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Biomedical subjects

D L Marquardt

Publications and source records attributed to D L Marquardt.

At least 19 recordsLinked to original sources
PubMed2000

Dependence of mast cell IgE-mediated cytokine production on nuclear factor-kappaB activity.

BACKGROUND: The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated in the regulation of a number of inflammatory cytokines and has been the proposed target for anti-inflammatory therapeutics. OBJECTIVE: Our purpose was to explore the role of NF-kappaB in the regulation of allergic inflammation. METHODS: To determine whether NF-kappaB is activated during IgE-mediated reactions and what types of mediators it regulates, a mutant form of IkappaB was used to block the ability of NF-kappaB to translocate to the nucleus and promote the transcription of selected genes. RESULTS: Mouse bone marrow-derived mast cells stimulated by IgE receptor cross-linking exhibited an activation of NF-kappaB as assessed by electrophoretic mobility shift assays. Transfected mast cells expressing the mutant IkappaB showed very little NF-kappaB activation. Both control and transfected cells released beta-hexosaminidase after specific antigen challenge, and this release could be potentiated by exogenous adenosine. Transfected mast cells that failed to develop NF-kappaB activation did not produce IL-6 messenger RNA or protein after IgE-mediated stimulation, but these cells retained the ability to produce transcripts for IL-4 and IL-5 in spite of the suppression of NF-kappaB activity. CONCLUSIONS: It appears that NF-kappaB is activated during IgE-mediated allergic inflammation and that this activity is necessary for the production of IL-6, but not IL-4 or IL-5. When considering the use of agents that target NF-kappaB to reduce inflammatory processes, it is important to know precisely which cytokines are under its control.

Animals↗
PubMed1997

Mast cell adenosine induced calcium mobilization via Gi3 and Gq proteins.

Adenosine is an important mediator of mast cell secretory responses. Adenosine appears to act through one or more adenosine receptor subtypes to activate several signal transduction pathways; however, the specific mechanisms involved are not clearly defined. We studied the pathways involved in adenosine receptor-mediated calcium fluxes in RBL-2H3 cells, a mucosal mast cell-like line. The role of endogenous heterotrimeric G proteins in adenosine mediated calcium mobilization was investigated by microinjection of inhibitory antibodies that block specific G protein subtype function. The calcium transients associated with adenosine and antigen stimulation were compared in noninjected cells and cells that were microinjected with affinity purified neutralizing antibodies to the alpha subunits of Gi3, Gq, or Gs. The percentage of cells responding to adenosine was decreased in the presence of antibodies to Gi3 and Gq, but not Gs. Pertussis toxin decreased the percentage of cells responding to adenosine, but not antigen. These studies demonstrated a functional requirement for the pertussis toxin sensitive Gi3 protein and the pertussis toxin insensitive Gq protein in adenosine mediated calcium mobilization in mast cells.

Adenosine↗
PubMed1996

The phosphatidylinositol 3-kinase inhibitor wortmannin blocks mast cell exocytosis but not IL-6 production.

Phosphatidylinositol 3-kinase (PI3-kinase) activity has been shown to be important in cellular signaling via receptors associated with tyrosine kinases and receptors coupled to small or heterotrimeric G proteins. The importance of this activity in mast cell degranulation, leukotriene C4 generation, and IL-6 production was examined in mouse bone marrow-derived mast cells stimulated by high affinity IgE receptor cross-linking, direct influx of calcium, and/or adenosine receptor agonist exposure. Wortmannin, a fungal metabolite that at nanomolar concentrations inhibits PI3-kinase relatively specifically, blocked the release of granule-associated mast cell mediators independent of the secretory stimulus used. This inhibition was most prominent after a 2- to 5-min preincubation with wortmannin and was equally effective in cells additionally treated with exogenous N-ethylcarboxamidoadenosine to potentiate preformed mediator release. Mast cell production of leukotriene C4 20 min after activation or IL-6 16 h after activation was unaffected by up to 100 nM of wortmannin exposure. Mast cells preincubated with wortmannin failed to develop the classic electronmicroscopic evidence of granule swelling and fusion, increased membrane ruffling, or exocytosis upon Ag challenge. Activation of PI3-kinase appears to be critical for mast cell degranulation but is not required for arachidonic acid metabolism or cytokine production to occur. Furthermore, the inhibition of mast cell secretion by wortmannin is not stimulus specific but is evident for both IgE receptor cross-linking and direct calcium influx.

