Biomedical subjects

C D Richards

Publications and source records attributed to C D Richards.

At least 19 recordsLinked to original sources

PubMed1998

Oncostatin M stimulates c-Fos to bind a transcriptionally responsive AP-1 element within the tissue inhibitor of metalloproteinase-1 promoter.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) can be regulated by gp130 cytokines such as IL-6 and oncostatin M (OSM). Polymerase chain reaction deletion analysis of the murine TIMP-1 proximal promoter in chloramphenicol acetyltransferase reporter gene constructs identified an AP-1 element (-59/-53) that allows maximal responsiveness to OSM in HepG2 cells. Fos and Jun nuclear factors bound constitutively to this site as identified by supershift analysis in electrophoretic mobility shift assays, and oncostatin M (but not IL-6) induced an additional "complex 2" that contained c-Fos and JunD. OSM stimulated a rapid and transient increase in c-Fos mRNA and nuclear protein that coincided with complex 2 formation. Phorbol 13-myristate 12-acetate could also induce c-Fos but could not regulate the TIMP-1 reporter gene constructs. Transfection studies also showed that 3'-deletion of sequences downstream of the transcriptional start site (+1/+47) markedly reduced OSM -fold induction. Nuclear factors bound to SP1 and Ets sequences were detected, but were not altered upon OSM stimulation. Although OSM and IL-6 induced STAT (signal transducers and activators of transcription) factors to bind a high affinity Sis-inducible element DNA probe, binding to homologous TIMP-1 promoter sequences was not detected. Thus, OSM (but not IL-6) stimulates c-Fos, which participates in maximal activation of TIMP-1 transcription, likely in cooperation with other factors such as SP1 or as yet unidentified mechanisms involving the +1 to +47 region of the promoter.

Animals↗

PubMed1998

Activation of group I metabotropic glutamate receptors elicits pH changes in cultured rat cortical glia and neurons.

Activation of metabotropic glutamate receptors is known to elicit a rise in intracellular Ca2+ and the present study was undertaken to see whether they also modulate the intracellular pH (pHi) of neurons and glia. Measurements of the pHi of neurons and astrocytes were made with the ratiometric fluorescent dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. In the absence of bicarbonate, stimulation with the specific metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid caused a fall in pHi in both astrocytes and neurons. In the presence of bicarbonate, stimulation with 25 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid elicited a rise in pHi in the astrocytes, while the neurons responded with a small acidification. The astrocytic alkalinization could also be elicited by the specific group I metabotropic glutamate receptor agonist (S)-3-hydroxyphenylglycine but not by the group II agonist (2S,1'S,2'S)-(2-carboxycyclopropyl)glycine or by the group III agonist L(+)-2-amino-4-phosphonobutyric acid. The alkalinization of glial cells could be reduced by preloading the cells with BAPTA, but not by removal of extracellular Ca2+. Depolarization of the astrocytes with potassium elicited a small alkalinization, but stimulation with 100 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid in high potassium medium elicited a further alkalinization. It is concluded that activation of group I metabotropic glutamate receptors leads to an alkalinization of astrocytes by a process that involves an elevation of intracellular Ca2+. The pHi changes that follow activation of the metabotropic glutamate receptors may play a role in initiation of glial proliferation following cerebral injury.

Animals↗

PubMed1997

Regulation of tissue inhibitor of metalloproteinase-1 in fibroblasts and acute phase proteins in hepatocytes in vitro by mouse oncostatin M, cardiotrophin-1, and IL-6.

Mouse oncostatin M (mOSM) has been recently cloned; however, its full spectrum of biologic functions has not been defined. To assess its potential role in inflammation, we have tested the activity of mOSM in vitro in regulation of fibroblasts and hepatic cells. At concentrations of 10 and 20 ng/ml, mOSM stimulates tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA in NIH-3T3 mouse embryonic fibroblasts, rat lung fibroblasts, and rat synovial fibroblasts, whereas mouse cardiotrophin-1 (mCT-1) or human OSM (hOSM) did not. Similarly, only mOSM was able to induce transcription of chloramphenicol acetyl-transferase (CAT) in NIH-3T3 cells transfected with a minimal TIMP-1 promoter/CAT construct. Mouse OSM had strong action inducing primary rat hepatocyte cultures to produce acute phase proteins; however, mOSM was very weak in its ability to stimulate acute phase protein synthesis in rat H35 cells or human HepG2 cells, which was consistent with weak STAT activation in H35 cells and HepG2 cells. Binding studies showed that NIH-3T3 cells possessed high affinity binding sites for mOSM, but rat H35 cells did not. On the other hand, mCT-1 and mouse IL-6 induced strong STAT activation as well as marked increases in acute phase protein production by H35 cells. These results indicate that mOSM does not share a functional receptor with mCT-1 or hOSM in mouse and rat cells and that hOSM does not activate the putatively specific OSM receptor on mouse or rat cells. These results also suggest that mOSM is an important cytokine in inflammation, through modulation of fibroblast function as well as hepatocyte responses.

