Methylxanthines with adenosine alter TNFα-primed PMN activation

Immunopharnlacology ELSEVIER

Immunopharmacology31 (1995) 19-29

Research Papers

Methylxanthines with adenosine alter TNFc -primed PMN activation Gail W. Sullivan *, L. Susan Luong, Holliday T. Carper, Ryan C. Barnes, Gerald L. Mandell Department of Medicine, Unit'ersiO' of Virginia, Charlottesl'ille, VA, 22908, USA

Received 12 December 1994; accepted 21 June 1995

Abstract Methylxanthines are best known as phosphodiesterase inhibitors that cause a rise in intracellular cAMP. One would expect the two methylxanthines, caffeine and pentoxifylline, to have similar actions on neutrophils (PMN). However, caffeine stimulated and pentoxifylline inhibited PMN oxidative activity. Micromolar concentrations of pentoxifylline decreased native and recombinant tumor necrosis factor-a (TNFa)-primed formyl met-leu-phe (fMLP)-stimulated PMN chemiluminescence, superoxide production and myeloperoxidase (MPO) release. In contrast, equal concentrations of caffeine increased chemiluminescence and MPO release with no effect on superoxide production. These activities of the methylxanthines were only observed in the presence of physiological concentrations of adenosine, and were abolished by the treatment of the PMN with adenosine deaminase. The activities of adenosine, pentoxifylline and caffeine on PMN activity could not be readily explained by changes in PMN [cAMP]. Thus for TNFa-primed PMN, pentoxifylline decreases PMN activity by enhancing the effect of adenosine on degranulation and superoxide production; whereas caffeine increases PMN activity by counteracting the effect of adenosine on degranulation. Keywords." Pentoxifylline; Caffeine; Adenosine; Neutrophil; Tumor necrosis factor; Superoxide

I. Introduction Lipopolysaccharides (LPS) from Gram-negative bacteria stimulate mononuclear leukocytes ( M N L ) to

Abbreviations: ADA, adenosine deaminase; ADO, adenosine; AU, arbitrary relative chemiluminescence units; cAMP, cyclic 3'-5' adenosine monophosphate; fMLP, formyl met-leu-phe; CGS 21680, 2-p-(2-Carboxyethyl)phenethylamino-5'N-ethylcarboxamido adenosine hydrochloride; CPA, N6-cyclopentyladenosine; HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; MNL, mononuclear leukocytes; MPO, myeloperoxidase; PMN, neutrophil; rhTNFot, recombinant human tumor necrosis factor o~; SOD, superoxide dismutase; TNFo~, tumor necrosis factor o~ * Corresponding author. Tel: + 1 804-924-9665.

release inflammatory cytokines including tumor necrosis factor-alpha ( T N F a ) (Couturier et al., 1991; Carswell et al., 1975). T N F a increases superoxide production and granule enzyme release such as myeloperoxidase (MPO) from PMN in response to a second stimulus (Klebanoff et al., 1986; Shalaby et al., 1987; Atkinson et al., 1988; Tennenberg and Solomkin, 1990; O'Flaherty et al., 1991). Although reactive oxygen intermediates and MPO are beneficial for host defense against infection they can also cause tissue damage and capillary leakage leading to septic shock and the acute respiratory distress syndrome (Tate and Repine, 1983; Tracey et al., 1986; Hogg, 1987; Mallick et al., 1989; Weiss, 1989).

0162-3109/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0162-3 109(95 )00030-5