Androstadienes↗
PubMed1995

Effects of tubercidin and its 5'-O-methyl ether on adenosine receptors and mediator release functions in mast cells.

Tubercidin (7-deazaadenosine, Tu) is a highly cytotoxic nucleoside xenobiotic that, as the nucleoside or nucleotide derivatives, closely mimics the actions of adenosine (or its corresponding nucleotides) in a wide variety of biochemical/biological systems. In light of its acceptance in these test systems as an adenosine (Ado) surrogate, it was postulated that the compound might interact with adenosine receptors. To test this hypothesis, a nonphosphorylatable derivative (5'-O-methyl tubercidin, MeTu) was prepared and evaluated in comparison with tubercidin and Ado in a variety of biological systems. In a cell culture assay using Chinese hamster ovary cells, MeTu is approximately one-third as cytotoxic as is Ado and 10(5)-fold less cytotoxic than Tu. Both Tu and MeTu inhibited the antigen-stimulated release of beta-hexosaminidase from mouse bone marrow derived mast cells in vitro, but only Tu was active in the in vivo PCA test. The inhibitory effect of MeTu on mast cell mediator release does not appear to involve interaction with adenosine receptors or to be the result of conversion to Tu per se.

Animals↗
PubMed1994

Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

Adenosine↗
PubMed1994

Inhibition of protein kinase A fails to alter mast cell adenosine responsiveness.

Adenosine activates adenylate cyclase and phospholipase C in mast cells and potentiates stimulated mediator release. To determine whether activation of adenylate cyclase is necessary for the effects of adenosine on the mast cell secretory process, a specific inhibitor of cAMP-dependent protein kinase, KT5720, was used. Antigen and adenosine each induced a rapid increase in mast cell cAMP-dependent protein kinase activity within 30 s. Preincubation with KT5720 (100 nM-10 microM) suppressed cAMP-dependent protein kinase activity and inhibited antigen-stimulated beta-hexosaminidase and leukotriene C4 releases. Adenosine retained its ability to potentiate beta-hexosaminidase release in antigen- and A23187-stimulated cells even in the presence of complete cAMP-dependent protein kinase inhibition. Mast cells rendered unresponsive to adenosine-related signals by preincubation with adenosine analogs maintained this hyporesponsiveness after incubation with KT5720. It appears that the abilities of adenosine to augment mast cell degranulation and induce receptor hyporesponsiveness are independent of changes in cAMP.

Adenosine↗
PubMed1993

Mast cell desensitization to IgE fails to induce a parallel adenosine receptor desensitization.

Desensitization induced by challenge of mast cells with antigen is specific for IgE-dependent signals. During the secretory process mast cells release adenosine, which can induce a desensitization of adenosine receptors. To determine whether adenosine receptors may be desensitized from a previous antigen challenge, mast cells were sensitized with anti-DNP IgE antibody, challenged with DNP-BSA antigen, returned to culture overnight, resensitized, and rechallenged. Previously challenged cells exhibited increased spontaneous beta-hexosaminidase release, but adenosine retained its ability to augment beta-hexosaminidase release. Adenosine enhanced A23187-stimulated release of beta-hexosaminidase in control and previously challenged cells. Leukotriene C4 generation followed a similar pattern. Mastoparan, a direct G protein activator and mast cells secretagogue, produced a doubling of beta-hexosaminidase release in previously challenged cells. Results using other G protein activators were equivocal. Degranulation alone is insufficient to induce adenosine receptor hyposensitization. Whether the hyperresponsiveness to mastoparan is a consequence of uncoupling of IgE receptors from G proteins remains uncertain.

