A short course of oral aspirin increases IL-18-induced interferon-g production in whole blood cultures.

ARTICLE

INTRODUCTION

Aspirin is the best known anti-inflammatory drug, acting to inhibit cyclooxygenase, leading to subsequent reduction in prostaglandin (PG) synthesis [1]. PG, especially PGE2, stimulate adenylcyclase, and cAMP inhibit the synthesis of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), two cytokines which contribute to inflammation [2]. Most studies investigating the effects of inhibitors of PG synthesis on cytokine production have shown an increase in the synthesis of TNF, interleukin-1beta (IL-1) or IFN-gamma, in vitro [3, 4] or in experimental models [5]. This is probably due to inhibition of PG as seen in in vitro culture conditions. Moreover, previous studies in human volunteers taking oral aspirin revealed an increased production of cytokines by isolated peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS), phytohemagglutinin (PHA) or cytokines themselves [6, 7].

After stimulation of mixed PBMC or unfractionated whole blood with bacterial products such as LPS, IFN-gamma production from T lymphocytes is the result of intermediary release of macrophage products such as IL-12 and the newly-described IL-18 [8, 9]. Despite the presence of IL-12, the requirement of IL-18 for the induction of IFN-gamma has been established in IL-18-deficient mice [10], mice which are unable to process the IL-18 precursor into mature active forms of IL-18 [11, 13], or after neutralization of IL-18 using antibodies or IL-18 binding protein [11, 14]. To date, the effect of blocking cyclooxygenase, on the IL-18 stimulation of IFN-gamma synthesis is not known. In the present study, IFN-gamma production was assessed using co-stimulation of IL-18 plus LPS in whole blood from healthy volunteers [15], before and after oral aspirin administration.

METHODS

Study population. The study was approved by the Colorado Multiple Institutional Review Board. Six, healthy, male volunteers participated in the study, and gave written, informed consent. The volunteers had not consumed NSAID or aspirin for at least 6 weeks prior to the study.

Study schedule. Blood was obtained for cytokine production determinations on the two consecutive days before initiating the aspirin treatment and the mean value was used as the pre-aspirin level: 325 mg of aspirin were taken orally, twice a day, for 3 days. Blood was collected again, one day after the last aspirin dose (day 4 of the study), after 7 days (day 11) and after 4 weeks (day 32) (Figure 1).

Whole blood assay. Six ml polypropylene tubes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) were prepared containing 1 ml of 20 ng/ml LPS (E. coli strain O55:B5, Sigma Chemical Co, St. Louis, MO, USA) in RPMI 1640 (Waukesha, WI, USA) with 10 mM HEPES, 10 mM glutamine, 100 U/ml penicillin, and 100 mug/ml streptomycin. A second set of tubes was prepared with a combination of IL-18 (5.0 nM, Peprotech Inc., Princeton, NJ, USA) plus 20 ng/ml LPS. All tubes were kept frozen at ­ 70° C, and thawed immediately prior to obtaining each blood sample. One ml of heparinized blood was mixed with each tube at specified times. The tubes containing LPS were used for assessment of TNF-alpha synthesis after incubation at 37° C for 8 hours, whereas the tubes containing IL-18 plus LPS were used for determination of IFN-gamma production after 48 hours. After the indicated incubation periods, a 200 mul sample was removed and lysed with 50 mul Triton X (final concentration 1%). All samples were kept at ­ 70° C until assay.

Cytokine measurements. TNF-alpha and IFN-gamma concentrations were measured using specific electrochemiluminescence assays [16]. Data are expressed as mean ± SEM. Groups were compared by analysis of variance (ANOVA) using Fisher's least significant difference.

