The effects of caffeine on bone mineral density and fracture risk.

Caffeine is a regular part of the diet of many adults (coffee, tea, soft drinks, and energy drinks). Multiple molecular effects of caffeine suggest that it may promote bone loss. Given the extensive consumption of caffeine worldwide, any impact of caffeine consumption on bone strength and/or density would have large population health implications. The most well-established pharmacological effect of caffeine is non-specific antagonism of adenosine receptors. Adenosine regulates bone metabolism in a complex manner, with in vitro studies suggesting that direct stimulation of adenosine A2A and A2B receptors induces bone formation by activating osteoblasts and suppressing osteoclast differentiation and function. Thus, competitive inhibition of adenosine A2 receptors by caffeine may inhibit bone formation and promote bone resorption. However, antagonism of adenosine A1 receptors may have opposing effects. Caffeine has also been suggested to affect bone through derangement of calcium metabolism, alteration of vitamin D responses, and other mechanisms. In clinical and population-based studies, the impact of caffeine consumption on bone metabolism offers a mixed picture, with some but not all studies suggesting a potential link between caffeine intake and reduced bone mineral density or increased fracture risk. Differences in methodology, selected populations, and duration/timing of the studies may account for study outcome discrepancies. The in vitro effects of caffeine on cells involved in bone metabolism suggest that caffeine intake may promote osteoporosis, and some but not all clinical studies support a modest adverse caffeine impact. Herein, we describe the basic biology of caffeine as it pertains to bone, review the clinical literature to date, and consider the implications of the current data on clinical practice and future studies.

Adenosine A1 receptor regulates osteoclast formation by altering TRAF6/TAK1 signaling.

Adenosine is an endogenous nucleoside that modulates many physiological processes through four receptor subtypes (A(1), A(2a), A(2b), A(3)). Previous work from our laboratory has uncovered a critical role for adenosine A(1) receptor (A(1) R) in osteoclastogenesis both in vivo and in vitro. Our current work focuses on understanding the details of how A(1) R modulates the receptor activator of NF-κB ligand (RANKL)-induced signaling in osteoclastogenesis. Osteoclasts were generated from mouse bone marrow precursors in the presence of RANKL and macrophage-colony stimulating factor. A pharmacological antagonist of A(1) R (DPCPX) inhibited RANKL-induced osteoclast differentiation, including osteoclast-specific genes (Acp5, MMP9, ß(3) Integrin, α(v) Integrin, and CTSK) and osteoclast-specific transcription factors such as c-fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) expression in a dose-dependent manner. DPCPX also inhibited RANKL-induced activation of NF-κB and JNK/c-Jun but had little effect on other mitogen-activated protein kinases (p38 and Erk). Finally, immunoprecipitation analysis showed that blockade of A(1)R resulted in disruption of the association of tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-ß-activated kinase 1 (TAK1), a signaling event that is important for activation of NF-κB and JNK, suggesting the participation of adenosine/A(1)R in early signaling of RANKL. Collectively, these data demonstrated an important role of adenosine, through A(1)R in RANKL-induced osteoclastogenesis.

Adenosine promotes neutrophil chemotaxis.

We have previously (1-4) demonstrated that adenosine, by engaging specific receptors on the surface of neutrophils, inhibits generation of toxic oxygen metabolites by activated neutrophils and prevents these activated neutrophils from injuring endothelial cells. We now report the surprising observation that engagement of these same neutrophil adenosine receptors promotes chemotaxis to C5 fragments (as zymosan-activated plasma [ZAP]) or to the bacterial chemoattractant FMLP. When chemotaxis was studied in a modified Boyden chamber, physiologic concentrations of adenosine promoted chemotaxis by as much as 60%. Adenosine receptor analogues, 5'N-ethylcarboxamidoadenosine (NECA) and N6-phenylisopropyladenosine (PIA), also promoted chemotaxis; the order of agonist potency was consistent with that of an A2 adenosine receptor (NECA greater than PIA greater than or equal to adenosine). A potent antagonist at adenosine receptors, 8-p-sulfophenyltheophylline (10 microM), completely reversed NECA enhancement of chemotaxis but did not affect chemotaxis by itself. Neither NECA nor 2-chloroadenosine, a nonselective adenosine receptor agonist, alone was chemotactic or chemokinetic by checkerboard analysis. NECA also promoted chemotaxis quantitated by a different technique, chemotaxis under agarose, to the surrogate bacterial chemoattractant FMLP. These data suggest that engagement of adenosine A2 receptors uniquely modulates neutrophil function so as to promote migration of neutrophils to sites of tissue damage while preventing the neutrophils from injuring healthy tissues en route.