Adenosine↗
PubMed1991

Antagonism of airway reactivity induced by ovalbumin antigen in guinea pigs by 5-amino-4-imidazolecarboxamide riboside.

The effect of 5-amino-4-imidazolecarboxamide riboside (AICA riboside), a modulator of purine metabolism, was studied on antigen-induced bronchospasm in ovalbumin (OA)-sensitized guinea pigs. In separate experiments, sodium cromoglycate (SCG) and terbutaline were used to compare their effectiveness with that of AICA riboside (wt/vol). AICA riboside and SCG were administered as an aerosol daily for a minimum of 2 weeks before OA aerosol challenge. Terbutaline, as an aerosol, was administered once 5 minutes before OA challenge. Airway reactivity was determined through the use of a whole-body plethysmography by monitoring specific airway resistance (SRaw). OA aerosol challenge of 0.05%, 0.1%, and 0.25% (wt/vol), administered for a period of 1 minute, increased SRaw. Each of the three agents attenuated the effect of OA on SRaw, although terbutaline demonstrated more consistency and potency as compared to either AICA riboside or SCG. However, at moderate degrees of OA challenge, AICA riboside appeared to be as effective as either agent. Although the mechanism of action of AICA riboside remains uncertain, it may have therapeutic benefit in the treatment of asthma or allergic diseases.

Aminoimidazole Carboxamide↗
PubMed1991

Lysophosphatidylcholine induces mast cell secretion and protein kinase C activation.

Lysophosphatidylcholine (lyso-PC), a natural product of phospholipase A2 activity, induced the secretion of both granule-associated beta-hexosaminidase and newly generated leukotriene C4 from mouse bone marrow-derived mast cells. Micromolar concentrations of lyso-PC potentiated the release of beta-hexosaminidase induced by specific antigen but not the calcium ionophore, A23187. Exogenous adenosine was relatively ineffective in enhancing beta-hexosaminidase release from cells challenged with lyso PC. Lyso-PC caused a marked increase in intracellular free-calcium levels and induced the activation of protein kinase C (PKC). These effects could not be abrogated by a prolonged preincubation with pertussis toxin. Staurosporine, an inhibitor of PKC, partially inhibited the abilities of antigen and A23187 to induce beta-hexosaminidase release but was ineffective when lyso-PC was the secretagogue. Lyso-PC appears to activate mast cell PKC, but its ability to stimulate mast cell mediator release appears to be related to its ability to elevate intracellular free calcium concentrations.

Adenosine↗
PubMed1990

Modulation of mast cell responses to adenosine by agents that alter protein kinase C activity.

The acute incubation of mouse bone marrow-derived mast cells with low concentrations of agents known to activate protein kinase C [phorbol myristate acetate (PMA), 1,2-dioctanoyl-sn-glycerol (diC8), and 1-oleoyl-2-acetyl-glycerol (OAG)] caused an enhancement of beta-hexosaminidase release stimulated by the calcium ionophore A23187. Higher concentrations of protein kinase C activators tended to inhibit A23187- or antigen-induced preformed mediator release. All concentrations studied induced a striking mast cell hyporesponsiveness to the mediator release augmenting effect of adenosine. Agents that have been reported to block protein kinase C activity [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7) and sphingosine] demonstrated diverse responses in this system. Up to 100 microM H-7 failed to affect mast cell beta-hexosaminidase release in the presence or absence of PMA and secretagogue. Sphingosine (10 microM) was a potent inhibitor of antigen- or A23187-induced mediator release as well as adenosine responsiveness. Sphingosine also blocked the effects of PMA noted above in a dose-dependent fashion. The generation of leukotriene C4 (LTC4) by stimulated mast cells surprisingly was not affected by concentrations of diC8 that significantly inhibited granule-associated mediator release. Translocation of protein kinase C activity from the cytosol to the mast cell membrane was evident in cells briefly pretreated with A23187, adenosine alone, and diC8 in the presence of Tyrode's buffer, A23187, or adenosine. These findings lend further support to the contention that signal transduction from mast cell adenosine receptors to processes that regulate degranulation may involve protein kinase C.