20

G. W. Sullil an et al. / lmmunopharmacology 31 (1995) 19-29

We have recently observed that endogenous concentrations of adenosine decrease superoxide production and degranulation of neutrophils (PMN) primed with tumor necrosis factor (TNFa) (Barnes et al., in preparation). Adenosine is released from the breakdown of adenylate nucleotides in damaged tissues (Berne, 1980; Cronstein et al., 1986; Kammer, 1987; Gunther and Herring, 1991). Physiological concentrations of adenosine have been found to inhibit respiratory burst activity in stimulated PMN (Cronstein et al., 1983). Thus, this endogenous adenosine has the potential to modulate the PMN inflammatory response by decreasing cytokine-primed PMN activity. Since methylxanthines are both adenosine receptor antagonists (Biaggioni et al., 1991) and phosphodiesterase inhibitors (Somani and Gupta, 1988), we examined the possibility that methylxanthines could alter adenosine-modulation of primed PMN activity. The methylxanthine pentoxifylline [3,7-dimethyl(5-oxo-hexyl)-xanthine] is an agent developed for the treatment of intermittent claudication (Ward and Clissold, 1987). Caffeine (1,3,7-trimethyl xanthine) is also a methylxanthine. Both pentoxifylline and caffeine have been shown to have effects on leukocyte function (Sullivan et al., 1984; Bessler et al., 1986; Strieter et al., 1988; Sullivan et al., 1988; Hand et al., 1989; Currie et al., 1990; Thiel et al., 1991; Crouch and Fletcher, 1992; Sullivan et al., 1992). In the present study, we observed divergent actions of pentoxifylline and caffeine on TNFa-primed PMN activity. Pentoxifylline enhanced the effect of adenosine to further lower PMN superoxide release and degranulation, and caffeine counteracted the modulating effect of adenosine on degranulation with no effect on superoxide production. In the absence of adenosine, neither pentoxifylline nor caffeine had any significant effect on primed PMN activity.

logical Laboratories (Campbell, CA), recombinant human TNFce (rhTNFa) (specific activity = ~ 600pg/U) was a gift from Dainippon Pharmaceutical Co. Ltd (Osaka, Japan), and Hanks Balanced Salt Solution (HBSS) was from Whittaker M.A. Bioproducts (Walkersville, MD). N-formyl-L-methionyl-Lleucyl-L-phenylalanine (fMLP), cytochrome c (type VI from horse heart), catalase, superoxide dismutase (SOD; from bovine liver), luminol, o-dianisidine, adenosine (ADO), and adenosine deaminase (ADA; type X) were from Sigma Chemical Co. (St. Louis, MO), CGS 21680 (2-p-[2-carboxyethyl]phenethylamino-5'N-ethylcarboxamido adenosine hydrochloride) and n6-cyclopentyladenosine (CPA) from Research Biochemicals Inc. (Natick, MA), Ficoll-Hypaque from ICN Biomedicals (Aurora, OH), Accurate Chemicals and Scientific (Westbury, NY) and Cardinal Associates (Santa Fe, NM).

2. Materials and methods

2.4. LPS-stimulated MNL conditioned medium

2.1. Materials

Conditioned medium was made by incubating mixed human peripheral blood mononuclear leukocytes ( ~ 80% lymphocytes and ~ 20% monocytes; 2 x 106/ml) isolated by Ficoll-Hypaque separation in HBSS containing 0.1% human serum albumin at 37°C with or without LPS (10 ng/ml) for 1 h. The

Pentoxifylline was a gift from Hoechst-Roussel Pharmaceuticals Inc. (Somersville, NJ) and caffeine was from Sigma Chemical (St. Louis, Mo). LPS (extracted from E. coli K 235) was from List Bio-

2.2. Neutrophil (PMN) preparation

Purified PMN ( ~ 98% PMN; > 95% viable as determined by trypan blue exclusion) containing < 50 pg/ml of LPS (as determined by limulus amebocyte lysate assay) were obtained from normal, heparinized (10 U/ml) venous human blood by a onestep Ficoll-Hypaque separation procedure (Ferrante and Thong, 1980). The PMN were washed three times with HBSS. Residual erythrocytes were removed by hypotonic lysis. 2.3. Mixed mononuclear leukocyte (MNL) preparation

MNL ( ~ 15-20% monocytes and 80-85% lymphocytes) were obtained from the Ficoll-Hypaque separation (see Section 2.1, 'PMN preparation', above). The MNL were washed three times with HBSS.

G.W. Sullivanet al. / Immunopharmacology31 (1995) 19-29 cells were removed by centrifugation and the cell-free supernatant was retained for use immediately in the chemiluminescence assays.

2.5. Assay for TNFa production Release of TNFa from LPS-stimulated monocytes was determined by incubating MNL (2 × 106/ml) for 60 rain in HBSS-0.1% HSA with or without LPS (10 ng/ml). Suspended leukocytes were removed by centrifugation (2000 rpm × 10 min), and TNFc~ released into the surrounding medium was assayed by ELISA with a kit purchased from Cistron Biotechnology (Pine Brook, N J).

