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Published ahead of print on August 8, 2002, doi:10.1164/rccm.200204-356OC
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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1055-1061, (2002)
© 2002 American Thoracic Society

Articles

Immunomodulatory Effects of Melatonin in Asthma

E. Rand Sutherland, Richard J. Martin, Misoo C. Ellison and Monica Kraft

Department of Medicine, National Jewish Medical and Research Center; and University of Colorado Health Sciences Center, Denver, Colorado

Correspondence and requests for reprints should be addressed to E. Rand Sutherland, M.D., 1400 Jackson Street, B-123, Denver, CO 80206. E-mail: sutherlande{at}njc.org

ABSTRACT

Patients with nocturnal asthma demonstrate circadian variations in airway inflammation. We hypothesized that melatonin, a circadian rhythm regulator, modulates circadian inflammatory variations in asthma. The effect of melatonin stimulation on peripheral blood mononuclear cell cytokine production was evaluated at 4:00 P.M. and 4:00 A.M. in normal control subjects, patients with nocturnal asthma, and patients with non-nocturnal asthma. Melatonin was proinflammatory, causing significantly increased production of interleukin-1, interleukin-6, and tumor necrosis factor-{alpha} at 4:00 P.M. and 4:00 A.M. in all subject groups (range, 12.8 ± 3.3 to 131.72 ± 16.4%, p <= 0.0003). The observed increases in cytokine production did not change between 4:00 P.M. and 4:00 A.M. in control subjects or in patients with nocturnal asthma (p > 0.05, both cases). At 4:00 P.M., the cytokine response to melatonin of patients with nocturnal asthma was greater than that of control subjects or patients with non-nocturnal asthma and did not change significantly at 4:00 A.M. At 4:00 P.M., the cytokine response of patients with non-nocturnal asthma was less than that of patients with nocturnal asthma and rose significantly at 4:00 A.M. (p = 0.0001, all comparisons). Melatonin is proinflammatory in both patients with asthma and healthy subjects. Patients with nocturnal asthma demonstrate the largest daytime cytokine response and cannot be further stimulated at 4:00 A.M., suggesting chronic overstimulation in vivo. These results suggest differential immunomodulatory effects of melatonin based on asthma clinical phenotype and may indicate an adverse effect of exogenous melatonin in asthma.

Key Words: hormones • inflammation • pathogenesis

Asthma is a clinical syndrome consisting of chronic airway inflammation, airway responsiveness, and expiratory airflow limitation that reverses after bronchodilator treatment. In patients with nocturnal asthma, circadian variations in airflow limitation are seen, with declines in measures of airflow such as peak expiratory flow rate and FEV1. The circadian worsening in nocturnal asthma is associated with increased airway inflammation at night (1), increased airways responsiveness at night (2), and worsened airflow limitation at night (3). In addition, circadian variations in multiple mediators of airway inflammation and function have been demonstrated in nocturnal asthma, including peripheral blood eosinophil number and function (4–7), alveolar tissue inflammation (8, 9), the intrinsic adrenergic hormonal milieu (10), and the affinity and activity of both glucocorticoid (11) and ß-adrenergic receptors (12).

Melatonin (N-acetyl-5-methoxytryptamine), a key regulator of circadian rhythm homeostasis in humans, is the principal hormone product of the pineal gland (13). Melatonin is also synthesized by human peripheral blood mononuclear cells (14), and it has been shown to have multiple immunomodulatory effects: T lymphocytes have cell surface G protein–linked and nuclear melatonin receptors, and melatonin has been shown to stimulate the production of helper T cell type 1 cytokines by T lymphocytes (15). In addition, melatonin has been shown to increase the production of tumor necrosis factor-{alpha} (TNF-{alpha}) by lipopolysaccharide-stimulated human monocytes (16). Melatonin has been shown to induce human lymphocytes and monocytes to synthesize a number of interleukins (ILs), including IL-2, IL-6, and IL-12 (17, 18).

