Inhibitory activity of 1,8-cineol (eucalyptol) on cytokine production in cultured human lymphocytes and monocytes

Pulmonary Pharmacology & Therapeutics 17 (2004) 281–287 www.elsevier.com/locate/ypupt

Inhibitory activity of 1,8-cineol (eucalyptol) on cytokine production in cultured human lymphocytes and monocytes Uwe R. Juergensa,*, Tanja Engelena, Kurt Racke´b, Meinolf Sto¨bera, Adrian Gillissenc, Hans Vettera a

Department of Pneumology, Allergology and Sleep Medicine, Medical Outpatient Clinic, Bonn University Hospital, Wilhelmestrasse 35-37, Bonn D-53111, Germany b Department of Pharmacology and Toxicology, University of Bonn, Germany c St George Medical Center, Robert-Koch-Hospital, Leipzig, Germany Received 18 December 2003; revised 6 June 2004; accepted 21 June 2004

Abstract Background: The therapeutic value of secretolytic agents in COPD and asthma is still disputed. For this reason, in a preclinical study we aimed to test the potential anti-inflammatory efficacy of 1,8-cineol (eucalyptol) in inhibiting polyclonal stimulated cytokine production by human unselected lymphocytes and LPS-stimulated monocytes. Methods: Cytokine production was determined following 20 h of incubation cells with 1,8-cineol simultaneously with the stimuli in culture supernatants by enzyme immunoassay. Results: Therapeutic concentrations of 1,8-cineol (1.5 mg/mlZ10K5 M) inhibited significantly (nZ13–19, pZ0.0001) cytokine production in lymphocytes of TNF-a O IL-1bO IL-4O IL-5 by 92, 84, 70, and 65%, respectively. Cytokine production in monocytes of TNF-a O IL1bO IL-6O IL-8 was also significantly (nZ7–16, p!0.001) inhibited by 99, 84, 76, and 65%, respectively. In the presence of 1,8-cineol (0.15 mg/mlZ10K6 M) production of TNF-aOIL-1b by monocytes and of IL-1bO TNF-a by lymph-ocytes was significantly inhibited by 77, 61 and by 36, 16%, respectively. 1,8-cineol (10K6 M) had a larger impact on TNF-a and IL-1b-production in monocytes compared to lymphocytes (p!0.03) and similar effects (pO0.59) at therapeutically relevant concentrations of 1,8-Cineol (10K5 M). Conclusion: These results characterize 1,8-cineol as strong inhibitor of TNF-a and IL-1b and suggest smaller effects on chemotactic cytokines. This is increasing evidence for the role of 1,8-cineol to control airway mucus hypersecretion by cytokine inhibition, suggesting long-term treatment to reduce exacerbations in asthma, sinusitis and COPD. q 2004 Elsevier Ltd. All rights reserved. Keywords: 1,8-Cineol (eucalyptol); Secretolysis; COPD; Asthma; Sinusitis

1. Introduction Alternative therapeutic options for early onset of antiinflammatory airway therapy remain entirely unknown despite to our increasing knowledge of side effects of inhaled glucocorticosteroids [1,2]. Most of the current alternative medications are used as secretolytic agents. They have in common that they are prescribed rather often with no proof of their efficacy in attenuating or halting the course * Corresponding author. Tel.: C49-228-287-2251; fax: C49-228-2872266. E-mail address: [email protected] (U.R. Juergens). 1094-5539/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2004.06.002

of the disease while being generally less effective compared to inhaled glucocorticosteroids [3]. Therefore, the widespread use of such agents cannot be recommended for treatment of COPD on the basis of present evidence (Evidence D, GOLD, NHLBI, 2001). In this regard, there is a need for non-steroid anti-inflammatory medications to reduce acute exacerbations, morbidity and healthcare costs, especially if inhaled glucocorticosteroids are still not used or recommended. Increased airway and mucus secretion often appears as the initial symptom in exacerbated COPD and asthma that are associated with a distinct pattern of stimulated mediator production from inflammatory cells that migrate to

