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Am J Physiol Regul Integr Comp Physiol 280: R1806-R1814, 2001;
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Vol. 280, Issue 6, R1806-R1814, June 2001

Cytokine- and microbially induced sleep responses of interleukin-10 deficient mice

Linda A. Toth1 and Mark R. Opp2

1 Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794; and 2 Department of Psychiatry and Behavioral Sciences, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interleukin (IL)-1 and tumor necrosis factor (TNF) promote slow-wave sleep (SWS), whereas IL-10 inhibits the synthesis of IL-1 and TNF and promotes waking. We evaluated the impact of endogenous IL-10 on sleep-wake behavior by studying mice that lack a functional IL-10 gene. Under baseline conditions, C57BL/6-IL-10 knockout (KO) mice spent more time in SWS during the dark phase of the light-dark cycle than did genetically intact C57BL/6 mice. The two strains of mice showed generally comparable responses to treatment with IL-1, IL-10, or influenza virus, but differed in their responses to lipopolysaccharide (LPS). In IL-10 KO mice, LPS induced an initial transient increase and a subsequent prolonged decrease in SWS, as well as profound hypothermia. These responses were not observed in LPS-treated C57BL/6 mice. These data demonstrate that in the absence of endogenous IL-10, spontaneous SWS is increased and the impact of LPS on vigilance states is altered. Collectively, these observations support a role for IL-10 in sleep regulation and provide further evidence for the involvement of cytokines in the regulation of sleep.

interleukin-1; tumor necrosis factor; lipopolysaccharide; thermoregulation; influenza


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALL CYTOKINES DESCRIBED to date influence multiple physiological systems and processes. Several cytokines influence the normal sleep patterns of experimental animals and humans; the best characterized of these are interleukin (IL)-1 and tumor necrosis factor (TNF) (reviewed in Ref. 33). IL-1 and TNF are also critical mediators of responses to immune challenge. IL-10 is a component of an endogenous regulatory feedback system that limits the production of IL-1 and TNF under some conditions (18). In contrast to IL-1 and TNF, which promote slow-wave sleep (SWS) (reviewed in Ref. 34), exogenous IL-10 reduces the percentage of time spent in SWS by normal rats (35) and rabbits (28). These observations suggest antagonistic roles for IL-10 and IL-1-TNF in the regulation of normal patterns of sleep and in the modulation of alterations in somnolence during microbial infections.

The potential modulatory effects of endogenous IL-10 on sleep under normal conditions and during states of infectious or inflammatory disease can be evaluated by characterizing the sleep patterns of mice with a genetic inability to produce functional IL-10. To that end, we compared sleep patterns of normal C57BL/6 mice to those of C57BL/6 mice that lack a functional IL-10 gene [IL-10 knockout (KO) mice] under normal conditions, after injection with IL-10 or IL-1beta , and after challenge with bacterial lipopolysaccharide (LPS) or influenza virus. We now report that IL-10 KO mice exhibit more spontaneous sleep than C57BL/6 mice and that responses to LPS differ dramatically between these two mouse strains.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Adult male C57BL/6J and C57BL/6J-Il10tm1Cgn (IL-10 KO) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Individual shipments of mice were subjected to 3-4 wk of quarantine and were serologically verified at a 95-99% confidence level to be free of antibodies to mycoplasma and common rodent viruses before release from quarantine. All procedures used in these studies were approved in advance by the Animal Care and Use Committee at St. Jude Children's Research Hospital, where these studies were performed.

Surgical procedures and data acquisition. Before experimental use, mice were surgically implanted with instrumentation to permit monitoring of the electroencephalogram (EEG), the electromyogram (EMG), locomotor activity, and core body temperature. Mice were anesthetized with a mixture of ketamine (50 mg/kg sc) and xylazine (50 mg/kg sc) and were supplemented with methoxyflurane or additional ketamine-xylazine mixture during surgery as needed. A bland ophthalmic ointment was applied to the eyes to prevent desiccation, and sterile saline (1 ml) was administered intraperitoneally to maintain hydration. Aseptic techniques were used for all surgical procedures. Four insulated stainless steel wires (Plastics One, Roanoke, VA) bared at the tips were positioned parallel to and under the skull in bilateral frontal and parietotemporal positions to serve as EEG electrodes. One of these was made continuous with cable shielding and served as a ground; two of the remaining three were referenced in the combination providing the best differentiation of three vigilance states [wakefulness, SWS, and rapid eye movement sleep (REMS)]. EMG electrodes (Plastics One) were placed subcutaneously overlying the nuchal muscles. All electrodes were inserted into a pedestal that was secured to the skull with dental acrylic. During the same surgery, mice were implanted with intraperitoneal transmitters (Data Sciences, St. Paul, MN) to telemetrically quantify locomotor activity and core temperature. After surgery, mice were housed in individual cages in a sound-attenuated temperature-controlled chamber under a 12:12-h light-dark cycle at 22 ± 1°C.

