Laura Selgas

Twenty-Four-Hour Rhythms in Immune Responses in Rat Submaxillary Lymph Nodes and Spleen: Effect of Cyclosporine

Twenty-four-hour variations in cellularity, lipopolysaccharide (LPS)- and concanavalin A (Con A)-induced cell proliferation, and natural killer (NK) activity were examined in submaxillary lymph nodes and spleen of rats injected with Freunds complete adjuvant or its vehicle and kept under light from 08:00 to 20:00 h daily. A significant daily variation in cellularity was detected, exhibiting maxima at 09:00 h in submaxillary lymph nodes (nonimmunized and immunized rats) and at 13:00 h in spleen (immunized rats only). Submaxillary lymph node LPS- and Con A-mitogenic effect displayed maximal activity during daytime (peak at 13:00-17:00 h). In spleen, the maxima for 24-h rhythm in LPS-induced cell proliferation and NK activity occurred at midnight and at early morning (09:00 h), respectively. Con A-induced spleen cell proliferation peaked at midday in nonimmunized rats only. Injection of the immunosuppressive drug cyclosporine decreased Freunds adjuvant-induced augmentation of LPS and Con A mitogenic effect in both tissues and diminished spleen cell number. Cyclosporine blunted circadian rhythms in submaxillary lymph node Con A response and cell number, while it shifted the maximum in LPS effect to peak at 01:00 h. Cyclosporine also suppressed the circadian changes in LPS- and Con A-induced spleen cell proliferation, but not those found in NK activity. The results indicate the existence of 24-h rhythms in immune responses of rat submaxillary lymph nodes and spleen with maxima at different times of the day and that were significantly affected by cyclosporine injection.

Twenty-four-hour rhythms of serum ACTH, prolactin, growth hormone, and thyroid-stimulating hormone, and of median-eminence norepinephrine, dopamine, and serotonin, in rats injected with Freund's adjuvant.

The effect of Freunds adjuvant injection on 24-h variation of circulating ACTH, prolactin, growth hormone (GH), and thyroid-stimulating hormone (TSH) levels, and of norepinephrine (NE) content, and dopamine (DA) and serotonin (5HT) turnover in median eminence, was examined in adult rats kept under light between 0800 and 2000 h daily. Groups of 6-10 animals received Freunds complete adjuvant or its vehicle at 1100 h 3 days before sacrifice and were killed by decapitation at six different time intervals throughout a 24-h cycle. In rats injected with adjuvants vehicle, serum ACTH and prolactin exhibited peak values around the light-dark transition (p < 0.0001 and < 0.04, respectively), while the maximum in TSH was found in the late afternoon (p < 0.0001, one-way ANOVA). GH levels did not vary on a 24-h basis. In Freunds adjuvant-injected rats, 24-h variations of TSH levels became blunted, while 24-h variations of prolactin and ACTH persisted. Freunds adjuvant augmented serum ACTH and prolactin levels, and decreased GH and TSH levels (p < 0.0007, factorial ANOVA). Median-eminence NE content, and turnover of DA, assessed by measuring dihydroxyphenylacetic acid, DOPAC/DA ratio, and of 5HT, assessed by measuring 5-hydroxyindoleacetic acid, HIAA/5HT ratio, varied on a 24-h basis in rats receiving adjuvants vehicle (p < 0.02). Median-eminence NE content attained its maximum at 1600-2000 h, while maxima in DOPA/DA and HIAA/5HT ratios occurred at 0400 h. Injection with Freunds adjuvant reduced the amplitude of the daily variation of NE content, shifted the maximum of DOPAC/DA ratio toward the light-dark transition, and blunted the daily variation in HIAA/5HT ratio in median eminence. The administration at 1200 of the immunosuppressant drug cyclosporine (5 mg/kg, 5 days) restored the augmented ACTH and prolactin levels (p < 0.0001, factorial ANOVA) and depressed GH and TSH levels (p < 0.02) found in Freunds adjuvant-injected rats. Cyclosporine was also effective in restoring 24-h rhythmicity of serum ACTH and TSH, but not of prolactin, levels. Cyclosporine did not modify the effect of Freunds adjuvant on time-of-day changes of median-eminence NE content, but it was effective in counteracting the changes of DA and 5HT turnover found after immunization. The results are compatible with a significant effect of immune-mediated inflammatory response at an early phase after Freunds adjuvant injection on ACTH, GH, prolactin, and TSH release, which is partially sensitive to immunosuppression by cyclosporine.

