INTRODUCTION
Sepsis is the third leading cause of death in developed societies and affects more than 18 million people worldwide, with an expected 1% increase of incidence per year (1 sepsis , which is a complex clinical syndrome resulting from excessive and uncontrolled host responses to infection characterized-at least initially-by an overactivation of the innate immune system with an overwhelming production of proinflammatory mediators (e.g., TNF-α). Recent insights into the complex pathophysiology of sepsis have revealed a biphasic inflammatory course. The initial hyperinflammation is followed by an anti-inflammatory later phase, leading to an "immune paralysis." Not only the innate but also the adaptive immune system is strongly involved in the pathogenesis of the septic disease; T cells amplify the inflammatory response with an initial TH 1-cell response (characterized by interferon γ [IFN-γ] and IL-12 production), which during sepsis diverts to a TH 2-cell response (characterized by IL-4, IL-5, IL-10, and IL-13) leading to severe immunosuppression (2, 3
Therapeutic strategies targeting specific proinflammatory cytokines during the early overwhelming inflammation have shown a beneficial effect in experimental models of sepsis (4, 5 sepsis have failed to obtain consistent results. This failure may be due to the complex and dynamic pathogenesis of sepsis , which involves a delicate balance between proinflammation and anti-inflammation and a different time course of both in each individual patient. Therefore, the modulation of immune processes rather than the complete blockade of single mediators may be the focus of future therapeutic approaches (6, 7
An agent that modulates inflammation is the phosphodiesterase inhibitor pentoxifylline (PTX). Pentoxifylline is well known to possess anti-inflammatory properties, most of them related to the documented downregulation of TNF-α synthesis. Furthermore, TNF-α-independent immunomodulatory effects of PTX have been described. Pentoxifylline attenuates the oxidative burst of polymorphonuclear (PMN) leukocytes, inhibits the proliferation of peripheral blood mononuclear cells, attenuates IL-12 release, and prevents adherence to the cell matrix and the endothelium. Moreover, PTX has been shown to inhibit T-cell cytotoxicity; to decrease IFN-γ, granulocyte-macrophage colony-stimulating factor, and IL-6 secretion; and to attenuate cell surface expression of the IL-2 receptor (8-12 sepsis , PTX has been shown to improve hemodynamics and to decrease serum levels of TNF-α (13 11, 14, 15 sepsis , several small studies suggest that the use of PTX reduces mortality without any adverse effects (16
The exact mechanism of action of PTX is not yet understood. Because of its properties as a phosphodiesterase inhibitor, PTX increases intracellular levels of the second messenger cyclic adenosine monophosphate (cAMP) with subsequent amplification of downstream signaling pathways. On the molecular level, a downregulation of nuclear factor κB activity has been described (17, 18
As PTX increases cellular cAMP levels, its effects may resultfrom the amplification of G protein-coupled receptor (GPCR) signaling, which exerts a multitude of biologic functions by coupling to stimulatory G proteins (Gs ), leading to the formation of cAMP. In the immune system, various GPCRs are known to mediate immunomodulatory effects. In particular, the adenosine A2A receptor (A2A R), which is expressed on all types of immune cells, is strongly involved in anti-inflammatory activities (19 2A R are well known as important mechanisms inhibiting overactive immune cells, limiting inflammation, and protecting normal tissues from excessive collateral damage. Activation of the A2A R was shown to decrease superoxide production in PMN leukocytes, TNF-α production by monocytes, and IL-2, TNF-α, and IFN-γ production of T cells. In sepsis , the occurring tissue hypoxia leads to significantly elevated adenosine levels, and animal experiments strongly suggest an important role of A2A R signaling in the modulation of the septic inflammatory processes (19 2A R pathways in immune cells could-at least partially-account for the immunomodulatory effects of PTX. In the current study, we investigated these issues in native and stimulated human PMN leukocytes and T cells. Our results show that the A2A R-signaling pathways mediate a considerable portion of the anti-inflammatory effects of PTX. In human PMN leukocytes and T cells, relevant anti-inflammatory effects of PTX (in therapeutically reachable concentrations) can be achieved only in the presence of sufficient adenosine concentrations.
