Immunology & Immunological Disorders Poster Session
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Introduction
In rats, Freund's adjuvant injection induced a hyperactivation of cellular immune functions that trigger the development of arthritis. Although the fact that immunization is associated with endocrine changes is well known (for a review see [1]), very few studies have addressed the question as to whether changes in circadian rhythms of hormones take place during the course of experimental arthritis [2-4]. The stimulation of the hypothalamo-pituitary-adrenal axis and a tendency to loss its rhythmicity seem to be generalized responses to immune-mediated inflammatory conditions. In previous studies we observed significant effects of immune-mediated inflammation on ACTH, GH, PRL and TSH release at an early phase (3 days later) after Freund's adjuvant injection to rats [5,6]. After mycobacterial adjuvant injection, 24-hour variations of TSH levels became blunted while 24-hour variations of PRL and ACTH persisted. Freund's adjuvant injection increased serum ACTH and PRL, and decreased serum GH and TSH levels. Since melatonin, because of its close relation to the circadian signal generated by the suprachiasmatic nuclei, had been proposed as a "chronobiotic" [7], we wished to assess whether early changes of circulating ACTH, GH, PRL and LH in immune-mediated inflammation could be affected by a pharmacological dose of melatonin. Recently we reported in groups of young and old rats studied the day before, and on days 6, 12 and 18 after mycobacterium adjuvant injection the pineal levels of melatonin. Old rats had the lowest pineal melatonin levels. On day 18th, a global depressive effect of arthritis development on pineal melatonin content was found [8].
Materials and Methods
Experiments were carried out in adult male Wistar rats (180-220 g), kept under light between 0800 and 2000 h daily. Rats received sc injections of melatonin (30 micrograms) in 0.1 ml of vehicle (10 % ethanol in saline), or its vehicle, 1 h before lights off for 12 days. On the 10th day of treatment the rats were sc injected with Freund's complete adjuvant (0.5 mg heat-killed Mycobacterium butyricum/rat) or its vehicle (0.5 ml paraffin oil containing 15% mannide monooleate) at 1100 h. On the third day after injection, groups of 6-8 rats were killed by decapitation at six different time intervals throughout a 24-hour cycle. Serum from the trunk blood was collected for hormone measurements by RIA using material kindly supplied by the NIDDK's National Hormone and Pituitary Program.
Results
Upper left panel of Figure shows the changes in serum PRL levels in rats injected with Freund's adjuvant or its vehicle, and subjected to melatonin or vehicle treatment. The changes caused by immunization and time of day, when analyzed as main factors in a factorial ANOVA, were significant (p= 0.0001 and p< 0.00001, respectively); in this case immunization increased circulating PRL. A significant 2-factor interaction for melatonin treatment and immunization was observed (p= 0.0006), i.e., melatonin injection resulted in a significant diurnal variation in Freund's adjuvant-injected rats only, with maxima not differing from those found in the remaining groups.
Figure. Twenty four-hour changes in serum PRL, ACTH, LH and GH levels of rats injected with Freund's complete adjuvant or adjuvant's vehicle and treated with melatonin or vehicle (control) as described in Methods. Shown are the means ± SEM. Analyzed as main factors in a factorial ANOVA, significant effects of immunization (F= 16.58, p= 0.0001) and time of day (F= 13.49, p< 0.00001) were found for serum PRL. Significant 2-factor interactions were found for immunization and melatonin treatment (F= 12.49, p= 0.0006) and for immunization and time of day (F= 3.17, p= 0.0099) to affect PRL. Analysis by a one-way ANOVA indicated time-related changes of serum PRL in adjuvant's vehicle-treated rats injected with vehicle (F= 8.56, p< 0.001) or melatonin (F= 4.24, p< 0.004) as well as in Freund's adjuvant-injected rats treated with vehicle (F= 4.83, p< 0.002) or melatonin (F= 3.74, p< 0.008). In the case of ACTH, analysis as main factors in a factorial ANOVA indicated significant effects of immunization (F= 5.85,
p= 0.016) and time of day (F= 13.15, p< 0.00001). Analysis by a one-way ANOVA indicated time-related changes of serum ACTH in adjuvant's vehicle- or Freund's treated rats (F= 5.40, p< 0.0001, and F= 2.91, p< 0.03, respectively). Melatonin-treated rats showed time-of-day-related changes in serum ACTH in non-immunized and immunized rats (F= 2.48, p< 0.05 and F= 5.09, p< 0.002, respectively). As far as LH, significant effects of melatonin (F= 8.00, p= 0.0054), immunization (F= 4.40, p= 0.037) and time of day (F= 10.14, p< 0.00001) were found when analyzed as main factors in a factorial ANOVA. In the case of LH, a one-way ANOVA indicated time-related changes for adjuvant's vehicle-treated rats subjected to vehicle (F= 5.47, p= 0.0009) or melatonin (F= 3.33, p< 0.02). After immunization, only melatonin-treated rats showed significant time-related variations in serum LH (F= 2.463, p= 0.05). In the case of GH, main factor analysis in a factorial ANOVA was significant for immunization alone (F= 12.75, p= 0.0005). Se
rum GH levels did not exhibit time-of-day-related changes in any of the experimental groups examined.
