Linda L. Walker

Accumulation of Peribronchial Mast Cells in a Mouse Model of Ovalbumin Allergen Induced Chronic Airway Inflammation: Modulation by Immunostimulatory DNA Sequences

Few peribronchial mast cells are noted either in the lungs of naive mice or in the lungs of OVA-sensitized mice challenged acutely with OVA by inhalation. In this study, we demonstrate that OVA-sensitized mice exposed to repetitive OVA inhalation for 1–6 mo have a significant accumulation of peribronchial mast cells. This accumulation of peribronchial mast cells is associated with increased expression of the Th2 cell-derived mast cell growth factors, including IL-4 and IL-9, but not with the non-Th2 cell-derived mast cell growth factor, stem cell factor. Pretreating mice with immunostimulatory sequences (ISS) of DNA containing a CpG motif significantly inhibited the accumulation of peribronchial mast cells and the expression of IL-4 and IL-9. To determine whether mast cells express Toll-like receptor-9 (TLR-9; the receptor for ISS), TLR-9 expression by mouse bone marrow-derived mast cells (MBMMCs) was assessed by RT-PCR. MBMMCs strongly expressed TLR-9 and bound rhodamine-labeled ISS. However, incubation of MBMMCs with ISS in vitro neither inhibited MBMMC proliferation nor inhibited Ag/IgE-mediated MBMMC degranulation, but they did induce IL-6. Overall these studies demonstrate that mice exposed to repetitive OVA challenge, but not acute OVA challenge, have an accumulation of peribronchial mast cells and express increased levels of mast cell growth factors in the lung. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro, suggesting that ISS inhibits accumulation of peribronchial mast cells in vivo by indirect mechanism(s), which include inhibiting the lung expression of Th2 cell-derived mast cell growth factors.

Cloned Cauliflower Mosaic Virus DNA Infects Turnips (Brassica rapa).

Cauliflower mosaic virus DNA cloned in the Sal I site of bacterial plasmid pBR322 infects turnip plants. The cloned viral DNA must be excised from the recombinant plasmid to infect, but need not be circularized and ligated in vitro. The cloned viral DNA lacks site-specific single-strand breaks found in DNA obtained directly from the virus. However, these breaks are reintroduced into the viral genome during multiplication of the virus in the plant host.

Informational complexity of the nuclear and chloroplast genomes of Chlamydomonas reinhardi.

DNA - DNA reassociation kinetics analyzed by hydroxylapatite chromatography have been used to determine the informational content of the Chlamydomonas reinhardi nuclear and chloroplast genomes. The kinetics indicate that nuclear DNA, with the exception of ribosomal cistrons, renatures as a single component with an informational complexity 25 times that of the Escherichia coli genome. The chloroplast genome has less than 0.3% of the informational complexity of the nuclear genome, but is present in about 50 copies in the vegetative cell. Chloroplast DNA shows about a 10-12% zero-time binding component.

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.

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 μM) suppressed cAMP-dependent protein kinase activity and inhibited antigen-stimulated β-hexosaminidase and leukotriene C4 releases. Adenosine retained its ability to potentiate β-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 induced receptor hyporesponsiveness are independent of changes in cAMP.

Transcription of the nuclear and chloroplast genomes during the vegetative cell cycle in Chlamydomonas reinhardi.

Abstract The fractions of the nuclear and chloroplast genomes represented by RNA transcripts in asynchronous or synchronous Chlamydomonas reinhardi cells at different cell cycle stages were measured using RNA-driven RNA:DNA hybridization reactions. The bulk of the nuclear and chloroplast DNA hybridization reactions were driven by a class of highly complex RNA transcripts present in few copies per cell. Hybridization saturation values obtained directly or by extrapolation with whole cell RNA from asynchronous cells (representing all cell cycle stages) showed that 12% of the nuclear genome (about 12,000 gene equivalents) and all of the chloroplast genome (about 200–300 gene equivalents) are transcribed. RNA extracted from synchronous cells at different cell cycle stages generally hybridized to lesser fractions of either genome than did RNA extracted from asynchronous cells. During the cell cycle, increasing fractions of both the nuclear and chloroplast genomes are transcribed at early cell cycle stages (light period) but later, near the time of cell division, lesser portions of either genome are represented as RNA transcripts in the cell. The fractions of the genomes transcribed at different cell cycle stages are partially additive, indicating that certain RNA transcripts are common to all cell cycle stages while others are not. These results indicate that C. reinhardi cells have a partly changing battery of highly complex RNA transcripts available at different cell cycle stages.

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 adenosines 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.

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 Tyrodes 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.

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.

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 α 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.