mRNA. Taken together, these results suggest that type I IL-1R are expressed and transduce IL-1 signals in both 70Z/3 and El-4 6.1 C10 cells.

Animals↗
PubMed1992

Pattern of IL-1 receptor gene expression suggests role in noninflammatory processes.

The cytokine IL-1 can be elaborated by most nucleated cell types and exerts its pleiotropic effects on a variety of tissues through binding to its cognate receptor. Two forms of IL-1R, designated types I and II, have been identified. These proteins share limited amino acid identity but both bind IL-1 alpha and IL-1 beta. A large number of cell types have been shown to possess IL-1R in vitro, but few studies have addressed the question of in vivo expression. Using in situ hybridization in normal adult BALB/c mice, we have examined the tissue distributions of both IL-1R types. The results demonstrate that although lymphoid tissues in healthy animals express low or nondetectable levels of receptor transcript, nonlymphoid tissues constitutively express readily detectable levels of IL-1R mRNA. Thymus and spleen samples were largely negative for both IL-1R types I and II, whereas half of the lymph nodes examined expressed IL-1R type I. In nonlymphoid tissues, IL-1R type I transcript was detected in the dentate gyrus of the brain, endocrine pancreas, cardiac endothelium, epidermis, dermis, hair follicle epithelium, uterine serosa, vaginal stroma, vaginal squamous epithelium, developing oocytes, and granulosa cells of ruptured follicles. In contrast, the IL-1R type II probe hybridized to epidermis, dermis, and vaginal basal epithelium. The two types of IL-1R were rarely, if ever, co-expressed by the same cell. The distributional segregation of the receptor types and the expression of IL-1R in normal nonlymphoid tissues has broad implications for the role of IL-1 in homeostatic physiology.

Animals↗
PubMed1992

Interleukin 1 participates in the development of anti-Listeria responses in normal and SCID mice.

Using T- and B-cell deficient C.B-17 mice with the scid mutation, we have previously documented the existence of a T-cell-independent but interferon gamma-dependent pathway of macrophage activation that confers upon the host partial resistance to the facultative intracellular bacterium Listeria monocytogenes. This pathway is operative in both normal and SCID mice and consists of at least four components: interferon gamma, tumor necrosis factor, macrophages, and natural killer cells. Here we demonstrate that interleukin 1 also participates in this pathway but at a different site of action. Using monoclonal antibodies that neutralize the biologic activities of interleukin 1 alpha and interleukin 1 beta, we document that interleukin 1 participates neither directly in the induction of interferon gamma from isolated SCID natural killer cells nor in the antigen-specific activation of CD4+ T cells derived from Listeria-immune C.B-17 mice. In contrast, injection of a mixture of anti-interleukin 1 alpha, anti-interleukin 1 beta, and a newly derived monoclonal antibody specific for the murine type I interleukin-1 receptor into either SCID or normal C.B-17 mice blocked the in vivo elaboration of class II major histocompatibility complex-positive macrophages after infection of the animals with Listeria. Moreover, SCID mice treated with the anti-interleukin-1 mixture failed to control the growth of Listeria in vivo and eventually succumbed to the infection. These results document that endogenously produced interleukin 1 plays an obligate role in the Listeria-dependent induction of activated macrophages in vivo and demonstrate that the action of interleukin 1 is distinct from the generation of natural killer cell-derived interferon gamma.

Animals↗
PubMed1992

Evidence that MAP (mitogen-activated protein) kinase activation may be a necessary but not sufficient signal for a restricted subset of responses in IL-1-treated epidermoid cells.

We have investigated the activation of mitogen-activated protein kinase (MAP-kinase) in KB human epidermoid carcinoma cells treated with interleukin 1 (IL-1). MAP-kinase activity was transient; the time required for activity to reach a maximal level was dependent upon the dose of IL-1, ranging from 15 minutes to 45 minutes. The level of kinase induction correlated well with dose-response curves for two characteristic IL-1-induced responses, PGE2 and IL-6 production. MAP-kinase activity returned to basal levels within 2 hours regardless of the amount of IL-1 added to the system. Exposure of KB cells to free IL-1 was accordingly restricted to periods of 2 hours or less, by replacing IL-1 with an excess of IL-1 receptor antagonist. Even after 2 hours exposure, the ability of IL-1 to induce IL-6 or PGE2 was still IL-1ra-inhibitable by more than 80%, suggesting that events downstream of, or parallel to MAP-kinase activation, requiring the continual formation of new IL-1 receptor complexes, are needed to fully elicit these responses. Two general serine/threonine kinase inhibitors, K252a and quercetin, were found to strongly inhibit MAP kinase in vivo with ED50s of c. 100 nM and 30 microM, respectively. At these concentrations, both compounds effectively inhibited IL-1-driven PGE2 and IL-6 induction without affecting general protein synthesis or secretion. Other non-selective kinase inhibitors had less effect on MAP-kinase activation or IL-1-induced biological responses. The transient activation of MAP-kinase induction correlated strikingly with activation of the transcription factor NF-kappa B. IL-1-induced NF-kappa B activation was, however, relatively insensitive to inhibition by K252a or quercetin. We suggest that MAP-kinase is likely to be a necessary, but not sufficient, intermediate in some (IL-6, PGE2 induction) but not all (NF-kappa B activation) IL-1 responses in these cells.

