Bruce N. Cronstein

Adenosine receptors: therapeutic aspects for inflammatory and immune diseases

Adenosine is a key endogenous molecule that regulates tissue function by activating four G-protein-coupled adenosine receptors: A1, A2A, A2B and A3. Cells of the immune system express these receptors and are responsive to the modulatory effects of adenosine in an inflammatory environment. Animal models of asthma, ischaemia, arthritis, sepsis, inflammatory bowel disease and wound healing have helped to elucidate the regulatory roles of the various adenosine receptors in dictating the development and progression of disease. This recent heightened awareness of the role of adenosine in the control of immune and inflammatory systems has generated excitement regarding the potential use of adenosine-receptor-based therapies in the treatment of infection, autoimmunity, ischaemia and degenerative diseases.

The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation.

Methotrexate, a folate antagonist, is a potent antiinflammatory agent when used weekly in low concentrations. We examined the hypothesis that the antiphlogistic effects of methotrexate result from its capacity to promote intracellular accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) that, under conditions of cell injury, increases local adenosine release. We now present the first evidence to establish this mechanism of action in an in vivo model of inflammation, the murine air pouch model. Mice were injected intraperitoneally with either methotrexate or saline for 3-4 wk during induction of air pouches. Pharmacologically relevant doses of methotrexate increased splenocyte AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air pouches, and markedly inhibited leukocyte accumulation in inflamed air pouches. The methotrexate-mediated reduction in leukocyte accumulation was partially reversed by injection of adenosine deaminase (ADA) into the air pouch, completely reversed by a specific adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), but not affected by an adenosine A1 receptor antagonist, 8-cyclopentyl-dipropylxanthine. Neither ADA nor DMPX affected leukocyte accumulation in the inflamed pouches of animals treated with either saline or the potent antiinflammatory steroid dexamethasone. These results indicate that methotrexate is a nonsteroidal antiinflammatory agent, the antiphlogistic action of which is due to increased adenosine release at inflamed sites.

Low-Dose Methotrexate: A Mainstay in the Treatment of Rheumatoid Arthritis

Methotrexate administered weekly in low doses is a mainstay in the therapy of rheumatoid arthritis. Although originally developed as a folate antagonist for the treatment of cancer, its mechanism of action in the therapy of rheumatoid arthritis remains less clear. Several mechanisms have been proposed including inhibition of T cell proliferation via its effects on purine and pyrimidine metabolism, inhibition of transmethylation reactions required for the prevention of T cell cytotoxicity, interference with glutathione metabolism leading to alterations in recruitment of monocytes and other cells to the inflamed joint, and promotion of the release of the endogenous anti-inflammatory mediator adenosine. These mechanisms of action and the role of methotrexate in the suppression of rheumatoid arthritis are reviewed.

Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial cells.

Since adenosine and its analogue 2-chloroadenosine prevent neutrophils from generating superoxide anion in response to chemoattractants, we sought to determine whether these agents could inhibit neutrophil-mediated injury of endothelial cells. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM) enhanced the adherence of neutrophils to endothelial cells twofold (18 +/- 2% vs. 39 +/- 3% adherence, P less than 0.001) and caused substantial neutrophil-mediated injury to endothelial cells (2 +/- 2% vs. 39 +/- 4% cytotoxicity, P less than 0.001). 2-Chloroadenosine (10 microM) not only inhibited the adherence of stimulated neutrophils by 60% (24 +/- 2% adherence, P less than 0.001) but also diminished the cytotoxicity by 51% (20 +/- 4% cytotoxicity, P less than 0.002). Furthermore, depletion of endogenously released adenosine from the medium by adenosine deaminase-enhanced injury to endothelial cells by stimulated neutrophils (from 39 +/- 4% to 69 +/- 3% cytotoxicity, P less than 0.001). Indeed, in the presence of adenosine deaminase, even unstimulated neutrophils injured endothelial cells (19 +/- 4% vs. 2 +/- 2% cytotoxicity, P less than 0.001). These data indicate that engagement of adenosine receptors prevents both the adhesion of neutrophils and the injury they cause to endothelial cells. Adenosine inhibits injury provoked not only by cells that have been stimulated by chemoattractants but also by unstimulated cells. Based on this model of acute vascular damage we suggest that adenosine is not only a potent vasodilator, but plays the additional role of protecting vascular endothelium from damage by neutrophils.

Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells.

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.

Methotrexate—how does it really work?

Methotrexate remains a cornerstone in the treatment of rheumatoid arthritis and other rheumatic diseases. Folate antagonism is known to contribute to the antiproliferative effects that are important in the action of methotrexate against malignant diseases, but concomitant administration of folic or folinic acid does not diminish the anti-inflammatory potential of this agent, which suggests that other mechanisms of action might be operative. Although no single mechanism is sufficient to account for all the anti-inflammatory activities of methotrexate, the release of adenosine from cells has been demonstrated both in vitro and in vivo. Methotrexate might also confer anti-inflammatory properties through the inhibition of polyamines. The biological effects on inflammation associated with adenosine release have provided insight into how methotrexate exerts its effects against inflammatory diseases and at the same time causes some of its well-known adverse effects. These activities contribute to the complex and multifaceted mechanisms that make methotrexate efficacious in the treatment of inflammatory disorders.