MATERIALS AND METHODS
Blood withdrawal was approved by the institutional ethics committee, and informed consent was obtained from healthy volunteers.
Materials
Fluorescence-activated cell sorting lysing solution was obtained from Becton Dickinson (Heidelberg, Germany). Hanks buffered salt solution (HBSS) was manufactured from soluble ingredients by the hospital's own pharmacy. Dihydrorhodamine 123 (DHR 123) was obtained from MoBiTec GmbH (Göttingen, Germany). The following agents were purchased from Sigma-Aldrich GmbH (Taufkirchen, Germany): Ficoll-Histopaque, fMLP, LPS, adenosine deaminase (ADA), the specific A2A R-agonist 2-[p -(2-carboxyethyl)-phenetyl-amino]-5′-N -ethylcarboxamidoadenosine (CGS21680), and adenosine. Pentoxifylline was from Aventis (Frankfurt, Germany).
Preparation of human blood peripheral neutrophils
Granulocytes were separated from whole blood of from healthy volunteers as previously described (20
Ex vivo stimulation of granulocytesCells were cultured in RPMI-1640 medium (Biofluids, Rockville, Md) supplemented with 10% (vol/vol) fetal calf serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 1 mM HEPES in 12-well plates at a density of 5 × 106 cells/well. For stimulation, 10 μg/mL LPS ± PTX ± ADA (1 U/mL) were added to each well. After incubation for 6 h, cells were harvested by centrifugation and washed twice with HBSS.
Analysis of granulocyte function
For measurement of hydrogen peroxide production by LPS-prestimulated human neutrophils, an assay assessing oxidation of DHR 123 to fluorescent rhodamine (R123) was used.
Isolated human PMN leukocytes (1 × 105 ) were incubated with DHR 123 (1 μM) in the presence or absence of different concentrations of adenosine or CGS21680 (10−9 to 10−4 M) and/or PTX (0.2 mM) in 1 mL of HBSS at 37°C for 5 min. Then PMN leukocytes were stimulated with fMLP (10−7 M). After 15 min, activation of cells was stopped by putting tubes on ice. Production of H2 O2 was determined by flow cytometry using a Becton Dickinson FACScan (Becton Dickinson, San Jose, Calif) equipped with an argon laser emitting light at 488 nm, as previously described. Dead cells were excluded from analyses after identification by propidium iodide staining (3 × 10−5 M).
Measurement of cAMP levels in human neutrophils
Cyclic AMP was determined by enzyme-linked immunosorbent assay (ELISA), as described in Kreth et al. (20 6 cells) were suspended in 1.0 mL HBSS, containing increasing concentrations of adenosine (10−9 to 10−6 M) in the presence or absence of PTX (0.2 mM) and were preincubated for 5 min in a shaking water bath at 37°C. Hereafter, fMLP in a concentration of 10−7 M was added, and the incubation was continued for another 2 min. The reaction was stopped by the addition of 12.5 μL 30% HCl and transferred to −70°C. After thawing and centrifugation at 10,000g to remove cellular debris, supernatants were assayed for cAMP using a cAMP EIA kit from Biomol (Hamburg, Germany).
Isolation of human T cells
Purified human T cells were obtained from blood by negative selection on MACS cell separator using the Pan T-cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) as per manufacturer's recommendation. Purified T cells were placed in RPMI-1640 (Biofluids) supplemented with 5% dialyzed fetal calf serum (heat inactivated) and 100 U/mL penicillin, 100 mg/mL streptomycin, 1 mM sodium pyruvate, 1 mM HEPES, and nonessential amino acids (RP5). Numbers and viability were assessed using a ViCell analyzer.
Ex vivo activation of T cellsPurified T cells were cultured in six-well plates. Culture volume was 2 mL per well containing 2 × 106 cells. For stimulation, 50 μL anti-CD3/CD28 beads (Invitrogen, Carlsbad, Calif) ± PTX (0.2 mM) ± adenosine (140 nM) ± ADA (1 U/mL) were added. Supernatants and cells were harvested 24 h later.
Cytokine measurements
Extracellular cytokine concentrations from cell culture supernatant were determined by ELISA kits (BD Pharmingen, Heidelberg, Germany) according to the manufacturer's instructions.