282

U.R. Juergens et al. / Pulmonary Pharmacology & Therapeutics 17 (2004) 281–287

the lungs. In these conditions, cytokines are secreted by resident tissue cells, recruited leukocytes, and cytokineactivated endothelial cells. Among these mediators, monocyte/macrophage and T cell-derived cytokines seem to be of importance, in particular TNF-a, IL-1b, IL-6, IL-8 and the Th2-type cytokines IL-4 and IL-5 known to induce IGEantibody synthesis [4] and maintain allergic eosinophilic inflammation [5]. In infectious and inflammatory conditions IL-1b and TNF-a stimulate production of cytokines and arachidonic acid metabolites in various airway cells [6,7]. IL-6 is the main stimulator of acute phase proteins, particularly when bacterial lipopolysaccharide (LPS) is the inflammatory stimulus [8]. During the initiation of inflammation, endothelial adhesive molecules are substantially upregulated by IL-1b and TNF-a, both known to stimulate also IL-8 by monocytes/macrophages, lymphocytes and endothelial cells. IL-8 is a specific neutrophil chemotaxin and important in the pathogenesis of COPD [9]. Interestingly, inhaled glucocorticosteroids were shown to reduce neutrophilic bronchial inflammation in these patients [10] and prevented virus-induced asthma exacerbations [11]. 1,8-Cineol, the major constituent of eucalyptus oil (eucalyptol), is known chemically as terpenoid oxide. In a double blind placebo-controlled trial (3!200 mg/day), this was recently shown to have an anti-inflammatory efficacy equivalent to 3.75 mg prednisone/day in patients with severe asthma [12]. In this report, we describe the potential mode of action underlying the anti-inflammatory activity of 1,8-cineol by investigating its efficacy in inhibiting Th1/Th2 associated cytokine production in human unfractionated lymphocytes compared to monocytes in vitro.

experiment from 50 ml of EDTA-blood by density gradient (1.068 g/ml) centrifugation (NycoPrep, Axis-Shield, Oslo, Norway) as recently described [13]. Platelet-free monocytes (O95%) purified by sequential centrifugation as assessed by light microscopy were used for the experiments. The average yield of vital monocytes was 2.8G1.2!106 cells. 2.3. Cell culture and cytokine induction Unselected lymphocytes were cultured (5!106 mlK1) at 37 8C for 20 h in RPMI 1640 (Life Technologies, Eggenstein, Germany) supplemented with 10% fetal calf serum and calcium 10K5 M on 24-well plates (Costar, Tecnorma, Fernwald, Germany) in a humidified air atmosphere containing 5% CO2. The cells were incubated without and with 1,8-cineol (10K9–10K5 M, Cassella-med, Cologne, Germany) simultaneously with the stimuli for 20 h to stabilize cell cultures. They were stimulated nonspecifically with a mixture of ionomycin (10 mM) and phorbol 12-myristate 13-acetate (PMA 10K8 M, Sigma) using optimized culture conditions in order to assure measurable cytokine production by direct assay. This was particularly important for measurement of IL-4 that is synthesized in vitro only by 1–3% of CD4C-lymphocytes [14,15]. Aliquots of isolated monocytes (105 mlK1) were stimulated with 10 mg/ml LPS (Sigma) for 20 h on 48-well culture plates (Costar) in the presence of RPMI 1640 containing 10% fetal calf serum as recently reported [16]. Because of the large amounts of IL-6 and IL-8 released, both cytokines were measured in cell culture supernatants of 2!104 monocytes. Different seeding densities of monocytes used in our human monocyte model did not affect the ratio of measured cytokines. Cells were cultured in medium alone as a negative control.

2. Materials and methods 2.4. Determination of cell viability 2.1. Subjects Nine healthy volunteers, without either personal or family history of atopy, gave their written informed consent to donate venous blood. All participants were non-smokers and had not taken any medication during the preceding 6 weeks. The institutional review board of Bonn University approved the protocol. 2.2. Isolation of peripheral blood lymphocytes and monocytes Unselected lymphocytes were isolated from 30 ml of venous EDTA-blood by Lymphoprep (1.077 g/ml) centrifugation (Axis-Shield, Oslo, Norway) using a standard protocol. The average yield of isolated lymphocytes (purity R90%) for each experiment was 85!106 consisting mainly of lymphocytes besides few amounts of contaminating monocytes (%10%) as determined by light microscopy. Peripheral blood monocytes were isolated for each