EEG and EMG signals were processed through an eight-channel Grass polygraph. The EEG signals were passed through delta (1-4 Hz) and theta (4-8 Hz) filters (Coulbourn Instruments, LeHigh Valley, PA) and into a data-acquisition system (Cambridge Electronics Design, Cambridge, UK) that samples at 100 Hz, digitizes, and stores the digitized signals. EMG signals were similarly processed without filtering. All data were continuously sampled and stored on computer. Computer-assisted scoring employing custom software was used to assign vigilance states to each 10-s epoch during the recording period. Initially, EEG tracings were visually examined to determine a threshold delta-wave amplitude (DWA) associated with SWS for each animal. Thresholds for EMG associated with periods of movement and for ratios of theta to delta band amplitudes associated with REMS were also determined. These thresholds were then used to score the data for each animal in 10-s intervals for the entire experiment. An animal was considered to be in a state of SWS whenever the average DWA for any two consecutive 10-s intervals exceeded the SWS threshold in association with a low-amplitude EMG signal. REMS was identified by low-amplitude EEG and EMG signals that occurred in association with a high ratio of theta to delta amplitudes. At all other times, the animal was considered to be awake. All computer-scored data were visually reviewed to verify the accuracy of the computerized scoring.

Experimental protocols. Before the initiation of data collection, mice were given 1 to 2 wk to recover from surgery and to acclimate to the housing conditions. To permit collection of EEG and EMG data, mice were tethered to a six-channel electrical commutator with a lightweight cable that permitted unrestricted movement. Mice were acclimated to the tether for at least 3 days. In past studies, this acclimation period has proven sufficient to permit stable recording of sleep on subsequent sequential days (46). Throughout all recording sessions, the mice could move about freely in their cages and had continuous access to food and water.

The sleep patterns of C57BL/6 mice (n = 9) and IL-10 KO mice (n = 11) were evaluated during sequential 24-h recording periods. Recording was initiated at dark onset. During the initial 24-h period, data were collected from undisturbed mice that received no experimental treatment. The next evening, immediately before dark onset, mice were injected intraperitoneally with 0.2 ml of sterile pyrogen-free saline (PFS). On the third evening, mice were injected intraperitoneally with 0.2 ml of PFS containing 0.4 µg of either murine recombinant IL-1beta (R & D Systems, Minneapolis, MN) or IL-10 (R & D Systems). The same mice were evaluated after treatment with each cytokine, with half of the animals receiving IL-1 initially and the other half receiving IL-10 initially. An interval of at least 1 wk was permitted between studies.

After a minimum interval of 1 wk after completion of the preceding protocol, sleep patterns of the same mice were evaluated for 2 sequential days without experimental treatment. Four naive C57BL/6 and three naive IL-10 KO mice were added to this portion of the study (final n = 13 and 14, respectively, for each strain). On the morning of the third day, mice were lightly anesthetized with methoxyflurane and were inoculated intranasally as previously described (46) with 50 µl of allantoic fluid containing ~1,000 hemagglutinating units of strain A/HKx31 influenza virus (44). Recordings continued for the next 96 h. The 24- to 48-h postinoculation interval (day 2 postinoculation) was chosen for evaluation because we (46) and others (15) find that this interval is characterized by robust increases in SWS in influenza-infected mice. To avoid characterizing sleep in moribund animals, we routinely excluded from analysis any mice that died on the following day. None of the mice we studied died during the 48- to 72-h period after influenza inoculation (day 2). Two of fourteen IL-10 KO mice were euthanized for humane reasons on day 4 at ~75 h postinoculation. Two of twelve C57BL/6 mice and one IL-10 KO mouse were found dead 96 h after inoculation (i.e., they died sometime during the night on day 4). All mice were killed by cervical dislocation under methoxyflurane anesthesia at the end of the recording period. Previous work has shown that intranasal inoculation with uninfected allantoic fluid does not influence the normal sleep-wake patterns of C57BL/6 mice (46). Therefore, the sleep patterns that developed after influenza inoculation were compared with those obtained during the baseline period for each animal.