3T3 Cells↗

PubMed1997

Antibodies to rat soluble IL-6 receptor stimulate B9 hybridoma cell proliferation.

Interleukin-6 mediates its pleiotropic effects by interacting with its membrane bound receptor (gp80) or the soluble counterpart gp54, resulting in activation of a complex that includes the transducer protein gp130. We have generated a polyclonal antibody against the rat soluble IL-6 receptor (anti-rat sIL-6R) in rabbits. By Western blot analysis we show that purified anti-rat sIL-6R IgG antibody reacts specifically with recombinant rat sIL-6R generated from E. coli, baculovirus or adenovirus expression systems. Anti-rat sIL-6R inhibited IL-6-induced acute phase protein synthesis in rat (H35) but not human (HepG2) hepatoma cells, and did not affect stimulation of those cells by Oncostatin-M. Conversely, on the mouse hybridoma B9 cell line, IgG anti-rat sIL-6R showed a dose-dependent stimulation of proliferation. Fab fragments of this antibody did not stimulate, but abrogated IL-6-mediated hepatoma cell stimulation and B9 cell proliferation. Gel shift analysis of STAT nuclear factors showed activation of STAT DNA binding in nuclei of B9 cells treated with IgG anti-rat sIL-6R, whereas in H35, NIH-3T3 and M1 cells, only IL-6 could trigger a similar STAT activation. Our data suggest that mechanisms of IL-6 receptor activation and signalling in mouse B9 hybridoma cells show subtle but important differences from other IL-6-responsive cells.

Acute-Phase Proteins↗

PubMed1997

Oncostatin M stimulates monocyte chemoattractant protein-1- and interleukin-1-induced matrix metalloproteinase-1 production by human synovial fibroblasts in vitro.

OBJECTIVE: To measure levels of oncostatin M (OSM) in the synovial fluid of rheumatoid arthritis (RA) patients and to examine the activities of human OSM in the regulation of human synovial fibroblast (HSF) production of chemokines and matrix metalloproteinases (MMP-1 and MMP-3) in vitro. METHODS: We examined the levels of OSM in the synovial fluids of patients with arthritis by an enzyme-linked immunosorbent assay (ELISA). ELISA of cell culture supernatants and Northern blots were used to assess responses of HSF to interleukin-1alpha (IL-1alpha), OSM, and other members of the IL-6/leukemia inhibitory factor (IL-6/LIF) family of cytokines. RESULTS: We detected variable levels of OSM antigen in 9 of 10 RA patient synovial fluids, but levels were not detectable in 9 of 10 osteoarthritis (OA) patient fluids. Upon examining the responses of HSF in culture, OSM stimulated monocyte chemoattractant protein 1 (MCP-1), whereas RANTES secretion (regulated upon activation, normal T expressed and presumably secreted) was not altered by OSM alone. In IL-1alpha-induced cells, OSM costimulation further enhanced MCP-1 release, but inhibited the release of RANTES and IL-8. Other members of the IL-6/LIF family of cytokines did not show these effects. OSM induced a small elevation of MMP-1 production over 2 and 3 days of stimulation (2-fold), and acted significantly to enhance IL-1alpha-induced production of MMP-1 (to 8-fold and 9-fold at 48 and 72 hours, respectively). No effect of OSM was seen on MMP-3 secretion, either alone or in IL-1alpha-costimulated cells. CONCLUSION: These results suggest that OSM has potentially important functions in the modulation of chemokine and metalloproteinase production by synovial cells of the joint.

