Mast Cell Adenosine Research Articles

Mast Cell Adenosine Receptors Function: A Focus on the A3 Adenosine Receptor and Inflammation

Adenosine is a metabolite, which has long been implicated in a variety of inflammatory processes. Inhaled adenosine provokes bronchoconstriction in asthmatics or chronic obstructive pulmonary disease patients, but not in non-asthmatics. This hyper responsiveness to adenosine appears to be mediated by mast cell activation. These observations have marked the receptor that mediates the bronchoconstrictor effect of adenosine on mast cells (MCs), as an attractive drug candidate. Four subtypes (A1, A2a, A2b, and A3) of adenosine receptors have been cloned and shown to display distinct tissue distributions and functions. Animal models have firmly established the ultimate role of the A3 adenosine receptor (A3R) in mediating hyper responsiveness to adenosine in MCs, although the influence of the A2b adenosine receptor was confirmed as well. In contrast, studies of the A3R in humans have been controversial. In this review, we summarize data on the role of different adenosine receptors in mast cell regulation of inflammation and pathology, with a focus on the common and distinct functions of the A3R in rodent and human MCs. The relevance of mouse studies to the human is discussed.

FcεRI β‐chain ITAM amplifies PI3K‐signaling to ensure synergistic degranulation response via FcεRI and adenosine receptors

AbstractSimultaneous stimulation with antigen and adenosine in mast cells induces a synergistic degranulation response at a low antigen dose that is insufficient to cause secretion by itself. This kind of stimulation is thought to be relevant to the immediate asthmatic response upon bronchial challenge with low‐dose allergen. In this context, FcεRI‐ and adenosine receptor‐mediated signalings cooperate to increase degranulation in mast cells. In the present study, we prepared mast cells that have mutations (Y219F/Y225F/Y229F) in three tyrosine residues of the FcεRI β‐chain (FcRβ)‐ITAM in order to elucidate the molecular mechanisms of degranulation response synergistically elicited by costimulation with low‐dose antigen and adenosine. Introduction of mutations in the FcRβ‐ITAM abolished the synergistic degranulation response. Upon costimulation with low‐dose antigen and adenosine, tyrosine phosphorylation of Grb2‐associated binder 2, which is located upstream of PI3K‐signaling, was significantly increased, but severely diminished in FcRβ‐ITAM mutant cells. These findings indicate that FcRβ acts as a critical element in mast cell synergistic degranulation response through FcεRI and adenosine receptors, and that PI3K‐signaling through FcRβ‐ITAM is a crucial participant in augmentation of FcεRI‐mediated degranulation by adenosine.

Effect of genetic A2B adenosine receptor ablation on proinflammatory actions of adenosine in mast cells

We have previously shown that A2B adenosine receptors (AR) play a pro‐inflammatory/pro‐angiogenic role, upregulating production of Th2 cytokines and angiogenic factors in human HMC‐1 mast cells.1,2 However, the pro‐inflammatory role of A2bAR in mast cells was recently questioned by a study that found an enhanced antigen‐induced degranulation of A2bAR knockout (A2BKO) mouse bone marrow‐derived mast cells (BMMCs).3 The authors interpreted this as evidence of anti‐inflammatory actions of A2BAR in BMMCs and suggested that A2BAR antagonists with inverse agonist activity could promote activation of mast cells. Here, we revisited these conclusions by studying the effect of genetic A2BAR ablation on adenosine‐dependent regulation of BMMCs. Our results show that genetic ablation of A2BAR had no effect on adenosine‐dependent potentiation of antigen‐induced degranulation, but abrogated adenosine‐dependent stimulation of IL‐13 and VEGF secretion. A2BAR antagonists with inverse agonist activity inhibited A2BAR‐dependent secretion, but did not mimic the enhanced antigen‐induced degranulation observed in A2BKO BMMCs. Thus, our study confirmed the pro‐inflammatory role of mast cell A2BAR and demonstrated anti‐inflammatory actions of A2BAR antagonists.Supported by NIH.

Mast cell adenosine receptor characteristics and signaling.

Adenosine appears to be an extremely important contributor to asthma and allergic inflammation. Mast cells, cells thought to be central to immediate hypersensitivity reactions, release markedly more histamine and other granule-associated mediators when activated in the presence of micromolar concentrations of adenosine.1,2 The release of newly-generated mediators such as the leukotrienes and prostaglandins is potentiated modestly by adenosine as well. Hypoxic lung and stimulated mast cells3 release adenosine, providing a ready local source of this molecule during allergic pulmonary inflammation.

Canine mast cell adenosine receptors: cloning and expression of the A3 receptor and evidence that degranulation is mediated by the A2B receptor.