Melatonin appears to have an important immunomodulatory effect in allergic diseases. In experiments designed to evaluate the importance of melatonin in regulating antigen-specific T cell response, Shaji and colleagues (19) demonstrated that melatonin enhanced the proliferation of ovalbumin-specific CD4-positive T lymphocytes. They also demonstrated that treatment with melatonin increased serum levels of IL-4 and reduced serum levels of IL-2 and interferon-{gamma}, while also inducing ovalbumin-specific IgG1 secretion and downregulating ovalbumin-specific IgG2a secretion (19). Experiments by Martins and colleagues in pinealectomized rats sensitized to ovalbumin demonstrated that pinealectomy significantly reduces bronchoalveolar lavage fluid inflammatory cell counts after ovalbumin inhalational challenge, and that bronchoalveolar lavage fluid cellularity is restored to control levels after treatment with melatonin (20). Although these experiments provide tentative evidence for an immunomodulatory effect of melatonin, the presentation and processing of antigen is complex and likely modified by a wide array of host and environmental factors (21). The contribution of melatonin to these processes remains to be fully elucidated.

Melatonin may also have a role in modulating airway function. Melatonin receptors have been shown to be present in the lungs of experimental animals (22, 23), and supraphysiologic concentrations of melatonin have been shown to cause airway smooth muscle contraction in isolated bovine airway smooth muscle rings (24). In anesthetized dogs, however, intravenous administration of melatonin results in bronchodilation, as reflected by a decrease in total lung resistance. This bronchodilator action of melatonin was demonstrated in both intact and vagotomized animals (25).

We hypothesized that melatonin may play an important role in the regulation of nocturnal asthma by altering the inflammatory characteristics of peripheral blood mononuclear cells (PBMCs) over the course of a 24-hour period. We further hypothesized that the response to melatonin would differ between patients with nocturnal asthma, patients with non-nocturnal asthma, and normal control subjects.

METHODS

All research was approved by the National Jewish Medical and Research Center (Denver, CO) Institutional Review Board. Subjects provided written informed consent.

Inclusion and Exclusion Criteria
Adult subjects (18 years of age or older) with nocturnal asthma or non-nocturnal asthma and normal subjects were recruited. Asthma was diagnosed in subjects with a suggestive clinical history who demonstrated airway hyperresponsiveness to methacholine. Nocturnal asthma was defined as a fall in peak flow of 15% or more, between bedtime and awakening, on four or more of seven sequential nights at home in a subject with asthma. Exclusion criteria included other concurrent lung disease, significant medical illness, age less than 18 years, pregnancy, and the use of corticosteroids or other antiinflammatory asthma medication within 6 weeks.

Circadian Rhythm Standardization and Subject Evaluation
Subjects maintained a 24-hour sleep–wake routine (bedtime at 10:00 P.M., awakening at 6:00 A.M.) for 7 days. Adherence with the routine was documented by acquisition of 24-hour activity data (ActiTrac; IM Systems, Baltimore, MD). On Day 8, subjects spent 24 hours in our clinical research laboratory, during which time the routine was maintained. Spirometry and phlebotomy were performed at 4:00 P.M. and 4:00 A.M.

PBMC Isolation
PBMCs were isolated from 60 cm3 of heparinized blood by centrifugation against a Ficoll-Paque gradient.

PBMC Stimulation
Three experimental conditions were established: (1) negative control: PBMCs alone; (2) positive control: PBMCs and stimulant (phytohemagglutinin for proliferation, zymosan for cytokine production); and (3) eight separate melatonin conditions: PBMCs plus stimulant plus melatonin (Sigma-Aldrich, St. Louis, MO) in concentrations between 10-12 and 10-5 M. This included the physiologic range of melatonin (10-9 to 10-7 M) (13).

Measurement of PBMC Proliferation
PBMCs were incubated at 37°C for 24 hours, pulsed with [3H]thymidine (10 µl of a 1:10 dilution), and incubated for an additional 6 hours. Cells were then frozen and rinsed, and incorporation of [3H]thymidine was measured (TopCount NXT; Packard BioScience, Meriden, CT).

Measurement of Cytokines
PBMCs were incubated at 37°C for 24 hours. Supernatant was removed for cytokine assay and concentrations of IL-1, IL-6, and TNF-{alpha} were assayed by ELISA. These cytokines were chosen because of prior research demonstrating their importance in asthma (26, 27). IL-1 has been shown to be synthesized by airway mononuclear cells in patients with nocturnal asthma (27), IL-6 production has been shown to be induced by melatonin (17), and altered steroid response at night in nocturnal asthma affects glucocorticoid-mediated suppression of TNF-{alpha} (26).

Statistical Analysis
Data are expressed as means ± standard error. A linear mixed effects model (28) was used to compare PBMC proliferation and IL-1, IL-6, and TNF-{alpha} as a percentage of positive control at different melatonin concentrations between 4:00 A.M. and 4:00 P.M. within each group and between groups at 4:00 A.M. and 4:00 P.M. This model accounts for within-group/between-subject variability and the correlation between the measurements within each subject. Pairwise comparisons for contrasting within and between groups at different melatonin concentrations were made via analysis of variance. A false discovery rate procedure (29) was adopted for multiple comparisons.