Adenosine: a physiological modulator of superoxide anion generation by human neutrophils.

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.

Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.

The complete healing of wounds is the final step in a highly regulated response to injury. Although many of the molecular mediators and cellular events of healing are known, their manipulation for the enhancement and acceleration of wound closure has not proven practical as yet. We and others have established that adenosine is a potent regulator of the inflammatory response, which is a component of wound healing. We now report that ligation of the G alpha s-linked adenosine receptors on the cells of an artificial wound dramatically alters the kinetics of wound closure. Excisional wound closure in normal, healthy mice was significantly accelerated by topical application of the specific A2A receptor agonist CGS-21680 (50% closure by day 2 in A2 receptor antagonists. In rats rendered diabetic (streptozotocin-induced diabetes mellitus) wound healing was impaired as compared to nondiabetic rats; CGS-21680 significantly increased the rate of wound healing in both nondiabetic and diabetic rats. Indeed, the rate of wound healing in the CGS-21680-treated diabetic rats was greater than or equal to that observed in untreated normal rats. These results appear to constitute the first evidence that a small molecule, such as an adenosine receptor agonist, accelerates wound healing in both normal animals and in animals with impaired wound healing.

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.

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.

The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O2 generation, respectively.

Occupancy of specific receptors on neutrophils by adenosine or its analogues diminishes the stimulated release of toxic oxygen metabolites from neutrophils, while paradoxically promoting chemotaxis. We now report evidence that two distinct adenosine receptors are found on neutrophils (presumably the A1 and A2 receptors of other cell types). These adenosine receptors modulate chemotaxis and O2- generation, respectively. N6-Cyclopentyladenosine (CPA), a selective A1 agonist, promoted neutrophil chemotaxis to the chemoattractant FMLP as well as or better than 5'N-ethylcarboxamidoadenosine (NECA). In contrast, CPA did not inhibit O2- generation stimulated by FMLP. Pertussis toxin completely abolished promotion of chemotaxis by CPA but enhanced inhibition by NECA of O2- generation. Disruption of microtubules by colchicine or vinblastine also abrogated the enhancement by NECA of chemotaxis whereas these agents did not markedly interfere with inhibition by NECA of O2- generation. FMLP receptors, once they have bound ligand, shift to a high affinity state and become associated with the cytoskeleton. NECA significantly increased association of [3H]FMLP with cytoskeletal preparations as it inhibited O2-. Disruption of microtubules did not prevent NECA from increasing association of [3H]FMLP with cytoskeletal preparations. Additionally, CPA (A1 agonist) did not increase binding of [3H]FMLP to the cytoskeleton as well as NECA (A2 agonist). These studies indicate that occupancy of one class of adenosine receptors (A1) promotes chemotaxis by a mechanism requiring intact microtubules and G proteins whereas engagement of a second class of receptors (A2) inhibits O2- generation. Signalling via A2 receptors is independent of microtubules, insensitive to pertussis toxin and is associated with binding of [3H]FMLP to cytoskeletal preparations.

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.

Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5'-nucleotidase-mediated conversion of adenine nucleotides.