Quantitative reverse transcriptase-polymerase chain reaction
Total RNA was purified from human T cells and granulocytes using the RNaequous Kit (Ambion, Austin, Tex) including DNAse treatment, following the manufacturer's protocol. Equal amounts of RNA (700 ng) from the different samples were transcribed into cDNA using mixed random and oligo-dT primers and Superscript III reverse transcriptase (Invitrogen), as per manufacturer's instructions.
Real-time quantitative polymerase chain reaction was performed in duplicates with the Light Cycler480 instrument (Roche Diagnostics, Mannheim, Germany), as described (21 21 22
Determination of adenosine concentrations
In vitro concentrations of the purine nucleoside adenosine were analyzed by dual-column switching high-affinity performance/reversed-phase high-performance liquid chromatography technique using an internal surface nitrophenylboronic acid precolumn as previously described (20
Statistical analysis
All data were analyzed using SigmaPlot 10.0 software (Systat Software). Unless stated otherwise, data are presented as mean ± SEM. Concentration-response curves and corresponding 50% inhibitory concentrations (IC50 ) were determined by nonlinear regression. Intergroup comparisons were performed by paired Student t test or Mann-Whitney U test, if data were normally or not normally distributed, respectively. Differences with P < 0.05 were considered to be statistically significant.
RESULTS
The adenosine-induced inhibition of H2 O2 production is potentiated by PTX in ex vivo stimulated human PMN leukocytes
We determined the effects of increasing adenosine concentrations on fMLP-stimulated H2 O2 production in LPS-treated human PMN leukocytes in the presence or absence of PTX. As there was significant interindividual variability in maximum concentrations, we expressed values as percentage of control maximum concentrations. As shown in Figure 1A , adenosine inhibited the generation of H2 O2 in a dose-dependent manner, reaching maximum inhibitory effects at concentrations in the range of 10−5 M without PTX, whereas maximum inhibition in PTX-treated PMN leukocytes was accomplished at concentrations of 10−6 M. Mean IC50 values of adenosine changed from 320 to 70 nM in the absence and presence of PTX, respectively. The use of the selective A2A R agonist CGS21680 exhibited identical results: dose-response curves shifted to the left and IC50 values-which are per se by factor 5 lower than for adenosine (23 Fig. 1B ), thereby confirming that the differences in stimulatory effects were essentially A2A R mediated.
Fig. 1: Pentoxifylline potentiates the adenosine-induced inhibition of the fMLP-stimulated H2 O2 production in LPS-treated human PMN leukocytes. H2 O2 production of LPS-treated (10 μg/mL, 6 h) PMN leukocytes was stimulated by fMLP (100 nM) and assessed by flow cytometry. Control activity was determined without addition of agonist (33 ± 8.4 mean fluorescence intensity [MFI]) and PTX (26.6 ± 6.4 MFI), respectively. A, Concentration-response curves and IC50 values for the inhibition of the H2 O2 production by increasing concentrations of adenosine in the presence (Δ) or absence (•) of PTX (0.2 mM) were determined by nonlinear regression analysis. Data points represent one of n = 6 independent experiments with similar results, each performed in triplicate. Dashed lines indicate IC50 values. Mean IC50 values of adenosine changed from 320 to 70 nM in the absence and presence of PTX, respectively (n = 6, P <0.05). B, Concentration-response curves and IC50 values for the inhibition of the H2 O2 production by increasing concentrations of CGS21680 in the presence (Δ) or absence (•) of PTX (0.2 mM) were determined by nonlinear regression analysis. Data points represent one of n = 3 independent experimentswith similar results, each performed in triplicate. Dashed lines indicate IC50 values. Mean IC50 values of CGS21680 changed from 85 to 19 nM in the absence and presence of PTX, respectively (n = 3, P < 0.05). C, In LPS-stimulated PMN leukocytes, the inhibition of the H2 O2 production by the combination of inhibitory concentrations of adenosine and PTX was greater than the calculated sum of the single effects (denoted as Σ). Data represent mean values in % of control ± SEM of n = 6 independent experiments performed in triplicate.