Arthritis, Rheumatoid↗

PubMed1997

Electrophysiological and immunocytochemical characterization of GABA and dopamine neurons in the substantia nigra of the rat.

Neurons in the substantia nigra pars reticulata and pars compacta of the rat were studied using a combination of intracellular electrophysiological recording in in vitro and subsequent immunocytochemical double and triple labelling techniques. The neurons recorded in the pars reticulata were identified as either GABA or dopamine neurons: neurons were considered to be GABA neurons if they were immunopositive for glutamate decarboxylase, whereas those neurons which were immunopositive for tyrosine hydroxylase were considered to be dopaminergic. The GABA neurons had short duration action potentials (0.45+/-0.03 ms halfwidth), no apparent rectifying currents, no low threshold calcium spikes, were spontaneously active (7.4+/-3.7 Hz), and could maintain high firing rates. The dopamine neurons had long duration action potentials (1.49+/-0.10 ms), displayed both anomalous inward and transient outward rectifying currents, and more than half (12/17 neurons) displayed a low threshold calcium spike. Their spontaneous firing rate was lower than that of the GABA neurons (2.3+/-1.0 Hz), and they displayed strong frequency adaptation. Morphological reconstruction of neurobiotin-filled neurons revealed that the pars reticulata GABA neurons had more extensive local dendritic arborization than the dopamine neurons from either the pars reticulata or the pars compacta. All of the neurons recorded from the pars compacta were dopamine neurons; they were found not to be different either electrophysiologically or morphologically from pars reticulata dopamine neurons. The electrophysiology of the GABA neurons suggests that input activity is translated linearly to spike frequency. These GABA neurons probably represent the projection neurons of the pars reticulata, and it is thus likely that this basal ganglia output is frequency coded. The close similarity between the dopamine neurons in the pars compacta, which give rise to the nigrostriatal pathway, and those in the pars reticulata supports the notion that the dopamine neurons in these two regions are part of the same neuronal population.

Animals↗

PubMed1997

Oncostatin M, but not interleukin-6 or leukemia inhibitory factor, stimulates expression of alpha1-proteinase inhibitor in A549 human alveolar epithelial cells.

Alpha-1 proteinase inhibitor (A1-Pi) is the main serine proteinase inhibitor found in human plasma and is a potent elastase inhibitor in various tissues, including lung. A1-Pi is expressed and induced in liver during inflammatory responses but can also be produced by epithelial cells. Since hepatocyte A1-Pi production is stimulated by interleukin-6 (IL-6) and other gp130-cytokines, such as leukemia inhibitory factor (LIF) and oncostatin M (OM), we investigated the role of these cytokines in regulating A1-Pi in lung epithelial cells. We show that OM, a monocyte and T cell product, can specifically and potently induce A1-Pi production in lung-derived A549 alveolar (epithelial) cells, as well as in liver-derived HepG2 cells. Both A1-Pi protein (as detected by ELISA and Western blots) and mRNA levels were enhanced 20-fold to 30-fold in A549 cells. OM was also able to stimulate the expression of tissue inhibitor of metalloproteinase-1 in these cells. Interestingly, other members of the IL-6 family (IL-6 and LIF) had little or no effect on A549 cells, and proinflammatory cytokines, such as IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) also had no stimulatory effect on A1-Pi synthesis in A549 cells. Costimulation with IL-1 beta resulted in a decrease in A1-Pi production from OM-stimulated A549 cells. However, IL-6 production was synergistically enhanced. OM was also able to stimulate A1-Pi production from a bronchial epithelial primary cell line, whereas an intestinal epithelial cell line HT29 responded to IL-6 but not OM. These results suggest that lung levels A1-Pi could be derived not only from liver and inflammatory cells but also from epithelial cells, which can be upregulated on stimulation by OM. This may have implications for regulation of local activity of human neutrophil elastase (HNE) in such diseases as emphysema and cystic fibrosis.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed1997

CD8+ T-cell-mediated suppression of HIV-1 long terminal repeat-driven gene expression is not modulated by the CC chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta.

