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production in human mononuclear cells by an adenosine kinase inhibitor
Division of Clinical Pharmacology, Medizinische Klinik, Klinikum Innenstadt, University of Munich, Germany; and
* Division of Rheumatology, University of California, San Diego School of Medicine, La Jolla
Correspondence: Stefan Endres, M.D., Clinical Pharmacology, Medizinische Klinik, University of Munich, Ziemssenstrasse 1, 80336 München, Germany. E-mail: EndresS{at}lrz.uni-muenchen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) synthesis in human peripheral blood mononuclear cells (PBMC)
or whole blood. Lipopolysaccharide (LPS; 10 ng/mL)-stimulated PBMC were
incubated in the absence or presence of these substances. GP515 alone
showed a dose-dependent suppression of TNF- production with an
IC50 of 80 µM. The TNF--inhibiting effects of
adenosine and GP515 were reversed in the presence of the cAMP
antagonist (Rp)-cAMPS, supporting the hypothesis of a cAMP-mediated
pathway. Combinations of GP515 with either adenosine or rolipram led to
an additive inhibition of TNF- synthesis. These experiments are the
first to demonstrate efficacy of an adenosine kinase inhibitor in
TNF- suppression in cells of human origin. The findings form a basis
to investigate these strategies in animal models of TNF--mediated
chronic inflammatory diseases.
Key Words: cytokines • lipopolysaccharide • second messengers
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-)
synthesis and induction of interleukin-10 (IL-10) production
[3
4
5
]. In studies of septic shock and pleural
inflammation in mouse and rat models, adenosine and adenosine analogs
revealed significant anti-inflammatory potency [6
].
However, systemic administration of adenosine is accompanied by severe
cardiovascular side effects limiting its therapeutic use. Recently, potent anti-inflammatory activities of an inhibitor of the adenosine kinase (GP515) could be demonstrated without systemic side effects [7 , 8 ]. By inhibiting the rephosphorylation of adenosine to AMP through the adenosine kinase, GP515 prolongs the half-life of endogenously produced adenosine at inflamed sites and thereby locally enhances the adenosine concentration. Application of GP515 to mice had beneficial effects in models of septic shock [7 ] and other acute and chronic inflammatory diseases [8 ]. In vitro, GP515 attenuates human neutrophil adhesion to endothelial cell monolayers [9 ] but its effects on cytokine production have not yet been investigated in cells of human origin.
We tested the effect of GP515 on lipopolysaccharide (LPS)-induced
TNF- and IL-10 synthesis in human peripheral blood mononuclear cells
(PBMC). Elevated levels of TNF- play a central role in the
pathogenesis of acute and chronic inflammatory diseases. Thus, its
suppression is of pivotal clinical significance. Treatment of patients
with rheumatoid arthritis, with inflammatory bowel disease, and with
Jarisch-Herxheimer reaction with anti-TNF- antibodies has shown a
significant decrease of disease activity [10
11
12
].
A number of endogenous and exogenous mediators of TNF- inhibition
have been identified in the last few years [13
]. Among
these, cAMP-elevating agents, i.e. adenosine and the specific
phosphodiesterase inhibitor rolipram, have been shown to additionally
induce synthesis of the anti-inflammatory cytokine IL-10 in
LPS-stimulated PBMC. IL-10 suppresses TNF- synthesis
[14
, 15
], inhibits superoxide anion
production [16
], and reduces expression of adhesion
molecules [17
].