RESULTS

Subject Characteristics
A total of 23 subjects were evaluated: 5 normal control subjects, 6 patients with nocturnal asthma, and 12 patients with non-nocturnal asthma. Subject characteristics are summarized in Table 1 .


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  		TABLE 1. Subject characteristics

 
Effect of Melatonin on the Production of IL-1, IL-6, and TNF-{alpha}
The effects of melatonin on PBMC cytokine production were evaluated in three ways. First, mean cytokine production in response to zymosan plus melatonin versus zymosan alone (positive control) was compared. Second, within-group comparisons were made between cytokine production at 4:00 P.M. versus 4:00 A.M. in normal control subjects, patients with nocturnal asthma, and patients with non-nocturnal asthma. Third, to further evaluate the cytokine response at differing levels of melatonin stimulation, cytokine production was compared in each group at 4:00 P.M. versus 4:00 A.M. at each individual experimental melatonin concentration.

IL-1.
At both 4:00 P.M. and 4:00 A.M. the addition of melatonin to zymosan-stimulated PBMCs caused significantly increased production of IL-1 in all three subject groups (Figure 1) . In normal control subjects, at 4:00 P.M. production of IL-1 increased 20.6 ± 4.6% versus positive control (p = 0.0001), and at 4:00 A.M. production of IL-1 increased 27.7 ± 4.7% (p = 0.0001 versus positive control). In patients with nocturnal asthma, the 4:00 P.M. increase was 82.5 ± 17.1% (p = 0.0001 versus positive control) and the 4:00 A.M. increase was 85.6 ± 17.1% (p = 0.0001 versus positive control). In patients with non-nocturnal asthma, an increase of 27.2 ± 3.8% (p = 0.0001 versus positive control) was noted at 4:00 P.M., with a 131.7 ± 16.4% increase (p = 0.0001 versus positive control) at 4:00 A.M.



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  		Figure 1. Daytime (4:00 P.M.) and nighttime (4:00 A.M.) percent change versus positive control in PBMC IL-1 production as a result of costimulation with melatonin (mean response across all experimental melatonin concentrations). *p < 0.05 For comparison with positive control. Horizontal dashed lines at the top indicate p values for within-group comparisons at 4:00 P.M. versus 4:00 A.M. NA = nocturnal asthma; NNA = non-nocturnal asthma.

 
Comparing mean responses to melatonin within subject groups at 4:00 P.M. versus 4:00 A.M. (Figure 1, dashed lines at top), normal subjects did not demonstrate a significantly different response at 4:00 P.M. versus 4:00 A.M. (20.6 ± 4.63 versus 27.7 ± 4.7%, p = 0.35). Patients with nocturnal asthma demonstrated a response at 4:00 P.M. that was greater than that seen in normal subjects, and this response was maintained and did not differ at 4:00 P.M. versus 4:00 A.M. (82.5 ± 17.1 versus 85.6 ± 17.1%, p = 0.91). In patients with non-nocturnal asthma, 4:00 P.M. IL-1 production was similar to that seen in normal subjects, but there was a significant increase in IL-1 production at 4:00 A.M. (27.2 ± 3.8 versus 131.7 ± 16.4%, p = 0.0001). At 4:00 A.M., IL-1 production was greater in patients with non-nocturnal asthma than in patients with nocturnal asthma (131.7 ± 16.4 versus 85.6 ± 17.1%, p = 0.03).

Four P.M. versus 4:00 A.M. IL-1 production at individual melatonin concentrations was then analyzed in all three groups (Figure 2) . There were no significant differences in IL-1 production as a percentage of positive control at 4:00 P.M. versus 4:00 A.M. across the entire experimental range of melatonin in normal subjects or subjects with nocturnal asthma. In subjects with non-nocturnal asthma, there was a significant increase in IL-1 production at 4:00 A.M. versus 4:00 P.M. at all melatonin concentrations between 10-9 and 10-6 M (Figure 2, bottom). This response occurred within the physiologic range of melatonin concentrations (10-9 to 10-7 M). There were no significant linear trends indicating a dose–response effect across the range of experimental melatonin concentrations (all p values for linear trend >= 0.2).