Base Sequence↗
PubMed1992

Molecular characterization of the interleukin-1 receptor (IL-1R) on monocytes and polymorphonuclear cells.

Primary human monocytes and monocytic cells express an interleukin 1 receptor (IL-1R) which is similar in molecular weight and IL-1 binding characteristics to the IL-1R expressed on B lymphocytes (type II). Northern blot analysis of monocytic cells using a cDNA probe from the recently isolated type II IL-1R indicates that this mRNA is detectable by 4 h and accumulates for at least 24 h following treatment with IL-1R inducing drugs. The time course of induction of this mRNA is slower than that of the type I IL-1R mRNA which is also transcribed in monocytic cells but does not appear to be translated. Sequence analysis of a monocyte-derived cDNA corresponding to the type II IL-1R mRNA shows that the monocyte and B-cell mRNAs are identical. Comparison of monocyte IL-1R peptide maps with those of the type II IL-1R suggests that the two surface IL-1R are identical. This was confirmed serologically using a polyclonal antiserum raised against the type II IL-1R. Data are presented which indicate that primary human neutrophils can also be induced to express abundant type II IL-1R.

Blotting, Northern↗
PubMed1991

Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases.

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.

3T3 Cells↗
PubMed1991

A serine/threonine kinase activity is closely associated with a 65-kDa phosphoprotein specifically recognized by the kappa B enhancer element.

The immunoglobulin kappa light chain enhancer, kappa B, is an important cis-acting transcriptional element. kappa B binds a number of proteins including the members of the ubiquitous NF-kappa B family of transcription factors. Agarose beads coupled to a double-stranded oligonucleotide containing the kappa B motif were used to isolate a 65-kDa predominantly nuclear phosphoprotein. Southwestern blot analysis demonstrated that this phosphoprotein can bind the kappa B element directly and specifically. This kappa B-associated protein was phosphorylated in vivo and in vitro by a nuclear serine/threonine kinase(s) which, in a number of different cell lines, appeared to be stimulated in response to interleukin-1 alpha and lipopolysaccharide treatment. In the B cell lines 70Z/3 and CH12 LX2B, and the T cell line EL-4 6.1 C10 the activity of the kappa B-associated kinase(s) correlated with the binding activity of nuclear NF-kappa B displayed in a gel shift assay. In vitro, the 65-kDa protein was phosphorylated in the absence of exogenously added kinase. The 65-kDa phosphoprotein and the kinase activity remained associated following sequential anion-exchange and hydrophobic interaction chromatography. These results suggest that the kappa B-associated phosphoprotein is either autophosphorylated or is phosphorylated by a closely associated kinase(s). Stimulation of a nuclear protein kinase which is closely associated with a sequence-specific DNA may reflect a novel mechanism by which growth factors regulate gene expression.

Base Sequence↗
PubMed1991

Phorbol ester induces phosphorylation of the 80 kilodalton murine interleukin 1 receptor at a single threonine residue.

The cytoplasmic domains of some cell surface receptors become phosphorylated in cells treated with phorbol esters. The present study was undertaken in order to determine whether this is also true of the 80 kDa interleukin 1 receptor (IL1R). Recombinant murine IL1R, transfected into chinese hamster ovary (CHO) cells or murine fibroblasts, was immunoprecipitated from [32P]orthophosphate-labelled cells. IL1R phosphorylation was only detected in cells pretreated with phorbol 12-myristate 13-acetate (PMA) and occurred solely on phosphothreonine. In contrast to a previous report, little or no IL1R phosphorylation occurred in response to IL1. By using a truncated receptor and receptors in which threonine residues were changed to alanines, we established that Thr537, near the carboxy-terminus, is the major site of PMA-induced phosphorylation. The human IL1R has a different sequence at this locus, and is apparently not phosphorylated. Binding studies showed that PMA-induced phosphorylation had no discernible effect on ligand binding or internalization.

Amino Acid Sequence↗
PubMed1991

Interleukin 1 beta induces rapid phosphorylation and redistribution of talin: a possible mechanism for modulation of fibroblast focal adhesion.

