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* Department of Neuroendocrinology, University of Lübeck, Germany;
Department of Internal Medicine, University of Lübeck, Germany;
Department of Medical Biochemistry and Immunology, The School of Medicine, Cardiff; University, Cardiff, Wales, UK; and
Department of Biochemistry, Christian-Albrechts University of Kiel, Kiel, Germany
1Correspondence: Department of Neuroendocrinology, University of Lübeck, Ratzeburger Allee 160, Haus 23a, Lübeck 23538, Germany. E-mail: born{at}kfg.uni-luebeck.de
SPECIFIC AIMS
Sleep is commonly considered to support immune defense. The underlying sleep-immune interaction appears to critically rely on cytokines, like interleukin-6 (IL-6), which influence both immune and neuronal functions. Studies outlined here aimed to define how sleep and circadian rhythm affect the capacity for IL-6 signaling. For this purpose, plasma concentrations of soluble IL-6 receptor (sIL-6R) and expression of membrane-bound IL-6 receptor (mIL-6R) were studied in conjunction with changes in IL-6 production by monocytes in healthy humans during 24 h of continuous wakefulness and during a regular sleep-wake cycle. Such analyses allow us to comment on the potential signaling capacity of IL-6 depending on sleep and to propose an involvement for sIL-6R-mediated signaling (a process termed IL-6 trans-signaling) during the normal sleep-wake cycle.
PRINCIPAL FINDINGS
1. mIL-6R density on leukocytes shows circadian variation but is not affected by sleep
Diurnal expression patterns of mIL-6R (CD126+) were studied by flow cytometry in leukocyte subpopulations. Granulocytes and monocytes, and 
80% of T cells, were positive for mIL-6R. Although sleep did not affect the surface expression of this receptor, expression of mIL-6R showed a distinct circadian fluctuation, with maximal expression observed during the latter half of the night.
2. Sleep strongly increases sIL-6R concentration without changing sgp130 concentration
In contrast to the circadian regulation of mIL-6R, plasma sIL-6R were significantly elevated during nocturnal sleep (Fig. 1
A). The temporal increase in sIL-6R was most pronounced during late sleep (after 2:00 h) and peaked shortly after awakening, when levels exceeded those during continuous wakefulness by >70% (P<0.01). Cosinor analyses confirmed the absence of any circadian rhythm during continuous wakefulness (P>0.41) but applied to the data of the sleep condition, a systematic diurnal variation was detected (P<0.01), with the fitted cosine curve showing a peak shortly after awakening from sleep. By contrast, plasma concentrations of soluble gp130 (sgp130), which antagonizes the IL-6/sIL-6R complex, remained unaffected by sleep (Fig. 1B
, P>0.1) and showed no evidence of regulation as a consequence of the circadian rhythm (P>0.13).
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3. Sleep-dependent regulation of sIL-6R concentration relates to enhanced proteolytic cleavage of the cognate IL-6R
Circulating levels of sIL-6R are generated through either proteolytic cleavage of the mIL-6R (PC-sIL-6R) or differential IL-6R mRNA splicing (DS-sIL-6R). To determine which of these two mechanisms contributes to the sleep-dependent elevation in sIL-6R, we quantified DS-sIL-6R levels using a monoclonal antibody (mAb) that recognizes the unique COOH-terminal amino acid sequence for DS-sIL-6R. Although the natural circadian rhythm did not affect the secretion of DS-sIL-6R (during continuous wakefulness (P>0.9), levels of this sIL-6R isoform were significantly enhanced by sleep (Fig. 1C
, P<0.05). These changes are consistent with the overall regulation of sIL-6R; however, the sleep-dependent increase in DS-sIL-6R levels was delayed by 6 h compared with that of total sIL-6R. Indeed, DS-sIL-6R levels did not peak until 14:00 h (P<0.01), suggesting that sleep coordinates a differential control of PC- and DS-sIL-6R secretion, with the release of PC-sIL-6R preceding that of DS-sIL-6R.
4. IL-6-producing monocyte counts show circadian but not sleep-dependent changes
To see whether the sleep-dependent increase in sIL-6R levels is paralleled by changes in IL-6 activity, we determined by flow cytometry the number of IL-6-producing monocytes. Monocytes represent the major cellular source of IL-6 in whole blood. The percentage of monocytes (expressed as proportion of the total number of monocytes) producing IL-6 showed a clear-cut 24 h variation, with maximum activity at 3:00 h (P<0.01, for respective cosinor analysis). This variation was independent of sleep and wakefulness (P>0.5, Fig. 1D
).