In native PMN leukocytes, no potentiation of PTX and adenosine could be detected, IC50 values did not shift, and only additive effects of PTX and adenosine were observed. At plasma adenosine concentrations found in patients with septic shock (140 nM; Table 1 ), adenosine alone had inhibitory effects of approximately 20%. Pentoxifylline alone in concentrations of 0.2 mM had an inhibitory effect of 18% ± 2%, whereas in combination with 140 nM adenosine, an inhibition of 56% ± 5% was obtained, which is clearly higher than expected when plainly adding the single inhibitory effects of the two substances (Fig. 1C ).
Table 1: Adenosine concentrations of cell culture supernatants and adenosine plasma levels of healthy volunteers and of patients with septic shock
The adenosine-induced stimulation of cAMP generation is potentiated by PTX in ex vivo stimulated human PMN leukocytes
To evaluate whether these overadditive effects of adenosine and PTX in LPS-stimulated PMN leukocytes also occur on the second-messenger level, we determined the stimulatory effects of adenosine alone and in combination with PTX on the adenylyl cyclase activity in LPS-treated neutrophils. In the presence of 0.2 mM PTX, potency of physiologically relevant adenosine levels (140 nM) to stimulate cAMP formation was more than 5-fold increased as compared with adenosine alone (Fig. 2 ). This effect is clearly overadditive, as the simple addition of stimulatory PTX and adenosine effects would result in a far less increase of cAMP formation (approximately 2-fold; Fig. 2 ).
Fig. 2: Pentoxifylline potentiates the adenosine-stimulated generation of cAMP in LPS-stimulated human PMN leukocytes. LPS-treated (10μg/mL, 6 h) PMN leukocytes were stimulated with increasing concentrations of adenosine in the presence (Δ) or absence (•) of PTX (0.2 mM), and cAMP levels were determined as described by ELISA. Results are expressed in % of maximum cAMP levels determined in the presence of 10−4 M adenosine 10−3 M PTX and represent mean values ± SEM of n = 6 independent experiments performed in triplicate. Cyclic AMP accumulation in response to the combination of pathophysiologically relevant concentrations of adenosine and PTX was greater than the calculated sum of the single effects (○).
In human T cells, PTX inhibits the production of proinflammatory cytokines in an adenosine-dependent manner
Next, we investigated whether interaction of PTX and the adenosine-A2A R pathway may also play an important role in the inhibition of effector functions of human T cells. Purified T cells were stimulated with anti-CD3/CD28 beads in the presence or absence of PTX combined with (i) 80 nM adenosine (produced by the T cells during 24 h of stimulation; Table 1 ), (ii) 140 nM adenosine, or (iii) without adenosine (removed by addition of ADA). We evaluated the production of the proinflammatory cytokines TNF-α and IFN-γ after stimulation for 24 h. Stimulated T cells without adenosine served as controls. First, inhibitory effects of adenosine alone were determined: 80 nM and 140 nM adenosine exerted an inhibition of IFN-γ generation of 9% ± 3% and 32% ± 4%, respectively, and an inhibition of TNF-α generation of 10% ± 3% and 17% ± 5%, respectively (Fig. 3 , A and B). In the presence of 80 and 140 nM adenosine, 0.2 mM PTX inhibited the production of IFN-γ by 21% ± 5% and 49% ± 2%, respectively. In the absence of adenosine, these inhibitory effects of PTX dramatically decreased to values less than 10% (Fig. 3A ). Regarding TNF-α, similar effects could be found. In the presence of 80 and 140 nM adenosine, 0.2 mM PTX inhibited the TNF-α generation by 50% ± 3% and 69% ± 6%, respectively, whereas in the absence of adenosine, an inhibition of only 31% ± 6% was detected (Fig. 3B ). In unstimulated T cells-per se producing only minimal levels of IFN-γ and TNF-α-significant inhibitory effects of PTX were not detected (data not shown).
Fig. 3: In human T cells, adenosine and PTX inhibit the production of IFN-γ and TNF-α in an overadditive manner. T cells were stimulated for24h with anti-CD3/CD28 beads in the presence or absence of adenosine(140 nM) ± PTX (0.2 mM) ± ADA (1 U/mL), as indicated. Endogenous adenosine production of stimulated T cells was 80 nM on average (
Table 1 ). Concentrations of IFN-γ (A) and TNF-α (B) in the supernatants were determined by ELISA. Results are expressed in % of the control (C) cytokine production of T cells stimulated in the presence of ADA (1 U/mL) and represent mean values ± SEM of n = 6 independent experiments; *
P < 0.05.
