Cooperation of adenosine and prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) in amplification of cAMP–PKA signaling and immunosuppression

INTRODUCTION: We hypothesize that adenosine and PGE(2) could have a complementary immunosuppressive effect that is mediated via common cAMP-PKA signaling. MATERIALS AND METHODS: To test this hypothesis, the effect of adenosine and PGE(2) on the cytotoxic activity and cytokine production of lymphokine activated killer (LAK) cells was investigated. RESULTS: PGE(2) and adenosine inhibited LAK cells cytotoxic activity and production of INF-gamma, GM-CSF and TNF-alpha. In combination they showed substantially higher inhibition than each modality used alone. Using agonists and antagonists specific for PGE(2) and adenosine receptors we found that cooperation of PGE(2) and adenosine in their inhibitory effects are mediated via EP(2) and A(2A) receptors, respectively. LAK cells have 35-fold higher expression of EP(2) than A(2A). Combined PGE(2) and adenosine treatment resulted in augmentation of cAMP production, PKA activity, CREB phosphorylation and inhibition of Akt phosphorylation. Wortmannin and LY294002 enhanced the suppressive effects of adenosine and PGE(2). In contrast, Rp-8-Br-cAMPS, an inhibitor of PKA type I blocked their immunosuppressive effects, suggesting that the inhibitory effects of PGE(2) and adenosine are mediated via common pathway with activation of cAMP-PKA and inhibition of Akt. CONCLUSION: In comparison to other immunosuppressive molecules (TGF-beta and IL-10), adenosine and PGE(2) are unique in their ability to inhibit the executive function of highly cytotoxic cells. High intratumor levels of adenosine and PGE(2) could protect tumor from immune-mediated destruction by inactivation of the tumor infiltrating functionally active immune cells.     

Effects of stable adenosine receptor agonists on bone marrow hematopoietic cells as inferred from the cytotoxic action of 5-fluorouracil

The aim of the study was to investigate the effects of stable adenosine receptor agonists on bone marrow hematopoiesis by utilizing the model of hematopoietic damage induced by 5-fluorouracil (5-FU), a cycle-specific cytotoxic agent. Effects of a non-selective agonist NECA activating all the known adenosine receptors (A1, A2A, A2B, A3) and of the selective agonists for A1 (CPA), A2A (CGS 21680), and A3 (IB-MECA) adenosine receptors were investigated. Experiments were performed with B10CBAF1 mice under in vivo conditions. Adenosine receptor agonists were given in single injections before 5-FU administration and the effects were determined 4 days later. The numbers of femoral marrow nucleated cells and hematopoietic progenitor cells (CFC-GM and BFU-E) were taken as indices of the effects. The non-selective agonist NECA given at a dose of 200 nmol/kg induced biphasic time-dependent effects, i.e. protection and sensitization, when given 10 h and 22 h before 5-FU administration, respectively. The use of isomolar doses of selective receptor agonists indicated that the protective effects of NECA were induced by activation of A2A and A2B receptors, while the sensitizing action of NECA was mediated via A3 receptors. In addition, it was observed that A1 receptors induced protection when activated by administration of CPA 22 h before 5-FU. These findings are discussed with respect to the action of adenosine receptor agonists on the cell cycle state and on the cell cycle-independent cellular protective mechanisms.     

Selective, high affinity A(2B) adenosine receptor antagonists: N-1 monosubstituted 8-(pyrazol-4-yl)xanthines

A series of N-1 monosubstituted 8-pyrazolyl xanthines have been synthesized and evaluated for their affinity for the adenosine receptors (AdoRs). We have discovered two compounds 18 (CVT-7124) and 28 (CVT-6694) that display good affinity for the A(2B) AdoR (K(i)=6 nM and 7 nM, respectively) and greater selectivity for the human A(1), A(2A), and A(3) AdoRs (>1000-, >830-, and >1500-fold; >850-, >700-, and >1280-fold, respectively). CVT-6694 has been shown to block the release of interleukin-6 and monocyte chemotactic protein-1 from bronchial smooth muscle cells (BSMC), a process believed to be promoted by activation of A(2B) AdoR.     