OBJECTIVE: To assess the role of RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta in modulation of HIV-1 long terminal repeat (LTR)-mediated gene expression and determine whether these chemokines share identity with CD8+ T-lymphocyte-derived HIV-1 LTR-suppressive factors. DESIGN: HIV-1 LTR-directed reporter gene expression is a model for transcription that is susceptible to inhibition by factors produced by CD8+ lymphocytes of HIV-1-infected individuals. The effect of recombinant chemokines on LTR-directed gene expression was examined. The ability of chemokines found to be present in CD8 supernatants to suppress HIV-1 LTR-mediated gene expression was determined by antibody inhibition assays. METHODS: The concentrations of RANTES, MIP-1 alpha and MIP-1 beta in a panel of CD8+ T-lymphocyte-derived supernatants were determined by enzyme-linked immunosorbent assay. Recombinant chemokines were added to freshly transfected (pLTR-CAT and pSV40-tat) human Jurkat T cells. Excessive polyclonal neutralizing antibodies to these chemokines were added to transfected Jurkat T cells cultured in the presence of strongly inhibitory CD8+ T-cell-derived supernatants with known chemokine concentrations. RESULTS: The concentrations of RANTES, MIP-1 alpha and MIP-1 beta in a panel of CD8+ lymphocyte-derived supernatants were found to correlate with their relative ability to suppress the LTR-mediated gene expression (r = 0.679, 0.764 and 0.48, respectively). The addition of recombinant CC chemokines had no effect over a broad range of doses on HIV-1 LTR-mediated gene expression. The CD8-suppressive effect on HIV-1 LTR-driven gene expression was not abrogated by a combination of antibodies of RANTES, MIP-1 alpha and MIP-1 beta. CONCLUSIONS: RANTES, MIP-1 alpha and MIP-1 beta do not alter HIV-1 LTR-directed gene expression at doses up to 100 ng/ml. Although present in varying concentrations in supernatants derived from CD8+ lymphocytes from HIV-positive individuals, these chemokines are not responsible for the powerful CD8-derived suppressive effect on HIV-1 LTR-mediated gene expression observed in our system.

CD8-Positive T-Lymphocytes↗

PubMed1996

Overexpression of RANTES using a recombinant adenovirus vector induces the tissue-directed recruitment of monocytes to the lung.

RANTES (regulated on activation, normal T cell expressed and secreted) is a member of the C-C superfamily of chemokines and is reported to function as a potent chemoattractant for monocytes, eosinophils, and a subpopulation of CD4+ T cells. Using a recombinant human type 5 adenovirus containing the murine RANTES cDNA (Ad5E3 mRANTES), which is capable of expressing biologically active cytokine upon infection, we initiated a study to characterize the biologic functions of RANTES cytokine in vivo. Intratracheal administration of Ad5E3 mRANTES targeted transient RANTES expression to the bronchial epithelium of the lung in Sprague-Dawley rats. Bronchoalveolar lavage fluids (BAL) collected at 24 h had increased chemotactic activity vs controls as measured in a murine CD4+ T cell Boyden chamber microchemotaxis assay. There was a dramatic increase in the number of cells (macrophage, monocytes, and neutrophils) recovered from BAL samples taken from Ad5E3 mRANTES-treated animals at 24 h, with a >50-fold increase in monocytes, indicating a proinflammatory effect for this cytokine in vivo. This effect on monocytes was transient, decreasing by 7 days, with evidence of increased eosinophils and lymphocytes at this time. Histologic examination of lung sections at 24 h revealed greatly increased numbers of mononuclear cells, primarily monocytes, within the lungs of Ad5E3 mRANTES-treated animals, with increased extravasation of monocytes around blood vessels, indicating an ongoing process of peripheral blood monocyte recruitment. This study provides further evidence for RANTES to be a monocyte chemoattractant in vivo.

Adenoviridae↗

PubMed1996

Oncostatin M inhibits IL-1-induced expression of IL-8 and granulocyte-macrophage colony-stimulating factor by synovial and lung fibroblasts.