Cell viability in cultures (20 h) of unselected lymphocytes and isolated monocytes was investigated by trypan blue exclusion and lactate dehydrogenase (LDH) activity after cell isolation, incubation with 1,8-cineol (10K5 M) for 20 h and co-incubation of 1,8-cineol with the cell stimuli (LPS or ionomycin/PMA). Using trypan blue exclusion (0.5% for 5 min) cell vitality was at all time points O98% for each experiment as established by light microscopy. LDH activity, determined after 20 h by the cytotoxicity assay (Boehringer Ingelheim, Germany) in the cell culture supernatants of unselected lymphocytes (5!106), was 58 U/l before and 62 U/l in the presence of 1,8-cineol (10K5 M). In stimulated lymphocytes (5!106/ml) LDH activity (nZ5–7) was 121 U/l before 1,8-cineol, and remained unchanged after 1,8-cineol 10K6 M (123 U/l) and 10K5 M (129 U/l). 1,8-cineol (10K5 M) also had no cytotoxic effect as measured by LDH activity in monocytes. As compared to the positive control (91 U/l) consisting of sonicated monocytes, LDH activity was 9 U/l in monocytes

U.R. Juergens et al. / Pulmonary Pharmacology & Therapeutics 17 (2004) 281–287

incubated for 20 h with 1,8-cineol (10K5 M). These data clearly rule out any cytotoxic effects of 1,8-cineol. 2.5. Quantification of cytokine production In all experiments, the profile of stimulated cytokine production of IL-1b, TNF-a, IL-4, IL-5 and of IL-1b, TNFa, IL-6, IL-8 was assayed by specific enzyme-linked immuno-sorbent assay (ELISA) in the same culture supernatants of isolated peripheral lymphocytes or monocytes, respectively [13,16]. It is assumed that the cytokines measured in the lymphocyte-enriched populations are derived from specific lymphocyte subsets. Direct ELISA (materials purchased from Cayman Chem. Corp., Ann Arbor, Michigan Arbor, USA; Amersham Inc., Freiburg, Germany; microtiter plates from Nunc, Kamstrup, Denmark) assayed all samples from each experiment twice on the same plate. Measurements were made with a computerized plate reader (SLT, Salzburg, Austria) running on a Peacock computer. The detection limits at 80% of maximum binding were: 1.5 pg/ml for IL-1b TNF-a and IL-6; 15 pg/ml for IL-4; 15.7 pg/ml for IL-5; 26 pg/ml for IL-8. According to the manufacturer, the antibodies used for all cytokines measured (100%) cross-reacted with each other !0.01%. 1,8-cineol (10K5 M) did not interfere with the cytokine assay systems used as determined by adequate measurement of known cytokine concentrations in the presence of 1,8-cineol. 1,8-Cineol also did not induce virtual cytokine detection in the absence of the cytokine. 2.6. Statistical analysis All results are expressed as the meanGSEM as compared to the control without 1,8-cineol. ANOVA analysis for multiple comparisons of different 1,8-cineol concentrations with the control and the Mann & Whitney U nonparametric test were used. ANOVA was also used for comparison of 1,8-cineol effects on cytokine production in lymphocytes and monocytes. Two-sided p-values were considered significant if !0.05. All analyses were performed using the StatView 5.01 (SAS Institute Inc., North Carolina, USA) on a Apple-Macintosh computer.

3. Results 3.1. Comparison of stimulated cytokine production by lymphocytes and monocytes before 1,8-cineol Spontaneous and stimulated cytokine production by unselected lymphocytes (5!106 mlK1, nZ13–17) and monocytes (105 mlK1, nZ9–15) was directly determined in the culture supernatants by ELISA. In unselected lymphocytes spontaneous production of IL-1b (143.3G 15 pg) and TNF-a (34.3G15 pg) was measured, whereas spontaneous IL-4 and IL-5 production was not detectable.