Both A1 and A2a Purine Receptors Regulate Striatal Acetylcholine Release

The receptors responsible for the adenosine-mediated control of acetylcholine release from immunoaffinity-purified rat striatal cholinergic nerve terminals have been characterized. The relative affinities of three analogues for the inhibitory receptor were (R)-phenylisopropyladenosine greater than cyclohexyladenosine greater than N-ethylcarboxamidoadenosine (NECA), with binding being dependent of the presence of Mg2+ and inhibited by 5'-guanylylimidodiphosphate [Gpp(NH)p] and adenosine receptor antagonists. Adenosine A1 receptor agonists inhibited forskolin-stimulated cholinergic adenylate cyclase activity, with an IC50 of 0.5 nM for (R)-phenylisopropyladenosine and 500 nM for (S)-phenylisopropyladenosine. A1 agonists inhibited acetylcholine release at concentrations approximately 10% of those required to inhibit the cholinergic adenylate cyclase. High concentrations (1 microM) of adenosine A1 agonists were less effective in inhibiting both adenylate cyclase and acetylcholine release, due to the presence of a lower affinity stimulatory A2 receptor. Blockade of the A1 receptor with 8-cyclopentyl-1,3-dipropylxanthine revealed a half-maximal stimulation by NECA of the adenylate cyclase at 10 nM, and of acetylcholine release at approximately 100 nM. NECA-stimulated adenylate cyclase activity copurified with choline acetyltransferase in the preparation of the cholinergic nerve terminals, suggesting that the striatal A2 receptor is localized to cholinergic neurones. The possible role of feedback inhibitory and stimulatory receptors on cholinergic nerve terminals is discussed.     

Adenosine as substrate and receptor agonist in the ovary

In the present study the possible dual effects of adenosine as substrate and adenosine receptor agonist in rat granulosa cells, cumulus-oocyte complexes, luteal cells and ovarian membranes are discussed. Adenosine is an indispensable compound in cell energy metabolism, as precursor to cofactors, second messenger and nucleic acids. Adenosine is also an agonist to adenosine receptors. The adenosine receptor can either inhibit (A1) or stimulate (A2) adenylate cyclase. Alternatively, in some cells adenosine receptor activation is linked to other cellular events like inhibition of Ca2+ fluxes. Adenosine is taken up by isolated preovulatory granulosa and luteal cells from pregnant mare serum gonadotropin-treated immature rats, but follicle stimulating hormone (FSH) decreases the uptake by granulosa cells. Adenosine, but not the non-metabolizable adenosine analogs 5'-(N-ethyl)carboxamide-adenosine (NECA), 2-chloro-adenosine (2-Clado), N6-(R-phenyl-isopropyl)-adenosine (R-PLA) and N6-(S-phenyl-isopropyl)-adenosine (S-PLA), increase granulosa cell ATP levels. FSH and luteinizing hormone (LH) decrease granulosa cell ATP levels in the presence or absence of adenosine. It has previously been shown that FSH and LH decrease oxygen consumption by cumulus-oocyte complexes and increase their lactate production. These effects have been suggested to be due to a competition of cofactors (e.g. ADP) common to glycolysis and the respiratory chain. The fact that adenosine reverse the gonadotropin-induced effects on oxygen consumption and lactate production support this theory. Adenosine and its analogs increase cAMP accumulation in luteal and granulosa cells only in the presence of gonadotropins, and this effect is antagonized by the adenosine receptor antagonist 8-phenyl-theophylline (8-PHT). Furthermore, adenylate cyclase is stimulated by adenosine analogs in membranes from non-luteinized and luteinized ovarian membranes and in luteal cell homogenates. The effect of NECA is antagonized by 8-PHT. In the membranes, the rank order of potency was NECA greater than 2-Clado greater than R-PLA greater than S-PLA, suggesting adenosine A2 receptors. In summary, it is suggested that adenosine can act both as a substrate to intracellular metabolism and as an adenosine A2 receptor agonist in granulosa and luteal cells. A paracrine short loop positive feedback model is proposed where extracellular adenosine, derived from a gonadotropin-induced extracellular increase in cAMP and a decrease in cellular ATP, enhances gonadotropin stimulation in granulosa and luteal cells.     

Receptor desensitization and blockade of the suppressive effects of prostaglandin E<Subscript>2</Subscript> and adenosine on the cytotoxic activity of human melanoma-infiltrating T lymphocytes

Previous studies document that PGE₂ and adenosine suppress production of inflammatory cytokines. The present study demonstrates for the first time that (1) PGE₂ and 2-chloroadenosine (CADO; a stable analog of adenosine) directly inhibit the cytolytic function of human tumor-infiltrating lymphocytes (TILs); (2) the combination PGE₂ and CADO have additive suppressive effects; and (3) the cooperative immunosuppressive actions of PGE₂ and CADO are mediated via EP2 receptors (EP2Rs) and A_2A receptors (A_2ARs) and are due to amplification of cAMP production, activation of protein kinase A (PKA) and T cell receptor (TCR) inhibitor Csk leading to inhibition of Lck, ZAP-70 and Akt phosphorylation. (4) During ex vivo expansion, TILs undergo three stages of differentiation converting from TILs with high cytotoxic activity and relative resistance to combined EP2R/A_2AR suppression (stage I) to TILs retaining high cytotoxicity and gaining sensitivity to combined suppression (stage II) and then to TILS that are less cytotoxic and very sensitive to combined suppression (stage III). (5) Finally, we find that pretreatment of TILs with non-inhibitory concentrations of EP2R agonists (such as PGE₂ or butaprost) or A_2AR agonists (such as CADO or CGS21680) increases their cytotoxic activity and induces resistance to EP2R and A_2AR inhibitory signaling (cross-resistance) due to homologous and heterologous desensitization and internalization of EP2Rs and A_2ARs, thus preventing their inhibitory signaling. We conclude that inducing resistance of TILs to the suppressive effects of PGE₂ and adenosine in the tumor microenvironment could represent a novel strategy for improving the efficacy of adoptive immunotherapy.     

Stimulation of glycogenolysis and vasoconstriction by adenosine and adenosine analogues in the perfused rat liver.

Infusion of adenosine into perfused rat livers resulted in transient increases in glucose output, portal-vein pressure, the effluent perfusate [lactate]/[pyruvate] ratio, and O2 consumption. 8-Phenyltheophylline (10 microM) inhibited adenosine responses, whereas dipyridamole (50 microM) potentiated the vasoconstrictive effect of adenosine. The order of potency for adenosine analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than L-phenylisopropyladenosine greater than cyclohexyladenosine greater than D-phenylisopropyladenosine greater than 2-chloroadenosine greater than adenosine, consistent with adenosine actions modulated through P1-purine receptors of the A2-subtype. Hepatic responses exhibited homologous desensitization in response to repeated infusion of adenosine. Adenosine effects on the liver were attenuated at lower perfusate Ca2+ concentrations. Indomethacin decreased hepatic responses to both adenosine and NECA. Whereas adenosine stimulated glycogen phosphorylase activity in isolated hepatocytes, NECA caused no effect in hepatocytes. The response to adenosine in hepatocytes was inhibited by dipyridamole (50 microM), but not 8-phenyltheophylline (10 microM). The present study indicates that, although adenosine has direct effects on parenchymal cells, indirect effects of adenosine, mediated through the A2-purinergic receptors on another hepatic cell type, appear to play a role in the perfused liver.     

The discovery of a selective, high affinity A(2B) adenosine receptor antagonist for the potential treatment of asthma

Adenosine has been suggested to play a role in asthma, possibly via activation of A(2B) adenosine receptors on mast cells and other pulmonary cells. We describe our initial efforts to discover a xanthine based selective A(2B) AdoR antagonist that resulted in the discovery of CVT-5440, a high affinity A(2B) AdoR antagonist with good selectivity (A(2B) AdoR K(i)=50 nM, selectivity A(1)>200: A(2A)>200: A(3)>167).     