The aims of the present study were as follows: first, to investigate
the effect of GP515 on TNF- and IL-10 synthesis in human PBMC;
second, to determine the role of cAMP in adenosine- and GP515-mediated
effects on TNF- synthesis by combination with the cAMP-antagonist
(Rp)-cAMPS; third, to study the effect of combinations of GP515 with
adenosine or rolipram on TNF- and IL-10 synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of the compounds
Adenosine (Adrekar®, Sanofi Winthrop, Munich, Germany) was
purchased as intravenous solution for clinical use and was diluted with
supplemented RPMI culture medium to the required concentrations. The
specific adenosine kinase inhibitor GP515
[4-amino-1-(5-amino-5-deoxy-1-b-D-ribofuranosyl)-3-bromopyrazolo[3,4-D]pyrimidine,
synthesized by Dr. Howard Cottam, UCSD School of Medicine, La Jolla,
CA] was dissolved in supplemented RPMI medium. GP515 is specific for
adenosine kinase and does not inhibit other enzymes involved in
adenosine metabolism, including adenosine deaminase and AMP deaminase,
and it does not bind to A1 and A2 receptors or the
nitrobenzylthioinosine-sensitive adenosine transporter
[7
]. Rolipram (specific type IV phosphodiesterase
inhibitor; kindly provided by Dr. Wachtel, Schering AG, Berlin,
Germany) and LPS (from Escherichia coli 055:B5; Sigma) were
freshly diluted from frozen aliquots with supplemented RPMI medium to
their respective final concentrations. (Rp)-cAMPS
(Rp-adenosine-3’,5’-cyclic phosphorothioate), a specific inhibitor of
cAMP-dependent protein kinase A (Biolog Life Science Institute, Bremen,
Germany), was purchased as lyophilisate and diluted in supplemented
RPMI to the required concentrations. Cells were preincubated with
(Rp)-cAMPS for 20 min before LPS stimulation and addition of the other
compounds.
Cell stimulation
Experiments were carried out in duplicate. For induction of
cytokines 100 µL LPS solution was added into wells of a 48-well
culture plate (Falcon, Becton Dickinson, Franklin Lakes, NJ) containing
100 µL of diluted compounds or supplemented culture medium as
described above. Subsequent addition of 200 µL PBMC suspension gave a
final volume of 400 µL with a final cell concentration of 2.5 x
106/mL and a final LPS concentration of 10 ng/mL. The
incubation periods of 4, 6, and 20 h at 37°C in 5%
CO2 and 90% humidified air were stopped by freezing plates
at -80°C to obtain combined lysate plus supernatant.
Whole blood stimulation
Supplemented RPMI culture medium containing LPS (20 ng/mL) and
diluted compounds was pipetted into 5-mL polypropylene tubes (1
mL/tube; Becton Dickinson). Heparinized whole blood was freshly drawn
from healthy volunteers and added at 1 mL/tube. Tubes were mixed gently
and incubated for 4 h at 37°C in 5% CO2 and 90%
humidified air. After centrifugation at 1200 g, 4°C for 5
min supernatant was separated from cell pellet, centrifuged at 15,000
g, 4°C for 5 min to obtain platelet-free supernatant, and
frozen at -80°C. Triton X-100 (500 µL; Boehringer, Mannheim,
Germany) diluted in 0.9% NaCl to a 2% solution was added to 500 µL
of the cell pellet to lyse cells, and the cell pellet was centrifuged
at 15,000 g, 4°C for 10 min. Supernatant was separated
from the pelleted cell debris and was stored at -80°C for
measurement of cell-associated cytokines.
Measurement of TNF-
TNF- protein levels were determined after three freeze-thaw
cycles with a human TNF- sandwich enzyme-linked immunosorbent assay
(ELISA) established in our laboratory.
Coating of microtiter plates
Microtiter plates (96-well; Nunc, Naperville, IL) were coated
with a monoclonal antibody specific for recombinant human TNF-
(Endogen, Woburn, MA). The monoclonal antibody was diluted with coating
buffer (distilled water adjusted to pH 9.6 by 50 mM
Na2HPO4 and 5 M NaOH) to a concentration of 1
µg/mL. The dilution (200 µL/well) was pipetted into the plates.
After a 48-h incubation period at 4°C plates were washed twice with
300 µL/well washing buffer [distilled water containing 50 mM Tris
(Boehringer) at pH 7.2, 0.2% Tween 20 and 0.0005% Thimerosal (both
from Sigma)] using an automatic washer (SLT Labinstruments,
Crailsheim, Germany) and were incubated for 1 h at room
temperature with 300 µL/well assay buffer (distilled water, 0.15 M
NaCl, 50 mM Tris at pH 7.75, 0.01% Tween 40, 0.5% calf serum albumin,
0.05% calf gamma-globulin, and 20 µM diethylenetriaminepentaacetic
acid, all from Sigma). Plates were washed once and kept at room
temperature for 1 h to dry. Plates were taped and stored at
-20°C.
Assay procedure
After having reached room temperature, plates were washed twice
with washing buffer and were filled with 100 µL/well assay buffer. A
standard curve was drawn from frozen aliquots [TNF- lyophilisate
(kindly supplied by the National Institute for Biologic Standards and
Control, Potters Bar, UK) previously diluted with assay buffer to the
required concentrations ranging from 31 to 2000 pg/mL]. Samples of the
experiments were diluted with assay buffer. Standards or diluted
samples (100 µL) were pipetted in duplicate into wells. Plates were
shaken for 2 h at room temperature and then washed three times. A
monoclonal Biotin-labeled antibody specific for human TNF- (Endogen,
Woburn, MA) was diluted to 25 ng/mL in assay buffer and pipetted at 200
µL/well followed by a similar 2-h incubation period and three washing
steps. Plates were then incubated as described above for 30 min with
200 µL/well of a 1:8000 dilution of horseradish peroxidase-conjugated
streptavidin (Zymed Laboratories, San Francisco, CA), which binds to
the Biotin-labeled antibody. After three washing steps plates were
incubated with tetra-methylbenzidine (200 µL/well; TMB; DAKO,
Carpinteria, CA). The chromogenic TMB is converted from a colorless
solution to a blue solution by streptavidin. Substrate conversion was
stopped after 15 min by adding 1 M H2SO4 (50
µL/well). An MRX Microplate Reader (Dynatech Laboratories,
Denkendorf, Germany) was used with dual wavelengths of 450 and 570 nm
to compare the absorbance of samples with that of TNF- standards for
determination of TNF- concentrations. Results were given as means of
duplicates.
Validity criteria
The lower detection limit of the TNF- ELISA was 25 pg/mL. The
intra-assay variation was 6.7% at 912 pg/mL TNF- (n = 27 values), the interassay variation was 5.7% at 378 pg/mL TNF-,
and 1.2% at 43 pg/mL TNF- (n = 29 assays). A
comparison of the established TNF- ELISA to a commercial TNF-
ELISA (Medgenix TNF- EASIATM kit; BioSource Europe, Fleurus,
Belgium) showed a correlation of r2 =
0.834.
Measurement of IL-10
IL-10 levels were quantified after one freeze-thaw cycle by a
commercial human IL-10 ELISA (Laboserv, Staufenberg, Germany).
Viability testing
PBMC were incubated at 37°C in 5% CO2 and 90%
humidified air in the presence or absence of LPS (10 ng/mL) and GP515
(0.2 µM up to 1 mM). After 1 h cells were assayed for viability
by their capacity to reduce
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT; Sigma) to MTT-formazan [20
]. Ten
microliters of 5 mg/mL MTT were added and after a 4-h incubation
period, samples were dissolved by the addition of 100 µL 10% sodium
dodecyl sulfate (Sigma). Overnight incubation was followed by
quantifying the reduced (blue) MTT at an optical density of 570 nm with
690 nm as the reference wavelength.
Flow cytometry
To identify subpopulations of PBMC phycoerythrin-labeled
monoclonal antibodies (Immunotech, Marseilles, France) were used as
described previously [21
]. The mononuclear cell fraction
contained monocytes (4.4%), T lymphocytes (64.7%), B lymphocytes (7.0
%), natural killer cells (16.0%), and other cells (7.9%) (means ± SEM of n = 3 donors). To assess toxicity
of the applied compounds after the experimental incubation period, PBMC
were washed three times with phosphate-buffered saline and stained with
propidium iodide (10 µg/mL; Sigma). Propidium iodide enters cells
with defective cell membranes. Flow cytometric data were obtained as
described [21
].
Statistical analysis
Results are presented as means ± SEM. The
paired two-tailed Student’s t test was performed for
comparisons of means of TNF- values. Differences were considered
statistically significant for P < 0.050. In Figure 1A and B
, and Figure 5, P values were corrected according to
Bonferroni’s method for multiple comparisons. Statistical analyses
were performed using Stat-View 5.4 software (Abacus Concepts,
Calabasas, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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synthesis by adenosine and by
GP515 synthesis in human PBMC. Our
experiments confirmed a dose-dependent suppression of TNF-
production (Fig. 1
). Stimulation of PBMC with 10 ng/mL LPS
led to a TNF- concentration of 5.2 ± 1.4 ng/mL. Significant
reduction of TNF- synthesis to 3.8 ± 1.0 ng/mL was achieved in
the presence of 1 µM adenosine. Ten micromolar adenosine showed
half-maximal suppression of TNF- production.
Addition of GP515 to LPS-stimulated human PBMC dose-dependently
suppressed TNF- production (Fig. 2
). Stimulation of PBMC with 10 ng/mL LPS alone for 20 h induced a
TNF- synthesis of 3.8 ± 0.7 ng/mL (means ±
SEM of n = 4 donors). Incubation in the
presence of 10 µM GP515 showed significant suppression of TNF-
production to 3.3 ± 0.6 ng/mL (P = 0.019). The
mean IC50 was calculated at 80 µM of GP515. In control
experiments TNF- concentrations in cells incubated without LPS were
below 0.04 ng/mL.
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synthesis in
human whole blood (data not shown). When 50 µM GP515 was added to
LPS-stimulated whole blood, significantly reduced TNF- production
from baseline stimulation of 2.2 ± 0.5 ng/mL to 1.4 ± 0.3
ng/mL (P = 0.018) after 4 h of incubation
(means ± SEM of n = 4 donors) was
found. The IC50 was calculated at 90 µM of GP515. To assess toxicity of GP515, we studied the ability of GP515-treated PBMC to reduce MTT to MTT-formazan. GP515 concentrations ranged from 0.2 µM up to 1 mM, incubation was carried out in the presence or absence of LPS (10 ng/mL). No changes in metabolic activity were detected, indicating viability of the cells (n = 2 donors; data not shown).
Reversal of adenosine- and GP515-mediated TNF- suppression by
the specific protein kinase A inhibitor (Rp)-cAMPS
To test the hypothesis of a cAMP-mediated pathway, LPS-stimulated
human PBMC were incubated in the presence of adenosine or GP515 alone
or in combination with 100 or 300 µM (Rp)-cAMPS, a cAMP antagonist
and specific inhibitor of protein kinase A (Fig. 3A and B
). TNF- levels reached 2.6 ± 0.3
ng/mL after a 4-h stimulation period with LPS alone. Adenosine (10
µM) reduced TNF- synthesis to 1.6 ± 0.3 ng/mL. This effect
was completely reversed to 2.6 ± 0.6 ng/mL TNF- by 300 µM
(Rp)-cAMPS. TNF- suppression to 2.0 ± 0.2 ng/mL by 100 µM
GP515 was equally reversed to 2.8 ± 0.5 ng/mL by 300 µM
(Rp)-cAMPS (means ± SEM of n = 3
donors).
|
production by the combination of
GP515 with adenosine production in LPS-stimulated
PBMC was investigated. The experiments were performed at different
incubation periods (4, 6, and 20 h). The combination of GP515 and
adenosine led to a more pronounced TNF- suppression than each of the
substances alone (measured at all time points investigated). The
maximal TNF- concentration 4 h after stimulation with 10 ng/mL
LPS was 3.5 ± 0.5 ng/mL (Fig. 4
). It was suppressed to 2.8 ± 0.5 ng/mL (P =
0.014) by 10 µM GP515, to 2.5 ± 0.5 ng/mL (P =
0.001) by 1 µM adenosine, and to 1.7 ± 0.3 ng/mL
(P < 0.001) by the combination of both (means ±
SEM of n = 6 donors). To exclude toxicity
of the compounds, samples of one donor were tested for propidium iodide
exclusion by flow cytometry. Cell viability of all samples was
confirmed by propidium iodide exclusion (0.4–0.5% of the mononuclear
cells were propidium iodide positive).
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synthesis than simultaneous incubation of
stimulus and inhibitors (n = 6 donors; data not shown).
Dose-dependent suppression of TNF- synthesis by rolipram
The specific type IV phosphodiesterase inhibitor rolipram is a
potent inhibitor of TNF- synthesis in human PBMC [22
,
23
]. In our experiments, rolipram (10 nM, 100 nM, and 1
µM) dose-dependently suppressed TNF- synthesis from 6.7 ±
1.6 ng/mL after stimulation with LPS alone to 5.3 ± 1.0, 4.0 ± 0.6, and 1.2 ± 0.2 ng/mL, respectively (Fig. 5
). The mean IC50 was calculated at 120 nM.