We and others have shown that an increased extracellular concentration of adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine both in vitro and in vivo, but the mechanism by which these drugs increase extracellular adenosine remains unclear. The results of the experiments reported here provide three distinct lines of evidence that adenosine results from the ecto-5'-nucleotidase- mediated conversion of adenine nucleotides to adenosine. First, pretreatment of a human microvascular endothelial cell line (HMEC-1) with methotrexate increases extracellular adenosine after exposure of the pretreated cells to activated neutrophils; the ecto-5'-nucleotidase inhibitor alpha, beta-methylene adenosine-5'-diphosphate (APCP) abrogates completely the increase in extracellular adenosine. Second, there is no methotrexate-mediated increase in extracellular adenosine concentration in the supernate of cells deficient in ecto-5'-nucleotidase, but there is a marked increase in extracellular adenosine concentration in the supernates of these cells after transfection and surface expression of the enzyme. Finally, as we have shown previously, adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine in the murine air pouch model of inflammation, and injection of APCP, the ecto-5'-nucleotidase inhibitor, abrogates completely the increase in adenosine and the decrement in inflammation in this in vivo model. These results not only show that ecto-5'-nucleotidase activity is a critical mediator of methotrexate- and sulfasalazine-induced antiinflammatory activity in vitro and in vivo but also indicate that adenine nucleotides, released from cells, are the source of extracellular adenosine.

Adenosine 5'-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation.

Human health is under constant threat of a wide variety of dangers, both self and nonself. The immune system is occupied with protecting the host against such dangers in order to preserve human health. For that purpose, the immune system is equipped with a diverse array of both cellular and non-cellular effectors that are in continuous communication with each other. The naturally occurring nucleotide adenosine 5'-triphosphate (ATP) and its metabolite adenosine (Ado) probably constitute an intrinsic part of this extensive immunological network through purinergic signaling by their cognate receptors, which are widely expressed throughout the body. This review provides a thorough overview of the effects of ATP and Ado on major immune cell types. The overwhelming evidence indicates that ATP and Ado are important endogenous signaling molecules in immunity and inflammation. Although the role of ATP and Ado during the course of inflammatory and immune responses in vivo appears to be extremely complex, we propose that their immunological role is both interdependent and multifaceted, meaning that the nature of their effects may shift from immunostimulatory to immunoregulatory or vice versa depending on extracellular concentrations as well as on expression patterns of purinergic receptors and ecto-enzymes. Purinergic signaling thus contributes to the fine-tuning of inflammatory and immune responses in such a way that the danger to the host is eliminated efficiently with minimal damage to healthy tissues.

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display.

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or changing the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5'-(N-ethylcarboxamido)adenosine (NECA, 10 microM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 +/- 0.02 (S.E.) to 1.80 +/- 0.02 (p less than 0.001) while inosine (10 microM), a poor adenosine receptor agonist, had no effect (1.73 +/- 0.04, p = n.s. vs. control, p less than 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA greater than adenosine = N6-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 microM) did not significantly change the number or affinity of [3H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.

Age-related changes in neutrophil structure and function.

The elderly suffer higher rates of morbidity and mortality from infectious disease than younger adults. Since neutrophils are the first line of defense against infection the vulnerability to infection of the elderly may be due, at least in part, to age-related changes in neutrophils (PMNs). Previous studies have suggested that there is an age-related increase in plasma membrane microviscosity in lymphocytes and neurons of the elderly. We have explored the hypothesis that there is a similar change in plasma membrane viscosity in the neutrophil and that this alteration in the neutrophil plasma membrane leads to diminished neutrophil function. When we studied plasma membrane viscosity of neutrophils by determining the fluorescence anisotropy of 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene labeled cells, we were surprised to find that plasma membrane viscosity is decreased in the neutrophils of the elderly as compared to young controls (microviscosity parameter, 1.33 +/- 0.07, in the elderly, n = 21, versus 1.62 +/- 0.06, in young controls, n = 19, P less than 0.004). As expected, stimulated (FMLP 0.1 microM) O2- generation by neutrophils from the elderly was significantly decreased compared with young controls (34 +/- 7% decrease, n = 6, P less than 0.04). Both resting and stimulated neutrophils demonstrated an age-related decline in adherence to a component of the extracellular matrix, denatured collagen (gelatin, P less than 0.04, n = 22 elderly subjects). In contrast, neutrophils from the elderly adhered to fibrinogen at least as well as neutrophils from young controls. Moreover, chemotaxis to activated complement components (in zymosan activated plasma) and FMLP did not change with increased age whether studied in the Boyden chamber (92 +/- 7% control, n = 14) or under agarose (90 +/- 13% control, n = 11). These studies suggest that an age-related decrease in plasma membrane viscosity is associated with a decrease in O2- production and adherence of neutrophils to components of the extracellular matrix. Thus, age-related alterations in neutrophil structure may result in diminished neutrophil function and increased susceptibility to infection with pyogenic bacteria.