Cross-regulation of carbon monoxide and the adenosine A2a receptor in macrophages

Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-alpha production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-alpha. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.     

Adenosine inhibits divalent cation influx across human neutrophil plasma membrane via surface adenosine A2 receptors.

Adenosine and its analogues inhibited increases in divalent cation influx stimulated by platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (FMLP) in a dose-dependent fashion. This effect was antagonized by theophylline, an adenosine receptor antagonist. When extracellular adenosine was removed by adenosine deaminase, the effect of adenosine was completely abolished. Two adenosine analogues with different affinities for adenosine receptor subtypes, 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (PIA), also inhibited divalent cation influx, NECA being more potent than PIA. These results suggest that adenosine and its analogues inhibit divalent cation influx across neutrophil plasma membranes via surface adenosine A2 receptors. Adenosine had little effect on the initial peaks of intracellular free calcium rises induced by chemoattractants, but it inhibited the subsequent rise in free calcium. Since calcium influx through the divalent cation channels or neutrophil plasma membranes is responsible for maintaining free calcium concentration following the initial peaks, we suggest that adenosine modulates neutrophil function by interfering with this calcium influx.     

Functional consequences of cAMP accumulation in human natural killer cells. Implications for IL-2 and IL-4 signal transduction

To approach the mechanisms whereby IL-2 activates human NK cells, we have compared the effects of IL-4 and of Bt2cAMP on this activation. Both agents block completely the proliferation induced by IL-2 on highly purified CD3-negative human NK cells. We also report that the net LAK response of PBL is inhibited by IL-4 and cAMP. However, kinetics analysis showed that IL-4 appears to inhibit an early stage of IL-2-induced activation of NK cells. IL-4 does not affect the cytotoxicity of freshly isolated NK cells against the K562 target and is ineffective on IL-2-preactivated cells. In contrast, cAMP primarily blocks the lytic effector phase, whether cells have been cultured in IL-2 or not, and its effect appears independent of time of addition. These differences between the activity of IL-4 and cAMP suggested that cAMP was not directly involved in IL-4 signal transduction in human NK cells. Consistent with this interpretation, we did not observe any variation in the level of intracellular cAMP concentrations when NK cells were stimulated with IL-4, or when they are stimulated with IL-2 or IL-2 plus IL-4. In addition, we also demonstrate that NK cell cytotoxic activation induced by IL-2 is still demonstrable in the presence of Bt2cAMP under conditions in which NK cell proliferation is blocked. These results clearly indicate that the differentiative effect of IL-2 on NK cells is independent of cell proliferation. Furthermore, the p70-75 IL-2R-initiated signal transduction pathway that leads to increased cytotoxicity appears not to be susceptible to inhibition by cAMP in human NK cells.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

Occupancy of adenosine receptors raises cyclic AMP alone and in synergy with occupancy of chemoattractant receptors and inhibits membrane depolarization.

We have recently demonstrated that adenosine, acting via adenosine A2 receptors, inhibits generation of superoxide anions (O2-) by stimulated neutrophils. To determine the mechanism(s) by which adenosine inhibits O2- generation stimulated by the chemoattractant N-formylmethionylleucylphenylalanine (FMLP), we examined cyclic AMP (cAMP) concentrations, stimulated membrane depolarization and Ca2+ movements. Neither adenosine nor 5'-N-ethylcarboxamidoadenosine (NECA), the most potent agonist at adenosine A2 receptors, increases neutrophil cAMP content. However in the presence of the non-methylxanthine phosphodiesterase inhibitor, Ro-20-1724, both adenosine and NECA elicit a reversible increase in intracellular cAMP concentration. The chemoattractant FMLP also elicits an increment in the neutrophil cAMP content. NECA, in the presence of Ro-20-1724, synergistically enhances the increment in cAMP following stimulation by FMLP. However Ro-20-1724 does not potentiate the inhibition of O2- generation by NECA. Unlike other agents which increase neutrophil cAMP concentrations, NECA, even in the presence of a phosphodiesterase inhibitor, only trivially inhibits degranulation. We also found that adenosine markedly inhibits stimulated membrane depolarization but does not affect the stimulated increment in free ionized intracellular calcium. Moreover, inhibition by adenosine of O2- generation does not vary with the concentration of extracellular calcium. These results fulfil the last criterion for the demonstration of an A2 receptor on human neutrophils, and indicate that adenosine occupies an A2 receptor on neutrophils to raise intracellular cAMP in synergy with occupancy of the FMLP receptor. The results reported here also indicate that cAMP is not the second messenger for inhibition of O2- generation by adenosine and its analogues.     