The role of oncostatin M (OM) in modulating production of cytokines by connective tissue cells is largely unexplored. We have examined the effects of stimulating fibroblast cultures derived from human synovium and from normal lung with OM alone or in combination with IL-1, IL-1 alpha (or IL-1 beta) at 1 or 5 ng/ml, stimulated production of high levels of granulocyte-macrophage CSF (GM-CSF), IL-8, and IL-6 protein. At various concentrations (0.1-50 ng/ml), OM alone failed to significantly enhance protein or mRNA levels of GM-CSF, IL-8, IL-6, or G-CSF after 18 h of stimulation. When combined with IL-1 alpha or -beta, OM caused a dose-dependent inhibition of the IL-1-induced level of IL-8 and GM-CSF protein and mRNA expression, whereas IL-6 production was simultaneously enhanced. In contrast, when IL-6 or leukemia inhibitory factor (two other cytokines that share gp130 receptor components with OM) were used in a similar fashion in combination with IL-1 alpha, neither cytokine consistently altered the IL-1-induced levels of IL-8, GM-CSF, or IL-6. In addition, only OM and not IL-6 or leukemia inhibitory factor was able to induce STAT-1 nuclear factor binding to DNA in stimulated fibroblast extracts as measured by electrophoretic mobility shift assay. These results suggest that OM can significantly alter cytokine profiles of stimulated fibroblasts and may play a unique role in modulating cytokine production by these cells at sites of inflammation.

Animals↗

PubMed1996

Murine cardiotrophin-1 stimulates the acute-phase response in rat hepatocytes and H35 hepatoma cells.

Mouse cardiotrophin-1 (CT-1) is a hypertrophy-inducing factor for cardiac myocytes and interacts with cell surface receptors that incorporate the signaling molecule gp130. Because other cytokines utilizing this receptor subunit stimulate acute-phase protein synthesis, we tested cardiotrophin-1 in in vitro assays of protein synthesis by primary rat hepatocytes, rat hepatoma cells (H35), and human hepatoma cells (HepG2). CT-1 showed a dose-dependent induction of protein synthesis by primary rat hepatocytes, with effective concentrations ranging from 0.1 to 100 ng/ml. Production of a number of acute-phase proteins, including alpha 1-cysteine proteinase inhibitor ( alpha 1-CPI), alpha 1-proteinase inhibitor (alpha 1-Pi), alpha 2-macroglobulin, and alpha 1-acid glycoprotein, was markedly increased at 48 and 72 h of cytokine stimulation. In rat H35 cells, CT-1 stimulated alpha 1-Pi and alpha 1-CPI protein production and upregulated alpha 1-CPI mRNA levels with similar potency. Compared with other IL-6-type human cytokines at optimal concentrations in parallel assays, CT-1 induced similar levels of acute-phase proteins as human oncostatin M (OM) and leukemia inhibitory factor (LIF), whereas human IL-6 induced the greatest levels of alpha 1-CPI or alpha 1-Pi production by H35 cells. When tested on human HepG2 cells, murine CT-1 was far less effective, in that it stimulated alpha 1-antichymotrypsin production only at very high concentrations (100 ng/ml) but did not alter haptoglobin or alpha 1-Pi. Human OM and IL-6 were effective at lower concentrations and induced much higher levels of acute-phase protein synthesis, whereas LIF activity was similar to that to CT-1. These results show that murine CT-1 is a strong acute-phase mediator for rat hepatocytes in vitro and its activity is similar to LIF on rat hepatocytes, H35 cells, and HepG2 cells.

Acute-Phase Reaction↗

PubMed1996

Intrinsic hydrogen ion buffering in rat CNS neurones maintained in culture.

The intrinsic proton buffering power (beta 1) of individual rat hippocampal and neocortical neurones maintained in culture has been investigated using the fluorescent dye 2', 7'-bis(carboxymethyl)-5, 6-(carboxyfluorescein) (BCECF). The steady-state intracellular pH (pH1) was estimated to be 7.03 +/- 0.04 (n = 22) in Hepes-buffered media and beta 1 estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-1 at pH1 values near to 7. Estimates of beta 1 made from butyric acid challenges were inconsistent with estimates made at the same pH1, using NH4Cl withdrawal. However, estimating beta 1 with butyrate in the presence of the monocarboxylate ion transport inhibitor alpha-cyano-hydroxy-cinnamate (CHC) yielded beta 1 values commensurate with those measured using NH4Cl. Application of CHC alone lead to a rapid fall in pH1, suggesting a significant contribution of the monocarboxylate transporter to pH1 regulation. beta 1 was also estimated from a step increase in extracellular P(CO2). This yielded a value of 11 mM at an average pH1 of 7.1, which is similar to that of the other estimates reported here. beta 1 was found to increase with decreasing pH1: each unit drop in pH1 increased buffering power by about 60%. Blockade of pH1 regulation did not significantly affect estimates of beta 1. The change in buffering power with pH could be closely modelled from the known concentrations of free amino acids and organic phosphates.