2.6. Chemiluminescence Luminol-enhanced chemiluminescence was employed as a measure of PMN oxidative activity. PMN with or without MNL were incubated with or without LPS (10 ng/ml), LPS-stimulated MNL conditioned medium, rhTNFa (1 U/ml), ADA (1 U/ml), pentoxifylline, caffeine, CGS 21680, CPA and adenosine at 37°C. Their relative continuous luminol-enhanced chemiluminescence was assayed at 37°C for 8 min after stimulation with fMLP (100 nM) in a Chronolog Photometer (Havertown, PA). Peak relative chemiluminescence was reported in arbitrary units (AU).

2.7. Superoxide production PMN (1 × 106/ml) with or without MNL (2 × 106/ml) were incubated at 37°C with or without LPS (10 ng/ml), rhTNFc~ (1 U/ml), caffeine, pentoxifylline, and adenosine. Cytochrome c (120/zM), catalase (0.062 m g / m l ) and fMLP (100 nM) were added and the samples were incubated for an additional 10 min at 37°C. SOD (200 U / m l ) was added to matched samples. The samples were then iced and centrifuged (2000 rpm × 10 min). The optical density of the supernatants were read at 550 nm against the matched SOD samples and the nmoles of SODinhibitable superoxide produced in 10 min were calculated using an extinction coefficient of 2.11 × 104/cm2/mmole (Van Gelder and Slate, 1962).

2.8. Degranulation: myeloperoxidase (MPO) release MPO was measured by a method adapted from Klebanoff (Klebanoff, 1968). The samples from the

21

chemiluminescence assay were iced and the cells removed by centrifugation. MPO was assayed by incubating 0.4 ml samples of cell-free supernatant with 0.6 ml fresh dye (0.5 ml of H202 [5 mM] + 0.1 ml of o-dianisidine [20 mg/ml]). The samples were incubated for 20 min at room temperature and then iced. The OD was read at 460 nm against a water blank, and the micrograms of MPO released were calculated from a standard curve which was determined by using purified human leukocyte MPO.

2.9. PMN [cAMP] PMN (5 × 106/ml) were incubated (30 min at 37°C) with rhTNFa (1 U / m l ) with or without ADA (1 U/ml), adenosine (30 nM), rolipram (100 nM), pentoxifylline (30 /zM) and caffeine (30 /xM) in a shaker water bath and then stimulated for an additional 30 s (37°C) with fMLP (100 nM). The samples were at once centrifuged (10,000g × 1 min), and the sedimented PMN cell-button was immediately extracted with 1 ml of 0.1 N HCI for 45 min at 24°C, and cell debris removed by centrifugation (3000g × 10 min). Cyclic AMP was assayed by the method of Brooker et al. (Brooker et al., 1976). The results are reported as pmoles of c A M P / 5 × 106 PMN.

2.10. Statistics Significance ( p < 0.050) was determined by the two-tailed paired Student's t-test. 3. Results

3.1. A comparison between pentoxifylline and caffeine on LPS-primed leukocyte chemiluminescence, superoxide production, and myeloperoxidase release LPS (10 ng/ml) without serum has no direct effect on PMN or MNL oxidative activity. LPS (10 ng/ml) without serum primed mixed leukocytes (PMN-MNL) for a 2 - 4 fold increase in chemiluminescence, superoxide production, and myeloperoxidase (MPO) release in response to fMLP (100 nM) suggesting the release of a primer from the LPSstimulated leukocytes (Fig. 1A, 2A and 2B).

Chemiluminescence Micromolar concentrations of pentoxifylline diminished LPS-primed PMN-MNL chemilumines-

22

G.W. Sullit,an et al. /Immunopharmacology 31 (1995) 19-29

12

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tion. Pentoxifylline (100 /zM) decreased superoxide production (Fig. 2A).

MPO Release Luminol-enhanced chemiluminescence is dependent upon both superoxide production and release of myeloperoxidase (MPO) from PMN primary granules (Rosen and Klebanoff, 1976). We examined the possibility that the difference between the superoxide and chemiluminescence results may be explained by the dependence of luminol-enhanced chemiluminescence on degranulation by measuring MPO release. The results from the MPO experiment paralleled the chemiluminescence results. Caffeine (100 /zM) significantly enhanced and pentoxifyiline (100 /zM) inhibited PMN-MNL MPO release (Fig. 2B).