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  		Figure 2. Plot of IL-1 production as a percentage of positive control versus melatonin concentration, at 4:00 A.M. (solid symbols) and 4:00 P.M. (open symbols) in normal control subjects, patients with nocturnal asthma (NA), and patients with non-nocturnal asthma (NNA). {dagger}p < 0.01 and {ddagger}p < 0.005. Dotted line represents 100% of positive control; solid bar represents the range of physiologic melatonin concentrations.

 
IL-6.
At both 4:00 P.M. and 4:00 A.M. the addition of melatonin to zymosan-stimulated PBMCs caused significantly increased production of IL-6 in all three subject groups (Figure 3) . In normal control subjects at 4:00 P.M., production of IL-6 increased 17.8 ± 4.8% versus positive control (p = 0.0003), and at 4:00 A.M. production of IL-6 increased 52.4 ± 10.6% (p = 0.0001 versus positive control). In patients with nocturnal asthma, the 4:00 P.M. increase was 105 ± 19.2% (p = 0.0001 versus positive control) and the 4:00 A.M. increase was 129.4 ± 22.5% (p = 0.0001 versus positive control). In patients with non-nocturnal asthma, an increase of 26.8 ± 5.2% (p = 0.0001 versus positive control) was noted at 4:00 P.M., with a 124.6 ± 14.7% increase (p = 0.0001 versus positive control) at 4:00 A.M.



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  		Figure 3. Daytime (4:00 P.M.) and nighttime (4:00 A.M.) percent change versus positive control in PBMC IL-6 production as a result of costimulation with melatonin (mean response across all experimental melatonin concentrations). *p < 0.05 For comparison with positive control. Horizontal dashed lines at the top indicate p values for within-group comparisons at 4:00 P.M. versus 4 A.M. NA = nocturnal asthma; NNA = non-nocturnal asthma.

 
Comparing mean responses to melatonin within subject groups at 4:00 P.M. versus 4:00 A.M. (Figure 3, dashed lines at top), normal subjects did demonstrate a small and significantly different response at 4:00 P.M. versus 4:00 A.M. (17.8 ± 4.8 versus 52.4 ± 10.6%, p = 0.02). Patients with nocturnal asthma demonstrated a response at 4:00 P.M. that was greater than that seen in normal subjects, and this response was maintained and did not differ at 4:00 P.M. versus 4:00 A.M. (105 ± 19.2 versus 129.4 ± 22.5%, p = 0.44). In patients with non-nocturnal asthma, 4:00 P.M. IL-6 production was similar to that seen in normal subjects, but there was a significant increase in IL-6 production at 4:00 A.M. (26.8 ± 5.2 versus 124.6 ± 14.7%, p = 0.0001). At 4:00 A.M., IL-6 production was equivalent in nocturnal and patients with non-nocturnal asthma (129.4 ± 22.5 versus 124.6 ± 14.7%, p = 0.84).

Four P.M. versus 4:00 A.M. IL-6 production at individual melatonin concentrations was then analyzed in all three groups (Figure 4) . There were no significant differences in IL-6 production as a percentage of positive control at 4:00 P.M. versus 4:00 A.M. across the entire experimental range of melatonin in normal subjects or patients with nocturnal asthma. In subjects with non-nocturnal asthma, there was a significant increase in IL-6 production at 4:00 A.M. versus 4:00 P.M. at all melatonin concentrations between 10-8 and 10-5 M (Figure 4, bottom). This response occurred in the upper portion of the physiologic range of melatonin concentrations (10-9 to 10-7 M). There were no significant linear trends indicating a dose–response effect across the range of experimental melatonin concentrations (all p values for linear trend >= 0.2).



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  		Figure 4. Plot of IL-6 production as a percentage of positive control versus melatonin concentration, at 4:00 A.M. (solid symbols) and 4:00 P.M. (open symbols) in normal control subjects, patients with nocturnal asthma (NA), and patients with non-nocturnal asthma (NNA). *p < 0.05 and {dagger}p < 0.01. Dotted line represents 100% of positive control; solid bar represents the range of physiologic melatonin concentrations.