Ammonium Chloride↗

PubMed1996

On the role of bicarbonate as a hydrogen ion buffer in rat CNS neurones.

The role of bicarbonate as a hydrogen ion buffer has been investigated using the fluorescent dye BCECF in individual rat cerebellar, hippocampal and neocortical neurones maintained in culture. The steady-state intracellular pH (pHi) was estimated to be 7.07 +/- 0.05 (n = 22) in CO2-HCO3(-)-buffered media. Buffering power (beta) estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-1 and was found to be similar in both CO2-HCO3(-)- and Hepes-buffered media. The membrane-permeant carbonic anhydrase inhibitor, acetazolamide (10-20 microM), did not affect estimates of beta. The results indicate that CO2-HCO3- does not act as an open buffer system in these neurones.

Acetazolamide↗

PubMed1996

Prostaglandin E2 enhances interleukin 8 (IL-8) and IL-6 but inhibits GMCSF production by IL-1 stimulated human synovial fibroblasts in vitro.

OBJECTIVE: To examine in vitro the effect of prostaglandin E2 (PGE2) on synovial cell cytokine production. METHODS: Human synovial fibroblasts were stimulated with PGE2 alone or PGE2 in combination with interleukin 1 alpha (IL-1 alpha) (5 ng/ml) and/or indomethacin (10(6) M) and assessed for the production of IL-8, IL-6, and granulocyte macrophage colony stimulating factor (GMCSF) at the protein and messenger RNA (mRNA) levels. RESULTS: PGE2 alone had little detectable effect on IL-8 or GMCSF; however, a small enhancement of both IL-6 mRNA and protein levels was seen. While all cytokines were markedly stimulated by IL-1 alpha), co-addition of the cyclooxygenase inhibitor indomethacin enhanced IL-8 and GMCSF levels, but caused a reduction in IL-6 expression. The addition of PGE2 to cultures stimulated with IL-1 alpha and indomethacin resulted increases in IL-6 mRNA and protein expression while causing a concomitant reduction in GMCSF protein and mRNA expression. PGE2 and illoprost (PGI2 analog) enhanced IL-8 production in stimulated cells. CONCLUSION: While PGE2 alone has limited effects on synovial cell production of IL-8 and GMCSF, its effects are significant in context of IL-1 alpha stimulation; endogenous PGE2 may alter cytokines secreted by mesenchymally derived cells. PGE2 may be an important modulator of cytokine driven inflammation.

Blotting, Northern↗

PubMed1995

Adenovirus vectors for cytokine gene expression.

Recombinant Adenovirus type 5 constructs containing IL-6 cDNA can be used to infect cells in vitro and obtain a high level of IL-6 expression and secretion into culture media. Furthermore, Ad5-IL-6 viruses can also be used to infect Balb/c mice or Sprague-Dawley rats and obtain a high level of IL-6 expression that is sustained over a period of 3-5 days. Intratracheal infection was accompanied by dramatic increases in virus-encoded IL-6 mRNA levels in rat lung tissue, raised levels of IL-6 detected in bronchoalveolar lavage fluids and in serum, and IL-6-dependent sequelae such as liver acute phase responses. This occurs in a tissue-specific manner, depending on routes of infection by the virus. Rat lungs showed a prominent expansion (10 fold in numbers) of all classes of lymphocytes, including B cells, T helper cells (CD4+) and CTL (CD8+) at day 7 after infection which resolved significantly by day 12. Thus the associated biological effects of viral vector mediated IL-6 over-expression was also transient in nature. Other tissues can be infected with Ad5 and thus can also be induced to express selected genes in a transient fashion. We are currently examining the potential for Ad recombinant cytokine vectors in therapy for cancer and for bone marrow reconstitution after transplantation. Thus the use of recombinant Ad5 vectors may have a broad application in the study of cytokine function and possibly in future therapy as a transient gene transfer approach.

Adenoviruses, Human↗

PubMed1995

Prostanoid enhancement of interleukin-6 production by rat peritoneal mast cells.