lb

methylxanthine~M] Fig. 1. Effects of caffeine and pentoxifylline on LPS-primed PMN plus MNL chemiluminescence, PMN (1X106/ml) and MNL (2× 106/ml) were incubated for 90 rain at 37°C with or without LPS (10 ng/ml), pentoxifylline (10-100 /xM), caffeine (10-100 /zM), and then stimulated with fMLP (100 nM) for an additional 10 min. (A) Caffeine enhanced and pentoxifylline inhibited the peak of fMLP-stimulated chemiluminescence of LPS-primed mixed leukocytes. (B) ADA (1 U/ml) decreased the effects of caffeine and pentoxifylline on LPS-primed fMLP-stimulated PMN-MNL chemiluminescence. Mean + SEM of 5 separate experiments. *p <0.05 activity was decreased compared to control without the methylxanthines. * *p < 0.05 activity was increased compared to control without the methylxanthines.

LPS

30 -oo

~ caffeine 20

pentoxifylline

10

~ no LPS

O0

Superoxide production In contrast to the chemiluminescence results, caffeine did not significantly affect superoxide produc-

160

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3.o.

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cence and the same concentrations of caffeine enhanced the activity (Fig. 1A). Addition of ADA (1 U/ml) (which converts adenosine to inosine) increased the chemiluminescence (50%) in the absence of the methylxanthines, and abolished the effects of both pentoxifylline and caffeine on the primed response indicating that pentoxifylline and caffeine act by altering an endogenous adenosine response (Fig. 1B).

1'0

~/I

~

pentoxi~lline

no LPS

1.0

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,0o

methylxanthine[pM] Fig. 2. Effects of caffeine and pentoxifylline on LPS-primed PMN plus MNL superoxide production and MPO release. (A) superoxide production: caffeine ( < 100 /zM) had no effect on fMLPstimulated superoxide production of LPS-stimulated mixed leukocytes and pentoxifylline (100 /xM) decreased superoxide production. (B) MPO release: caffeine (10-100 gM) augmented and pentoxifylline (100 /xM) diminished fMLP-stimulated MPO release of mixed leukocytes. Mean +SEM of 4-5 separate experiments. *p < 0.05 activity was decreased compared to control without the methylxanthines. * *p < 0.05 activity was increased compared to control without the methylxanthines.

G.W. Sullivan et aL/ Immunopharmacology 31 (1995) 19-29 10

1TIT

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+ LPS-stim. MNL cond. med.

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Fig. 3. The effects of pentoxifylline and caffeine on cell-free LPS-stimulated MNL conditioned medium-primedPMN chemiluminescence in response to fMLP. Pentoxifylline inhibited and caffeine augmentedthe priming of PMN by LPS-stimulated MNL conditioned medium as measured by chemiluminescence. PMN (1 × 106/ml) and LPS-stimulatedMNL conditionedmedium were incubated at 37°C for 30 min with or without caffeine (100 /zM) and pentoxifylline(100/xM) and then stimulated with fMLP(100 nM). Mean+_SEM of 7-18 separate experiments. ~ p < 0.001 activity was increased compared to PMN without LPS-stimulated MNL conditioned medium. 11 p < 0.001 activity was decreased compared to PMN primed with LPS-stimulated MNL conditioned medium, fr f p < 0.001 activity was increased comparedto LPSstim. MNL conditioned medium primed PMN.

3.2. Pentoxifylline decreases and caffeine increases chemiluminescence of PMN primed with LPS-stimulated MNL conditioned medium LPS-stimulated cell-free MNL conditioned medium primed PMN (1 × 106/ml) for increased chemiluminescence in response to fMLP (100 nM) (from 0.6 to 5.4 AU; p < 0.001). The following experiments were designed to determine the effect of pentoxifylline and caffeine on cell-free LPS-stimulated MNL conditioned medium-primed PMN chemiluminescence. We observed that pentoxifylline (100 /xM) significantly inhibited the enhanced chemiluminescence of PMN primed with LPSstimulated MNL conditioned medium ( p < 0.001). In contrast, caffeine (100 ~M) significantly increased chemiluminescence ( p < 0 . 0 0 1 ) (Fig. 3). Thus, the effect of pentoxifylline and caffeine on the chemiluminescence of PMN primed with LPSstimulated MNL conditioned was similar to that observed in the mixed PMN-MNL leukocyte preparations.