283

Spontaneous production of IL-1b (244.7G46 pg) and TNF-a (45.1G12 pg) by monocytes was similar compared to unselected lymphocytes, but monocytes produced spontaneously much more of IL-6 (492.5G118 pg) and IL-8 (6390G520 pg). Measurements of stimulated cytokine production by lymphocytes and monocytes are displayed in Tables 1 and 2. In consequence of the huge IL-8 and IL-6 production by monocytes cultures for determination of these cytokines were set up with 2!104 cells while IL-1b and TNF-a were measured in cultures of 105 monocytes. Following adjustment for the quantity of cells used in these cultures the profile of cytokine production in LPSstimulated monocytes (IL-8OIL-6OIL-1bOTNF-a) was different compared to ionomycin/PMA-stimulated lymphocytes (TNF-a OIL-1bOIL-4OIL-5). This may indicate that the noted contamination with monocytes in the lymphocyte cultures had no evident impact on TNF-a measurements. However, the noted contamination with monocytes (%10%) in the lymphocyte cultures may have had an impact on IL-1b measurements due to the relative high production of IL-1b by monocytes compared to lymphocytes. Total cytokine production (calculated as ng/5!106 cells) in monocytes was about 1000-fold higher (basically reflected by huge amounts of IL-8) compared to lymphocyte cultures that produced relatively small amounts of IL-4 and IL-5 in vitro. 3.2. Effects of 1,8-cineol in lymphocytes In cultures of ionomycin/PMA-stimulated lymphocytes Th1 associated production of TNF-a and IL1-b was significantly inhibited by small concentrations of 1,8-cineol (10K6 M), whereas Th2 cell associated production of IL-4 and IL-5 was not significantly suppressed (Fig. 1). However, increasing concentrations of 1,8-cineol (10K5 M) inhibited in a concentration-dependent manner and to similar degree production of measured cytokines (Table 1). 3.3. Effects of 1,8-cineol in monocytes LPS-stimulated production of TNF-a and IL1-b was inhibited in cultured monocytes in a concentration-dependent manner (Fig. 2). By contrast, small amounts of 1,8-cineol (10K6 M) had no effect on the production of IL-6 and IL-8. However, production of IL-6 and IL-8 was significantly inhibited in the presence of 1,8-cineol (10K5 M) with a trend to smaller inhibition of IL-8 compared to IL-1b and TNF-a production (Table 2). 3.4. Comparison of 1,8-cineol effects on Th1-lymphocyte associated and monocyte cytokine production (TNF-a and IL-1b) Th1 cell associated production of TNF-a and IL-1b compared to monocytes was inhibited to a similar degree (ANOVA, pO0.59) by therapeutic concentrations of

284

Table 1 Inhibition of cytokine production by 1,8-cineol in human lymphocytes in vitro Cineol (M)

TNF-a 6

IL-4

IL-5

n

pg/5!10

Effect (%)

p-value

n

pg/5!10

Effect (%)

p-value

n

pg/5!10

Effect (%)

p-value

n

pg/5!106

Effect (%)

p-value

13 13 13 13

1158G171 1271G217 740G104 182G18

–

– 0.8980 0.0483 0.0001

17 17 17 17

4158G354 3876G357 3489G347 311G64

– K7G9 K16G10 K92G20

– 0.6920 0.0367 0.0001

16 16 16 16

183G15 167G16 145G18 56G10

– K8G9 K21G12 K70G18

– 0.4299 0.083 0.0001

16 14 17 19

68G6 76G7 61G5 24G4

–

– 0.2684 0.2962 0.0001

10G17 K36G14 K84G10

6

6

12G9 K10G8 K65G17

Table 2 Inhibition of cytokine production by 1,8-cineol in human monocytes in vitro Cineol (M)

Control 10K7 10K6 10K5

IL-1b

TNF-a

IL-6

IL-8

n

pg/105

Effect (%)

p-value

n

pg/105

Effect (%)

p-value

n

pg/2!104

Effect (%)

p-value

9 9 9 7

5367G406 4926G567 2074G458 846G261

– K8G11 K61G22 K84G31

– 0.2332 0.0003 0.0009

12 12 11 11

216G45 122G 50G14 2G1

– K44G77 K77G29 K99G2

– 0.1659 0.0007 0.0001

10 11 7 11

1667G119 1629G121 1661G103 402G40

– K2G7 0G6 K76G10

–

n

15 0.7248 16 O0.999 16 0.0001 16

pg/2!104

Effect (%)