We cloned and characterized the canine A3 adenosine receptor (AR) and examined AR-induced degranulation of the BR line of canine mastocytoma cells. Canine A3AR transcript is found predominantly in spleen, lung, liver, and testes and encodes a 314-amino acid heptahelical receptor. 125I-N6-Aminobenzyladenosine binds to two affinity states of canine A3AR with KD values of 0.7 +/- 0.1 and 16 +/- 0.8 nM, reflecting G protein-coupled and -uncoupled receptors, respectively. Xanthine antagonists bind with similar affinities to human, canine, and rabbit receptors but with 80-400-fold lower affinities to rat A3AR. Although canine BR mastocytoma cells contain A1AR, A2BAR, and A3AR, degranulation seems to be mediated primarily by A2BARs stimulated by the nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA) but not by the A3-selective agonist N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide. NECA-stimulated degranulation is not prevented by pertussis toxin and is blocked by enprofylline (Ki = 7 microM), an antiasthmatic xanthine with low affinity (Ki > 100 microM) for A1AR, A2AAR, and A3AR. NECA increases canine mastocytoma cell cAMP, Ca2+, and inositol trisphosphate levels; these responses are antagonized half-maximally by 7-15 microM enprofylline. The results suggest that (i) the cloned canine A3AR is structurally and pharmacologically more similar to human than to rat A3AR; (ii) the A2BAR, and not the A1AR or A3AR, is principally responsible for adenosine-mediated degranulation of canine BR mastocytoma cells; and (iii) the BR cell A2BAR couples to both Ca2+ mobilization and cAMP accumulation. Although A2B receptors play a major role in the regulation of BR mast cell degranulation, multiple AR subtypes and G proteins may influence mast cell functions.

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.

323 Importance of G q protein function in mast cell adenosine receptor mediated calcium mobilization

323 Importance of G q protein function in mast cell adenosine receptor mediated calcium mobilization

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.

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.

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 β-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-methylpiperazine dihydrochloride (H-7) and sphingosine] demonstrated diverse responses in this system. Up to 100 μM H-7 failed to affect mast cell β-hexosaminidase release in the presence or absence of PMA and secretagogue. Sphingosine (10 μM) 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 C 4 (LTC 4) 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.

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.

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 2hr exhibited an inhibition of antigen-induced β-hexosaminidase and leukotriene C 4 release. The ability of adenosine to potentiate β-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 β-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.

83 PHORBOL ESTERS ABROGATE MAST CELL ADENOSINE RESPONSIVENESS

Adenosine potentiates the release of preformed mediators such as β-hexosaminidase (β-hex) from stimulated mast cells by a mechanism that is poorly understood. Mouse bone marrow-derived mast cells incubated for 2 hours with nanomolar concentrations of 4β-phorbol 12β-myristate 13αacetate (PMA) exhibited a marked suppression of the ability of adenosine to augment β-hex release induced by specific antigen or the calcium ionophore, A23187. Antigen-induced mediator release itself was also suppressed by PMA, whereas PMA and A23187 caused a synergistic enhancement of β-hex release. Overnight PMA treatment of mast cells produced a similar hyporesponsiveness to adenosine as well as a striking inhibition of the secretory response to antigen or A23187. The generation of leukotriene C4 was unaffected by overnight PMA. The ability of adenosine to augment mast cell cyclic AMP concentrations was only modestly inhibited by 2-hour or overnight PMA exposure. PMA induced an enhancement of antigen-stimulated intracellular free calcium levels as determined utilizing fura-2, but the ability of adenosine to potentiate this calcium response was completely abrogated. Either protein kinase C activation or a PMA effect on adenosine receptor expression or recycling in some way abolishes the ability of adenosine to augment mediator release. The inability of PMA to affect LTC4 generation underscores the dissociation between preformed and newly generated mediator releases. These data suggest that protein kinase C activity may be a key component of the mechanism of action of adenosine in mast cells.

170 MAST CELL ADENOSINE RECEPTORS, G PROTEINS, AND ADENYLATE CYCLASE INTERACTIONS

Forskolin, cholera toxin, and pertussis toxin may alter various aspects of cell function. Mouse bone marrow-derived mast cells exposed to micromolar concentrations of forskolin, an agent thought to directly activate adenylate cyclase, exhibited an inhibition of antigen-induced β-hexosaminidase (β-hex) release but retained the ability of adenosine to potentiate this release. Forskolin alone augmented resting mast cell cyclic AMP levels in a dose-dependent fashion, but adenosine enhanced cyclic AMP concentrations in both control and forskolin-treated cells. Cholera toxin, an agent that ADP-ribosylates and activates Gs proteins in many cell types, failed to alter β-hex release from antigen-challenged mast cells in the presence or absence of 10uM adenosine. Although cholera toxin caused an elevation of mast cell cyclic AMP levels, adenosine enhanced cyclic AMP to a similar extent in control and toxin-treated cells. Mast cells cultured with pertussis toxin, an agent that ADP-ribosylates the Gi protein in some cells, exhibited an attenuation of antigen-stimulated β-hex release and an inhibition of adenosine responsiveness as well. Although pertussis toxin did not alter resting mast cell cyclic AMP levels, high doses of the toxin inhibited the ability of adenosine to increase cyclic AMP. These data suggest that many agents that alter mast cell cyclic AMP concentrations fail to alter adenosine responsiveness. Pertussis toxin appears to be an exception to this finding, although its exact mechanism of action on mast cell adenosine receptors is uncertain.