23

3.3. Physiological concentrations of adenosine decrease PMN chemiluminescence primed with rhTNFa LPS-stimulated MNL conditioned medium contained TNFa (1 U / m l ) and adenosine (30 nM) (Barnes et al., submitted manuscript). Recombinant human TNFa (rhTNFa) is a potent primer of PMN chemiluminescence in response to fMLP (100 nM). Recombinant human TNFa (1 U / m l ) was found to prime PMN for increased chemiluminescence (from 0.6 + 0.3 to 8.2 ___1.3 AU; p = 0.002), and adenosine at a concentration equal to that observed in the LPS-stimulated MNL conditioned medium (30 nM) decreased rhTNFa-primed PMN chemiluminescence (from 8.2 + 1.3 to 5.0 ± 1.3 AU; p = 0.001).

3.4. Pentoxifylline and caffeine hat,e little effect on PMN chemiluminescence primed with recombinant human TNFa We examined the behavior of pentoxifylline (30 /zM) and caffeine (30 /zM) on the chemiluminescence of PMN primed with rhTNFa (1 U / m l ) and stimulated with fMLP (100 nM). Pentoxifylline and caffeine were found to have only a small effect on the priming of PMN by rhTNFa. Pentoxifylline (30 /xM) decreased rhTNFa-primed activity from 8.2 + 1.3 to 6.9 + 1.5 ( p = 0.016), and caffeine (30 /zM) increased rhTNFa-primed activity from 8.2 + 1.3 to 9.5 + 1.3 ( p = 0.050).

3.5. Pentoxifylline potentiates and caffeine counteracts the modulating effect of adenosine on rhTNFaprimed PMN chemiluminescence Micromolar concentrations of caffeine increased PMN chemiluminescence primed by rhTNFa and modulated by adenosine (30 nM) (Fig. 4A), but not modulated by a much higher concentration adenosine (10 /zM; 1.19_+0.42 AU). In contrast, pentoxifylline decreased the PMN chemiluminescence (Fig. 4A). To further investigate the role of adenosine in caffeine and pentoxifylline altered PMN oxidative activity, we examined the effect of removing adenosine from these assays with adenosine deaminase (ADA). ADA removed the modulating effect of adenosine (30 nM) ( p = 0 . 0 0 3 ) , increasing the chemiluminescence to an activity comparable to

G.W. Sulliuan et al. / lmmunopharmacology 31 (1995) 19-29

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3.6. Neither adenosine nor the methylxanthines affected P M N [cAMP] A l t h o u g h adenosine (30 n M ) halved TNFo~-primed P M N c h e m i l u m i n e s c e n c e , it did not measurably affect P M N [cAMP]. A l t h o u g h the n o n - x a n t h i n e phosphodiesterase inhibitor rolipram markedly increased

4-

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receptors. C P A binds to A 1 receptors with a K i of 0.6 n M and to A 2 receptors with a K i of 462nM. In contrast, C G S 21680 binds to A 1 receptors with a K i of 2600 n M and A 2 receptors with a Ki of 15 n M (reviewed by Collis and H o u r a n i (1993)). C G S 21680 (100 n M ) was a more potent inhibitor of rhTNFceprimed f M L P - s t i m u l a t e d activity than C P A (100 n M ) indicating that primed c h e m i l u m i n e s c e n c e is modulated by b i n d i n g to A e adenosine receptors. Caffeine (30 /zM) increased and pentoxifylline (30 /xM) decreased primed c h e m i l u m i n e s c e n c e modulated by both C G S 21680 and C P A (Fig. 5).

I-Ino TNF no ADO ,,2 0

;

10

100

methylxanthine [i.tM] Fig. 4. The effect of caffeine and pentoxifylline with and without adenosine on rhTNFa primed-PMN fMLP-stimulated chemiluminescence. (A) Pentoxifylline potentiated and caffeine counteracted the effect of adenosine on rhTNFo~-primedactivity. (B) ADA (1 U/ml) decreased the effects of pentoxifylline and caffeine on rhTNFot-primed activity. PMN (1 × 106/ml)were incubated for 30 min at 37°C with or without adenosine (ADO; 30 nM), ADA (1 U/ml), pentoxifylline (1 /zM-30/zM), caffeine (1 /zM-30/zM), and rhTNFa (1 U/ml) and then stimulated with fMLP (100 nM). Mean _+SEM of 4-9 separate experiments. #p = 0.001 decrease in activity compared to rhTNFot-primed PMN. *p = 0.030 decrease in activity compared to adenosine-modulated rhTNFot-primed PMN in absence of a methylxanthine. * *p < 0.005 increase in activity compared to adenosine-modulated rhTNFot-primed PMN without methylxanthine.