PTX does not influence the mRNA transcription of the A2A R in human PMN leukocytes and T cells
In a previous study, we have already shown that adenosine receptors are equally distributed in native PMN leukocytes, whereas in LPS-stimulated PMN leukocytes, the A2A R is the predominant receptor subtype. As adenosine-dependent inhibitory effects of PTX in human PMN leukocytes and T cells occurred only in stimulated cells, we next determined the mRNA expression of the A2A R in resting and stimulated human T cells. Furthermore, as an elevation of cAMP levels may lead to transcriptional changes even of the receptors themselves, which might influence the experimental results, we determined the mRNA expression of the A2A R in (i) native, (ii) stimulated, and (iii) stimulated and PTX-treated human PMN leukocytes and T cells. In T cells, we detected a 3.5-fold increase of A2A R mRNA upon stimulation with anti-CD3/CD28 beads (4.5 ± 0.21). Addition of 0.2 mM PTX did neither in PMN leukocytes nor in T cells significantly affect the observed changes in transcript levels upon stimulation (Fig. 4 ). Addition of ADA to all cell incubations did not change the receptor expression, thereby excluding effects of adenosine itself.
Fig. 4: Stimulation of human T cells and PMN leukocytes increases the mRNA transcription of the A2A R independently of PTX. Adenosine A2A receptor mRNA levels in native and stimulated PMN leukocytes (LPS 10μg/mL, 6 h) and in native and stimulated T cells (anti-CD3/CD28 beads, 24 h) in the presence or absence of PTX (0.2 mM) were determined by quantitative reverse transcriptase-polymerase chain reaction. Relative mRNA expression was calculated using an efficiency-corrected algorithm with standard curves and reference gene normalization against SDHA and TBP. Results are expressed as fold change relative to native cells and represent the mean ± SEM of n = 5 independent experiments performed in duplicate; *P < 0.05 vs. native cells.
DISCUSSION
Understanding the network of proinflammatory and anti-inflammatory pathways driving the pathogenesis of sepsis and identification of drugs positively influencing the course of the disease still remains a challenge. A long known but still actual substance exerting a wide range of documented immunomodulatory properties is the phosphodiesterase inhibitor PTX, which is used in the attenuation of inflammatory responses in patients with sepsis , adult respiratory distress syndrome (ARDS), and after cardiosurgery (15, 24-26 14, 27 28 2A R pathway. Pentoxifylline treatment of LPS-stimulated human PMN leukocytes led to a 4.5-fold decrease of the IC50 of adenosine on the H2 O2 production, indicating a significant pharmacological potentiation. Both IC50 values were in the range of the binding affinity of adenosine to an A2A R site. Although adenosine and PTX alone (in physiological/therapeutic concentrations) exerted only a weak respiratory burst suppression, both substances administered in combination interacted in an overadditive manner leading to strong inhibitory effects. This means that only in the presence of sufficiently high adenosine levels that PTX can provoke a clinically relevant suppression of the H2 O2 production.
In human T cells, an even more impressive synergism of adenosine and PTX was found. In the absence of adenosine, only marginal inhibitory effects of PTX on the production IFN-γ could be detected, and the inhibition of TNF-α reached less than 50% of the combined effect of PTX and adenosine.
All experiments were performed with PTX concentrations that are therapeutically achieved in patients treated with PTX standard protocols (15 20 in vivo was thereby mimicked as realistically as possible, and artifacts resulting from unphysiological/supratherapeutic levels of adenosine and/or PTX have been excluded.