Adenosine↗
PubMed1989

Pretreatment with phorbol esters abrogates mast cell adenosine responsiveness.

Adenosine potentiates preformed mediator release from mouse bone marrow-derived mast cells stimulated with specific Ag or the calcium ionophore A23187. When these mast cells were cultured for 30 to 120 min with the phorbol ester PMA (10(-8) or 10(-7) M), protein kinase C activity was increased and Ag-stimulated beta-hexosaminidase release was modestly inhibited, whereas A23187-stimulated release was synergistically enhanced. However, in both cases, exogenous adenosine failed to augment beta-hexosaminidase release. Overnight PMA exposure produced a decrease in protein kinase C activity and a decrease in both Ag- and A23187-stimulated preformed mediator release, as well as a lack of responsiveness to adenosine. This hyporesponsiveness could be reversed by 24 h after washing the cells free of PMA. The generation of the arachidonic acid metabolite leukotriene C4 was not altered by mast cell PMA exposure. The ability of adenosine to increase intracellular cAMP concentrations was modestly blunted by high doses of PMA, and PMA abrogated the increase in intracellular free calcium levels usually observed in cells stimulated with Ag in the presence of 10(-5) M adenosine. PMA exposure induces a hyporesponsiveness to adenosine in mast cells, either by a direct effect on protein kinase C activity and/or by an effect on adenosine receptor expression or recycling.

Adenosine↗
PubMed1988

Alteration of mast cell responsiveness to adenosine by pertussis toxin.

Adenosine potentiates mouse bone marrow-derived mast cell mediator release by a mechanism that appears to involve cell surface adenosine receptors. In an attempt to explore possible interactions between G proteins and adenosine receptors, mast cells were incubated with activated pertussis toxin, an agent that ADP-ribosylates and inactivates some G protein subtypes, prior to challenge with specific antigen or the calcium ionophore A23187. Mast cells preincubated with 10 ng/ml pertussis toxin for at least 2 hr exhibited an inhibition of antigen-induced beta-hexosaminidase and leukotriene C4 release. The ability of adenosine to potentiate beta-hexosaminidase release was attenuated to an even greater degree by pertussis toxin. A23187-stimulated mediator release was not altered by pertussis toxin, although a modest inhibition of the ability of adenosine to enhance A23187-induced beta-hexosaminidase release was observed in pertussis toxin-treated mast cells. Although up to 24-hr exposure to 100 ng/ml pertussis toxin did not alter resting mast cell cyclic AMP levels, the ability of adenosine to elevate cell cyclic AMP concentrations was diminished markedly by doses of the toxin higher than those required to affect mediator release. Neither antigen-stimulated intracellular free calcium level augmentation alone nor the additional potentiation of these levels by adenosine was changed by pertussis toxin treatment. Inositol trisphosphate was generated by mast cells stimulated by IgE-mediated mechanisms, but a preincubation with pertussis toxin did not influence its generation. In summary, adenosine appeared to produce some of its alterations in mast cell biochemical events by a mechanism that was partially inhibited by pertussis toxin. The nature of the G protein linked to the mast cell adenosine receptor is yet to be determined.

Adenosine↗
PubMed1987

Inhibition of mast cell adenosine responsiveness by chronic exposure to adenosine receptor agonists.