T N F - p r i m e d activity in the absence of adenosine. No significant effect of either pentoxifylline or caffeine on the rhTNFc~-primed response was observed in the presence of A D A indicating again that pentoxifylline and caffeine have activity only in the presence of adenosine (Fig. 4B). The two adenosine analogs C P A and C G S 21680 are not internalized or metabolized by cells and have differing b i n d i n g capacities to adenosine A 1 and A 2

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Fig. 5. The effect of caffeine and pentoxifylline with and without the adenosine analogs CGS 21680 and CPA on rhTNFa primedPMN fMLP-stimulated chemiluminescence. Pentoxifylline potentiated and caffeine counteracted the effect of CGS 21680 and CPA on rhTNFot-primed activity. PMN (1 x 106/ml) were incubated for 30 min at 37°C with ADA (1 U/ml) and with or without CGS 21680 (100 nM) or CPA (100 nM), pentoxifyUine (30 /zM), caffeine (30 /xM), and rhTNFot (1 U/ml) and then stimulated with fMLP (100 nM). Mean ±SEM of 4 separate experiments. * p < 0.050 increase in activity compared to adenosine analog-modulated rhTNFa-primed PMN without a methylxanthine. * * p < 0.050 decrease in activity compared to adenosine analog-modulated rhTNFa-primed PMN in absence of a methylxanthine.

G. W. Sullican et al. / lmmunopharmacology 3l (1995) 19-29

no Rolipram

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primed PMN-MNL and rhTNFa-primed pure PMN plus adenosine. Caffeine (100 IzM) had no effect on the superoxide production of both mixed PMN-MNL primed with LPS ( p = 0.249) and pure PMN plus adenosine (100 nM) primed with rhTNFa ( p = 0.542) (Fig. 7A and Fig. 7B).

-6 ~

~

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2 0 Medium

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ADO + PTX

ADO + CAF

Fig. 6. The effect of adenosine, pentoxifylline and caffeine on PMN [cAMP]. Adenosine (30 nM), pentoxifylline (30 /zM) and caffeine (30 /.tM) did not affect PMN [cAMP] either in the presence or absence of ADA (1 U / m l ) or rolipram (100 nM). PMN (5 × 106/ml) were incubated (30 min at 37°C) with rhTNFo~ (1 U / m l ) with or without ADA (1 U / m l ) , adenosine (30 nM), pentoxifylline (30 /xM) and caffeine (30 /xM) in a shaker water bath and then stimulated for 30 s more (37°C) with fMLP (100 nM). Mean + SEM of 3 - 4 separate experiments.

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the amount of cAMP detected in the samples, the presence of rolipram did not significantly affect the amount of cAMP in adenosine treated cells ( p = 0.091). Also, pentoxifylline and caffeine (30 /~M) with or without adenosine, ADA, or rolipram did not affect the amount of PMN [cAMP] measured, suggesting that cAMP does not mediate the responses of adenosine and methylxanthines at these low concentrations (Fig. 6).

3.7. Comparison of pentoxifylline and caffeine effects on LPS-and rhTNFa-primed leukocyte chemiluminescence, superoxide production, and MPO release We compared the effects of caffeine and pentoxifylline on chemiluminescence, superoxide production, and MPO release of mixed PMN-MNL primed with LPS (Fig. 7A) and on pure PMN (plus exogenous adenosine [100 nM]) primed with rhTNFo~ (Fig. 7B). Control activity was the activity that was seen in the absence of caffeine and pentoxifylline.