Pentoxifylline exerts its various biologic effects by increasing intracellular levels of the second messenger cAMP, which is generated by activation of GPCRs coupled to stimulatory G proteins. In other words, PTX amplifies signaling pathways downstream of Gs -coupled GPCRs, and its effects are determined by the type of GPCRs expressed in the respective cell type. In immune cells, several members of the GPCR family have been found, and some of them may play a role in immunomodulation (29, 30 s -coupled adenosine A2A R is an important "target" of PTX, and the amplification of the cAMP answer provoked by activation of the A2A R might even be the main cause for the anti-inflammatory effects of PTX. Activation of the A2A R has conclusively been shown to be involved in the natural anti-inflammatory mechanisms and is well known to inhibit overactive immune cells during acute inflammation (31-34 2A Rs in human sepsis patients, and we have shown that A2A R expression in PMN leukocytes significantly increases upon stimulation (35 2A R increases significantly upon stimulation. Assuming that A2A Rs indeed are the main mediators of anti-inflammatory PTX effects, the divergent findings regarding the effects of PTX in native versus stimulated PMN leukocytes/T cells may thereby be explained. Significant anti-inflammatory effects of PTX need sufficient expression and signaling of the A2A R as a prerequisite, which is fulfilled only in stimulated cells, whereas in native cells, the abundance of the A2A R is too low to provoke significant PTX effects.
Regarding the molecular ways of action underlying the link between cAMP elevation and immunosuppression, only fragmentary knowledge exists. Stimulation of certain GPCRs-with the A2A R being the best characterized member within this group-has been reported to exert immunosuppressive effects on various cell types (e.g., including PMN leukocytes, T cells, and macrophages), provoked by the elevation of intracellular cAMP through the activation of adenylyl cyclase (36 37 38 39 40, 41 39, 42-45
In this context, the different extent of IFN-γ and TNF-α inhibition by PTX in human T cells may be explained. Whereas IFN-γ production is regulated via the protein kinase A-cAMP response element-binding protein pathway (46, 47 42 2A R) by PTX is translated into different signaling pathways, leading to a differential modulation of biologic responses. Noteworthy, the expression of the A2A R itself is not influenced by PTX treatment.
Our results suggest that sufficient effects of PTX depend on sufficient levels of adenosine, which might have implications for the immunomodulatory PTX therapy in sepsis patients. As only 50% of sepsis patients exhibit adenosine levels determined as sufficient in our study, one may speculate that those patients with lower adenosine levels may turn out as nonresponders to PTX therapy, which might explain the observed heterogeneity of PTX treatment responses (11, 14, 15 48 in vitro findings needs to be investigated in septic patients.
In conclusion, we propose that, in human immune cells, a large portion of immunomodulatory effects of PTX is mediated via adenosine-dependent pathways. Sufficient adenosine levels might be a prerequisite for the accessibility of sepsis patients to treatment with PTX; however, further studies are necessary to prove the clinical relevance of this hypothesis.
ACKNOWLEDGMENTS
The authors thank Gabriele Groeger and Jessica Rink for expert technical assistance.
REFERENCES
1. Hotchkiss RS, Karl IE: The pathophysiology and treatment of
sepsis .
N Engl J Med 348:138-150, 2003.
2. Shimaoka M, Park EJ: Advances in understanding
sepsis .
Eur J Anaesthesiol Suppl 42:146-153, 2008.
3. Rittirsch D, Flierl MA, Ward PA: Harmful molecular mechanisms in
sepsis .
Nat Rev Immunol 8:776-787, 2008.
4. Marshall JC: Such stuff as dreams are made on: mediator-directed therapy in
sepsis .
Nat Rev Drug Discov 2:391-405, 2003.
5. Russell JA: Management of
sepsis .
N Engl J Med 355:1699-1713, 2006.
6. Parrish WR, Gallowitsch-Puerta M, Czura CJ, Tracey KJ: Experimental therapeutic strategies for severe
sepsis : mediators and mechanisms.
Ann N Y Acad Sci 1144:210-236, 2008.
7. Ulloa L, Tracey KJ: The "cytokine profile": a code for
sepsis .
Trends Mol Med 11:56-63, 2005.
8. Deree J, Lall R, Melbostad H, Grant M, Hoyt DB, Coimbra R: Neutrophil degranulation and the effects of phosphodiesterase inhibition.
J Surg Res 133:22-28, 2006.
9. Zhang R, Bharadwaj U, Li M, Chen C, Yao Q: Effects of pentoxifylline on differentiation, maturation, and function of human CD14
+ monocyte-derived dendritic cells.
J Immunother 30:89-95, 2007.