 
TNF-{alpha}.
At both 4:00 P.M. and 4:00 A.M., the addition of melatonin to zymosan-stimulated PBMCs caused significantly increased production of TNF-{alpha} in all three subject groups (Figure 5) . In normal control subjects at 4:00 P.M., production of TNF-{alpha} increased 14.3 ± 2.8% versus positive control (p = 0.0001), and at 4:00 A.M., production of TNF-{alpha} increased 14 ± 3.2% (p = 0.0001 versus positive control). In patients with nocturnal asthma, the 4:00 P.M. increase was 81 ± 16.9% (p = 0.0001 versus positive control) and the 4:00 A.M. increase was 46.6 ± 10% (p = 0.0001 versus positive control). In patients with non-nocturnal asthma, an increase of 12.8 ± 3.3% (p = 0.0002 versus positive control) was noted at 4:00 P.M., with a 51.8 ± 8.1% increase (p = 0.0001 versus positive control) at 4:00 A.M.



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  		Figure 5. Daytime (4:00 P.M.) and nighttime (4:00 A.M.) percent change versus positive control in PBMC TNF-{alpha} production as a result of costimulation with melatonin (mean response across all experimental melatonin concentrations). Horizontal interrupted lines at the top indicate p values for within-group comparisons at 4:00 P.M. versus 4:00 A.M. *p < 0.05 For comparison with positive control. NA = nocturnal asthma; NNA = non-nocturnal asthma.

 
Comparing mean responses to melatonin within subject groups at 4:00 P.M. versus 4:00 A.M. (Figure 5, dashed lines at top), normal subjects did not demonstrate a significantly different response (14.3 ± 2.8% at 4:00 P.M. versus 14 ± 3.2% at 4:00 A.M., p = 0.94). Patients with nocturnal asthma demonstrated a response that was greater than that seen in normal subjects, and this response was maintained and did not differ significantly at 4:00 P.M. versus 4:00 A.M. (81 ± 16.9 versus 46.6 ± 10%, p = 0.09). In patients with non-nocturnal asthma, 4:00 P.M. TNF-{alpha} production was similar to that seen in normal subjects, but there was a significant increase in TNF-{alpha} production at 4:00 A.M. (12.8 ± 3.3% at 4:00 P.M. versus 51.8 ± 8.1% at 4:00 A.M., p = 0.0001). At 4:00 A.M., TNF-{alpha} production was equivalent in nocturnal and patients with non-nocturnal asthma (46.6 ± 10% versus 51.8 ± 8.1%, p = 0.64).

Four P.M. versus 4:00 A.M. TNF-{alpha} production at individual melatonin concentrations was then analyzed in all three groups (Figure 6) . There were no significant differences in TNF-{alpha} production as a percentage of positive control across the entire experimental range of melatonin in normal subjects. In patients with nocturnal asthma, significant increases in TNF-{alpha} at 4:00 P.M. versus 4:00 A.M. occurred at melatonin concentrations of 10-12 and 10-10 M. In subjects with non-nocturnal asthma, there was a significant increase in TNF-{alpha} production at 4:00 A.M. versus 4:00 P.M. at all melatonin concentrations between 10-7 and 10-5 M (Figure 6, bottom). This response again occurred within the upper portion of the physiologic range of melatonin concentrations (10-9 to 10-7 M). There were no significant linear trends indicating a dose–response effect across the range of experimental melatonin concentrations (all p values for linear trend >= 0.2).



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  		Figure 6. Plot of TNF-{alpha} production as a percentage of positive control versus melatonin concentration, at 4:00 A.M. (solid symbols) and 4:00 P.M. (open symbols) in normal control subjects, patients with nocturnal asthma (NA), and patients with non-nocturnal asthma (NNA). *p < 0.05, {dagger}p < 0.01, and {ddagger}p < 0.005. Dotted line represents 100% of positive control; solid bar represents the range of physiologic melatonin concentrations.

 
DISCUSSION

A number of conclusions can be drawn from the results of our experiments: first, the addition of melatonin to zymosan-stimulated PBMCs increases the production of IL-1, IL-6, and TNF-{alpha} when compared with zymosan stimulation alone, indicating that melatonin is proinflammatory. Although there are a number of reports that melatonin is in fact proinflammatory (16–18), controversy persists in the literature, where antiinflammatory properties have also been attributed to melatonin (30–32). The degree of cytokine production as a result of melatonin stimulation does not appear to be dependent on the concentration of melatonin used as a stimulus, as there was no evidence of a linear dose–response trend with increasing melatonin concentration.