The Adenosine System Selectively Inhibits TLR-Mediated TNF-α Production in the Human Newborn

Human newborns are susceptible to microbial infection and mount poor vaccine responses, yet the mechanisms underlying their susceptibility are incompletely defined. We have previously reported that despite normal basal expression of TLRs and associated signaling intermediates, human neonatal cord blood monocytes demonstrate severe impairment in TNF-α production in response to triacylated (TLR 2/1) and diacylated (TLR 2/6) bacterial lipopeptides (BLPs). We now demonstrate that in marked contrast, BLP-induced synthesis of IL-6, a cytokine with anti-inflammatory and Th2-polarizing properties, is actually greater in neonates than adults. Remarkably, newborn blood plasma confers substantially reduced BLP-induced monocyte synthesis of TNF-α, while preserving IL-6 synthesis, reflecting the presence in neonatal blood plasma of a soluble, low molecular mass inhibitory factor (<10 kDa) that we identify as adenosine, an endogenous purine metabolite with immunomodulatory properties. The neonatal adenosine system also inhibits TNF-α production in response to whole microbial particles known to express TLR2 agonist activity, including Listeria monocytogenes, Escherichia coli (that express BLPs), and zymosan particles. Selective inhibition of neonatal TNF-α production is due to the distinct neonatal adenosine system, including relatively high adenosine concentrations in neonatal blood plasma and heightened sensitivity of neonatal mononuclear cells to adenosine A3 receptor-mediated accumulation of cAMP, a second messenger that inhibits TLR-mediated TNF-α synthesis but preserves IL-6 production. We conclude that the distinct adenosine system of newborns polarizes TLR-mediated cytokine production during the perinatal period and may thereby modulate their innate and adaptive immune responses.

Colchicine alters the quantitative and qualitative display of selectins on endothelial cells and neutrophils.

Since colchicine-sensitive microtubules regulate the expression and topography of surface glycoproteins on a variety of cells, we sought evidence that colchicine interferes with neutrophil-endothelial interactions by altering the number and/or distribution of selectins on endothelial cells and neutrophils. Extremely low, prophylactic, concentrations of colchicine (IC50 = 3 nM) eliminated the E-selectin-mediated increment in endothelial adhesiveness for neutrophils in response to IL-1 (P < 0.001) or TNF alpha (P < 0.001) by changing the distribution, but not the number, of E-selectin molecules on the surface of the endothelial cells. Colchicine inhibited stimulated endothelial adhesiveness via its effects on microtubules since vinblastine, an agent which perturbs microtubule function by other mechanisms, diminished adhesiveness whereas the photoinactivated colchicine derivative gamma-lumicolchicine was inactive. Colchicine had no effect on cell viability. At higher, therapeutic, concentrations colchicine (IC50 = 300 nM, P < 0.001) also diminished the expression of L-selectin on the surface of neutrophils (but not lymphocytes) without affecting expression of the beta 2-integrin CD11b/CD18. In confirmation, L-selectin expression was strikingly reduced (relative to CD11b/CD18 expression) on neutrophils from two individuals who had ingested therapeutic doses of colchicine. These results suggest that colchicine may exert its prophylactic effects on cytokine-provoked inflammation by diminishing the qualitative expression of E-selectin on endothelium, and its therapeutic effects by diminishing the quantitative expression of L-selectin on neutrophils.

THE MECHANISM OF ACTION OF METHOTREXATE

Because of methotrexates well-documented efficacy in the treatment of rheumatoid arthritis, it is important that we understand the mechanism of action of this drug. There are two biochemical mechanisms by which methotrexate may modulate inflammation: (1) promotion of adenosine release and (2) inhibition of transmethylation reactions. Evidence is reviewed that favors the notion that the endogenous anti-inflammatory autocoid adenosine mediates the anti-inflammatory effects of methotrexate. This insight should aid in the design of new agents for the treatment of rheumatoid arthritis and other inflammatory diseases.

Adenosine A2A receptors play a role in the pathogenesis of hepatic cirrhosis

1 Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. 2 As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl−1) and methotrexate (100 nM). 3 Adenosine A2A receptors are expressed on rat and human hepatic stellate cell lines and adenosine A2A receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl4) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg−1 in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. 4 Adenosine A2A receptor‐deficient, but not wild‐type or A3 receptor‐deficient, mice are protected from development of hepatic fibrosis following CCl4 or thioacetamide exposure. 5 Similarly, caffeine (50 mg kg−1 day−1, po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg−1 bid), a more selective antagonist of the adenosine A2A receptor, diminished hepatic fibrosis in wild‐type mice exposed to either CCl4 or thioacetamide. 6 These results demonstrate that hepatic adenosine A2A receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis.