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(Journal of Leukocyte Biology. 2000;68:194-200.)
© 2000 by Society for Leukocyte Biology

Effects of theophylline on human eosinophil functions: comparative study with neutrophil functions

Kozo Yasui, Kazunaga Agematsu, Koji Shinozaki, Sho Hokibara, Haruo Nagumo, Shinji Yamada, Norimoto Kobayashi and Atsushi Komiyama

Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan

Correspondence: Kozo Yasui, Department of Pediatrics, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Japan. E-mail: koyasui{at}gipac.shinshu-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
The understanding of theophylline as a bronchodilator has been reconsidered in recent years. We undertook to determine its immunomodulatory actions in granulocytes and elucidate their mechanism. Preincubation of neutrophils with theophylline (10-5 to 5 x 10-3 M) had a biphasic effect on O2- production stimulated with N-formyl-methionyl-leucyl-phenylalanine or C5a. Theophylline potentiates O2- production via adenosine A2A receptor antagonism induced by receptor-linked agonists from neutrophils, but not from eosinophils. The addition of theophylline caused a significant decline in neutrophil chemotaxis at lower concentrations than those for eosinophil motility. Theophylline reduces neutrophil chemotaxis via adenosine A1 receptor antagonism. At high concentrations, with an intracellular cAMP accumulation as a result of phosphodiesterase (PDE) inhibition, theophylline also exerts an inhibitory effect on the O2- production and chemotaxis of both types of cells. The difference in theophylline’s effect on neutrophils and eosinophils appears to depend on the existence of specific adenosine receptors. Theophylline thus modulates granulocyte functions in association with specific adenosine receptor antagonism and cAMP-PDE inhibition.

Key Words: superoxide anion production • chemotaxis • adenosine • receptor antagonism • airway inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
Theophylline has been widely used in the treatment of patients with bronchial asthma. The primary pharmacological mechanism of theophylline has long been considered to be bronchodilation due to the inhibition of cyclic nucleotide phosphodiesterase (PDE) isoenzymes, leading to accumulation of adenosine 3’,5’-cyclic monophosphate (cAMP) in the airway to relax smooth muscle. However, this view has come under review in recent years. The hypothesis was challenged on the basis of the observation that the concentrations of theophylline required to induce airway smooth muscle relaxation in vitro are higher than the therapeutic concentrations usually achieved in plasma (5–20 µg/mL; 3 to 11 x 10-5 M), so that therapeutic concentrations of theophylline can produce a slight inhibition of PDE activity [1 2 3 ]. These findings suggest that theophylline has a beneficial effect on asthma control that is not related to PDE inhibition or mild bronchodilator action. This effect is thought to involve the suppression of the inflammatory processes underlying the development and exacerbation of asthma.

There is increasing evidence that theophylline has anti-asthma properties other than bronchodilation, which include anti-inflammatory actions [4 , 5 ]. The inhibition of a late asthmatic response by theophylline has been demonstrated in conjunction with significant reduction of inflammatory cells in the airway [6 , 7 ], and these effects were seen at plasma concentrations below those traditionally regarded as therapeutically useful [6 ]. Bronchial hyper-responsiveness is an important feature in asthma, while inflammation associated with granulocyte (neutrophil as well as eosinophil) infiltration is involved in its development. Granulocytes are thought to damage the normal airway tissue through releasing toxic substances such as superoxide and harmful proteins.

Because neutrophil and eosinophil activation is associated with late-phase asthmatic reactions [3 , 6 , 7 ], to elucidate the effects of theophylline on these cells may be of importance.

Several studies have been published on the effects of theophylline on neutrophil functions such as generation of oxygen metabolites and chemotactic movement [8 9 10 11 12 ]. It has been established that the physiological mechanism for the effect of theophylline on the modulation of neutrophil functions may be dependent on adenosine receptor antagonism and/or on PDE inhibition. Adenosine, which is normally present in plasma at concentrations up to 3 x 10-7 M [13 , 14 ], is one of the pharmacological and hormonal agents that modulate neutrophil functions in vitro [10 , 14 15 16 17 18 ]. Neutrophils release a certain amount of adenosine [13 ], and adenosine A1 and A2A but not A2B receptors are known to be strongly expressed on neutrophils [19 , 20 ]. Theophylline (an A1, A2A, and A2B antagonist) is believed to modulate neutrophil functions at least in part through selective adenosine receptor antagonism. However, very limited information on the effects and mechanism of theophylline on eosinophil functions is available compared with those on neutrophils, and little confused [21 22 23 24 ].

This study was undertaken to determine the involvement of adenosine receptor antagonism and PDE inhibition in the effect of theophylline on eosinophil functions and to compare it with that on neutrophils to reach a consolidated view of the functions and mechanisms of theophylline.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 REFERENCES
 
Reagents
Dextran T500 was purchased from Pharmacia (Uppsala, Sweden). N-formyl-methionyl-leucyl-phenylalanine (fMLP), complement C5a, phorbol 12-myristate 13-acetate (PMA), cytochrome c (type VI), N-ethylmalemide, superoxide dismutase, theophylline, dibutyryl-adenosine 3’,5’-monophosphate (dbcAMP), adenosine deaminase (ADA; type V), N6-cyclohexyladenosine (CHA; A1 >> A2 agonist), 5’-N-ethylcarboxamidoadenosine (NECA; A2A and A2B > A1), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A1 and A2B receptor antagonist), dimethyl sulfoxide (DMSO), Histopaque, HEPES, and propidium iodide (PI) were purchased from Sigma Chemical (St. Louis, MO). 1-Deoxyl-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-ß-D-ribofuranuronamide (IB-MECA; A3 agonist), 8-p-sulfophenyltheophylline (8-SPT) and 3-n-propylxanthine (enprofylline) were obtained from RBI (Natick, MA). 2-p-(2-carboxyethyl) phenethylamino-5’-N-ethylcarboxamidoadenosine (CGS21680; a selective adenosine A2A receptor agonist) [20 ] was acquired from Tocris Cookson (Bristol, UK). (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KF-17837; a selective adenosine A2A receptor antagonist with a low affinity for A2B receptors) [25 ] was provided by Kyowa Hakko (Tokyo, Japan). Anti-CD16 mAb-coated immunologic magnetic beads and the magnetic cell sorter system were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany).

Preparation of cells
Heparinized venous blood was obtained from healthy volunteers. Neutrophils were isolated by using dextran sedimentation and centrifugation on a Histopaque gradient (without endotoxin) as previously described [26 ]. Contaminating red blood cells were removed by means of cold hypotonic water lysis. Cells were then subjected to another density cut using centrifugation over a Percoll gradient in order to isolate neutrophils from contaminating eosinophils. Eosinophils were purified from the peripheral blood of slightly allergic donors (mild rhinitis) with the aid of gradient centrifugation and negative selection with anti-CD16 mAb-coated immunologic magnetic beads and a magnetic cell sorter. The purity of the isolate was assessed by preparing cytocentrifuged smears and staining with May-Grünwald-Giemsa (Merck, Darmstadt, Germany) stain. The purity of the neutrophils was >97% and that of the eosinophil preparations was >98%, whereas the granulocyte viability was >99% as determined by trypan blue dye exclusion (Sigma). Purified cells were suspended in Hanks’ balanced salt solution (HBSS) without calcium chloride or magnesium sulfate.

Superoxide anion (O2-) production
Superoxide production was determined at 37°C, with the modified method of Cohen and Chovaniec [26 , 27 ]. The amount of superoxide released was determined with a Hitachi spectrophotometer U 2000 (Tokyo, Japan) as the change in absorbance at 550 nm resulting from superoxide dismutase (SOD)-inhibitable cytochrome c reduction. The reaction was carried out for 5 min with fMLP (10-6 M), C5a (10-7 M), and PMA (5 ng/mL), after which the reaction was stopped by the addition of 0.5 mM N-ethylmalemide. The generation of superoxide was calculated by subtracting the change in absorbance in the presence of SOD (1 mM) from that in its absence, and then dividing this value by 21.1 x 103/M/cm for the molar extinction coefficient.

Cell motility (chemotaxis)
Cell motility was measured with the agarose method [26 , 28 ]. Briefly, 5 mL of 1.2% agarose dissolved in HBSS containing Ca2+ and Mg2+ and supplemented with 10% fetal calf serum (FCS) was placed in 60 x 15-mm Petri dishes. A 10-µL aliquot of cell suspension, containing 5 x 105 cells, was placed in the center well of the plate, and equal volumes of the chemoattractant (2 x 10-7 M fMLP) and HBSS were placed in the outer and inner wells, respectively. The wells were 3 mm in diameter, and the outer and inner wells were located at a distance of 7 mm from the center one. After incubation at 37°C in 5% CO2 for 2 h, the plates were fixed with ethanol and formalin and the cells stained with Wright’s stain (Sigma). Cell motility was defined as the linear distance the cells moved from the center well in the direction of the well containing the chemoattractant (chemotaxis), and the migration distance was compared to that of controls. All results represent the averages of duplicate measurements.

Statistical analysis
All data are presented as means ± SD. Comparisons between paired conditions were made with the aid of paired t tests. One-way factorial analysis of variance and multiple comparison tests (Fisher’s PLSD) were used for comparative analyses of data for several groups. P < 0.05 was considered significant in all cases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
REFERENCES
 
Effects of theophylline on superoxide anion production
Isolated neutrophils (1 x 107 cells/mL) and eosinophils (2 x 106 cells/mL) in HBSS were preincubated with various concentrations of theophylline at 37°C for 15 min. Control O2- production stimulated with 1 x 10-6 M fMLP ranged from 60 to 100 nmol cytochrome c reduced/107 cells for 5 min (86 ± 14 nmol; mean ± SD of five individual donors) in neutrophils, and from 49 to 63 nmol cytochrome c reduced/2 x 106 cells for 5 min (55 ± 6 nmol; mean ± SD of four individual donors) in eosinophils. This concentration of fMLP caused the strongest activation for O2- production. As shown in Figure 1 , preincubation of neutrophils with 10-5 to 5 x 10-3 M theophylline had a biphasic, concentration-related effect on O2- production stimulated with fMLP. Theophylline potentiated O2- production by 20–50% at a clinically relevant concentration range of 10-5 to 10-4 M, and maximal potentiation was observed at a concentration of 5 x 10-5 M (P < 0.002), whereas theophylline at higher concentrations (10-3 to 5 x 10-3 M) inhibited O2- production from 25 to 60% of control value (5 x 10-3 M; P < 0.0001). Theophylline induced similar degrees of potentiation and biphasic influence on O2- production stimulated with C5a (1 x 10-7 M) in neutrophils (control value of 103 ± 18 nmol; mean ± SD, n = 4), but no significant change in O2- production was in PMA (5 ng/mL)-stimulated neutrophils (control value of 83 ± 12 nmol, n = 4).



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  		Figure 1. Effects of theophylline on superoxide anion (O2-) production from neutrophils (107 cells/mL) and eosinophils (2 x 106 cells/mL) stimulated with several agents. Cells were preincubated with buffer or the indicated concentrations of theophylline for 15 min at 37°C before addition of stimulants, after which O2- production was determined. The reaction was conducted for 5 min. Results from five donors were normalized to obtain percentages of control values and are shown as the mean ± SD. *Significant differences from control values: P < 0.05.

 
The effect of theophylline on O2- production in eosinophils was substantially different from that in neutrophils because no potentiation was observed at any concentration of theophylline. Preincubation of eosinophils with 10-4 to 5 x 10-3 M theophylline significantly inhibited O2- production stimulated with fMLP and C5a (P < 0.01, n = 3) but not with PMA.

Effects of theophylline on chemotaxis
The maximum linear distance that cells had migrated from the margin of the center well was measured and compared with that in control experiments (Fig. 2 ). The control migration distance for four individual donors was 3120 ± 160 µm in neutrophils, and 2250 ± 150 µm in eosinophils. As shown, theophylline caused a concentration-dependent reduction in the chemotactic response of both cells. The addition of theophylline to neutrophils caused a significant decline in chemotaxis at lower concentrations than those for eosinophils.



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  		Figure 2. Effects of theophylline on fMLP (2 x 10-7 M)-induced chemotaxis of neutrophils and eosinophils. Chemotaxis was defined as the maximum distance cells migrated under agarose (2 h) from the edge of the center well. Results are shown as means ± SD from three separate experiments and normalized as control percentage. *Significant differences from control values: P < 0.05.

 
Effects of 8-p-sulfophenyltheophylline and enprofylline on superoxide anion production
Theophylline had a biphasic influence on fMLP-stimulated generation of superoxide anions in neutrophils (Fig. 1) . In contrast to theophylline, 8-SPT produced only potentiation of O2- production (Fig. 3 ). Major potentiation of more than 100% was observed at a concentration of 5 x 10-5 M (P < 0.0001, n = 3) when stimulated with fMLP. The same effect of 8-SPT on O2- production was observed in neutrophils stimulated with C5a (data not shown) but not with enprofylline. Incubation with 0.1 U/mL of adenosine deaminase (ADA) or 1 µM of KF-17837 (10 min, 37°C) also augmented superoxide production more than twice as much as in control experiments, but DPCPX had no effect (data not shown). In contrast, none of the effects seen in neutrophils, i.e., potentiation by 8-SPT, ADA, or KF-17837, was observed in eosinophils. Incubation of neutrophils at various concentrations of enprofylline (5 x 10-6 to 10-3 M) caused a concentration-dependent inhibition of O2- production. A similar inhibition was seen in eosinophils treated with enprofylline.



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  		Figure 3. Effects of 8-sulfophenyltheophylline (8-SPT) or enprofylline on O2- production from neutrophils (A) and eosinophils (B) stimulated with fMLP (10-6 M). Cells were preincubated with buffer or the indicated concentrations of drugs for 15 min at 37°C before addition of the stimulant, after which O2- production was determined. The reaction was conducted for 5 min. The cells were also incubated with 0.1 U/mL ADA (filled triangles) or 1 µM KF-17837 (open triangles). Results are shown as means ± SD from three separate experiments and normalized as control percentage. *Significant differences from control values: P < 0.05.

 
Effects of 8-p-sulfophenyltheophylline and enprofylline on chemotaxis
As shown in Figure 4 , the addition of 8-SPT or enprofylline caused a concentration-dependent reduction in the chemotactic response in neutrophils (Fig. 4A) , but significant reduction was not observed at lower concentrations of enprofylline (<=10-4 M). Preincubation with 0.1 U/mL of ADA also significantly inhibited neutrophil chemotaxis, whereas treatment of eosinophils with 8-SPT or ADA (Fig. 4B) had no effect on eosinophil chemotaxis. Inhibitory effect was observed in eosinophils treated with enprofylline at higher concentrations.



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  		Figure 4. Effects of 8-SPT or enprofylline on fMLP (2 x 10-7 M)-induced chemotaxis of neutrophils (A) and eosinophils (B). Chemotaxis was defined as the maximum distance cells migrated under agarose (2 h) from the edge of the center well. The cells were also treated with 0.1 U/mL ADA (filled triangles) or 1 µM KF-17837 (open triangles). Results are shown as means ± SD from three separate experiments and normalized as percentages of control values. *Significant differences from control values: P < 0.05.

 
Effects of adenosine analogs on superoxide anion production
To determine what class of adenosine receptor exists on neutrophils or eosinophils and affects their functions, we studied the effects of several adenosine analogs on O2- production in these granulocytes.

All of the adenosine analogs inhibited O2- production from neutrophils stimulated with fMLP (Fig. 5A ), with a rank order for potency obtained with selective adenosine receptor agonists of CGS-21680 (A2A) > NECA (A1 and A2A, A2B) > CHA (A1) > IB-MECA (A3). CGS-21680 was most potent in adenosine analogs, suggesting that an A2A-receptor was involved in neutrophil events. Exposure to 5 x 10-7 M CGS-21680 for 15 min resulted in a 50% inhibition of O2- production from neutrophils.



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  		Figure 5. Effects of adenosine analogs on O2- production from neutrophils (A) or eosinophils (B) stimulated with fMLP (10-6 M). Neutrophils (107 cells/mL) and eosinophils (2 x 106 cells/mL) were preincubated with adenosine analogs (final DMSO concentration < 0.1%) for 15 min at 37°C. Representative data are shown. Essentially the same results were obtained in three similarly designed experiments.

 
IB-MECA was the most potent inhibitor of superoxide production in eosinophils. Exposure to 1 x 10-6 M IB-MECA resulted in a 50% inhibition of O2- production from eosinophils.