A separate group of mice (C57BL/6, n = 7; IL-10 KO, n = 15) was used to evaluate the effects of LPS on sleep-wake behavior. For this study, recordings were obtained for 24 h after the administration of 0.2 ml of PFS and then for 48 h after administration of 10 µg of LPS (Escherichia coli serotype O111:B4; Sigma, St. Louis, MO) in 0.2 ml of PFS. All injections were given intraperitoneally and were administered immediately before dark onset. These mice were used for only one experimental treatment and were killed by cervical dislocation under methoxyflurane anesthesia at the end of the recording period.

Statistical analyses. All values presented are means ± SE for indicated sample sizes. Spontaneous sleep-wake patterns of IL-10 KO mice and C57BL/6 mice were compared using one-way ANOVA in which the percentage of time spent in each vigilance state (SWS, REMS, and wakefulness), DWA during SWS, core body temperature, and locomotor activity were the dependent variables. Values were analyzed for the 12-h time blocks that comprised either the light or the dark phase of the light-dark cycle. The main effect in this analysis was mouse strain (i.e., the background C57BL/6 strain vs. the IL-10 KO strain). Comparison of the effects of test substances on subsequent sleep-wake patterns were also made using one-way ANOVAs, but in these analyses the main effect was manipulation (baseline or vehicle, test substance); comparisons were made within strains. Strain differences in responses to test substances were assessed based on difference scores calculated for each parameter, subject, and time period. Difference scores were obtained by subtracting values obtained during baseline recordings or after administration of vehicle from those obtained after administration of test substances. These difference scores were the dependent variables in one-way ANOVAs, with the main effect being mouse strain. An alpha -level of P < 0.05 was considered to indicate a statistically significant difference between groups.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline patterns of sleep, temperature, and locomotor activity. Sleep patterns of untreated and undisturbed C57BL/6 mice and IL-10 KO mice were evaluated for 24 h. Patterns of core body temperature, locomotor activity, REMS, and DWA during SWS did not differ between strains. However, during the dark phase of the light-dark cycle, IL-10 KO mice spent significantly more time in SWS and less time in waking than did C57BL/6 mice (Fig. 1).


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  		Fig. 1.   Spontaneous patterns of slow-wave sleep (SWS), rapid eye movement sleep (REMS), waking, delta wave amplitudes (DWA) during SWS, core body temperature (Tcore) and locomotor activity of C57BL/6 and IL-10 knockout (KO) mice. After acclimation to the recording conditions, C57BL/6 (n = 9, open circle ) and IL-10 KO (n = 11, ) mice were monitored for 24-h without disturbance. Horizontal bars and *, time periods when SWS and waking amounts differed between the 2 strains. Data points are the means ± SE. Black bars on the x-axis depict the dark phase of the light-dark cycle.

Effects of IL-1. Intraperitoneal administration of 0.4 µg of IL-1beta to C57BL/6 or IL-10 KO mice immediately before dark onset did not alter the duration of SWS, REMS, or wakefulness across 12-h recording periods (Table 1). However, when restricted to the first 2-h postinjection, ANOVA revealed IL-1-induced alterations in vigilance state duration for both mouse strains; SWS increased (C57BL/6: F1,16 = 12.9, P = 0.002; IL-10 KO: F1,20 = 16.6, P = 0.001) and wakefulness was reduced (C57BL/6: F1,16 = 11.4, P = 0.004; IL-10 KO: F1,20 = 16.5, P = 0.001; Fig. 2). In addition, DWA during SWS and locomotor activity were depressed for the 12-h dark phase immediately after IL-1 administration (Fig. 2, Table 1). Core body temperature was modestly reduced during the dark phase and slightly increased thereafter. Effects on all parameters measured were similar in magnitude and duration in both mouse strains.

                              
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  		Table 1.   Effects of interleukin-1 or A/HKx31 influenza virus on amount of time spent in vigilance states, DWA during SWS, Tcore, and locomotor activity of C57BL/6 and IL-10 KO mice



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  		Fig. 2.   Effects of interleukin (IL)-1beta on sleep-wake behavior, DWA during SWS, Tcore, and locomotor activity. C57BL/6 (n = 9, open circle ) and IL-10 KO mice (n = 11, ) were injected intraperitoneally with 0.2 ml of pyrogen-free saline (vehicle) or with 0.4 µg of IL-1 immediately before dark onset. The values presented are mean differences ± SE from values obtained after vehicle administration, represented by the zero line. Horizontal bars and *, time periods when responses of IL-1-treated C57BL/6 and IL-10 KO mice differed from those of the same mice treated with vehicle. There were no statistically significant differences between strains in responses to IL-1. Black bars on the x-axis depict the dark phase of the light-dark cycle.

