ELLIOT D. ROSENSTEIN, JOLANTA KUNICKA, NEIL KRAMER, and GIDEON GOLDSTEIN ..... Abramson SB: Treatment of gout and crystal arthropathies and.
Modification of Cytokine Production by Piroxicam ELLIOT D. ROSENSTEIN, ABSTRACT.
JOLANTA KUNICKA,
NEIL KRAMER,
Objective. Nonsteroidal antiinflammatory drugs have been thought to act by inhibiting the enzyme, cyclooxygenase (prostaglandin H synthetase). We sought to demonstrate additional biologic actions of this class of drugs including effects on cytokine production. Methods. We administered the nonsteroidal antiinflammatory drug piroxicam 20 mg to 6 healthy volunteers daily for 7 days. Before and for one week after drug administration, concentrations of interleukin 1 (IL-I), IL-2, IL-4, IL-6, tumor necrosis factor ex (TNFex) and interferon-y (IFN--y), produced by anti-CD3 stimulated peripheral blood mononuclear cells, were measured. Results. Piroxicam treatment resulted in elevation of levels of IL-2, depression of IL-I, IL-6, TNFex and IFN--y, and no consistent effect on IL-4. Conclusion. Piroxicam modulates production of various cytokines in a complex fashion when administered to healthy individuals. (J Rheumatol 1994;21:901-4) Key Indexing Terms: PIROXICAM PROSTAGLANDINS
NONSTEROIDAL
Early studies of the antiinflammatory effects of nonsteroidal antiinflammatory drugs (NSAID) concentrated on their collective ability to inhibit generation of prostaglandins (PG), via the cyclooxygenase pathway of cell membrane arachidonate metabolism. Subsequent investigations support additional mechanisms for the antiinflammatory effects of these agents, including inhibition of transmembrane anion transport; inhibition of oxidative phosphorylation in mitochondria; insertion into the cell membrane lipid bilayer, disruption of signal transduction and thus interference with mobilization of intracellular calcium and the activation of protein kinase C; and inhibition of neutrophil aggregation in vivo and in vitro'», More recent reports indicate that NSAlD may mediate anti-: inflammatory effects through alterations in levels of cytokines. Several NSAID have been shown to raise interleukin 2 (lL-2) levels->. These effects occur at doses that inhibit prostaglandin E, (PGE2) generation and can be reversed by the addition of exogenous PGE2. Additional in vitro studies have documented inhibitory effects of NSAID on levels of IL-l, IL-6, and tumor necrosis factor (TNF). However, these effects are not clearly linked to inhibition of PG synthesis. Due to cell-cell interactions and self-regulatory influences among cytokines, an ex vivo study design was felt to most accurately reflect in vivo responses to drug administration. To evaluate these effects, we studied levels of the cytokines, IL-l, IL-2, IL-4, IL-6, TNFa and interferon-v (lFN-y), produced by anti-CD3 stimulated peripheral blood From the Immunobiology Research Institute, Annandale, and New Jersey Center for Rheumatic Diseases, Livingston, NJ, USA. E.D. Rosenstein, MD; J. Kunicka, Goldstein, MD, PhD.
PhD; N. Kramer, MD; G.
Address reprint requests to Dr. G. Goldstein, lmmunobiology Research Institute, Route 22, PO Box 999, Annandale, NJ 08801-0999. Submiued
Rosenstein,
March ll,
and GIDEON GOLDSTEIN
1993 revision accepted September 20, 1993.
et at: Piroxicam
modulates
cytokine production
ANTIINFLAMMATORY DRUGS CYTOKINES
mononuclear cells (PBMC) of healthy individuals, after short term administration of the NSAID, piroxicam. MATERIALS
AND METHODS
Subject selection. Six healthy male volunteers, between the ages of 20-47, were selected, and informed consent obtained after review board approval. No subject had taken aspirin or other NSAID for 30 days before selection. Before drug administration, blood was obtained for baseline determination of cytokine production. All subjects were then given piroxicam 20 mg orally for a total of7 days. At 4 h, and at 1, 2, 3, and 7 days after discontinuation of piroxicam, blood was again collected for determination of cytokine production. Preparation of cells. Venous blood was drawn into heparin (30 Vlml), centrifuged at 300 x g for 10 min to remove platelet rich plasma, diluted 1: 1 with Hank's balanced salt solution (HBSS) (Gibco, Grand Island, NY) and layered over Lymphocyte Separation Medium (LSM) (Sigma, SI. Louis, MO) in the proportion of I part LSM and 2 parts of diluted blood. After centrifugation at 400 x g at room temperature for 30 min, the interphase cells were collected and washed and resuspended 3 times in HBSS at 400 x g for 10 min. The cells were then resuspended at a concentration of 2 x 196 cellslml in AIM-V serum free culture medium (Gibco). Cell culture. PBMC were cultured in 2 ml portions in 24·well flat bottom tissue culture plates (Corning, Corning, NY), at 3rC, 5% CO2 in a humidified atmosphere. As an antigen-like stimulator ofT cells, anti-CD3 (OKT3, azide-free, Ortho Pharmaceuticals, Raritan, NJ) was added to the cultures for 24 h , at a concentration of 10 ng/ml. This concentration was optimal for cytokine secretion. Cell culture supernatants were collected, microcentrifuged for 5 min, aliquoted to cryovials and frozen at -80°C until cytokine assays were performed. Cytokine assays. The concentrations of IL-2, IL-4, IL-6, TNFO:' (R&D Systems, Minneapolis, MN), IL-Ill (Cistron Biotechnology, Pine Brook, NJ), and IFN-I' (Centocor Diagnostics, Malvern, PA) in the supernatant were measured by commercially available enzyme immunoassay (ELISA) in accordance with the manufacturer's protocols. All samples were tested in duplicate. Statistical analysis. The significance of a linear trend was analyzed based on a repeated measures model which considers that samples measured within each subject were not totally independentv-".