Comparative study between theophylline and 8-SPT in O2- production from neutrophils
A likely explanation of the inhibitory effects of O2- production at high concentrations of theophylline (>10-3 M) can be found in the intracellular cAMP accumulation as a result of PDE inhibition. On the basis of the finding that 8-SPT does not inhibit PDE activity [10 , 29 ], we used 8-SPT and an excess of dbcAMP to reproduce the biphasic effect of theophylline on O2- production from neutrophils stimulated with fMLP (10-6 M). dbcAMP was used as a cell-permeant cAMP analog that can modulate intracellular signaling and cellular function. The results are shown in Figure 6 . The potentiation of O2- production from neutrophils by 8-SPT was reversed by the addition of dbcAMP, and an excess of dbcAMP (5 x 10-4 M) significantly inhibited O2- production from neutrophils in comparison to that seen in controls (ANOVA: F3,9 = 60.642, P < 0.0001). In other words, the biphasic effect of theophylline could be reproduced with these chemicals.



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  		Figure 6. Effects of 8-SPT and dbcAMP on O2- production from neutrophils. Neutrophils (107 cells/mL) were preincubated for 15 min at 37°C with buffer and the indicated concentrations of 8-SPT alone or in the presence of dbcAMP. O2- production was conducted for 5 min under stimulation with fMLP (10-6 M). Results are shown as means ± SD from three separate experiments and normalized as percentages of control.

 
DISCUSSION
Our results confirm that theophylline has a biphasic effect on O2- production from neutrophils to receptor-linked agonists such as fMLP and C5a as previously reported [10 , 11 , 17 ]. Superoxide anion production was potentiated by 50% at concentrations of 10-5 to 10-4 M of theophylline, which are within the therapeutic range. We also observed that a specific adenosine A2A receptor agonist, CGS-21680, has the strongest inhibitory effect on O2- production in neutrophils, and that this potentiation can also be induced with the A2A receptor-selective antagonist KF-17837 or adenosine deaminase but not with DPCPX (an A1 and A2B receptor antagonist) or enprofylline (A1 and A2B antagonist [11 , 30 ]). These results strongly suggest that A2A receptors are involved in the modulation of superoxide production in neutrophils. A recent study has shown that a selective A2A agonist reduces superoxide release from neutrophils and inhibits inflammation in a rat model of meningitis [31 ]. These findings are in agreement with the previous observation that adenosine acts on A2 receptors and inhibits some neutrophil functions [32 ]. A2A receptor activation is linked to stimulation of adenylate cyclase, and adenosine increases intracellular cAMP levels in neutrophils [25 ]. Consequently, the elevated cAMP levels have been postulated as representing the second messenger mediating neutrophil functions with adenosine A2A agonists. Meanwhile, a serine/threonine protein phosphatase has been suggested as an alternative candidate [33 ], but more detailed information is needed to reach a definitive conclusion.

It should be noted that a different effect of theophylline was observed in eosinophils, where neither theophylline nor A2A-specific adenosine analog had any potentiating effect on O2- production. This appears to be consistent with the results of receptor binding studies that few adenosine A2A receptors occur in eosinophils [20 , 34 ]. The inhibitory effect of theophylline on O2- production from eosinophils was previously reported by several investigators [21 , 23 , 24 ]. With regard to the effect of theophylline, contradictory findings to ours have been reported by different parties [21 , 24 ]. They reported that lower concentrations of theophylline may potentiate O2- production from eosinophils. Yukawa and colleagues [25 ] reported that eosinophil activation is caused by competing with circulating adenosine for eosinophil A2 receptors. One explanation for this clear discrepancy could be the insufficient purity of eosinophils. Although high-purity eosinophils should have been obtained for their functional study, the centrifugation over Percoll density gradients was used, resulting in low-purity eosinophils (80%). In contrast, the magnetic bead method is recommended for the isolation of eosinophils with a high purity (>98%) without functional priming [35 ]. It can therefore be assumed that the eosinophils used in the study contained 20% neutrophils; the potentiation of superoxide production by theophylline could therefore be the result of neutrophil contamination. In a recent study, however, Ezeamuzie and Al-Hage [24 ] again reported a biphasic effect of theophylline on the activation of highly purified eosinophils. Furthermore, they observed significant inhibition of O2- production from eosinophils at much lower concentrations (10-6 and 10-5 M). These findings are contrary to other studies [21 , 23 ], but so far the causes of these discrepancies are not fully understood.