The majority of interleukin 1 (IL-1) receptors in human fibroblasts has been shown to be localized at focal adhesions. This study describes rapid alterations caused by IL-1 beta/IL-1-receptor interaction at these sites. Fibroblast monolayers, incubated with IL-1 beta and prepared for electron microscopy, showed successive loss of cell-substratum contact and fewer and less-pronounced processes. Immunocytochemistry revealed loss and redistribution of the talin staining initially observed after 5-15 min of IL-1 beta incubation. Similarly, the cytoskeleton showed a decrease in staining and a disorganization starting from 15 to 30 min after IL-1 addition, whereas extracellular fibronectin appeared largely unaffected. Prelabeling with [32P]phosphate showed a 2- to 3-fold increase in the level of talin phosphorylation, peaking at 15 min. Phospho amino acid analyses revealed a higher level of serine and threonine phosphorylation. The data suggest that the action of IL-1 beta on fibroblasts may be partially mediated by direct phosphorylation of talin via activation of a protein serine/threonine kinase, leading to changes in transmembrane linkage proteins and the cytoskeleton. Such alterations at focal adhesions may provide a mechanism by which IL-1 can rapidly modulate cell-matrix interactions during inflammation and wound healing.

Cell Adhesion↗
PubMed1991

Evidence that interleukin-1 and phorbol esters activate NF-kappa B by different pathways: role of protein kinase C.

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor that affects expression of many genes, including immunoglobulin kappa (kappa), the interleukin-2 receptor alpha chain, and two genes in HIV-1. NF-kappa B can be activated by a number of stimuli, including pharmacological stimulation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and treatment in vitro with either protein kinase C or protein kinase A. This has lead to the proposal that these kinases are key enzymes in the physiological activation of NF-kappa B as well. We have used a murine B cell line, 70Z/3, and T cell line, EL-4 6.1 C10, to study the activation of NF-kappa B by two physiological activators, interleukin-1 alpha (IL-1) and lipopolysaccharide (LPS). There are four reasons to propose that these agents activate pathways that do not include protein kinase C as a major component in these cell lines. First, the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) strongly inhibited PMA-induced activation of NF-kappa B in 70Z/3 cells but had no effect on NF-kappa B activated by IL-1 or LPS. Second, depletion of protein kinase C by prolonged growth of 70Z/3 in PMA abrogated the capacity of the cells to activate NF-kappa B in response to further PMA treatment. However, these same cells activated NF-kappa B normally after either IL-1 or LPS treatment. Third, IL-1 effectively activated NF-kappa B in EL-4 6.1 C10 cells, but PMA did not. Fourth, interferon-gamma is a potent activator of protein kinase C in 70Z/3 cells, but is completely inactive in the mobilization of NF-kappa B. These results suggest that the physiological inducers IL-1 and LPS activate NF-kappa B by pathways independent of protein kinase C in both 70Z/3 and EL-4 6.1 C10 cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA.

Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.

Cell Line↗
PubMed1990

Heterogeneity in interleukin (IL)-1 receptors expressed on human B cell lines. Differences in the molecular properties of IL-1 alpha and IL-1 beta binding sites.

IL-1 alpha and IL-1 beta although distantly related at the primary sequence level, bind to the same Mr 80,000 IL-1 receptor on various cell types. Several lines of evidence indicate, however, that the IL-1 receptor on B cells and T cells differ. By binding experiments with 125I-IL-1, marked heterogeneity in IL-1 receptor binding was observed in 13 of 24 B cell lines studied. This was classified into three categories: (I) in nine cell lines, 125I-IL-1 alpha binding revealed high (kD = 10(-10) M) and low affinity (kD = 10(-8) M) IL-1 alpha receptors, whereas 125I-IL-1 beta binding showed one class only with intermediate affinity (kD = 10(-9) M); (II) in three cell lines selective binding with 125I-IL-1 beta was observed; (III) in one cell line only, 125IL-1 alpha and 125I-IL-1 beta bind to a single class of IL-1 receptors as has been described for most cell types. Cross-linking with 125I-IL-1 alpha or 125I-IL-1 beta demonstrated their specific binding to Mr 80,000 and to Mr 68,000 in cell lines in categories I and III, whereas for those in category II, binding to the IL-1 receptor was confined to 125I-IL-1 beta. The expression of two subsets of IL-1 alpha receptors but only one class of IL-1 beta receptors was further confirmed in kinetic studies. Internalization at 37 degrees C demonstrated that only 19% of IL-1 beta was internalized and that binding with IL-1 alpha was entirely cell surface. Flow cytometry studies showed that IL-1 alpha and IL-1 beta do not influence B cell surface antigen expression, suggesting that the ability of IL-1 to influence B cell proliferation is not mediated via direct binding to the IL-1 receptor only.