DAILY VARIATIONS OF AMINO ACID CONCENTRATION IN MEDIOBASAL HYPOTHALAMUS, IN RATS INJECTED WITH FREUND'S ADJUVANT. EFFECT OF CYCLOSPORINE

Although the existence of central responses to inflammatory injuries was already reported, the existence of hypothalamic amino acid responses has been less explored. The present study was designed to characterize the 24-h changes in mediobasal hypothalamic excitatory and inhibitory amino acid neurotransmitter contents and to analyze the effect of Freunds complete adjuvant administration on these patterns. Also the effects of the immunosuppressant drug Cyclosporine was studied. The content of aspartate, glutamate, glutamine, GABA and taurine was measured by HPLC with fluorimetric detection. The results show the existence of specific daily rhythms of aspartate, glutamate, glutamine, GABA and taurine contents in the mediobasal hypothalamus of control rats. Maxima for these amino acids was found at midnight, although another peak of lesser magnitude, occurred during the light phase of the photoperiod, except for TAU in which both peaks were of similar magnitude. Freunds complete adjuvant administration did not modify the 24-h pattern of any amino acid studied. It reduced the midnight peak of glutamate, glutamine and GABA and increased that of taurine. Moreover, it increased and extended the midday peak of glutamate. Besides, Freunds adjuvant did not modify aspartate content at any time point studied. Cyclosporine pretreatment did not prevent the inhibitory effects of Freunds complete adjuvant on glutamate, glutamine and GABA midnight peaks. However, the drug blocked the increase in the content of taurine at midnight and increased its midday peak. Moreover, cyclosporine administration abolished the variations of ASP during the scotophase, as compared to control animals and shift delayed both peaks of glutamate. The results indicate the existence of a significant effect of immune-mediated inflammatory response of the mediobasal hypothalamic amino acids studied, at an early phase after Freunds adjuvant administration, and that these changes were partially sensitive to the immunosuppression induced by cyclosporine.

Circadian Rhythms in Adenohypophysial Hormone Levels and Hypothalamic Monoamine Turnover in Mycobacterial-Adjuvant-Injected Rats

The effect of Freund’s adjuvant injection on 24-hour variation in circulating ACTH, prolactin, growth hormone (GH) and thyroid-stimulating hormone (TSH) levels, and of hypothalamic norepinephrine (NE) content and dopamine (DA) and serotonin (5HT) turnover was examined in adult rats. In control rats, serum ACTH and prolactin exhibited peak values at the light-dark transition while the maximum in TSH was found in the late afternoon. GH levels did not vary on a 24-hour basis. In Freund’s-adjuvant-injected rats, 24-hour variations in TSH levels became blunted while 24-hour variations in prolactin and ACTH persisted. Freund’s adjuvant treatment augmented serum ACTH and prolactin levels, and decreased GH and TSH levels. Hypothalamic NE content, and turnover of DA and 5HT varied on a 24-hour basis in rats receiving adjuvant’s vehicle. The NE content of the anterior, medial and posterior hypothalamus peaked at 04.00 h, while that of the median eminence attained its maximum at 16.00–20.00 h. Maxima in hypothalamic DA and 5HT turnover occurred at 04.00 h regardless of the region examined. In Freund’s-adjuvant-injected rats, reduced amplitude of daily variations of NE content in the median eminence and anterior and medial hypothalamus, as well as a phase advance in the 24-hour rhythm of the posterior hypothalamic NE content were seen. Mycobacterial adjuvant injection also reduced the amplitude of circadian rhythm in hypothalamic 5HT turnover, shifted the maximum in median eminence DA turnover towards light-dark transition, and decreased the amplitude of DA turnover rhythm in the anterior, medial and posterior hypothalamus. Administration of the immunosuppressant drug cyclosporine restored the augmented ACTH and prolactin levels and the depressed GH and TSH levels found in Freund’s-adjuvant-injected rats. Cyclosporine was also effective to restore 24-hour rhythmicity of serum ACTH and TSH, but not of prolactin levels. Immunosuppression restored rhythmicity of NE content and of DA and 5HT turnover in anterior, medial and posterior hypothalamic regions. Cyclosporine did not modify the effect of Freund’s adjuvant on median eminence but in was able to counteract the changes in the DA and 5HT turnover in the median eminence found after immunization. The results are in accord with a significant effect of immune-mediated inflammatory response at an early phase after Freund’s adjuvant injection on ACTH, GH, prolactin and TSH release mechanisms, which was partially sensitive to immunosuppression induced by cyclosporine.

Diurnal rhythm in ornithine decar☐ylase activity and noradrenergic and cholinergic markers in rat submaxillary lymph nodes

Diurnal variations in lymph node ornithine decarboxylase activity were examined in submaxillary lymph nodes of rats injected with Freunds complete adjuvant or its vehicle. After immunization, lymph node ornithine decarboxylase activity increased by about 10-fold. Both in immunized and non-immunized rats, a significant diurnal variation in ornithine decarboxylase activity was found, with a maximal activity at early (i.e. 13.00 h, vehicle) or late afternoon (i.e. 17.00 h, Freunds adjuvant). Injection of Freunds adjuvant during daylight or at night resulted in similar day-night differences in submaxillary lymph node ornithine decarboxylase activity. In rats subjected to the sympathetic postganglionic denervation (by ipsilateral superior cervical ganglionectomy) or the preganglionic parasympathetic decentralization (by chorda tympani section) of submaxillary lymph nodes, nyctohemeral variations in ornithine decarboxylase were still present, showing a maximum at 17.00 h. Superior cervical ganglionectomy augmented lymph node ornithine decarboxylase while chorda tympani section decreased it. When a unilateral superior cervical ganglionectomy plus chorda tympani section was performed, the diurnal changes in ornithine decarboxylase were abolished. [3H]Norepinephrine uptake and tyrosine hydroxylase activity attained their maxima in submaxillary lymph nodes at early night. After immunization, these two presynaptic indicators of sympathetic activity in submaxillary lymph nodes augmented significantly. Neuronal [3H]choline uptake and [3H]choline conversion into acetylcholine (two indicators of cholinergic activity) also augmented in lymph nodes of rats injected with Freunds adjuvant. In immunized rats, maxima in [3H]choline uptake and [3H]acetylcholine synthesis were found at 13.00-17.00 h while in non-immunized rats, a maximum in acetylcholine synthesis was found at 17.00 h. The results are compatible with the view that the autonomic nervous system plays a role in circadian changes of immune responsiveness in lymphoid tissue and that a significant augmentation of presynaptic autonomic activity takes place during immunization in lymphoid tissue.