Mast cell adenosine receptors are up-regulated functionally and numerically by chronic exposure to receptor antagonists, but their response to long-term treatment with receptor agonists has not been studied. To address this issue cultured mouse bone marrow-derived mast cells were exposed to N-ethylcarboxamide adenosine (NECA), an adenosine receptor agonist that augments stimulated mast cell mediator release. Cells grown for 3 days in 1 nM NECA responded normally to A23187 or antigen in releasing beta-hexosaminidase, but the ability of exogenous adenosine to potentiate this mediator release was attenuated markedly. This inhibition of adenosine responsiveness was partially present after 10 min of 1 microM NECA exposure and complete after 4 hr. The inhibitory effects could be reversed by washing NECA-exposed cells and returning them to culture for more than 4 hr. The adenosine present in the fetal calf serum coupled with deoxycoformycin attenuated mast cell adenosine responsiveness. The NECA-treated cells also exhibited a hyporesponsiveness to adenosine's augmentation of cell cyclic AMP content. This hyporesponsiveness was specific for adenosine receptors in that exogenous isoproterenol was able to increase cyclic AMP levels to a similar degree in both control and NECA-treated cells. Thus, chronic NECA exposure induces a homologous desensitization of mast cell adenosine receptors.

Adenosine↗
PubMed1987

Ribavirin inhibits mast cell mediator release.

Ribavirin (1-beta-D-ribofuranosyl-1,2-4-triazole-3-carboxamide) is a promising antiviral agent as well as a structural analog of guanosine. Although at different concentrations it has been reported to induce either immunosuppression or immune stimulation, its effects upon immediate hypersensitivity reactions are largely unknown. Because purine metabolism appears to be important in mast cell secretion, the effects of ribavirin on mouse bone marrow-derived mast cell functions were investigated. When ribavirin was added to mast cells at the time of stimulation with A23187 or specific antigen, no effect on the release of beta-hexosaminidase, a preformed mediator, was evident. However, mast cells cultured in 1 to 20 microM ribavirin for 1 to 7 days exhibited dose- and time-dependent inhibitions of stimulated beta-hexosaminidase and leukotriene C4 releases without altering mast cell mediator content. This inhibition occurred even when ribavirin had no effect on cell growth. A concomitant decrease in antigen-challenged mast cell intracellular Ca concentration was also observed after ribavirin treatment. Chronic ribavirin exposure in vitro inhibits mast cell secretory processes stimulated by both immunoglobulin E- and nonimmunoglobulin E-related signals. Its precise mechanism of action and any potential efficacy as an antiallergic agent remain to be elucidated.

Animals↗
PubMed1986

Inhibition of mast cell mediator release by 5-amino-4-imidazolecarboxamide riboside.

Stimulated mast cells produce and release adenosine, and the release of mast cell mediators is potentiated by adenosine, yet very little is known regarding mast cell purine metabolism. Because 5-amino-4-imidazolecarboxamide riboside (AICA riboside) has been shown to alter adenosine metabolism and accelerate the repletion of ATP pools in other tissues, its effects on mast cell function were examined. Neither simultaneous addition of A23187 and AICA riboside nor a 1-hr preincubation with AICA riboside altered mast cell beta-hexosaminidase release to an appreciable degree. However, mouse bone marrow-derived mast cells cultured for 2 or more days in the presence of 1-100 microM AICA riboside exhibited a markedly attenuated mediator release response to A23187 compared to control cells with or without the additional presence of adenosine. IgE-mediated leukotriene C4 generation from AICA riboside-exposed mast cells was even more profoundly inhibited without affecting cell viability or resting mediator content. An unusual ribonucleotide triphosphate previously identified in folate-depleted cells, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-triphosphate (ZTP), has been identified in AICA riboside-treated mast cells as well. Although the mechanism of this global inhibition of mast cell mediator release by chronic AICA riboside treatment is not clear, alterations in mast cell purine metabolism may prove to be important in the treatment of allergic diseases.

Adenosine Triphosphate↗