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synthesis by the combination of
GP515 with rolipram suppression was not
additive after 4 h and additive after 20 h. After 20 h
of incubation (Fig. 6
), LPS-stimulated TNF- synthesis of 3.8 ± 0.7 ng/mL was reduced
to 3.3 ± 0.6 ng/mL (P = 0.019) by 10 µM GP515
and to 2.0 ± 0.5 ng/mL (P = 0.012) by 100 nM
rolipram. Simultaneous incubation with both substances inhibited
TNF- production to 1.3 ± 0.2 ng/mL (P = 0.010;
means ± SEM of n = 4 donors). The
samples of one donor were assayed for cell viability by quantifying
propidium iodide uptake. Between 0.3 and 0.5% of the mononuclear cells
were propidium iodide positive.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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synthesis by GP515 in cells
of human origin. GP515 led to significant TNF- inhibition in both
human PBMC and human whole blood. Cytotoxicity of GP515 was excluded by
MTT assay. GP515- and adenosine-mediated suppression of TNF-
synthesis was reversed in the presence of the cAMP antagonist
(Rp)-cAMPS, indicating that an increase of cAMP and activation of
protein kinase A are involved in the signal transduction pathway.
Combinations of GP515 with adenosine or with rolipram achieved additive
suppression of TNF- production in PBMC. Furthermore, GP515
suppressed IL-10 formation.
We could demonstrate reduced TNF- synthesis by LPS-stimulated PBMC
incubated in the presence of GP515. These findings are consistent with
the results of a model of endotoxic shock in mice by Firestein et al.
in which GP515 reduced plasma TNF- concentrations
[7
]. Furthermore, GP515 suppressed carrageenan-induced
TNF- synthesis in air pouches on BALB/c mice in a study by Cronstein
et al. [25
]. GP515 has been shown to enhance endogenous
adenosine concentrations [2
, 9
,
25
]. Adenosine is generated as an end-product of ATP
catabolism. In conditions of cellular stress such as inflammation or
hypoxia the breakdown of ATP is enhanced and adenosine concentrations
increase. In humans, adenosine has a short plasma half-life of less
than 2 s [26
], which is caused by rapid cellular
uptake. Adenosine is metabolized intracellularly by both adenosine
deaminase and by adenosine kinase, adenosine kinase being the major
adenosine-metabolizing enzyme in conditions of physiological adenosine
concentrations [3
, 26
]. The adenosine
kinase inhibitor GP515 inhibits the rephosphorylation of adenosine to
AMP and thereby prolongs the half-life of adenosine. GP515 itself is
not metabolized by adenosine deaminase [9
].
The exact mechanism by which adenosine suppresses TNF- synthesis in
PBMC has not yet been elucidated. One hypothesized pathway includes
binding to the adenosine A2 receptor [3
,
27
] followed by elevation of the intracellular second
messenger cAMP [3
, 5
, 27
,
28
], which activates cAMP-dependent protein kinases A,
resulting in TNF- inhibition [29
30
31
32
]. Another
hypothesis suggests an A3 receptor-mediated and cAMP-independent
pathway [33
]. To investigate whether elevation of cAMP
accounts for adenosine-mediated suppression of TNF- synthesis, we
studied the effect of the cAMP antagonist (Rp)-cAMPS in
adenosine-treated PBMC. (Rp)-cAMPS specifically binds to type I and II
protein kinases A and competitively inhibits their cAMP-induced
activation [34
]. In our experiments, (Rp)-cAMPS
completely reversed adenosine-induced TNF--inhibition. These
findings confirm the hypothesized signal transduction pathway.
Furthermore, (Rp)-cAMPS similarly reversed GP515-mediated TNF-
suppression indicating the same pathway. GP515 itself does not bind to
A2 receptors [7
], but A2 receptors have been shown to
mediate effects of GP515 [8
, 9
,
25
]. Thus, our results indicate that the effect of GP515
on TNF- suppression in PBMC is caused by increased endogenous
adenosine concentrations that act via cAMP-dependent protein kinase A.