Mast cells are traditionally associated with an acute response involving the short-term release of mediators such as histamine. We have shown previously that mast cells can produce IL-6 without prior histamine release. In this study we examined the hypothesis that mast cell IL-6 production can be selectively regulated by PGs. Highly purified rat peritoneal mast cells were cultured in the presence of PGE1, PGE2, or PGD2 alone or in combination with anti-IgE or bacterial LPS. Histamine release was assessed after 10 min; IL-6 and TNF-alpha production was measured in supernatants after 18 h. Mast cell IL-6 production was induced by PGE1 and PGE2 to a similar level to that observed in anti-IgE-activated cells. In contrast, constitutive production of TNF-alpha was inhibited by PGE1 and PGE2, but not by PGD2. PGE2 had a synergistic effect, inducing IL-6 in the presence of LPS, whereas an additive effect was observed in the presence of anti-IgE. None of the prostanoids alone induced significant histamine release at the 10-min time point. However, PGE2 significantly increased histamine release when added concurrently with anti-IgE. Flurbiprofen in the context of anti-IgE or LPS activation did not alter mast cell IL-6 or TNF-alpha production. IL-6 production in response to each of the stimuli was significantly inhibited by the corticosteroid dexamethasone. These observations of selective modulation of mast cell cytokine production are important to understand the mechanisms by which mast cells interact with other cells during an inflammatory process involving prostanoid synthesis.

Alprostadil↗

PubMed1995

Cytokines oncostatin M and interleukin 1 regulate the expression of the IL-6 receptor (gp80, gp130).

The steady-state mRNA levels of the interleukin 6 receptor (IL-6R, gp80) and its signal transducing molecule, gp130, were examined in the rat hepatoma cell line, H-35, stimulated by cytokines IL-6, IL-1, oncostatin M (OSM) and/or Dexamethasone (Dex). In contrast to our previous findings in vivo [Geisterfer et al., 1993, Cytokine, 5:1] in vitro Dex seemed to be the major stimulator of IL-6R mRNA expression, whereas IL-6 seemed to have little effect on the expression of its own receptor mRNA levels. However, the presence of other cytokines influenced the Dex mediated stimulation of IL-6R expression. OSM stimulated IL-6R mRNA levels. At 6 h, cells stimulated with OSM showed a 2.1-fold increase in IL-6R mRNA expression. This stimulation was additive with the Dex-mediated stimulation of IL-6R mRNA levels. In contrast, IL-1 inhibited the Dex-mediated stimulation of IL-6R mRNA. At the same time, IL-1 stimulated the presence of a second smaller mRNA transcript. This mRNA species contained the extracellular domain but lacked both the transmembrane and cytoplasmic domains of the IL-6R, suggesting alternate splicing, possibly coding for a soluble form of gp80. Unlike the gp80 IL-6R molecule, the expression of the gp130 molecule normally expressed as two species of mRNA was not regulated to any major extent in vitro. IL-1 and OSM stimulated both mRNA bands (7.5 and 9.0 kb) approximately 2-fold, whereas IL-6 stimulated mainly the upper 9.0 kb mRNA band.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Proteins↗

PubMed1995

Anaesthetic modulation of nicotinic ion channel kinetics in bovine chromaffin cells.

1. We have investigated the action of the anaesthetics methoxyflurane, methohexitone and etomidate on the nicotinic acetylcholine receptor channel of bovine adrenal chromaffin cells using the whole cell patch clamp technique. 2. Spectral analysis of macroscopic currents evoked by 25 microM carbachol revealed that each of the agents tested reduced the lifetime of the channel open state in a dose-dependent manner. The whole cell current was inhibited in a concentration-dependent fashion by each agent. 3. Channel gating parameters were calculated from single channel studies and the results used to test models explaining the modulation of nicotinic acetylcholine receptor channels by anaesthetics. 4. Each of the agents studied reduced the mean channel open time in a concentration-dependent manner. Anaesthetic concentrations reducing mean open time by 50% were: 370 microM methoxyflurane, 30 microM methohexitone or 23 microM etomidate. 5. Methohexitone and etomidate produced an increase in the number of brief closures within bursts, while no such increase was observed with methoxyflurane. Despite these inter-burst gaps, mean burst length was reduced by each of the agents tested. 6. It is concluded that a simple sequential blocking model fails to account for the action of these anaesthetics. An extended model, in which blocked channels can close, may be applicable.

Adrenal Medulla↗