10. Zhang M, Xu YJ, Saini HK, Turan B, Liu PP, Dhalla NS: Pentoxifylline attenuates cardiac dysfunction and reduces TNF-alpha level in ischemic-reperfused heart.
Am J Physiol Heart Circ Physiol 289:H832-H839, 2005.
11. Wang W, Zolty E, Falk S, Basava V, Reznikov L, Schrier R: Pentoxifylline protects against endotoxin-induced acute renal failure in mice.
Am J Physiol Renal Physiol 291:F1090-F1095, 2006.
12. D'Hellencourt CL, Diaw L, Cornillet P, Guenounou M: Differential regulation of TNF alpha, IL-1 beta, IL-6, IL-8, TNF beta, and IL-10 by pentoxifylline.
Int J Immunopharmacol 18:739-748, 1996.
13. Strieter RM, Remick DG, Ward PA, Spengler RN, Lynch JP, Larrick J: Cellular and molecular regulation of tumor necrosis factor-alpha production by pentoxifylline.
Biochem Biophys Res Commun 155:1230-1236, 1988.
14. Randomized, placebo-controlled trial of lisofylline for early treatment of acute lung injury and acute respiratory distress syndrome.
Crit Care Med 30:1-6, 2002.
15. Staubach KH, Schroder J, Stuber F, Gehrke K, Traumann E, Zabel P: Effect of pentoxifylline in severe
sepsis : results of a randomized, double-blind, placebo-controlled study.
Arch Surg 133:94-100, 1998.
16. Zimmerman JJ: Appraising the potential of pentoxifylline in septic premies.
Crit Care Med 27:695-697, 1999.
17. Coimbra R, Melbostad H, Loomis W, Tobar M, Hoyt DB: Phosphodiesterase inhibition decreases nuclear factor-kappaB activation and shifts the cytokine response toward anti-inflammatory activity in acute endotoxemia.
J Trauma 59:575-582, 2005.
18. Coimbra R, Melbostad H, Loomis W, Porcides RD, Wolf P, Tobar M: LPS-induced acute lung injury is attenuated by phosphodiesterase inhibition: effects on proinflammatory mediators, metalloproteinases, NF-kappaB, and ICAM-1 expression.
J Trauma 60:115-125, 2006.
19. Sitkovsky MV, Lukashev D, Apasov S, Kojima H, Koshiba M, Caldwell C, Ohta A, Thiel M: Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors.
Annu Rev Immunol 22:657-682, 2004.
20. Kreth S, Kaufmann I, Ledderose C, Luchting B, Thiel M: Reduced ligand affinity leads to an impaired function of the adenosine A2A receptor of human granulocytes in
sepsis .
J Cell Mol Med 13:985-994, 2009.
21. Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR.
Nucleic Acids Res 29:e45, 2001.
22. Andersen CL, Jensen JL, Orntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets.
Cancer Res 64:5245-5250, 2004.
23. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, Linden J: International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors.
Pharmacol Rev 53:527-552, 2001.
24. Sliwa K, Woodiwiss A, Kone VN, Candy G, Badenhorst D, Norton G: Therapy of ischemic cardiomyopathy with the immunomodulating agent pentoxifylline: results of a randomized study.
Circulation 109:750-755, 2004.
25. Shaw SM, Shah MK, Williams SG, Fildes JE: Immunological mechanisms of pentoxifylline in chronic heart failure.
Eur J Heart Fail 11:113-118, 2009.
26. Sliwa K, Woodiwiss A, Candy G, Badenhorst D, Libhaber C, Norton G: Effects of pentoxifylline on cytokine profiles and left ventricular performance in patients with decompensated congestive heart failure secondary to idiopathic dilated cardiomyopathy.
Am J Cardiol 90:1118-1122, 2002.
27. Zeni F, Pain P, Vindimian M, Gay JP, Gery P, Bertrand M: Effects of pentoxifylline on circulating cytokine concentrations and hemodynamics in patients with septic shock: results from a double-blind, randomized, placebo-controlled study.
Crit Care Med 24:207-214, 1996.
28. Ridings PC, Windsor AC, Sugerman HJ, Kennedy E, Sholley MM, Blocher CR: Beneficial cardiopulmonary effects of pentoxifylline in experimental
sepsis are lost once septic shock is established.
Arch Surg 129:1144-1152, 1994.
29. Lombardi MS, Kavelaars A, Heijnen CJ: Role and modulation of G protein-coupled receptor signaling in inflammatory processes.
Crit Rev Immunol 22:141-163, 2002.
30. Kehrl JH: G-protein-coupled receptor signaling, RGS proteins, and lymphocyte function.
Crit Rev Immunol 24:409-423, 2004.
31. Thiel M, Caldwell CC, Sitkovsky MV: The critical role of adenosine A2A receptors in downregulation of inflammation and immunity in the pathogenesis of infectious diseases.
Microbes Infect 5:515-526, 2003.
32. Ohta A, Sitkovsky M: Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage.
Nature 414:916-920, 2001.
33. Lappas CM, Sullivan GW, Linden J: Adenosine A2A agonists in development for the treatment of inflammation.
Expert Opin Investig Drugs 14:797-806, 2005.
34. Moore CC, Martin EN, Lee GH, Obrig T, Linden J, Scheld WM: An A2A adenosine receptor agonist, ATL313, reduces inflammation and improves survival in murine
sepsis models.
BMC Infect Dis 8:141, 2008.
35. Kreth S, Ledderose C, Kaufmann I, Groeger G, Thiel M: Differential expression of 5'-UTR splice variants of the adenosine A2A receptor gene in human granulocytes: identification, characterization, and functional impact on activation.
FASEB J 22:3276-3286, 2008.
36. Hasko G, Linden J, Cronstein B, Pacher P: Adenosine receptors: therapeutic aspects for inflammatory and immune diseases.
Nat Rev Drug Discov 7:759-770, 2008.
37. Rowe J, Finlay-Jones JJ, Nicholas TE, Bowden J, Morton S, Hart PH: Inability of histamine to regulate TNF-alpha production by human alveolar macrophages.
Am J Respir Cell Mol Biol 17:218-226, 1997.
38. Dent G, Giembycz MA, Rabe KF, Wolf B, Barnes PJ, Magnussen H: Theophylline suppresses human alveolar macrophage respiratory burst through phosphodiesterase inhibition.
Am J Respir Cell Mol Biol 10:565-572, 1994.
39. Sands WA, Palmer TM: Regulating gene transcription in response to cyclic AMP elevation.
Cell Signal 20:460-466, 2008.
40. Kamthong PJ, Wu M: Inhibitor of nuclear factor-kappaB induction by cAMP antagonizes interleukin-1-induced human macrophage-colony-stimulating-factor expression.
Biochem J 356:525-530, 2001.
41. Minguet S, Huber M, Rosenkranz L, Schamel WW, Reth M, Brummer T: Adenosine and cAMP are potent inhibitors of the NF-kappa B pathway downstream of immunoreceptors.
Eur J Immunol 35:31-41, 2005.
42. Koga K, Takaesu G, Yoshida R, Nakaya M, Kobayashi T, Kinjyo I: Cyclic adenosine monophosphate suppresses the transcription of proinflammatory cytokines via the phosphorylated c-Fos protein.
Immunity 30:372-383, 2009.
43. Wang W, Tam WF, Hughes CC, Rath S, Sen R: c-Rel is a target of pentoxifylline-mediated inhibition of T lymphocyte activation.
Immunity 6:165-174, 1997.
44. Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A: Nucleotide receptors: an emerging family of regulatory molecules in blood cells.
Blood 97:587-600, 2001.
45. Pozo D: VIP- and PACAP-mediated immunomodulation as prospective therapeutic tools.
Trends Mol Med 9:211-217, 2003.
46. Samten B, Townsend JC, Weis SE, Bhoumik A, Klucar P, Shams H: CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen.
J Immunol 181:2056-2064, 2008.
47. Mayr B, Montminy M: Transcriptional regulation by the phosphorylation-dependent factor CREB.
Nat Rev Mol Cell Biol 2:599-609, 2001.
48. Ramakers BP, Pickkers P, Deussen A, Rongen GA, van den Broek P, van der Hoeven JG, Smits P, Riksen NP: Measurement of the endogenous adenosine concentration in humans in vivo: methodological considerations.
Curr Drug Metab 9:679-685, 2008.