20388G672 – 20364G712 0G3 19231G1650 K6G8 7032G656 K65G9

p-value – O0.999 0.9056 0.0001

U.R. Juergens et al. / Pulmonary Pharmacology & Therapeutics 17 (2004) 281–287

Control 10K7 10K6 10K5

IL-1b

U.R. Juergens et al. / Pulmonary Pharmacology & Therapeutics 17 (2004) 281–287

Fig. 1. Inhibition of Th1/Th2 cytokines by 1,8-cineol in human lymphocytes. Unselected lymphocytes were incubated with ionomycin (10 mM)/PMA (10K8 M) for 20 h together with 1,8-cineol. 1,8-Cineol (10K6 M) significantly (*p!0.05) inhibited Th1 associated production of TNF-a (16G10%, nZ17) and IL-1(36G14%, nZ13) from a baseline of 4158G354 pg/5!106 cells and 1158G171 pg/5!106, respectively. Cytokine production by Th1/Th2 lymphocytes of TNF-a (92G20%) and IL-1b (84G18%) was strongly inhibited (**pZ0.0001) by increasing concentrations of 1,8-cineol 10K5 M. Production of IL-4 (183G15 pg/5! 106 cells, nZ16) and IL-5 (68G6 pg/5!106 cells, nZ17) was measured in the same culture supernatants before 1,8-cineol. 1,8-cineol (10K5 M) significantly (pZ0.0001) suppressed production of IL-4 (70G18%) and IL-5 (65G17%). 1,8-Cineol (10K6 M) did not significantly inhibit production of IL-4 (21G12%, pZ0.083) and of IL-5 (10G8%, pZ0.2962).

285

Fig. 3. Comparison of TNF-a and IL-1b inhibition in Th1-lymphocytes and monocytes by 1,8-cineol. 1,8-cineol (10K6 M, 0.15 mg/ml) significantly inhibited TNF-a and IL-1b production in monocytes (*p%0.0009) and lymphocytes (*p!0.05) as compared to the control. At this concentration 1,8-cineol revealed a larger impact on TNF-a and IL-1b-production in monocytes compared to lymphocytes (ANOVA, p!0.03). The effect of therapeutic concentrations of 1,8-cineol (10K5 M, 1.5 mg/ml) on TNF-a and IL-1b-production was similar as compared to the control (*p%0.0009) and not significantly different in both cell types (ANOVA, pO0.59).

1,8-cineol (10K5 M). However, production of measured cytokine was only significantly inhibited by 1,8-cineol (10K6 M) in monocytes, whereas at this concentration no effects could be demonstrated on Th1 cell associated cytokine production (Fig. 3). These data suggest stronger activity of 1,8-cineol in cultured monocytes compared to lymphocytes.

4. Discussion

Fig. 2. Differential effects of 1,8-cineol on cytokine production in human monocytes. Before 1,8-cineol LPS-stimulated production of TNF-a (216G45 pg, nZ12), IL-1b (5367G406 pg, nZ9) and of IL-6 (1667G376 pg, nZ7–11), IL-8 (20388G672 pg, nZ16) was determined in monocyte culture supernatants of 105 cells/ml and 2!104 cells/ml, respectively. Cineol (10K5 M) significantly inhibited production of TNF-a (99G29%, pZ0.0001) and IL-1b (84G31%, pZ0.0009) as compared to the control. 1,8-cineol 10K6 M suppressed also significantly (*p!0.0001) production of TNF-a (77G29%) and IL-1b (61G 22%). By contrast production of IL-6 (76G10%) and IL-8 (65G9%) was significantly suppressed only by 1,8-cineol 10K5 M.

In this study, we report on the first evidence that 1,8cineol inhibits cytokine production by normal human peripheral lymphocytes in vitro. These results demonstrate that 1,8-cineol is a surprisingly strong inhibitor of TNF-a and IL-1b production in stimulated lymphocytes and monocytes. We also report for the first time that 1,8-cineol at known therapeutic blood concentrations has inhibitory effects on the chemotactic cytokine of IL-8 and IL-5 and may have additional anti-allergic activity by blocking production of IL-4. Therefore, these results corroborate the evidence, that systemic treatment with 1,8-cineol may be useful to control activation of the inflammatory processes underlying the exacerbation of asthma and COPD. We studied in vitro cytokine production by normal human unselected lymphocytes (O90%) compared to monocytes (O95%) using optimal culture conditions. Since LPS did not stimulate IL-4 and IL-5 production in lymphocytes different stimuli were used in this study and therefore we cannot