Because anti-inflammatory effects of GP515 are dependent on the presence of endogenous adenosine, GP515 acts preferentially at sites of inflammation where adenosine concentration is elevated. Thus, anti-inflammatory effects as well as possible side effects might be confined to the inflamed target tissue. This hypothesis is supported by a study by Rosengren et al. who applied GP515 in models of rat paw swelling and rat skin lesion [8 ]. Although GP515 exerted beneficial anti-inflammatory effects, no detrimental systemic side effects were observed. GP515 did not exhibit a reduction in heart rate or blood pressure in the treated animals as would have occurred after infusion of exogenous adenosine.
Rolipram is a highly specific and potent inhibitor of phosphodiesterase
(PDE) type IV, which is the predominant PDE isoenzyme in monocytes. The
TNF--suppressing activity of rolipram is supposed to be mediated by
accumulation of intracellular cAMP [32
,
35
]. In a previous study we could demonstrate that
rolipram-induced TNF- inhibition is reversed by adenosine deaminase
and is therefore dependent on the presence of adenosine
[5
]. Thus, similar to GP515, rolipram might exert
locally enhanced effects at sites with elevated endogenous adenosine
concentrations. Combinations of GP515 with adenosine and of GP515 with
rolipram showed additive suppression of TNF- synthesis. Combinations
of drugs frequently reveal clinical advantages because lower doses of
the individual compounds are required with reduced side effects.
Although adenosine induces IL-10 synthesis in human PBMC, it was
surprising that GP515 suppressed IL-10 production. However, it is known
that significant increase of IL-10 synthesis in LPS-stimulated
monocytes occurs only at high concentrations of adenosine (100 µM),
as demonstrated by Le Moine et al. [4
]. During infection
or inflammation, adenosine is formed locally but remains in the
nanomolar range or reaches low micromolar concentrations
[27
]. After application of GP515 in a murine air pouch
model of inflammation adenosine did not exceed nanomolar concentrations
[25
]. In a whole blood model GP515 led to elevated
adenosine concentrations of about 14 µM [2
]. Thus, it
is presumable that the amount of adenosine released from LPS-stimulated
PBMC after incubation with LPS and GP515 was not sufficient to induce
IL-10 synthesis. The mechanism of adenosine-mediated effects on IL-10
synthesis has not yet been clarified. Indeed, it has been shown that
enhancement of IL-10 by adenosine is not caused by activation of A2
receptors [4
]. Cytotoxic effects of GP515 leading to an
unspecific down-regulation of cytokine production seem unlikely because
reduction of MTT by PBMC incubated with GP515 showed no decrease in
metabolic activity of the cells. Also, the reversal of GP515-mediated
TNF- suppression in the presence of (Rp)-cAMPS contradicts toxicity
of the compound.
TNF- is a necessary mediator of various inflammatory disease
processes. Beneficial effects of anti-TNF- antibodies in the
treatment of patients with rheumatoid arthritis, inflammatory bowel
disease, and Jarisch-Herxheimer reaction have demonstrated the proof of
principle of anti-TNF- strategies in clinical therapy
[10
11
12
]. Repeated administration of anti-TNF-
antibodies is, however, accompanied by adverse effects through
endogenous antibody production against the antibodies applied.
Adenosine, GP515, and rolipram interfere with the synthesis of TNF-
and represent an alternative approach to curtail TNF--mediated
effects. The strategies of the present in vitro study are
now under investigation in a mouse model of chronic colitis;
preliminary results suggest a marked amelioration of disease activity
[36
].
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ACKNOWLEDGEMENTS |
|---|
Received September 7, 1999; revised February 12, 2000; accepted February 15, 2000.
|
REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(cA2) versus placebo in rheumatoid arthritis Lancet 344,1105-1110[Medline]
N. Engl. J. Med. 335,311-315
and tumor necrosis factor production during experimental endotoxemia Eur. J. Immunol. 24,1167-1171[Medline]
production by human mononuclear cells Int. J. Immunopharmacol. 15,409-413[Medline]
generation from human peripheral blood monocytes by inhibitors of phosphodiesterase 4: interaction with stimulants of adenylyl cyclase Mol. Pharmacol. 48,747-757[Abstract]
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expression by adenosine: role of A3 adenosine receptors J. Immunol. 156,3435-3442[Abstract]
in LPS-stimulated human peripheral blood mononuclear cells Immunopharmacology 26,259-264[Medline]
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