Upper right panel of Figure shows the changes in serum ACTH concentration in rats injected with Freund's adjuvant or its vehicle, and subjected to melatonin treatment. Analyzed as main factors in a factorial ANOVA, changes due to immunization and time of day were significant (p= 0.016 and p< 0.00001, respectively), Freund's adjuvant injection augmented ACTH. Melatonin-treated, immunized rats showed a significantly higher amplitude than control, Freund's adjuvant-injected animals (p < 0.05, Table 1).
Lower left panel of Figure depicts LH levels. Immunization decreased circulating LH significantly (p= 0.037, factorial ANOVA) and suppressed the 24-hour rhythm found in controls (one-way ANOVA). The effect of Freund's adjuvant administration on 24-hour LH rhythm was counteracted by melatonin.
Serum GH levels in the same group of rats are shown in lower right panel of Figure. In a factorial ANOVA, only changes due to immunization were significant, with a significant decrease of circulating GH being seen after Freund's adjuvant injection (p= 0.0005 for main factor analysis). As shown in a one-way ANOVA, time-of-day-related changes of serum GH did not attain significance in any of the experimental groups.
Discussion and Conclusion
The foregoing data indicate that significant changes took place in circulating levels of serum PRL, ACTH, LH and GH at an early phase after mycobacterial adjuvant's injection. Immunization increased circulating PRL while conserving its diurnal rhythmicity. After immunization, an increase in serum ACTH levels with maintenance of the diurnal rhythm of ACTH was found, while immunization decreased circulating LH and suppressed its diurnal rhythmicity. As reported previously [5], we were unable to detect the presence of circadian variations of GH in immunized or control rats; only changes due to immunization were significant, with a decrease of circulating GH after immunization.
Another aim of the present study was to check whether administration of pharmacological amounts of melatonin could restore some of the endocrine changes observed after immunization. Our foregoing results indicate that in rats receiving sc injections of melatonin (30 micrograms), 1 h before lights off for 12 days, an augmentation of the amplitude of serum ACTH rhythm could be detected, an indirect evaluation of the total amount of ACTH secreted during the day. Melatonin effectively counteracted the decreased circulating LH and restored its 24-hour rhythmicity in immunized rats. With regard to PRL, melatonin injection produced a significant diurnal variation in immunized rats only. Therefore, several early changes in levels and 24-hour rhythms of circulating ACTH, PRL and LH in Freund's adjuvant-injected rats were sensitive to treatment with pharmacological amounts of melatonin. The present results are in line with the hypothesis that melatonin could be a useful chronobiotic to treat circadian sequelae of infla mmation. Whether this occurs under physiological conditions (i.e., melatonin suppression and replacement in physiological amounts) remains to be defined.
Acknowledgements
This work was supported by grants from Dirección General de Investigación Científica y Técnica (DGICYT, PB94-0260), Spain, the University of Buenos Aires (TM 07), the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina (PIP 4156) and the Agencia Nacional de Promoción Científica y Tecnológica, Argentina (A97B01 and PICT 2350).
References
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