286

U.R. Juergens et al. / Pulmonary Pharmacology & Therapeutics 17 (2004) 281–287

entirely exclude that reported differences in responsiveness were due to the nature of the stimulus or to cell type. LPS-stimulated monocytes predominantly produced IL-8 and IL-1b [16] but only small amounts of TNF-a whereas PMA/ionomycin-activated unfractionated peripheral lymphocytes primarily produced the Th1 associated TNF-a and very small amounts of the Th2-type cytokine IL-4 and IL-5 [15,17]. This mediator profile suggests T-lymphocytes as the major cytokine source of TNF-a, but we cannot completely neglect IL-1b production by contaminating monocytes in our experiments [18]. Small production of IL4 and IL-5 was also reported in T-cells after optimal in vitro polyclonal cell activation as a consequence of rather limited IL-4 mRNA expression of 1–2% in unfractionated T-cells and of !5% in CD4C T-cells [14,15]. The efficacy of 1,8-cineol (10K5 M) in suppressing TNFa and IL-1b production in monocytes and unselected lymphocytes was comparable. However, in the presence of 1,8-cineol (10K6 M), there was a significant (p!0.03, ANOVA) stronger inhibition of IL-1b and TNF-a in monocytes suggesting particular activity of 1,8-cineol in these cells. Th2 cytokines IL-4, IL-5, measured in supernatants of unselected lymphocytes and monocyte derived cytokines of IL-6 and IL-8 were surprisingly not suppressed by a 10-fold smaller concentration of 1,8-cineol (10K6 M). Inhibition of cytokines by 1,8-cineol could be mediated by effects on transcription and transcription activity as mechanism of action of 1,8-cineol, that still needs to be provided. Due to the known high lipophilic affinity of monoterpenes we expect cineol to bind on cell membranes non-specifically and to enter the cytosome. 1,8-Cineol at the known therapeutic concentration of 1.5 mg/ml (10K5 M) had a strong and significant inhibitory effect in a range of 65–99% on all cytokines measured in cultures of unselected lymphocytes and monocytes. The maximum molar concentration of 1,8-cineol used in our experiments was the optimal therapeutic concentration (1.5 mg/mlZ10K5 M) of 1,8-cineol, which had no toxic effects in cultured monocytes or lymphocytes as determined by measurement of LDH-activity. This therapeutic concentration was determined in a phase I study in normal subjects (nZ40) after single dosing (200 mg) and 6 days after treatment with small gut soluble capsules containing 100 mg of 1,8-cineol (3!2 capsules/day) (Pidgeon, unpublished Phase I Study, 1993). 1,8-cineol, in small gut soluble capsules (3!200 mg/day) is known since 20 years in Germany as a licensed medicinal product (Soledumw capsules, Cassella-med. Cologne) for the treatment of bronchitis, sinusitis and common colds. It is known as monoterpen cyclic ether originated from eucalyptus oil and is tolerated well without any cell activating properties or steroid-like side effects. Furthermore, it was reported to display an inhibitory effect on some types of experimental inflammation in rats and demonstrated an antinociceptive effect that is potentially due to inhibition of PGE2 [19,20].