Diurnal rhythms in ornithine decarboxylase activity and norepinephrine and acetylcholine synthesis in submaxillary lymph nodes and spleen of young and aged rats during Freund's adjuvant-induced arthritis

Aging has been associated with attenuation of amplitude and changes in period of many circadian rhythms. The present study was carried out to examine, in young (50 days old) and old (18 months old) rats, whether 24-h rhythms of cell proliferation (as assessed by measuring ornithine decarboxylase activity) and of presynaptic adrenergic and cholinergic markers change in lymph nodes and spleen during Freunds adjuvant-induced arthritis. Groups of young and old Sprague-Dawley rats were studied the day before, and on days 6, 12 and 18 after Freunds adjuvant injection. On day 16 after adjuvant injection, inflammation of hind paws, mainly in the ankle joints, was less marked in old than in young rats. Lymph node and splenic ornithine decarboxylase activity exhibited significant 24-h variations with maximal activity during daily hours. Before treatment, enzyme activity values were significantly lower in old rats in both tissues examined. During the immune reaction, lymph node and splenic ornithine decarboxylase augmented 8-10-fold, with progressively smaller amplitude of daily variations as arthritis developed. In every case, mesor and amplitude of ornithine decarboxylase activity were lowest in old rats. Submaxillary lymph node and splenic tyrosine hydroxylase activity attained maximal values at night. At every time interval after mycobacterium adjuvant injection, amplitude and mesor of tyrosine hydroxylase activity rhythm were lowest in old rats. A maximum in submaxillary lymph node 3H-acetylcholine synthesis occurred at the afternoon. On day 6 and 12 after Freunds adjuvant injection, lymph node 3H-acetylcholine synthesis was significantly smaller in old rats. Day-night differences in submaxillary lymph node or splenic ornithine decarboxylase and tyrosine hydroxylase activities, or in submaxillary lymph node 3H-acetylcholine synthesis, of rats treated with the adjuvants vehicle, did not differ significantly from those seen in untreated controls. The results are compatible with an age-dependent decline of immune-mediated inflammatory responses. The activity of the central circadian oscillator, driven to the organs in part via the autonomic nervous system, seems also to deteriorate during aging.

Annual Physiology Symposium 1999

pp. 193–220 of this journal issue entitled: Annual Physiology Symposium 1999, Hong Kong, China, May 21–22, 1999

Diurnal changes in cyclosporine effect on ornithine decarboxylase and noradrenergic and cholinergic activities in submaxillary lymph nodes

Diurnal changes in cyclosporine efficacy to affect ornithine decarboxylase activity, [3H]norepinephrine uptake and [3H]choline conversion into [3H]acetylcholine were examined in rat submaxillary lymph nodes. Cyclosporine (5 or 20 mg/kg) caused a dose-dependent decrease in lymph node ornithine decarboxylase, being active at 5 or 20 mg/kg in Freunds adjuvant-treated rats, and at 20 mg/kg in rats treated with the adjuvants vehicle. In immunized rats the lower cyclosporine dose was ineffective when injected during the night. Cyclosporine increased lymph node [3H]norepinephrine uptake dose dependently, with significant differences between the 20 mg/kg dose and controls in vehicle-treated rats and between 5 or 20 mg/kg and controls in Freunds adjuvant-treated rats. In immunized rats, 5 mg/kg cyclosporine increased [3H]norepinephrine uptake when injected at 13:00 or 17:00 h. Both doses of cyclosporine augmented lymph node synthesis of [3H]acetylcholine to a similar extent. In immunized and non-immunized rats cyclosporine suppressed the diurnal rhythm of lymph node adrenergic and cholinergic activity found in controls. After unilateral sympathetic denervation (by superior cervical ganglionectomy) and/or unilateral parasympathetic decentralization (by chorda tympani section), cyclosporine (5 mg/kg) decreased Freunds adjuvant-induced activation of lymph node ornithine decarboxylase when injected at 17:00 or 01:00 h. On the sham-operated side, cyclosporine was effective when injected at 17:00 h only. Decentralization, or a combined ganglionectomy plus decentralization, decreased lymph node ornithine decarboxylase activity. The results active regulation of the effects of cyclosporine in submaxillary lymph nodes by local autonomic nerves.