Adenosine, acting at its receptors, particularly A2A receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A2A receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680), a selective A2A receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A2A receptor antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-α (100 U/ml) but markedly attenuated in cells pretreated with IFN-γ (100 U/ml). Similarly, IL-1 and TNF-α treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-γ treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-α-treated cells but not in control or IFN-γ-treated cells. Both IL-1 and TNF-α increased A2A receptor mRNA and protein. In parallel with its effect on A2A receptor function, IFN-γ down-regulated A2A receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.

We have reported previously that interleukin-1 and tumor necrosis factor (TNF)-␣ increase expression and function of adenosine A 2A receptors (A 2A Rs), although the increased function is disproportionate to the increment in expression. We therefore studied the effect of TNF-␣ on A 2A R function and desensitization in human monocytoid THP-1 cells. We observed that TNF-␣ regulates activity of A 2A Rs and other G protein-coupled receptors (GPCRs) by altering their ligand-mediated desensitization. Pretreatment of resting cells with the A 2A R agonist 2-[p-(2-carboxyethyl)phenethylamino]-5Ј-N-ethylcarboxamidoadenosine (CGS 21680) or the pan-adenosine receptor agonist 5Ј-N-ethylcarboxamidoadenosine quickly desensitized cAMP responses to CGS 21680 restimulation, but TNF-␣ treatment prevented A 2A R desensitization. As expected, A 2A R occupancy induced translocation of GPCR kinase-2 (GRK2) to the plasma membrane (PM). We were surprised to find that after TNF-␣ treatment, A 2A R occupancy not only failed to induce GRK2 translocation to PM but also decreased GRK2 association with PM. TNF-␣ altered GRK2 translocation in response to the ␤-adrenergic receptor agonist isoproterenol in a similar manner. Similar to GRK2, ␤-arrestin associated with PM after A 2A R stimulation in control cells but not in TNF-␣-treated cells. C 2 -ceramide, a downstream mediator in the sphingomyelinase (SMase)-dependent pathway, mimicked the effect of TNF-␣ on GRK2 translocation. Moreover, inhibitors of the SMases and an inhibitor of c-Jun NH 2 -terminal kinase, also a downstream effector in the SMase pathway, reversed TNF-␣-mediated effects on GRK2 translocation and A 2A R desensitization. These results suggest a novel form of cross-talk between TNF-␣ receptors and GPCRs; TNF-␣ enhances GPCR function by preventing agonist-induced desensitization of GPCRs by diminishing agonist-dependent recruitment of GRK2 and ␤-arrestin to PM by a SMase pathway-mediated mechanism.

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Scrub typhus, caused by the intracellular bacterium Orientia tsutsugamushi, is a major cause of febrile illness in the Asia/Pacific region. Here, we implemented a novel real-time PCR and determined the relation of DNA target gene concentration with serum cytokine levels. The limit of detection of the novel real-time PCR was 1,062 DNA copies per ml of EDTA whole blood. Specificity was excellent as determined on a panel of blood-and skin-borne bacteria, including Rickettsia spp. as well as healthy Vietnamese blood donors. Bacterial DNA concentrations after 9 to 12 days from symptoms onset were significantly higher than in earlier or later periods (P < 0.05). Significantly higher concentrations of gamma interferon (IFN-␥) and interleukin-10 (IL-10) occurred during the acute phase of disease (<10 days from onset) as opposed to the convalescent phase (P < 0.05). No significant differences were observed between the acute and the convalescent phases for tumor necrosis factor alpha (TNF-␣) and IL-1␤ concentrations. Regression analysis of DNA concentrations and cytokine levels identified a significant positive relationship for IL-10 (P < 0.0182) but not for IFN-␥, TNF-␣, and IL-1␤. In conclusion, proinflammatory cytokines and IL-10 were differentially related to human bacteremia. They may thus be induced by different constituents of O. tsutsugamushi. As a future prospect in a clinical diagnostic laboratory, quantitative real-time PCR may serve as a reliable tool to monitor therapy and to detect treatment failure.

We and others have shown that adenosine, acting at its receptors, is a potent modulator of inflammation and angiogenesis. To better understand the regulation of adenosine receptors during these processes we studied the effects of IL-1, TNF-α, and IFN-γ on expression and function of adenosine receptors and select members of their coupling G proteins in human dermal microvascular endothelial cells (HMVEC). HMVEC expressed message and protein for A2A and A2B, but not A1 or A3 receptors. IL-1 and TNF-α treatment increased message and protein expression of A2A and A2B receptor. IFN-γ treatment also increased the expression of A2B receptors, but decreased expression of A2A receptors. Resting HMVEC and IFN-γ-treated cells showed minimal cAMP response to the selective A2A receptor agonist 2-[2-(4-chlorophenyl)ethoxy]adenosine (MRE0094). In contrast, MRE0094 stimulated a dose-dependent increase in cAMP levels in TNF-α-treated cells that was almost completely blocked by the A2A receptor antagonist ZM-241385 (4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol). The nonselective adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine increased cAMP levels in both TNF-α- and IFN-γ-treated cells, but not control cells, and its effect was only partially reversed by ZM-241385 in TNF-α-treated cells and not affected in IFN-γ-treated cells. HMVEC expressed a higher level of G protein β1 isoform than β4 isoform. Although none of the cytokines tested affected Gβ1 expression, both IL-1 and TNF-α significantly up-regulated Gβ4 expression. These findings indicate that inflammatory cytokines modulate adenosine receptor expression and function on HMVECs and suggest that the interaction between proinflammatory cytokines and adenosine receptors may affect therapeutic responses to anti-inflammatory drugs that act via adenosine-dependent mechanisms.

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Congenital hyperinsulinism (CHI) is related to two main histological pancreas anomalies: focal adenomatous hyperplasia and diffuse beta-cell hypersecretion. Pharmacological tests to measure acute insulin responses (AIR) to peripheral i.v. injections of glucose, calcium, and tolbutamide have been reported as potential means to distinguish between these histological forms. In patients with defects in ATP-sensitive potassium channels, tolbutamide will fail to induce insulin release in affected portions of the pancreas, whereas calcium gluconate will enhance insulin release through spontaneously active voltage-gated Ca(2+) channels. Consequently, in focal CHI patients, calcium should promote AIRs from the lesion, whereas tolbutamide should act to promote insulin secretion from the healthy region of the pancreas (outside the focal hyperplasia). We therefore studied AIRs to calcium and tolbutamide stimulation tests in 16 children with focal (n = 9) or diffuse (n = 7) CHI before pancreatic surgery. We found hypervariable AIRs to glucose and calcium stimulation in both focal and diffuse CHI patients. AIRs to tolbutamide stimulation were found modest in focal CHI patients, which might account for beta-cell quiescence in the healthy portion of the pancreas of these patients. We conclude that AIRs to calcium and tolbutamide stimulation tests are not sufficient to differentiate the focal from the diffuse CHI patients.