Higher concentrations (>=10-3 M) of theophylline caused significant inhibition of O2- production from both neutrophils and eosinophils. Our findings showed that theophylline analog 8-SPT, more potent A1 and A2A receptor antagonists, which lacks PDE inhibitory activity [10 , 29 ], had only a potentiating effect on O2- production in neutrophils. On the other hand, enprofylline, which is known as an adenosine A1 and A2B antagonist [11 , 30 ] with PDE inhibitory activity, inhibited O2- production from both cells. This suggests that the difference in effects between theophylline and enprofylline might be due to the presence or absence of A2A antagonism and that the inhibitory effects of these xanthine derivatives on O2- production may be due to PDE inhibition. The inhibitory effects of theophylline and enprofylline on cAMP-PDE are already well known [11 , 36 ]. In addition, we were able to reproduce the biphasic effect of theophylline on O2- production from neutrophils by using 8-SPT and an excess of dbcAMP. The addition of dbcAMP reversed the potentiation of O2- production from neutrophils by 8-SPT. Selective PDE IV inhibitors have been found to be very effective for the inhibition of superoxide production from activated eosinophils [14 , 23 , 37 ]. These findings support our contention that the inhibitory effects of O2- production at a high concentration of theophylline can be explained by intracellular cAMP accumulation as a result of PDE inhibition.

The elevation of intracellular cAMP inhibits activation of several functions of human granulocytes including O2- production induced by receptor-linked agonists [38 ]. In contrast, when PMA was used to activate cells, theophylline had no effect on O2- production from either neutrophils or eosinophils. cAMP PDE inhibitor in combination with prostaglandin E1 markedly inhibits superoxide production from neutrophils stimulated with fMLP, but not from those stimulated with phorbol ester [39 ], whereas inactive C5b67 inhibits C5a- and fMLP-, but not PMA-induced superoxide production [40 ]. These results indicate that such inhibitions occur independent of protein kinase C activation, and that cAMP levels represent the second messenger interfering with intracellular signaling stimulated with receptor-linked agonists and not with PMA in granulocytes.

The addition of theophylline to neutrophils caused a significant decline in chemotaxis at lower concentrations than those used for eosinophils. The effect thus seems to depend on the existence of a specific receptor for adenosine. A1 receptor activation in neutrophils is known to be associated with augmentation of chemotaxis [15 ]. Theophylline’s antagonism does not discriminate between A1 and A2A receptor subtypes [20 ], and neutrophils express both A1 and A2A receptors [15 , 19 ]. The difference in effects of 8-SPT and enprofylline on neutrophil chemotaxis was according to their affinities for adenosine A1 receptor [20 ]. On the other hand, eosinophils possess fewer binding sites for adenosine A1 and A2A agonists [34 ] and cAMP is known as an inhibitory modulator of leukocyte chemotaxis [41 ]; the inhibitory effects on eosinophil chemotaxis at a high concentration of enprofylline and theophylline can be explained by intracellular cAMP accumulation as a result of PDE inhibition.

Recently, it was found that human eosinophils express higher levels of adenosine A3 receptors [34 ], and that A3 receptor stimulation modulates the functions of eosinophils [30 , 42 , 43 ]. We also confirmed that a selective A3 receptor agonist (IB-MECA) reduces O2- production in eosinophils most effectively [43 ]. Furthermore, theophylline did not influence the effect of IB-MECA (data not shown), which may be due to the lack of A3 receptor antagonism.

The primary aim of this study was to organize the currently available information on the effects of theophylline on granulocyte functions, and to understand the mechanism that mediates cellular functions. We can conclude that theophylline modulates granulocyte functions in conjunction with adenosine receptor antagonism (A1 and A2A) and cAMP-PDE inhibition. Furthermore, the difference in effect on neutrophils and eosinophils appears to depend on the existence of specific adenosine receptors. In connection with the anti-inflammatory actions of theophylline, the results presented here may raise fears that neutrophils subjected to theophylline therapy may exhibit enhanced superoxide anion production and cause normal tissue damage. However, the priming effect of theophylline on O2- production is very small compared to that of inflammatory cytokines and is normalized by the inhibitory action of PDE at therapeutic concentrations. In addition, accelerated neutrophil apoptosis by theophylline [44 ] may well prevent any prolonged airway inflammation. In conclusion, the anti-inflammatory effects of theophylline on granulocyte functions are partly the result of inhibition of the release of toxic oxygen metabolites from eosinophils and/or suppression of chemotactic migration by both neutrophils and eosinophils.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare and a grant from the Ministry of Culture (12670738) of Japan.

Received November 22, 1999; revised February 29, 2000; accepted March 1, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 

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