Cell activation is generally known for essential oils that consist of natural mixtures of various monoterpenes. Due to their cell activating properties and stimulation of inflammatory mediator production, essential oils activate mucus and goblet cells to induce mucus and airway hypersecretion rather than simple secretolysis. But bronchospastic reactions were reported in sensitive subjects that are caused by stimulation of prostaglandin production in the presence of very small concentrations [16]. By contrast, the isolated monoterpene 1,8-cineol has no cell activating potency and was shown to inhibit inflammatory mediator production. Inflammatory mediators play a potential role in initiating and perpetuating mucus and airway hypersecretion in consequence of the inflammatory responses in asthma and in particular of COPD exacerbated by respiratory infections [21–23]. Under these conditions, TNF-a promotes secretion of IL-1b, IL-6 and GM-CSF by mononuclear cells, primes eosinophiles for leukotriene release and activates epithelial cells to release IL-8 for the recruitment of neutrophils important in the pathogenesis of COPD [24]. Inhibition of IL1-b by 1,8-cineol plays a central role: IL1-b is known to induce mucus and airway secretion as well as activating lymphocytes and monocytes/macrophages [25]. Previously, we reported that 1,8-cineol inhibited IL-1b-stimulated production of TNF-a [26] and LPS-stimulated production of IL-1b in monocytes to a similar extent as compared to budenoside [20]. The only difference between the two substances was in the potency in that a 100- to 1000-fold higher concentration of cineol was necessary to produce the same degree of inhibition of PGE2 and IL1-b, respectively. This correlates with the therapeutic molar airway concentration of budenoside (10K8 M) and 1,8-cineol (10K5 M). For this reason, systemic therapy with 1,8-cineol seems to be favorable with regard to the group-related lipophilicity of the monoterpenes and their predominant excretion by exhalation. Additional therapy with 1,8-cineol in small gut soluble capsules (3!200 mg/day) of patients with asthma improved lung function [27] and inhibited monocyte ex vivo production of LTB4 by 40% [28]. This effect subsided completely 4 days after withdrawal of 1,8-cineol. Remarkably, in this previous study in a small group of normal subjects ex vivo IL-1b production was also suppressed to a similar extent in monocytes following 3 days of therapy but did not increase after withdrawal. These ex vivo-data suggest stronger effects of 1,8-cineol on cytokine production and support the relevance of this vitro-study with 1,8-cineol. To summarize, these results support new evidence that 1,8-cineol blocks cytokine production in lymphocytes and monocytes. The reduction of cytokine production suggests an anti-inflammatory mode of action and consequently inhibition of cytokine induced airway mucus hypersecretion rather than simple secretolytic activity. Further research should be directed to the underlying mechanism of action of 1,8-cineol and other related terpenes. These data support our previous reports and contribute to identifying 1,8-cineol as alternative drug with potentially steroid-like anti-inflammatory

U.R. Juergens et al. / Pulmonary Pharmacology & Therapeutics 17 (2004) 281–287

activity [12]. Therefore, isolated monoterpenes, such as 1,8-cineol, may offer a new opportunity for initial and longterm treatment of COPD and asthma, particularly, if inhaled glucocorticosteroids have still not been used. With the inconsistant effects of glucocorticosteroids on the underlying inflammatory process in COPD research on monoterpenes, such as with 1,8-cineol, should also be directed into nonsteroid dependent mechanisms of airway inflammation in COPD and asthma. This potential role of 1,8-cineol in patients who rarely achieve treatment goals established in the Guidelines of COPD and asthma with regular use of a single controller medication to effectively treat the disease needs to be further investigated in clinical studies.

References [1] Allen DB. Growth suppression by glucocorticoid therapy. Endocrinol Metab Clin North Am 1996;25:699–717. [2] Wise R, Connett J, Weinmann G, Scanlon P, Skeans M. “The Lung Health Study Research Group”. Effect of inhaled triamcinolone on the decline in pulmonary function in chronic obstructive pulmonary disease. N Engl J Med 2000;343:1902–9. [3] NHLBI. Global initiative for chronic obstructive lung disease. NIH Publication March 2001; No 2701A. [4] Ricci M, Matucci A, Rossi O. Recent advances in the pathogenetic mechanisms and genetic aspects of atopic diseases. Allergy Clin Immunol News 1994;6:103–8. [5] Bell SJ, Metzger WJ, Welch CA, Gilmour MI. A role for Th2 T-memory cells in early airway obstruction. Cell Immunol 1996;170: 185–94. [6] Vilcek J, Lee TH. Tumor necrosis factor. J Biol Chem 1991;266: 7313–6. [7] Panuska JR, Midulla F, Cirino NM, Villani A, Gilbert IA, Mc Fadden Jr. ER, Huang YT. Virus induced alterations in macrophage production of tumor necrosis factor and prostaglandin E2. Am J Physiol 1990;259:L396–L402. [8] Fattori E, Cappelletti M, Costa P, Sellito C, Cantoni L, Carelli M, Faggioni R, Fantuzzi G, Ghezzi P, Poli V. Defective inflammatory response in interleukin-6 deficient mice. J Exp Med 1994;180:1243–50. [9] White JR, Lee JM, Young PR. Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits interleukin-8-induced neutrophil migration. J Biol Chem 1998;273:10095–8. [10] Confalonieri M, Mainardi E, Della Porta R. Inhaled corticosteroids reduce neutrophilic bronchial inflammation in patients with chronic obstructive pulmonary disease. Thorax 1998;53:583–5. [11] Connett G, Lenney W. Prevention of viral induced asthma attacks using inhaled budenoside. Arch Dis Child 1993;68:85–7. [12] Juergens UR, Dethlefsen U, Steinkamp G, Gillissen A, Repges R, Vetter H. Anti-inflammatory activity of eucalyptol (1,8-cineol) in bronchial asthma: a double blind placebo-controlled trial. Respir Med 2003;97:250–6.