901
RESULTS Quantitation of cytokine production by anti-CD3 stimulated PBMC is summarized in Table 1. The time course of cytokine production during the 14-day duration of our study is illustrated in Figure 1 (all cytokines except IL-4). IL-IB, TNF, and IL-6 are synthesized primarily by stimulated macrophages and fibroblasts". Treatment with piroxicarn resulted in suppression of IL-6 levels during the period of drug administration, which was sustained and, in fact, maximal at Day 7 of posttreatment observation. Levels of IL-IB and TNF exhibited no substantial change during the period of drug administration, However, during the posttreatment observation, statistically significant decrements in levels of all 3 cytokines were observed. IL-2 and IFN--y are produced primarily by a subpopulation of T helper cells, characterized as naive/virgin or suppressor-inducer". In contrast to the effects on cytokines of macrophage/monocyte origin, anti-CD3 stimulated production of IL-2 by PBMC resulted in significant elevation of levels following the period of piroxicam administration. However, in our study, contrasting results were exhibitTable 1. Cytokine production T
F-a (pg/ml)
Baseline Day 7 8
9
IL-I (pg/ml)
10 14 Baseline Day 7
8
[L-2 (fmol/tube)
9 10 14 Baseline Day 7 8
9
lL-4 (pg/ml)
10 14 Baseline Day 7
8
[L-6 (pg/ml)
[FN--y (u/ml)
* p Value
902
9 10 14 Baseline Day 7 8 9 10 14 Baseline Day 7 8 9 10 14
< 0.05 based on a linear trend analysis.
2281 2622 2211 1961 1768 1625 1353 996 974 1079 616 446 8.4 8.7 9.0 12.3 11.7 12.8 16.7 18.7 17.5 32.7 21.6 7.6 471 274 207 285 132 91 84 71 58 68 48 29
± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ±
± ± ±
± ± ± ± ± ± ± ±
404 416 507 359 355 221* 179 178 272 159 155 88* 1.0 0.6 1.4 0.3 0.3 0.4* 5.0 6.5 4.4 6.7 8.3 7.6 102 45 42 55 20 13* 28 21 23 23 19 11*
CYTOKINE
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40 30
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LEVELS
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«>: --"--'.:.-·'.....:·-:.'0-. More recent studies, using more specific immunoassays, documented that when indomethacin was administered to recipients of renal allografts, in an attempt to minimize the adverse reactions associated with administration of anti-CD3 (Orthoclone OKT3), no attenuation of the 'rise in IL-\ B, TNF, or IL-6 was seen-", Endres, et aL established an increase in IL-IB levels among 12 healthy subjects taking various NSAID, after LPS stimulation of PBMC30. Subsequently, this group showed that oral ibuprofen or aspirin increased the production of IL-I Band TNF after IL-l a stimulation of PBMC3I. Enhanced release of TNF, IL-6, and IL-8 was also seen in healthy subjects administered endotoxin after pretreatment with ibuprofenv-". Contrarily, exposure to flurbiprofen caused moderate reduction of TN Fa and IL-IB in LPS-stimulated human monocytes and marked reduction of TNFa, IL-IB, and IL-6 in human histiocytic and monocytic cell lines>'. Similarly, tenidap, an investigational cyclooxygenase/5-lipoxygenase inhibitor, markedly inhibited production of TNFa, IL-IB, and IL-6 in LPS-stimulated human monocytes. However, naproxen showed only moderate activity in inhibiting production of IL-J B alone>. By using an ex vivo experimental design, the contributions of T cell and monocyte/macrophage effects are not examined in isolation. Rather, it is felt that the observed effects are reflections of the interdependence of different cell types and the mutual influences of cytokines. Using these techniques, it was hoped to more closely mimic conditions resembling those found in vivo. Further investigation will be necessary to assess the full duration of piroxicam's effects on cytokine production and to firmly establish whether the observed results are direct effects of the medication or the consequences of drug withdrawal. Additional investigations must also assess whether the effects of piroxicam are unique to this particular drug or are shared by other members of the NSAID class. Conceivably, the influence of piroxicam on levels of some cytokines may be independent of its effects on cyclooxygenase and PO metabolism. Further definition of the influences on cytokine production could provide insight into the mechanism whereby piroxicam, and other NSAID, may affect the course of inflammatory conditions such as rheumatoid arthritis36,37.
903
ACKNOWLEDGMENT
20.
The authors thank Alan Fisher and Ching-Chang Hwang for their statistical analysis and Cathleen Muraski for her administrative assistance. 21.
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The Journal of Rheumatology
1994; 21:5