Adenosine: a novel ulcer modulator in stomachs.

Adenosine has been demonstrated for its actions on gastric secretion and stress-induced gastric ulceration in animals. We examined the pharmacological actions of adenosine on ethanol-evoked gastric lesions and gastric mucosal blood flow (GMBF) in rats, because both of them are closely related. Adenosine pretreatment, in dose of 7.5 mg/kg increased GMBF and protected against ethanol-evoked gastric lesion formation. However, this antiulcer action was followed by an aggravation of gastric lesions and reduction in GMBF. We further investigated whether these actions could act through the adenosine A1 or A2 receptors, therefore L-phenylisopropyladenosine (L-PIA) or N-ethylcarboxamidoadenosine (NECA), the adenosine A1 or A2 receptor agonists, respectively, were used. The drugs given in doses of 10 or 50 micrograms/kg for L-PIA and 1 or 5 micrograms/kg for NECA, dose-dependently inhibited GMBF and potentiated ethanol-induced gastric damage. When the two drugs were given together to animals, they did not further aggravate the severity of ulceration and reduction of GMBF. These findings indicate that the antiulcer action of adenosine is not mediated via the adenosine A1 and A2 receptors but if acts through different adenosine receptor subtypes. It was because the lesion worsening effects of adenosine at the second stage of the biphasic responses were similar to the actions of L-PIA and NECA, the ulcer potentiating effect is probably acting through adenosine A1 and A2 receptors in anaesthetised rats.     

Adenosine and its analogues, possibly acting at A2 receptors, stimulate mouse sperm fertilizing ability during early stages of capacitation

Earlier studies have provided indirect evidence that the availability of endogenous adenosine can modulate the fertilizing ability of mouse spermatozoa during capacitation. More direct evidence has been sought by evaluating the effect of exogenous adenosine present during the early stages of capacitation. A concentration-dependent stimulation of in-vitro fertilizing ability was observed, with 10 microM- and 100 microM-adenosine significantly increasing the proportion of eggs fertilized compared with drug-free controls. The adenosine-induced stimulation was observed in the presence of 0.01 microM- and 0.1 microM-dipyridamole, an inhibitor of adenosine uptake, suggesting that adenosine is acting at an external site. Comparison of adenosine with its analogues 2'-deoxyadenosine and 2-chloroadenosine indicated that the analogues at 10 microM were able to stimulate fertilization in a manner similar to adenosine. While neither adenosine nor 2'-deoxyadenosine was consistently effective at 1 microM, 2-chloroadenosine significantly stimulated fertilization at both 1 microM and 0.1 microM. In addition, 5'-N-ethylcarboxamidoadenosine (NECA) and (R)-N6-phenylisopropyladenosine (R-PIA), potent analogues in somatic cell systems, proved to be so with mouse sperm suspensions, NECA being stimulatory at greater than or equal to 0.01 microM and R-PIA at greater than or equal to 0.1 microM. Subjective evaluation of motility patterns indicated that more cells exhibited hyperactivated motility in the presence of stimulatory concentrations of adenosine or analogues. Assessment of capacitation state using chlortetracycline fluorescence patterns indicated that incubation in 2'-deoxyadenosine resulted in significantly fewer cells expressing the uncapacitated F pattern and significantly more cells with the capacitated AR (acrosome-reacted) pattern, compared with drug-free counterparts. It is concluded that adenosine promotes capacitation by interacting with externally-directed receptors, possibly on adenylate cyclase to increase the intracellular availability of cyclic adenosine monophosphate (cAMP); cAMP is known to stimulate mouse sperm fertilizing ability. The greater sensitivity to NECA, 2-chloroadenosine and R-PIA, relative to adenosine and 2'-deoxyadenosine, is consistent with interaction at stimulatory A2 adenosine receptors.     