Caffeine Caffeine (100 /xM) significantly increased the chemiluminescence and MPO release of both LPS-

< 150 "E

8

100

"6 50 0 Chemilum

Superoxide

MPO

Fig. 7. Effects of caffeine and pentoxifylline on (A) PMN-MNL and (B) PMN+adenosine (100 nM) modulated chemiluminescence, superoxide production, and MPO release. (A) Caffeine enhanced both chemiluminescence and MPO release of mixed PMN-MNL primed with LPS while having no effect on superoxide production. Pentoxifylline inhibited chemiluminescence, superoxidc production, and MPO release of mixed PMN-MNL primed with LPS. The activity is displayed as % of control activity of LPS-primed mixed leukocytes in the absence of methylxanthines. Chemiluminescence control = 7.5 + 1.1 AU, superoxide - 26.1_+4.2 nmol, MPO - 2.7_+0.1 /xg. (B) Similar to LPSprimed PMN-MNL results, caffeine enhanced both chemiluminescence and MPO release of adenosine-modulated pure PMN primed with rhTNFa while having no effect on superoxide production. Likewise, pentoxifylline decreased chemiluminescence, superoxide production, and MPO release. The control was the activity of adenosine-modulated PMN primed with rhTNFa in the absence of caffeine and pentoxifylline. Chemiluminescence control = 2.6_+ 0.3 AU, superoxide = 15.6_+ 1.0 nmol, and MPO 1.2_+0.2 #g. {} p < 0.05 activity was increased compared to control without the methylxanthines. 1~ p < 0.05 activity was decreased compared to control without the methylxanthines.

26

G. W. Sullivan et aL / lmmunopharmacology 31 (1995) 19-29

Pentoxifylline The effect of pentoxifylline on chemiluminescence, superoxide production, and MPO release of mixed PMN-MNL primed with LPS (no added exogenous adenosine) was similar to that of pure PMN plus adenosine primed with rhTNFc~. Pentoxifylline (100 /xM) inhibited chemiluminescence, superoxide, and MPO release of both mixed PMN-MNL primed with LPS and pure PMN plus adenosine (100 nM) primed with rhTNFoz (Fig. 7A and Fig. 7B).

4. Discussion

We found that the two methylxanthines, pentoxifylline and caffeine, have divergent effects on primed PMN oxidative activity as measured by chemiluminescence. Opposite effects of pentoxifylline and caffeine were observed with preparations of PMN plus MNL primed with LPS, pure PMN primed with recombinant human TNFot, and pure PMN primed with cell-free LPS-stimulated MNL conditioned medium. A prominent priming factor in the LPSstimulated conditioned medium was TNFa, and the LPS-stimulated MNL conditioned medium contained adenosine (30 nM). The activities of both methylxanthines were dependent upon the presence of adenosine. The divergent effects of pentoxifylline and caffeine on primed-chemiluminescence were only observed in the presence of adenosine. When micromolar concentrations of pentoxifylline or caffeine were added to pure PMN (containing about 6 nM endogenous adenosine) there was little effect on rhTNFaprimed chemiluminescence. In contrast, when 30 nM adenosine was added to the TNFo~-primed PMN the divergent effects of pentoxifylline and caffeine were very evident, and removal of the added adenosine with ADA completely abolished the effects of both pentoxifylline and caffeine on TNFa-primed PMN chemiluminescence. Similar results were observed when ADA was added to the adenosine containing ( ~ 30 nM) mixed leukocytes (PMN-MNL) primed with LPS. We therefore conclude that adenosine is necessary for the actions of both pentoxifylline and caffeine on TNFa-primed PMN activity, and that methylxanthines can modulate the activity of physiological concentrations of adenosine.

Why do the two methylxanthines cause opposite effects? One possible target location for caffeine and pentoxifylline actions is the adenosine receptor. Caffeine, has been demonstrated to block adenosine receptor binding and activity (Biaggioni et al., 1991). Hence, when adenosine is removed from the PMN, the chemiluminescent activity is higher and there is no effect of caffeine suggesting that caffeine acts by blocking the effect of adenosine. In contrast, pentoxifylline does not to act via adenosine receptors in the inhibition of PMN superoxide production (Hand et al., 1989; Thiel et al., 1991). However, pentoxifylline decreases adenosine uptake in activated PMN (Hand et al., 1989), thus increasing the availability of adenosine to adenosine receptors on the cell surface. In the present experiments, removal of adenosine with ADA abolished the effect of pentoxifylline on both PMN primed with rhTNFa and mixed PMN and MNL primed with LPS indicating that the activity of pentoxifylline is dependent on the presence of adenosine. Since pentoxifylline also enhanced the activity of CGS 21680 and CPA two adenosine analogs which are neither internalized nor metabolized is evidence that pentoxifylline can act by means other than affecting adenosine uptake. Luminol-enhanced chemiluminescence is a measure of oxidative activity which is dependent upon both PMN production of reactive oxygen species and mobilization of MPO from PMN granules (Rosen and Klebanoff, 1976). Caffeine affected chemilUminescence by potentiating the degranulation of MPO with little effect on superoxide production, and pentoxifylline augmented the diminishing effect of adenosine on chemiluminescence by decreasing both MPO release and superoxide production. There is evidence that cAMP can down regulate PMN oxidative activity (Sedgwick et al., 1985; Gibson-Berry et al., 1993), and that adenosine especially in the presence of phosphodiesterase inhibitors can stimulate measurable concentrations of PMN cAMP (Cronstein et al., 1992; Nielson et al., 1992). The modulating effect of adenosine on the PMN oxidative burst has been attributed to the binding of adenosine to adenosine A 2 receptors (Cronstein et al., 1985; Cronstein et al., 1992). Adenosine when it binds to A 2 receptors stimulates the production of cAMP. Phosphodiesterase activity in the cell breaks down cAMP to 5'-AMP. Methylxanthines can have