RESULTS AND DISCUSSION

Cytokine determinations for each period were performed in a single assay for TNF-alpha and IFN-gamma, respectively, to avoid assay differences on separate days. Twenty-four hours after the last dose of aspirin (day 4 of the study), a significant 3-fold (p < 0.05) increase in TNF-alpha production was already present (Figure 1). The same day, there was a trend towards higher IFN-gamma using stimulation with IL-18 plus LPS, but the difference was not statistically significant (Figure 1). On day 11, the TNFalpha production was increased 4-fold over the baseline level (p = 0.025), and was accompanied by a 70 % higher synthesis of IFN-gamma (p = 0.048) (Figure 1). After a 4 weeks wash-out period, cytokines were measured on day 32, and both TNF-alpha and IFN-gamma production returned to the levels measured prior to the short aspirin regimen.

The results of the present study demonstrate that oral aspirin increases the production of TNF-alpha and IFN-gamma in whole blood cultures. The increased TNF-alpha production after the short course of aspirin administration is consistent with other published data showing an increase in LPS-stimulated PBMC production of TNF-alpha following oral aspirin [6]. A similar stimulatory effect was reported after oral ibuprofen administration during an infusion of endotoxin into healthy subjects [17]. An important and new observation of the present study is the increased IFN-gamma production after aspirin intake, when blood was stimulated with a combination of IL-18 plus LPS. IL-18 is a newly described cytokine [8], its main function being the costimulatory activity on IFN-gamma production [9]. The capacity of whole blood to produce IFN-gamma is increased 10 to 20-fold by addition of IL-18 to LPS stimulation [15]. This optimal stimulation of IFN-gamma was significantly enhanced after 3 days of aspirin, and was further increased 7 days after stopping the drug. Therefore, the stimulatory activity of IL-18 on IFN-gamma production is also under PG regulation, which is in agreement with the studies showing that IFN-gamma induced by PHA or LPS is down-regulated by PG [4, 7].

The effects of the short aspirin course on cytokine production were long-lasting, with elevated TNF-alpha and IFN-gamma levels exhibiting an even greater increase on day 11 of the study compared to day 4. Because the half-life of aspirin in the circulation is only approximately 15 min, residual concentrations of aspirin in the bloodstream cannot account for this effect. It is likely that the persistance of the aspirin effect is due to acetylation of cyclooxygenase in cytokine-producing leukocytes in the circulation and in the bone marrow [3]. After a 4-week wash-out period, TNF-alpha and IFN-gamma production returned to pre-aspirin levels.

The stimulatory effect of aspirin on elevating TNF-alpha and IFN-gamma production may provide an explanation for the deleterious consequences of cyclooxygenase inhibition in inflammatory bowel diseases (IBD). Unlike rheumatoid arthritis, where the antiinflammatory effects through PG inhibition by NSAID improve the clinical course, in IBD, cyclooxygenase inhibition consistently worsens disease in animal models [18] and in IBD patients [19]. Given the up-regulation of IL-18 expression in IBD patients [20], and the mounting evidence supporting a role of proinflammatory cytokines such as TNF-alpha and IFN-gamma in the pathogenesis of human IBD [21, 22], the increased production of these cytokines after aspirin may represent a mechanism through which this class of drugs adversely influences the course of the disease.

The importance of the present study is also related to the large body of evidence suggesting a beneficial effect of aspirin for the prevention of colon cancer [23, 24]. Because both TNF and IFN-gamma have anti-tumor activity against colorectal adenocarcinoma [25], the potentiation of IL-18 -induced TNF and IFN-gamma production suggests an alternative pathway of cancer prevention by long term aspirin use, in addition to induction of cell cycle arrest, stimulation of apoptosis of malignant cells, and inhibition of angiogenesis [26, 27]. Indeed, Pages and colleagues have recently shown decreased or abolished synthesis of IL-18 in human colon adenocarcinomas compared with normal mucosa, resulting in the absence of IFN-gamma expression [28]. This suggests that IL-18 -induced production of IFN-gamma plays an important role in tumor immune surveillance, and aspirin may beneficially influence this pathway through potentiation of cytokine production.

CONCLUSION

Acknowledgements. These studies were supported by NIH Grant AI 15614.
Mihai G. Netea was supported by a grant from Van Walree Fonds, Dutch Royal Academy of Sciences.

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