Interleukin 2 induces human acute lymphocytic leukemia cells to manifest lymphokine-activated-killer (LAK) cytotoxicity

Lymphokine-activated killer cells (LAK) were originally distinguished from natural killers (NK) and cytotoxic T lymphocytes. Recently, however, IL 2-activated NK cells were suggested as the major source of LAK reactivity in human peripheral blood (PBL). Because certain T cell acute lymphoblastic leukemia (T-ALL) cells are phenotypically similar to LAK precursors, we have asked whether these leukemic cells can be induced toward LAK-cytotoxicity and express NK reactivity before stimulation. Five out of seven T-ALL preparations were induced by IL 2 to kill target cells. The cytotoxicity of the leukemic-LAK cells resembled that of normal LAK effectors as they lysed efficiently the NK-resistant target Daudi, as well as fresh human sarcoma, carcinoma, and renal cancer cells but not normal PBL. The ALL-LAK precursors phenotype was T3-, T4-, T8-, and T11+, similar to most normal LAK precursors. In contrast to normal PBL that generated LAK effectors when their proliferation was inhibited, the irradiated, nonproliferating T-ALL leukemic cells did not respond to IL 2. Therefore, the T-ALL LAK cytotoxicity was attributed to the leukemic cells rather than to residual normal lymphocytes. The IL 2-responding T-ALL cells did not express autonomous NK type cytotoxicity, suggesting that they reflect LAK precursors of non-NK origin. The homogeneous leukemic preparations with inducible LAK cytotoxicity described herein provide a model system for studying normal LAK cells.     

Adenosine modulates LPS-induced cytokine production in porcine monocytes

Adenosine plays an important role during inflammation, particularly through modulation of monocyte function. The objective of the present study was to evaluate the effect of synthetic adenosine analogs on cytokine production by porcine monocytes. The LPS-stimulated cytokine production was measured by flow cytometry and quantitative real-time PCR. Adenosine receptor expression was measured by quantitative real-time PCR. The present study demonstrates that adenosine analog N-ethylcarboxyamidoadenosine (NECA) down-regulates TNF-α production and up-regulates IL-8 production by LPS-stimulated porcine monocytes. The effect was more pronounced in CD163^? subset of monocytes compared to the CD163⁺ subset. Although both monocyte subsets express mRNA for A1, A2A, A2B and A3 adenosine receptors, the treatment of monocytes with various adenosine receptor agonists and antagonists proved that the effect of adenosine is mediated preferentially via A2A adenosine receptor. Moreover, the study suggests that the effect of NECA on porcine monocytes alters the levels of the cytokines which could play a role in the differentiation of naive T cells into Th17 cells. The results suggest that adenosine plays an important role in modulation of cytokine production by porcine monocytes.     

Modification of cytokine milieu by A2A adenosine receptor signaling--possible application for inflammatory diseases

Pro-inflammatory cytokine TNF-alpha (TNF) production from in vitro lipopolysaccharide (LPS)-stimulated human peripheral blood CD14+ cells (PB-CD14) was inhibited by A2A adenosine receptor (AdoR) (A2AR) or beta2 adrenergic receptor (ADR) (beta2R) signaling in a concentration-dependent manner. These inhibitory effects were presumably mediated by the increase in intracellular cAMP. Furthermore A2AR agonist and beta2R agonist synergistically inhibited the TNF production of LPS-stimulated PB-CD14 cells. These results suggest that the anti-inflammatory effect of extracellular adenosine is, at least in part, due to the modification of the cytokine milieu via A2A signaling, and that the targeting of both A2AR and beta2R may have strong therapeutic potential for the inflammatory diseases.