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 β-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 μM 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.

Aminophylline exposure alters mouse bone marrow-derived mast cell adenosine responsiveness

Aminophylline-treated mouse bone marrow-derived mast cells exhibit a hyperresponsiveness to adenosine addition (10 −6 to 10 −4 mol/L) at the time of secretagogue challenge coincident with an up regulation of adenosine receptor numbers. This effect on β-hexosaminidase release is maximal at 100 μmol/L of aminophylline, evident after 5 days of aminophylline exposure, and reversed by 6 days after washing. Neither radiolabeled arachidonic acid nor leukotriene C 4 release in the absence or presence of adenosine was altered by aminophylline treatment. Although cAMP levels were dynamically changed by adenosine or secretagogue, these changes were not different in the two cell populations; resting and challenged mast cell adenosine levels were similarly unaffected by aminophylline. The long-term action of aminophylline on mouse bone marrow-derived mast cells appears to be primarily on adenosine receptors and thereby on preformed mediators, and this action may have some importance in the pathophysiology and treatment of asthma.

3H]Adenosine binding to rat mast cells--pharmacologic and functional characterization.

Rat serosal mast cell adenosine receptors were characterized by [3H]adenosine binding to cell membrane particulates, and functional changes in mast cell mediator release and cyclic AMP levels were assessed, utilizing various adenosine analogs. [3H]adenosine binding to sonicated mast cell membrane preparations at 0 degrees C in the presence of deoxycoformycin is linear with initial cell number, rapid and reversible. The cells display 16,400 +/- 1600 high affinity [3H]adenosine binding sites/cell, equivalent to 118 fmol bound/mg protein, with an equilibrium dissociation constant of 27.97 +/- 3.0 nM. Competition studies reveal that adenosine greater than 2-chloroadenosine greater than NECA greater than L-PIA greater than D-PIA in competing for [3H]adenosine binding sites and that aminophylline and cromolyn sodium also bind to the putative receptor. Adenosine and its analogs, NECA, and L-PIA, appear to activate adenylate cyclase in resting mast cells by raising cyclic AMP, suggesting an Ra cell surface adenosine receptor subtype; these same analogs potentiate mast cell B-hexosaminidase release stimulated by specific antigen. The identification of rat mast cell [3H]adenosine binding sites whose stimulation augments resting cell cyclic AMP levels and antigen-induced mediator release suggests that these receptors may be important in the biochemical mechanisms of allergic diseases. The ability to assess the number and affinity of mast cell adenosine receptors will enable one to monitor receptor alterations during pharmacologic manipulation and in disease states.

Modulation of Cyclic AMP in Purified Rat Mast Cells

Abstract Changes in intracellular and extracellular rat mast cell adenosine 3′:5′ monophosphate (cAMP) concentrations during stimulation of histamine release by 48/80 were studied. There was a rapid and progressive fall in intracellular cAMP beginning within 10 sec after the addition of 48/80. The lowest cAMP values were obtained at 10 min, with return to control levels by 30 min. The fall in cAMP was dose-related with progressive decreases in 10-min cAMP measurements as the 48/80 concentration was increased from 0.25 to 1.00 µg/ml. There was a graded increase in histamine release over the same concentration range. Attempts to demonstrate significant amounts of cAMP in the medium during 48/80 stimulation were unsuccessful, indicating that the changes in cAMP intracellularly are not due to altered cellular permeability. There was a general correlation between the ability of pharmacologic agents to sustain high intracellular levels of cAMP in the presence of 48/80, and inhibition of histamine release. Theophylline (20 mM) which increased cAMP levels 2-3-fold prevented a detectable decrease in cAMP after 1 µg/ml 48/80 (measured at 10 min) and almost completely inhibited histamine release. Prostaglandin E1 (27 µM) also raised cAMP levels, decreased the 48/80-induced fall in cAMP (by 31%), and partially inhibited histamine release (by 42%). Epinephrine increased mast cell cAMP levels, but did not prevent the subsequent 48/80-induced decrease in cAMP and did not inhibit histamine release. Carbamylcholine (1 nM), adenine (1 µM), and diazoxide (10 µM) lowered mast cell cAMP and potentiated 48/80 induced release. In view of previous studies from this laboratory indicating that 48/80 stimulates mast cell phosphodiesterase, it seems likely that the 48/80-induced fall in cAMP is due, at least in part, to increased cAMP destruction. Since agents which prevent the fall in cAMP inhibit histamine release, it is apparent that cAMP is an important part of the control mechanism of histamine secretion. On the other hand, it cannot be concluded that a decrease in cAMP alone is sufficient to produce a response since carbamylcholine, diazoxide, and adenine which lower cAMP do not alter histamine release unless 48/80 is also present.