Effects of IL-10. Intraperitoneal administration of 0.4 µg of IL-10 immediately before dark onset did not significantly alter any measured parameter in either C57BL/6 or IL-10 KO mice (data not shown).

Effects of influenza infection. Data collected on the day before influenza inoculation were compared with data collected during the 24- to 48-h period after influenza inoculation. Previous studies indicate that this postinoculation interval is associated with significant nocturnal sleep enhancement in C57BL/6 mice (44, 46). Inoculation of C57BL/6 mice with influenza virus was associated with significant alterations in SWS, REMS, and waking compared with sleep during the baseline period (Fig. 3, Table 1). During the dark portion of the light-dark cycle, the amount of time spent in SWS was significantly increased, and the amount of wakefulness was reduced, whereas the amount of REMS was reduced during the 12-h light phase. Locomotor activity, DWA during SWS, and core body temperature were reduced during the entire postinoculation period (Fig. 3, Table 1).


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  		Fig. 3.   Effects of influenza virus on sleep-wake behavior, Tcore, and locomotor activity of C57BL/6 and IL-10 KO mice. Immediately after light onset, C57BL/6 (n = 13, open circle ) and IL-10 KO (n = 14, ) were lightly anesthetized with methoxyflurane and inoculated intranasally with 50 µl of allantoic fluid containing ~1,000 hemagglutinating units of strain A/HK×31 influenza virus. The values presented are mean differences ± SE between values obtained during the 24- to 48-h period after inoculation and those obtained during corresponding baseline periods (represented by the zero line). Black bars on the x-axis depict the dark phase of the light-dark cycle. *P < 0.05 for within-strain comparisons (i.e., comparison of infected vs. uninfected mice of the same strain); #P < 0.05 for between-strain comparisons (i.e., C57BL/6 vs. IL-10 KO mice after infection).

The sleep patterns of influenza-infected IL-10 KO mice were qualitatively similar to those of C57BL/6 mice, although the SWS enhancement during the dark phase was somewhat lower in magnitude (Fig. 3). Alterations in REMS, waking, core body temperature, and locomotor activity were similar in both strains. However, the dark-phase reduction in DWA during SWS was greater in IL-10 KO than in C57BL/6 mice (Fig. 3).

Effects of LPS administration. Administration of 10 µg of LPS to C57BL/6 mice immediately before dark onset altered both SWS and REMS (Fig. 4, Table 2). The amount of time spent in SWS increased significantly during the initial 12 h after injection, whereas the amount of REMS was reduced during the first 24-h period after injection. Locomotor activity and DWA during SWS were also reduced during the initial 24 h, but core body temperature was not greatly affected (Fig. 4, Table 2). All parameters had returned to normal or near-normal levels by ~28 to 30 h postinjection.


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  		Fig. 4.   Effects of lipopolysaccharide (LPS) on sleep-wake behavior, Tcore, and locomotor activity. C57BL/6 (n = 7, open circle ) and IL-10 KO (n = 15, ) mice were injected intraperitoneally with 0.2 ml of pyrogen-free saline (vehicle) or with 10 µg of LPS (Escherichia coli serotype O111:B4) immediately before dark onset. The values presented are mean differences ± SE between values obtained after administration of LPS and those obtained after administration of vehicle (represented by the zero line). Black bars on the x-axis depict the dark phase of the light-dark cycle. *P < 0.05 for between-strain comparisons (i.e., C57BL/6 vs. IL-10 KO mice after challenge).


                              
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  		Table 2.   Effects of LPS on amount of time spent in vigilance states, DWA during SW, Tcore, and locomotor activity of C57BL/6 and IL-10 KO mice