287

[13] Juergens UR, Christiansen SC, Stevenson DD, Zuraw BL. Arachidonic acid metabolism in monocytes of aspirin-sensitive asthmatic patients before and after oral aspirin challenge. J Allergy Clin Immunol 1992;90:636–45. [14] Andersson U, Andersson J, Lindfors A, Wagner K, Mo¨ller G, Heusser CH. Simultaneous production of interleukin 2, interleukin 4 and inferferon-g by activated human blood monocytes. Eur J Immunol 1990;20:1591–6. [15] Lewis DB, Prickett KS, Larsen A, Grabstein K, Weaver M, Wilson CB. Restricted production of interleukin 4 by activated human T cells. Proc Natl Acad Sci USA 1988;85:9743–7. [16] Juergens UR, Sto¨ber M, Vetter H. The anti-inflammatory activity of L-menthol compared to mint oil in human monocytes in vitro: a novel perspective for its therapeutic use in inflammatory diseases. Eur J Med Res 1998;3:539–45. [17] Chatila T, Silverman L, Miller R, Geha R. Mechanisms of T cell activation by the calcium ionophore ionomycin. J Immunol 1989;143: 1283–9. [18] Munos-Bellido FJ, Monteseirin FJ, Escribano MM, Delgado J, Vela´zquez E, Conde J. Effect of seasonal exposure to pollen on nonspecific interleukin-4, interleukin-5, and interferon-gamma in vitro release by peripheral blood mononuclear cells from subjects with pollinosis. Allergy 1998;53:420–5. [19] Santos FA, Roa VS. Antiinflammatory and antinocieptive effects of 1,8- cineol a terpenoid oxide present in many essential oils. Phytother Res 2000;14(4):240–4. [20] Juergens UR, Sto¨ber M, Vetter N. Steroid-like inhibition of monocyte arachidonic acid metabolism and IL-1b production by eucalyptol (1,8-cineol) (in German). Atemw-Lungenkrkh 1998;24: 3–11. [21] Lundgren JD, Shelhammer JH. Pathogenesis of airway mucus hypersecretion. J Allergy Clin Immunol 1990;85:399–419. [22] Marom Z, Shelhamer JH, Bach MK, Morton DR, Kaliner M. Slowreacting substances, leukotrienes C4 and D4, increase the release of mucus from human airways in vitro. Am Rev Respir Dis 1982;126: 449–51. [23] Marom Z, Shelhamer JH, Sun F, Kaliner M. Human airway monohydroxyeicosa-tetraenoic acid generation and mucus release. J Clin Invest 1983;72:122–7. [24] Calhoun WJ, Kelly J. Pharmacology of the respiratory tract: clinical and experimental. In: Chung F, Barnes P, editors. Cytokines in the respiratory tract. New York: Marcel Dekker; 1993. p. 253–88. [25] Cohan VL, Scott AL, Dinarello CA, Prendergast RA. Interleukin 1 is a mucus secretagogue. Cell Immunol 1991;136:425–34. [26] Juergens UR, Sto¨ber M, Vetter H. Inhibition of cytokine production and arachidonic acid metabolism by eucalyptol (1,8-cineol) in human blood monocytes in vitro. Eur J Med Res 1998;3:508–10. [27] Wittmann M, Petro W, Kaspar P, Repges R, Dethlefsen U. Therapy with expectorants: a double-blind randomised study comparing ambroxol and cineol. Atemw-Lungenkrkh 1998;24(2):67–74 [in German]. [28] Juergens UR, Sto¨ber M, Schmidt-Schilling L, Kleuver T, Vetter H. Anti-inflammatory effects of eucalyptol (1,8-cineol) in bronchial asthma: inhibition of arachidonic acid metabolism in human blood monocytes ex vivo. Eur Med Res 1998;3:407–12.