Serum complement values (C3 and C4) to differentiate between systemic lupus activity and pre-eclampsia.

It is often difficult to differentiate between an exacerbation of systemic lupus erythematosus (SLE) and intercurrent pre-eclampsia in a patient with SLE since the manifestations of both entities include proteinuria and hypertension. This study was undertaken to determine wether serum C3 and C4 values can help distinguish SLE activity from pre-eclampsia. In 21 nonpregnant women of child-bearing age, the mean C3 level was 124 +/- 5 mg/dl and the mean C4 was 31 +/- 1 mg/dl. In 24 normal women in the third trimester of pregnancy, the C3 and C4 levels were elevated (165 +/- 4 mg/dl, p less than 0.001 versus nonpregnant control women; 37 +/- 2 mg/dl, p less than 0.01 versus nonpregnant control women, respectively). In 17 women in the third trimester of pregnancy with documented pre-eclampsia, the mean C3 level was 162 +/- 4 mg/dl, no different from that in normal pregnant women (p less than 0.001 versus nonpregnant control women; p = NS versus normal pregnant women), and the mean C4 was 29 +/- 3 mg/dl, lower than that found in normal pregnant women (p less than 0.02 versus normal pregnant women). Antinuclear antibody was absent at titers of less than 1:20 in all of these pre-eclamptic patients. In contrast, pregnant women with SLE has significantly lower C3 (103 +/- 13 mg/dl) and C4 (15.3 +/- 3.6 mg/dl) values during the third trimester of pregnancy than either normal pregnant women (p less than 0.001 for C3 and C4) or women with pre-eclampsia (p less than 0.001 for C3 and p less than 0.004 for C4) during the third trimester of pregnancy. Of the eight women with SLE in whom serial complement values were determined, three had falling C3 or C4 levels, and in each, there was a flare of SLE activity either during or immediately after pregnancy. None of the five patients with a rising C3 concentration had a flare of disease activity; however, pre-eclampsia developed in one of these patients, characterized by hypertension and proteinuria. Thus, measurement of serum C3 and C4 can help differentiate between SLE activity and pre-eclampsia, since both C3 and C4 are significantly lower in women with SLE than women with pre-eclampsia, and serum C3 and C4 concentrations rise during uncomplicated or pre-eclamptic pregnancy in women with SLE.

The mechanism of action of methotrexate.

Because of methotrexate's 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, an endogenous anti-inflammatory agent.

Adenosine receptors are present on most cells and organs, yet, although the physiological effects of adenosine were first described over 60 years ago, the potential therapeutic uses of adenosine have only been recognized and realized recently. A decade ago the potent anti-inflammatory effects of adenosine were first described; adenosine, acting at specific A2 receptors, inhibits some, but not all, neutrophil functions. Adenosine inhibits phagocytosis, generation of toxic oxygen metabolites, and adhesion (to some surfaces and to endothelial cells) but does not inhibit degranulation or chemotaxis. Occupancy of adenosine A2 receptors modulates leukocyte function by a novel mechanism. Although adenosine A2 receptors are classically linked to heterotrimeric GS signaling proteins and stimulation of adenylate cyclase, adenosine 3',5'-cyclic monophosphate does not act as the second messenger for inhibition of leukocyte function. By a mechanism that still remains obscure, occupancy of adenosine A2 receptors on neutrophils "uncouples" chemoattractant receptors from their stimulus-transduction proteins. The concentrations of adenosine that inhibit inflammatory cell function are similar to those observed in vivo and suggest a role for adenosine in the modulation of inflammation in vivo. Indeed, recent studies indicate that nonmetabolized adenosine receptor agonists are potent anti-inflammatory agents, and other studies indicate that methotrexate, a commonly used anti-inflammatory agent, diminishes inflammation by increasing adenosine release at inflamed sites. The observations reviewed here suggest that adenosine and agents that act through adenosine are excellent candidates for development as anti-inflammatory agents.

Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors.

Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H2O2, O2- or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O2- generation and O2 consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, O2- generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of O2- generation is similar in the presence or absence of cytochalasin B (IC50 = 53 +/- 19 and 16 +/- 5 nM, respectively, P = NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O2 consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O2 consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O2- release and O2 consumption (10 +/- 5 and 5 +/- 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O2 consumption in parallel with inhibition of O2- release.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonsteroidal antiinflammatory agents inhibit stimulated neutrophil adhesion to endothelium: adenosine dependent and independent mechanisms.

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil aggregation (homotypic cell-cell adhesion) and do so without affecting expression of CD11b/CD18. Since the first step in acute inflammation is a critical interaction between neutrophils and the vascular endothelium (heterotypic cell-cell adhesion), we determined whether NSAIDs diminish the adherence of neutrophils to the endothelium. At antiinflammatory concentrations (0.5-5 mM) sodium salicylate, an NSAID that does not inhibit prostaglandin synthesis, inhibited stimulated but not unstimulated neutrophil adherence to endothelial cells (IC50 < 1 mM, P < 0.00001). Salicylates have previously been shown to inhibit oxidative phosphorylation and, predictably, sodium salicylate inhibited oxidative phosphorylation, as evidenced by depletion of ATP stores (875 +/- 75 pmol/10(6) PMN, [2.92 +/- 0.25 mM]) in stimulated (FMLP, 0.1 microM) but not resting neutrophils treated with antiinflammatory doses of sodium salicylate (EC50 = 1 mM, P < 0.00001). Indomethacin and piroxicam (10 and 30 microM) only minimally decreased ATP concentrations in stimulated and resting neutrophils. ATP is metabolized to adenosine, and we have previously demonstrated that both endogenously released (180-200 nM) and exogenous adenosine (IC50 = 250 nM) inhibit stimulated neutrophil adherence to endothelial cells. To determine whether the increased metabolism of ATP and the resultant increase in adenosine release were responsible for inhibition of neutrophil adhesion to endothelium, we determined whether addition of adenosine deaminase (ADA, 0.125 IU/ml), an enzyme that converts extracellular adenosine to its inactive metabolite, inosine, affected inhibition of neutrophil adhesion to endothelium by stimulated neutrophils. ADA significantly reversed inhibition of neutrophil adherence to endothelium by sodium salicylate (0.5-5 mM, P < 0.00001). This suggests that sodium salicylate inhibits neutrophil adherence by increasing adenosine release. Whereas indomethacin and piroxicam (10-50 microM) also inhibited stimulated neutrophil adherence to endothelial cells, ADA did not affect their inhibition of adherence. These studies demonstrate a heretofore unexpected antiinflammatory mechanism for salicylates: salicylates increase ATP hydrolysis and thereby enhance release of adenosine. Moreover, these data are consistent with the hypothesis that NSAIDs differ from one another with respect to their mechanisms of action.

The antiinflammatory effects of methotrexate are mediated by adenosine.

In summary, intermittent, low dose methotrexate treatment is: 1.) antiinflammatory in the murine air pouch model of inflammation; 2.) selectively increases intracellular AICAR concentration; 3.) increases adenosine concentration in an inflammatory exudate; and, 4.) inhibits leukocyte accumulation at an inflamed site by a mechanism that is specifically reversed by adenosine deaminase and the adenosine A2 receptor antagonist DMPX but not the A1 antagonist DPCPX. In conclusion, we have demonstrated a novel mechanism for the antiinflammatory action of methotrexate; methotrexate is a nonsteroidal antiinflammatory agent that acts by promoting the release of adenosine which engages A2 receptors on inflammatory cells.