IL-10 KO mice injected with 10 µg of LPS developed a transient increase in SWS during the initial 2 h after injection but then showed profound decreases in the percentage of time spent in both SWS and REMS (Fig. 4, Table 2). Marked reductions in DWA during SWS, locomotor activity, and core body temperature developed with a similar time course. All parameters were reduced throughout the entire 48-h postinjection observation period, with the exception of locomotor activity, which was reduced for only 36 h. The LPS-induced alterations in sleep-wake behavior, DWA during SWS, and in core body temperature of IL-10 KO mice differed from those of C57BL/6 mice for most time periods, whereas changes in locomotor activity were similar in both strains (Fig. 4, Table 2).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented here demonstrate that the inability to produce IL-10 is associated with altered sleep patterns in otherwise normal mice and in mice undergoing some types of immune challenge. Untreated IL-10 KO mice spend significantly more time in SWS and less time in wakefulness than do genetically intact C57BL/6 mice, although the magnitude of these differences is modest. A tendency toward increased SWS and reduced waking also occurs in vehicle-treated IL-KO mice but is not apparent in influenza-infected mice that had previously received cytokine treatment, suggesting that prior manipulation could prevent or obscure this subtle difference. The increased sleep propensity seen in untreated IL-10 KO mice is consistent with previous demonstrations that central administration of IL-10 reduces sleep in rats and rabbits (28, 35). Sleep is also reduced in rabbits after central administration of IL-4 (29), a cytokine that shares many properties with IL-10, including the ability to inhibit production of IL-1 and TNF (30). These findings support the hypothesis that interactive cytokine networks contribute to the normal regulation of sleep.

The dramatic behavioral and physiological responses of IL-10 KO mice to LPS administration are likely to reflect their previously reported sensitivity to the toxic effects of LPS (5). The impact of endogenous IL-10 on sensitivity to LPS is perhaps best illustrated by the markedly divergent temperature responses of the genetically intact and the IL-10 KO mice. Core body temperature of C57BL/6 mice is not substantively affected by the dose of LPS used in this study, whereas IL-10 KO mice develop profound and prolonged hypothermia, an observation previously reported (31). Similarly, LPS-treated C57BL/6 mice, similar to other species (26, 27, 36), develop an increase in the percentage of time spent in SWS, whereas LPS-treated IL-10 KO mice show only a transient increase followed by a profound decrease. The hypothermia and marked reduction in time spent in SWS and in DWA during SWS of IL-10 KO mice treated with this dose of LPS may reflect endotoxic shock. Profound sleep and DWA suppression also occurs in rabbits with fatal microbial infections (47), in mice with fatal rabies encephalitis (21), and in aged mice before spontaneous death (52). Such responses would be anticipated in C57BL/6 mice if treated with a substantially higher dose of LPS. Conversely, treatment of IL-10 KO mice with a lower dose of LPS elicits less severe effects on core body temperature and other physiological parameters (31). By rendering mice more sensitive to endotoxic shock, the genetic absence of IL-10 appears to promote the development of sleep patterns that are associated with fatality, rather than recuperation. Our data therefore corroborate previous reports that IL-10 provides powerful protection against the severity of endotoxemia (23, 50).

Because IL-10 inhibits the synthesis of IL-1 and TNF under some conditions (18, 24), it is generally viewed as an endogenous antagonist to these cytokines (1). Without the counterregulatory control normally provided by IL-10, responses to some challenges are exacerbated. For example, in a model of chronic Pseudomonas pneumonia, IL-10 KO mice exhibit more severe weight loss and greater pulmonary inflammation than do wild-type mice, and intraperitoneal administration of exogenous IL-10 improves survival and reduces inflammation (8). In a model of Borrelia arthritis, IL-10 KO mice develop higher bacterial titers and more severe arthritis than wild-type mice, and strains of mice that produce greater amounts of endogenous IL-10 have less severe symptoms than those that produce lower amounts (7). IL-10 also inhibits hepatic injury induced by LPS, concanavalin-A, and staphylococcal enterotoxin B (11, 32). Furthermore, central injection of IL-10 antagonizes the suppressive effects of central or systemic LPS on behavior (6). In influenza-infected mice, IL-10 production is increased in lung homogenates and bronchoalveolar lavage cells for up to 10 days after challenge (4, 37, 39, 40). However, in contrast to the marked alterations in LPS-induced somnolence associated with IL-10 deficiency, this genetic manipulation has little impact on the somnogenic effects of influenza virus.