Antigens, CD↗
PubMed1990

Role of cAMP in interleukin-1-induced kappa light chain gene expression in murine B cell line.

The murine lymphoid cell line 70Z/3 has been extensively used to study the intracellular mechanisms of interleukin-1 (IL-1) action. In these cells IL-1 is known to induce kappa gene expression but the signal transduction pathway has yet to be defined. IL-1-induced kappa expression is associated with stimulation of Na+/H+ exchange and activation of protein kinase C, but these events are not sufficient to trigger kappa expression. Thus, other signals must be present. Because cAMP is a well recognized second messenger, we sought to determine whether cAMP is the signal that triggers IL-1-induced kappa expression. To that end we first measured intracellular levels of cAMP following IL-1 treatment. The results showed that exposure of 70Z/3 cells to IL-1 alpha induced a rapid and a transient increase in cAMP, it peaked at 5 min and was back to base-line level at 20 min. Prostaglandin E2 (PGE2) also increased cAMP with similar kinetics to IL-1 alpha but the increased levels were far greater. IL-1 alpha-induced increase in cAMP proved not be a sufficient signal because an increase in intracellular cAMP by N6,O2'-dibutyryl cAMP (Bt2cAMP) or PGE2 failed to increase surface IgM or to increase kappa mRNA level. Although when used alone they had no effect, Bt2cAMP and PGE2 were found to amplify the IL-1 alpha-induced kappa expression. IL-1 alpha transiently activated NF-kappa B transcription factor. But this effect could not be simulated by Bt2cAMP or PGE2. This observation provides further evidence that cAMP is not a trigger of kappa expression. Although Bt2cAMP or PGE2 when used alone had no effect, they did consistently modify the level of NF-kappa B activity induced by IL-1 alpha. Results of this study show that cAMP is not sufficient to induce NF-kappa B or kappa expression. Therefore, the role of cAMP may not be trigger but rather to modulate the IL-1 alpha-induced kappa expression. Regulation of the response could occur at one or a number of points along the signal pathway. Such a regulatory role is supported by the observation that cAMP modulates the IL-1 alpha-induced NF-kappa B activity.

Animals↗
PubMed1990

A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins.

Tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) bind surface receptors on a variety of cell types to mediate a wide range of immunological responses, inflammatory reactions, and anti-tumor effects. A cDNA clone encoding an integral membrane protein of 461 amino acids was isolated from a human lung fibroblast library by direct expression screening with radiolabeled TNF-alpha. The encoded receptor was also able to bind TNF-beta. The predicted cysteine-rich extracellular domain has extensive sequence similarity with five proteins, including nerve growth factor receptor and a transcriptionally active open reading frame from Shope fibroma virus, and thus defines a family of receptors.

Amino Acid Sequence↗
PubMed1990

Human B cells express two types of interleukin-1 receptors.

Several reports demonstrate that interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta), although distantly related at the primary sequence level, bind to the same receptor. This raises the question of what separation, if any, there might be between their biologic activities. Receptor binding assays and cross-competition studies in three Epstein Barr virus (EBV) positive cell lines showed that a subset of IL-1 receptors may bind IL-1 alpha preferentially and that a separate subset may bind IL-1 beta preferentially. Crosslinking studies with 125I-IL-1 alpha or 125I-IL-1 beta demonstrated the expression of both relative molecular mass (Mr) 80,500 IL-1 receptor, known to be T cell-specific, and Mr 69,500 IL-1 receptor, recently detected on B-cell lineage only. Whether the high affinity binding sites correspond to Mr 80,500 and the low affinity binding sites to Mr 69,500 remains to be elucidated.

B-Lymphocytes↗
PubMed1990

Regulation of alloreactivity in vivo by a soluble form of the interleukin-1 receptor.

In vitro studies have shown that cytokines are involved in the regulation of the immune response, but their role in vivo is less well defined. Specific cytokine antagonists enable the identification of particular cytokines involved in the response and offer a means for modifying it. Systemic administration of a soluble, extracellular portion of the receptor for interleukin-1 (sIL-1R) had profound inhibitory effects on the development of in vivo alloreactivity. Survival of heterotopic heart allografts was prolonged from 12 days in controls to 17 days in mice treated with sIL-1R. Lymph node hyperplasia in response to a localized injection of allogeneic cells was completely blocked by sIL-1R treatment. The inhibition was overcome by simultaneous administration of interleukin-1 (IL-1); thus, sIL-1R acts by neutralizing IL-1. These results implicate IL-1 as a regulator of allograft rejection and demonstrate the in vivo biological efficacy of a soluble cytokine receptor.

Animals↗