G. W. Sullivan et al. / Immunopharmacology 3l (1995) 19-29

phosphodiesterase inhibitory activity. Therefore, we hypothesized that cAMP production stimulated by adenosine might be the mediator that decreases TNFo~-primed PMN activity, and that the effects of pentoxifylline and caffeine might be explained by the differential effects of these two methylxanthines on adenosine binding to A 2 receptors a n d / o r their inhibitory effects on cAMP phosphodiesterase activity. As mentioned above caffeine and not pentoxifylline acts as an adenosine receptor antagonist. Both pentoxifylline and caffeine have phosphodiesterase inhibitory activity, but only at concentrations above > 100/zM (Sawynok and Yaksh, 1993). We observed that adenosine (30 nM) with and without the presence of methylxanthines had no measurable effect on PMN [cAMP]. Thus, as others have observed adenosine and phosphodiesterase inhibitors can affect cellular activity at concentrations less than those that cause measurable changes in [cAMP] (Cronstein et al., 1990; Sinha et al., 1994). Two possible explanations for the observations are the existence of non-cAMP dependent adenosine and the methylxanthines effects a n d / o r temporal or spatial distributions on cAMP in the cell not detectable by our assays (Pryzwansky et al., 1981). Our study indicates that pentoxifylline can be considered as a possible therapeutic agent that not only decreases TNFo~ production (Strieter et al., 1988; Han et al., 1990; Endres et al., 1991), but also decreases TNFo~-primed PMN activity when combined with adenosine. Oral administration of a 400 mg sustained release dose of pentoxifylline results in a peak serum concentration of pentoxifylline and its metabolites of 1 /zM at 3 h. Although, the concentrations of pentoxifylline used in our experiments cannot be obtained in the plasma, plasma concentrations have been found to have similar effects on PMN oxidative activity when measured ex vivo. A plasma concentration of pentoxifylline of ~ 300 nM has been found to be capable of diminishing PMN superoxide production as measured ex vivo from subjects who had ingested pentoxifylline (400 mg) (Crouch and Fletcher, 1992). Crouch et al. have indicated that it is metabolites and not pentoxifylline itself that are effective at reducing PMN respiratory burst activity. It has also been reported recently that an early unstable metabolite of pentoxifylline, 1-[5R-hydroxy-hexyl]-3,7-dimethylxanthine, is also a potent

27

anti-inflammatory agent (Rice et al., 1994). Thus, the existence of highly active metabolites may explain the greater than expected activity of pentoxifylline than can be predicted from in vitro studies. Concentrations of adenosine found in plasma ( ~ 0.2 to 0.4 /~M) were effective in our experiments with pentoxifylline. Ingestion of 60 to 180 mg of caffeine results in a peak serum concentrations of 30 to 5 0 / x M (Sawynok and Yaksh, 1993). In our experiments, caffeine at physiologically obtainable concentrations increased PMN chemiluminescence and MPO release, indicating the immunostimulating properties of caffeine. The difference in the PMN response to caffeine and pentoxifylline indicates that not all methylxanthines have the same effect on PMN activity. Further experiments are needed to determine if this difference in in vitro activity can predict the behavior of these two methylxanthines in vivo.

Acknowledgements This study was supported in part by Public Health Service grant RO1AI09504 from the National Institutes of Health.

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