Previous studies in which IL-10 was administered centrally demonstrate that exogenous IL-10 reduces spontaneous sleep in rats and rabbits (28, 35), supporting the hypothesis of cytokine involvement in the regulation of sleep. The well-documented enhancement of SWS by IL-1 and TNF, together with data demonstrating an inhibitory effect of IL-10 on the synthesis of these cytokines, suggests inhibition of IL-1 and/or TNF synthesis as one mechanism for IL-10-induced sleep reduction. On the basis of those observations, we expected that exogenous IL-10 would also reduce sleep in mice, particularly during the light phase of the light-dark cycle, when their sleep amounts are relatively high. However, the dose of IL-10 used in this study did not influence any parameter we measured in either C57BL/6 or IL-10 KO mice. In this present study we tested only one dose of IL-10, which was administered immediately before dark onset. Thus either the dose or the circadian timing of administration may have been inappropriate to alter sleep. Previous work has demonstrated that a low dose of IL-10 induces sleep suppression within a few hours of administration, whereas a higher dose reduces sleep only after a 24-h latency (35). Thus a detectable reduction in sleep might have been apparent had we administered IL-10 at light onset immediately before the circadian phase in which the percentage of time spent in SWS is normally high. Differences in the species tested and the route of IL-10 administration could also account for the differences between our findings and previous reports (28, 35). Previous studies administered IL-10 directly into the central nervous system via intraventricular cannulas, whereas in the present study IL-10 was injected peripherally. Although systemic IL-10 can inhibit LPS-induced TNF production in the mouse brain (12, 13), systemic IL-10 may not alter basal cytokine production in brain to the extent necessary to cause detectable alterations in the behavior of normal, unchallenged animals. The intravenous administration of IL-10 increases mRNA expression of basic fibroblast growth factor, but not nerve growth factor, in the rat hypothalamus (53). However, central administration of basic fibroblast growth factor does not alter sleep in rats or rabbits (22).

In our study, IL-1-induced sleep enhancement of mice persisted for only 2 h, whereas Fang and colleagues (17) report increased somnolence lasting for 6 h after injection of the same dose of IL-1. These differences in the duration of the effect could be related to the use of different mouse strains or different environmental temperature. Fang and colleagues (17) used mice with a mixed C57BL/6/129 genetic background, whereas we studied inbred C57BL/6 mice. Different genetic backgrounds can significantly influence the expression of many behavioral traits (20), including sleep (10, 19, 25, 38, 41, 43, 48, 49). In addition, mice studied in the previous report were housed at 30 ± 1°C, whereas our mice were housed at 22 ± 1°C. Higher ambient temperatures are associated with greater amounts of sleep in mice (38). Finally, even subtle variations in experimental technique can influence the measurement and assessment of complex behavioral phenotypes (9). Thus the results obtained in different studies may be influenced by the array of recording devices implanted to permit the assessment of sleep and by the method used to assign vigilance state (i.e., the scoring algorithm) (45, 51).

Physiological and behavioral responses to IL-1 administration were similar in IL-10 KO and genetically intact C57BL/6 mice. Developmental or physiological compensation for the missing endogenous substrate could underlie these similarities (20). In addition, IL-10 has been more frequently identified as a physiological antagonist of TNF-mediated, rather than IL-1-mediated, responses. For example, systemic administration of IL-10 inhibits the production of TNF in brain of LPS-treated mice (12, 14). IL-10 also inhibits TNF mRNA expression in mouse brain induced by LPS or staphylococcal enterotoxin B (32). Conversely, endogenous TNF alters the production of IL-10 in LPS-treated mice (2, 3). Mice that lack the TNF 55-kDa receptor show less spontaneous sleep than do normal mice and do not develop sleep enhancement in response to peripheral administration of TNF (16); however, these mice do show sleep enhancement in response to IL-1 administration (42). The lack of a functional IL-10 gene may thus impact TNF-mediated processes more than IL-1-mediated processes. Our observation that C57BL/6 and IL-10 KO mice show similar responses to IL-1 supports this conclusion, although additional experiments would be necessary to directly test this hypothesis.

In conclusion, the data presented here demonstrate that spontaneous sleep is increased and wakefulness reduced in mice that lack functional IL-10. In addition, the impact of LPS on vigilance states, similar to its impact on survival, is profoundly altered in the absence of IL-10. These observations support a role for IL-10 in the regulation of normal patterns of sleep and in the modulation of alterations in somnolence during microbial challenge.


    ACKNOWLEDGEMENTS

The authors thank J. Raucci and E. Kuliyev for technical assistance.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants NS-26429 (to L. A. Toth), MH-54976 (to M. R. Opp), and CA-21765 (to L. A. Toth) and the American Lebanese Syrian Associated Charities (to L. A. Toth).

Address for reprint requests and other correspondence: M. R. Opp. Dept. of Anesthesiology, M-7433 Medical Sciences Bldg. 1, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0615 (E-mail: mopp{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9 November 2000; accepted in final form 22 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 280(6):R1806-R1814
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