Of interest is the variable intensity of the inflammatory response at 4:00 P.M. versus 4:00 A.M. in normal subjects, patients with nocturnal asthma, and patients with non-nocturnal asthma (Figures 1, 3, and 5). Normal subjects have small percent increases in cytokines at 4:00 P.M. (range, 14.34 ± 2.84 to 20.56 ± 4.63%) that, except in the case of IL-6, do not increase significantly at 4:00 A.M. Patients with nocturnal asthma, by contrast, have a heightened response at both 4:00 P.M. and 4:00 A.M., but this response does not differ at the two time points, possibly indicating an inability of these cells to further respond to inflammatory stimuli in vitro. Patients with non-nocturnal asthma show a response at 4:00 P.M. that is similar to that seen in normal subjects, but in these subjects a markedly increased response to melatonin is seen at 4:00 A.M.

Although our research was not designed to determine the mechanisms behind these between-group differences, several hypotheses can be generated. First, because there are clear differences between normal subjects and subjects with asthma, the differences in PBMC cytokine production could possibly be explained on the basis of asthma severity alone. As indicated in Table 1, subjects with nocturnal asthma have more severe expiratory airflow limitation than patients with non-nocturnal asthma during both the day and night. Using the severity of airflow limitation as a surrogate marker for severity of chronic airway and systemic inflammation, it may be that the PBMCs of patients with more severe asthma are subjected to heightened inflammatory stimuli in vivo, thereby reducing the ability of these cells to respond in a circadian fashion to the inflammatory stimulus of elevated night-time melatonin levels. In patients with non-nocturnal asthma, a process of priming or activation may occur at night only, thereby facilitating the PBMC cytokine response. Alternatively, a differential reduction in endogenous cortisol levels at night may facilitate this response. Of additional interest is the fact that the effect of melatonin does not appear to be dose-dependent. This may indicate that small amounts of melatonin are enough either to fully activate and/or to saturate the melatonin receptor. Further research will be required to further understand the kinetics of melatonin–receptor interactions in asthma.

Although not evaluated as part of this research, PBMC melatonin receptor affinity may differ at 4:00 A.M. and 4:00 P.M. in subjects with varying asthma phenotypes. Alternatively, melatonin receptor affinity may be similar, but postreceptor signaling mechanisms such as transcription regulation may differ between subjects with different asthma phenotypes, resulting in varying effects on cytokine production. We have previously shown that patients with nocturnal asthma demonstrate polymorphisms or functional alterations in glucocorticoid (11) and ß-adrenergic (12, 33) receptors that affect their activity, and further research will be required to evaluate the potential importance of altered melatonin receptor number or function in nocturnal asthma.

Our research is limited by the fact that sampling of mononuclear cells was restricted to the peripheral blood. If PBMCs were to demonstrate different inflammatory phenotype characteristics than lung mononuclear cells, or if there were homing to the lung of a subset of PBMCs more likely to produce cytokines in nocturnal asthma, this could explain the paradoxical differences observed between PBMCs from patients with nocturnal asthma and those from patients with non-nocturnal asthma. Previous research in our laboratory has demonstrated that there is a nocturnal influx of lymphocytes, macrophages, and eosinophils into the lung in nocturnal asthma (9). Although the origin of these infiltrating cells remains unknown, one potential explanation would be that these cells move from the peripheral blood into the lung. If this were also to be the case with mononuclear cells in nocturnal asthma, this could explain the differences in the inflammatory phenotype of these cells in nocturnal versus non-nocturnal asthma. Support for this hypothesis may be derived from the research of Martins and colleagues, who showed that in a pinealectomized mouse model of allergic asthma, the absence of melatonin reduced inflammatory cell influx into the lung after allergen challenge, a phenomenon that was abolished with melatonin replacement (20). Future research should be directed at evaluating differences in peripheral and lung monocyte inflammatory phenotypes in nocturnal and non-nocturnal asthma, for example, by examining the effect of melatonin on mononuclear cells derived from bronchoalveolar lavage.

The clinical relevance of these observations requires formal evaluation. Our results indicate that melatonin causes increased PBMC production of selected cytokines in vitro, a finding that may have clinical relevance in patients who use over-the-counter pharmaceutical preparations containing melatonin. Data about the number of people using melatonin are scarce, but millions of Americans are reported to use melatonin (32), and a proportion of these individuals presumably suffer from asthma. For these patients, avoidance of melatonin may be appropriate until further information about the clinical effect of melatonin in asthma becomes available.

Acknowledgments

The authors acknowledge the research subjects and staff of the Felt Laboratory for Asthma Research at the National Jewish Medical and Research Center for contributions to this research.

FOOTNOTES

This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form April 22, 2002; accepted in final form August 5, 2002

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