5. Sleep-induced changes in sIL-6R concentrations correlate with time in specific sleep stages and sleep-associated hormone release
To explore whether the sleep-dependent increase in sIL-6R plasma concentrations was linked to specific sleep stages or hormonal release, we calculated Pearson correlation coefficients to evaluate potential links between sIL-6R release and 1) the time spent in different sleep stages, and 2) sleep-associated hormone concentrations. A substantial negative correlation was observed between sIL-6R levels seen during early sleep (0:30–2:00 h) and the time in slow wave sleep (SWS, r=–0.66, P<0.01), as well as with the average GH concentration observed during this period (r=–0.52, P<0.05). sIL-6R levels during late night (3:30–8:00 h) were positively correlated with the time in rapid eye movement sleep (REM, r=0.54, P<0.05).
CONCLUSIONS AND SIGNIFICANCE
Data presented here emphasize a tight association between sleep and the temporal fluctuation of inflammatory mediator concentrations in normal healthy individuals. Specifically, comparative studies using volunteers either presenting with a normal sleep pattern or subjected to continuous wakefulness demonstrated that sleep, together with circadian rhythm, influence the potential control of IL-6 responses (Fig. 2
). These events appear to be highly orchestrated, with increases in sIL-6R typically bridging the late period of sleep dominated by REM sleep and the first hours after morning awaking. Regular sleep did not affect membrane-bound IL-6R expression or promote a concomitant increase in the proportion of IL-6 secreting monocytes. These parameters did, however, increase during the nighttime, suggesting they are governed as part of a circadian rhythm. Consequently, sleep-induced increases in sIL-6R levels coincide with a circadian increase in IL-6 levels allowing for increased formation of IL-6/sIL-6R complexes. Such changes would potentially enhance the capacity for IL-6 trans-signaling in cell types that do not inherently respond to IL-6 itself. To substantiate this claim, plasma sgp130 concentrations were monitored. Soluble gp130 acts as the natural antagonist for IL-6 trans-signaling, and circulating levels of this negative regulator remained unaffected by sleep or by circadian rhythm.
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Using the comparative approaches designed to delineate between sleep and circadian-induced changes of IL-6 receptor components, it is evident that regulation of the cognate mIL-6R within leukocyte subpopulations is distinct from that of sIL-6R liberation. Specifically, our results show a circadian increase in leukocyte IL-6R expression that peaked sometime after that of IL-6, whereas modulation in sIL-6R was completely reliant on sleep. Such processes may point to a distinction in manner by which classical IL-6 signaling and IL-6 trans-signaling is governed. The mechanisms and consequence for this divergent regulation remain unresolved.
Another level of complexity is offered by the differential control of the sIL-6R isoforms (termed PC-sIL-6R and DS-sIL-6R) as a consequence of sleep. Although both sIL-6R variants showed no evidence of circadian regulation, the temporal emergence of sleep-dependent increases in PC-sIL-6R and DS-sIL-6R levels was distinct. In particular, sleep-induced increases in total sIL-6R concentrations (containing to the greatest part the PC variant) peaked around morning awakening, whereas maximal DS-sIL-6R levels were not detected until 6 h (14:00 h) later. Although the mechanisms underlying this delay remain obscure, the profile of PC-sIL-6R and DS-sIL-6R release is consistent with the known kinetics of production and their potential regulation by defined mediators. Activators of sIL-6R shedding are typically rapid, and significant increases in PC-sIL-6R can be seen within 30–120 min of stimulation. In contrast, secretion of DS-sIL-6R is relatively slow, needs de novo synthesis, and secretion of this isoform typically does not occur until 8–24 h after activation. Thus, sleep might stimulate production and release of these receptor isoforms at an early stage, which, according to the different mechanism and speed of synthesis, evolves into increased circulating receptor concentrations at different times. To date, the biological significance of these two forms remain unclear, but both promote IL-6 trans-signaling events.
To identify possible mediators involved in the regulation of sIL-6R during sleep, we measured several hormones with known sleep-dependent patterns of release. Of these, the sleep-dependent peak in GH concentration showed a negative correlation with sIL-6R concentrations during early sleep, which is consistent with reports that GH suppresses sIL-6R levels. Regulation of sIL-6R by GH is further supported by the fact that sIL-6R concentrations show a negative correlation with SWS, typically associated with enhanced somatotropic secretory activity.
In conclusion, we propose that sleep under normal conditions specifically increases sIL-6R and thus enhances IL-6 trans-signaling, i.e., the effects of IL-6 on all cells expressing membrane gp130 (in the absence of mIL-6R). These data present a defined relationship between sleep and cytokine control of homeostatic housekeeping functions. However, given the involvement of IL-6 and IL-6 trans-signaling in various inflammatory diseases, sleep may also act to extend or enhance IL-6-mediated processes. Consequently, sleep may contribute not only to the basal control of homeostatic processes in multiple organ systems, but within an inflammatory context may ultimately dictate the manner by which cytokine responses are governed. Such regulation may, for instance, relate to the early morning joint stiffness seen in arthritic patients in which IL-6 (via sIL-6R) is known to perform a detrimental role.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5754fje
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